Research ArticlePharmacokinetics Tissue Distribution and Metabolism Study ofIcariin in Rat

Shunjun Xu1 Jiejing Yu2 Jingjing Zhan2 Liu Yang12 Longgang Guo1 and Yijuan Xu1

1Guangzhou ImVin Pharmaceutical Co Ltd Guangzhou 510663 China2Guangdong Provincial Hospital of Chinese Medicine Guangzhou University of Chinese Medicine Guangzhou 510120 China

Correspondence should be addressed to Liu Yang yangliu979hotmailcom

Received 6 June 2017 Accepted 4 October 2017 Published 13 November 2017

Academic Editor Stephen H Safe

Copyright copy 2017 Shunjun Xu et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Icariin is one of the predominant flavonoids contained in Herba Epimedii (Yin-yang-huo in Chinese) a well-known Chinesemedicine for the treatment of cancers and immune system diseases Although Herba Epimedii has been widely used in China andthere are so many and various research reports on the herbal drug and its main flavones very limited data is available on the tissuedistribution and biotransformation of icariin In the present study a liquid chromatographic method combined with electrosprayionization tandem mass spectrometry was developed to quantify the concentration of icariin in rat plasma and various tissuescollected at different time points after oral administration of the total flavonoid extract of Herba Epimedii at a dose of 069 gkg(corresponding to 42mgg icariin) Biological samples were processed by simple protein precipitation Genistein was chosen asinternal standard The method was successfully applied to plasma pharmacokinetic and tissue distribution studies of icariin inrat As a result it was worth noting that the tissue distribution characteristics of icariin exhibited a significant gender differenceMoreover in vivometabolism of icariin was also investigated A total of 11 potential metabolites were found in rat feces collected indifferent time periods after oral and intramuscular administration of icariin In vivometabolic pathwayswere involved in hydrolysisdemethylation oxidation and conjugation The preclinical data would be useful for fully understanding in vivo disposition of thiscompound and interpreting the mechanism of its biological response

1 Introduction

Herba Epimedii the fresh or dried aerial parts of Epimediumgenus has been used not only as a kind of food but also asa Chinese herbal drug for thousands of years It is officiallylisted in all versions of Chinese pharmacopoeia with thename ldquoYin-yang-huordquo which originates from 4 species ofEpimedium that is E brevicornum Maxim E sagittatumMaxim E pubescensMaxim and E koreanumNakai HerbaEpimedii possesses estrogen-like activity and prevention ofosteoporosis [1ndash3] improvement of sexual function [4 5]regulation of immune response [6 7] and putative anticanceractivity [8] Icariin is themost abundant bioactive componentof this herbal drug There has been a lot of literatureshowing that icariin has various biological activities such asanticancer and immune regulation prevention and treatmentof osteoporosis prolongation of lifespan and improvementof sexual function [9ndash14] Therefore icariin has received

more and more attention from medical and pharmaceuticalresearchers

Despite icariin displaying a wide variety of bioactivitiesit has been reported that this compound has very low oralbioavailability Chen et al [15] reported that prenylatedflavonoids in Herba Epimedii such as icariin epimedin Aepimedin B and epimedin C should be hydrolyzed intosecondary glycosides in small intestine prior to absorptionOur study [16] also demonstrated that the recovery of icariinin urine was less than 0425permil suggesting that most of icariinshould be metabolized and excreted as metabolites Clearlyit is necessary to investigate the in vivo drug disposition oficariin and to identify its metabolites which is useful forunderstanding the biological activity of icariin

Thepharmacokinetics of icariin in rat has been previouslyinvestigated and reported Lu et al [17] developed a UPLC-MSMS method to quantify icariin in rat plasma after oraladministration of a Chinese herbal preparation and the

HindawiBioMed Research InternationalVolume 2017 Article ID 4684962 17 pageshttpsdoiorg10115520174684962

2 BioMed Research International

O

OOH

O

O

O

O

OH

OH

OH

OHOH

HO

HO

OCH3

(a)

O

OOH

HO

OH(b)

Figure 1 Chemical structure of icariin (a) and genistein (internal standard b)

results indicated that there is no statistic difference in themain pharmacokinetic parameters of icariin between maleand female ratsWang et al [18] established anHPLCmethodfor the determination of epimedins A B and C and icariin inplasma with peak concentrations of 045 plusmn 001 053 plusmn 002411 plusmn 008 and 131 plusmn 007 120583gmL respectively about 025 hafter oral administration of Herba Epimedii extract (10 gkgbody weight) to rats Although these articles separatelyfocused on pharmacological plasma pharmacokinetic andmetabolism studies of icariin the systemic in vivodispositionespecially tissue distribution and complete metabolic profileof this compound has not been summarized so far

In the present study we established and validated aquantitative LC-MSMS method of icariin in rat plasmaand various tissue homogenate and successfully applied themethod to pharmacokinetic and tissue distribution studies oficariin We also investigated the tissue distribution featuresof icariin in male and female rats and minutely summarizedthe similarities and differences of icariin distribution betweenmale and female rats Moreover we analyzed the completemetabolic profiles of this compound in rat feces and urinefollowing oral and intramuscular administration Accord-ingly the metabolites of icariin were identified or tentativelycharacterized and the potential metabolic pathways of icariinwere also described in this paper

2 Materials and Methods

21 Chemicals and Reagents Icariin (purity 98) and genis-tein (IS purity 98) were purchased from the NationalInstitute for the Control of Pharmaceutical and Biolog-ical Products (Beijing China) Icaritin was provided byShanghai Winherb Medical Technology Co Ltd (ShanghaiChina) Icariside II baohuoside II sagittatoside B and 210158401015840-O-rhamnosyl-icariside II were purchased from Baoji HerbestBio-Tech Co Ltd (Baoji China) Their purities were gt98determined by HPLC-UV analysis The chemical structuresof icariin and IS are shown in Figure 1 Herba Epimediiextract originating from E sagittatumMaxim was producedand provided by Guangzhou ImVin Pharmaceutical CoLtd (Guangzhou China) LC grade methanol and acetoni-trile were obtained from Fisher Company (Fisher Scientific

Fairlawn NJ) HPLC-grade formic acid was purchased fromTedia Company Inc (Fairfield OH USA) Water was puri-fied using aMillipore purification system (MilliporeMilfordMA USA) All of other chemicals and reagents were ofanalytical grade and were commercially available

22 Chromatographic Conditions For plasma pharmacoki-netic and tissue distribution studies the LC-MSMS systemconsisted of a Shimadzu LC-20A HPLC system and anApplied Biosystems Sciex API 4000+ (MDS-Sciex ConcordCanada) equipped with a Turbo Ion Spray Data acquisitionand processing were performed using Analyst 163 software(Applied Biosystems Foster City CA USA) Chromato-graphic separation was carried out on an Agilent EclipseXDB-C18 (21 times 150mm 5 120583m) and the column temperaturewas maintained at 40∘C The mobile phase was composed ofacetonitrile-water containing 01 formic acid (32 68 vv)The flow rate was set at 03mLmin The autosampler was setat 4∘C and the injection volume was 5 120583L

In the study on metabolism of icariin metabolites wereidentified by LTQ-Orbitrap XL mass spectrometer (ThermoElectron Bremen Germany) coupled with an ESI sourceAn Agilent Eclipse SB-C18 column (21 times 100mm 18 120583m)was employed and column temperature was maintainedat 40∘C Mobile phase was composed of A (01 formicacid aqueous solution) and B (acetonitrile) with a gradientelution as follows 0ndash35min 15ndash30 B 35ndash55min 30ndash50B 55ndash70min 50 B 70ndash80min 50ndash100 B 80ndash90min100 B The flow rate of mobile phase was 02mLmin Theinjection volume of samples was 5 120583L

23 Mass Spectrometer Conditions For plasma pharmacoki-netic and tissue distribution studies mass spectrometer wasoperated in negative mode for the detection of icariin andIS Quantification was performed with multiple reactionmonitoring (MRM) mode and the fragmentation transitionwas119898119911 7357rarr 5135 for icariin ([M+CH3COOH-H]minus) and119898119911 2690rarr 1332 for IS ESI-MSMS operation parameterswere as follows ion spray (IS) voltage minus4500V sourcetemperature (TME) 350∘C Gas 1 Gas 2 curtain gas andcollision gas (nitrogen) were separately set at 50 50 45 and12 psi

BioMed Research International 3

To study the metabolism profiles of icariin optimizedoperating parameters in negative-ion mode were as followssheath gas flow rate 40 arbitrary units auxiliary gas flowrate 5 arbitrary units electrospray voltage 35 kV capillaryvoltage minus32V capillary temperature 270∘C

24 Standard and Sample Preparation

241 Preparation of Stock Solutions Calibration StandardsandQuality Controls The stock solution (1mgmL) of icariinand IS was prepared with methanol respectively The stocksolution of icariin was diluted withmethanol to make a seriesof working solutions of 5 50 500 and 5000 ngmLThe stocksolution of IS was diluted with methanol to make a workingsolution of 1 120583gmL All stock and working solutions werekept at minus20∘C

A series of calibration standards containing 100 ngmL ofIS were separately prepared to contain icariin concentrationsof 1 2 5 10 20 50 100 200 and 500 ngmL by freshly spikingthe working solution of icariin and IS into blank biologicalmatrix Quality control (QC) samples at three concentrationlevels (low 2 ngmL medium 20 ngmL high 400 ngmL)were respectively prepared in the same way All of thecalibration standards and QC samples were freshly preparedbefore use

242 Sample Preparation For pharmacokinetic and distri-bution studies blank plasma was obtained from drug-freerats (Sprague-Dawley rats) Feces and urine were collected24 hours before and after the time of administration Blanktissue homogenate was prepared by homogenizing the tissueof drug-free rat and then diluted with three times the volumeof saline solution

Microcentrifuge tubes containing 15 120583L of IS and 150 120583Lof each calibration standard and the QC samples were driedunder a gentle stream of nitrogen An aliquot of 150 120583L ofdrug-free plasma or blank tissue homogenate was addedto these tubes and then followed by a single-step proteinprecipitation by adding 600120583L of methanol vigorouslyshaking on a vortex-mixer for 10min and then centrifugingthe tubes at 14800 rpm for 10minThe supernatant consistingof the organic solvent was transferred to a fresh tube anddried under nitrogenThe residue was reconstituted in 150120583Lof methanol and centrifuged to filter by a microspin filtertube (022 120583m nylon Alltech) at 14800 rpm for 1min A 5 120583Laliquot of the filtrate was injected into RRLC-MSMS foranalysis

In the metabolism study rat feces samples were pul-verized with a mortar and pestle Methanol (1 5 wv) wasadded and homogenized with the pulverized feces Mixturewas extracted by ultrasound for 90min and centrifuged at14800 rpm for 10min The supernatant fluid was transferredto an autosampler vial for analysis For urine sample 2mLof rat urine flowed through a pretreatment C18 SPE cartridgewith gravityThe solid-phase cartridge was washed with 2mLwater and then eluted with 2mL methanol The methanoleluate was evaporated to dryness by water bath at 60∘CResidue was dissolved into 500 120583Lmethanol for analysisTheinjection volume of urine sample was 5 120583L

25 Method Validation The method was validated in termsof specificity linearity sensitivity precision and accuracystability recovery and matrix effect

251 Specificity Specificity was assessed by analyzing blankbiological samples blank biological matrix spiked withicariin and IS and real biological samples from rat afteroral administration of the total flavonoid extract of HerbaEpimedii

252 Linearity and Sensitivity Calibration curves wereobtained by plotting the peak area ratios (y-axis) of icariin toIS against the nominal concentration (x-axis) of icariin andassessed by weighted least-squares linear regression using11199092 as weighting factor Concentration of QC and biologicalsamples were calculated using the regression equation of thecalibration curve

LLOQ was defined as the lowest concentration on thestandard curve at which the standard deviation was within20 and accuracy was within 100 plusmn 20 and it wasestablished using five samples independent from the standardcurve

253 Precision and Accuracy Intraday precision and accu-racy were evaluated by analyzing five reduplicate QC samplesat three concentrations on the same day Interday precisionand accuracy were established by three analytical batcheson three consecutive days The accuracy was expressedas (observed concentrationnominal concentration)times100Intra- and interday precision was obtained by one-way analy-sis of variance (ANOVA) testing andwas expressed as relativestandard deviation (RSD) The accuracy was required to bewithin 85ndash115 and the precision should not exceed 15

254 Stability The stability of icariin was evaluated withquintuplicate spiked biological samples at three QC levelswhich were tested under the following conditions (1) thefreeze-thaw stability of icariin in rat biological samplesthrough three freeze-thaw (minus80∘C to room temperature)cycles (2) the short-term stability of icariin in rat biologicalsamples at room temperature for 6 h (3) the long-termstability of icariin in rat biological samples stored atminus80∘C for30 days and (4) the postpreparative stability of icariin duringstorage in the autosampler at 4∘C for 24 h

255 Recovery and Matrix Effects Extraction recovery andmatrix effect were determined by comparing the peak areas ofanalyte between three types of samples (1) biological samplesspiked with known amount of analyte before sample prepa-ration (2) biological samples spiked with known amount ofanalyte after sample preparation (3) pure standard solutionof analyte The difference in peak areas between samples 1and 2 reflects the extraction recovery while the difference inpeak areas between samples 2 and 3 reflects the matrix effectThe accuracy of the method was calculated by comparingtheoretically and experimentally measured analyte levels

26 Pharmacokinetic Distribution and Metabolism Studiesof Icariin In the present study the experimentation on rats

4 BioMed Research International

gained the approval of an independent ethics committeeat Guangdong Provincial Hospital of Chinese MedicineThe experiment was performed in an SPF level laboratoryauthorized by Guangdong Provincial Government

261 Plasma Pharmacokinetics Six male Sprague-Dawley(SD) rats (250 plusmn 20 g) were obtained from GuangdongProvince Laboratory Animal Center and kept in an envi-ronmentally controlled breeding room for 3 days beforethe experiment started The rats were orally given a singledose of 069 gkg of total flavonoid extract from HerbaEpimedii (corresponding icariin approximately at 42mgg)Blood samples were serially withdrawn from ratrsquos retroorbitalplexus into heparin lithium-anticoagulant tubes at 0 (beforedose) 0083 025 05 075 1 15 2 3 4 6 8 10 12 and24 h after administration The collected samples were thencentrifuged at 3000 rpm for 15min and supernatant plasmawas transferred into another tube and stored at minus80∘C untilanalysis

262 Tissue Distribution Study Thirty-six SD rats (220 plusmn20 g 18 males and 18 females) were orally given a single doseof 069 gkg of total flavonoid extract from Herba Epimedii(approximately containing 42mgg of icariin) Animals wereeuthanized at the time points of 025 05 1 2 4 and 6 hafter dose (119899 = 6 at each time point) Blood was col-lected via cardiac puncture (about 1000 120583L) and immediatelytransferred into heparinized tube The collected sample wascentrifuged at 3000 rpm for 15min and supernatant plasmawas transferred into another tube and stored at minus80∘C untilanalysis Nine kinds of rat tissues including liver heartspleen lung kidney brain testicle uterus and ovary wereimmediately removed and rinsed with ice-cold saline toremove extraneous blood and blot-dried and then stored inpreweighted and labeled vials at minus80∘C until use On the dayof analysis the tissue samples were thawed and weighed toobtain the tissue weight expressed as the difference betweenthe pre- and postvial weights

263 Metabolism Study of Icariin in Rat Urine and Feces Inthis study six SD rats (220 plusmn 20 g male) were individuallyplaced in metabolic cages and halved into two groups atrandom The reference standard of icariin (60mg) wasdissolved in 8mL of sterile water for injection containing40 1 3-propanediol (vv) The intramuscular group wasdosed with icariin (20mgkg) by intramuscular injection ofthis solution Oral administration of icariin (50mgkg) wasconducted on the other group Feces and urine samples wereseparately collected at 24 h after administration of icariin Allthe samples were stored at minus80∘C until use

27 Data Analysis Plasma concentration versus time profileswas obtained from each individual rat and the calculationof pharmacokinetic parameters was performed via DAS 211software (Mathematical Pharmacology Professional Com-mittee of China Shanghai China) with noncompartmentalpharmacokinetic analysis All data were expressed as mean plusmnstandard deviation (SD)

3 Results and Discussions

31 Optimization of Sample Preparation To obtain betterextraction efficiency and less endogenous interference differ-ent extraction procedures including solid-phase extractionand liquid-liquid extraction with different solvents weretested But all the extraction recoveries were not satisfactoryThus a simpler and less time-consuming method proteinprecipitation with methanol was finally used which wastestified to be rapid repeatable and sensitive enough for thedetermination of biological samples in the present study

32 Optimization of Chromatography andMass-SpectrometricConditions It is very important to pick a suitable IS toget high accuracy and precision when a mass spectrometeris equipped with LC as a detector In this study genisteinwas chosen as the IS because of its similar basic structurephysicochemical property and mass-spectrometric behaviorto those of icariin

The mass-spectrometric behavior of icariin and IS wasinvestigated using both positive- and negative-ion ESI Itwas found that both icariin and IS had good responses innegative-ion detection mode with low background noiselevel Multiple reaction monitoring (MRM) scan type wasused to improve specificity Detection was finally operated innegative-ion mode Quantification was performed by MRMmode and the selectedmonitor ion was119898119911 7357rarr 5135 foricariin ([M+CH3COOH-H]minus) and 119898119911 2690rarr 1332 for ISThe full scan andMSMS spectra of icariin and ISwere shownin Figure 2

In order to improve the peak shape and signal response ofanalytes and to reduce run time different analytical columnsand mobile phase compositions were tried to achieve goodresolution and symmetric peak shape for icariin and IS Bycomparison with Shiseido capcell pak C18 (150 times 20mmid 5 120583m) column Agilent XDB C18 (150 times 21mm id5 120583m) column could obtain better chromatographic behaviorand higher signal response for the two analytes Thus thiscolumn was finally selected for chromatographic analysisThe modifier of the mobile phase composed of acetonitrile-water binary solvent systemwas screened among ammoniumacetate acetic acid and formic acid As a result the mobilephase consisting of acetonitrile-water containing 01 formicacid could improve the symmetry of peak shape and enhancethe signal response The flow rate of mobile phase was alsooptimized and finally set at 03mLmin Under the above-mentioned HPLC conditions the retention time of icariinand IS was within 6min and no interfering substance wasdetected at the retention time of analytes in blank rat plasmaand tissue samples (Figure 3)

33 Method Validation

331 Specificity The selectivity of the method was examinedby comparing the chromatograms of blank and correspond-ing spiked biological matrices The retention time of icariinand IS was about 26 and 50min respectively No interferingpeak of endogenous substances was observed at themeasured

BioMed Research International 5

Table 1 Regression equations of icariin in rat plasma and tissue samples (119899 = 3)

Sample Equation Correlation coefficient Linear rangengmL

Plasma 119884 = (00060 plusmn 00002)119883 + (00011 plusmn 00006) 09973 plusmn 00013 100ndash500Liver 119884 = (00053 plusmn 00001)119883 + (00014 plusmn 00008) 09986 plusmn 00008 100ndash500Heart 119884 = (00062 plusmn 00003)119883 + (00006 plusmn 00002) 09979 plusmn 00013 100ndash500Spleen 119884 = (00053 plusmn 00006)119883 + (00010 plusmn 00008) 09979 plusmn 00009 100ndash500Lung 119884 = (00060 plusmn 00006)119883 + (00015 plusmn 00008) 09974 plusmn 00015 100ndash500Kidney 119884 = (00045 plusmn 00003)119883 + (00006 plusmn 00002) 09986 plusmn 00004 100ndash500Brain 119884 = (00033 plusmn 00002)119883 + (00011 plusmn 00003) 09985 plusmn 00005 100ndash500Testicle 119884 = (00066 plusmn 00001)119883 + (00015 plusmn 00001) 09977 plusmn 00001 100ndash500

(a-1) (a-2)

7356

5136

765652972549

400 600 800 1000200mz (Da)

00

20e7

40e7

60e7

80e7

10e8

12e8

14e8

Inte

nsity

(cps

)

1332

26901067 1347633

2242

00

10e6

20e6

30e6

40e6

50e6

60e6

70e6

80e6

Inte

nsity

(cps

)

50 150 200 250 300 350 400 450 500100mz (Da)

(b-1) (b-2)

2690

2852

00

50e6

10e7

15e7

20e7

25e7

29e7

Inte

nsity

(cps

)

150 200 250 300 350 400 450 500100mz (Da)

5135

7357

529500

50e6

10e7

15e7

20e7

23e7

Inte

nsity

(cps

)

400 600 800 1000200mz (Da)

Figure 2 Full scan mass spectrum of icariin (a-1) and internal standard (genistein b-1) product ion spectrum of icariin (a-2) and internalstandard (genistein b-2)

mass transitions and retention time of icariin and IS in blankbiological matrix samples

332 Linearity and Sensitivity The assay was found to belinear over the concentration range of 1ndash500 ngmL forplasma and tissue homogenate The mean linear regressionequations were listed in Table 1 with correlations coefficientover 099 in which119883 corresponds to nominal concentrations

and 119884 corresponds to peak area ratios LLOQs with an 119878119873ratio of gt20 was 1 ngmL which was sensitive enough for thepharmacokinetic and tissue distribution studies of icariin

333 Precision and Accuracy Inter- and intra-assay vari-ability at three different concentrations HQC (400 ngmL)MQC (20 ngmL) LQC (2 ngmL) was determined in plasmaand tissue homogenate with five replicates on each day for

6 BioMed Research International

(a-1)

0

20

40

60

80

100

120

Inte

nsity

(cps

)

20 30 40 50 6010Time (min)

(a-2)

0

20

40

60

80

100

Inte

nsity

(cps

)

20 30 40 50 6010Time (min)

(b-2)

501

005000010e4

15e4

20e4

25e4

30e4

35e4

Inte

nsity

(cps

)

20 30 40 50 6010Time (min)

(c-2)

258

0

1000

2000

3000

4000

5000

60006787

Inte

nsity

(cps

)

20 30 40 50 6010Time (min)

(d-2)

260

0500

100015002000250030003500

Inte

nsity

(cps

)

20 30 40 50 6010Time (min)

(d-1)

260

20 30 40 50 6010Time (min)

0

100

200

300

400

500597

Inte

nsity

(cps

)

(c-1)

261

20 30 40 50 6010Time (min)

0

50

100

150

200

250

Inte

nsity

(cps

)

(b-1)

500

00

50000

10e4

15e4

20e422e4

Inte

nsity

(cps

)

20 30 40 50 6010Time (min)

Figure 3 Representative MRM chromatograms of blank plasma (a-1) blank liver (a-2) blank plasma spiked with IS (b-1) blank liver spikedwith IS (b-2) blank plasma spiked with icariin at LOQ (c-1) blank liver spiked with icariin at LOQ (d-1) rat plasma sample obtained at 05 hafter administration (c-2) and rat liver sample obtained at 05 h after administration (d-2)

BioMed Research International 7

Table 2 Intra- and interday accuracy and precision for the determination of icariin in plasma and tissue samples (119899 = 18 6 replicates perday for 3 days)

c Conc Interday IntradayngmL RE () CV () RE () CV ()

Plasma2 minus0394 134 minus0887 35020 minus518 156 minus485 225400 minus328 150 minus276 247

Liver2 0690 155 minus0197 46420 minus0528 959 minus0198 185400 minus103 104 minus117 162

Heart2 minus0309 425 minus483 32220 409 161 495 246400 minus365 219 minus257 159

Spleen2 minus214 900 minus335 76320 396 509 346 225400 minus0313 475 0 198

Lung2 minus680 137 minus133 47220 minus205 141 minus852 267400 minus930 102 minus126 111

Kidney2 473 536 394 53620 399 133 743 277400 minus649 136 minus573 149

Brain2 minus131 948 minus123 47420 327 559 0792 140400 minus328 356 minus336 212

Testicle2 minus420 325 minus236 46020 0538 493 307 269400 minus0379 579 257 402

three separate days The detailed results of the inter- andintraday precision and accuracy of icariin in biologicalmatrixwere summarized in Table 2

334 Recovery and Matrix Effects As shown in Table 3 theextraction recovery of icariin was in the range of 825ndash1006which suggested that the recovery of this method was consis-tent and reproducible And the suppression of ionization inplasma and tissue homogenate was differentThe interferenceof ionization caused by kidney homogenate was significantwhile that of brain homogenate was much lower

335 Stability Five replicates of QC at three different con-centration levels were used to assess the stability of icariinunder various conditions The results were summarized inTable 4 which indicated that icariin was stable in autosam-pler (24 h) at 4∘C bench-top (6 h) at room temperature threefreeze-thaw cycles and frozen condition atminus80∘C for 30 daysas the RE values were within plusmn15 for the low medium andhigh concentrations

336 Plasma Pharmacokinetics After a single oral admin-istration of the total flavonoid extract of Herba Epimediithe concentration of icariin in plasma was successfully deter-mined by LC-MSMS method described above The meanplasma concentration-time profile of icariin was shown in

Icariin

10 1550Time (h)

0

50

100

150

Con

cent

ratio

n (n

gm

L)

Figure 4 Plasma concentration versus time profile of icariin in rats(119899 = 6) following a single oral dose of Herba Epimedii extract(corresponding 42mgg icariin)

Figure 4 Pharmacokinetic parameters were estimated usingtheDAS software and performed by noncompartmental anal-ysis Peak plasma concentration (119862max) and the time at whichit occurred (119879max) were obtained from the visual inspection

8 BioMed Research International

Table 3 Matrix effect and extraction recovery for the assay of icariin in plasma and tissue samples (119899 = 5)

Matrix ConcngmL

Matrix effectMean plusmn SD ()

Extraction efficiencyMean plusmn SD ()

Plasma2 712 plusmn 29 869 plusmn 60

20 690 plusmn 20 841 plusmn 47

400 667 plusmn 10 849 plusmn 58

Liver2 715 plusmn 33 939 plusmn 75

20 722 plusmn 41 903 plusmn 19

400 703 plusmn 31 905 plusmn 22

Heart2 679 plusmn 38 889 plusmn 39

20 688 plusmn 26 922 plusmn 23

400 699 plusmn 18 931 plusmn 17

Spleen2 692 plusmn 53 887 plusmn 84

20 701 plusmn 37 835 plusmn 39

400 714 plusmn 38 825 plusmn 14

Lung2 710 plusmn 22 880 plusmn 59

20 712 plusmn 24 854 plusmn 45

400 738 plusmn 16 838 plusmn 19

Kidney2 577 plusmn 62 865 plusmn 67

20 555 plusmn 53 900 plusmn 37

400 666 plusmn 152 853 plusmn 61

Brain2 906 plusmn 58 897 plusmn 57

20 893 plusmn 19 949 plusmn 39

400 870 plusmn 23 990 plusmn 23

Testicle2 802 plusmn 35 915 plusmn 67

20 773 plusmn 21 964 plusmn 22

400 777 plusmn 22 1006 plusmn 19

of the plasma concentration-time curve Elimination rateconstant (119870119890) was determined from the least-square fittedterminal log-linear portion of the plasma concentration-timeprofile Elimination half-life (11990512) was calculated by 0693KeTotal area under the plasma concentration-time curve oficariin from time zero to infinity (AUC0ndashinfin) was calculatedas the sumofAUC0ndash12 the area up to the last quantifiable timepoint (12 h) and the area from the 12 h time point to infinityAUC0ndash12 was calculated using the trapezoidal rule and theextrapolated area was calculated from the last measurableconcentration 119862119905 divided by 119870119890 The major pharmacokineticparameters of icariin were summarized in Table 5

34 Tissue Distribution The tissue distribution of icariin infemale and male rats was investigated following a single oraldose of total flavonoid extract from Herba Epimedii (cor-responding 42mgg icariin) The mean concentration-timeprofile of each tissue was presented in Figure 5 indicatingthat icariin was poorly absorbed after oral administrationbecause all the tissue concentrations were kept at a verylow level The peak concentration of icariin in most ofcollected tissues appeared at 1 h after administration except

for liver and female reproductive organs in which theconcentration reached the peak at 05 h demonstrating thaticariin underwent a rapid and wide distribution Moreoverthe tissue concentrations in male rat were generally muchhigher than those in female rat The tissue area under thecurve from time zero to infinity was in the following orderliver gt lung gt spleen gt heart gt kidney gt brain gt testiclefor mail rat and liver gt uterus gt heart gt ovary gt lung gtkidney gt spleen gt brain for female rats As shown in Figure 5the concentration levels in liver and lung were much higherthan those in other tissues illustrating that liver and lungare the main target organs of icariin in male rat after oraladministration of the total flavonoid extract Furthermorethe concentration level in uteruswasmuchhigher than that inany other tissues of female rat demonstrating that the gender-related difference of icariin in tissue distribution do existand the reproductive organs are the main target organs oficariin for female rat Meanwhile icariin was found with verylow AUC0ndashinfin in brain which indicated that icariin could notefficiently cross the blood-brain barrier Over time the maintrend of the mean concentration of icariin in all tissues wasreduced and the concentration dropped very quickly at 4 h

BioMed Research International 9

Table 4 Stability of icariin in plasma and tissue samples (119899 = 6)

Matrix Conc Mean plusmn SDngmL Bench-topa Freeze-thawb Autosamplerc Long-termd

Plasma2 975 plusmn 37 968 plusmn 57 1047 plusmn 81 993 plusmn 59

20 945 plusmn 59 1013 plusmn 31 955 plusmn 37 943 plusmn 16

400 977 plusmn 39 1030 plusmn 19 945 plusmn 26 955 plusmn 37

Liver2 987 plusmn 63 1015 plusmn 21 1114 plusmn 44 986 plusmn 54

20 949 plusmn 14 900 plusmn 13 1060 plusmn 16 957 plusmn 21

400 875 plusmn 13 897 plusmn 13 894 plusmn 14 1125 plusmn 22

Heart2 1086 plusmn 33 1002 plusmn 43 955 plusmn 22 1150 plusmn 30

20 1056 plusmn 17 996 plusmn 10 973 plusmn 13 1002 plusmn 42

400 1026 plusmn 15 942 plusmn 29 892 plusmn 09 907 plusmn 50

Spleen2 954 plusmn 92 1018 plusmn 44 955 plusmn 76 1013 plusmn 59

20 989 plusmn 14 1094 plusmn 11 928 plusmn 09 1123 plusmn 34

400 967 plusmn 27 1032 plusmn 04 871 plusmn 12 974 plusmn 26

Lung2 959 plusmn 35 856 plusmn 30 1032 plusmn 48 983 plusmn 43

20 1017 plusmn 80 923 plusmn 40 1094 plusmn 46 1102 plusmn 18

400 925 plusmn 18 932 plusmn 15 997 plusmn 04 1098 plusmn 17

Kidney2 1012 plusmn 34 916 plusmn 40 951 plusmn 22 984 plusmn 48

20 1025 plusmn 28 974 plusmn 18 955 plusmn 16 934 plusmn 22

400 920 plusmn 14 911 plusmn 08 899 plusmn 10 1095 plusmn 22

Brain2 858 plusmn 60 974 plusmn 73 966 plusmn 32 1094 plusmn 32

20 1033 plusmn 41 1035 plusmn 31 1030 plusmn 35 974 plusmn 12

400 1002 plusmn 29 990 plusmn 31 978 plusmn 21 1102 plusmn 30

Testicle2 1058 plusmn 39 853 plusmn 24 997 plusmn 55 933 plusmn 41

20 1040 plusmn 14 870 plusmn 13 1016 plusmn 19 1065 plusmn 15

400 1034 plusmn 39 984 plusmn 16 995 plusmn 33 1068 plusmn 38aAt least 1 h at room temperature bAt least 3 freeze-thaw cycles cAt least 24 h at 4∘C dAt least 4 weeks at minus80 plusmn 5∘C

Table 5 Pharmacokinetic parameters of icariin in rat after asingle oral dose of Herba Epimedii extract (corresponding 42mggicariin)

Parameter IcariinAUC0ndashinfin (ugLsdoth) 164155 plusmn 144129

AUMC0ndashinfin (ugLsdoth) 538424 plusmn 262081

MRT0ndashinfin (h) 3990 plusmn 1987

11990512 (h) 3149 plusmn 2364

119879max (h) 0458 plusmn 0246

CL119911119865 (Lhkg) 389043 plusmn 204472

after administration suggesting no accumulation in tissuesand a rapid elimination of this compound

35 Metabolism Study of Icariin in Rat Urine and Feces

351 Metabolite Profile of Rat Urine and Feces In thisstudy urine and feces samples were both analyzed by LC-MSMS By comparing the total ion chromatograms of drug-containing urine sample and predose control urine sampleneither parent drug nor metabolites were observed in raturine following two routes of administration of icariinHowever a total of 11 icariin metabolites were detected in ratfeces including 10 metabolites after oral administration and

9 following intramuscular introduction The representativechromatograms of the metabolites of 0ndash24 h rat feces wereshown in Figure 6 No additional metabolites were observedin the feces of 24ndash48 h collection period Results showedthat the major metabolites of icariin were M8 and M11whose total amount accounted for over 90 of total peakarea in the LC-UV chromatogram of 0ndash24 h feces And theconcentration of metabolites in feces rapidly declined over24ndash48 h collection period The MS2 and MS3 spectra oficariin and its metabolites were exhibited in Figure 7 andthe observed and calculatedmasses formula retention timesmass errors and MS data of these analytes were presented inTable 6 Proposed metabolic pathways for icariin in rat feceswere shown in Figure 8

352 Identification of Metabolites Known compounds wereidentified by comparison with the chromatographic behaviorand MS fragmentation pattern of reference standards Forunknown constituents their structures were characterizedbased on their retention behavior and their MS119899 spectraobtained on-line Moreover under the optimized LC-MSconditions all the in vivometabolites of icariin could give theprecursor ions of sufficient abundance in order to facilitatethe analysis of multistage mass spectrometry Because allmetabolites originated from icariin and their MS fragmen-tation patterns were largely similar to that of icariin it was

10 BioMed Research International

Heart Spleen Lung Kidney Brain TesticleLiver0

10

20

30

40

50C

once

ntra

tion

(ng

g)

025 Ｂ

05 Ｂ

1 Ｂ

2 Ｂ

4 Ｂ

6 Ｂ

(a)

0

10

20

30

40

50

Con

cent

ratio

n (n

gg)

Heart Spleen Lung Kidney Brain Uterus OvaryLiver

025 Ｂ

05 Ｂ

1 Ｂ

2 Ｂ

4 Ｂ

6 Ｂ

(b)

Figure 5 Tissue distribution of icariin in rats after a single oral dose of Herba Epimedii extract (corresponding 42mgg icariin (a) male rat(b) female rat)

663517

5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 900Time (min)

0

50

100

(a)

6155

512665

6637520419520

20

40

60

80

100

5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 900Time (min)

52 54 56 58500

50 M11

M10

M8

M7

M6M5

M4

M3M2

M1

M9

(b)

658

61465278

506

0

20

40

60

80

100

5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 900Time (min)

M11

M3 M8

(c)

Figure 6 Representative chromatograms of icariin metabolites in rat feces of 0ndash24 h (a) blank feces sample (b) feces sample after oraladministration and (c) feces sample after intramuscular administration

BioMed Research International 11

36724

6755435214

Icariin

513320

20

40

60

80

100

Rela

tive a

bund

ance

400 600 800200mz

35218

Icariin

367250

20

40

60

80

100

Rela

tive a

bund

ance

400 600200mz

35211

36722

29727

0

20

40

60

80

100

Rela

tive a

bund

ance

400300mz

2811329595

26725 3090935216

25506

0

20

40

60

80

100

Rela

tive a

bund

ance

300 400200mz

28408

35323

25512

0

20

40

60

80

100

Rela

tive a

bund

ance

400 600200mz

35217

36722

0

20

40

60

80

100

Rela

tive a

bund

ance

300 400 500200mz

36722

6892932124

400 600 800200mz

0

20

40

60

80

100

Rela

tive a

bund

ance

35318

49919

0

20

40

60

80

100

Rela

tive a

bund

ance

400 600200mz

35215

48116 64516

400 600 800200mz

0

20

40

60

80

100

Rela

tive a

bund

ance

400 600 800 1000mz

36723

83734

79088

0

20

40

60

80

100

Rela

tive a

bund

ance

M1M1

M2

M2

M3

M3

M4

M4

MS2

MS3

MS2

MS3

MS3

MS2

MS2

MS2

MS3

MS3

Figure 7 Continued

12 BioMed Research International

38218

36714 5293950114

0

20

40

60

80

100

Rela

tive a

bund

ance

400 500 600300mz

M5

MS2

36722

33916 382180

20

40

60

80

100

Rela

tive a

bund

ance

300 400 500200mz

M5

MS3

35217

36711

0

20

40

60

80

100

Rela

tive a

bund

ance

300 400 500200mz

M6

MS3

36724

35227 6452032309

0

20

40

60

80

100

Rela

tive a

bund

ance

400 600 800200mz

M6

MS2

36614

6592335118

0

20

40

60

80

100

Rela

tive a

bund

ance

400 600 800200mz

M7

MS2

35116

32317

31112

36607

0

20

40

60

80

100

Rela

tive a

bund

ance

300 400 500200mz

M7

MS3

35109

32308

31113

3660629520

0

20

40

60

80

100

Rela

tive a

bund

ance

300 400 500200mz

M8

MS3

36615

51313

400 600200mz

0

20

40

60

80

100

Rela

tive a

bund

ance M8

MS2

36514

35528 38328

33929

0

20

40

60

80

100

Rela

tive a

bund

ance

400300mz

M9

MS2

35019

36508

33724

0

20

40

60

80

100

Rela

tive a

bund

ance

400200mz

M9

MS3

Figure 7 Continued

BioMed Research International 13

25308

16310

27902

29817

0

20

40

60

80

100

Rela

tive a

bund

ance

400200mz

M10

MS3

29810

35312

21913

400200mz

0

20

40

60

80

100

Rela

tive a

bund

ance M10

MS2

35208

36723

309280

20

40

60

80

100

Rela

tive a

bund

ance

400 600200mz

M11

MS2

30911

2810435215

400200mz

0

20

40

60

80

100

Rela

tive a

bund

ance M11

MS3

Figure 7 Representative LC-MS119899 spectra of icariin and its metabolites M1ndashM11 in rat feces

necessary to characterize the MS119899 behavior of icariin toidentify and assign the metabolites of the constituent Thedeprotonated molecule [MndashH]minus of icariin appears at 119898119911675 which readily eliminates its glucosyl and rhamnosylgroups to produce an [MndashHndash308]minus ion at 119898119911 367 The[MndashHndash308]minus ion yields a fragment at 119898119911 352 by losing amethyl radical And of theMS4 spectrum of [MndashHndash308ndash15]minusion the fragment at 119898119911 323 can be reasonably attributed tothe loss of 3-COH of flavone skeletonrsquos C ring

M1 as aminormetabolite of icariin was only found in thefeces of the oral group Its quasimolecular ion occurs at 119898119911837 (C39H49O20) whose molecular mass is 162Da more thanthat of icariin And its major MS119899 fragment ions are the sameas those of icariinThemost abundantMS2 andMS3 fragmentions also appear at 119898119911 367 and 352 indicating the loss ofone rhamnosyl and two glucosyl moieties and the sequentialloss of methyl group respectively Although the molecularweight of M1 is equal to that of epimedin A there existsa significant difference on their chromatographic retentionbehavior Therefore M1 is tentatively identified as an isomerof epimedin A

Both M2 and M6 show the same quasimolecular ion[M minus H]minus at 119898119911 645 (C32H37O14) with the elution time at5044 and 5743min respectively M2 was only observed infeces after intramuscular introduction of icariin while M6was found in feces following both routes of administrationM6 is unambiguously identified as sagittatoside B incomparison with the reference standard Analogous to theMS119899 fragments of icariin (see Figure 7 and Table 6) M2also yielded the major fragment ions at 119898119911 352 323 309296 281 and 268 strongly suggesting that M2 should be

a conjunction metabolite of icariin Thus M2 is tentativelyassigned as sagittatoside B isomer

M3 was found in rat feces after both intramuscular andoral administration of icariin Its MS spectrum exhibits adeprotonated molecule [MndashH]minus at 119898119911 499 (C26H27O10)176Da less than that of icariin By comparison with thereference standard it is identified as baohuoside II TheMSMS spectrum shows the most abundant fragment ionat 119898119911 353 indicating the loss of rhamnose moiety TheMS3 spectrum displays the major fragment ions at 119898119911 325298 and 284 separately corresponding to the loss of COisobutenyl and isopentenyl radicals

M4 is a minor metabolite of icariin detected in the fecesof rats in the oral administration group Its deprotonatedmolecule (C33H37O16) is at 119898119911 689 And the major MSMSfragment at 119898119911 367 results from the loss of glucuronicacid and rhamnose moieties Its MS3 spectrum produces afragment at119898119911 352 (the loss of a methyl group) Thus M4 istentatively characterized as icariin glucuronide

M5ndashM11 of icariin metabolites were observed in rat fecesafter both intramuscular and oral administration of icariin

M5 has a deprotonated molecular ion at 119898119911 529(C27H29O11) in its MS spectrum The MS119899 spectra show themajor fragment ions at119898119911 501 (the loss of CO) 382 (the lossof rhamnosyl group) 367 (the loss of methyl radical from theion at119898119911 382) and 339 (the successive loss of methyl radicaland CO) Therefore M5 is tentatively identified as hydroxylicariside II

M7 is identified as 210158401015840-O-rhamnosyl icariside II based onthe reference standard and related mass spectral pattern The

14 BioMed Research International

Table6Ch

aracteriz

ationof

metaboliteso

ficariinin

ratfeces

Num

ber

Com

poun

dRT

Form

ula

Observedmass

RDB

Error

LCM

S119899data

[MndashH]minus

(ppm

)

PlowastIcariin

5519

C 33H39O15

67522888

145

0790

MS2[675]367(100)352(25)

MS3[675-367]352(100)

MS4[675-367-352]323(

30)309(100)296(45)281

(65)268

(50)255

(20)

M1

Epim

edin

Aiso

mer

a5038

C 39H49O20

8372

819

155

0824

MS2[837]790(50)367

(100)

MS3[837-367]352(100)297(20)

M2

Sagitta

tosid

eBiso

mer

b5044

C 32H37O14

6452186

145

1314

MS2[645]481(20)352(100)

MS3[645-352]323(40)309

(60)296

(90)281

(100)268(60)

M3

lowastBa

ohuo

sideIIa

b5278

C 26H27O10

4991

605

135

1295

MS2[499]353(100)

MS3[499-353]325(20)298

(40)284

(100)255(30)

M4

Icariin

glucuron

idea

5404

C 33H37O16

6892

084

155

1202

MS2[689]367(100)321(60)

MS3[689-367]352(100)

M5

Hydroxylicaris

ideIIa

b5412

C 27H29O11

5291

716

135

1132

MS2[529]501(20)382(100)367(40)

MS3[529-382]367(100)339(10)

M6

lowastSagitta

tosid

eBab

5743

C 32H37O14

6452184

145

1020

MS2[645]367(100)352(30)

MS3[645-367]352(100)

M7

lowast210158401015840-O

-Rhamno

syl-icaris

ideIIa

b5756

C 33H39O14

6592

344

145

1529

MS2[659]366(100)351(20)323(15)

MS3[659-366]351(100)323

(80)311(60)305

(20)

M8

lowastIcariside

IIab

6146

C 27H29O10

5131760

135

0851

MS2[513]366(100)

MS3[513-366]351(100)323

(80)311(50)305

(20)

M9

Hydroxylicaritin

ab6233

C 21H19O7

3831137

125

3160

MS2[383]365(100)355(60)339

(40)

MS3[383-365]350(100)321(20)230(40)

M10

Desmethylicaritin

ab6636

C 20H17O6

3531024

125

1119

MS2[353]298(100)219(10)

MS3[353-298]279(60)269

(40)253

(100)241(30)164(70)

M11

lowastIcaritinab

8265

C 21H19O6

3671183

125

1921

MS2[367]352(100)309(20)297

(20)

MS3[367-352]309(100)297(60)

lowastCom

paredwith

ther

eference

standardaObservedin

ratfeces

after

oraladministratio

nb O

bservedin

ratfeces

after

intram

usculara

dministratio

n

BioMed Research International 15

O

OO

O

O

O

OHOH

OH

OH

OH OHOH

OH

OHOH

OHOH

OH

OHOH OH

OH

OH

OH

OHOH

OH

OH

OH

OH

OH

OH

OH

OHOH

OH

OH

OH

OH

OHOH

OHOH

OHOH

OH

OH

OH

OH

OHOH

HO HO

HO

HO

HO

HOHO

HO

HO

HO

OH

O

OO

O

OOHOOC

O

OO

O

O

OO

OO

O

O

OO

OO

O

O

OO

O

O

OO

O

O

O

O

O

O

O

icariin

epimedin A isomer

minus rh

a

minus methyl

+ rhaOxidation

minus methyl

Icariin glucuronide

+ xyl

+ glcGlucuronidation

minus rha

Oxidation

minus rha

+ xyl

Sagittatoside B isomer

minus glc

M1

M4

M2

OCH3 OCH3

OCH3

OCH3

OCH3

OCH3

OCH3

OCH3

M7 2-O-rhamnosyl icariside II

M5 hydroxyl icariside II

M3 baohuoside II

M8 icariside II

M6 sagittatoside B

M9 hydroxyl icaritinM10 desmethyl icaritin

M11 icaritin

Figure 8 Proposed metabolic pathways for icariin in rat feces

MS spectrum of M7 shows a quasimolecular ion [MndashH]minusat 119898119911 659 (C33H39O14) The major fragment ions at 119898119911366 351 and 323 result from the concurrent loss of therhamnose chain the successive loss of a methyl group andCO respectively

As shownFigure 7M8 is themost abundantmetabolite oficariin By comparison with the reference standard it is iden-tified as icariside II Its MS spectrum shows a deprotonatedmolecular ion at 119898119911 513 (C27H29O10) And similar to the

MS119899 pattern of icariin the major fragment ions at 119898119911 366351 and 323 separately match the loss of rhamnose moietythe sequential loss of rhamnose and methyl groups and theloss of CO

M9 has a quasimolecular ion [MndashH]minus at 119898119911 383(C21H19O7) 146Da less than that of M5 (hydroxyl icarisideII) It is tentatively identified as hydroxyl icaritin Its MSMSspectrum shows themain fragments of119898119911 365 355 and 339respectively corresponding to the neutral loss of H2O CO

16 BioMed Research International

and CO2 MS3 spectrum the fragment ions at 119898119911 350 and321 individually result from the neutral loss of methyl radicaland CO2

The quasimolecular ion [MndashH]minus of M10 is at 119898119911 353146Da less than that of M3 (baohuoside II) The majorfragment ion in its MSMS spectrum is at119898119911 298 matchingthe loss of isobutenyl radical The fragment at 119898119911 219 ispossibly produced by the neutral loss of C8H6O2 (134Da) dueto the retro-Diels-Alder (RDA) fragmentation of the flavonersquosC ring Moreover the MS3 spectrum affords fragment ionsat 119898119911 279 269 253 and 164 resulting from the loss ofH2O CO CO2 and C8H6O2 (134Da) respectively Thus thismetabolite is reasonably identified as desmethyl icaritin

M11 was observed as anothermajormetabolite in rat fecesof both two administration groups (Figure 7) By comparisonwith the reference standard it is identified as icaritin Its MSspectrum yields a quasimolecular ion at119898119911 367 (C21H19O6)And themajor fragment ion at119898119911 352 is characterized as theloss of methyl group The MS3 fragment at 119898119911 309 resultsfrom the loss of methyl group and CO and ion at 119898119911 297is the outcome of the neutral loss of isopentene (70Da) fromthe deprotonated molecule

4 Conclusion

Themenopausal syndrome improvement and possible cancerpreventive activity of Herba Epimedii is receiving more andmore attention Information on the in vivo disposition ofHerba Epimedii components is important for better under-standing the biological effects of this herbal drug For thispurpose our investigation was designed to obtain informa-tion on the plasma pharmacokinetics and tissue distributionof icariin in rat and the excretion of icariin and itsmetabolitesIn this study we firstly established and validated a rapidsensitive and reliable LC-MS method for the determinationof icariin in various biological samples collected at differenttime points and then the method was applied to the plasmapharmacokinetics and tissue distribution study of icariin inrats after oral administration of the total flavonoid extractof Herba Epimedii In addition the differences of the tissuedistribution of icariin in between male and female rats werestudied after oral and intramuscular administration of icariinAnd the metabolic pattern of icariin was also investigatedThe data showed the plasma and tissue concentration oficariin were kept at a very low level at all collection pointswhich suggests icariin is poorly absorbed after oral adminis-trationThepeak concentration in plasma and collected tissuesamples appeared at 05ndash1 h and then quickly dropped at 4 hafter administration demonstrating that a small amount oficariin can be rapidly absorbed and eliminated and there isno accumulation in rat tissues Moreover the concentrationin liver lung and reproductive organs was much higherthan that in other tissues indicating that liver lung andreproductive organs are the main target sites of icariin in rats

The gender-related difference of icariin in tissue distribu-tion has not been reported in the past published literatureOur study demonstrated that the distribution features oficariin are similar in most of tissues between male andfemale rats However there exist a few remarkable gender

differences such that thewhole tissue concentration of icariinin male rats was much higher than female rats except in gen-ital organs (high in female and low in male) In China HerbaEpimedii is a famous tonic and aphrodisiac to invigoratekidney and strengthenYang therefore it is commonly used totreat male sexual functional decline and female menopausalsyndrome As one of the predominant constitutes in HerbaEpimedii icariin plays a very crucial role in exerting effectsso the relatively high icariin concentration in female genitalorgans actually supports the above-mentioned traditionalChinese medicine theory and clinical practice and makesthis compound a good future prospect for treating womenrsquosmenopausal related diseases

In the present study the complete metabolic profile oficariin has also been investigated The results illustrate thatfeces should be the major excretion way for both oral andintramuscular administration of icariin Based on LC-UVand LC-MS119899 analyses eleven metabolites were detected infecal samples and were further identified Among them10 metabolites appeared in rat feces of oral administrationgroup and 9 in the feces of intramuscular introduction groupThe major metabolic routes of clearance are proposed to behydrolysis demethylation oxidation and conjugation

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

Authorsrsquo Contributions

Shunjun Xu and Jiejing Yu contributed equally to this work

References

[1] X Wang Y He B Guo et al ldquoIn vivo screening for anti-osteo-porotic fraction from extract of herbal formula xianlinggubaoin ovariectomized micerdquo PLoS ONE vol 10 no 2 Article IDe0118184 2015

[2] R-H Liu X Kang L-P Xu et al ldquoEffects of the combinedextracts of Herba Epimedii and Fructus Ligustri Lucidi on bonemineral content and bone turnover in osteoporotic ratsrdquo BMCComplementary and Alternative Medicine vol 15 no 1 article112 2015

[3] B C L Chan H Y Lee W S Siu et al ldquoSuppression of mastcell activity contributes to the osteoprotective effect of an herbalformula containing Herba Epimedii Fructus Ligustri Lucidiand Fructus Psoraleaerdquo Journal of Pharmacy and Pharmacologyvol 66 no 3 pp 437ndash444 2014

[4] S CW Sze Y Tong T B Ng C L Y Cheng andH P CheungldquoHerba Epimedii anti-oxidative properties and its medicalimplicationsrdquoMolecules vol 15 no 11 pp 7861ndash7870 2010

[5] R Niu ldquoAction of the drug Herba Epimedii on testosterone ofthe mouse plasma and its accessory sexual organ before andafter processingrdquo China Journal of Chinese Materia Medica vol14 no 9 pp 530ndash574 1989

[6] F-X Liu S Sun and Z-Z Cui ldquoAnalysis of immunologi-cal enhancement of immunosuppressed chickens by Chineseherbal extractsrdquo Journal of Ethnopharmacology vol 127 no 2pp 251ndash256 2010

BioMed Research International 17

[7] J-H Kim Y-J Mun S-J Im J-H Han H-S Lee and W-HWoo ldquoEffects of the aqueous extract of Epimedii Herba on theantibody responses in micerdquo International Immunopharmacol-ogy vol 1 no 5 pp 935ndash944 2001

[8] X Yu Y Tong X-Q Han et al ldquoAnti-angiogenic activity ofHerba Epimedii on zebrafish embryos in vivo and HUVECs invitrordquo Phytotherapy Research vol 27 no 9 pp 1368ndash1375 2013

[9] D-C Zhang J-L Liu Y-B Ding J-G Xia and G-Y ChenldquoIcariin potentiates the antitumor activity of gemcitabine ingallbladder cancer by suppressingNF-120581BrdquoActa PharmacologicaSinica vol 34 no 2 pp 301ndash308 2013

[10] L Kong J Liu J Wang et al ldquoIcariin inhibits TNF-120572IFN-120574induced inflammatory response via inhibition of the substanceP and p38-MAPK signaling pathway in human keratinocytesrdquoInternational Immunopharmacology vol 29 no 2 pp 401ndash4072015

[11] H Shao J Shen M Wang et al ldquoIcariin protects against tita-nium particle-induced osteolysis and inflammatory response ina mouse calvarial modelrdquo Biomaterials vol 60 pp 92ndash99 2015

[12] Q Wei J Zhang G Hong et al ldquoIcariin promotes osteogenicdifferentiation of rat bone marrow stromal cells by activatingthe ER120572-Wnt120573-catenin signaling pathwayrdquo Biomedicine ampPharmacotherapy vol 84 pp 931ndash939 2016

[13] S-Q Zhang W-J Cai J-H Huang et al ldquoIcariin a naturalflavonol glycoside extends healthspan in micerdquo ExperimentalGerontology vol 69 pp 226ndash235 2015

[14] M N Makarova O N Pozharitskaya A N Shikov S VTesakova V G Makarov and V P Tikhonov ldquoEffect of lipid-based suspension of Epimedium koreanum Nakai extract onsexual behavior in ratsrdquo Journal of Ethnopharmacology vol 114no 3 pp 412ndash416 2007

[15] Y Chen J Wang X Jia X Tan and M Hu ldquoRole of intestinalhydrolase in the absorption of prenylated flavonoids present inyinyanghuordquoMolecules vol 16 no 2 pp 1336ndash1348 2011

[16] J J Yu Y J Liu S J Xu S Sun Y J Xu and L Yang ldquoSimulta-neous quantitative determination of four epimediumflavonoidsin rat urine by LC-MSMSrdquo Traditional Chinese Drug Researchamp Clinical Pharmacology vol 27 no 4 pp 537ndash541 2016

[17] Y Lu N Li Y Deng et al ldquoSimultaneous determination oficariin naringin and osthole in rat plasma by UPLC-MSMSand its application for pharmacokinetic study after oral admin-istration of Gushudan capsulesrdquo Journal of Chromatography Bvol 993-994 pp 75ndash80 2015

[18] N Wang X Huang X Wang Y Zhang R Wu and D ShouldquoPipette tip solid-phase extraction and high-performance liquidchromatography for the determination of flavonoids fromEpimedii herba in rat serum and application of the techniqueto pharmacokinetic studiesrdquo Journal of Chromatography B vol990 pp 64ndash72 2015

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

BioMed Research International 3

To study the metabolism profiles of icariin optimizedoperating parameters in negative-ion mode were as followssheath gas flow rate 40 arbitrary units auxiliary gas flowrate 5 arbitrary units electrospray voltage 35 kV capillaryvoltage minus32V capillary temperature 270∘C

24 Standard and Sample Preparation

241 Preparation of Stock Solutions Calibration StandardsandQuality Controls The stock solution (1mgmL) of icariinand IS was prepared with methanol respectively The stocksolution of icariin was diluted withmethanol to make a seriesof working solutions of 5 50 500 and 5000 ngmLThe stocksolution of IS was diluted with methanol to make a workingsolution of 1 120583gmL All stock and working solutions werekept at minus20∘C

A series of calibration standards containing 100 ngmL ofIS were separately prepared to contain icariin concentrationsof 1 2 5 10 20 50 100 200 and 500 ngmL by freshly spikingthe working solution of icariin and IS into blank biologicalmatrix Quality control (QC) samples at three concentrationlevels (low 2 ngmL medium 20 ngmL high 400 ngmL)were respectively prepared in the same way All of thecalibration standards and QC samples were freshly preparedbefore use

242 Sample Preparation For pharmacokinetic and distri-bution studies blank plasma was obtained from drug-freerats (Sprague-Dawley rats) Feces and urine were collected24 hours before and after the time of administration Blanktissue homogenate was prepared by homogenizing the tissueof drug-free rat and then diluted with three times the volumeof saline solution

Microcentrifuge tubes containing 15 120583L of IS and 150 120583Lof each calibration standard and the QC samples were driedunder a gentle stream of nitrogen An aliquot of 150 120583L ofdrug-free plasma or blank tissue homogenate was addedto these tubes and then followed by a single-step proteinprecipitation by adding 600120583L of methanol vigorouslyshaking on a vortex-mixer for 10min and then centrifugingthe tubes at 14800 rpm for 10minThe supernatant consistingof the organic solvent was transferred to a fresh tube anddried under nitrogenThe residue was reconstituted in 150120583Lof methanol and centrifuged to filter by a microspin filtertube (022 120583m nylon Alltech) at 14800 rpm for 1min A 5 120583Laliquot of the filtrate was injected into RRLC-MSMS foranalysis

In the metabolism study rat feces samples were pul-verized with a mortar and pestle Methanol (1 5 wv) wasadded and homogenized with the pulverized feces Mixturewas extracted by ultrasound for 90min and centrifuged at14800 rpm for 10min The supernatant fluid was transferredto an autosampler vial for analysis For urine sample 2mLof rat urine flowed through a pretreatment C18 SPE cartridgewith gravityThe solid-phase cartridge was washed with 2mLwater and then eluted with 2mL methanol The methanoleluate was evaporated to dryness by water bath at 60∘CResidue was dissolved into 500 120583Lmethanol for analysisTheinjection volume of urine sample was 5 120583L

25 Method Validation The method was validated in termsof specificity linearity sensitivity precision and accuracystability recovery and matrix effect

251 Specificity Specificity was assessed by analyzing blankbiological samples blank biological matrix spiked withicariin and IS and real biological samples from rat afteroral administration of the total flavonoid extract of HerbaEpimedii

252 Linearity and Sensitivity Calibration curves wereobtained by plotting the peak area ratios (y-axis) of icariin toIS against the nominal concentration (x-axis) of icariin andassessed by weighted least-squares linear regression using11199092 as weighting factor Concentration of QC and biologicalsamples were calculated using the regression equation of thecalibration curve

LLOQ was defined as the lowest concentration on thestandard curve at which the standard deviation was within20 and accuracy was within 100 plusmn 20 and it wasestablished using five samples independent from the standardcurve

253 Precision and Accuracy Intraday precision and accu-racy were evaluated by analyzing five reduplicate QC samplesat three concentrations on the same day Interday precisionand accuracy were established by three analytical batcheson three consecutive days The accuracy was expressedas (observed concentrationnominal concentration)times100Intra- and interday precision was obtained by one-way analy-sis of variance (ANOVA) testing andwas expressed as relativestandard deviation (RSD) The accuracy was required to bewithin 85ndash115 and the precision should not exceed 15

254 Stability The stability of icariin was evaluated withquintuplicate spiked biological samples at three QC levelswhich were tested under the following conditions (1) thefreeze-thaw stability of icariin in rat biological samplesthrough three freeze-thaw (minus80∘C to room temperature)cycles (2) the short-term stability of icariin in rat biologicalsamples at room temperature for 6 h (3) the long-termstability of icariin in rat biological samples stored atminus80∘C for30 days and (4) the postpreparative stability of icariin duringstorage in the autosampler at 4∘C for 24 h

255 Recovery and Matrix Effects Extraction recovery andmatrix effect were determined by comparing the peak areas ofanalyte between three types of samples (1) biological samplesspiked with known amount of analyte before sample prepa-ration (2) biological samples spiked with known amount ofanalyte after sample preparation (3) pure standard solutionof analyte The difference in peak areas between samples 1and 2 reflects the extraction recovery while the difference inpeak areas between samples 2 and 3 reflects the matrix effectThe accuracy of the method was calculated by comparingtheoretically and experimentally measured analyte levels

26 Pharmacokinetic Distribution and Metabolism Studiesof Icariin In the present study the experimentation on rats

4 BioMed Research International

gained the approval of an independent ethics committeeat Guangdong Provincial Hospital of Chinese MedicineThe experiment was performed in an SPF level laboratoryauthorized by Guangdong Provincial Government

261 Plasma Pharmacokinetics Six male Sprague-Dawley(SD) rats (250 plusmn 20 g) were obtained from GuangdongProvince Laboratory Animal Center and kept in an envi-ronmentally controlled breeding room for 3 days beforethe experiment started The rats were orally given a singledose of 069 gkg of total flavonoid extract from HerbaEpimedii (corresponding icariin approximately at 42mgg)Blood samples were serially withdrawn from ratrsquos retroorbitalplexus into heparin lithium-anticoagulant tubes at 0 (beforedose) 0083 025 05 075 1 15 2 3 4 6 8 10 12 and24 h after administration The collected samples were thencentrifuged at 3000 rpm for 15min and supernatant plasmawas transferred into another tube and stored at minus80∘C untilanalysis

262 Tissue Distribution Study Thirty-six SD rats (220 plusmn20 g 18 males and 18 females) were orally given a single doseof 069 gkg of total flavonoid extract from Herba Epimedii(approximately containing 42mgg of icariin) Animals wereeuthanized at the time points of 025 05 1 2 4 and 6 hafter dose (119899 = 6 at each time point) Blood was col-lected via cardiac puncture (about 1000 120583L) and immediatelytransferred into heparinized tube The collected sample wascentrifuged at 3000 rpm for 15min and supernatant plasmawas transferred into another tube and stored at minus80∘C untilanalysis Nine kinds of rat tissues including liver heartspleen lung kidney brain testicle uterus and ovary wereimmediately removed and rinsed with ice-cold saline toremove extraneous blood and blot-dried and then stored inpreweighted and labeled vials at minus80∘C until use On the dayof analysis the tissue samples were thawed and weighed toobtain the tissue weight expressed as the difference betweenthe pre- and postvial weights

263 Metabolism Study of Icariin in Rat Urine and Feces Inthis study six SD rats (220 plusmn 20 g male) were individuallyplaced in metabolic cages and halved into two groups atrandom The reference standard of icariin (60mg) wasdissolved in 8mL of sterile water for injection containing40 1 3-propanediol (vv) The intramuscular group wasdosed with icariin (20mgkg) by intramuscular injection ofthis solution Oral administration of icariin (50mgkg) wasconducted on the other group Feces and urine samples wereseparately collected at 24 h after administration of icariin Allthe samples were stored at minus80∘C until use

27 Data Analysis Plasma concentration versus time profileswas obtained from each individual rat and the calculationof pharmacokinetic parameters was performed via DAS 211software (Mathematical Pharmacology Professional Com-mittee of China Shanghai China) with noncompartmentalpharmacokinetic analysis All data were expressed as mean plusmnstandard deviation (SD)

3 Results and Discussions

31 Optimization of Sample Preparation To obtain betterextraction efficiency and less endogenous interference differ-ent extraction procedures including solid-phase extractionand liquid-liquid extraction with different solvents weretested But all the extraction recoveries were not satisfactoryThus a simpler and less time-consuming method proteinprecipitation with methanol was finally used which wastestified to be rapid repeatable and sensitive enough for thedetermination of biological samples in the present study

32 Optimization of Chromatography andMass-SpectrometricConditions It is very important to pick a suitable IS toget high accuracy and precision when a mass spectrometeris equipped with LC as a detector In this study genisteinwas chosen as the IS because of its similar basic structurephysicochemical property and mass-spectrometric behaviorto those of icariin

The mass-spectrometric behavior of icariin and IS wasinvestigated using both positive- and negative-ion ESI Itwas found that both icariin and IS had good responses innegative-ion detection mode with low background noiselevel Multiple reaction monitoring (MRM) scan type wasused to improve specificity Detection was finally operated innegative-ion mode Quantification was performed by MRMmode and the selectedmonitor ion was119898119911 7357rarr 5135 foricariin ([M+CH3COOH-H]minus) and 119898119911 2690rarr 1332 for ISThe full scan andMSMS spectra of icariin and ISwere shownin Figure 2

In order to improve the peak shape and signal response ofanalytes and to reduce run time different analytical columnsand mobile phase compositions were tried to achieve goodresolution and symmetric peak shape for icariin and IS Bycomparison with Shiseido capcell pak C18 (150 times 20mmid 5 120583m) column Agilent XDB C18 (150 times 21mm id5 120583m) column could obtain better chromatographic behaviorand higher signal response for the two analytes Thus thiscolumn was finally selected for chromatographic analysisThe modifier of the mobile phase composed of acetonitrile-water binary solvent systemwas screened among ammoniumacetate acetic acid and formic acid As a result the mobilephase consisting of acetonitrile-water containing 01 formicacid could improve the symmetry of peak shape and enhancethe signal response The flow rate of mobile phase was alsooptimized and finally set at 03mLmin Under the above-mentioned HPLC conditions the retention time of icariinand IS was within 6min and no interfering substance wasdetected at the retention time of analytes in blank rat plasmaand tissue samples (Figure 3)

33 Method Validation

331 Specificity The selectivity of the method was examinedby comparing the chromatograms of blank and correspond-ing spiked biological matrices The retention time of icariinand IS was about 26 and 50min respectively No interferingpeak of endogenous substances was observed at themeasured

BioMed Research International 5

Table 1 Regression equations of icariin in rat plasma and tissue samples (119899 = 3)

Sample Equation Correlation coefficient Linear rangengmL

Plasma 119884 = (00060 plusmn 00002)119883 + (00011 plusmn 00006) 09973 plusmn 00013 100ndash500Liver 119884 = (00053 plusmn 00001)119883 + (00014 plusmn 00008) 09986 plusmn 00008 100ndash500Heart 119884 = (00062 plusmn 00003)119883 + (00006 plusmn 00002) 09979 plusmn 00013 100ndash500Spleen 119884 = (00053 plusmn 00006)119883 + (00010 plusmn 00008) 09979 plusmn 00009 100ndash500Lung 119884 = (00060 plusmn 00006)119883 + (00015 plusmn 00008) 09974 plusmn 00015 100ndash500Kidney 119884 = (00045 plusmn 00003)119883 + (00006 plusmn 00002) 09986 plusmn 00004 100ndash500Brain 119884 = (00033 plusmn 00002)119883 + (00011 plusmn 00003) 09985 plusmn 00005 100ndash500Testicle 119884 = (00066 plusmn 00001)119883 + (00015 plusmn 00001) 09977 plusmn 00001 100ndash500

(a-1) (a-2)

7356

5136

765652972549

400 600 800 1000200mz (Da)

00

20e7

40e7

60e7

80e7

10e8

12e8

14e8

Inte

nsity

(cps

)

1332

26901067 1347633

2242

00

10e6

20e6

30e6

40e6

50e6

60e6

70e6

80e6

Inte

nsity

(cps

)

50 150 200 250 300 350 400 450 500100mz (Da)

(b-1) (b-2)

2690

2852

00

50e6

10e7

15e7

20e7

25e7

29e7

Inte

nsity

(cps

)

150 200 250 300 350 400 450 500100mz (Da)

5135

7357

529500

50e6

10e7

15e7

20e7

23e7

Inte

nsity

(cps

)

400 600 800 1000200mz (Da)

Figure 2 Full scan mass spectrum of icariin (a-1) and internal standard (genistein b-1) product ion spectrum of icariin (a-2) and internalstandard (genistein b-2)

mass transitions and retention time of icariin and IS in blankbiological matrix samples

332 Linearity and Sensitivity The assay was found to belinear over the concentration range of 1ndash500 ngmL forplasma and tissue homogenate The mean linear regressionequations were listed in Table 1 with correlations coefficientover 099 in which119883 corresponds to nominal concentrations

and 119884 corresponds to peak area ratios LLOQs with an 119878119873ratio of gt20 was 1 ngmL which was sensitive enough for thepharmacokinetic and tissue distribution studies of icariin

333 Precision and Accuracy Inter- and intra-assay vari-ability at three different concentrations HQC (400 ngmL)MQC (20 ngmL) LQC (2 ngmL) was determined in plasmaand tissue homogenate with five replicates on each day for

6 BioMed Research International

(a-1)

0

20

40

60

80

100

120

Inte

nsity

(cps

)

20 30 40 50 6010Time (min)

(a-2)

0

20

40

60

80

100

Inte

nsity

(cps

)

20 30 40 50 6010Time (min)

(b-2)

501

005000010e4

15e4

20e4

25e4

30e4

35e4

Inte

nsity

(cps

)

20 30 40 50 6010Time (min)

(c-2)

258

0

1000

2000

3000

4000

5000

60006787

Inte

nsity

(cps

)

20 30 40 50 6010Time (min)

(d-2)

260

0500

100015002000250030003500

Inte

nsity

(cps

)

20 30 40 50 6010Time (min)

(d-1)

260

20 30 40 50 6010Time (min)

0

100

200

300

400

500597

Inte

nsity

(cps

)

(c-1)

261

20 30 40 50 6010Time (min)

0

50

100

150

200

250

Inte

nsity

(cps

)

(b-1)

500

00

50000

10e4

15e4

20e422e4

Inte

nsity

(cps

)

20 30 40 50 6010Time (min)

Figure 3 Representative MRM chromatograms of blank plasma (a-1) blank liver (a-2) blank plasma spiked with IS (b-1) blank liver spikedwith IS (b-2) blank plasma spiked with icariin at LOQ (c-1) blank liver spiked with icariin at LOQ (d-1) rat plasma sample obtained at 05 hafter administration (c-2) and rat liver sample obtained at 05 h after administration (d-2)

BioMed Research International 7

Table 2 Intra- and interday accuracy and precision for the determination of icariin in plasma and tissue samples (119899 = 18 6 replicates perday for 3 days)

c Conc Interday IntradayngmL RE () CV () RE () CV ()

Plasma2 minus0394 134 minus0887 35020 minus518 156 minus485 225400 minus328 150 minus276 247

Liver2 0690 155 minus0197 46420 minus0528 959 minus0198 185400 minus103 104 minus117 162

Heart2 minus0309 425 minus483 32220 409 161 495 246400 minus365 219 minus257 159

Spleen2 minus214 900 minus335 76320 396 509 346 225400 minus0313 475 0 198

Lung2 minus680 137 minus133 47220 minus205 141 minus852 267400 minus930 102 minus126 111

Kidney2 473 536 394 53620 399 133 743 277400 minus649 136 minus573 149

Brain2 minus131 948 minus123 47420 327 559 0792 140400 minus328 356 minus336 212

Testicle2 minus420 325 minus236 46020 0538 493 307 269400 minus0379 579 257 402

three separate days The detailed results of the inter- andintraday precision and accuracy of icariin in biologicalmatrixwere summarized in Table 2

334 Recovery and Matrix Effects As shown in Table 3 theextraction recovery of icariin was in the range of 825ndash1006which suggested that the recovery of this method was consis-tent and reproducible And the suppression of ionization inplasma and tissue homogenate was differentThe interferenceof ionization caused by kidney homogenate was significantwhile that of brain homogenate was much lower

335 Stability Five replicates of QC at three different con-centration levels were used to assess the stability of icariinunder various conditions The results were summarized inTable 4 which indicated that icariin was stable in autosam-pler (24 h) at 4∘C bench-top (6 h) at room temperature threefreeze-thaw cycles and frozen condition atminus80∘C for 30 daysas the RE values were within plusmn15 for the low medium andhigh concentrations

336 Plasma Pharmacokinetics After a single oral admin-istration of the total flavonoid extract of Herba Epimediithe concentration of icariin in plasma was successfully deter-mined by LC-MSMS method described above The meanplasma concentration-time profile of icariin was shown in

Icariin

10 1550Time (h)

0

50

100

150

Con

cent

ratio

n (n

gm

L)

Figure 4 Plasma concentration versus time profile of icariin in rats(119899 = 6) following a single oral dose of Herba Epimedii extract(corresponding 42mgg icariin)

Figure 4 Pharmacokinetic parameters were estimated usingtheDAS software and performed by noncompartmental anal-ysis Peak plasma concentration (119862max) and the time at whichit occurred (119879max) were obtained from the visual inspection

8 BioMed Research International

Table 3 Matrix effect and extraction recovery for the assay of icariin in plasma and tissue samples (119899 = 5)

Matrix ConcngmL

Matrix effectMean plusmn SD ()

Extraction efficiencyMean plusmn SD ()

Plasma2 712 plusmn 29 869 plusmn 60

20 690 plusmn 20 841 plusmn 47

400 667 plusmn 10 849 plusmn 58

Liver2 715 plusmn 33 939 plusmn 75

20 722 plusmn 41 903 plusmn 19

400 703 plusmn 31 905 plusmn 22

Heart2 679 plusmn 38 889 plusmn 39

20 688 plusmn 26 922 plusmn 23

400 699 plusmn 18 931 plusmn 17

Spleen2 692 plusmn 53 887 plusmn 84

20 701 plusmn 37 835 plusmn 39

400 714 plusmn 38 825 plusmn 14

Lung2 710 plusmn 22 880 plusmn 59

20 712 plusmn 24 854 plusmn 45

400 738 plusmn 16 838 plusmn 19

Kidney2 577 plusmn 62 865 plusmn 67

20 555 plusmn 53 900 plusmn 37

400 666 plusmn 152 853 plusmn 61

Brain2 906 plusmn 58 897 plusmn 57

20 893 plusmn 19 949 plusmn 39

400 870 plusmn 23 990 plusmn 23

Testicle2 802 plusmn 35 915 plusmn 67

20 773 plusmn 21 964 plusmn 22

400 777 plusmn 22 1006 plusmn 19

of the plasma concentration-time curve Elimination rateconstant (119870119890) was determined from the least-square fittedterminal log-linear portion of the plasma concentration-timeprofile Elimination half-life (11990512) was calculated by 0693KeTotal area under the plasma concentration-time curve oficariin from time zero to infinity (AUC0ndashinfin) was calculatedas the sumofAUC0ndash12 the area up to the last quantifiable timepoint (12 h) and the area from the 12 h time point to infinityAUC0ndash12 was calculated using the trapezoidal rule and theextrapolated area was calculated from the last measurableconcentration 119862119905 divided by 119870119890 The major pharmacokineticparameters of icariin were summarized in Table 5

34 Tissue Distribution The tissue distribution of icariin infemale and male rats was investigated following a single oraldose of total flavonoid extract from Herba Epimedii (cor-responding 42mgg icariin) The mean concentration-timeprofile of each tissue was presented in Figure 5 indicatingthat icariin was poorly absorbed after oral administrationbecause all the tissue concentrations were kept at a verylow level The peak concentration of icariin in most ofcollected tissues appeared at 1 h after administration except

for liver and female reproductive organs in which theconcentration reached the peak at 05 h demonstrating thaticariin underwent a rapid and wide distribution Moreoverthe tissue concentrations in male rat were generally muchhigher than those in female rat The tissue area under thecurve from time zero to infinity was in the following orderliver gt lung gt spleen gt heart gt kidney gt brain gt testiclefor mail rat and liver gt uterus gt heart gt ovary gt lung gtkidney gt spleen gt brain for female rats As shown in Figure 5the concentration levels in liver and lung were much higherthan those in other tissues illustrating that liver and lungare the main target organs of icariin in male rat after oraladministration of the total flavonoid extract Furthermorethe concentration level in uteruswasmuchhigher than that inany other tissues of female rat demonstrating that the gender-related difference of icariin in tissue distribution do existand the reproductive organs are the main target organs oficariin for female rat Meanwhile icariin was found with verylow AUC0ndashinfin in brain which indicated that icariin could notefficiently cross the blood-brain barrier Over time the maintrend of the mean concentration of icariin in all tissues wasreduced and the concentration dropped very quickly at 4 h

BioMed Research International 9

Table 4 Stability of icariin in plasma and tissue samples (119899 = 6)

Matrix Conc Mean plusmn SDngmL Bench-topa Freeze-thawb Autosamplerc Long-termd

Plasma2 975 plusmn 37 968 plusmn 57 1047 plusmn 81 993 plusmn 59

20 945 plusmn 59 1013 plusmn 31 955 plusmn 37 943 plusmn 16

400 977 plusmn 39 1030 plusmn 19 945 plusmn 26 955 plusmn 37

Liver2 987 plusmn 63 1015 plusmn 21 1114 plusmn 44 986 plusmn 54

20 949 plusmn 14 900 plusmn 13 1060 plusmn 16 957 plusmn 21

400 875 plusmn 13 897 plusmn 13 894 plusmn 14 1125 plusmn 22

Heart2 1086 plusmn 33 1002 plusmn 43 955 plusmn 22 1150 plusmn 30

20 1056 plusmn 17 996 plusmn 10 973 plusmn 13 1002 plusmn 42

400 1026 plusmn 15 942 plusmn 29 892 plusmn 09 907 plusmn 50

Spleen2 954 plusmn 92 1018 plusmn 44 955 plusmn 76 1013 plusmn 59

20 989 plusmn 14 1094 plusmn 11 928 plusmn 09 1123 plusmn 34

400 967 plusmn 27 1032 plusmn 04 871 plusmn 12 974 plusmn 26

Lung2 959 plusmn 35 856 plusmn 30 1032 plusmn 48 983 plusmn 43

20 1017 plusmn 80 923 plusmn 40 1094 plusmn 46 1102 plusmn 18

400 925 plusmn 18 932 plusmn 15 997 plusmn 04 1098 plusmn 17

Kidney2 1012 plusmn 34 916 plusmn 40 951 plusmn 22 984 plusmn 48

20 1025 plusmn 28 974 plusmn 18 955 plusmn 16 934 plusmn 22

400 920 plusmn 14 911 plusmn 08 899 plusmn 10 1095 plusmn 22

Brain2 858 plusmn 60 974 plusmn 73 966 plusmn 32 1094 plusmn 32

20 1033 plusmn 41 1035 plusmn 31 1030 plusmn 35 974 plusmn 12

400 1002 plusmn 29 990 plusmn 31 978 plusmn 21 1102 plusmn 30

Testicle2 1058 plusmn 39 853 plusmn 24 997 plusmn 55 933 plusmn 41

20 1040 plusmn 14 870 plusmn 13 1016 plusmn 19 1065 plusmn 15

400 1034 plusmn 39 984 plusmn 16 995 plusmn 33 1068 plusmn 38aAt least 1 h at room temperature bAt least 3 freeze-thaw cycles cAt least 24 h at 4∘C dAt least 4 weeks at minus80 plusmn 5∘C

Table 5 Pharmacokinetic parameters of icariin in rat after asingle oral dose of Herba Epimedii extract (corresponding 42mggicariin)

Parameter IcariinAUC0ndashinfin (ugLsdoth) 164155 plusmn 144129

AUMC0ndashinfin (ugLsdoth) 538424 plusmn 262081

MRT0ndashinfin (h) 3990 plusmn 1987

11990512 (h) 3149 plusmn 2364

119879max (h) 0458 plusmn 0246

CL119911119865 (Lhkg) 389043 plusmn 204472

after administration suggesting no accumulation in tissuesand a rapid elimination of this compound

35 Metabolism Study of Icariin in Rat Urine and Feces

351 Metabolite Profile of Rat Urine and Feces In thisstudy urine and feces samples were both analyzed by LC-MSMS By comparing the total ion chromatograms of drug-containing urine sample and predose control urine sampleneither parent drug nor metabolites were observed in raturine following two routes of administration of icariinHowever a total of 11 icariin metabolites were detected in ratfeces including 10 metabolites after oral administration and

9 following intramuscular introduction The representativechromatograms of the metabolites of 0ndash24 h rat feces wereshown in Figure 6 No additional metabolites were observedin the feces of 24ndash48 h collection period Results showedthat the major metabolites of icariin were M8 and M11whose total amount accounted for over 90 of total peakarea in the LC-UV chromatogram of 0ndash24 h feces And theconcentration of metabolites in feces rapidly declined over24ndash48 h collection period The MS2 and MS3 spectra oficariin and its metabolites were exhibited in Figure 7 andthe observed and calculatedmasses formula retention timesmass errors and MS data of these analytes were presented inTable 6 Proposed metabolic pathways for icariin in rat feceswere shown in Figure 8

352 Identification of Metabolites Known compounds wereidentified by comparison with the chromatographic behaviorand MS fragmentation pattern of reference standards Forunknown constituents their structures were characterizedbased on their retention behavior and their MS119899 spectraobtained on-line Moreover under the optimized LC-MSconditions all the in vivometabolites of icariin could give theprecursor ions of sufficient abundance in order to facilitatethe analysis of multistage mass spectrometry Because allmetabolites originated from icariin and their MS fragmen-tation patterns were largely similar to that of icariin it was

10 BioMed Research International

Heart Spleen Lung Kidney Brain TesticleLiver0

10

20

30

40

50C

once

ntra

tion

(ng

g)

025 Ｂ

05 Ｂ

1 Ｂ

2 Ｂ

4 Ｂ

6 Ｂ

(a)

0

10

20

30

40

50

Con

cent

ratio

n (n

gg)

Heart Spleen Lung Kidney Brain Uterus OvaryLiver

025 Ｂ

05 Ｂ

1 Ｂ

2 Ｂ

4 Ｂ

6 Ｂ

(b)

Figure 5 Tissue distribution of icariin in rats after a single oral dose of Herba Epimedii extract (corresponding 42mgg icariin (a) male rat(b) female rat)

663517

5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 900Time (min)

0

50

100

(a)

6155

512665

6637520419520

20

40

60

80

100

5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 900Time (min)

52 54 56 58500

50 M11

M10

M8

M7

M6M5

M4

M3M2

M1

M9

(b)

658

61465278

506

0

20

40

60

80

100

5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 900Time (min)

M11

M3 M8

(c)

Figure 6 Representative chromatograms of icariin metabolites in rat feces of 0ndash24 h (a) blank feces sample (b) feces sample after oraladministration and (c) feces sample after intramuscular administration

BioMed Research International 11

36724

6755435214

Icariin

513320

20

40

60

80

100

Rela

tive a

bund

ance

400 600 800200mz

35218

Icariin

367250

20

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100

Rela

tive a

bund

ance

400 600200mz

35211

36722

29727

0

20

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100

Rela

tive a

bund

ance

400300mz

2811329595

26725 3090935216

25506

0

20

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100

Rela

tive a

bund

ance

300 400200mz

28408

35323

25512

0

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Rela

tive a

bund

ance

400 600200mz

35217

36722

0

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tive a

bund

ance

300 400 500200mz

36722

6892932124

400 600 800200mz

0

20

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Rela

tive a

bund

ance

35318

49919

0

20

40

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Rela

tive a

bund

ance

400 600200mz

35215

48116 64516

400 600 800200mz

0

20

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Rela

tive a

bund

ance

400 600 800 1000mz

36723

83734

79088

0

20

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Rela

tive a

bund

ance

M1M1

M2

M2

M3

M3

M4

M4

MS2

MS3

MS2

MS3

MS3

MS2

MS2

MS2

MS3

MS3

Figure 7 Continued

12 BioMed Research International

38218

36714 5293950114

0

20

40

60

80

100

Rela

tive a

bund

ance

400 500 600300mz

M5

MS2

36722

33916 382180

20

40

60

80

100

Rela

tive a

bund

ance

300 400 500200mz

M5

MS3

35217

36711

0

20

40

60

80

100

Rela

tive a

bund

ance

300 400 500200mz

M6

MS3

36724

35227 6452032309

0

20

40

60

80

100

Rela

tive a

bund

ance

400 600 800200mz

M6

MS2

36614

6592335118

0

20

40

60

80

100

Rela

tive a

bund

ance

400 600 800200mz

M7

MS2

35116

32317

31112

36607

0

20

40

60

80

100

Rela

tive a

bund

ance

300 400 500200mz

M7

MS3

35109

32308

31113

3660629520

0

20

40

60

80

100

Rela

tive a

bund

ance

300 400 500200mz

M8

MS3

36615

51313

400 600200mz

0

20

40

60

80

100

Rela

tive a

bund

ance M8

MS2

36514

35528 38328

33929

0

20

40

60

80

100

Rela

tive a

bund

ance

400300mz

M9

MS2

35019

36508

33724

0

20

40

60

80

100

Rela

tive a

bund

ance

400200mz

M9

MS3

Figure 7 Continued

BioMed Research International 13

25308

16310

27902

29817

0

20

40

60

80

100

Rela

tive a

bund

ance

400200mz

M10

MS3

29810

35312

21913

400200mz

0

20

40

60

80

100

Rela

tive a

bund

ance M10

MS2

35208

36723

309280

20

40

60

80

100

Rela

tive a

bund

ance

400 600200mz

M11

MS2

30911

2810435215

400200mz

0

20

40

60

80

100

Rela

tive a

bund

ance M11

MS3

Figure 7 Representative LC-MS119899 spectra of icariin and its metabolites M1ndashM11 in rat feces

necessary to characterize the MS119899 behavior of icariin toidentify and assign the metabolites of the constituent Thedeprotonated molecule [MndashH]minus of icariin appears at 119898119911675 which readily eliminates its glucosyl and rhamnosylgroups to produce an [MndashHndash308]minus ion at 119898119911 367 The[MndashHndash308]minus ion yields a fragment at 119898119911 352 by losing amethyl radical And of theMS4 spectrum of [MndashHndash308ndash15]minusion the fragment at 119898119911 323 can be reasonably attributed tothe loss of 3-COH of flavone skeletonrsquos C ring

M1 as aminormetabolite of icariin was only found in thefeces of the oral group Its quasimolecular ion occurs at 119898119911837 (C39H49O20) whose molecular mass is 162Da more thanthat of icariin And its major MS119899 fragment ions are the sameas those of icariinThemost abundantMS2 andMS3 fragmentions also appear at 119898119911 367 and 352 indicating the loss ofone rhamnosyl and two glucosyl moieties and the sequentialloss of methyl group respectively Although the molecularweight of M1 is equal to that of epimedin A there existsa significant difference on their chromatographic retentionbehavior Therefore M1 is tentatively identified as an isomerof epimedin A

Both M2 and M6 show the same quasimolecular ion[M minus H]minus at 119898119911 645 (C32H37O14) with the elution time at5044 and 5743min respectively M2 was only observed infeces after intramuscular introduction of icariin while M6was found in feces following both routes of administrationM6 is unambiguously identified as sagittatoside B incomparison with the reference standard Analogous to theMS119899 fragments of icariin (see Figure 7 and Table 6) M2also yielded the major fragment ions at 119898119911 352 323 309296 281 and 268 strongly suggesting that M2 should be

a conjunction metabolite of icariin Thus M2 is tentativelyassigned as sagittatoside B isomer

M3 was found in rat feces after both intramuscular andoral administration of icariin Its MS spectrum exhibits adeprotonated molecule [MndashH]minus at 119898119911 499 (C26H27O10)176Da less than that of icariin By comparison with thereference standard it is identified as baohuoside II TheMSMS spectrum shows the most abundant fragment ionat 119898119911 353 indicating the loss of rhamnose moiety TheMS3 spectrum displays the major fragment ions at 119898119911 325298 and 284 separately corresponding to the loss of COisobutenyl and isopentenyl radicals

M4 is a minor metabolite of icariin detected in the fecesof rats in the oral administration group Its deprotonatedmolecule (C33H37O16) is at 119898119911 689 And the major MSMSfragment at 119898119911 367 results from the loss of glucuronicacid and rhamnose moieties Its MS3 spectrum produces afragment at119898119911 352 (the loss of a methyl group) Thus M4 istentatively characterized as icariin glucuronide

M5ndashM11 of icariin metabolites were observed in rat fecesafter both intramuscular and oral administration of icariin

M5 has a deprotonated molecular ion at 119898119911 529(C27H29O11) in its MS spectrum The MS119899 spectra show themajor fragment ions at119898119911 501 (the loss of CO) 382 (the lossof rhamnosyl group) 367 (the loss of methyl radical from theion at119898119911 382) and 339 (the successive loss of methyl radicaland CO) Therefore M5 is tentatively identified as hydroxylicariside II

M7 is identified as 210158401015840-O-rhamnosyl icariside II based onthe reference standard and related mass spectral pattern The

14 BioMed Research International

Table6Ch

aracteriz

ationof

metaboliteso

ficariinin

ratfeces

Num

ber

Com

poun

dRT

Form

ula

Observedmass

RDB

Error

LCM

S119899data

[MndashH]minus

(ppm

)

PlowastIcariin

5519

C 33H39O15

67522888

145

0790

MS2[675]367(100)352(25)

MS3[675-367]352(100)

MS4[675-367-352]323(

30)309(100)296(45)281

(65)268

(50)255

(20)

M1

Epim

edin

Aiso

mer

a5038

C 39H49O20

8372

819

155

0824

MS2[837]790(50)367

(100)

MS3[837-367]352(100)297(20)

M2

Sagitta

tosid

eBiso

mer

b5044

C 32H37O14

6452186

145

1314

MS2[645]481(20)352(100)

MS3[645-352]323(40)309

(60)296

(90)281

(100)268(60)

M3

lowastBa

ohuo

sideIIa

b5278

C 26H27O10

4991

605

135

1295

MS2[499]353(100)

MS3[499-353]325(20)298

(40)284

(100)255(30)

M4

Icariin

glucuron

idea

5404

C 33H37O16

6892

084

155

1202

MS2[689]367(100)321(60)

MS3[689-367]352(100)

M5

Hydroxylicaris

ideIIa

b5412

C 27H29O11

5291

716

135

1132

MS2[529]501(20)382(100)367(40)

MS3[529-382]367(100)339(10)

M6

lowastSagitta

tosid

eBab

5743

C 32H37O14

6452184

145

1020

MS2[645]367(100)352(30)

MS3[645-367]352(100)

M7

lowast210158401015840-O

-Rhamno

syl-icaris

ideIIa

b5756

C 33H39O14

6592

344

145

1529

MS2[659]366(100)351(20)323(15)

MS3[659-366]351(100)323

(80)311(60)305

(20)

M8

lowastIcariside

IIab

6146

C 27H29O10

5131760

135

0851

MS2[513]366(100)

MS3[513-366]351(100)323

(80)311(50)305

(20)

M9

Hydroxylicaritin

ab6233

C 21H19O7

3831137

125

3160

MS2[383]365(100)355(60)339

(40)

MS3[383-365]350(100)321(20)230(40)

M10

Desmethylicaritin

ab6636

C 20H17O6

3531024

125

1119

MS2[353]298(100)219(10)

MS3[353-298]279(60)269

(40)253

(100)241(30)164(70)

M11

lowastIcaritinab

8265

C 21H19O6

3671183

125

1921

MS2[367]352(100)309(20)297

(20)

MS3[367-352]309(100)297(60)

lowastCom

paredwith

ther

eference

standardaObservedin

ratfeces

after

oraladministratio

nb O

bservedin

ratfeces

after

intram

usculara

dministratio

n

BioMed Research International 15

O

OO

O

O

O

OHOH

OH

OH

OH OHOH

OH

OHOH

OHOH

OH

OHOH OH

OH

OH

OH

OHOH

OH

OH

OH

OH

OH

OH

OH

OHOH

OH

OH

OH

OH

OHOH

OHOH

OHOH

OH

OH

OH

OH

OHOH

HO HO

HO

HO

HO

HOHO

HO

HO

HO

OH

O

OO

O

OOHOOC

O

OO

O

O

OO

OO

O

O

OO

OO

O

O

OO

O

O

OO

O

O

O

O

O

O

O

icariin

epimedin A isomer

minus rh

a

minus methyl

+ rhaOxidation

minus methyl

Icariin glucuronide

+ xyl

+ glcGlucuronidation

minus rha

Oxidation

minus rha

+ xyl

Sagittatoside B isomer

minus glc

M1

M4

M2

OCH3 OCH3

OCH3

OCH3

OCH3

OCH3

OCH3

OCH3

M7 2-O-rhamnosyl icariside II

M5 hydroxyl icariside II

M3 baohuoside II

M8 icariside II

M6 sagittatoside B

M9 hydroxyl icaritinM10 desmethyl icaritin

M11 icaritin

Figure 8 Proposed metabolic pathways for icariin in rat feces

MS spectrum of M7 shows a quasimolecular ion [MndashH]minusat 119898119911 659 (C33H39O14) The major fragment ions at 119898119911366 351 and 323 result from the concurrent loss of therhamnose chain the successive loss of a methyl group andCO respectively

As shownFigure 7M8 is themost abundantmetabolite oficariin By comparison with the reference standard it is iden-tified as icariside II Its MS spectrum shows a deprotonatedmolecular ion at 119898119911 513 (C27H29O10) And similar to the

MS119899 pattern of icariin the major fragment ions at 119898119911 366351 and 323 separately match the loss of rhamnose moietythe sequential loss of rhamnose and methyl groups and theloss of CO

M9 has a quasimolecular ion [MndashH]minus at 119898119911 383(C21H19O7) 146Da less than that of M5 (hydroxyl icarisideII) It is tentatively identified as hydroxyl icaritin Its MSMSspectrum shows themain fragments of119898119911 365 355 and 339respectively corresponding to the neutral loss of H2O CO

16 BioMed Research International

and CO2 MS3 spectrum the fragment ions at 119898119911 350 and321 individually result from the neutral loss of methyl radicaland CO2

The quasimolecular ion [MndashH]minus of M10 is at 119898119911 353146Da less than that of M3 (baohuoside II) The majorfragment ion in its MSMS spectrum is at119898119911 298 matchingthe loss of isobutenyl radical The fragment at 119898119911 219 ispossibly produced by the neutral loss of C8H6O2 (134Da) dueto the retro-Diels-Alder (RDA) fragmentation of the flavonersquosC ring Moreover the MS3 spectrum affords fragment ionsat 119898119911 279 269 253 and 164 resulting from the loss ofH2O CO CO2 and C8H6O2 (134Da) respectively Thus thismetabolite is reasonably identified as desmethyl icaritin

M11 was observed as anothermajormetabolite in rat fecesof both two administration groups (Figure 7) By comparisonwith the reference standard it is identified as icaritin Its MSspectrum yields a quasimolecular ion at119898119911 367 (C21H19O6)And themajor fragment ion at119898119911 352 is characterized as theloss of methyl group The MS3 fragment at 119898119911 309 resultsfrom the loss of methyl group and CO and ion at 119898119911 297is the outcome of the neutral loss of isopentene (70Da) fromthe deprotonated molecule

4 Conclusion

Themenopausal syndrome improvement and possible cancerpreventive activity of Herba Epimedii is receiving more andmore attention Information on the in vivo disposition ofHerba Epimedii components is important for better under-standing the biological effects of this herbal drug For thispurpose our investigation was designed to obtain informa-tion on the plasma pharmacokinetics and tissue distributionof icariin in rat and the excretion of icariin and itsmetabolitesIn this study we firstly established and validated a rapidsensitive and reliable LC-MS method for the determinationof icariin in various biological samples collected at differenttime points and then the method was applied to the plasmapharmacokinetics and tissue distribution study of icariin inrats after oral administration of the total flavonoid extractof Herba Epimedii In addition the differences of the tissuedistribution of icariin in between male and female rats werestudied after oral and intramuscular administration of icariinAnd the metabolic pattern of icariin was also investigatedThe data showed the plasma and tissue concentration oficariin were kept at a very low level at all collection pointswhich suggests icariin is poorly absorbed after oral adminis-trationThepeak concentration in plasma and collected tissuesamples appeared at 05ndash1 h and then quickly dropped at 4 hafter administration demonstrating that a small amount oficariin can be rapidly absorbed and eliminated and there isno accumulation in rat tissues Moreover the concentrationin liver lung and reproductive organs was much higherthan that in other tissues indicating that liver lung andreproductive organs are the main target sites of icariin in rats

The gender-related difference of icariin in tissue distribu-tion has not been reported in the past published literatureOur study demonstrated that the distribution features oficariin are similar in most of tissues between male andfemale rats However there exist a few remarkable gender

differences such that thewhole tissue concentration of icariinin male rats was much higher than female rats except in gen-ital organs (high in female and low in male) In China HerbaEpimedii is a famous tonic and aphrodisiac to invigoratekidney and strengthenYang therefore it is commonly used totreat male sexual functional decline and female menopausalsyndrome As one of the predominant constitutes in HerbaEpimedii icariin plays a very crucial role in exerting effectsso the relatively high icariin concentration in female genitalorgans actually supports the above-mentioned traditionalChinese medicine theory and clinical practice and makesthis compound a good future prospect for treating womenrsquosmenopausal related diseases

In the present study the complete metabolic profile oficariin has also been investigated The results illustrate thatfeces should be the major excretion way for both oral andintramuscular administration of icariin Based on LC-UVand LC-MS119899 analyses eleven metabolites were detected infecal samples and were further identified Among them10 metabolites appeared in rat feces of oral administrationgroup and 9 in the feces of intramuscular introduction groupThe major metabolic routes of clearance are proposed to behydrolysis demethylation oxidation and conjugation

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

Authorsrsquo Contributions

Shunjun Xu and Jiejing Yu contributed equally to this work

References

[1] X Wang Y He B Guo et al ldquoIn vivo screening for anti-osteo-porotic fraction from extract of herbal formula xianlinggubaoin ovariectomized micerdquo PLoS ONE vol 10 no 2 Article IDe0118184 2015

[2] R-H Liu X Kang L-P Xu et al ldquoEffects of the combinedextracts of Herba Epimedii and Fructus Ligustri Lucidi on bonemineral content and bone turnover in osteoporotic ratsrdquo BMCComplementary and Alternative Medicine vol 15 no 1 article112 2015

[3] B C L Chan H Y Lee W S Siu et al ldquoSuppression of mastcell activity contributes to the osteoprotective effect of an herbalformula containing Herba Epimedii Fructus Ligustri Lucidiand Fructus Psoraleaerdquo Journal of Pharmacy and Pharmacologyvol 66 no 3 pp 437ndash444 2014

[4] S CW Sze Y Tong T B Ng C L Y Cheng andH P CheungldquoHerba Epimedii anti-oxidative properties and its medicalimplicationsrdquoMolecules vol 15 no 11 pp 7861ndash7870 2010

[5] R Niu ldquoAction of the drug Herba Epimedii on testosterone ofthe mouse plasma and its accessory sexual organ before andafter processingrdquo China Journal of Chinese Materia Medica vol14 no 9 pp 530ndash574 1989

[6] F-X Liu S Sun and Z-Z Cui ldquoAnalysis of immunologi-cal enhancement of immunosuppressed chickens by Chineseherbal extractsrdquo Journal of Ethnopharmacology vol 127 no 2pp 251ndash256 2010

BioMed Research International 17

[7] J-H Kim Y-J Mun S-J Im J-H Han H-S Lee and W-HWoo ldquoEffects of the aqueous extract of Epimedii Herba on theantibody responses in micerdquo International Immunopharmacol-ogy vol 1 no 5 pp 935ndash944 2001

[8] X Yu Y Tong X-Q Han et al ldquoAnti-angiogenic activity ofHerba Epimedii on zebrafish embryos in vivo and HUVECs invitrordquo Phytotherapy Research vol 27 no 9 pp 1368ndash1375 2013

[9] D-C Zhang J-L Liu Y-B Ding J-G Xia and G-Y ChenldquoIcariin potentiates the antitumor activity of gemcitabine ingallbladder cancer by suppressingNF-120581BrdquoActa PharmacologicaSinica vol 34 no 2 pp 301ndash308 2013

[10] L Kong J Liu J Wang et al ldquoIcariin inhibits TNF-120572IFN-120574induced inflammatory response via inhibition of the substanceP and p38-MAPK signaling pathway in human keratinocytesrdquoInternational Immunopharmacology vol 29 no 2 pp 401ndash4072015

[11] H Shao J Shen M Wang et al ldquoIcariin protects against tita-nium particle-induced osteolysis and inflammatory response ina mouse calvarial modelrdquo Biomaterials vol 60 pp 92ndash99 2015

[12] Q Wei J Zhang G Hong et al ldquoIcariin promotes osteogenicdifferentiation of rat bone marrow stromal cells by activatingthe ER120572-Wnt120573-catenin signaling pathwayrdquo Biomedicine ampPharmacotherapy vol 84 pp 931ndash939 2016

[13] S-Q Zhang W-J Cai J-H Huang et al ldquoIcariin a naturalflavonol glycoside extends healthspan in micerdquo ExperimentalGerontology vol 69 pp 226ndash235 2015

[14] M N Makarova O N Pozharitskaya A N Shikov S VTesakova V G Makarov and V P Tikhonov ldquoEffect of lipid-based suspension of Epimedium koreanum Nakai extract onsexual behavior in ratsrdquo Journal of Ethnopharmacology vol 114no 3 pp 412ndash416 2007

[15] Y Chen J Wang X Jia X Tan and M Hu ldquoRole of intestinalhydrolase in the absorption of prenylated flavonoids present inyinyanghuordquoMolecules vol 16 no 2 pp 1336ndash1348 2011

[16] J J Yu Y J Liu S J Xu S Sun Y J Xu and L Yang ldquoSimulta-neous quantitative determination of four epimediumflavonoidsin rat urine by LC-MSMSrdquo Traditional Chinese Drug Researchamp Clinical Pharmacology vol 27 no 4 pp 537ndash541 2016

[17] Y Lu N Li Y Deng et al ldquoSimultaneous determination oficariin naringin and osthole in rat plasma by UPLC-MSMSand its application for pharmacokinetic study after oral admin-istration of Gushudan capsulesrdquo Journal of Chromatography Bvol 993-994 pp 75ndash80 2015

[18] N Wang X Huang X Wang Y Zhang R Wu and D ShouldquoPipette tip solid-phase extraction and high-performance liquidchromatography for the determination of flavonoids fromEpimedii herba in rat serum and application of the techniqueto pharmacokinetic studiesrdquo Journal of Chromatography B vol990 pp 64ndash72 2015

Submit your manuscripts athttpswwwhindawicom

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Medicinal ChemistryInternational Journal of

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Pharmaceutics

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MEDIATORSINFLAMMATION

of

4 BioMed Research International

gained the approval of an independent ethics committeeat Guangdong Provincial Hospital of Chinese MedicineThe experiment was performed in an SPF level laboratoryauthorized by Guangdong Provincial Government

261 Plasma Pharmacokinetics Six male Sprague-Dawley(SD) rats (250 plusmn 20 g) were obtained from GuangdongProvince Laboratory Animal Center and kept in an envi-ronmentally controlled breeding room for 3 days beforethe experiment started The rats were orally given a singledose of 069 gkg of total flavonoid extract from HerbaEpimedii (corresponding icariin approximately at 42mgg)Blood samples were serially withdrawn from ratrsquos retroorbitalplexus into heparin lithium-anticoagulant tubes at 0 (beforedose) 0083 025 05 075 1 15 2 3 4 6 8 10 12 and24 h after administration The collected samples were thencentrifuged at 3000 rpm for 15min and supernatant plasmawas transferred into another tube and stored at minus80∘C untilanalysis

262 Tissue Distribution Study Thirty-six SD rats (220 plusmn20 g 18 males and 18 females) were orally given a single doseof 069 gkg of total flavonoid extract from Herba Epimedii(approximately containing 42mgg of icariin) Animals wereeuthanized at the time points of 025 05 1 2 4 and 6 hafter dose (119899 = 6 at each time point) Blood was col-lected via cardiac puncture (about 1000 120583L) and immediatelytransferred into heparinized tube The collected sample wascentrifuged at 3000 rpm for 15min and supernatant plasmawas transferred into another tube and stored at minus80∘C untilanalysis Nine kinds of rat tissues including liver heartspleen lung kidney brain testicle uterus and ovary wereimmediately removed and rinsed with ice-cold saline toremove extraneous blood and blot-dried and then stored inpreweighted and labeled vials at minus80∘C until use On the dayof analysis the tissue samples were thawed and weighed toobtain the tissue weight expressed as the difference betweenthe pre- and postvial weights

263 Metabolism Study of Icariin in Rat Urine and Feces Inthis study six SD rats (220 plusmn 20 g male) were individuallyplaced in metabolic cages and halved into two groups atrandom The reference standard of icariin (60mg) wasdissolved in 8mL of sterile water for injection containing40 1 3-propanediol (vv) The intramuscular group wasdosed with icariin (20mgkg) by intramuscular injection ofthis solution Oral administration of icariin (50mgkg) wasconducted on the other group Feces and urine samples wereseparately collected at 24 h after administration of icariin Allthe samples were stored at minus80∘C until use

27 Data Analysis Plasma concentration versus time profileswas obtained from each individual rat and the calculationof pharmacokinetic parameters was performed via DAS 211software (Mathematical Pharmacology Professional Com-mittee of China Shanghai China) with noncompartmentalpharmacokinetic analysis All data were expressed as mean plusmnstandard deviation (SD)

3 Results and Discussions

31 Optimization of Sample Preparation To obtain betterextraction efficiency and less endogenous interference differ-ent extraction procedures including solid-phase extractionand liquid-liquid extraction with different solvents weretested But all the extraction recoveries were not satisfactoryThus a simpler and less time-consuming method proteinprecipitation with methanol was finally used which wastestified to be rapid repeatable and sensitive enough for thedetermination of biological samples in the present study

32 Optimization of Chromatography andMass-SpectrometricConditions It is very important to pick a suitable IS toget high accuracy and precision when a mass spectrometeris equipped with LC as a detector In this study genisteinwas chosen as the IS because of its similar basic structurephysicochemical property and mass-spectrometric behaviorto those of icariin

The mass-spectrometric behavior of icariin and IS wasinvestigated using both positive- and negative-ion ESI Itwas found that both icariin and IS had good responses innegative-ion detection mode with low background noiselevel Multiple reaction monitoring (MRM) scan type wasused to improve specificity Detection was finally operated innegative-ion mode Quantification was performed by MRMmode and the selectedmonitor ion was119898119911 7357rarr 5135 foricariin ([M+CH3COOH-H]minus) and 119898119911 2690rarr 1332 for ISThe full scan andMSMS spectra of icariin and ISwere shownin Figure 2

In order to improve the peak shape and signal response ofanalytes and to reduce run time different analytical columnsand mobile phase compositions were tried to achieve goodresolution and symmetric peak shape for icariin and IS Bycomparison with Shiseido capcell pak C18 (150 times 20mmid 5 120583m) column Agilent XDB C18 (150 times 21mm id5 120583m) column could obtain better chromatographic behaviorand higher signal response for the two analytes Thus thiscolumn was finally selected for chromatographic analysisThe modifier of the mobile phase composed of acetonitrile-water binary solvent systemwas screened among ammoniumacetate acetic acid and formic acid As a result the mobilephase consisting of acetonitrile-water containing 01 formicacid could improve the symmetry of peak shape and enhancethe signal response The flow rate of mobile phase was alsooptimized and finally set at 03mLmin Under the above-mentioned HPLC conditions the retention time of icariinand IS was within 6min and no interfering substance wasdetected at the retention time of analytes in blank rat plasmaand tissue samples (Figure 3)

33 Method Validation

331 Specificity The selectivity of the method was examinedby comparing the chromatograms of blank and correspond-ing spiked biological matrices The retention time of icariinand IS was about 26 and 50min respectively No interferingpeak of endogenous substances was observed at themeasured

BioMed Research International 5

Table 1 Regression equations of icariin in rat plasma and tissue samples (119899 = 3)

Sample Equation Correlation coefficient Linear rangengmL

Plasma 119884 = (00060 plusmn 00002)119883 + (00011 plusmn 00006) 09973 plusmn 00013 100ndash500Liver 119884 = (00053 plusmn 00001)119883 + (00014 plusmn 00008) 09986 plusmn 00008 100ndash500Heart 119884 = (00062 plusmn 00003)119883 + (00006 plusmn 00002) 09979 plusmn 00013 100ndash500Spleen 119884 = (00053 plusmn 00006)119883 + (00010 plusmn 00008) 09979 plusmn 00009 100ndash500Lung 119884 = (00060 plusmn 00006)119883 + (00015 plusmn 00008) 09974 plusmn 00015 100ndash500Kidney 119884 = (00045 plusmn 00003)119883 + (00006 plusmn 00002) 09986 plusmn 00004 100ndash500Brain 119884 = (00033 plusmn 00002)119883 + (00011 plusmn 00003) 09985 plusmn 00005 100ndash500Testicle 119884 = (00066 plusmn 00001)119883 + (00015 plusmn 00001) 09977 plusmn 00001 100ndash500

(a-1) (a-2)

7356

5136

765652972549

400 600 800 1000200mz (Da)

00

20e7

40e7

60e7

80e7

10e8

12e8

14e8

Inte

nsity

(cps

)

1332

26901067 1347633

2242

00

10e6

20e6

30e6

40e6

50e6

60e6

70e6

80e6

Inte

nsity

(cps

)

50 150 200 250 300 350 400 450 500100mz (Da)

(b-1) (b-2)

2690

2852

00

50e6

10e7

15e7

20e7

25e7

29e7

Inte

nsity

(cps

)

150 200 250 300 350 400 450 500100mz (Da)

5135

7357

529500

50e6

10e7

15e7

20e7

23e7

Inte

nsity

(cps

)

400 600 800 1000200mz (Da)

Figure 2 Full scan mass spectrum of icariin (a-1) and internal standard (genistein b-1) product ion spectrum of icariin (a-2) and internalstandard (genistein b-2)

mass transitions and retention time of icariin and IS in blankbiological matrix samples

332 Linearity and Sensitivity The assay was found to belinear over the concentration range of 1ndash500 ngmL forplasma and tissue homogenate The mean linear regressionequations were listed in Table 1 with correlations coefficientover 099 in which119883 corresponds to nominal concentrations

and 119884 corresponds to peak area ratios LLOQs with an 119878119873ratio of gt20 was 1 ngmL which was sensitive enough for thepharmacokinetic and tissue distribution studies of icariin

333 Precision and Accuracy Inter- and intra-assay vari-ability at three different concentrations HQC (400 ngmL)MQC (20 ngmL) LQC (2 ngmL) was determined in plasmaand tissue homogenate with five replicates on each day for

6 BioMed Research International

(a-1)

0

20

40

60

80

100

120

Inte

nsity

(cps

)

20 30 40 50 6010Time (min)

(a-2)

0

20

40

60

80

100

Inte

nsity

(cps

)

20 30 40 50 6010Time (min)

(b-2)

501

005000010e4

15e4

20e4

25e4

30e4

35e4

Inte

nsity

(cps

)

20 30 40 50 6010Time (min)

(c-2)

258

0

1000

2000

3000

4000

5000

60006787

Inte

nsity

(cps

)

20 30 40 50 6010Time (min)

(d-2)

260

0500

100015002000250030003500

Inte

nsity

(cps

)

20 30 40 50 6010Time (min)

(d-1)

260

20 30 40 50 6010Time (min)

0

100

200

300

400

500597

Inte

nsity

(cps

)

(c-1)

261

20 30 40 50 6010Time (min)

0

50

100

150

200

250

Inte

nsity

(cps

)

(b-1)

500

00

50000

10e4

15e4

20e422e4

Inte

nsity

(cps

)

20 30 40 50 6010Time (min)

Figure 3 Representative MRM chromatograms of blank plasma (a-1) blank liver (a-2) blank plasma spiked with IS (b-1) blank liver spikedwith IS (b-2) blank plasma spiked with icariin at LOQ (c-1) blank liver spiked with icariin at LOQ (d-1) rat plasma sample obtained at 05 hafter administration (c-2) and rat liver sample obtained at 05 h after administration (d-2)

BioMed Research International 7

Table 2 Intra- and interday accuracy and precision for the determination of icariin in plasma and tissue samples (119899 = 18 6 replicates perday for 3 days)

c Conc Interday IntradayngmL RE () CV () RE () CV ()

Plasma2 minus0394 134 minus0887 35020 minus518 156 minus485 225400 minus328 150 minus276 247

Liver2 0690 155 minus0197 46420 minus0528 959 minus0198 185400 minus103 104 minus117 162

Heart2 minus0309 425 minus483 32220 409 161 495 246400 minus365 219 minus257 159

Spleen2 minus214 900 minus335 76320 396 509 346 225400 minus0313 475 0 198

Lung2 minus680 137 minus133 47220 minus205 141 minus852 267400 minus930 102 minus126 111

Kidney2 473 536 394 53620 399 133 743 277400 minus649 136 minus573 149

Brain2 minus131 948 minus123 47420 327 559 0792 140400 minus328 356 minus336 212

Testicle2 minus420 325 minus236 46020 0538 493 307 269400 minus0379 579 257 402

three separate days The detailed results of the inter- andintraday precision and accuracy of icariin in biologicalmatrixwere summarized in Table 2

334 Recovery and Matrix Effects As shown in Table 3 theextraction recovery of icariin was in the range of 825ndash1006which suggested that the recovery of this method was consis-tent and reproducible And the suppression of ionization inplasma and tissue homogenate was differentThe interferenceof ionization caused by kidney homogenate was significantwhile that of brain homogenate was much lower

335 Stability Five replicates of QC at three different con-centration levels were used to assess the stability of icariinunder various conditions The results were summarized inTable 4 which indicated that icariin was stable in autosam-pler (24 h) at 4∘C bench-top (6 h) at room temperature threefreeze-thaw cycles and frozen condition atminus80∘C for 30 daysas the RE values were within plusmn15 for the low medium andhigh concentrations

336 Plasma Pharmacokinetics After a single oral admin-istration of the total flavonoid extract of Herba Epimediithe concentration of icariin in plasma was successfully deter-mined by LC-MSMS method described above The meanplasma concentration-time profile of icariin was shown in

Icariin

10 1550Time (h)

0

50

100

150

Con

cent

ratio

n (n

gm

L)

Figure 4 Plasma concentration versus time profile of icariin in rats(119899 = 6) following a single oral dose of Herba Epimedii extract(corresponding 42mgg icariin)

Figure 4 Pharmacokinetic parameters were estimated usingtheDAS software and performed by noncompartmental anal-ysis Peak plasma concentration (119862max) and the time at whichit occurred (119879max) were obtained from the visual inspection

8 BioMed Research International

Table 3 Matrix effect and extraction recovery for the assay of icariin in plasma and tissue samples (119899 = 5)

Matrix ConcngmL

Matrix effectMean plusmn SD ()

Extraction efficiencyMean plusmn SD ()

Plasma2 712 plusmn 29 869 plusmn 60

20 690 plusmn 20 841 plusmn 47

400 667 plusmn 10 849 plusmn 58

Liver2 715 plusmn 33 939 plusmn 75

20 722 plusmn 41 903 plusmn 19

400 703 plusmn 31 905 plusmn 22

Heart2 679 plusmn 38 889 plusmn 39

20 688 plusmn 26 922 plusmn 23

400 699 plusmn 18 931 plusmn 17

Spleen2 692 plusmn 53 887 plusmn 84

20 701 plusmn 37 835 plusmn 39

400 714 plusmn 38 825 plusmn 14

Lung2 710 plusmn 22 880 plusmn 59

20 712 plusmn 24 854 plusmn 45

400 738 plusmn 16 838 plusmn 19

Kidney2 577 plusmn 62 865 plusmn 67

20 555 plusmn 53 900 plusmn 37

400 666 plusmn 152 853 plusmn 61

Brain2 906 plusmn 58 897 plusmn 57

20 893 plusmn 19 949 plusmn 39

400 870 plusmn 23 990 plusmn 23

Testicle2 802 plusmn 35 915 plusmn 67

20 773 plusmn 21 964 plusmn 22

400 777 plusmn 22 1006 plusmn 19

of the plasma concentration-time curve Elimination rateconstant (119870119890) was determined from the least-square fittedterminal log-linear portion of the plasma concentration-timeprofile Elimination half-life (11990512) was calculated by 0693KeTotal area under the plasma concentration-time curve oficariin from time zero to infinity (AUC0ndashinfin) was calculatedas the sumofAUC0ndash12 the area up to the last quantifiable timepoint (12 h) and the area from the 12 h time point to infinityAUC0ndash12 was calculated using the trapezoidal rule and theextrapolated area was calculated from the last measurableconcentration 119862119905 divided by 119870119890 The major pharmacokineticparameters of icariin were summarized in Table 5

34 Tissue Distribution The tissue distribution of icariin infemale and male rats was investigated following a single oraldose of total flavonoid extract from Herba Epimedii (cor-responding 42mgg icariin) The mean concentration-timeprofile of each tissue was presented in Figure 5 indicatingthat icariin was poorly absorbed after oral administrationbecause all the tissue concentrations were kept at a verylow level The peak concentration of icariin in most ofcollected tissues appeared at 1 h after administration except

for liver and female reproductive organs in which theconcentration reached the peak at 05 h demonstrating thaticariin underwent a rapid and wide distribution Moreoverthe tissue concentrations in male rat were generally muchhigher than those in female rat The tissue area under thecurve from time zero to infinity was in the following orderliver gt lung gt spleen gt heart gt kidney gt brain gt testiclefor mail rat and liver gt uterus gt heart gt ovary gt lung gtkidney gt spleen gt brain for female rats As shown in Figure 5the concentration levels in liver and lung were much higherthan those in other tissues illustrating that liver and lungare the main target organs of icariin in male rat after oraladministration of the total flavonoid extract Furthermorethe concentration level in uteruswasmuchhigher than that inany other tissues of female rat demonstrating that the gender-related difference of icariin in tissue distribution do existand the reproductive organs are the main target organs oficariin for female rat Meanwhile icariin was found with verylow AUC0ndashinfin in brain which indicated that icariin could notefficiently cross the blood-brain barrier Over time the maintrend of the mean concentration of icariin in all tissues wasreduced and the concentration dropped very quickly at 4 h

BioMed Research International 9

Table 4 Stability of icariin in plasma and tissue samples (119899 = 6)

Matrix Conc Mean plusmn SDngmL Bench-topa Freeze-thawb Autosamplerc Long-termd

Plasma2 975 plusmn 37 968 plusmn 57 1047 plusmn 81 993 plusmn 59

20 945 plusmn 59 1013 plusmn 31 955 plusmn 37 943 plusmn 16

400 977 plusmn 39 1030 plusmn 19 945 plusmn 26 955 plusmn 37

Liver2 987 plusmn 63 1015 plusmn 21 1114 plusmn 44 986 plusmn 54

20 949 plusmn 14 900 plusmn 13 1060 plusmn 16 957 plusmn 21

400 875 plusmn 13 897 plusmn 13 894 plusmn 14 1125 plusmn 22

Heart2 1086 plusmn 33 1002 plusmn 43 955 plusmn 22 1150 plusmn 30

20 1056 plusmn 17 996 plusmn 10 973 plusmn 13 1002 plusmn 42

400 1026 plusmn 15 942 plusmn 29 892 plusmn 09 907 plusmn 50

Spleen2 954 plusmn 92 1018 plusmn 44 955 plusmn 76 1013 plusmn 59

20 989 plusmn 14 1094 plusmn 11 928 plusmn 09 1123 plusmn 34

400 967 plusmn 27 1032 plusmn 04 871 plusmn 12 974 plusmn 26

Lung2 959 plusmn 35 856 plusmn 30 1032 plusmn 48 983 plusmn 43

20 1017 plusmn 80 923 plusmn 40 1094 plusmn 46 1102 plusmn 18

400 925 plusmn 18 932 plusmn 15 997 plusmn 04 1098 plusmn 17

Kidney2 1012 plusmn 34 916 plusmn 40 951 plusmn 22 984 plusmn 48

20 1025 plusmn 28 974 plusmn 18 955 plusmn 16 934 plusmn 22

400 920 plusmn 14 911 plusmn 08 899 plusmn 10 1095 plusmn 22

Brain2 858 plusmn 60 974 plusmn 73 966 plusmn 32 1094 plusmn 32

20 1033 plusmn 41 1035 plusmn 31 1030 plusmn 35 974 plusmn 12

400 1002 plusmn 29 990 plusmn 31 978 plusmn 21 1102 plusmn 30

Testicle2 1058 plusmn 39 853 plusmn 24 997 plusmn 55 933 plusmn 41

20 1040 plusmn 14 870 plusmn 13 1016 plusmn 19 1065 plusmn 15

400 1034 plusmn 39 984 plusmn 16 995 plusmn 33 1068 plusmn 38aAt least 1 h at room temperature bAt least 3 freeze-thaw cycles cAt least 24 h at 4∘C dAt least 4 weeks at minus80 plusmn 5∘C

Table 5 Pharmacokinetic parameters of icariin in rat after asingle oral dose of Herba Epimedii extract (corresponding 42mggicariin)

Parameter IcariinAUC0ndashinfin (ugLsdoth) 164155 plusmn 144129

AUMC0ndashinfin (ugLsdoth) 538424 plusmn 262081

MRT0ndashinfin (h) 3990 plusmn 1987

11990512 (h) 3149 plusmn 2364

119879max (h) 0458 plusmn 0246

CL119911119865 (Lhkg) 389043 plusmn 204472

after administration suggesting no accumulation in tissuesand a rapid elimination of this compound

35 Metabolism Study of Icariin in Rat Urine and Feces

351 Metabolite Profile of Rat Urine and Feces In thisstudy urine and feces samples were both analyzed by LC-MSMS By comparing the total ion chromatograms of drug-containing urine sample and predose control urine sampleneither parent drug nor metabolites were observed in raturine following two routes of administration of icariinHowever a total of 11 icariin metabolites were detected in ratfeces including 10 metabolites after oral administration and

9 following intramuscular introduction The representativechromatograms of the metabolites of 0ndash24 h rat feces wereshown in Figure 6 No additional metabolites were observedin the feces of 24ndash48 h collection period Results showedthat the major metabolites of icariin were M8 and M11whose total amount accounted for over 90 of total peakarea in the LC-UV chromatogram of 0ndash24 h feces And theconcentration of metabolites in feces rapidly declined over24ndash48 h collection period The MS2 and MS3 spectra oficariin and its metabolites were exhibited in Figure 7 andthe observed and calculatedmasses formula retention timesmass errors and MS data of these analytes were presented inTable 6 Proposed metabolic pathways for icariin in rat feceswere shown in Figure 8

352 Identification of Metabolites Known compounds wereidentified by comparison with the chromatographic behaviorand MS fragmentation pattern of reference standards Forunknown constituents their structures were characterizedbased on their retention behavior and their MS119899 spectraobtained on-line Moreover under the optimized LC-MSconditions all the in vivometabolites of icariin could give theprecursor ions of sufficient abundance in order to facilitatethe analysis of multistage mass spectrometry Because allmetabolites originated from icariin and their MS fragmen-tation patterns were largely similar to that of icariin it was

10 BioMed Research International

Heart Spleen Lung Kidney Brain TesticleLiver0

10

20

30

40

50C

once

ntra

tion

(ng

g)

025 Ｂ

05 Ｂ

1 Ｂ

2 Ｂ

4 Ｂ

6 Ｂ

(a)

0

10

20

30

40

50

Con

cent

ratio

n (n

gg)

Heart Spleen Lung Kidney Brain Uterus OvaryLiver

025 Ｂ

05 Ｂ

1 Ｂ

2 Ｂ

4 Ｂ

6 Ｂ

(b)

Figure 5 Tissue distribution of icariin in rats after a single oral dose of Herba Epimedii extract (corresponding 42mgg icariin (a) male rat(b) female rat)

663517

5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 900Time (min)

0

50

100

(a)

6155

512665

6637520419520

20

40

60

80

100

5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 900Time (min)

52 54 56 58500

50 M11

M10

M8

M7

M6M5

M4

M3M2

M1

M9

(b)

658

61465278

506

0

20

40

60

80

100

5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 900Time (min)

M11

M3 M8

(c)

Figure 6 Representative chromatograms of icariin metabolites in rat feces of 0ndash24 h (a) blank feces sample (b) feces sample after oraladministration and (c) feces sample after intramuscular administration

BioMed Research International 11

36724

6755435214

Icariin

513320

20

40

60

80

100

Rela

tive a

bund

ance

400 600 800200mz

35218

Icariin

367250

20

40

60

80

100

Rela

tive a

bund

ance

400 600200mz

35211

36722

29727

0

20

40

60

80

100

Rela

tive a

bund

ance

400300mz

2811329595

26725 3090935216

25506

0

20

40

60

80

100

Rela

tive a

bund

ance

300 400200mz

28408

35323

25512

0

20

40

60

80

100

Rela

tive a

bund

ance

400 600200mz

35217

36722

0

20

40

60

80

100

Rela

tive a

bund

ance

300 400 500200mz

36722

6892932124

400 600 800200mz

0

20

40

60

80

100

Rela

tive a

bund

ance

35318

49919

0

20

40

60

80

100

Rela

tive a

bund

ance

400 600200mz

35215

48116 64516

400 600 800200mz

0

20

40

60

80

100

Rela

tive a

bund

ance

400 600 800 1000mz

36723

83734

79088

0

20

40

60

80

100

Rela

tive a

bund

ance

M1M1

M2

M2

M3

M3

M4

M4

MS2

MS3

MS2

MS3

MS3

MS2

MS2

MS2

MS3

MS3

Figure 7 Continued

12 BioMed Research International

38218

36714 5293950114

0

20

40

60

80

100

Rela

tive a

bund

ance

400 500 600300mz

M5

MS2

36722

33916 382180

20

40

60

80

100

Rela

tive a

bund

ance

300 400 500200mz

M5

MS3

35217

36711

0

20

40

60

80

100

Rela

tive a

bund

ance

300 400 500200mz

M6

MS3

36724

35227 6452032309

0

20

40

60

80

100

Rela

tive a

bund

ance

400 600 800200mz

M6

MS2

36614

6592335118

0

20

40

60

80

100

Rela

tive a

bund

ance

400 600 800200mz

M7

MS2

35116

32317

31112

36607

0

20

40

60

80

100

Rela

tive a

bund

ance

300 400 500200mz

M7

MS3

35109

32308

31113

3660629520

0

20

40

60

80

100

Rela

tive a

bund

ance

300 400 500200mz

M8

MS3

36615

51313

400 600200mz

0

20

40

60

80

100

Rela

tive a

bund

ance M8

MS2

36514

35528 38328

33929

0

20

40

60

80

100

Rela

tive a

bund

ance

400300mz

M9

MS2

35019

36508

33724

0

20

40

60

80

100

Rela

tive a

bund

ance

400200mz

M9

MS3

Figure 7 Continued

BioMed Research International 13

25308

16310

27902

29817

0

20

40

60

80

100

Rela

tive a

bund

ance

400200mz

M10

MS3

29810

35312

21913

400200mz

0

20

40

60

80

100

Rela

tive a

bund

ance M10

MS2

35208

36723

309280

20

40

60

80

100

Rela

tive a

bund

ance

400 600200mz

M11

MS2

30911

2810435215

400200mz

0

20

40

60

80

100

Rela

tive a

bund

ance M11

MS3

Figure 7 Representative LC-MS119899 spectra of icariin and its metabolites M1ndashM11 in rat feces

necessary to characterize the MS119899 behavior of icariin toidentify and assign the metabolites of the constituent Thedeprotonated molecule [MndashH]minus of icariin appears at 119898119911675 which readily eliminates its glucosyl and rhamnosylgroups to produce an [MndashHndash308]minus ion at 119898119911 367 The[MndashHndash308]minus ion yields a fragment at 119898119911 352 by losing amethyl radical And of theMS4 spectrum of [MndashHndash308ndash15]minusion the fragment at 119898119911 323 can be reasonably attributed tothe loss of 3-COH of flavone skeletonrsquos C ring

M1 as aminormetabolite of icariin was only found in thefeces of the oral group Its quasimolecular ion occurs at 119898119911837 (C39H49O20) whose molecular mass is 162Da more thanthat of icariin And its major MS119899 fragment ions are the sameas those of icariinThemost abundantMS2 andMS3 fragmentions also appear at 119898119911 367 and 352 indicating the loss ofone rhamnosyl and two glucosyl moieties and the sequentialloss of methyl group respectively Although the molecularweight of M1 is equal to that of epimedin A there existsa significant difference on their chromatographic retentionbehavior Therefore M1 is tentatively identified as an isomerof epimedin A

Both M2 and M6 show the same quasimolecular ion[M minus H]minus at 119898119911 645 (C32H37O14) with the elution time at5044 and 5743min respectively M2 was only observed infeces after intramuscular introduction of icariin while M6was found in feces following both routes of administrationM6 is unambiguously identified as sagittatoside B incomparison with the reference standard Analogous to theMS119899 fragments of icariin (see Figure 7 and Table 6) M2also yielded the major fragment ions at 119898119911 352 323 309296 281 and 268 strongly suggesting that M2 should be

a conjunction metabolite of icariin Thus M2 is tentativelyassigned as sagittatoside B isomer

M3 was found in rat feces after both intramuscular andoral administration of icariin Its MS spectrum exhibits adeprotonated molecule [MndashH]minus at 119898119911 499 (C26H27O10)176Da less than that of icariin By comparison with thereference standard it is identified as baohuoside II TheMSMS spectrum shows the most abundant fragment ionat 119898119911 353 indicating the loss of rhamnose moiety TheMS3 spectrum displays the major fragment ions at 119898119911 325298 and 284 separately corresponding to the loss of COisobutenyl and isopentenyl radicals

M4 is a minor metabolite of icariin detected in the fecesof rats in the oral administration group Its deprotonatedmolecule (C33H37O16) is at 119898119911 689 And the major MSMSfragment at 119898119911 367 results from the loss of glucuronicacid and rhamnose moieties Its MS3 spectrum produces afragment at119898119911 352 (the loss of a methyl group) Thus M4 istentatively characterized as icariin glucuronide

M5ndashM11 of icariin metabolites were observed in rat fecesafter both intramuscular and oral administration of icariin

M5 has a deprotonated molecular ion at 119898119911 529(C27H29O11) in its MS spectrum The MS119899 spectra show themajor fragment ions at119898119911 501 (the loss of CO) 382 (the lossof rhamnosyl group) 367 (the loss of methyl radical from theion at119898119911 382) and 339 (the successive loss of methyl radicaland CO) Therefore M5 is tentatively identified as hydroxylicariside II

M7 is identified as 210158401015840-O-rhamnosyl icariside II based onthe reference standard and related mass spectral pattern The

14 BioMed Research International

Table6Ch

aracteriz

ationof

metaboliteso

ficariinin

ratfeces

Num

ber

Com

poun

dRT

Form

ula

Observedmass

RDB

Error

LCM

S119899data

[MndashH]minus

(ppm

)

PlowastIcariin

5519

C 33H39O15

67522888

145

0790

MS2[675]367(100)352(25)

MS3[675-367]352(100)

MS4[675-367-352]323(

30)309(100)296(45)281

(65)268

(50)255

(20)

M1

Epim

edin

Aiso

mer

a5038

C 39H49O20

8372

819

155

0824

MS2[837]790(50)367

(100)

MS3[837-367]352(100)297(20)

M2

Sagitta

tosid

eBiso

mer

b5044

C 32H37O14

6452186

145

1314

MS2[645]481(20)352(100)

MS3[645-352]323(40)309

(60)296

(90)281

(100)268(60)

M3

lowastBa

ohuo

sideIIa

b5278

C 26H27O10

4991

605

135

1295

MS2[499]353(100)

MS3[499-353]325(20)298

(40)284

(100)255(30)

M4

Icariin

glucuron

idea

5404

C 33H37O16

6892

084

155

1202

MS2[689]367(100)321(60)

MS3[689-367]352(100)

M5

Hydroxylicaris

ideIIa

b5412

C 27H29O11

5291

716

135

1132

MS2[529]501(20)382(100)367(40)

MS3[529-382]367(100)339(10)

M6

lowastSagitta

tosid

eBab

5743

C 32H37O14

6452184

145

1020

MS2[645]367(100)352(30)

MS3[645-367]352(100)

M7

lowast210158401015840-O

-Rhamno

syl-icaris

ideIIa

b5756

C 33H39O14

6592

344

145

1529

MS2[659]366(100)351(20)323(15)

MS3[659-366]351(100)323

(80)311(60)305

(20)

M8

lowastIcariside

IIab

6146

C 27H29O10

5131760

135

0851

MS2[513]366(100)

MS3[513-366]351(100)323

(80)311(50)305

(20)

M9

Hydroxylicaritin

ab6233

C 21H19O7

3831137

125

3160

MS2[383]365(100)355(60)339

(40)

MS3[383-365]350(100)321(20)230(40)

M10

Desmethylicaritin

ab6636

C 20H17O6

3531024

125

1119

MS2[353]298(100)219(10)

MS3[353-298]279(60)269

(40)253

(100)241(30)164(70)

M11

lowastIcaritinab

8265

C 21H19O6

3671183

125

1921

MS2[367]352(100)309(20)297

(20)

MS3[367-352]309(100)297(60)

lowastCom

paredwith

ther

eference

standardaObservedin

ratfeces

after

oraladministratio

nb O

bservedin

ratfeces

after

intram

usculara

dministratio

n

BioMed Research International 15

O

OO

O

O

O

OHOH

OH

OH

OH OHOH

OH

OHOH

OHOH

OH

OHOH OH

OH

OH

OH

OHOH

OH

OH

OH

OH

OH

OH

OH

OHOH

OH

OH

OH

OH

OHOH

OHOH

OHOH

OH

OH

OH

OH

OHOH

HO HO

HO

HO

HO

HOHO

HO

HO

HO

OH

O

OO

O

OOHOOC

O

OO

O

O

OO

OO

O

O

OO

OO

O

O

OO

O

O

OO

O

O

O

O

O

O

O

icariin

epimedin A isomer

minus rh

a

minus methyl

+ rhaOxidation

minus methyl

Icariin glucuronide

+ xyl

+ glcGlucuronidation

minus rha

Oxidation

minus rha

+ xyl

Sagittatoside B isomer

minus glc

M1

M4

M2

OCH3 OCH3

OCH3

OCH3

OCH3

OCH3

OCH3

OCH3

M7 2-O-rhamnosyl icariside II

M5 hydroxyl icariside II

M3 baohuoside II

M8 icariside II

M6 sagittatoside B

M9 hydroxyl icaritinM10 desmethyl icaritin

M11 icaritin

Figure 8 Proposed metabolic pathways for icariin in rat feces

MS spectrum of M7 shows a quasimolecular ion [MndashH]minusat 119898119911 659 (C33H39O14) The major fragment ions at 119898119911366 351 and 323 result from the concurrent loss of therhamnose chain the successive loss of a methyl group andCO respectively

As shownFigure 7M8 is themost abundantmetabolite oficariin By comparison with the reference standard it is iden-tified as icariside II Its MS spectrum shows a deprotonatedmolecular ion at 119898119911 513 (C27H29O10) And similar to the

MS119899 pattern of icariin the major fragment ions at 119898119911 366351 and 323 separately match the loss of rhamnose moietythe sequential loss of rhamnose and methyl groups and theloss of CO

M9 has a quasimolecular ion [MndashH]minus at 119898119911 383(C21H19O7) 146Da less than that of M5 (hydroxyl icarisideII) It is tentatively identified as hydroxyl icaritin Its MSMSspectrum shows themain fragments of119898119911 365 355 and 339respectively corresponding to the neutral loss of H2O CO

16 BioMed Research International

and CO2 MS3 spectrum the fragment ions at 119898119911 350 and321 individually result from the neutral loss of methyl radicaland CO2

The quasimolecular ion [MndashH]minus of M10 is at 119898119911 353146Da less than that of M3 (baohuoside II) The majorfragment ion in its MSMS spectrum is at119898119911 298 matchingthe loss of isobutenyl radical The fragment at 119898119911 219 ispossibly produced by the neutral loss of C8H6O2 (134Da) dueto the retro-Diels-Alder (RDA) fragmentation of the flavonersquosC ring Moreover the MS3 spectrum affords fragment ionsat 119898119911 279 269 253 and 164 resulting from the loss ofH2O CO CO2 and C8H6O2 (134Da) respectively Thus thismetabolite is reasonably identified as desmethyl icaritin

M11 was observed as anothermajormetabolite in rat fecesof both two administration groups (Figure 7) By comparisonwith the reference standard it is identified as icaritin Its MSspectrum yields a quasimolecular ion at119898119911 367 (C21H19O6)And themajor fragment ion at119898119911 352 is characterized as theloss of methyl group The MS3 fragment at 119898119911 309 resultsfrom the loss of methyl group and CO and ion at 119898119911 297is the outcome of the neutral loss of isopentene (70Da) fromthe deprotonated molecule

4 Conclusion

Themenopausal syndrome improvement and possible cancerpreventive activity of Herba Epimedii is receiving more andmore attention Information on the in vivo disposition ofHerba Epimedii components is important for better under-standing the biological effects of this herbal drug For thispurpose our investigation was designed to obtain informa-tion on the plasma pharmacokinetics and tissue distributionof icariin in rat and the excretion of icariin and itsmetabolitesIn this study we firstly established and validated a rapidsensitive and reliable LC-MS method for the determinationof icariin in various biological samples collected at differenttime points and then the method was applied to the plasmapharmacokinetics and tissue distribution study of icariin inrats after oral administration of the total flavonoid extractof Herba Epimedii In addition the differences of the tissuedistribution of icariin in between male and female rats werestudied after oral and intramuscular administration of icariinAnd the metabolic pattern of icariin was also investigatedThe data showed the plasma and tissue concentration oficariin were kept at a very low level at all collection pointswhich suggests icariin is poorly absorbed after oral adminis-trationThepeak concentration in plasma and collected tissuesamples appeared at 05ndash1 h and then quickly dropped at 4 hafter administration demonstrating that a small amount oficariin can be rapidly absorbed and eliminated and there isno accumulation in rat tissues Moreover the concentrationin liver lung and reproductive organs was much higherthan that in other tissues indicating that liver lung andreproductive organs are the main target sites of icariin in rats

The gender-related difference of icariin in tissue distribu-tion has not been reported in the past published literatureOur study demonstrated that the distribution features oficariin are similar in most of tissues between male andfemale rats However there exist a few remarkable gender

differences such that thewhole tissue concentration of icariinin male rats was much higher than female rats except in gen-ital organs (high in female and low in male) In China HerbaEpimedii is a famous tonic and aphrodisiac to invigoratekidney and strengthenYang therefore it is commonly used totreat male sexual functional decline and female menopausalsyndrome As one of the predominant constitutes in HerbaEpimedii icariin plays a very crucial role in exerting effectsso the relatively high icariin concentration in female genitalorgans actually supports the above-mentioned traditionalChinese medicine theory and clinical practice and makesthis compound a good future prospect for treating womenrsquosmenopausal related diseases

In the present study the complete metabolic profile oficariin has also been investigated The results illustrate thatfeces should be the major excretion way for both oral andintramuscular administration of icariin Based on LC-UVand LC-MS119899 analyses eleven metabolites were detected infecal samples and were further identified Among them10 metabolites appeared in rat feces of oral administrationgroup and 9 in the feces of intramuscular introduction groupThe major metabolic routes of clearance are proposed to behydrolysis demethylation oxidation and conjugation

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

Authorsrsquo Contributions

Shunjun Xu and Jiejing Yu contributed equally to this work

References

[1] X Wang Y He B Guo et al ldquoIn vivo screening for anti-osteo-porotic fraction from extract of herbal formula xianlinggubaoin ovariectomized micerdquo PLoS ONE vol 10 no 2 Article IDe0118184 2015

[2] R-H Liu X Kang L-P Xu et al ldquoEffects of the combinedextracts of Herba Epimedii and Fructus Ligustri Lucidi on bonemineral content and bone turnover in osteoporotic ratsrdquo BMCComplementary and Alternative Medicine vol 15 no 1 article112 2015

[3] B C L Chan H Y Lee W S Siu et al ldquoSuppression of mastcell activity contributes to the osteoprotective effect of an herbalformula containing Herba Epimedii Fructus Ligustri Lucidiand Fructus Psoraleaerdquo Journal of Pharmacy and Pharmacologyvol 66 no 3 pp 437ndash444 2014

[4] S CW Sze Y Tong T B Ng C L Y Cheng andH P CheungldquoHerba Epimedii anti-oxidative properties and its medicalimplicationsrdquoMolecules vol 15 no 11 pp 7861ndash7870 2010

[5] R Niu ldquoAction of the drug Herba Epimedii on testosterone ofthe mouse plasma and its accessory sexual organ before andafter processingrdquo China Journal of Chinese Materia Medica vol14 no 9 pp 530ndash574 1989

[6] F-X Liu S Sun and Z-Z Cui ldquoAnalysis of immunologi-cal enhancement of immunosuppressed chickens by Chineseherbal extractsrdquo Journal of Ethnopharmacology vol 127 no 2pp 251ndash256 2010

BioMed Research International 17

[7] J-H Kim Y-J Mun S-J Im J-H Han H-S Lee and W-HWoo ldquoEffects of the aqueous extract of Epimedii Herba on theantibody responses in micerdquo International Immunopharmacol-ogy vol 1 no 5 pp 935ndash944 2001

[8] X Yu Y Tong X-Q Han et al ldquoAnti-angiogenic activity ofHerba Epimedii on zebrafish embryos in vivo and HUVECs invitrordquo Phytotherapy Research vol 27 no 9 pp 1368ndash1375 2013

[9] D-C Zhang J-L Liu Y-B Ding J-G Xia and G-Y ChenldquoIcariin potentiates the antitumor activity of gemcitabine ingallbladder cancer by suppressingNF-120581BrdquoActa PharmacologicaSinica vol 34 no 2 pp 301ndash308 2013

[10] L Kong J Liu J Wang et al ldquoIcariin inhibits TNF-120572IFN-120574induced inflammatory response via inhibition of the substanceP and p38-MAPK signaling pathway in human keratinocytesrdquoInternational Immunopharmacology vol 29 no 2 pp 401ndash4072015

[11] H Shao J Shen M Wang et al ldquoIcariin protects against tita-nium particle-induced osteolysis and inflammatory response ina mouse calvarial modelrdquo Biomaterials vol 60 pp 92ndash99 2015

[12] Q Wei J Zhang G Hong et al ldquoIcariin promotes osteogenicdifferentiation of rat bone marrow stromal cells by activatingthe ER120572-Wnt120573-catenin signaling pathwayrdquo Biomedicine ampPharmacotherapy vol 84 pp 931ndash939 2016

[13] S-Q Zhang W-J Cai J-H Huang et al ldquoIcariin a naturalflavonol glycoside extends healthspan in micerdquo ExperimentalGerontology vol 69 pp 226ndash235 2015

[14] M N Makarova O N Pozharitskaya A N Shikov S VTesakova V G Makarov and V P Tikhonov ldquoEffect of lipid-based suspension of Epimedium koreanum Nakai extract onsexual behavior in ratsrdquo Journal of Ethnopharmacology vol 114no 3 pp 412ndash416 2007

[15] Y Chen J Wang X Jia X Tan and M Hu ldquoRole of intestinalhydrolase in the absorption of prenylated flavonoids present inyinyanghuordquoMolecules vol 16 no 2 pp 1336ndash1348 2011

[16] J J Yu Y J Liu S J Xu S Sun Y J Xu and L Yang ldquoSimulta-neous quantitative determination of four epimediumflavonoidsin rat urine by LC-MSMSrdquo Traditional Chinese Drug Researchamp Clinical Pharmacology vol 27 no 4 pp 537ndash541 2016

[17] Y Lu N Li Y Deng et al ldquoSimultaneous determination oficariin naringin and osthole in rat plasma by UPLC-MSMSand its application for pharmacokinetic study after oral admin-istration of Gushudan capsulesrdquo Journal of Chromatography Bvol 993-994 pp 75ndash80 2015

[18] N Wang X Huang X Wang Y Zhang R Wu and D ShouldquoPipette tip solid-phase extraction and high-performance liquidchromatography for the determination of flavonoids fromEpimedii herba in rat serum and application of the techniqueto pharmacokinetic studiesrdquo Journal of Chromatography B vol990 pp 64ndash72 2015

Submit your manuscripts athttpswwwhindawicom

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ToxinsJournal of

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Pharmaceutics

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MEDIATORSINFLAMMATION

of

BioMed Research International 5

Table 1 Regression equations of icariin in rat plasma and tissue samples (119899 = 3)

Sample Equation Correlation coefficient Linear rangengmL

Plasma 119884 = (00060 plusmn 00002)119883 + (00011 plusmn 00006) 09973 plusmn 00013 100ndash500Liver 119884 = (00053 plusmn 00001)119883 + (00014 plusmn 00008) 09986 plusmn 00008 100ndash500Heart 119884 = (00062 plusmn 00003)119883 + (00006 plusmn 00002) 09979 plusmn 00013 100ndash500Spleen 119884 = (00053 plusmn 00006)119883 + (00010 plusmn 00008) 09979 plusmn 00009 100ndash500Lung 119884 = (00060 plusmn 00006)119883 + (00015 plusmn 00008) 09974 plusmn 00015 100ndash500Kidney 119884 = (00045 plusmn 00003)119883 + (00006 plusmn 00002) 09986 plusmn 00004 100ndash500Brain 119884 = (00033 plusmn 00002)119883 + (00011 plusmn 00003) 09985 plusmn 00005 100ndash500Testicle 119884 = (00066 plusmn 00001)119883 + (00015 plusmn 00001) 09977 plusmn 00001 100ndash500

(a-1) (a-2)

7356

5136

765652972549

400 600 800 1000200mz (Da)

00

20e7

40e7

60e7

80e7

10e8

12e8

14e8

Inte

nsity

(cps

)

1332

26901067 1347633

2242

00

10e6

20e6

30e6

40e6

50e6

60e6

70e6

80e6

Inte

nsity

(cps

)

50 150 200 250 300 350 400 450 500100mz (Da)

(b-1) (b-2)

2690

2852

00

50e6

10e7

15e7

20e7

25e7

29e7

Inte

nsity

(cps

)

150 200 250 300 350 400 450 500100mz (Da)

5135

7357

529500

50e6

10e7

15e7

20e7

23e7

Inte

nsity

(cps

)

400 600 800 1000200mz (Da)

Figure 2 Full scan mass spectrum of icariin (a-1) and internal standard (genistein b-1) product ion spectrum of icariin (a-2) and internalstandard (genistein b-2)

mass transitions and retention time of icariin and IS in blankbiological matrix samples

332 Linearity and Sensitivity The assay was found to belinear over the concentration range of 1ndash500 ngmL forplasma and tissue homogenate The mean linear regressionequations were listed in Table 1 with correlations coefficientover 099 in which119883 corresponds to nominal concentrations

and 119884 corresponds to peak area ratios LLOQs with an 119878119873ratio of gt20 was 1 ngmL which was sensitive enough for thepharmacokinetic and tissue distribution studies of icariin

333 Precision and Accuracy Inter- and intra-assay vari-ability at three different concentrations HQC (400 ngmL)MQC (20 ngmL) LQC (2 ngmL) was determined in plasmaand tissue homogenate with five replicates on each day for

6 BioMed Research International

(a-1)

0

20

40

60

80

100

120

Inte

nsity

(cps

)

20 30 40 50 6010Time (min)

(a-2)

0

20

40

60

80

100

Inte

nsity

(cps

)

20 30 40 50 6010Time (min)

(b-2)

501

005000010e4

15e4

20e4

25e4

30e4

35e4

Inte

nsity

(cps

)

20 30 40 50 6010Time (min)

(c-2)

258

0

1000

2000

3000

4000

5000

60006787

Inte

nsity

(cps

)

20 30 40 50 6010Time (min)

(d-2)

260

0500

100015002000250030003500

Inte

nsity

(cps

)

20 30 40 50 6010Time (min)

(d-1)

260

20 30 40 50 6010Time (min)

0

100

200

300

400

500597

Inte

nsity

(cps

)

(c-1)

261

20 30 40 50 6010Time (min)

0

50

100

150

200

250

Inte

nsity

(cps

)

(b-1)

500

00

50000

10e4

15e4

20e422e4

Inte

nsity

(cps

)

20 30 40 50 6010Time (min)

Figure 3 Representative MRM chromatograms of blank plasma (a-1) blank liver (a-2) blank plasma spiked with IS (b-1) blank liver spikedwith IS (b-2) blank plasma spiked with icariin at LOQ (c-1) blank liver spiked with icariin at LOQ (d-1) rat plasma sample obtained at 05 hafter administration (c-2) and rat liver sample obtained at 05 h after administration (d-2)

BioMed Research International 7

Table 2 Intra- and interday accuracy and precision for the determination of icariin in plasma and tissue samples (119899 = 18 6 replicates perday for 3 days)

c Conc Interday IntradayngmL RE () CV () RE () CV ()

Plasma2 minus0394 134 minus0887 35020 minus518 156 minus485 225400 minus328 150 minus276 247

Liver2 0690 155 minus0197 46420 minus0528 959 minus0198 185400 minus103 104 minus117 162

Heart2 minus0309 425 minus483 32220 409 161 495 246400 minus365 219 minus257 159

Spleen2 minus214 900 minus335 76320 396 509 346 225400 minus0313 475 0 198

Lung2 minus680 137 minus133 47220 minus205 141 minus852 267400 minus930 102 minus126 111

Kidney2 473 536 394 53620 399 133 743 277400 minus649 136 minus573 149

Brain2 minus131 948 minus123 47420 327 559 0792 140400 minus328 356 minus336 212

Testicle2 minus420 325 minus236 46020 0538 493 307 269400 minus0379 579 257 402

three separate days The detailed results of the inter- andintraday precision and accuracy of icariin in biologicalmatrixwere summarized in Table 2

334 Recovery and Matrix Effects As shown in Table 3 theextraction recovery of icariin was in the range of 825ndash1006which suggested that the recovery of this method was consis-tent and reproducible And the suppression of ionization inplasma and tissue homogenate was differentThe interferenceof ionization caused by kidney homogenate was significantwhile that of brain homogenate was much lower

335 Stability Five replicates of QC at three different con-centration levels were used to assess the stability of icariinunder various conditions The results were summarized inTable 4 which indicated that icariin was stable in autosam-pler (24 h) at 4∘C bench-top (6 h) at room temperature threefreeze-thaw cycles and frozen condition atminus80∘C for 30 daysas the RE values were within plusmn15 for the low medium andhigh concentrations

336 Plasma Pharmacokinetics After a single oral admin-istration of the total flavonoid extract of Herba Epimediithe concentration of icariin in plasma was successfully deter-mined by LC-MSMS method described above The meanplasma concentration-time profile of icariin was shown in

Icariin

10 1550Time (h)

0

50

100

150

Con

cent

ratio

n (n

gm

L)

Figure 4 Plasma concentration versus time profile of icariin in rats(119899 = 6) following a single oral dose of Herba Epimedii extract(corresponding 42mgg icariin)

Figure 4 Pharmacokinetic parameters were estimated usingtheDAS software and performed by noncompartmental anal-ysis Peak plasma concentration (119862max) and the time at whichit occurred (119879max) were obtained from the visual inspection

8 BioMed Research International

Table 3 Matrix effect and extraction recovery for the assay of icariin in plasma and tissue samples (119899 = 5)

Matrix ConcngmL

Matrix effectMean plusmn SD ()

Extraction efficiencyMean plusmn SD ()

Plasma2 712 plusmn 29 869 plusmn 60

20 690 plusmn 20 841 plusmn 47

400 667 plusmn 10 849 plusmn 58

Liver2 715 plusmn 33 939 plusmn 75

20 722 plusmn 41 903 plusmn 19

400 703 plusmn 31 905 plusmn 22

Heart2 679 plusmn 38 889 plusmn 39

20 688 plusmn 26 922 plusmn 23

400 699 plusmn 18 931 plusmn 17

Spleen2 692 plusmn 53 887 plusmn 84

20 701 plusmn 37 835 plusmn 39

400 714 plusmn 38 825 plusmn 14

Lung2 710 plusmn 22 880 plusmn 59

20 712 plusmn 24 854 plusmn 45

400 738 plusmn 16 838 plusmn 19

Kidney2 577 plusmn 62 865 plusmn 67

20 555 plusmn 53 900 plusmn 37

400 666 plusmn 152 853 plusmn 61

Brain2 906 plusmn 58 897 plusmn 57

20 893 plusmn 19 949 plusmn 39

400 870 plusmn 23 990 plusmn 23

Testicle2 802 plusmn 35 915 plusmn 67

20 773 plusmn 21 964 plusmn 22

400 777 plusmn 22 1006 plusmn 19

of the plasma concentration-time curve Elimination rateconstant (119870119890) was determined from the least-square fittedterminal log-linear portion of the plasma concentration-timeprofile Elimination half-life (11990512) was calculated by 0693KeTotal area under the plasma concentration-time curve oficariin from time zero to infinity (AUC0ndashinfin) was calculatedas the sumofAUC0ndash12 the area up to the last quantifiable timepoint (12 h) and the area from the 12 h time point to infinityAUC0ndash12 was calculated using the trapezoidal rule and theextrapolated area was calculated from the last measurableconcentration 119862119905 divided by 119870119890 The major pharmacokineticparameters of icariin were summarized in Table 5

34 Tissue Distribution The tissue distribution of icariin infemale and male rats was investigated following a single oraldose of total flavonoid extract from Herba Epimedii (cor-responding 42mgg icariin) The mean concentration-timeprofile of each tissue was presented in Figure 5 indicatingthat icariin was poorly absorbed after oral administrationbecause all the tissue concentrations were kept at a verylow level The peak concentration of icariin in most ofcollected tissues appeared at 1 h after administration except

for liver and female reproductive organs in which theconcentration reached the peak at 05 h demonstrating thaticariin underwent a rapid and wide distribution Moreoverthe tissue concentrations in male rat were generally muchhigher than those in female rat The tissue area under thecurve from time zero to infinity was in the following orderliver gt lung gt spleen gt heart gt kidney gt brain gt testiclefor mail rat and liver gt uterus gt heart gt ovary gt lung gtkidney gt spleen gt brain for female rats As shown in Figure 5the concentration levels in liver and lung were much higherthan those in other tissues illustrating that liver and lungare the main target organs of icariin in male rat after oraladministration of the total flavonoid extract Furthermorethe concentration level in uteruswasmuchhigher than that inany other tissues of female rat demonstrating that the gender-related difference of icariin in tissue distribution do existand the reproductive organs are the main target organs oficariin for female rat Meanwhile icariin was found with verylow AUC0ndashinfin in brain which indicated that icariin could notefficiently cross the blood-brain barrier Over time the maintrend of the mean concentration of icariin in all tissues wasreduced and the concentration dropped very quickly at 4 h

BioMed Research International 9

Table 4 Stability of icariin in plasma and tissue samples (119899 = 6)

Matrix Conc Mean plusmn SDngmL Bench-topa Freeze-thawb Autosamplerc Long-termd

Plasma2 975 plusmn 37 968 plusmn 57 1047 plusmn 81 993 plusmn 59

20 945 plusmn 59 1013 plusmn 31 955 plusmn 37 943 plusmn 16

400 977 plusmn 39 1030 plusmn 19 945 plusmn 26 955 plusmn 37

Liver2 987 plusmn 63 1015 plusmn 21 1114 plusmn 44 986 plusmn 54

20 949 plusmn 14 900 plusmn 13 1060 plusmn 16 957 plusmn 21

400 875 plusmn 13 897 plusmn 13 894 plusmn 14 1125 plusmn 22

Heart2 1086 plusmn 33 1002 plusmn 43 955 plusmn 22 1150 plusmn 30

20 1056 plusmn 17 996 plusmn 10 973 plusmn 13 1002 plusmn 42

400 1026 plusmn 15 942 plusmn 29 892 plusmn 09 907 plusmn 50

Spleen2 954 plusmn 92 1018 plusmn 44 955 plusmn 76 1013 plusmn 59

20 989 plusmn 14 1094 plusmn 11 928 plusmn 09 1123 plusmn 34

400 967 plusmn 27 1032 plusmn 04 871 plusmn 12 974 plusmn 26

Lung2 959 plusmn 35 856 plusmn 30 1032 plusmn 48 983 plusmn 43

20 1017 plusmn 80 923 plusmn 40 1094 plusmn 46 1102 plusmn 18

400 925 plusmn 18 932 plusmn 15 997 plusmn 04 1098 plusmn 17

Kidney2 1012 plusmn 34 916 plusmn 40 951 plusmn 22 984 plusmn 48

20 1025 plusmn 28 974 plusmn 18 955 plusmn 16 934 plusmn 22

400 920 plusmn 14 911 plusmn 08 899 plusmn 10 1095 plusmn 22

Brain2 858 plusmn 60 974 plusmn 73 966 plusmn 32 1094 plusmn 32

20 1033 plusmn 41 1035 plusmn 31 1030 plusmn 35 974 plusmn 12

400 1002 plusmn 29 990 plusmn 31 978 plusmn 21 1102 plusmn 30

Testicle2 1058 plusmn 39 853 plusmn 24 997 plusmn 55 933 plusmn 41

20 1040 plusmn 14 870 plusmn 13 1016 plusmn 19 1065 plusmn 15

400 1034 plusmn 39 984 plusmn 16 995 plusmn 33 1068 plusmn 38aAt least 1 h at room temperature bAt least 3 freeze-thaw cycles cAt least 24 h at 4∘C dAt least 4 weeks at minus80 plusmn 5∘C

Table 5 Pharmacokinetic parameters of icariin in rat after asingle oral dose of Herba Epimedii extract (corresponding 42mggicariin)

Parameter IcariinAUC0ndashinfin (ugLsdoth) 164155 plusmn 144129

AUMC0ndashinfin (ugLsdoth) 538424 plusmn 262081

MRT0ndashinfin (h) 3990 plusmn 1987

11990512 (h) 3149 plusmn 2364

119879max (h) 0458 plusmn 0246

CL119911119865 (Lhkg) 389043 plusmn 204472

after administration suggesting no accumulation in tissuesand a rapid elimination of this compound

35 Metabolism Study of Icariin in Rat Urine and Feces

351 Metabolite Profile of Rat Urine and Feces In thisstudy urine and feces samples were both analyzed by LC-MSMS By comparing the total ion chromatograms of drug-containing urine sample and predose control urine sampleneither parent drug nor metabolites were observed in raturine following two routes of administration of icariinHowever a total of 11 icariin metabolites were detected in ratfeces including 10 metabolites after oral administration and

9 following intramuscular introduction The representativechromatograms of the metabolites of 0ndash24 h rat feces wereshown in Figure 6 No additional metabolites were observedin the feces of 24ndash48 h collection period Results showedthat the major metabolites of icariin were M8 and M11whose total amount accounted for over 90 of total peakarea in the LC-UV chromatogram of 0ndash24 h feces And theconcentration of metabolites in feces rapidly declined over24ndash48 h collection period The MS2 and MS3 spectra oficariin and its metabolites were exhibited in Figure 7 andthe observed and calculatedmasses formula retention timesmass errors and MS data of these analytes were presented inTable 6 Proposed metabolic pathways for icariin in rat feceswere shown in Figure 8

352 Identification of Metabolites Known compounds wereidentified by comparison with the chromatographic behaviorand MS fragmentation pattern of reference standards Forunknown constituents their structures were characterizedbased on their retention behavior and their MS119899 spectraobtained on-line Moreover under the optimized LC-MSconditions all the in vivometabolites of icariin could give theprecursor ions of sufficient abundance in order to facilitatethe analysis of multistage mass spectrometry Because allmetabolites originated from icariin and their MS fragmen-tation patterns were largely similar to that of icariin it was

10 BioMed Research International

Heart Spleen Lung Kidney Brain TesticleLiver0

10

20

30

40

50C

once

ntra

tion

(ng

g)

025 Ｂ

05 Ｂ

1 Ｂ

2 Ｂ

4 Ｂ

6 Ｂ

(a)

0

10

20

30

40

50

Con

cent

ratio

n (n

gg)

Heart Spleen Lung Kidney Brain Uterus OvaryLiver

025 Ｂ

05 Ｂ

1 Ｂ

2 Ｂ

4 Ｂ

6 Ｂ

(b)

Figure 5 Tissue distribution of icariin in rats after a single oral dose of Herba Epimedii extract (corresponding 42mgg icariin (a) male rat(b) female rat)

663517

5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 900Time (min)

0

50

100

(a)

6155

512665

6637520419520

20

40

60

80

100

5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 900Time (min)

52 54 56 58500

50 M11

M10

M8

M7

M6M5

M4

M3M2

M1

M9

(b)

658

61465278

506

0

20

40

60

80

100

5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 900Time (min)

M11

M3 M8

(c)

Figure 6 Representative chromatograms of icariin metabolites in rat feces of 0ndash24 h (a) blank feces sample (b) feces sample after oraladministration and (c) feces sample after intramuscular administration

BioMed Research International 11

36724

6755435214

Icariin

513320

20

40

60

80

100

Rela

tive a

bund

ance

400 600 800200mz

35218

Icariin

367250

20

40

60

80

100

Rela

tive a

bund

ance

400 600200mz

35211

36722

29727

0

20

40

60

80

100

Rela

tive a

bund

ance

400300mz

2811329595

26725 3090935216

25506

0

20

40

60

80

100

Rela

tive a

bund

ance

300 400200mz

28408

35323

25512

0

20

40

60

80

100

Rela

tive a

bund

ance

400 600200mz

35217

36722

0

20

40

60

80

100

Rela

tive a

bund

ance

300 400 500200mz

36722

6892932124

400 600 800200mz

0

20

40

60

80

100

Rela

tive a

bund

ance

35318

49919

0

20

40

60

80

100

Rela

tive a

bund

ance

400 600200mz

35215

48116 64516

400 600 800200mz

0

20

40

60

80

100

Rela

tive a

bund

ance

400 600 800 1000mz

36723

83734

79088

0

20

40

60

80

100

Rela

tive a

bund

ance

M1M1

M2

M2

M3

M3

M4

M4

MS2

MS3

MS2

MS3

MS3

MS2

MS2

MS2

MS3

MS3

Figure 7 Continued

12 BioMed Research International

38218

36714 5293950114

0

20

40

60

80

100

Rela

tive a

bund

ance

400 500 600300mz

M5

MS2

36722

33916 382180

20

40

60

80

100

Rela

tive a

bund

ance

300 400 500200mz

M5

MS3

35217

36711

0

20

40

60

80

100

Rela

tive a

bund

ance

300 400 500200mz

M6

MS3

36724

35227 6452032309

0

20

40

60

80

100

Rela

tive a

bund

ance

400 600 800200mz

M6

MS2

36614

6592335118

0

20

40

60

80

100

Rela

tive a

bund

ance

400 600 800200mz

M7

MS2

35116

32317

31112

36607

0

20

40

60

80

100

Rela

tive a

bund

ance

300 400 500200mz

M7

MS3

35109

32308

31113

3660629520

0

20

40

60

80

100

Rela

tive a

bund

ance

300 400 500200mz

M8

MS3

36615

51313

400 600200mz

0

20

40

60

80

100

Rela

tive a

bund

ance M8

MS2

36514

35528 38328

33929

0

20

40

60

80

100

Rela

tive a

bund

ance

400300mz

M9

MS2

35019

36508

33724

0

20

40

60

80

100

Rela

tive a

bund

ance

400200mz

M9

MS3

Figure 7 Continued

BioMed Research International 13

25308

16310

27902

29817

0

20

40

60

80

100

Rela

tive a

bund

ance

400200mz

M10

MS3

29810

35312

21913

400200mz

0

20

40

60

80

100

Rela

tive a

bund

ance M10

MS2

35208

36723

309280

20

40

60

80

100

Rela

tive a

bund

ance

400 600200mz

M11

MS2

30911

2810435215

400200mz

0

20

40

60

80

100

Rela

tive a

bund

ance M11

MS3

Figure 7 Representative LC-MS119899 spectra of icariin and its metabolites M1ndashM11 in rat feces

necessary to characterize the MS119899 behavior of icariin toidentify and assign the metabolites of the constituent Thedeprotonated molecule [MndashH]minus of icariin appears at 119898119911675 which readily eliminates its glucosyl and rhamnosylgroups to produce an [MndashHndash308]minus ion at 119898119911 367 The[MndashHndash308]minus ion yields a fragment at 119898119911 352 by losing amethyl radical And of theMS4 spectrum of [MndashHndash308ndash15]minusion the fragment at 119898119911 323 can be reasonably attributed tothe loss of 3-COH of flavone skeletonrsquos C ring

M1 as aminormetabolite of icariin was only found in thefeces of the oral group Its quasimolecular ion occurs at 119898119911837 (C39H49O20) whose molecular mass is 162Da more thanthat of icariin And its major MS119899 fragment ions are the sameas those of icariinThemost abundantMS2 andMS3 fragmentions also appear at 119898119911 367 and 352 indicating the loss ofone rhamnosyl and two glucosyl moieties and the sequentialloss of methyl group respectively Although the molecularweight of M1 is equal to that of epimedin A there existsa significant difference on their chromatographic retentionbehavior Therefore M1 is tentatively identified as an isomerof epimedin A

Both M2 and M6 show the same quasimolecular ion[M minus H]minus at 119898119911 645 (C32H37O14) with the elution time at5044 and 5743min respectively M2 was only observed infeces after intramuscular introduction of icariin while M6was found in feces following both routes of administrationM6 is unambiguously identified as sagittatoside B incomparison with the reference standard Analogous to theMS119899 fragments of icariin (see Figure 7 and Table 6) M2also yielded the major fragment ions at 119898119911 352 323 309296 281 and 268 strongly suggesting that M2 should be

a conjunction metabolite of icariin Thus M2 is tentativelyassigned as sagittatoside B isomer

M3 was found in rat feces after both intramuscular andoral administration of icariin Its MS spectrum exhibits adeprotonated molecule [MndashH]minus at 119898119911 499 (C26H27O10)176Da less than that of icariin By comparison with thereference standard it is identified as baohuoside II TheMSMS spectrum shows the most abundant fragment ionat 119898119911 353 indicating the loss of rhamnose moiety TheMS3 spectrum displays the major fragment ions at 119898119911 325298 and 284 separately corresponding to the loss of COisobutenyl and isopentenyl radicals

M4 is a minor metabolite of icariin detected in the fecesof rats in the oral administration group Its deprotonatedmolecule (C33H37O16) is at 119898119911 689 And the major MSMSfragment at 119898119911 367 results from the loss of glucuronicacid and rhamnose moieties Its MS3 spectrum produces afragment at119898119911 352 (the loss of a methyl group) Thus M4 istentatively characterized as icariin glucuronide

M5ndashM11 of icariin metabolites were observed in rat fecesafter both intramuscular and oral administration of icariin

M5 has a deprotonated molecular ion at 119898119911 529(C27H29O11) in its MS spectrum The MS119899 spectra show themajor fragment ions at119898119911 501 (the loss of CO) 382 (the lossof rhamnosyl group) 367 (the loss of methyl radical from theion at119898119911 382) and 339 (the successive loss of methyl radicaland CO) Therefore M5 is tentatively identified as hydroxylicariside II

M7 is identified as 210158401015840-O-rhamnosyl icariside II based onthe reference standard and related mass spectral pattern The

14 BioMed Research International

Table6Ch

aracteriz

ationof

metaboliteso

ficariinin

ratfeces

Num

ber

Com

poun

dRT

Form

ula

Observedmass

RDB

Error

LCM

S119899data

[MndashH]minus

(ppm

)

PlowastIcariin

5519

C 33H39O15

67522888

145

0790

MS2[675]367(100)352(25)

MS3[675-367]352(100)

MS4[675-367-352]323(

30)309(100)296(45)281

(65)268

(50)255

(20)

M1

Epim

edin

Aiso

mer

a5038

C 39H49O20

8372

819

155

0824

MS2[837]790(50)367

(100)

MS3[837-367]352(100)297(20)

M2

Sagitta

tosid

eBiso

mer

b5044

C 32H37O14

6452186

145

1314

MS2[645]481(20)352(100)

MS3[645-352]323(40)309

(60)296

(90)281

(100)268(60)

M3

lowastBa

ohuo

sideIIa

b5278

C 26H27O10

4991

605

135

1295

MS2[499]353(100)

MS3[499-353]325(20)298

(40)284

(100)255(30)

M4

Icariin

glucuron

idea

5404

C 33H37O16

6892

084

155

1202

MS2[689]367(100)321(60)

MS3[689-367]352(100)

M5

Hydroxylicaris

ideIIa

b5412

C 27H29O11

5291

716

135

1132

MS2[529]501(20)382(100)367(40)

MS3[529-382]367(100)339(10)

M6

lowastSagitta

tosid

eBab

5743

C 32H37O14

6452184

145

1020

MS2[645]367(100)352(30)

MS3[645-367]352(100)

M7

lowast210158401015840-O

-Rhamno

syl-icaris

ideIIa

b5756

C 33H39O14

6592

344

145

1529

MS2[659]366(100)351(20)323(15)

MS3[659-366]351(100)323

(80)311(60)305

(20)

M8

lowastIcariside

IIab

6146

C 27H29O10

5131760

135

0851

MS2[513]366(100)

MS3[513-366]351(100)323

(80)311(50)305

(20)

M9

Hydroxylicaritin

ab6233

C 21H19O7

3831137

125

3160

MS2[383]365(100)355(60)339

(40)

MS3[383-365]350(100)321(20)230(40)

M10

Desmethylicaritin

ab6636

C 20H17O6

3531024

125

1119

MS2[353]298(100)219(10)

MS3[353-298]279(60)269

(40)253

(100)241(30)164(70)

M11

lowastIcaritinab

8265

C 21H19O6

3671183

125

1921

MS2[367]352(100)309(20)297

(20)

MS3[367-352]309(100)297(60)

lowastCom

paredwith

ther

eference

standardaObservedin

ratfeces

after

oraladministratio

nb O

bservedin

ratfeces

after

intram

usculara

dministratio

n

BioMed Research International 15

O

OO

O

O

O

OHOH

OH

OH

OH OHOH

OH

OHOH

OHOH

OH

OHOH OH

OH

OH

OH

OHOH

OH

OH

OH

OH

OH

OH

OH

OHOH

OH

OH

OH

OH

OHOH

OHOH

OHOH

OH

OH

OH

OH

OHOH

HO HO

HO

HO

HO

HOHO

HO

HO

HO

OH

O

OO

O

OOHOOC

O

OO

O

O

OO

OO

O

O

OO

OO

O

O

OO

O

O

OO

O

O

O

O

O

O

O

icariin

epimedin A isomer

minus rh

a

minus methyl

+ rhaOxidation

minus methyl

Icariin glucuronide

+ xyl

+ glcGlucuronidation

minus rha

Oxidation

minus rha

+ xyl

Sagittatoside B isomer

minus glc

M1

M4

M2

OCH3 OCH3

OCH3

OCH3

OCH3

OCH3

OCH3

OCH3

M7 2-O-rhamnosyl icariside II

M5 hydroxyl icariside II

M3 baohuoside II

M8 icariside II

M6 sagittatoside B

M9 hydroxyl icaritinM10 desmethyl icaritin

M11 icaritin

Figure 8 Proposed metabolic pathways for icariin in rat feces

MS spectrum of M7 shows a quasimolecular ion [MndashH]minusat 119898119911 659 (C33H39O14) The major fragment ions at 119898119911366 351 and 323 result from the concurrent loss of therhamnose chain the successive loss of a methyl group andCO respectively

As shownFigure 7M8 is themost abundantmetabolite oficariin By comparison with the reference standard it is iden-tified as icariside II Its MS spectrum shows a deprotonatedmolecular ion at 119898119911 513 (C27H29O10) And similar to the

MS119899 pattern of icariin the major fragment ions at 119898119911 366351 and 323 separately match the loss of rhamnose moietythe sequential loss of rhamnose and methyl groups and theloss of CO

M9 has a quasimolecular ion [MndashH]minus at 119898119911 383(C21H19O7) 146Da less than that of M5 (hydroxyl icarisideII) It is tentatively identified as hydroxyl icaritin Its MSMSspectrum shows themain fragments of119898119911 365 355 and 339respectively corresponding to the neutral loss of H2O CO

16 BioMed Research International

and CO2 MS3 spectrum the fragment ions at 119898119911 350 and321 individually result from the neutral loss of methyl radicaland CO2

The quasimolecular ion [MndashH]minus of M10 is at 119898119911 353146Da less than that of M3 (baohuoside II) The majorfragment ion in its MSMS spectrum is at119898119911 298 matchingthe loss of isobutenyl radical The fragment at 119898119911 219 ispossibly produced by the neutral loss of C8H6O2 (134Da) dueto the retro-Diels-Alder (RDA) fragmentation of the flavonersquosC ring Moreover the MS3 spectrum affords fragment ionsat 119898119911 279 269 253 and 164 resulting from the loss ofH2O CO CO2 and C8H6O2 (134Da) respectively Thus thismetabolite is reasonably identified as desmethyl icaritin

M11 was observed as anothermajormetabolite in rat fecesof both two administration groups (Figure 7) By comparisonwith the reference standard it is identified as icaritin Its MSspectrum yields a quasimolecular ion at119898119911 367 (C21H19O6)And themajor fragment ion at119898119911 352 is characterized as theloss of methyl group The MS3 fragment at 119898119911 309 resultsfrom the loss of methyl group and CO and ion at 119898119911 297is the outcome of the neutral loss of isopentene (70Da) fromthe deprotonated molecule

4 Conclusion

Themenopausal syndrome improvement and possible cancerpreventive activity of Herba Epimedii is receiving more andmore attention Information on the in vivo disposition ofHerba Epimedii components is important for better under-standing the biological effects of this herbal drug For thispurpose our investigation was designed to obtain informa-tion on the plasma pharmacokinetics and tissue distributionof icariin in rat and the excretion of icariin and itsmetabolitesIn this study we firstly established and validated a rapidsensitive and reliable LC-MS method for the determinationof icariin in various biological samples collected at differenttime points and then the method was applied to the plasmapharmacokinetics and tissue distribution study of icariin inrats after oral administration of the total flavonoid extractof Herba Epimedii In addition the differences of the tissuedistribution of icariin in between male and female rats werestudied after oral and intramuscular administration of icariinAnd the metabolic pattern of icariin was also investigatedThe data showed the plasma and tissue concentration oficariin were kept at a very low level at all collection pointswhich suggests icariin is poorly absorbed after oral adminis-trationThepeak concentration in plasma and collected tissuesamples appeared at 05ndash1 h and then quickly dropped at 4 hafter administration demonstrating that a small amount oficariin can be rapidly absorbed and eliminated and there isno accumulation in rat tissues Moreover the concentrationin liver lung and reproductive organs was much higherthan that in other tissues indicating that liver lung andreproductive organs are the main target sites of icariin in rats

The gender-related difference of icariin in tissue distribu-tion has not been reported in the past published literatureOur study demonstrated that the distribution features oficariin are similar in most of tissues between male andfemale rats However there exist a few remarkable gender

differences such that thewhole tissue concentration of icariinin male rats was much higher than female rats except in gen-ital organs (high in female and low in male) In China HerbaEpimedii is a famous tonic and aphrodisiac to invigoratekidney and strengthenYang therefore it is commonly used totreat male sexual functional decline and female menopausalsyndrome As one of the predominant constitutes in HerbaEpimedii icariin plays a very crucial role in exerting effectsso the relatively high icariin concentration in female genitalorgans actually supports the above-mentioned traditionalChinese medicine theory and clinical practice and makesthis compound a good future prospect for treating womenrsquosmenopausal related diseases

In the present study the complete metabolic profile oficariin has also been investigated The results illustrate thatfeces should be the major excretion way for both oral andintramuscular administration of icariin Based on LC-UVand LC-MS119899 analyses eleven metabolites were detected infecal samples and were further identified Among them10 metabolites appeared in rat feces of oral administrationgroup and 9 in the feces of intramuscular introduction groupThe major metabolic routes of clearance are proposed to behydrolysis demethylation oxidation and conjugation

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

Authorsrsquo Contributions

Shunjun Xu and Jiejing Yu contributed equally to this work

References

[1] X Wang Y He B Guo et al ldquoIn vivo screening for anti-osteo-porotic fraction from extract of herbal formula xianlinggubaoin ovariectomized micerdquo PLoS ONE vol 10 no 2 Article IDe0118184 2015

[2] R-H Liu X Kang L-P Xu et al ldquoEffects of the combinedextracts of Herba Epimedii and Fructus Ligustri Lucidi on bonemineral content and bone turnover in osteoporotic ratsrdquo BMCComplementary and Alternative Medicine vol 15 no 1 article112 2015

[3] B C L Chan H Y Lee W S Siu et al ldquoSuppression of mastcell activity contributes to the osteoprotective effect of an herbalformula containing Herba Epimedii Fructus Ligustri Lucidiand Fructus Psoraleaerdquo Journal of Pharmacy and Pharmacologyvol 66 no 3 pp 437ndash444 2014

[4] S CW Sze Y Tong T B Ng C L Y Cheng andH P CheungldquoHerba Epimedii anti-oxidative properties and its medicalimplicationsrdquoMolecules vol 15 no 11 pp 7861ndash7870 2010

[5] R Niu ldquoAction of the drug Herba Epimedii on testosterone ofthe mouse plasma and its accessory sexual organ before andafter processingrdquo China Journal of Chinese Materia Medica vol14 no 9 pp 530ndash574 1989

[6] F-X Liu S Sun and Z-Z Cui ldquoAnalysis of immunologi-cal enhancement of immunosuppressed chickens by Chineseherbal extractsrdquo Journal of Ethnopharmacology vol 127 no 2pp 251ndash256 2010

BioMed Research International 17

[7] J-H Kim Y-J Mun S-J Im J-H Han H-S Lee and W-HWoo ldquoEffects of the aqueous extract of Epimedii Herba on theantibody responses in micerdquo International Immunopharmacol-ogy vol 1 no 5 pp 935ndash944 2001

[8] X Yu Y Tong X-Q Han et al ldquoAnti-angiogenic activity ofHerba Epimedii on zebrafish embryos in vivo and HUVECs invitrordquo Phytotherapy Research vol 27 no 9 pp 1368ndash1375 2013

[9] D-C Zhang J-L Liu Y-B Ding J-G Xia and G-Y ChenldquoIcariin potentiates the antitumor activity of gemcitabine ingallbladder cancer by suppressingNF-120581BrdquoActa PharmacologicaSinica vol 34 no 2 pp 301ndash308 2013

[10] L Kong J Liu J Wang et al ldquoIcariin inhibits TNF-120572IFN-120574induced inflammatory response via inhibition of the substanceP and p38-MAPK signaling pathway in human keratinocytesrdquoInternational Immunopharmacology vol 29 no 2 pp 401ndash4072015

[11] H Shao J Shen M Wang et al ldquoIcariin protects against tita-nium particle-induced osteolysis and inflammatory response ina mouse calvarial modelrdquo Biomaterials vol 60 pp 92ndash99 2015

[12] Q Wei J Zhang G Hong et al ldquoIcariin promotes osteogenicdifferentiation of rat bone marrow stromal cells by activatingthe ER120572-Wnt120573-catenin signaling pathwayrdquo Biomedicine ampPharmacotherapy vol 84 pp 931ndash939 2016

[13] S-Q Zhang W-J Cai J-H Huang et al ldquoIcariin a naturalflavonol glycoside extends healthspan in micerdquo ExperimentalGerontology vol 69 pp 226ndash235 2015

[14] M N Makarova O N Pozharitskaya A N Shikov S VTesakova V G Makarov and V P Tikhonov ldquoEffect of lipid-based suspension of Epimedium koreanum Nakai extract onsexual behavior in ratsrdquo Journal of Ethnopharmacology vol 114no 3 pp 412ndash416 2007

[15] Y Chen J Wang X Jia X Tan and M Hu ldquoRole of intestinalhydrolase in the absorption of prenylated flavonoids present inyinyanghuordquoMolecules vol 16 no 2 pp 1336ndash1348 2011

[16] J J Yu Y J Liu S J Xu S Sun Y J Xu and L Yang ldquoSimulta-neous quantitative determination of four epimediumflavonoidsin rat urine by LC-MSMSrdquo Traditional Chinese Drug Researchamp Clinical Pharmacology vol 27 no 4 pp 537ndash541 2016

[17] Y Lu N Li Y Deng et al ldquoSimultaneous determination oficariin naringin and osthole in rat plasma by UPLC-MSMSand its application for pharmacokinetic study after oral admin-istration of Gushudan capsulesrdquo Journal of Chromatography Bvol 993-994 pp 75ndash80 2015

[18] N Wang X Huang X Wang Y Zhang R Wu and D ShouldquoPipette tip solid-phase extraction and high-performance liquidchromatography for the determination of flavonoids fromEpimedii herba in rat serum and application of the techniqueto pharmacokinetic studiesrdquo Journal of Chromatography B vol990 pp 64ndash72 2015

Submit your manuscripts athttpswwwhindawicom

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ToxinsJournal of

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Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

6 BioMed Research International

(a-1)

0

20

40

60

80

100

120

Inte

nsity

(cps

)

20 30 40 50 6010Time (min)

(a-2)

0

20

40

60

80

100

Inte

nsity

(cps

)

20 30 40 50 6010Time (min)

(b-2)

501

005000010e4

15e4

20e4

25e4

30e4

35e4

Inte

nsity

(cps

)

20 30 40 50 6010Time (min)

(c-2)

258

0

1000

2000

3000

4000

5000

60006787

Inte

nsity

(cps

)

20 30 40 50 6010Time (min)

(d-2)

260

0500

100015002000250030003500

Inte

nsity

(cps

)

20 30 40 50 6010Time (min)

(d-1)

260

20 30 40 50 6010Time (min)

0

100

200

300

400

500597

Inte

nsity

(cps

)

(c-1)

261

20 30 40 50 6010Time (min)

0

50

100

150

200

250

Inte

nsity

(cps

)

(b-1)

500

00

50000

10e4

15e4

20e422e4

Inte

nsity

(cps

)

20 30 40 50 6010Time (min)

Figure 3 Representative MRM chromatograms of blank plasma (a-1) blank liver (a-2) blank plasma spiked with IS (b-1) blank liver spikedwith IS (b-2) blank plasma spiked with icariin at LOQ (c-1) blank liver spiked with icariin at LOQ (d-1) rat plasma sample obtained at 05 hafter administration (c-2) and rat liver sample obtained at 05 h after administration (d-2)

BioMed Research International 7

Table 2 Intra- and interday accuracy and precision for the determination of icariin in plasma and tissue samples (119899 = 18 6 replicates perday for 3 days)

c Conc Interday IntradayngmL RE () CV () RE () CV ()

Plasma2 minus0394 134 minus0887 35020 minus518 156 minus485 225400 minus328 150 minus276 247

Liver2 0690 155 minus0197 46420 minus0528 959 minus0198 185400 minus103 104 minus117 162

Heart2 minus0309 425 minus483 32220 409 161 495 246400 minus365 219 minus257 159

Spleen2 minus214 900 minus335 76320 396 509 346 225400 minus0313 475 0 198

Lung2 minus680 137 minus133 47220 minus205 141 minus852 267400 minus930 102 minus126 111

Kidney2 473 536 394 53620 399 133 743 277400 minus649 136 minus573 149

Brain2 minus131 948 minus123 47420 327 559 0792 140400 minus328 356 minus336 212

Testicle2 minus420 325 minus236 46020 0538 493 307 269400 minus0379 579 257 402

three separate days The detailed results of the inter- andintraday precision and accuracy of icariin in biologicalmatrixwere summarized in Table 2

334 Recovery and Matrix Effects As shown in Table 3 theextraction recovery of icariin was in the range of 825ndash1006which suggested that the recovery of this method was consis-tent and reproducible And the suppression of ionization inplasma and tissue homogenate was differentThe interferenceof ionization caused by kidney homogenate was significantwhile that of brain homogenate was much lower

335 Stability Five replicates of QC at three different con-centration levels were used to assess the stability of icariinunder various conditions The results were summarized inTable 4 which indicated that icariin was stable in autosam-pler (24 h) at 4∘C bench-top (6 h) at room temperature threefreeze-thaw cycles and frozen condition atminus80∘C for 30 daysas the RE values were within plusmn15 for the low medium andhigh concentrations

336 Plasma Pharmacokinetics After a single oral admin-istration of the total flavonoid extract of Herba Epimediithe concentration of icariin in plasma was successfully deter-mined by LC-MSMS method described above The meanplasma concentration-time profile of icariin was shown in

Icariin

10 1550Time (h)

0

50

100

150

Con

cent

ratio

n (n

gm

L)

Figure 4 Plasma concentration versus time profile of icariin in rats(119899 = 6) following a single oral dose of Herba Epimedii extract(corresponding 42mgg icariin)

Figure 4 Pharmacokinetic parameters were estimated usingtheDAS software and performed by noncompartmental anal-ysis Peak plasma concentration (119862max) and the time at whichit occurred (119879max) were obtained from the visual inspection

8 BioMed Research International

Table 3 Matrix effect and extraction recovery for the assay of icariin in plasma and tissue samples (119899 = 5)

Matrix ConcngmL

Matrix effectMean plusmn SD ()

Extraction efficiencyMean plusmn SD ()

Plasma2 712 plusmn 29 869 plusmn 60

20 690 plusmn 20 841 plusmn 47

400 667 plusmn 10 849 plusmn 58

Liver2 715 plusmn 33 939 plusmn 75

20 722 plusmn 41 903 plusmn 19

400 703 plusmn 31 905 plusmn 22

Heart2 679 plusmn 38 889 plusmn 39

20 688 plusmn 26 922 plusmn 23

400 699 plusmn 18 931 plusmn 17

Spleen2 692 plusmn 53 887 plusmn 84

20 701 plusmn 37 835 plusmn 39

400 714 plusmn 38 825 plusmn 14

Lung2 710 plusmn 22 880 plusmn 59

20 712 plusmn 24 854 plusmn 45

400 738 plusmn 16 838 plusmn 19

Kidney2 577 plusmn 62 865 plusmn 67

20 555 plusmn 53 900 plusmn 37

400 666 plusmn 152 853 plusmn 61

Brain2 906 plusmn 58 897 plusmn 57

20 893 plusmn 19 949 plusmn 39

400 870 plusmn 23 990 plusmn 23

Testicle2 802 plusmn 35 915 plusmn 67

20 773 plusmn 21 964 plusmn 22

400 777 plusmn 22 1006 plusmn 19

of the plasma concentration-time curve Elimination rateconstant (119870119890) was determined from the least-square fittedterminal log-linear portion of the plasma concentration-timeprofile Elimination half-life (11990512) was calculated by 0693KeTotal area under the plasma concentration-time curve oficariin from time zero to infinity (AUC0ndashinfin) was calculatedas the sumofAUC0ndash12 the area up to the last quantifiable timepoint (12 h) and the area from the 12 h time point to infinityAUC0ndash12 was calculated using the trapezoidal rule and theextrapolated area was calculated from the last measurableconcentration 119862119905 divided by 119870119890 The major pharmacokineticparameters of icariin were summarized in Table 5

34 Tissue Distribution The tissue distribution of icariin infemale and male rats was investigated following a single oraldose of total flavonoid extract from Herba Epimedii (cor-responding 42mgg icariin) The mean concentration-timeprofile of each tissue was presented in Figure 5 indicatingthat icariin was poorly absorbed after oral administrationbecause all the tissue concentrations were kept at a verylow level The peak concentration of icariin in most ofcollected tissues appeared at 1 h after administration except

for liver and female reproductive organs in which theconcentration reached the peak at 05 h demonstrating thaticariin underwent a rapid and wide distribution Moreoverthe tissue concentrations in male rat were generally muchhigher than those in female rat The tissue area under thecurve from time zero to infinity was in the following orderliver gt lung gt spleen gt heart gt kidney gt brain gt testiclefor mail rat and liver gt uterus gt heart gt ovary gt lung gtkidney gt spleen gt brain for female rats As shown in Figure 5the concentration levels in liver and lung were much higherthan those in other tissues illustrating that liver and lungare the main target organs of icariin in male rat after oraladministration of the total flavonoid extract Furthermorethe concentration level in uteruswasmuchhigher than that inany other tissues of female rat demonstrating that the gender-related difference of icariin in tissue distribution do existand the reproductive organs are the main target organs oficariin for female rat Meanwhile icariin was found with verylow AUC0ndashinfin in brain which indicated that icariin could notefficiently cross the blood-brain barrier Over time the maintrend of the mean concentration of icariin in all tissues wasreduced and the concentration dropped very quickly at 4 h

BioMed Research International 9

Table 4 Stability of icariin in plasma and tissue samples (119899 = 6)

Matrix Conc Mean plusmn SDngmL Bench-topa Freeze-thawb Autosamplerc Long-termd

Plasma2 975 plusmn 37 968 plusmn 57 1047 plusmn 81 993 plusmn 59

20 945 plusmn 59 1013 plusmn 31 955 plusmn 37 943 plusmn 16

400 977 plusmn 39 1030 plusmn 19 945 plusmn 26 955 plusmn 37

Liver2 987 plusmn 63 1015 plusmn 21 1114 plusmn 44 986 plusmn 54

20 949 plusmn 14 900 plusmn 13 1060 plusmn 16 957 plusmn 21

400 875 plusmn 13 897 plusmn 13 894 plusmn 14 1125 plusmn 22

Heart2 1086 plusmn 33 1002 plusmn 43 955 plusmn 22 1150 plusmn 30

20 1056 plusmn 17 996 plusmn 10 973 plusmn 13 1002 plusmn 42

400 1026 plusmn 15 942 plusmn 29 892 plusmn 09 907 plusmn 50

Spleen2 954 plusmn 92 1018 plusmn 44 955 plusmn 76 1013 plusmn 59

20 989 plusmn 14 1094 plusmn 11 928 plusmn 09 1123 plusmn 34

400 967 plusmn 27 1032 plusmn 04 871 plusmn 12 974 plusmn 26

Lung2 959 plusmn 35 856 plusmn 30 1032 plusmn 48 983 plusmn 43

20 1017 plusmn 80 923 plusmn 40 1094 plusmn 46 1102 plusmn 18

400 925 plusmn 18 932 plusmn 15 997 plusmn 04 1098 plusmn 17

Kidney2 1012 plusmn 34 916 plusmn 40 951 plusmn 22 984 plusmn 48

20 1025 plusmn 28 974 plusmn 18 955 plusmn 16 934 plusmn 22

400 920 plusmn 14 911 plusmn 08 899 plusmn 10 1095 plusmn 22

Brain2 858 plusmn 60 974 plusmn 73 966 plusmn 32 1094 plusmn 32

20 1033 plusmn 41 1035 plusmn 31 1030 plusmn 35 974 plusmn 12

400 1002 plusmn 29 990 plusmn 31 978 plusmn 21 1102 plusmn 30

Testicle2 1058 plusmn 39 853 plusmn 24 997 plusmn 55 933 plusmn 41

20 1040 plusmn 14 870 plusmn 13 1016 plusmn 19 1065 plusmn 15

400 1034 plusmn 39 984 plusmn 16 995 plusmn 33 1068 plusmn 38aAt least 1 h at room temperature bAt least 3 freeze-thaw cycles cAt least 24 h at 4∘C dAt least 4 weeks at minus80 plusmn 5∘C

Table 5 Pharmacokinetic parameters of icariin in rat after asingle oral dose of Herba Epimedii extract (corresponding 42mggicariin)

Parameter IcariinAUC0ndashinfin (ugLsdoth) 164155 plusmn 144129

AUMC0ndashinfin (ugLsdoth) 538424 plusmn 262081

MRT0ndashinfin (h) 3990 plusmn 1987

11990512 (h) 3149 plusmn 2364

119879max (h) 0458 plusmn 0246

CL119911119865 (Lhkg) 389043 plusmn 204472

after administration suggesting no accumulation in tissuesand a rapid elimination of this compound

35 Metabolism Study of Icariin in Rat Urine and Feces

351 Metabolite Profile of Rat Urine and Feces In thisstudy urine and feces samples were both analyzed by LC-MSMS By comparing the total ion chromatograms of drug-containing urine sample and predose control urine sampleneither parent drug nor metabolites were observed in raturine following two routes of administration of icariinHowever a total of 11 icariin metabolites were detected in ratfeces including 10 metabolites after oral administration and

9 following intramuscular introduction The representativechromatograms of the metabolites of 0ndash24 h rat feces wereshown in Figure 6 No additional metabolites were observedin the feces of 24ndash48 h collection period Results showedthat the major metabolites of icariin were M8 and M11whose total amount accounted for over 90 of total peakarea in the LC-UV chromatogram of 0ndash24 h feces And theconcentration of metabolites in feces rapidly declined over24ndash48 h collection period The MS2 and MS3 spectra oficariin and its metabolites were exhibited in Figure 7 andthe observed and calculatedmasses formula retention timesmass errors and MS data of these analytes were presented inTable 6 Proposed metabolic pathways for icariin in rat feceswere shown in Figure 8

352 Identification of Metabolites Known compounds wereidentified by comparison with the chromatographic behaviorand MS fragmentation pattern of reference standards Forunknown constituents their structures were characterizedbased on their retention behavior and their MS119899 spectraobtained on-line Moreover under the optimized LC-MSconditions all the in vivometabolites of icariin could give theprecursor ions of sufficient abundance in order to facilitatethe analysis of multistage mass spectrometry Because allmetabolites originated from icariin and their MS fragmen-tation patterns were largely similar to that of icariin it was

10 BioMed Research International

Heart Spleen Lung Kidney Brain TesticleLiver0

10

20

30

40

50C

once

ntra

tion

(ng

g)

025 Ｂ

05 Ｂ

1 Ｂ

2 Ｂ

4 Ｂ

6 Ｂ

(a)

0

10

20

30

40

50

Con

cent

ratio

n (n

gg)

Heart Spleen Lung Kidney Brain Uterus OvaryLiver

025 Ｂ

05 Ｂ

1 Ｂ

2 Ｂ

4 Ｂ

6 Ｂ

(b)

Figure 5 Tissue distribution of icariin in rats after a single oral dose of Herba Epimedii extract (corresponding 42mgg icariin (a) male rat(b) female rat)

663517

5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 900Time (min)

0

50

100

(a)

6155

512665

6637520419520

20

40

60

80

100

5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 900Time (min)

52 54 56 58500

50 M11

M10

M8

M7

M6M5

M4

M3M2

M1

M9

(b)

658

61465278

506

0

20

40

60

80

100

5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 900Time (min)

M11

M3 M8

(c)

Figure 6 Representative chromatograms of icariin metabolites in rat feces of 0ndash24 h (a) blank feces sample (b) feces sample after oraladministration and (c) feces sample after intramuscular administration

BioMed Research International 11

36724

6755435214

Icariin

513320

20

40

60

80

100

Rela

tive a

bund

ance

400 600 800200mz

35218

Icariin

367250

20

40

60

80

100

Rela

tive a

bund

ance

400 600200mz

35211

36722

29727

0

20

40

60

80

100

Rela

tive a

bund

ance

400300mz

2811329595

26725 3090935216

25506

0

20

40

60

80

100

Rela

tive a

bund

ance

300 400200mz

28408

35323

25512

0

20

40

60

80

100

Rela

tive a

bund

ance

400 600200mz

35217

36722

0

20

40

60

80

100

Rela

tive a

bund

ance

300 400 500200mz

36722

6892932124

400 600 800200mz

0

20

40

60

80

100

Rela

tive a

bund

ance

35318

49919

0

20

40

60

80

100

Rela

tive a

bund

ance

400 600200mz

35215

48116 64516

400 600 800200mz

0

20

40

60

80

100

Rela

tive a

bund

ance

400 600 800 1000mz

36723

83734

79088

0

20

40

60

80

100

Rela

tive a

bund

ance

M1M1

M2

M2

M3

M3

M4

M4

MS2

MS3

MS2

MS3

MS3

MS2

MS2

MS2

MS3

MS3

Figure 7 Continued

12 BioMed Research International

38218

36714 5293950114

0

20

40

60

80

100

Rela

tive a

bund

ance

400 500 600300mz

M5

MS2

36722

33916 382180

20

40

60

80

100

Rela

tive a

bund

ance

300 400 500200mz

M5

MS3

35217

36711

0

20

40

60

80

100

Rela

tive a

bund

ance

300 400 500200mz

M6

MS3

36724

35227 6452032309

0

20

40

60

80

100

Rela

tive a

bund

ance

400 600 800200mz

M6

MS2

36614

6592335118

0

20

40

60

80

100

Rela

tive a

bund

ance

400 600 800200mz

M7

MS2

35116

32317

31112

36607

0

20

40

60

80

100

Rela

tive a

bund

ance

300 400 500200mz

M7

MS3

35109

32308

31113

3660629520

0

20

40

60

80

100

Rela

tive a

bund

ance

300 400 500200mz

M8

MS3

36615

51313

400 600200mz

0

20

40

60

80

100

Rela

tive a

bund

ance M8

MS2

36514

35528 38328

33929

0

20

40

60

80

100

Rela

tive a

bund

ance

400300mz

M9

MS2

35019

36508

33724

0

20

40

60

80

100

Rela

tive a

bund

ance

400200mz

M9

MS3

Figure 7 Continued

BioMed Research International 13

25308

16310

27902

29817

0

20

40

60

80

100

Rela

tive a

bund

ance

400200mz

M10

MS3

29810

35312

21913

400200mz

0

20

40

60

80

100

Rela

tive a

bund

ance M10

MS2

35208

36723

309280

20

40

60

80

100

Rela

tive a

bund

ance

400 600200mz

M11

MS2

30911

2810435215

400200mz

0

20

40

60

80

100

Rela

tive a

bund

ance M11

MS3

Figure 7 Representative LC-MS119899 spectra of icariin and its metabolites M1ndashM11 in rat feces

necessary to characterize the MS119899 behavior of icariin toidentify and assign the metabolites of the constituent Thedeprotonated molecule [MndashH]minus of icariin appears at 119898119911675 which readily eliminates its glucosyl and rhamnosylgroups to produce an [MndashHndash308]minus ion at 119898119911 367 The[MndashHndash308]minus ion yields a fragment at 119898119911 352 by losing amethyl radical And of theMS4 spectrum of [MndashHndash308ndash15]minusion the fragment at 119898119911 323 can be reasonably attributed tothe loss of 3-COH of flavone skeletonrsquos C ring

M1 as aminormetabolite of icariin was only found in thefeces of the oral group Its quasimolecular ion occurs at 119898119911837 (C39H49O20) whose molecular mass is 162Da more thanthat of icariin And its major MS119899 fragment ions are the sameas those of icariinThemost abundantMS2 andMS3 fragmentions also appear at 119898119911 367 and 352 indicating the loss ofone rhamnosyl and two glucosyl moieties and the sequentialloss of methyl group respectively Although the molecularweight of M1 is equal to that of epimedin A there existsa significant difference on their chromatographic retentionbehavior Therefore M1 is tentatively identified as an isomerof epimedin A

Both M2 and M6 show the same quasimolecular ion[M minus H]minus at 119898119911 645 (C32H37O14) with the elution time at5044 and 5743min respectively M2 was only observed infeces after intramuscular introduction of icariin while M6was found in feces following both routes of administrationM6 is unambiguously identified as sagittatoside B incomparison with the reference standard Analogous to theMS119899 fragments of icariin (see Figure 7 and Table 6) M2also yielded the major fragment ions at 119898119911 352 323 309296 281 and 268 strongly suggesting that M2 should be

a conjunction metabolite of icariin Thus M2 is tentativelyassigned as sagittatoside B isomer

M3 was found in rat feces after both intramuscular andoral administration of icariin Its MS spectrum exhibits adeprotonated molecule [MndashH]minus at 119898119911 499 (C26H27O10)176Da less than that of icariin By comparison with thereference standard it is identified as baohuoside II TheMSMS spectrum shows the most abundant fragment ionat 119898119911 353 indicating the loss of rhamnose moiety TheMS3 spectrum displays the major fragment ions at 119898119911 325298 and 284 separately corresponding to the loss of COisobutenyl and isopentenyl radicals

M4 is a minor metabolite of icariin detected in the fecesof rats in the oral administration group Its deprotonatedmolecule (C33H37O16) is at 119898119911 689 And the major MSMSfragment at 119898119911 367 results from the loss of glucuronicacid and rhamnose moieties Its MS3 spectrum produces afragment at119898119911 352 (the loss of a methyl group) Thus M4 istentatively characterized as icariin glucuronide

M5ndashM11 of icariin metabolites were observed in rat fecesafter both intramuscular and oral administration of icariin

M5 has a deprotonated molecular ion at 119898119911 529(C27H29O11) in its MS spectrum The MS119899 spectra show themajor fragment ions at119898119911 501 (the loss of CO) 382 (the lossof rhamnosyl group) 367 (the loss of methyl radical from theion at119898119911 382) and 339 (the successive loss of methyl radicaland CO) Therefore M5 is tentatively identified as hydroxylicariside II

M7 is identified as 210158401015840-O-rhamnosyl icariside II based onthe reference standard and related mass spectral pattern The

14 BioMed Research International

Table6Ch

aracteriz

ationof

metaboliteso

ficariinin

ratfeces

Num

ber

Com

poun

dRT

Form

ula

Observedmass

RDB

Error

LCM

S119899data

[MndashH]minus

(ppm

)

PlowastIcariin

5519

C 33H39O15

67522888

145

0790

MS2[675]367(100)352(25)

MS3[675-367]352(100)

MS4[675-367-352]323(

30)309(100)296(45)281

(65)268

(50)255

(20)

M1

Epim

edin

Aiso

mer

a5038

C 39H49O20

8372

819

155

0824

MS2[837]790(50)367

(100)

MS3[837-367]352(100)297(20)

M2

Sagitta

tosid

eBiso

mer

b5044

C 32H37O14

6452186

145

1314

MS2[645]481(20)352(100)

MS3[645-352]323(40)309

(60)296

(90)281

(100)268(60)

M3

lowastBa

ohuo

sideIIa

b5278

C 26H27O10

4991

605

135

1295

MS2[499]353(100)

MS3[499-353]325(20)298

(40)284

(100)255(30)

M4

Icariin

glucuron

idea

5404

C 33H37O16

6892

084

155

1202

MS2[689]367(100)321(60)

MS3[689-367]352(100)

M5

Hydroxylicaris

ideIIa

b5412

C 27H29O11

5291

716

135

1132

MS2[529]501(20)382(100)367(40)

MS3[529-382]367(100)339(10)

M6

lowastSagitta

tosid

eBab

5743

C 32H37O14

6452184

145

1020

MS2[645]367(100)352(30)

MS3[645-367]352(100)

M7

lowast210158401015840-O

-Rhamno

syl-icaris

ideIIa

b5756

C 33H39O14

6592

344

145

1529

MS2[659]366(100)351(20)323(15)

MS3[659-366]351(100)323

(80)311(60)305

(20)

M8

lowastIcariside

IIab

6146

C 27H29O10

5131760

135

0851

MS2[513]366(100)

MS3[513-366]351(100)323

(80)311(50)305

(20)

M9

Hydroxylicaritin

ab6233

C 21H19O7

3831137

125

3160

MS2[383]365(100)355(60)339

(40)

MS3[383-365]350(100)321(20)230(40)

M10

Desmethylicaritin

ab6636

C 20H17O6

3531024

125

1119

MS2[353]298(100)219(10)

MS3[353-298]279(60)269

(40)253

(100)241(30)164(70)

M11

lowastIcaritinab

8265

C 21H19O6

3671183

125

1921

MS2[367]352(100)309(20)297

(20)

MS3[367-352]309(100)297(60)

lowastCom

paredwith

ther

eference

standardaObservedin

ratfeces

after

oraladministratio

nb O

bservedin

ratfeces

after

intram

usculara

dministratio

n

BioMed Research International 15

O

OO

O

O

O

OHOH

OH

OH

OH OHOH

OH

OHOH

OHOH

OH

OHOH OH

OH

OH

OH

OHOH

OH

OH

OH

OH

OH

OH

OH

OHOH

OH

OH

OH

OH

OHOH

OHOH

OHOH

OH

OH

OH

OH

OHOH

HO HO

HO

HO

HO

HOHO

HO

HO

HO

OH

O

OO

O

OOHOOC

O

OO

O

O

OO

OO

O

O

OO

OO

O

O

OO

O

O

OO

O

O

O

O

O

O

O

icariin

epimedin A isomer

minus rh

a

minus methyl

+ rhaOxidation

minus methyl

Icariin glucuronide

+ xyl

+ glcGlucuronidation

minus rha

Oxidation

minus rha

+ xyl

Sagittatoside B isomer

minus glc

M1

M4

M2

OCH3 OCH3

OCH3

OCH3

OCH3

OCH3

OCH3

OCH3

M7 2-O-rhamnosyl icariside II

M5 hydroxyl icariside II

M3 baohuoside II

M8 icariside II

M6 sagittatoside B

M9 hydroxyl icaritinM10 desmethyl icaritin

M11 icaritin

Figure 8 Proposed metabolic pathways for icariin in rat feces

MS spectrum of M7 shows a quasimolecular ion [MndashH]minusat 119898119911 659 (C33H39O14) The major fragment ions at 119898119911366 351 and 323 result from the concurrent loss of therhamnose chain the successive loss of a methyl group andCO respectively

As shownFigure 7M8 is themost abundantmetabolite oficariin By comparison with the reference standard it is iden-tified as icariside II Its MS spectrum shows a deprotonatedmolecular ion at 119898119911 513 (C27H29O10) And similar to the

MS119899 pattern of icariin the major fragment ions at 119898119911 366351 and 323 separately match the loss of rhamnose moietythe sequential loss of rhamnose and methyl groups and theloss of CO

M9 has a quasimolecular ion [MndashH]minus at 119898119911 383(C21H19O7) 146Da less than that of M5 (hydroxyl icarisideII) It is tentatively identified as hydroxyl icaritin Its MSMSspectrum shows themain fragments of119898119911 365 355 and 339respectively corresponding to the neutral loss of H2O CO

16 BioMed Research International

and CO2 MS3 spectrum the fragment ions at 119898119911 350 and321 individually result from the neutral loss of methyl radicaland CO2

The quasimolecular ion [MndashH]minus of M10 is at 119898119911 353146Da less than that of M3 (baohuoside II) The majorfragment ion in its MSMS spectrum is at119898119911 298 matchingthe loss of isobutenyl radical The fragment at 119898119911 219 ispossibly produced by the neutral loss of C8H6O2 (134Da) dueto the retro-Diels-Alder (RDA) fragmentation of the flavonersquosC ring Moreover the MS3 spectrum affords fragment ionsat 119898119911 279 269 253 and 164 resulting from the loss ofH2O CO CO2 and C8H6O2 (134Da) respectively Thus thismetabolite is reasonably identified as desmethyl icaritin

M11 was observed as anothermajormetabolite in rat fecesof both two administration groups (Figure 7) By comparisonwith the reference standard it is identified as icaritin Its MSspectrum yields a quasimolecular ion at119898119911 367 (C21H19O6)And themajor fragment ion at119898119911 352 is characterized as theloss of methyl group The MS3 fragment at 119898119911 309 resultsfrom the loss of methyl group and CO and ion at 119898119911 297is the outcome of the neutral loss of isopentene (70Da) fromthe deprotonated molecule

4 Conclusion

Themenopausal syndrome improvement and possible cancerpreventive activity of Herba Epimedii is receiving more andmore attention Information on the in vivo disposition ofHerba Epimedii components is important for better under-standing the biological effects of this herbal drug For thispurpose our investigation was designed to obtain informa-tion on the plasma pharmacokinetics and tissue distributionof icariin in rat and the excretion of icariin and itsmetabolitesIn this study we firstly established and validated a rapidsensitive and reliable LC-MS method for the determinationof icariin in various biological samples collected at differenttime points and then the method was applied to the plasmapharmacokinetics and tissue distribution study of icariin inrats after oral administration of the total flavonoid extractof Herba Epimedii In addition the differences of the tissuedistribution of icariin in between male and female rats werestudied after oral and intramuscular administration of icariinAnd the metabolic pattern of icariin was also investigatedThe data showed the plasma and tissue concentration oficariin were kept at a very low level at all collection pointswhich suggests icariin is poorly absorbed after oral adminis-trationThepeak concentration in plasma and collected tissuesamples appeared at 05ndash1 h and then quickly dropped at 4 hafter administration demonstrating that a small amount oficariin can be rapidly absorbed and eliminated and there isno accumulation in rat tissues Moreover the concentrationin liver lung and reproductive organs was much higherthan that in other tissues indicating that liver lung andreproductive organs are the main target sites of icariin in rats

The gender-related difference of icariin in tissue distribu-tion has not been reported in the past published literatureOur study demonstrated that the distribution features oficariin are similar in most of tissues between male andfemale rats However there exist a few remarkable gender

differences such that thewhole tissue concentration of icariinin male rats was much higher than female rats except in gen-ital organs (high in female and low in male) In China HerbaEpimedii is a famous tonic and aphrodisiac to invigoratekidney and strengthenYang therefore it is commonly used totreat male sexual functional decline and female menopausalsyndrome As one of the predominant constitutes in HerbaEpimedii icariin plays a very crucial role in exerting effectsso the relatively high icariin concentration in female genitalorgans actually supports the above-mentioned traditionalChinese medicine theory and clinical practice and makesthis compound a good future prospect for treating womenrsquosmenopausal related diseases

In the present study the complete metabolic profile oficariin has also been investigated The results illustrate thatfeces should be the major excretion way for both oral andintramuscular administration of icariin Based on LC-UVand LC-MS119899 analyses eleven metabolites were detected infecal samples and were further identified Among them10 metabolites appeared in rat feces of oral administrationgroup and 9 in the feces of intramuscular introduction groupThe major metabolic routes of clearance are proposed to behydrolysis demethylation oxidation and conjugation

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

Authorsrsquo Contributions

Shunjun Xu and Jiejing Yu contributed equally to this work

References

[1] X Wang Y He B Guo et al ldquoIn vivo screening for anti-osteo-porotic fraction from extract of herbal formula xianlinggubaoin ovariectomized micerdquo PLoS ONE vol 10 no 2 Article IDe0118184 2015

[2] R-H Liu X Kang L-P Xu et al ldquoEffects of the combinedextracts of Herba Epimedii and Fructus Ligustri Lucidi on bonemineral content and bone turnover in osteoporotic ratsrdquo BMCComplementary and Alternative Medicine vol 15 no 1 article112 2015

[3] B C L Chan H Y Lee W S Siu et al ldquoSuppression of mastcell activity contributes to the osteoprotective effect of an herbalformula containing Herba Epimedii Fructus Ligustri Lucidiand Fructus Psoraleaerdquo Journal of Pharmacy and Pharmacologyvol 66 no 3 pp 437ndash444 2014

[4] S CW Sze Y Tong T B Ng C L Y Cheng andH P CheungldquoHerba Epimedii anti-oxidative properties and its medicalimplicationsrdquoMolecules vol 15 no 11 pp 7861ndash7870 2010

[5] R Niu ldquoAction of the drug Herba Epimedii on testosterone ofthe mouse plasma and its accessory sexual organ before andafter processingrdquo China Journal of Chinese Materia Medica vol14 no 9 pp 530ndash574 1989

[6] F-X Liu S Sun and Z-Z Cui ldquoAnalysis of immunologi-cal enhancement of immunosuppressed chickens by Chineseherbal extractsrdquo Journal of Ethnopharmacology vol 127 no 2pp 251ndash256 2010

BioMed Research International 17

[7] J-H Kim Y-J Mun S-J Im J-H Han H-S Lee and W-HWoo ldquoEffects of the aqueous extract of Epimedii Herba on theantibody responses in micerdquo International Immunopharmacol-ogy vol 1 no 5 pp 935ndash944 2001

[8] X Yu Y Tong X-Q Han et al ldquoAnti-angiogenic activity ofHerba Epimedii on zebrafish embryos in vivo and HUVECs invitrordquo Phytotherapy Research vol 27 no 9 pp 1368ndash1375 2013

[9] D-C Zhang J-L Liu Y-B Ding J-G Xia and G-Y ChenldquoIcariin potentiates the antitumor activity of gemcitabine ingallbladder cancer by suppressingNF-120581BrdquoActa PharmacologicaSinica vol 34 no 2 pp 301ndash308 2013

[10] L Kong J Liu J Wang et al ldquoIcariin inhibits TNF-120572IFN-120574induced inflammatory response via inhibition of the substanceP and p38-MAPK signaling pathway in human keratinocytesrdquoInternational Immunopharmacology vol 29 no 2 pp 401ndash4072015

[11] H Shao J Shen M Wang et al ldquoIcariin protects against tita-nium particle-induced osteolysis and inflammatory response ina mouse calvarial modelrdquo Biomaterials vol 60 pp 92ndash99 2015

[12] Q Wei J Zhang G Hong et al ldquoIcariin promotes osteogenicdifferentiation of rat bone marrow stromal cells by activatingthe ER120572-Wnt120573-catenin signaling pathwayrdquo Biomedicine ampPharmacotherapy vol 84 pp 931ndash939 2016

[13] S-Q Zhang W-J Cai J-H Huang et al ldquoIcariin a naturalflavonol glycoside extends healthspan in micerdquo ExperimentalGerontology vol 69 pp 226ndash235 2015

[14] M N Makarova O N Pozharitskaya A N Shikov S VTesakova V G Makarov and V P Tikhonov ldquoEffect of lipid-based suspension of Epimedium koreanum Nakai extract onsexual behavior in ratsrdquo Journal of Ethnopharmacology vol 114no 3 pp 412ndash416 2007

[15] Y Chen J Wang X Jia X Tan and M Hu ldquoRole of intestinalhydrolase in the absorption of prenylated flavonoids present inyinyanghuordquoMolecules vol 16 no 2 pp 1336ndash1348 2011

[16] J J Yu Y J Liu S J Xu S Sun Y J Xu and L Yang ldquoSimulta-neous quantitative determination of four epimediumflavonoidsin rat urine by LC-MSMSrdquo Traditional Chinese Drug Researchamp Clinical Pharmacology vol 27 no 4 pp 537ndash541 2016

[17] Y Lu N Li Y Deng et al ldquoSimultaneous determination oficariin naringin and osthole in rat plasma by UPLC-MSMSand its application for pharmacokinetic study after oral admin-istration of Gushudan capsulesrdquo Journal of Chromatography Bvol 993-994 pp 75ndash80 2015

[18] N Wang X Huang X Wang Y Zhang R Wu and D ShouldquoPipette tip solid-phase extraction and high-performance liquidchromatography for the determination of flavonoids fromEpimedii herba in rat serum and application of the techniqueto pharmacokinetic studiesrdquo Journal of Chromatography B vol990 pp 64ndash72 2015

Submit your manuscripts athttpswwwhindawicom

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MEDIATORSINFLAMMATION

of

BioMed Research International 7

Table 2 Intra- and interday accuracy and precision for the determination of icariin in plasma and tissue samples (119899 = 18 6 replicates perday for 3 days)

c Conc Interday IntradayngmL RE () CV () RE () CV ()

Plasma2 minus0394 134 minus0887 35020 minus518 156 minus485 225400 minus328 150 minus276 247

Liver2 0690 155 minus0197 46420 minus0528 959 minus0198 185400 minus103 104 minus117 162

Heart2 minus0309 425 minus483 32220 409 161 495 246400 minus365 219 minus257 159

Spleen2 minus214 900 minus335 76320 396 509 346 225400 minus0313 475 0 198

Lung2 minus680 137 minus133 47220 minus205 141 minus852 267400 minus930 102 minus126 111

Kidney2 473 536 394 53620 399 133 743 277400 minus649 136 minus573 149

Brain2 minus131 948 minus123 47420 327 559 0792 140400 minus328 356 minus336 212

Testicle2 minus420 325 minus236 46020 0538 493 307 269400 minus0379 579 257 402

three separate days The detailed results of the inter- andintraday precision and accuracy of icariin in biologicalmatrixwere summarized in Table 2

334 Recovery and Matrix Effects As shown in Table 3 theextraction recovery of icariin was in the range of 825ndash1006which suggested that the recovery of this method was consis-tent and reproducible And the suppression of ionization inplasma and tissue homogenate was differentThe interferenceof ionization caused by kidney homogenate was significantwhile that of brain homogenate was much lower

335 Stability Five replicates of QC at three different con-centration levels were used to assess the stability of icariinunder various conditions The results were summarized inTable 4 which indicated that icariin was stable in autosam-pler (24 h) at 4∘C bench-top (6 h) at room temperature threefreeze-thaw cycles and frozen condition atminus80∘C for 30 daysas the RE values were within plusmn15 for the low medium andhigh concentrations

336 Plasma Pharmacokinetics After a single oral admin-istration of the total flavonoid extract of Herba Epimediithe concentration of icariin in plasma was successfully deter-mined by LC-MSMS method described above The meanplasma concentration-time profile of icariin was shown in

Icariin

10 1550Time (h)

0

50

100

150

Con

cent

ratio

n (n

gm

L)

Figure 4 Plasma concentration versus time profile of icariin in rats(119899 = 6) following a single oral dose of Herba Epimedii extract(corresponding 42mgg icariin)

Figure 4 Pharmacokinetic parameters were estimated usingtheDAS software and performed by noncompartmental anal-ysis Peak plasma concentration (119862max) and the time at whichit occurred (119879max) were obtained from the visual inspection

8 BioMed Research International

Table 3 Matrix effect and extraction recovery for the assay of icariin in plasma and tissue samples (119899 = 5)

Matrix ConcngmL

Matrix effectMean plusmn SD ()

Extraction efficiencyMean plusmn SD ()

Plasma2 712 plusmn 29 869 plusmn 60

20 690 plusmn 20 841 plusmn 47

400 667 plusmn 10 849 plusmn 58

Liver2 715 plusmn 33 939 plusmn 75

20 722 plusmn 41 903 plusmn 19

400 703 plusmn 31 905 plusmn 22

Heart2 679 plusmn 38 889 plusmn 39

20 688 plusmn 26 922 plusmn 23

400 699 plusmn 18 931 plusmn 17

Spleen2 692 plusmn 53 887 plusmn 84

20 701 plusmn 37 835 plusmn 39

400 714 plusmn 38 825 plusmn 14

Lung2 710 plusmn 22 880 plusmn 59

20 712 plusmn 24 854 plusmn 45

400 738 plusmn 16 838 plusmn 19

Kidney2 577 plusmn 62 865 plusmn 67

20 555 plusmn 53 900 plusmn 37

400 666 plusmn 152 853 plusmn 61

Brain2 906 plusmn 58 897 plusmn 57

20 893 plusmn 19 949 plusmn 39

400 870 plusmn 23 990 plusmn 23

Testicle2 802 plusmn 35 915 plusmn 67

20 773 plusmn 21 964 plusmn 22

400 777 plusmn 22 1006 plusmn 19

of the plasma concentration-time curve Elimination rateconstant (119870119890) was determined from the least-square fittedterminal log-linear portion of the plasma concentration-timeprofile Elimination half-life (11990512) was calculated by 0693KeTotal area under the plasma concentration-time curve oficariin from time zero to infinity (AUC0ndashinfin) was calculatedas the sumofAUC0ndash12 the area up to the last quantifiable timepoint (12 h) and the area from the 12 h time point to infinityAUC0ndash12 was calculated using the trapezoidal rule and theextrapolated area was calculated from the last measurableconcentration 119862119905 divided by 119870119890 The major pharmacokineticparameters of icariin were summarized in Table 5

34 Tissue Distribution The tissue distribution of icariin infemale and male rats was investigated following a single oraldose of total flavonoid extract from Herba Epimedii (cor-responding 42mgg icariin) The mean concentration-timeprofile of each tissue was presented in Figure 5 indicatingthat icariin was poorly absorbed after oral administrationbecause all the tissue concentrations were kept at a verylow level The peak concentration of icariin in most ofcollected tissues appeared at 1 h after administration except

for liver and female reproductive organs in which theconcentration reached the peak at 05 h demonstrating thaticariin underwent a rapid and wide distribution Moreoverthe tissue concentrations in male rat were generally muchhigher than those in female rat The tissue area under thecurve from time zero to infinity was in the following orderliver gt lung gt spleen gt heart gt kidney gt brain gt testiclefor mail rat and liver gt uterus gt heart gt ovary gt lung gtkidney gt spleen gt brain for female rats As shown in Figure 5the concentration levels in liver and lung were much higherthan those in other tissues illustrating that liver and lungare the main target organs of icariin in male rat after oraladministration of the total flavonoid extract Furthermorethe concentration level in uteruswasmuchhigher than that inany other tissues of female rat demonstrating that the gender-related difference of icariin in tissue distribution do existand the reproductive organs are the main target organs oficariin for female rat Meanwhile icariin was found with verylow AUC0ndashinfin in brain which indicated that icariin could notefficiently cross the blood-brain barrier Over time the maintrend of the mean concentration of icariin in all tissues wasreduced and the concentration dropped very quickly at 4 h

BioMed Research International 9

Table 4 Stability of icariin in plasma and tissue samples (119899 = 6)

Matrix Conc Mean plusmn SDngmL Bench-topa Freeze-thawb Autosamplerc Long-termd

Plasma2 975 plusmn 37 968 plusmn 57 1047 plusmn 81 993 plusmn 59

20 945 plusmn 59 1013 plusmn 31 955 plusmn 37 943 plusmn 16

400 977 plusmn 39 1030 plusmn 19 945 plusmn 26 955 plusmn 37

Liver2 987 plusmn 63 1015 plusmn 21 1114 plusmn 44 986 plusmn 54

20 949 plusmn 14 900 plusmn 13 1060 plusmn 16 957 plusmn 21

400 875 plusmn 13 897 plusmn 13 894 plusmn 14 1125 plusmn 22

Heart2 1086 plusmn 33 1002 plusmn 43 955 plusmn 22 1150 plusmn 30

20 1056 plusmn 17 996 plusmn 10 973 plusmn 13 1002 plusmn 42

400 1026 plusmn 15 942 plusmn 29 892 plusmn 09 907 plusmn 50

Spleen2 954 plusmn 92 1018 plusmn 44 955 plusmn 76 1013 plusmn 59

20 989 plusmn 14 1094 plusmn 11 928 plusmn 09 1123 plusmn 34

400 967 plusmn 27 1032 plusmn 04 871 plusmn 12 974 plusmn 26

Lung2 959 plusmn 35 856 plusmn 30 1032 plusmn 48 983 plusmn 43

20 1017 plusmn 80 923 plusmn 40 1094 plusmn 46 1102 plusmn 18

400 925 plusmn 18 932 plusmn 15 997 plusmn 04 1098 plusmn 17

Kidney2 1012 plusmn 34 916 plusmn 40 951 plusmn 22 984 plusmn 48

20 1025 plusmn 28 974 plusmn 18 955 plusmn 16 934 plusmn 22

400 920 plusmn 14 911 plusmn 08 899 plusmn 10 1095 plusmn 22

Brain2 858 plusmn 60 974 plusmn 73 966 plusmn 32 1094 plusmn 32

20 1033 plusmn 41 1035 plusmn 31 1030 plusmn 35 974 plusmn 12

400 1002 plusmn 29 990 plusmn 31 978 plusmn 21 1102 plusmn 30

Testicle2 1058 plusmn 39 853 plusmn 24 997 plusmn 55 933 plusmn 41

20 1040 plusmn 14 870 plusmn 13 1016 plusmn 19 1065 plusmn 15

400 1034 plusmn 39 984 plusmn 16 995 plusmn 33 1068 plusmn 38aAt least 1 h at room temperature bAt least 3 freeze-thaw cycles cAt least 24 h at 4∘C dAt least 4 weeks at minus80 plusmn 5∘C

Table 5 Pharmacokinetic parameters of icariin in rat after asingle oral dose of Herba Epimedii extract (corresponding 42mggicariin)

Parameter IcariinAUC0ndashinfin (ugLsdoth) 164155 plusmn 144129

AUMC0ndashinfin (ugLsdoth) 538424 plusmn 262081

MRT0ndashinfin (h) 3990 plusmn 1987

11990512 (h) 3149 plusmn 2364

119879max (h) 0458 plusmn 0246

CL119911119865 (Lhkg) 389043 plusmn 204472

after administration suggesting no accumulation in tissuesand a rapid elimination of this compound

35 Metabolism Study of Icariin in Rat Urine and Feces

351 Metabolite Profile of Rat Urine and Feces In thisstudy urine and feces samples were both analyzed by LC-MSMS By comparing the total ion chromatograms of drug-containing urine sample and predose control urine sampleneither parent drug nor metabolites were observed in raturine following two routes of administration of icariinHowever a total of 11 icariin metabolites were detected in ratfeces including 10 metabolites after oral administration and

9 following intramuscular introduction The representativechromatograms of the metabolites of 0ndash24 h rat feces wereshown in Figure 6 No additional metabolites were observedin the feces of 24ndash48 h collection period Results showedthat the major metabolites of icariin were M8 and M11whose total amount accounted for over 90 of total peakarea in the LC-UV chromatogram of 0ndash24 h feces And theconcentration of metabolites in feces rapidly declined over24ndash48 h collection period The MS2 and MS3 spectra oficariin and its metabolites were exhibited in Figure 7 andthe observed and calculatedmasses formula retention timesmass errors and MS data of these analytes were presented inTable 6 Proposed metabolic pathways for icariin in rat feceswere shown in Figure 8

352 Identification of Metabolites Known compounds wereidentified by comparison with the chromatographic behaviorand MS fragmentation pattern of reference standards Forunknown constituents their structures were characterizedbased on their retention behavior and their MS119899 spectraobtained on-line Moreover under the optimized LC-MSconditions all the in vivometabolites of icariin could give theprecursor ions of sufficient abundance in order to facilitatethe analysis of multistage mass spectrometry Because allmetabolites originated from icariin and their MS fragmen-tation patterns were largely similar to that of icariin it was

10 BioMed Research International

Heart Spleen Lung Kidney Brain TesticleLiver0

10

20

30

40

50C

once

ntra

tion

(ng

g)

025 Ｂ

05 Ｂ

1 Ｂ

2 Ｂ

4 Ｂ

6 Ｂ

(a)

0

10

20

30

40

50

Con

cent

ratio

n (n

gg)

Heart Spleen Lung Kidney Brain Uterus OvaryLiver

025 Ｂ

05 Ｂ

1 Ｂ

2 Ｂ

4 Ｂ

6 Ｂ

(b)

Figure 5 Tissue distribution of icariin in rats after a single oral dose of Herba Epimedii extract (corresponding 42mgg icariin (a) male rat(b) female rat)

663517

5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 900Time (min)

0

50

100

(a)

6155

512665

6637520419520

20

40

60

80

100

5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 900Time (min)

52 54 56 58500

50 M11

M10

M8

M7

M6M5

M4

M3M2

M1

M9

(b)

658

61465278

506

0

20

40

60

80

100

5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 900Time (min)

M11

M3 M8

(c)

Figure 6 Representative chromatograms of icariin metabolites in rat feces of 0ndash24 h (a) blank feces sample (b) feces sample after oraladministration and (c) feces sample after intramuscular administration

BioMed Research International 11

36724

6755435214

Icariin

513320

20

40

60

80

100

Rela

tive a

bund

ance

400 600 800200mz

35218

Icariin

367250

20

40

60

80

100

Rela

tive a

bund

ance

400 600200mz

35211

36722

29727

0

20

40

60

80

100

Rela

tive a

bund

ance

400300mz

2811329595

26725 3090935216

25506

0

20

40

60

80

100

Rela

tive a

bund

ance

300 400200mz

28408

35323

25512

0

20

40

60

80

100

Rela

tive a

bund

ance

400 600200mz

35217

36722

0

20

40

60

80

100

Rela

tive a

bund

ance

300 400 500200mz

36722

6892932124

400 600 800200mz

0

20

40

60

80

100

Rela

tive a

bund

ance

35318

49919

0

20

40

60

80

100

Rela

tive a

bund

ance

400 600200mz

35215

48116 64516

400 600 800200mz

0

20

40

60

80

100

Rela

tive a

bund

ance

400 600 800 1000mz

36723

83734

79088

0

20

40

60

80

100

Rela

tive a

bund

ance

M1M1

M2

M2

M3

M3

M4

M4

MS2

MS3

MS2

MS3

MS3

MS2

MS2

MS2

MS3

MS3

Figure 7 Continued

12 BioMed Research International

38218

36714 5293950114

0

20

40

60

80

100

Rela

tive a

bund

ance

400 500 600300mz

M5

MS2

36722

33916 382180

20

40

60

80

100

Rela

tive a

bund

ance

300 400 500200mz

M5

MS3

35217

36711

0

20

40

60

80

100

Rela

tive a

bund

ance

300 400 500200mz

M6

MS3

36724

35227 6452032309

0

20

40

60

80

100

Rela

tive a

bund

ance

400 600 800200mz

M6

MS2

36614

6592335118

0

20

40

60

80

100

Rela

tive a

bund

ance

400 600 800200mz

M7

MS2

35116

32317

31112

36607

0

20

40

60

80

100

Rela

tive a

bund

ance

300 400 500200mz

M7

MS3

35109

32308

31113

3660629520

0

20

40

60

80

100

Rela

tive a

bund

ance

300 400 500200mz

M8

MS3

36615

51313

400 600200mz

0

20

40

60

80

100

Rela

tive a

bund

ance M8

MS2

36514

35528 38328

33929

0

20

40

60

80

100

Rela

tive a

bund

ance

400300mz

M9

MS2

35019

36508

33724

0

20

40

60

80

100

Rela

tive a

bund

ance

400200mz

M9

MS3

Figure 7 Continued

BioMed Research International 13

25308

16310

27902

29817

0

20

40

60

80

100

Rela

tive a

bund

ance

400200mz

M10

MS3

29810

35312

21913

400200mz

0

20

40

60

80

100

Rela

tive a

bund

ance M10

MS2

35208

36723

309280

20

40

60

80

100

Rela

tive a

bund

ance

400 600200mz

M11

MS2

30911

2810435215

400200mz

0

20

40

60

80

100

Rela

tive a

bund

ance M11

MS3

Figure 7 Representative LC-MS119899 spectra of icariin and its metabolites M1ndashM11 in rat feces

necessary to characterize the MS119899 behavior of icariin toidentify and assign the metabolites of the constituent Thedeprotonated molecule [MndashH]minus of icariin appears at 119898119911675 which readily eliminates its glucosyl and rhamnosylgroups to produce an [MndashHndash308]minus ion at 119898119911 367 The[MndashHndash308]minus ion yields a fragment at 119898119911 352 by losing amethyl radical And of theMS4 spectrum of [MndashHndash308ndash15]minusion the fragment at 119898119911 323 can be reasonably attributed tothe loss of 3-COH of flavone skeletonrsquos C ring

M1 as aminormetabolite of icariin was only found in thefeces of the oral group Its quasimolecular ion occurs at 119898119911837 (C39H49O20) whose molecular mass is 162Da more thanthat of icariin And its major MS119899 fragment ions are the sameas those of icariinThemost abundantMS2 andMS3 fragmentions also appear at 119898119911 367 and 352 indicating the loss ofone rhamnosyl and two glucosyl moieties and the sequentialloss of methyl group respectively Although the molecularweight of M1 is equal to that of epimedin A there existsa significant difference on their chromatographic retentionbehavior Therefore M1 is tentatively identified as an isomerof epimedin A

Both M2 and M6 show the same quasimolecular ion[M minus H]minus at 119898119911 645 (C32H37O14) with the elution time at5044 and 5743min respectively M2 was only observed infeces after intramuscular introduction of icariin while M6was found in feces following both routes of administrationM6 is unambiguously identified as sagittatoside B incomparison with the reference standard Analogous to theMS119899 fragments of icariin (see Figure 7 and Table 6) M2also yielded the major fragment ions at 119898119911 352 323 309296 281 and 268 strongly suggesting that M2 should be

a conjunction metabolite of icariin Thus M2 is tentativelyassigned as sagittatoside B isomer

M3 was found in rat feces after both intramuscular andoral administration of icariin Its MS spectrum exhibits adeprotonated molecule [MndashH]minus at 119898119911 499 (C26H27O10)176Da less than that of icariin By comparison with thereference standard it is identified as baohuoside II TheMSMS spectrum shows the most abundant fragment ionat 119898119911 353 indicating the loss of rhamnose moiety TheMS3 spectrum displays the major fragment ions at 119898119911 325298 and 284 separately corresponding to the loss of COisobutenyl and isopentenyl radicals

M4 is a minor metabolite of icariin detected in the fecesof rats in the oral administration group Its deprotonatedmolecule (C33H37O16) is at 119898119911 689 And the major MSMSfragment at 119898119911 367 results from the loss of glucuronicacid and rhamnose moieties Its MS3 spectrum produces afragment at119898119911 352 (the loss of a methyl group) Thus M4 istentatively characterized as icariin glucuronide

M5ndashM11 of icariin metabolites were observed in rat fecesafter both intramuscular and oral administration of icariin

M5 has a deprotonated molecular ion at 119898119911 529(C27H29O11) in its MS spectrum The MS119899 spectra show themajor fragment ions at119898119911 501 (the loss of CO) 382 (the lossof rhamnosyl group) 367 (the loss of methyl radical from theion at119898119911 382) and 339 (the successive loss of methyl radicaland CO) Therefore M5 is tentatively identified as hydroxylicariside II

M7 is identified as 210158401015840-O-rhamnosyl icariside II based onthe reference standard and related mass spectral pattern The

14 BioMed Research International

Table6Ch

aracteriz

ationof

metaboliteso

ficariinin

ratfeces

Num

ber

Com

poun

dRT

Form

ula

Observedmass

RDB

Error

LCM

S119899data

[MndashH]minus

(ppm

)

PlowastIcariin

5519

C 33H39O15

67522888

145

0790

MS2[675]367(100)352(25)

MS3[675-367]352(100)

MS4[675-367-352]323(

30)309(100)296(45)281

(65)268

(50)255

(20)

M1

Epim

edin

Aiso

mer

a5038

C 39H49O20

8372

819

155

0824

MS2[837]790(50)367

(100)

MS3[837-367]352(100)297(20)

M2

Sagitta

tosid

eBiso

mer

b5044

C 32H37O14

6452186

145

1314

MS2[645]481(20)352(100)

MS3[645-352]323(40)309

(60)296

(90)281

(100)268(60)

M3

lowastBa

ohuo

sideIIa

b5278

C 26H27O10

4991

605

135

1295

MS2[499]353(100)

MS3[499-353]325(20)298

(40)284

(100)255(30)

M4

Icariin

glucuron

idea

5404

C 33H37O16

6892

084

155

1202

MS2[689]367(100)321(60)

MS3[689-367]352(100)

M5

Hydroxylicaris

ideIIa

b5412

C 27H29O11

5291

716

135

1132

MS2[529]501(20)382(100)367(40)

MS3[529-382]367(100)339(10)

M6

lowastSagitta

tosid

eBab

5743

C 32H37O14

6452184

145

1020

MS2[645]367(100)352(30)

MS3[645-367]352(100)

M7

lowast210158401015840-O

-Rhamno

syl-icaris

ideIIa

b5756

C 33H39O14

6592

344

145

1529

MS2[659]366(100)351(20)323(15)

MS3[659-366]351(100)323

(80)311(60)305

(20)

M8

lowastIcariside

IIab

6146

C 27H29O10

5131760

135

0851

MS2[513]366(100)

MS3[513-366]351(100)323

(80)311(50)305

(20)

M9

Hydroxylicaritin

ab6233

C 21H19O7

3831137

125

3160

MS2[383]365(100)355(60)339

(40)

MS3[383-365]350(100)321(20)230(40)

M10

Desmethylicaritin

ab6636

C 20H17O6

3531024

125

1119

MS2[353]298(100)219(10)

MS3[353-298]279(60)269

(40)253

(100)241(30)164(70)

M11

lowastIcaritinab

8265

C 21H19O6

3671183

125

1921

MS2[367]352(100)309(20)297

(20)

MS3[367-352]309(100)297(60)

lowastCom

paredwith

ther

eference

standardaObservedin

ratfeces

after

oraladministratio

nb O

bservedin

ratfeces

after

intram

usculara

dministratio

n

BioMed Research International 15

O

OO

O

O

O

OHOH

OH

OH

OH OHOH

OH

OHOH

OHOH

OH

OHOH OH

OH

OH

OH

OHOH

OH

OH

OH

OH

OH

OH

OH

OHOH

OH

OH

OH

OH

OHOH

OHOH

OHOH

OH

OH

OH

OH

OHOH

HO HO

HO

HO

HO

HOHO

HO

HO

HO

OH

O

OO

O

OOHOOC

O

OO

O

O

OO

OO

O

O

OO

OO

O

O

OO

O

O

OO

O

O

O

O

O

O

O

icariin

epimedin A isomer

minus rh

a

minus methyl

+ rhaOxidation

minus methyl

Icariin glucuronide

+ xyl

+ glcGlucuronidation

minus rha

Oxidation

minus rha

+ xyl

Sagittatoside B isomer

minus glc

M1

M4

M2

OCH3 OCH3

OCH3

OCH3

OCH3

OCH3

OCH3

OCH3

M7 2-O-rhamnosyl icariside II

M5 hydroxyl icariside II

M3 baohuoside II

M8 icariside II

M6 sagittatoside B

M9 hydroxyl icaritinM10 desmethyl icaritin

M11 icaritin

Figure 8 Proposed metabolic pathways for icariin in rat feces

MS spectrum of M7 shows a quasimolecular ion [MndashH]minusat 119898119911 659 (C33H39O14) The major fragment ions at 119898119911366 351 and 323 result from the concurrent loss of therhamnose chain the successive loss of a methyl group andCO respectively

As shownFigure 7M8 is themost abundantmetabolite oficariin By comparison with the reference standard it is iden-tified as icariside II Its MS spectrum shows a deprotonatedmolecular ion at 119898119911 513 (C27H29O10) And similar to the

MS119899 pattern of icariin the major fragment ions at 119898119911 366351 and 323 separately match the loss of rhamnose moietythe sequential loss of rhamnose and methyl groups and theloss of CO

M9 has a quasimolecular ion [MndashH]minus at 119898119911 383(C21H19O7) 146Da less than that of M5 (hydroxyl icarisideII) It is tentatively identified as hydroxyl icaritin Its MSMSspectrum shows themain fragments of119898119911 365 355 and 339respectively corresponding to the neutral loss of H2O CO

16 BioMed Research International

and CO2 MS3 spectrum the fragment ions at 119898119911 350 and321 individually result from the neutral loss of methyl radicaland CO2

The quasimolecular ion [MndashH]minus of M10 is at 119898119911 353146Da less than that of M3 (baohuoside II) The majorfragment ion in its MSMS spectrum is at119898119911 298 matchingthe loss of isobutenyl radical The fragment at 119898119911 219 ispossibly produced by the neutral loss of C8H6O2 (134Da) dueto the retro-Diels-Alder (RDA) fragmentation of the flavonersquosC ring Moreover the MS3 spectrum affords fragment ionsat 119898119911 279 269 253 and 164 resulting from the loss ofH2O CO CO2 and C8H6O2 (134Da) respectively Thus thismetabolite is reasonably identified as desmethyl icaritin

M11 was observed as anothermajormetabolite in rat fecesof both two administration groups (Figure 7) By comparisonwith the reference standard it is identified as icaritin Its MSspectrum yields a quasimolecular ion at119898119911 367 (C21H19O6)And themajor fragment ion at119898119911 352 is characterized as theloss of methyl group The MS3 fragment at 119898119911 309 resultsfrom the loss of methyl group and CO and ion at 119898119911 297is the outcome of the neutral loss of isopentene (70Da) fromthe deprotonated molecule

4 Conclusion

Themenopausal syndrome improvement and possible cancerpreventive activity of Herba Epimedii is receiving more andmore attention Information on the in vivo disposition ofHerba Epimedii components is important for better under-standing the biological effects of this herbal drug For thispurpose our investigation was designed to obtain informa-tion on the plasma pharmacokinetics and tissue distributionof icariin in rat and the excretion of icariin and itsmetabolitesIn this study we firstly established and validated a rapidsensitive and reliable LC-MS method for the determinationof icariin in various biological samples collected at differenttime points and then the method was applied to the plasmapharmacokinetics and tissue distribution study of icariin inrats after oral administration of the total flavonoid extractof Herba Epimedii In addition the differences of the tissuedistribution of icariin in between male and female rats werestudied after oral and intramuscular administration of icariinAnd the metabolic pattern of icariin was also investigatedThe data showed the plasma and tissue concentration oficariin were kept at a very low level at all collection pointswhich suggests icariin is poorly absorbed after oral adminis-trationThepeak concentration in plasma and collected tissuesamples appeared at 05ndash1 h and then quickly dropped at 4 hafter administration demonstrating that a small amount oficariin can be rapidly absorbed and eliminated and there isno accumulation in rat tissues Moreover the concentrationin liver lung and reproductive organs was much higherthan that in other tissues indicating that liver lung andreproductive organs are the main target sites of icariin in rats

The gender-related difference of icariin in tissue distribu-tion has not been reported in the past published literatureOur study demonstrated that the distribution features oficariin are similar in most of tissues between male andfemale rats However there exist a few remarkable gender

differences such that thewhole tissue concentration of icariinin male rats was much higher than female rats except in gen-ital organs (high in female and low in male) In China HerbaEpimedii is a famous tonic and aphrodisiac to invigoratekidney and strengthenYang therefore it is commonly used totreat male sexual functional decline and female menopausalsyndrome As one of the predominant constitutes in HerbaEpimedii icariin plays a very crucial role in exerting effectsso the relatively high icariin concentration in female genitalorgans actually supports the above-mentioned traditionalChinese medicine theory and clinical practice and makesthis compound a good future prospect for treating womenrsquosmenopausal related diseases

In the present study the complete metabolic profile oficariin has also been investigated The results illustrate thatfeces should be the major excretion way for both oral andintramuscular administration of icariin Based on LC-UVand LC-MS119899 analyses eleven metabolites were detected infecal samples and were further identified Among them10 metabolites appeared in rat feces of oral administrationgroup and 9 in the feces of intramuscular introduction groupThe major metabolic routes of clearance are proposed to behydrolysis demethylation oxidation and conjugation

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

Authorsrsquo Contributions

Shunjun Xu and Jiejing Yu contributed equally to this work

References

[1] X Wang Y He B Guo et al ldquoIn vivo screening for anti-osteo-porotic fraction from extract of herbal formula xianlinggubaoin ovariectomized micerdquo PLoS ONE vol 10 no 2 Article IDe0118184 2015

[2] R-H Liu X Kang L-P Xu et al ldquoEffects of the combinedextracts of Herba Epimedii and Fructus Ligustri Lucidi on bonemineral content and bone turnover in osteoporotic ratsrdquo BMCComplementary and Alternative Medicine vol 15 no 1 article112 2015

[3] B C L Chan H Y Lee W S Siu et al ldquoSuppression of mastcell activity contributes to the osteoprotective effect of an herbalformula containing Herba Epimedii Fructus Ligustri Lucidiand Fructus Psoraleaerdquo Journal of Pharmacy and Pharmacologyvol 66 no 3 pp 437ndash444 2014

[4] S CW Sze Y Tong T B Ng C L Y Cheng andH P CheungldquoHerba Epimedii anti-oxidative properties and its medicalimplicationsrdquoMolecules vol 15 no 11 pp 7861ndash7870 2010

[5] R Niu ldquoAction of the drug Herba Epimedii on testosterone ofthe mouse plasma and its accessory sexual organ before andafter processingrdquo China Journal of Chinese Materia Medica vol14 no 9 pp 530ndash574 1989

[6] F-X Liu S Sun and Z-Z Cui ldquoAnalysis of immunologi-cal enhancement of immunosuppressed chickens by Chineseherbal extractsrdquo Journal of Ethnopharmacology vol 127 no 2pp 251ndash256 2010

BioMed Research International 17

[7] J-H Kim Y-J Mun S-J Im J-H Han H-S Lee and W-HWoo ldquoEffects of the aqueous extract of Epimedii Herba on theantibody responses in micerdquo International Immunopharmacol-ogy vol 1 no 5 pp 935ndash944 2001

[8] X Yu Y Tong X-Q Han et al ldquoAnti-angiogenic activity ofHerba Epimedii on zebrafish embryos in vivo and HUVECs invitrordquo Phytotherapy Research vol 27 no 9 pp 1368ndash1375 2013

[9] D-C Zhang J-L Liu Y-B Ding J-G Xia and G-Y ChenldquoIcariin potentiates the antitumor activity of gemcitabine ingallbladder cancer by suppressingNF-120581BrdquoActa PharmacologicaSinica vol 34 no 2 pp 301ndash308 2013

[10] L Kong J Liu J Wang et al ldquoIcariin inhibits TNF-120572IFN-120574induced inflammatory response via inhibition of the substanceP and p38-MAPK signaling pathway in human keratinocytesrdquoInternational Immunopharmacology vol 29 no 2 pp 401ndash4072015

[11] H Shao J Shen M Wang et al ldquoIcariin protects against tita-nium particle-induced osteolysis and inflammatory response ina mouse calvarial modelrdquo Biomaterials vol 60 pp 92ndash99 2015

[12] Q Wei J Zhang G Hong et al ldquoIcariin promotes osteogenicdifferentiation of rat bone marrow stromal cells by activatingthe ER120572-Wnt120573-catenin signaling pathwayrdquo Biomedicine ampPharmacotherapy vol 84 pp 931ndash939 2016

[13] S-Q Zhang W-J Cai J-H Huang et al ldquoIcariin a naturalflavonol glycoside extends healthspan in micerdquo ExperimentalGerontology vol 69 pp 226ndash235 2015

[14] M N Makarova O N Pozharitskaya A N Shikov S VTesakova V G Makarov and V P Tikhonov ldquoEffect of lipid-based suspension of Epimedium koreanum Nakai extract onsexual behavior in ratsrdquo Journal of Ethnopharmacology vol 114no 3 pp 412ndash416 2007

[15] Y Chen J Wang X Jia X Tan and M Hu ldquoRole of intestinalhydrolase in the absorption of prenylated flavonoids present inyinyanghuordquoMolecules vol 16 no 2 pp 1336ndash1348 2011

[16] J J Yu Y J Liu S J Xu S Sun Y J Xu and L Yang ldquoSimulta-neous quantitative determination of four epimediumflavonoidsin rat urine by LC-MSMSrdquo Traditional Chinese Drug Researchamp Clinical Pharmacology vol 27 no 4 pp 537ndash541 2016

[17] Y Lu N Li Y Deng et al ldquoSimultaneous determination oficariin naringin and osthole in rat plasma by UPLC-MSMSand its application for pharmacokinetic study after oral admin-istration of Gushudan capsulesrdquo Journal of Chromatography Bvol 993-994 pp 75ndash80 2015

[18] N Wang X Huang X Wang Y Zhang R Wu and D ShouldquoPipette tip solid-phase extraction and high-performance liquidchromatography for the determination of flavonoids fromEpimedii herba in rat serum and application of the techniqueto pharmacokinetic studiesrdquo Journal of Chromatography B vol990 pp 64ndash72 2015

Submit your manuscripts athttpswwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom

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ToxinsJournal of

VaccinesJournal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

8 BioMed Research International

Table 3 Matrix effect and extraction recovery for the assay of icariin in plasma and tissue samples (119899 = 5)

Matrix ConcngmL

Matrix effectMean plusmn SD ()

Extraction efficiencyMean plusmn SD ()

Plasma2 712 plusmn 29 869 plusmn 60

20 690 plusmn 20 841 plusmn 47

400 667 plusmn 10 849 plusmn 58

Liver2 715 plusmn 33 939 plusmn 75

20 722 plusmn 41 903 plusmn 19

400 703 plusmn 31 905 plusmn 22

Heart2 679 plusmn 38 889 plusmn 39

20 688 plusmn 26 922 plusmn 23

400 699 plusmn 18 931 plusmn 17

Spleen2 692 plusmn 53 887 plusmn 84

20 701 plusmn 37 835 plusmn 39

400 714 plusmn 38 825 plusmn 14

Lung2 710 plusmn 22 880 plusmn 59

20 712 plusmn 24 854 plusmn 45

400 738 plusmn 16 838 plusmn 19

Kidney2 577 plusmn 62 865 plusmn 67

20 555 plusmn 53 900 plusmn 37

400 666 plusmn 152 853 plusmn 61

Brain2 906 plusmn 58 897 plusmn 57

20 893 plusmn 19 949 plusmn 39

400 870 plusmn 23 990 plusmn 23

Testicle2 802 plusmn 35 915 plusmn 67

20 773 plusmn 21 964 plusmn 22

400 777 plusmn 22 1006 plusmn 19

of the plasma concentration-time curve Elimination rateconstant (119870119890) was determined from the least-square fittedterminal log-linear portion of the plasma concentration-timeprofile Elimination half-life (11990512) was calculated by 0693KeTotal area under the plasma concentration-time curve oficariin from time zero to infinity (AUC0ndashinfin) was calculatedas the sumofAUC0ndash12 the area up to the last quantifiable timepoint (12 h) and the area from the 12 h time point to infinityAUC0ndash12 was calculated using the trapezoidal rule and theextrapolated area was calculated from the last measurableconcentration 119862119905 divided by 119870119890 The major pharmacokineticparameters of icariin were summarized in Table 5

34 Tissue Distribution The tissue distribution of icariin infemale and male rats was investigated following a single oraldose of total flavonoid extract from Herba Epimedii (cor-responding 42mgg icariin) The mean concentration-timeprofile of each tissue was presented in Figure 5 indicatingthat icariin was poorly absorbed after oral administrationbecause all the tissue concentrations were kept at a verylow level The peak concentration of icariin in most ofcollected tissues appeared at 1 h after administration except

for liver and female reproductive organs in which theconcentration reached the peak at 05 h demonstrating thaticariin underwent a rapid and wide distribution Moreoverthe tissue concentrations in male rat were generally muchhigher than those in female rat The tissue area under thecurve from time zero to infinity was in the following orderliver gt lung gt spleen gt heart gt kidney gt brain gt testiclefor mail rat and liver gt uterus gt heart gt ovary gt lung gtkidney gt spleen gt brain for female rats As shown in Figure 5the concentration levels in liver and lung were much higherthan those in other tissues illustrating that liver and lungare the main target organs of icariin in male rat after oraladministration of the total flavonoid extract Furthermorethe concentration level in uteruswasmuchhigher than that inany other tissues of female rat demonstrating that the gender-related difference of icariin in tissue distribution do existand the reproductive organs are the main target organs oficariin for female rat Meanwhile icariin was found with verylow AUC0ndashinfin in brain which indicated that icariin could notefficiently cross the blood-brain barrier Over time the maintrend of the mean concentration of icariin in all tissues wasreduced and the concentration dropped very quickly at 4 h

BioMed Research International 9

Table 4 Stability of icariin in plasma and tissue samples (119899 = 6)

Matrix Conc Mean plusmn SDngmL Bench-topa Freeze-thawb Autosamplerc Long-termd

Plasma2 975 plusmn 37 968 plusmn 57 1047 plusmn 81 993 plusmn 59

20 945 plusmn 59 1013 plusmn 31 955 plusmn 37 943 plusmn 16

400 977 plusmn 39 1030 plusmn 19 945 plusmn 26 955 plusmn 37

Liver2 987 plusmn 63 1015 plusmn 21 1114 plusmn 44 986 plusmn 54

20 949 plusmn 14 900 plusmn 13 1060 plusmn 16 957 plusmn 21

400 875 plusmn 13 897 plusmn 13 894 plusmn 14 1125 plusmn 22

Heart2 1086 plusmn 33 1002 plusmn 43 955 plusmn 22 1150 plusmn 30

20 1056 plusmn 17 996 plusmn 10 973 plusmn 13 1002 plusmn 42

400 1026 plusmn 15 942 plusmn 29 892 plusmn 09 907 plusmn 50

Spleen2 954 plusmn 92 1018 plusmn 44 955 plusmn 76 1013 plusmn 59

20 989 plusmn 14 1094 plusmn 11 928 plusmn 09 1123 plusmn 34

400 967 plusmn 27 1032 plusmn 04 871 plusmn 12 974 plusmn 26

Lung2 959 plusmn 35 856 plusmn 30 1032 plusmn 48 983 plusmn 43

20 1017 plusmn 80 923 plusmn 40 1094 plusmn 46 1102 plusmn 18

400 925 plusmn 18 932 plusmn 15 997 plusmn 04 1098 plusmn 17

Kidney2 1012 plusmn 34 916 plusmn 40 951 plusmn 22 984 plusmn 48

20 1025 plusmn 28 974 plusmn 18 955 plusmn 16 934 plusmn 22

400 920 plusmn 14 911 plusmn 08 899 plusmn 10 1095 plusmn 22

Brain2 858 plusmn 60 974 plusmn 73 966 plusmn 32 1094 plusmn 32

20 1033 plusmn 41 1035 plusmn 31 1030 plusmn 35 974 plusmn 12

400 1002 plusmn 29 990 plusmn 31 978 plusmn 21 1102 plusmn 30

Testicle2 1058 plusmn 39 853 plusmn 24 997 plusmn 55 933 plusmn 41

20 1040 plusmn 14 870 plusmn 13 1016 plusmn 19 1065 plusmn 15

400 1034 plusmn 39 984 plusmn 16 995 plusmn 33 1068 plusmn 38aAt least 1 h at room temperature bAt least 3 freeze-thaw cycles cAt least 24 h at 4∘C dAt least 4 weeks at minus80 plusmn 5∘C

Table 5 Pharmacokinetic parameters of icariin in rat after asingle oral dose of Herba Epimedii extract (corresponding 42mggicariin)

Parameter IcariinAUC0ndashinfin (ugLsdoth) 164155 plusmn 144129

AUMC0ndashinfin (ugLsdoth) 538424 plusmn 262081

MRT0ndashinfin (h) 3990 plusmn 1987

11990512 (h) 3149 plusmn 2364

119879max (h) 0458 plusmn 0246

CL119911119865 (Lhkg) 389043 plusmn 204472

after administration suggesting no accumulation in tissuesand a rapid elimination of this compound

35 Metabolism Study of Icariin in Rat Urine and Feces

351 Metabolite Profile of Rat Urine and Feces In thisstudy urine and feces samples were both analyzed by LC-MSMS By comparing the total ion chromatograms of drug-containing urine sample and predose control urine sampleneither parent drug nor metabolites were observed in raturine following two routes of administration of icariinHowever a total of 11 icariin metabolites were detected in ratfeces including 10 metabolites after oral administration and

9 following intramuscular introduction The representativechromatograms of the metabolites of 0ndash24 h rat feces wereshown in Figure 6 No additional metabolites were observedin the feces of 24ndash48 h collection period Results showedthat the major metabolites of icariin were M8 and M11whose total amount accounted for over 90 of total peakarea in the LC-UV chromatogram of 0ndash24 h feces And theconcentration of metabolites in feces rapidly declined over24ndash48 h collection period The MS2 and MS3 spectra oficariin and its metabolites were exhibited in Figure 7 andthe observed and calculatedmasses formula retention timesmass errors and MS data of these analytes were presented inTable 6 Proposed metabolic pathways for icariin in rat feceswere shown in Figure 8

352 Identification of Metabolites Known compounds wereidentified by comparison with the chromatographic behaviorand MS fragmentation pattern of reference standards Forunknown constituents their structures were characterizedbased on their retention behavior and their MS119899 spectraobtained on-line Moreover under the optimized LC-MSconditions all the in vivometabolites of icariin could give theprecursor ions of sufficient abundance in order to facilitatethe analysis of multistage mass spectrometry Because allmetabolites originated from icariin and their MS fragmen-tation patterns were largely similar to that of icariin it was

10 BioMed Research International

Heart Spleen Lung Kidney Brain TesticleLiver0

10

20

30

40

50C

once

ntra

tion

(ng

g)

025 Ｂ

05 Ｂ

1 Ｂ

2 Ｂ

4 Ｂ

6 Ｂ

(a)

0

10

20

30

40

50

Con

cent

ratio

n (n

gg)

Heart Spleen Lung Kidney Brain Uterus OvaryLiver

025 Ｂ

05 Ｂ

1 Ｂ

2 Ｂ

4 Ｂ

6 Ｂ

(b)

Figure 5 Tissue distribution of icariin in rats after a single oral dose of Herba Epimedii extract (corresponding 42mgg icariin (a) male rat(b) female rat)

663517

5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 900Time (min)

0

50

100

(a)

6155

512665

6637520419520

20

40

60

80

100

5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 900Time (min)

52 54 56 58500

50 M11

M10

M8

M7

M6M5

M4

M3M2

M1

M9

(b)

658

61465278

506

0

20

40

60

80

100

5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 900Time (min)

M11

M3 M8

(c)

Figure 6 Representative chromatograms of icariin metabolites in rat feces of 0ndash24 h (a) blank feces sample (b) feces sample after oraladministration and (c) feces sample after intramuscular administration

BioMed Research International 11

36724

6755435214

Icariin

513320

20

40

60

80

100

Rela

tive a

bund

ance

400 600 800200mz

35218

Icariin

367250

20

40

60

80

100

Rela

tive a

bund

ance

400 600200mz

35211

36722

29727

0

20

40

60

80

100

Rela

tive a

bund

ance

400300mz

2811329595

26725 3090935216

25506

0

20

40

60

80

100

Rela

tive a

bund

ance

300 400200mz

28408

35323

25512

0

20

40

60

80

100

Rela

tive a

bund

ance

400 600200mz

35217

36722

0

20

40

60

80

100

Rela

tive a

bund

ance

300 400 500200mz

36722

6892932124

400 600 800200mz

0

20

40

60

80

100

Rela

tive a

bund

ance

35318

49919

0

20

40

60

80

100

Rela

tive a

bund

ance

400 600200mz

35215

48116 64516

400 600 800200mz

0

20

40

60

80

100

Rela

tive a

bund

ance

400 600 800 1000mz

36723

83734

79088

0

20

40

60

80

100

Rela

tive a

bund

ance

M1M1

M2

M2

M3

M3

M4

M4

MS2

MS3

MS2

MS3

MS3

MS2

MS2

MS2

MS3

MS3

Figure 7 Continued

12 BioMed Research International

38218

36714 5293950114

0

20

40

60

80

100

Rela

tive a

bund

ance

400 500 600300mz

M5

MS2

36722

33916 382180

20

40

60

80

100

Rela

tive a

bund

ance

300 400 500200mz

M5

MS3

35217

36711

0

20

40

60

80

100

Rela

tive a

bund

ance

300 400 500200mz

M6

MS3

36724

35227 6452032309

0

20

40

60

80

100

Rela

tive a

bund

ance

400 600 800200mz

M6

MS2

36614

6592335118

0

20

40

60

80

100

Rela

tive a

bund

ance

400 600 800200mz

M7

MS2

35116

32317

31112

36607

0

20

40

60

80

100

Rela

tive a

bund

ance

300 400 500200mz

M7

MS3

35109

32308

31113

3660629520

0

20

40

60

80

100

Rela

tive a

bund

ance

300 400 500200mz

M8

MS3

36615

51313

400 600200mz

0

20

40

60

80

100

Rela

tive a

bund

ance M8

MS2

36514

35528 38328

33929

0

20

40

60

80

100

Rela

tive a

bund

ance

400300mz

M9

MS2

35019

36508

33724

0

20

40

60

80

100

Rela

tive a

bund

ance

400200mz

M9

MS3

Figure 7 Continued

BioMed Research International 13

25308

16310

27902

29817

0

20

40

60

80

100

Rela

tive a

bund

ance

400200mz

M10

MS3

29810

35312

21913

400200mz

0

20

40

60

80

100

Rela

tive a

bund

ance M10

MS2

35208

36723

309280

20

40

60

80

100

Rela

tive a

bund

ance

400 600200mz

M11

MS2

30911

2810435215

400200mz

0

20

40

60

80

100

Rela

tive a

bund

ance M11

MS3

Figure 7 Representative LC-MS119899 spectra of icariin and its metabolites M1ndashM11 in rat feces

necessary to characterize the MS119899 behavior of icariin toidentify and assign the metabolites of the constituent Thedeprotonated molecule [MndashH]minus of icariin appears at 119898119911675 which readily eliminates its glucosyl and rhamnosylgroups to produce an [MndashHndash308]minus ion at 119898119911 367 The[MndashHndash308]minus ion yields a fragment at 119898119911 352 by losing amethyl radical And of theMS4 spectrum of [MndashHndash308ndash15]minusion the fragment at 119898119911 323 can be reasonably attributed tothe loss of 3-COH of flavone skeletonrsquos C ring

M1 as aminormetabolite of icariin was only found in thefeces of the oral group Its quasimolecular ion occurs at 119898119911837 (C39H49O20) whose molecular mass is 162Da more thanthat of icariin And its major MS119899 fragment ions are the sameas those of icariinThemost abundantMS2 andMS3 fragmentions also appear at 119898119911 367 and 352 indicating the loss ofone rhamnosyl and two glucosyl moieties and the sequentialloss of methyl group respectively Although the molecularweight of M1 is equal to that of epimedin A there existsa significant difference on their chromatographic retentionbehavior Therefore M1 is tentatively identified as an isomerof epimedin A

Both M2 and M6 show the same quasimolecular ion[M minus H]minus at 119898119911 645 (C32H37O14) with the elution time at5044 and 5743min respectively M2 was only observed infeces after intramuscular introduction of icariin while M6was found in feces following both routes of administrationM6 is unambiguously identified as sagittatoside B incomparison with the reference standard Analogous to theMS119899 fragments of icariin (see Figure 7 and Table 6) M2also yielded the major fragment ions at 119898119911 352 323 309296 281 and 268 strongly suggesting that M2 should be

a conjunction metabolite of icariin Thus M2 is tentativelyassigned as sagittatoside B isomer

M3 was found in rat feces after both intramuscular andoral administration of icariin Its MS spectrum exhibits adeprotonated molecule [MndashH]minus at 119898119911 499 (C26H27O10)176Da less than that of icariin By comparison with thereference standard it is identified as baohuoside II TheMSMS spectrum shows the most abundant fragment ionat 119898119911 353 indicating the loss of rhamnose moiety TheMS3 spectrum displays the major fragment ions at 119898119911 325298 and 284 separately corresponding to the loss of COisobutenyl and isopentenyl radicals

M4 is a minor metabolite of icariin detected in the fecesof rats in the oral administration group Its deprotonatedmolecule (C33H37O16) is at 119898119911 689 And the major MSMSfragment at 119898119911 367 results from the loss of glucuronicacid and rhamnose moieties Its MS3 spectrum produces afragment at119898119911 352 (the loss of a methyl group) Thus M4 istentatively characterized as icariin glucuronide

M5ndashM11 of icariin metabolites were observed in rat fecesafter both intramuscular and oral administration of icariin

M5 has a deprotonated molecular ion at 119898119911 529(C27H29O11) in its MS spectrum The MS119899 spectra show themajor fragment ions at119898119911 501 (the loss of CO) 382 (the lossof rhamnosyl group) 367 (the loss of methyl radical from theion at119898119911 382) and 339 (the successive loss of methyl radicaland CO) Therefore M5 is tentatively identified as hydroxylicariside II

M7 is identified as 210158401015840-O-rhamnosyl icariside II based onthe reference standard and related mass spectral pattern The

14 BioMed Research International

Table6Ch

aracteriz

ationof

metaboliteso

ficariinin

ratfeces

Num

ber

Com

poun

dRT

Form

ula

Observedmass

RDB

Error

LCM

S119899data

[MndashH]minus

(ppm

)

PlowastIcariin

5519

C 33H39O15

67522888

145

0790

MS2[675]367(100)352(25)

MS3[675-367]352(100)

MS4[675-367-352]323(

30)309(100)296(45)281

(65)268

(50)255

(20)

M1

Epim

edin

Aiso

mer

a5038

C 39H49O20

8372

819

155

0824

MS2[837]790(50)367

(100)

MS3[837-367]352(100)297(20)

M2

Sagitta

tosid

eBiso

mer

b5044

C 32H37O14

6452186

145

1314

MS2[645]481(20)352(100)

MS3[645-352]323(40)309

(60)296

(90)281

(100)268(60)

M3

lowastBa

ohuo

sideIIa

b5278

C 26H27O10

4991

605

135

1295

MS2[499]353(100)

MS3[499-353]325(20)298

(40)284

(100)255(30)

M4

Icariin

glucuron

idea

5404

C 33H37O16

6892

084

155

1202

MS2[689]367(100)321(60)

MS3[689-367]352(100)

M5

Hydroxylicaris

ideIIa

b5412

C 27H29O11

5291

716

135

1132

MS2[529]501(20)382(100)367(40)

MS3[529-382]367(100)339(10)

M6

lowastSagitta

tosid

eBab

5743

C 32H37O14

6452184

145

1020

MS2[645]367(100)352(30)

MS3[645-367]352(100)

M7

lowast210158401015840-O

-Rhamno

syl-icaris

ideIIa

b5756

C 33H39O14

6592

344

145

1529

MS2[659]366(100)351(20)323(15)

MS3[659-366]351(100)323

(80)311(60)305

(20)

M8

lowastIcariside

IIab

6146

C 27H29O10

5131760

135

0851

MS2[513]366(100)

MS3[513-366]351(100)323

(80)311(50)305

(20)

M9

Hydroxylicaritin

ab6233

C 21H19O7

3831137

125

3160

MS2[383]365(100)355(60)339

(40)

MS3[383-365]350(100)321(20)230(40)

M10

Desmethylicaritin

ab6636

C 20H17O6

3531024

125

1119

MS2[353]298(100)219(10)

MS3[353-298]279(60)269

(40)253

(100)241(30)164(70)

M11

lowastIcaritinab

8265

C 21H19O6

3671183

125

1921

MS2[367]352(100)309(20)297

(20)

MS3[367-352]309(100)297(60)

lowastCom

paredwith

ther

eference

standardaObservedin

ratfeces

after

oraladministratio

nb O

bservedin

ratfeces

after

intram

usculara

dministratio

n

BioMed Research International 15

O

OO

O

O

O

OHOH

OH

OH

OH OHOH

OH

OHOH

OHOH

OH

OHOH OH

OH

OH

OH

OHOH

OH

OH

OH

OH

OH

OH

OH

OHOH

OH

OH

OH

OH

OHOH

OHOH

OHOH

OH

OH

OH

OH

OHOH

HO HO

HO

HO

HO

HOHO

HO

HO

HO

OH

O

OO

O

OOHOOC

O

OO

O

O

OO

OO

O

O

OO

OO

O

O

OO

O

O

OO

O

O

O

O

O

O

O

icariin

epimedin A isomer

minus rh

a

minus methyl

+ rhaOxidation

minus methyl

Icariin glucuronide

+ xyl

+ glcGlucuronidation

minus rha

Oxidation

minus rha

+ xyl

Sagittatoside B isomer

minus glc

M1

M4

M2

OCH3 OCH3

OCH3

OCH3

OCH3

OCH3

OCH3

OCH3

M7 2-O-rhamnosyl icariside II

M5 hydroxyl icariside II

M3 baohuoside II

M8 icariside II

M6 sagittatoside B

M9 hydroxyl icaritinM10 desmethyl icaritin

M11 icaritin

Figure 8 Proposed metabolic pathways for icariin in rat feces

MS spectrum of M7 shows a quasimolecular ion [MndashH]minusat 119898119911 659 (C33H39O14) The major fragment ions at 119898119911366 351 and 323 result from the concurrent loss of therhamnose chain the successive loss of a methyl group andCO respectively

As shownFigure 7M8 is themost abundantmetabolite oficariin By comparison with the reference standard it is iden-tified as icariside II Its MS spectrum shows a deprotonatedmolecular ion at 119898119911 513 (C27H29O10) And similar to the

MS119899 pattern of icariin the major fragment ions at 119898119911 366351 and 323 separately match the loss of rhamnose moietythe sequential loss of rhamnose and methyl groups and theloss of CO

M9 has a quasimolecular ion [MndashH]minus at 119898119911 383(C21H19O7) 146Da less than that of M5 (hydroxyl icarisideII) It is tentatively identified as hydroxyl icaritin Its MSMSspectrum shows themain fragments of119898119911 365 355 and 339respectively corresponding to the neutral loss of H2O CO

16 BioMed Research International

and CO2 MS3 spectrum the fragment ions at 119898119911 350 and321 individually result from the neutral loss of methyl radicaland CO2

The quasimolecular ion [MndashH]minus of M10 is at 119898119911 353146Da less than that of M3 (baohuoside II) The majorfragment ion in its MSMS spectrum is at119898119911 298 matchingthe loss of isobutenyl radical The fragment at 119898119911 219 ispossibly produced by the neutral loss of C8H6O2 (134Da) dueto the retro-Diels-Alder (RDA) fragmentation of the flavonersquosC ring Moreover the MS3 spectrum affords fragment ionsat 119898119911 279 269 253 and 164 resulting from the loss ofH2O CO CO2 and C8H6O2 (134Da) respectively Thus thismetabolite is reasonably identified as desmethyl icaritin

M11 was observed as anothermajormetabolite in rat fecesof both two administration groups (Figure 7) By comparisonwith the reference standard it is identified as icaritin Its MSspectrum yields a quasimolecular ion at119898119911 367 (C21H19O6)And themajor fragment ion at119898119911 352 is characterized as theloss of methyl group The MS3 fragment at 119898119911 309 resultsfrom the loss of methyl group and CO and ion at 119898119911 297is the outcome of the neutral loss of isopentene (70Da) fromthe deprotonated molecule

4 Conclusion

Themenopausal syndrome improvement and possible cancerpreventive activity of Herba Epimedii is receiving more andmore attention Information on the in vivo disposition ofHerba Epimedii components is important for better under-standing the biological effects of this herbal drug For thispurpose our investigation was designed to obtain informa-tion on the plasma pharmacokinetics and tissue distributionof icariin in rat and the excretion of icariin and itsmetabolitesIn this study we firstly established and validated a rapidsensitive and reliable LC-MS method for the determinationof icariin in various biological samples collected at differenttime points and then the method was applied to the plasmapharmacokinetics and tissue distribution study of icariin inrats after oral administration of the total flavonoid extractof Herba Epimedii In addition the differences of the tissuedistribution of icariin in between male and female rats werestudied after oral and intramuscular administration of icariinAnd the metabolic pattern of icariin was also investigatedThe data showed the plasma and tissue concentration oficariin were kept at a very low level at all collection pointswhich suggests icariin is poorly absorbed after oral adminis-trationThepeak concentration in plasma and collected tissuesamples appeared at 05ndash1 h and then quickly dropped at 4 hafter administration demonstrating that a small amount oficariin can be rapidly absorbed and eliminated and there isno accumulation in rat tissues Moreover the concentrationin liver lung and reproductive organs was much higherthan that in other tissues indicating that liver lung andreproductive organs are the main target sites of icariin in rats

The gender-related difference of icariin in tissue distribu-tion has not been reported in the past published literatureOur study demonstrated that the distribution features oficariin are similar in most of tissues between male andfemale rats However there exist a few remarkable gender

differences such that thewhole tissue concentration of icariinin male rats was much higher than female rats except in gen-ital organs (high in female and low in male) In China HerbaEpimedii is a famous tonic and aphrodisiac to invigoratekidney and strengthenYang therefore it is commonly used totreat male sexual functional decline and female menopausalsyndrome As one of the predominant constitutes in HerbaEpimedii icariin plays a very crucial role in exerting effectsso the relatively high icariin concentration in female genitalorgans actually supports the above-mentioned traditionalChinese medicine theory and clinical practice and makesthis compound a good future prospect for treating womenrsquosmenopausal related diseases

In the present study the complete metabolic profile oficariin has also been investigated The results illustrate thatfeces should be the major excretion way for both oral andintramuscular administration of icariin Based on LC-UVand LC-MS119899 analyses eleven metabolites were detected infecal samples and were further identified Among them10 metabolites appeared in rat feces of oral administrationgroup and 9 in the feces of intramuscular introduction groupThe major metabolic routes of clearance are proposed to behydrolysis demethylation oxidation and conjugation

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

Authorsrsquo Contributions

Shunjun Xu and Jiejing Yu contributed equally to this work

References

[1] X Wang Y He B Guo et al ldquoIn vivo screening for anti-osteo-porotic fraction from extract of herbal formula xianlinggubaoin ovariectomized micerdquo PLoS ONE vol 10 no 2 Article IDe0118184 2015

[2] R-H Liu X Kang L-P Xu et al ldquoEffects of the combinedextracts of Herba Epimedii and Fructus Ligustri Lucidi on bonemineral content and bone turnover in osteoporotic ratsrdquo BMCComplementary and Alternative Medicine vol 15 no 1 article112 2015

[3] B C L Chan H Y Lee W S Siu et al ldquoSuppression of mastcell activity contributes to the osteoprotective effect of an herbalformula containing Herba Epimedii Fructus Ligustri Lucidiand Fructus Psoraleaerdquo Journal of Pharmacy and Pharmacologyvol 66 no 3 pp 437ndash444 2014

[4] S CW Sze Y Tong T B Ng C L Y Cheng andH P CheungldquoHerba Epimedii anti-oxidative properties and its medicalimplicationsrdquoMolecules vol 15 no 11 pp 7861ndash7870 2010

[5] R Niu ldquoAction of the drug Herba Epimedii on testosterone ofthe mouse plasma and its accessory sexual organ before andafter processingrdquo China Journal of Chinese Materia Medica vol14 no 9 pp 530ndash574 1989

[6] F-X Liu S Sun and Z-Z Cui ldquoAnalysis of immunologi-cal enhancement of immunosuppressed chickens by Chineseherbal extractsrdquo Journal of Ethnopharmacology vol 127 no 2pp 251ndash256 2010

BioMed Research International 17

[7] J-H Kim Y-J Mun S-J Im J-H Han H-S Lee and W-HWoo ldquoEffects of the aqueous extract of Epimedii Herba on theantibody responses in micerdquo International Immunopharmacol-ogy vol 1 no 5 pp 935ndash944 2001

[8] X Yu Y Tong X-Q Han et al ldquoAnti-angiogenic activity ofHerba Epimedii on zebrafish embryos in vivo and HUVECs invitrordquo Phytotherapy Research vol 27 no 9 pp 1368ndash1375 2013

[9] D-C Zhang J-L Liu Y-B Ding J-G Xia and G-Y ChenldquoIcariin potentiates the antitumor activity of gemcitabine ingallbladder cancer by suppressingNF-120581BrdquoActa PharmacologicaSinica vol 34 no 2 pp 301ndash308 2013

[10] L Kong J Liu J Wang et al ldquoIcariin inhibits TNF-120572IFN-120574induced inflammatory response via inhibition of the substanceP and p38-MAPK signaling pathway in human keratinocytesrdquoInternational Immunopharmacology vol 29 no 2 pp 401ndash4072015

[11] H Shao J Shen M Wang et al ldquoIcariin protects against tita-nium particle-induced osteolysis and inflammatory response ina mouse calvarial modelrdquo Biomaterials vol 60 pp 92ndash99 2015

[12] Q Wei J Zhang G Hong et al ldquoIcariin promotes osteogenicdifferentiation of rat bone marrow stromal cells by activatingthe ER120572-Wnt120573-catenin signaling pathwayrdquo Biomedicine ampPharmacotherapy vol 84 pp 931ndash939 2016

[13] S-Q Zhang W-J Cai J-H Huang et al ldquoIcariin a naturalflavonol glycoside extends healthspan in micerdquo ExperimentalGerontology vol 69 pp 226ndash235 2015

[14] M N Makarova O N Pozharitskaya A N Shikov S VTesakova V G Makarov and V P Tikhonov ldquoEffect of lipid-based suspension of Epimedium koreanum Nakai extract onsexual behavior in ratsrdquo Journal of Ethnopharmacology vol 114no 3 pp 412ndash416 2007

[15] Y Chen J Wang X Jia X Tan and M Hu ldquoRole of intestinalhydrolase in the absorption of prenylated flavonoids present inyinyanghuordquoMolecules vol 16 no 2 pp 1336ndash1348 2011

[16] J J Yu Y J Liu S J Xu S Sun Y J Xu and L Yang ldquoSimulta-neous quantitative determination of four epimediumflavonoidsin rat urine by LC-MSMSrdquo Traditional Chinese Drug Researchamp Clinical Pharmacology vol 27 no 4 pp 537ndash541 2016

[17] Y Lu N Li Y Deng et al ldquoSimultaneous determination oficariin naringin and osthole in rat plasma by UPLC-MSMSand its application for pharmacokinetic study after oral admin-istration of Gushudan capsulesrdquo Journal of Chromatography Bvol 993-994 pp 75ndash80 2015

[18] N Wang X Huang X Wang Y Zhang R Wu and D ShouldquoPipette tip solid-phase extraction and high-performance liquidchromatography for the determination of flavonoids fromEpimedii herba in rat serum and application of the techniqueto pharmacokinetic studiesrdquo Journal of Chromatography B vol990 pp 64ndash72 2015

Submit your manuscripts athttpswwwhindawicom

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ToxinsJournal of

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Pharmaceutics

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MEDIATORSINFLAMMATION

of

BioMed Research International 9

Table 4 Stability of icariin in plasma and tissue samples (119899 = 6)

Matrix Conc Mean plusmn SDngmL Bench-topa Freeze-thawb Autosamplerc Long-termd

Plasma2 975 plusmn 37 968 plusmn 57 1047 plusmn 81 993 plusmn 59

20 945 plusmn 59 1013 plusmn 31 955 plusmn 37 943 plusmn 16

400 977 plusmn 39 1030 plusmn 19 945 plusmn 26 955 plusmn 37

Liver2 987 plusmn 63 1015 plusmn 21 1114 plusmn 44 986 plusmn 54

20 949 plusmn 14 900 plusmn 13 1060 plusmn 16 957 plusmn 21

400 875 plusmn 13 897 plusmn 13 894 plusmn 14 1125 plusmn 22

Heart2 1086 plusmn 33 1002 plusmn 43 955 plusmn 22 1150 plusmn 30

20 1056 plusmn 17 996 plusmn 10 973 plusmn 13 1002 plusmn 42

400 1026 plusmn 15 942 plusmn 29 892 plusmn 09 907 plusmn 50

Spleen2 954 plusmn 92 1018 plusmn 44 955 plusmn 76 1013 plusmn 59

20 989 plusmn 14 1094 plusmn 11 928 plusmn 09 1123 plusmn 34

400 967 plusmn 27 1032 plusmn 04 871 plusmn 12 974 plusmn 26

Lung2 959 plusmn 35 856 plusmn 30 1032 plusmn 48 983 plusmn 43

20 1017 plusmn 80 923 plusmn 40 1094 plusmn 46 1102 plusmn 18

400 925 plusmn 18 932 plusmn 15 997 plusmn 04 1098 plusmn 17

Kidney2 1012 plusmn 34 916 plusmn 40 951 plusmn 22 984 plusmn 48

20 1025 plusmn 28 974 plusmn 18 955 plusmn 16 934 plusmn 22

400 920 plusmn 14 911 plusmn 08 899 plusmn 10 1095 plusmn 22

Brain2 858 plusmn 60 974 plusmn 73 966 plusmn 32 1094 plusmn 32

20 1033 plusmn 41 1035 plusmn 31 1030 plusmn 35 974 plusmn 12

400 1002 plusmn 29 990 plusmn 31 978 plusmn 21 1102 plusmn 30

Testicle2 1058 plusmn 39 853 plusmn 24 997 plusmn 55 933 plusmn 41

20 1040 plusmn 14 870 plusmn 13 1016 plusmn 19 1065 plusmn 15

400 1034 plusmn 39 984 plusmn 16 995 plusmn 33 1068 plusmn 38aAt least 1 h at room temperature bAt least 3 freeze-thaw cycles cAt least 24 h at 4∘C dAt least 4 weeks at minus80 plusmn 5∘C

Table 5 Pharmacokinetic parameters of icariin in rat after asingle oral dose of Herba Epimedii extract (corresponding 42mggicariin)

Parameter IcariinAUC0ndashinfin (ugLsdoth) 164155 plusmn 144129

AUMC0ndashinfin (ugLsdoth) 538424 plusmn 262081

MRT0ndashinfin (h) 3990 plusmn 1987

11990512 (h) 3149 plusmn 2364

119879max (h) 0458 plusmn 0246

CL119911119865 (Lhkg) 389043 plusmn 204472

after administration suggesting no accumulation in tissuesand a rapid elimination of this compound

35 Metabolism Study of Icariin in Rat Urine and Feces

351 Metabolite Profile of Rat Urine and Feces In thisstudy urine and feces samples were both analyzed by LC-MSMS By comparing the total ion chromatograms of drug-containing urine sample and predose control urine sampleneither parent drug nor metabolites were observed in raturine following two routes of administration of icariinHowever a total of 11 icariin metabolites were detected in ratfeces including 10 metabolites after oral administration and

9 following intramuscular introduction The representativechromatograms of the metabolites of 0ndash24 h rat feces wereshown in Figure 6 No additional metabolites were observedin the feces of 24ndash48 h collection period Results showedthat the major metabolites of icariin were M8 and M11whose total amount accounted for over 90 of total peakarea in the LC-UV chromatogram of 0ndash24 h feces And theconcentration of metabolites in feces rapidly declined over24ndash48 h collection period The MS2 and MS3 spectra oficariin and its metabolites were exhibited in Figure 7 andthe observed and calculatedmasses formula retention timesmass errors and MS data of these analytes were presented inTable 6 Proposed metabolic pathways for icariin in rat feceswere shown in Figure 8

352 Identification of Metabolites Known compounds wereidentified by comparison with the chromatographic behaviorand MS fragmentation pattern of reference standards Forunknown constituents their structures were characterizedbased on their retention behavior and their MS119899 spectraobtained on-line Moreover under the optimized LC-MSconditions all the in vivometabolites of icariin could give theprecursor ions of sufficient abundance in order to facilitatethe analysis of multistage mass spectrometry Because allmetabolites originated from icariin and their MS fragmen-tation patterns were largely similar to that of icariin it was

10 BioMed Research International

Heart Spleen Lung Kidney Brain TesticleLiver0

10

20

30

40

50C

once

ntra

tion

(ng

g)

025 Ｂ

05 Ｂ

1 Ｂ

2 Ｂ

4 Ｂ

6 Ｂ

(a)

0

10

20

30

40

50

Con

cent

ratio

n (n

gg)

Heart Spleen Lung Kidney Brain Uterus OvaryLiver

025 Ｂ

05 Ｂ

1 Ｂ

2 Ｂ

4 Ｂ

6 Ｂ

(b)

Figure 5 Tissue distribution of icariin in rats after a single oral dose of Herba Epimedii extract (corresponding 42mgg icariin (a) male rat(b) female rat)

663517

5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 900Time (min)

0

50

100

(a)

6155

512665

6637520419520

20

40

60

80

100

5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 900Time (min)

52 54 56 58500

50 M11

M10

M8

M7

M6M5

M4

M3M2

M1

M9

(b)

658

61465278

506

0

20

40

60

80

100

5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 900Time (min)

M11

M3 M8

(c)

Figure 6 Representative chromatograms of icariin metabolites in rat feces of 0ndash24 h (a) blank feces sample (b) feces sample after oraladministration and (c) feces sample after intramuscular administration

BioMed Research International 11

36724

6755435214

Icariin

513320

20

40

60

80

100

Rela

tive a

bund

ance

400 600 800200mz

35218

Icariin

367250

20

40

60

80

100

Rela

tive a

bund

ance

400 600200mz

35211

36722

29727

0

20

40

60

80

100

Rela

tive a

bund

ance

400300mz

2811329595

26725 3090935216

25506

0

20

40

60

80

100

Rela

tive a

bund

ance

300 400200mz

28408

35323

25512

0

20

40

60

80

100

Rela

tive a

bund

ance

400 600200mz

35217

36722

0

20

40

60

80

100

Rela

tive a

bund

ance

300 400 500200mz

36722

6892932124

400 600 800200mz

0

20

40

60

80

100

Rela

tive a

bund

ance

35318

49919

0

20

40

60

80

100

Rela

tive a

bund

ance

400 600200mz

35215

48116 64516

400 600 800200mz

0

20

40

60

80

100

Rela

tive a

bund

ance

400 600 800 1000mz

36723

83734

79088

0

20

40

60

80

100

Rela

tive a

bund

ance

M1M1

M2

M2

M3

M3

M4

M4

MS2

MS3

MS2

MS3

MS3

MS2

MS2

MS2

MS3

MS3

Figure 7 Continued

12 BioMed Research International

38218

36714 5293950114

0

20

40

60

80

100

Rela

tive a

bund

ance

400 500 600300mz

M5

MS2

36722

33916 382180

20

40

60

80

100

Rela

tive a

bund

ance

300 400 500200mz

M5

MS3

35217

36711

0

20

40

60

80

100

Rela

tive a

bund

ance

300 400 500200mz

M6

MS3

36724

35227 6452032309

0

20

40

60

80

100

Rela

tive a

bund

ance

400 600 800200mz

M6

MS2

36614

6592335118

0

20

40

60

80

100

Rela

tive a

bund

ance

400 600 800200mz

M7

MS2

35116

32317

31112

36607

0

20

40

60

80

100

Rela

tive a

bund

ance

300 400 500200mz

M7

MS3

35109

32308

31113

3660629520

0

20

40

60

80

100

Rela

tive a

bund

ance

300 400 500200mz

M8

MS3

36615

51313

400 600200mz

0

20

40

60

80

100

Rela

tive a

bund

ance M8

MS2

36514

35528 38328

33929

0

20

40

60

80

100

Rela

tive a

bund

ance

400300mz

M9

MS2

35019

36508

33724

0

20

40

60

80

100

Rela

tive a

bund

ance

400200mz

M9

MS3

Figure 7 Continued

BioMed Research International 13

25308

16310

27902

29817

0

20

40

60

80

100

Rela

tive a

bund

ance

400200mz

M10

MS3

29810

35312

21913

400200mz

0

20

40

60

80

100

Rela

tive a

bund

ance M10

MS2

35208

36723

309280

20

40

60

80

100

Rela

tive a

bund

ance

400 600200mz

M11

MS2

30911

2810435215

400200mz

0

20

40

60

80

100

Rela

tive a

bund

ance M11

MS3

Figure 7 Representative LC-MS119899 spectra of icariin and its metabolites M1ndashM11 in rat feces

necessary to characterize the MS119899 behavior of icariin toidentify and assign the metabolites of the constituent Thedeprotonated molecule [MndashH]minus of icariin appears at 119898119911675 which readily eliminates its glucosyl and rhamnosylgroups to produce an [MndashHndash308]minus ion at 119898119911 367 The[MndashHndash308]minus ion yields a fragment at 119898119911 352 by losing amethyl radical And of theMS4 spectrum of [MndashHndash308ndash15]minusion the fragment at 119898119911 323 can be reasonably attributed tothe loss of 3-COH of flavone skeletonrsquos C ring

M1 as aminormetabolite of icariin was only found in thefeces of the oral group Its quasimolecular ion occurs at 119898119911837 (C39H49O20) whose molecular mass is 162Da more thanthat of icariin And its major MS119899 fragment ions are the sameas those of icariinThemost abundantMS2 andMS3 fragmentions also appear at 119898119911 367 and 352 indicating the loss ofone rhamnosyl and two glucosyl moieties and the sequentialloss of methyl group respectively Although the molecularweight of M1 is equal to that of epimedin A there existsa significant difference on their chromatographic retentionbehavior Therefore M1 is tentatively identified as an isomerof epimedin A

Both M2 and M6 show the same quasimolecular ion[M minus H]minus at 119898119911 645 (C32H37O14) with the elution time at5044 and 5743min respectively M2 was only observed infeces after intramuscular introduction of icariin while M6was found in feces following both routes of administrationM6 is unambiguously identified as sagittatoside B incomparison with the reference standard Analogous to theMS119899 fragments of icariin (see Figure 7 and Table 6) M2also yielded the major fragment ions at 119898119911 352 323 309296 281 and 268 strongly suggesting that M2 should be

a conjunction metabolite of icariin Thus M2 is tentativelyassigned as sagittatoside B isomer

M3 was found in rat feces after both intramuscular andoral administration of icariin Its MS spectrum exhibits adeprotonated molecule [MndashH]minus at 119898119911 499 (C26H27O10)176Da less than that of icariin By comparison with thereference standard it is identified as baohuoside II TheMSMS spectrum shows the most abundant fragment ionat 119898119911 353 indicating the loss of rhamnose moiety TheMS3 spectrum displays the major fragment ions at 119898119911 325298 and 284 separately corresponding to the loss of COisobutenyl and isopentenyl radicals

M4 is a minor metabolite of icariin detected in the fecesof rats in the oral administration group Its deprotonatedmolecule (C33H37O16) is at 119898119911 689 And the major MSMSfragment at 119898119911 367 results from the loss of glucuronicacid and rhamnose moieties Its MS3 spectrum produces afragment at119898119911 352 (the loss of a methyl group) Thus M4 istentatively characterized as icariin glucuronide

M5ndashM11 of icariin metabolites were observed in rat fecesafter both intramuscular and oral administration of icariin

M5 has a deprotonated molecular ion at 119898119911 529(C27H29O11) in its MS spectrum The MS119899 spectra show themajor fragment ions at119898119911 501 (the loss of CO) 382 (the lossof rhamnosyl group) 367 (the loss of methyl radical from theion at119898119911 382) and 339 (the successive loss of methyl radicaland CO) Therefore M5 is tentatively identified as hydroxylicariside II

M7 is identified as 210158401015840-O-rhamnosyl icariside II based onthe reference standard and related mass spectral pattern The

14 BioMed Research International

Table6Ch

aracteriz

ationof

metaboliteso

ficariinin

ratfeces

Num

ber

Com

poun

dRT

Form

ula

Observedmass

RDB

Error

LCM

S119899data

[MndashH]minus

(ppm

)

PlowastIcariin

5519

C 33H39O15

67522888

145

0790

MS2[675]367(100)352(25)

MS3[675-367]352(100)

MS4[675-367-352]323(

30)309(100)296(45)281

(65)268

(50)255

(20)

M1

Epim

edin

Aiso

mer

a5038

C 39H49O20

8372

819

155

0824

MS2[837]790(50)367

(100)

MS3[837-367]352(100)297(20)

M2

Sagitta

tosid

eBiso

mer

b5044

C 32H37O14

6452186

145

1314

MS2[645]481(20)352(100)

MS3[645-352]323(40)309

(60)296

(90)281

(100)268(60)

M3

lowastBa

ohuo

sideIIa

b5278

C 26H27O10

4991

605

135

1295

MS2[499]353(100)

MS3[499-353]325(20)298

(40)284

(100)255(30)

M4

Icariin

glucuron

idea

5404

C 33H37O16

6892

084

155

1202

MS2[689]367(100)321(60)

MS3[689-367]352(100)

M5

Hydroxylicaris

ideIIa

b5412

C 27H29O11

5291

716

135

1132

MS2[529]501(20)382(100)367(40)

MS3[529-382]367(100)339(10)

M6

lowastSagitta

tosid

eBab

5743

C 32H37O14

6452184

145

1020

MS2[645]367(100)352(30)

MS3[645-367]352(100)

M7

lowast210158401015840-O

-Rhamno

syl-icaris

ideIIa

b5756

C 33H39O14

6592

344

145

1529

MS2[659]366(100)351(20)323(15)

MS3[659-366]351(100)323

(80)311(60)305

(20)

M8

lowastIcariside

IIab

6146

C 27H29O10

5131760

135

0851

MS2[513]366(100)

MS3[513-366]351(100)323

(80)311(50)305

(20)

M9

Hydroxylicaritin

ab6233

C 21H19O7

3831137

125

3160

MS2[383]365(100)355(60)339

(40)

MS3[383-365]350(100)321(20)230(40)

M10

Desmethylicaritin

ab6636

C 20H17O6

3531024

125

1119

MS2[353]298(100)219(10)

MS3[353-298]279(60)269

(40)253

(100)241(30)164(70)

M11

lowastIcaritinab

8265

C 21H19O6

3671183

125

1921

MS2[367]352(100)309(20)297

(20)

MS3[367-352]309(100)297(60)

lowastCom

paredwith

ther

eference

standardaObservedin

ratfeces

after

oraladministratio

nb O

bservedin

ratfeces

after

intram

usculara

dministratio

n

BioMed Research International 15

O

OO

O

O

O

OHOH

OH

OH

OH OHOH

OH

OHOH

OHOH

OH

OHOH OH

OH

OH

OH

OHOH

OH

OH

OH

OH

OH

OH

OH

OHOH

OH

OH

OH

OH

OHOH

OHOH

OHOH

OH

OH

OH

OH

OHOH

HO HO

HO

HO

HO

HOHO

HO

HO

HO

OH

O

OO

O

OOHOOC

O

OO

O

O

OO

OO

O

O

OO

OO

O

O

OO

O

O

OO

O

O

O

O

O

O

O

icariin

epimedin A isomer

minus rh

a

minus methyl

+ rhaOxidation

minus methyl

Icariin glucuronide

+ xyl

+ glcGlucuronidation

minus rha

Oxidation

minus rha

+ xyl

Sagittatoside B isomer

minus glc

M1

M4

M2

OCH3 OCH3

OCH3

OCH3

OCH3

OCH3

OCH3

OCH3

M7 2-O-rhamnosyl icariside II

M5 hydroxyl icariside II

M3 baohuoside II

M8 icariside II

M6 sagittatoside B

M9 hydroxyl icaritinM10 desmethyl icaritin

M11 icaritin

Figure 8 Proposed metabolic pathways for icariin in rat feces

MS spectrum of M7 shows a quasimolecular ion [MndashH]minusat 119898119911 659 (C33H39O14) The major fragment ions at 119898119911366 351 and 323 result from the concurrent loss of therhamnose chain the successive loss of a methyl group andCO respectively

As shownFigure 7M8 is themost abundantmetabolite oficariin By comparison with the reference standard it is iden-tified as icariside II Its MS spectrum shows a deprotonatedmolecular ion at 119898119911 513 (C27H29O10) And similar to the

MS119899 pattern of icariin the major fragment ions at 119898119911 366351 and 323 separately match the loss of rhamnose moietythe sequential loss of rhamnose and methyl groups and theloss of CO

M9 has a quasimolecular ion [MndashH]minus at 119898119911 383(C21H19O7) 146Da less than that of M5 (hydroxyl icarisideII) It is tentatively identified as hydroxyl icaritin Its MSMSspectrum shows themain fragments of119898119911 365 355 and 339respectively corresponding to the neutral loss of H2O CO

16 BioMed Research International

and CO2 MS3 spectrum the fragment ions at 119898119911 350 and321 individually result from the neutral loss of methyl radicaland CO2

The quasimolecular ion [MndashH]minus of M10 is at 119898119911 353146Da less than that of M3 (baohuoside II) The majorfragment ion in its MSMS spectrum is at119898119911 298 matchingthe loss of isobutenyl radical The fragment at 119898119911 219 ispossibly produced by the neutral loss of C8H6O2 (134Da) dueto the retro-Diels-Alder (RDA) fragmentation of the flavonersquosC ring Moreover the MS3 spectrum affords fragment ionsat 119898119911 279 269 253 and 164 resulting from the loss ofH2O CO CO2 and C8H6O2 (134Da) respectively Thus thismetabolite is reasonably identified as desmethyl icaritin

M11 was observed as anothermajormetabolite in rat fecesof both two administration groups (Figure 7) By comparisonwith the reference standard it is identified as icaritin Its MSspectrum yields a quasimolecular ion at119898119911 367 (C21H19O6)And themajor fragment ion at119898119911 352 is characterized as theloss of methyl group The MS3 fragment at 119898119911 309 resultsfrom the loss of methyl group and CO and ion at 119898119911 297is the outcome of the neutral loss of isopentene (70Da) fromthe deprotonated molecule

4 Conclusion

Themenopausal syndrome improvement and possible cancerpreventive activity of Herba Epimedii is receiving more andmore attention Information on the in vivo disposition ofHerba Epimedii components is important for better under-standing the biological effects of this herbal drug For thispurpose our investigation was designed to obtain informa-tion on the plasma pharmacokinetics and tissue distributionof icariin in rat and the excretion of icariin and itsmetabolitesIn this study we firstly established and validated a rapidsensitive and reliable LC-MS method for the determinationof icariin in various biological samples collected at differenttime points and then the method was applied to the plasmapharmacokinetics and tissue distribution study of icariin inrats after oral administration of the total flavonoid extractof Herba Epimedii In addition the differences of the tissuedistribution of icariin in between male and female rats werestudied after oral and intramuscular administration of icariinAnd the metabolic pattern of icariin was also investigatedThe data showed the plasma and tissue concentration oficariin were kept at a very low level at all collection pointswhich suggests icariin is poorly absorbed after oral adminis-trationThepeak concentration in plasma and collected tissuesamples appeared at 05ndash1 h and then quickly dropped at 4 hafter administration demonstrating that a small amount oficariin can be rapidly absorbed and eliminated and there isno accumulation in rat tissues Moreover the concentrationin liver lung and reproductive organs was much higherthan that in other tissues indicating that liver lung andreproductive organs are the main target sites of icariin in rats

The gender-related difference of icariin in tissue distribu-tion has not been reported in the past published literatureOur study demonstrated that the distribution features oficariin are similar in most of tissues between male andfemale rats However there exist a few remarkable gender

differences such that thewhole tissue concentration of icariinin male rats was much higher than female rats except in gen-ital organs (high in female and low in male) In China HerbaEpimedii is a famous tonic and aphrodisiac to invigoratekidney and strengthenYang therefore it is commonly used totreat male sexual functional decline and female menopausalsyndrome As one of the predominant constitutes in HerbaEpimedii icariin plays a very crucial role in exerting effectsso the relatively high icariin concentration in female genitalorgans actually supports the above-mentioned traditionalChinese medicine theory and clinical practice and makesthis compound a good future prospect for treating womenrsquosmenopausal related diseases

In the present study the complete metabolic profile oficariin has also been investigated The results illustrate thatfeces should be the major excretion way for both oral andintramuscular administration of icariin Based on LC-UVand LC-MS119899 analyses eleven metabolites were detected infecal samples and were further identified Among them10 metabolites appeared in rat feces of oral administrationgroup and 9 in the feces of intramuscular introduction groupThe major metabolic routes of clearance are proposed to behydrolysis demethylation oxidation and conjugation

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

Authorsrsquo Contributions

Shunjun Xu and Jiejing Yu contributed equally to this work

References

[1] X Wang Y He B Guo et al ldquoIn vivo screening for anti-osteo-porotic fraction from extract of herbal formula xianlinggubaoin ovariectomized micerdquo PLoS ONE vol 10 no 2 Article IDe0118184 2015

[2] R-H Liu X Kang L-P Xu et al ldquoEffects of the combinedextracts of Herba Epimedii and Fructus Ligustri Lucidi on bonemineral content and bone turnover in osteoporotic ratsrdquo BMCComplementary and Alternative Medicine vol 15 no 1 article112 2015

[3] B C L Chan H Y Lee W S Siu et al ldquoSuppression of mastcell activity contributes to the osteoprotective effect of an herbalformula containing Herba Epimedii Fructus Ligustri Lucidiand Fructus Psoraleaerdquo Journal of Pharmacy and Pharmacologyvol 66 no 3 pp 437ndash444 2014

[4] S CW Sze Y Tong T B Ng C L Y Cheng andH P CheungldquoHerba Epimedii anti-oxidative properties and its medicalimplicationsrdquoMolecules vol 15 no 11 pp 7861ndash7870 2010

[5] R Niu ldquoAction of the drug Herba Epimedii on testosterone ofthe mouse plasma and its accessory sexual organ before andafter processingrdquo China Journal of Chinese Materia Medica vol14 no 9 pp 530ndash574 1989

[6] F-X Liu S Sun and Z-Z Cui ldquoAnalysis of immunologi-cal enhancement of immunosuppressed chickens by Chineseherbal extractsrdquo Journal of Ethnopharmacology vol 127 no 2pp 251ndash256 2010

BioMed Research International 17

[7] J-H Kim Y-J Mun S-J Im J-H Han H-S Lee and W-HWoo ldquoEffects of the aqueous extract of Epimedii Herba on theantibody responses in micerdquo International Immunopharmacol-ogy vol 1 no 5 pp 935ndash944 2001

[8] X Yu Y Tong X-Q Han et al ldquoAnti-angiogenic activity ofHerba Epimedii on zebrafish embryos in vivo and HUVECs invitrordquo Phytotherapy Research vol 27 no 9 pp 1368ndash1375 2013

[9] D-C Zhang J-L Liu Y-B Ding J-G Xia and G-Y ChenldquoIcariin potentiates the antitumor activity of gemcitabine ingallbladder cancer by suppressingNF-120581BrdquoActa PharmacologicaSinica vol 34 no 2 pp 301ndash308 2013

[10] L Kong J Liu J Wang et al ldquoIcariin inhibits TNF-120572IFN-120574induced inflammatory response via inhibition of the substanceP and p38-MAPK signaling pathway in human keratinocytesrdquoInternational Immunopharmacology vol 29 no 2 pp 401ndash4072015

[11] H Shao J Shen M Wang et al ldquoIcariin protects against tita-nium particle-induced osteolysis and inflammatory response ina mouse calvarial modelrdquo Biomaterials vol 60 pp 92ndash99 2015

[12] Q Wei J Zhang G Hong et al ldquoIcariin promotes osteogenicdifferentiation of rat bone marrow stromal cells by activatingthe ER120572-Wnt120573-catenin signaling pathwayrdquo Biomedicine ampPharmacotherapy vol 84 pp 931ndash939 2016

[13] S-Q Zhang W-J Cai J-H Huang et al ldquoIcariin a naturalflavonol glycoside extends healthspan in micerdquo ExperimentalGerontology vol 69 pp 226ndash235 2015

[14] M N Makarova O N Pozharitskaya A N Shikov S VTesakova V G Makarov and V P Tikhonov ldquoEffect of lipid-based suspension of Epimedium koreanum Nakai extract onsexual behavior in ratsrdquo Journal of Ethnopharmacology vol 114no 3 pp 412ndash416 2007

[15] Y Chen J Wang X Jia X Tan and M Hu ldquoRole of intestinalhydrolase in the absorption of prenylated flavonoids present inyinyanghuordquoMolecules vol 16 no 2 pp 1336ndash1348 2011

[16] J J Yu Y J Liu S J Xu S Sun Y J Xu and L Yang ldquoSimulta-neous quantitative determination of four epimediumflavonoidsin rat urine by LC-MSMSrdquo Traditional Chinese Drug Researchamp Clinical Pharmacology vol 27 no 4 pp 537ndash541 2016

[17] Y Lu N Li Y Deng et al ldquoSimultaneous determination oficariin naringin and osthole in rat plasma by UPLC-MSMSand its application for pharmacokinetic study after oral admin-istration of Gushudan capsulesrdquo Journal of Chromatography Bvol 993-994 pp 75ndash80 2015

[18] N Wang X Huang X Wang Y Zhang R Wu and D ShouldquoPipette tip solid-phase extraction and high-performance liquidchromatography for the determination of flavonoids fromEpimedii herba in rat serum and application of the techniqueto pharmacokinetic studiesrdquo Journal of Chromatography B vol990 pp 64ndash72 2015

Submit your manuscripts athttpswwwhindawicom

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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

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Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

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Autoimmune Diseases

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ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

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Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

10 BioMed Research International

Heart Spleen Lung Kidney Brain TesticleLiver0

10

20

30

40

50C

once

ntra

tion

(ng

g)

025 Ｂ

05 Ｂ

1 Ｂ

2 Ｂ

4 Ｂ

6 Ｂ

(a)

0

10

20

30

40

50

Con

cent

ratio

n (n

gg)

Heart Spleen Lung Kidney Brain Uterus OvaryLiver

025 Ｂ

05 Ｂ

1 Ｂ

2 Ｂ

4 Ｂ

6 Ｂ

(b)

Figure 5 Tissue distribution of icariin in rats after a single oral dose of Herba Epimedii extract (corresponding 42mgg icariin (a) male rat(b) female rat)

663517

5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 900Time (min)

0

50

100

(a)

6155

512665

6637520419520

20

40

60

80

100

5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 900Time (min)

52 54 56 58500

50 M11

M10

M8

M7

M6M5

M4

M3M2

M1

M9

(b)

658

61465278

506

0

20

40

60

80

100

5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 900Time (min)

M11

M3 M8

(c)

Figure 6 Representative chromatograms of icariin metabolites in rat feces of 0ndash24 h (a) blank feces sample (b) feces sample after oraladministration and (c) feces sample after intramuscular administration

BioMed Research International 11

36724

6755435214

Icariin

513320

20

40

60

80

100

Rela

tive a

bund

ance

400 600 800200mz

35218

Icariin

367250

20

40

60

80

100

Rela

tive a

bund

ance

400 600200mz

35211

36722

29727

0

20

40

60

80

100

Rela

tive a

bund

ance

400300mz

2811329595

26725 3090935216

25506

0

20

40

60

80

100

Rela

tive a

bund

ance

300 400200mz

28408

35323

25512

0

20

40

60

80

100

Rela

tive a

bund

ance

400 600200mz

35217

36722

0

20

40

60

80

100

Rela

tive a

bund

ance

300 400 500200mz

36722

6892932124

400 600 800200mz

0

20

40

60

80

100

Rela

tive a

bund

ance

35318

49919

0

20

40

60

80

100

Rela

tive a

bund

ance

400 600200mz

35215

48116 64516

400 600 800200mz

0

20

40

60

80

100

Rela

tive a

bund

ance

400 600 800 1000mz

36723

83734

79088

0

20

40

60

80

100

Rela

tive a

bund

ance

M1M1

M2

M2

M3

M3

M4

M4

MS2

MS3

MS2

MS3

MS3

MS2

MS2

MS2

MS3

MS3

Figure 7 Continued

12 BioMed Research International

38218

36714 5293950114

0

20

40

60

80

100

Rela

tive a

bund

ance

400 500 600300mz

M5

MS2

36722

33916 382180

20

40

60

80

100

Rela

tive a

bund

ance

300 400 500200mz

M5

MS3

35217

36711

0

20

40

60

80

100

Rela

tive a

bund

ance

300 400 500200mz

M6

MS3

36724

35227 6452032309

0

20

40

60

80

100

Rela

tive a

bund

ance

400 600 800200mz

M6

MS2

36614

6592335118

0

20

40

60

80

100

Rela

tive a

bund

ance

400 600 800200mz

M7

MS2

35116

32317

31112

36607

0

20

40

60

80

100

Rela

tive a

bund

ance

300 400 500200mz

M7

MS3

35109

32308

31113

3660629520

0

20

40

60

80

100

Rela

tive a

bund

ance

300 400 500200mz

M8

MS3

36615

51313

400 600200mz

0

20

40

60

80

100

Rela

tive a

bund

ance M8

MS2

36514

35528 38328

33929

0

20

40

60

80

100

Rela

tive a

bund

ance

400300mz

M9

MS2

35019

36508

33724

0

20

40

60

80

100

Rela

tive a

bund

ance

400200mz

M9

MS3

Figure 7 Continued

BioMed Research International 13

25308

16310

27902

29817

0

20

40

60

80

100

Rela

tive a

bund

ance

400200mz

M10

MS3

29810

35312

21913

400200mz

0

20

40

60

80

100

Rela

tive a

bund

ance M10

MS2

35208

36723

309280

20

40

60

80

100

Rela

tive a

bund

ance

400 600200mz

M11

MS2

30911

2810435215

400200mz

0

20

40

60

80

100

Rela

tive a

bund

ance M11

MS3

Figure 7 Representative LC-MS119899 spectra of icariin and its metabolites M1ndashM11 in rat feces

necessary to characterize the MS119899 behavior of icariin toidentify and assign the metabolites of the constituent Thedeprotonated molecule [MndashH]minus of icariin appears at 119898119911675 which readily eliminates its glucosyl and rhamnosylgroups to produce an [MndashHndash308]minus ion at 119898119911 367 The[MndashHndash308]minus ion yields a fragment at 119898119911 352 by losing amethyl radical And of theMS4 spectrum of [MndashHndash308ndash15]minusion the fragment at 119898119911 323 can be reasonably attributed tothe loss of 3-COH of flavone skeletonrsquos C ring

M1 as aminormetabolite of icariin was only found in thefeces of the oral group Its quasimolecular ion occurs at 119898119911837 (C39H49O20) whose molecular mass is 162Da more thanthat of icariin And its major MS119899 fragment ions are the sameas those of icariinThemost abundantMS2 andMS3 fragmentions also appear at 119898119911 367 and 352 indicating the loss ofone rhamnosyl and two glucosyl moieties and the sequentialloss of methyl group respectively Although the molecularweight of M1 is equal to that of epimedin A there existsa significant difference on their chromatographic retentionbehavior Therefore M1 is tentatively identified as an isomerof epimedin A

Both M2 and M6 show the same quasimolecular ion[M minus H]minus at 119898119911 645 (C32H37O14) with the elution time at5044 and 5743min respectively M2 was only observed infeces after intramuscular introduction of icariin while M6was found in feces following both routes of administrationM6 is unambiguously identified as sagittatoside B incomparison with the reference standard Analogous to theMS119899 fragments of icariin (see Figure 7 and Table 6) M2also yielded the major fragment ions at 119898119911 352 323 309296 281 and 268 strongly suggesting that M2 should be

a conjunction metabolite of icariin Thus M2 is tentativelyassigned as sagittatoside B isomer

M3 was found in rat feces after both intramuscular andoral administration of icariin Its MS spectrum exhibits adeprotonated molecule [MndashH]minus at 119898119911 499 (C26H27O10)176Da less than that of icariin By comparison with thereference standard it is identified as baohuoside II TheMSMS spectrum shows the most abundant fragment ionat 119898119911 353 indicating the loss of rhamnose moiety TheMS3 spectrum displays the major fragment ions at 119898119911 325298 and 284 separately corresponding to the loss of COisobutenyl and isopentenyl radicals

M4 is a minor metabolite of icariin detected in the fecesof rats in the oral administration group Its deprotonatedmolecule (C33H37O16) is at 119898119911 689 And the major MSMSfragment at 119898119911 367 results from the loss of glucuronicacid and rhamnose moieties Its MS3 spectrum produces afragment at119898119911 352 (the loss of a methyl group) Thus M4 istentatively characterized as icariin glucuronide

M5ndashM11 of icariin metabolites were observed in rat fecesafter both intramuscular and oral administration of icariin

M5 has a deprotonated molecular ion at 119898119911 529(C27H29O11) in its MS spectrum The MS119899 spectra show themajor fragment ions at119898119911 501 (the loss of CO) 382 (the lossof rhamnosyl group) 367 (the loss of methyl radical from theion at119898119911 382) and 339 (the successive loss of methyl radicaland CO) Therefore M5 is tentatively identified as hydroxylicariside II

M7 is identified as 210158401015840-O-rhamnosyl icariside II based onthe reference standard and related mass spectral pattern The

14 BioMed Research International

Table6Ch

aracteriz

ationof

metaboliteso

ficariinin

ratfeces

Num

ber

Com

poun

dRT

Form

ula

Observedmass

RDB

Error

LCM

S119899data

[MndashH]minus

(ppm

)

PlowastIcariin

5519

C 33H39O15

67522888

145

0790

MS2[675]367(100)352(25)

MS3[675-367]352(100)

MS4[675-367-352]323(

30)309(100)296(45)281

(65)268

(50)255

(20)

M1

Epim

edin

Aiso

mer

a5038

C 39H49O20

8372

819

155

0824

MS2[837]790(50)367

(100)

MS3[837-367]352(100)297(20)

M2

Sagitta

tosid

eBiso

mer

b5044

C 32H37O14

6452186

145

1314

MS2[645]481(20)352(100)

MS3[645-352]323(40)309

(60)296

(90)281

(100)268(60)

M3

lowastBa

ohuo

sideIIa

b5278

C 26H27O10

4991

605

135

1295

MS2[499]353(100)

MS3[499-353]325(20)298

(40)284

(100)255(30)

M4

Icariin

glucuron

idea

5404

C 33H37O16

6892

084

155

1202

MS2[689]367(100)321(60)

MS3[689-367]352(100)

M5

Hydroxylicaris

ideIIa

b5412

C 27H29O11

5291

716

135

1132

MS2[529]501(20)382(100)367(40)

MS3[529-382]367(100)339(10)

M6

lowastSagitta

tosid

eBab

5743

C 32H37O14

6452184

145

1020

MS2[645]367(100)352(30)

MS3[645-367]352(100)

M7

lowast210158401015840-O

-Rhamno

syl-icaris

ideIIa

b5756

C 33H39O14

6592

344

145

1529

MS2[659]366(100)351(20)323(15)

MS3[659-366]351(100)323

(80)311(60)305

(20)

M8

lowastIcariside

IIab

6146

C 27H29O10

5131760

135

0851

MS2[513]366(100)

MS3[513-366]351(100)323

(80)311(50)305

(20)

M9

Hydroxylicaritin

ab6233

C 21H19O7

3831137

125

3160

MS2[383]365(100)355(60)339

(40)

MS3[383-365]350(100)321(20)230(40)

M10

Desmethylicaritin

ab6636

C 20H17O6

3531024

125

1119

MS2[353]298(100)219(10)

MS3[353-298]279(60)269

(40)253

(100)241(30)164(70)

M11

lowastIcaritinab

8265

C 21H19O6

3671183

125

1921

MS2[367]352(100)309(20)297

(20)

MS3[367-352]309(100)297(60)

lowastCom

paredwith

ther

eference

standardaObservedin

ratfeces

after

oraladministratio

nb O

bservedin

ratfeces

after

intram

usculara

dministratio

n

BioMed Research International 15

O

OO

O

O

O

OHOH

OH

OH

OH OHOH

OH

OHOH

OHOH

OH

OHOH OH

OH

OH

OH

OHOH

OH

OH

OH

OH

OH

OH

OH

OHOH

OH

OH

OH

OH

OHOH

OHOH

OHOH

OH

OH

OH

OH

OHOH

HO HO

HO

HO

HO

HOHO

HO

HO

HO

OH

O

OO

O

OOHOOC

O

OO

O

O

OO

OO

O

O

OO

OO

O

O

OO

O

O

OO

O

O

O

O

O

O

O

icariin

epimedin A isomer

minus rh

a

minus methyl

+ rhaOxidation

minus methyl

Icariin glucuronide

+ xyl

+ glcGlucuronidation

minus rha

Oxidation

minus rha

+ xyl

Sagittatoside B isomer

minus glc

M1

M4

M2

OCH3 OCH3

OCH3

OCH3

OCH3

OCH3

OCH3

OCH3

M7 2-O-rhamnosyl icariside II

M5 hydroxyl icariside II

M3 baohuoside II

M8 icariside II

M6 sagittatoside B

M9 hydroxyl icaritinM10 desmethyl icaritin

M11 icaritin

Figure 8 Proposed metabolic pathways for icariin in rat feces

MS spectrum of M7 shows a quasimolecular ion [MndashH]minusat 119898119911 659 (C33H39O14) The major fragment ions at 119898119911366 351 and 323 result from the concurrent loss of therhamnose chain the successive loss of a methyl group andCO respectively

As shownFigure 7M8 is themost abundantmetabolite oficariin By comparison with the reference standard it is iden-tified as icariside II Its MS spectrum shows a deprotonatedmolecular ion at 119898119911 513 (C27H29O10) And similar to the

MS119899 pattern of icariin the major fragment ions at 119898119911 366351 and 323 separately match the loss of rhamnose moietythe sequential loss of rhamnose and methyl groups and theloss of CO

M9 has a quasimolecular ion [MndashH]minus at 119898119911 383(C21H19O7) 146Da less than that of M5 (hydroxyl icarisideII) It is tentatively identified as hydroxyl icaritin Its MSMSspectrum shows themain fragments of119898119911 365 355 and 339respectively corresponding to the neutral loss of H2O CO

16 BioMed Research International

and CO2 MS3 spectrum the fragment ions at 119898119911 350 and321 individually result from the neutral loss of methyl radicaland CO2

The quasimolecular ion [MndashH]minus of M10 is at 119898119911 353146Da less than that of M3 (baohuoside II) The majorfragment ion in its MSMS spectrum is at119898119911 298 matchingthe loss of isobutenyl radical The fragment at 119898119911 219 ispossibly produced by the neutral loss of C8H6O2 (134Da) dueto the retro-Diels-Alder (RDA) fragmentation of the flavonersquosC ring Moreover the MS3 spectrum affords fragment ionsat 119898119911 279 269 253 and 164 resulting from the loss ofH2O CO CO2 and C8H6O2 (134Da) respectively Thus thismetabolite is reasonably identified as desmethyl icaritin

M11 was observed as anothermajormetabolite in rat fecesof both two administration groups (Figure 7) By comparisonwith the reference standard it is identified as icaritin Its MSspectrum yields a quasimolecular ion at119898119911 367 (C21H19O6)And themajor fragment ion at119898119911 352 is characterized as theloss of methyl group The MS3 fragment at 119898119911 309 resultsfrom the loss of methyl group and CO and ion at 119898119911 297is the outcome of the neutral loss of isopentene (70Da) fromthe deprotonated molecule

4 Conclusion

Themenopausal syndrome improvement and possible cancerpreventive activity of Herba Epimedii is receiving more andmore attention Information on the in vivo disposition ofHerba Epimedii components is important for better under-standing the biological effects of this herbal drug For thispurpose our investigation was designed to obtain informa-tion on the plasma pharmacokinetics and tissue distributionof icariin in rat and the excretion of icariin and itsmetabolitesIn this study we firstly established and validated a rapidsensitive and reliable LC-MS method for the determinationof icariin in various biological samples collected at differenttime points and then the method was applied to the plasmapharmacokinetics and tissue distribution study of icariin inrats after oral administration of the total flavonoid extractof Herba Epimedii In addition the differences of the tissuedistribution of icariin in between male and female rats werestudied after oral and intramuscular administration of icariinAnd the metabolic pattern of icariin was also investigatedThe data showed the plasma and tissue concentration oficariin were kept at a very low level at all collection pointswhich suggests icariin is poorly absorbed after oral adminis-trationThepeak concentration in plasma and collected tissuesamples appeared at 05ndash1 h and then quickly dropped at 4 hafter administration demonstrating that a small amount oficariin can be rapidly absorbed and eliminated and there isno accumulation in rat tissues Moreover the concentrationin liver lung and reproductive organs was much higherthan that in other tissues indicating that liver lung andreproductive organs are the main target sites of icariin in rats

The gender-related difference of icariin in tissue distribu-tion has not been reported in the past published literatureOur study demonstrated that the distribution features oficariin are similar in most of tissues between male andfemale rats However there exist a few remarkable gender

differences such that thewhole tissue concentration of icariinin male rats was much higher than female rats except in gen-ital organs (high in female and low in male) In China HerbaEpimedii is a famous tonic and aphrodisiac to invigoratekidney and strengthenYang therefore it is commonly used totreat male sexual functional decline and female menopausalsyndrome As one of the predominant constitutes in HerbaEpimedii icariin plays a very crucial role in exerting effectsso the relatively high icariin concentration in female genitalorgans actually supports the above-mentioned traditionalChinese medicine theory and clinical practice and makesthis compound a good future prospect for treating womenrsquosmenopausal related diseases

In the present study the complete metabolic profile oficariin has also been investigated The results illustrate thatfeces should be the major excretion way for both oral andintramuscular administration of icariin Based on LC-UVand LC-MS119899 analyses eleven metabolites were detected infecal samples and were further identified Among them10 metabolites appeared in rat feces of oral administrationgroup and 9 in the feces of intramuscular introduction groupThe major metabolic routes of clearance are proposed to behydrolysis demethylation oxidation and conjugation

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

Authorsrsquo Contributions

Shunjun Xu and Jiejing Yu contributed equally to this work

References

[1] X Wang Y He B Guo et al ldquoIn vivo screening for anti-osteo-porotic fraction from extract of herbal formula xianlinggubaoin ovariectomized micerdquo PLoS ONE vol 10 no 2 Article IDe0118184 2015

[2] R-H Liu X Kang L-P Xu et al ldquoEffects of the combinedextracts of Herba Epimedii and Fructus Ligustri Lucidi on bonemineral content and bone turnover in osteoporotic ratsrdquo BMCComplementary and Alternative Medicine vol 15 no 1 article112 2015

[3] B C L Chan H Y Lee W S Siu et al ldquoSuppression of mastcell activity contributes to the osteoprotective effect of an herbalformula containing Herba Epimedii Fructus Ligustri Lucidiand Fructus Psoraleaerdquo Journal of Pharmacy and Pharmacologyvol 66 no 3 pp 437ndash444 2014

[4] S CW Sze Y Tong T B Ng C L Y Cheng andH P CheungldquoHerba Epimedii anti-oxidative properties and its medicalimplicationsrdquoMolecules vol 15 no 11 pp 7861ndash7870 2010

[5] R Niu ldquoAction of the drug Herba Epimedii on testosterone ofthe mouse plasma and its accessory sexual organ before andafter processingrdquo China Journal of Chinese Materia Medica vol14 no 9 pp 530ndash574 1989

[6] F-X Liu S Sun and Z-Z Cui ldquoAnalysis of immunologi-cal enhancement of immunosuppressed chickens by Chineseherbal extractsrdquo Journal of Ethnopharmacology vol 127 no 2pp 251ndash256 2010

BioMed Research International 17

[7] J-H Kim Y-J Mun S-J Im J-H Han H-S Lee and W-HWoo ldquoEffects of the aqueous extract of Epimedii Herba on theantibody responses in micerdquo International Immunopharmacol-ogy vol 1 no 5 pp 935ndash944 2001

[8] X Yu Y Tong X-Q Han et al ldquoAnti-angiogenic activity ofHerba Epimedii on zebrafish embryos in vivo and HUVECs invitrordquo Phytotherapy Research vol 27 no 9 pp 1368ndash1375 2013

[9] D-C Zhang J-L Liu Y-B Ding J-G Xia and G-Y ChenldquoIcariin potentiates the antitumor activity of gemcitabine ingallbladder cancer by suppressingNF-120581BrdquoActa PharmacologicaSinica vol 34 no 2 pp 301ndash308 2013

[10] L Kong J Liu J Wang et al ldquoIcariin inhibits TNF-120572IFN-120574induced inflammatory response via inhibition of the substanceP and p38-MAPK signaling pathway in human keratinocytesrdquoInternational Immunopharmacology vol 29 no 2 pp 401ndash4072015

[11] H Shao J Shen M Wang et al ldquoIcariin protects against tita-nium particle-induced osteolysis and inflammatory response ina mouse calvarial modelrdquo Biomaterials vol 60 pp 92ndash99 2015

[12] Q Wei J Zhang G Hong et al ldquoIcariin promotes osteogenicdifferentiation of rat bone marrow stromal cells by activatingthe ER120572-Wnt120573-catenin signaling pathwayrdquo Biomedicine ampPharmacotherapy vol 84 pp 931ndash939 2016

[13] S-Q Zhang W-J Cai J-H Huang et al ldquoIcariin a naturalflavonol glycoside extends healthspan in micerdquo ExperimentalGerontology vol 69 pp 226ndash235 2015

[14] M N Makarova O N Pozharitskaya A N Shikov S VTesakova V G Makarov and V P Tikhonov ldquoEffect of lipid-based suspension of Epimedium koreanum Nakai extract onsexual behavior in ratsrdquo Journal of Ethnopharmacology vol 114no 3 pp 412ndash416 2007

[15] Y Chen J Wang X Jia X Tan and M Hu ldquoRole of intestinalhydrolase in the absorption of prenylated flavonoids present inyinyanghuordquoMolecules vol 16 no 2 pp 1336ndash1348 2011

[16] J J Yu Y J Liu S J Xu S Sun Y J Xu and L Yang ldquoSimulta-neous quantitative determination of four epimediumflavonoidsin rat urine by LC-MSMSrdquo Traditional Chinese Drug Researchamp Clinical Pharmacology vol 27 no 4 pp 537ndash541 2016

[17] Y Lu N Li Y Deng et al ldquoSimultaneous determination oficariin naringin and osthole in rat plasma by UPLC-MSMSand its application for pharmacokinetic study after oral admin-istration of Gushudan capsulesrdquo Journal of Chromatography Bvol 993-994 pp 75ndash80 2015

[18] N Wang X Huang X Wang Y Zhang R Wu and D ShouldquoPipette tip solid-phase extraction and high-performance liquidchromatography for the determination of flavonoids fromEpimedii herba in rat serum and application of the techniqueto pharmacokinetic studiesrdquo Journal of Chromatography B vol990 pp 64ndash72 2015

Submit your manuscripts athttpswwwhindawicom

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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

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ToxicologyJournal of

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Advances in Pharmacological Sciences

Tropical MedicineJournal of

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Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

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BioMed Research International

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Journal of

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Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

BioMed Research International 11

36724

6755435214

Icariin

513320

20

40

60

80

100

Rela

tive a

bund

ance

400 600 800200mz

35218

Icariin

367250

20

40

60

80

100

Rela

tive a

bund

ance

400 600200mz

35211

36722

29727

0

20

40

60

80

100

Rela

tive a

bund

ance

400300mz

2811329595

26725 3090935216

25506

0

20

40

60

80

100

Rela

tive a

bund

ance

300 400200mz

28408

35323

25512

0

20

40

60

80

100

Rela

tive a

bund

ance

400 600200mz

35217

36722

0

20

40

60

80

100

Rela

tive a

bund

ance

300 400 500200mz

36722

6892932124

400 600 800200mz

0

20

40

60

80

100

Rela

tive a

bund

ance

35318

49919

0

20

40

60

80

100

Rela

tive a

bund

ance

400 600200mz

35215

48116 64516

400 600 800200mz

0

20

40

60

80

100

Rela

tive a

bund

ance

400 600 800 1000mz

36723

83734

79088

0

20

40

60

80

100

Rela

tive a

bund

ance

M1M1

M2

M2

M3

M3

M4

M4

MS2

MS3

MS2

MS3

MS3

MS2

MS2

MS2

MS3

MS3

Figure 7 Continued

12 BioMed Research International

38218

36714 5293950114

0

20

40

60

80

100

Rela

tive a

bund

ance

400 500 600300mz

M5

MS2

36722

33916 382180

20

40

60

80

100

Rela

tive a

bund

ance

300 400 500200mz

M5

MS3

35217

36711

0

20

40

60

80

100

Rela

tive a

bund

ance

300 400 500200mz

M6

MS3

36724

35227 6452032309

0

20

40

60

80

100

Rela

tive a

bund

ance

400 600 800200mz

M6

MS2

36614

6592335118

0

20

40

60

80

100

Rela

tive a

bund

ance

400 600 800200mz

M7

MS2

35116

32317

31112

36607

0

20

40

60

80

100

Rela

tive a

bund

ance

300 400 500200mz

M7

MS3

35109

32308

31113

3660629520

0

20

40

60

80

100

Rela

tive a

bund

ance

300 400 500200mz

M8

MS3

36615

51313

400 600200mz

0

20

40

60

80

100

Rela

tive a

bund

ance M8

MS2

36514

35528 38328

33929

0

20

40

60

80

100

Rela

tive a

bund

ance

400300mz

M9

MS2

35019

36508

33724

0

20

40

60

80

100

Rela

tive a

bund

ance

400200mz

M9

MS3

Figure 7 Continued

BioMed Research International 13

25308

16310

27902

29817

0

20

40

60

80

100

Rela

tive a

bund

ance

400200mz

M10

MS3

29810

35312

21913

400200mz

0

20

40

60

80

100

Rela

tive a

bund

ance M10

MS2

35208

36723

309280

20

40

60

80

100

Rela

tive a

bund

ance

400 600200mz

M11

MS2

30911

2810435215

400200mz

0

20

40

60

80

100

Rela

tive a

bund

ance M11

MS3

Figure 7 Representative LC-MS119899 spectra of icariin and its metabolites M1ndashM11 in rat feces

necessary to characterize the MS119899 behavior of icariin toidentify and assign the metabolites of the constituent Thedeprotonated molecule [MndashH]minus of icariin appears at 119898119911675 which readily eliminates its glucosyl and rhamnosylgroups to produce an [MndashHndash308]minus ion at 119898119911 367 The[MndashHndash308]minus ion yields a fragment at 119898119911 352 by losing amethyl radical And of theMS4 spectrum of [MndashHndash308ndash15]minusion the fragment at 119898119911 323 can be reasonably attributed tothe loss of 3-COH of flavone skeletonrsquos C ring

M1 as aminormetabolite of icariin was only found in thefeces of the oral group Its quasimolecular ion occurs at 119898119911837 (C39H49O20) whose molecular mass is 162Da more thanthat of icariin And its major MS119899 fragment ions are the sameas those of icariinThemost abundantMS2 andMS3 fragmentions also appear at 119898119911 367 and 352 indicating the loss ofone rhamnosyl and two glucosyl moieties and the sequentialloss of methyl group respectively Although the molecularweight of M1 is equal to that of epimedin A there existsa significant difference on their chromatographic retentionbehavior Therefore M1 is tentatively identified as an isomerof epimedin A

Both M2 and M6 show the same quasimolecular ion[M minus H]minus at 119898119911 645 (C32H37O14) with the elution time at5044 and 5743min respectively M2 was only observed infeces after intramuscular introduction of icariin while M6was found in feces following both routes of administrationM6 is unambiguously identified as sagittatoside B incomparison with the reference standard Analogous to theMS119899 fragments of icariin (see Figure 7 and Table 6) M2also yielded the major fragment ions at 119898119911 352 323 309296 281 and 268 strongly suggesting that M2 should be

a conjunction metabolite of icariin Thus M2 is tentativelyassigned as sagittatoside B isomer

M3 was found in rat feces after both intramuscular andoral administration of icariin Its MS spectrum exhibits adeprotonated molecule [MndashH]minus at 119898119911 499 (C26H27O10)176Da less than that of icariin By comparison with thereference standard it is identified as baohuoside II TheMSMS spectrum shows the most abundant fragment ionat 119898119911 353 indicating the loss of rhamnose moiety TheMS3 spectrum displays the major fragment ions at 119898119911 325298 and 284 separately corresponding to the loss of COisobutenyl and isopentenyl radicals

M4 is a minor metabolite of icariin detected in the fecesof rats in the oral administration group Its deprotonatedmolecule (C33H37O16) is at 119898119911 689 And the major MSMSfragment at 119898119911 367 results from the loss of glucuronicacid and rhamnose moieties Its MS3 spectrum produces afragment at119898119911 352 (the loss of a methyl group) Thus M4 istentatively characterized as icariin glucuronide

M5ndashM11 of icariin metabolites were observed in rat fecesafter both intramuscular and oral administration of icariin

M5 has a deprotonated molecular ion at 119898119911 529(C27H29O11) in its MS spectrum The MS119899 spectra show themajor fragment ions at119898119911 501 (the loss of CO) 382 (the lossof rhamnosyl group) 367 (the loss of methyl radical from theion at119898119911 382) and 339 (the successive loss of methyl radicaland CO) Therefore M5 is tentatively identified as hydroxylicariside II

M7 is identified as 210158401015840-O-rhamnosyl icariside II based onthe reference standard and related mass spectral pattern The

14 BioMed Research International

Table6Ch

aracteriz

ationof

metaboliteso

ficariinin

ratfeces

Num

ber

Com

poun

dRT

Form

ula

Observedmass

RDB

Error

LCM

S119899data

[MndashH]minus

(ppm

)

PlowastIcariin

5519

C 33H39O15

67522888

145

0790

MS2[675]367(100)352(25)

MS3[675-367]352(100)

MS4[675-367-352]323(

30)309(100)296(45)281

(65)268

(50)255

(20)

M1

Epim

edin

Aiso

mer

a5038

C 39H49O20

8372

819

155

0824

MS2[837]790(50)367

(100)

MS3[837-367]352(100)297(20)

M2

Sagitta

tosid

eBiso

mer

b5044

C 32H37O14

6452186

145

1314

MS2[645]481(20)352(100)

MS3[645-352]323(40)309

(60)296

(90)281

(100)268(60)

M3

lowastBa

ohuo

sideIIa

b5278

C 26H27O10

4991

605

135

1295

MS2[499]353(100)

MS3[499-353]325(20)298

(40)284

(100)255(30)

M4

Icariin

glucuron

idea

5404

C 33H37O16

6892

084

155

1202

MS2[689]367(100)321(60)

MS3[689-367]352(100)

M5

Hydroxylicaris

ideIIa

b5412

C 27H29O11

5291

716

135

1132

MS2[529]501(20)382(100)367(40)

MS3[529-382]367(100)339(10)

M6

lowastSagitta

tosid

eBab

5743

C 32H37O14

6452184

145

1020

MS2[645]367(100)352(30)

MS3[645-367]352(100)

M7

lowast210158401015840-O

-Rhamno

syl-icaris

ideIIa

b5756

C 33H39O14

6592

344

145

1529

MS2[659]366(100)351(20)323(15)

MS3[659-366]351(100)323

(80)311(60)305

(20)

M8

lowastIcariside

IIab

6146

C 27H29O10

5131760

135

0851

MS2[513]366(100)

MS3[513-366]351(100)323

(80)311(50)305

(20)

M9

Hydroxylicaritin

ab6233

C 21H19O7

3831137

125

3160

MS2[383]365(100)355(60)339

(40)

MS3[383-365]350(100)321(20)230(40)

M10

Desmethylicaritin

ab6636

C 20H17O6

3531024

125

1119

MS2[353]298(100)219(10)

MS3[353-298]279(60)269

(40)253

(100)241(30)164(70)

M11

lowastIcaritinab

8265

C 21H19O6

3671183

125

1921

MS2[367]352(100)309(20)297

(20)

MS3[367-352]309(100)297(60)

lowastCom

paredwith

ther

eference

standardaObservedin

ratfeces

after

oraladministratio

nb O

bservedin

ratfeces

after

intram

usculara

dministratio

n

BioMed Research International 15

O

OO

O

O

O

OHOH

OH

OH

OH OHOH

OH

OHOH

OHOH

OH

OHOH OH

OH

OH

OH

OHOH

OH

OH

OH

OH

OH

OH

OH

OHOH

OH

OH

OH

OH

OHOH

OHOH

OHOH

OH

OH

OH

OH

OHOH

HO HO

HO

HO

HO

HOHO

HO

HO

HO

OH

O

OO

O

OOHOOC

O

OO

O

O

OO

OO

O

O

OO

OO

O

O

OO

O

O

OO

O

O

O

O

O

O

O

icariin

epimedin A isomer

minus rh

a

minus methyl

+ rhaOxidation

minus methyl

Icariin glucuronide

+ xyl

+ glcGlucuronidation

minus rha

Oxidation

minus rha

+ xyl

Sagittatoside B isomer

minus glc

M1

M4

M2

OCH3 OCH3

OCH3

OCH3

OCH3

OCH3

OCH3

OCH3

M7 2-O-rhamnosyl icariside II

M5 hydroxyl icariside II

M3 baohuoside II

M8 icariside II

M6 sagittatoside B

M9 hydroxyl icaritinM10 desmethyl icaritin

M11 icaritin

Figure 8 Proposed metabolic pathways for icariin in rat feces

MS spectrum of M7 shows a quasimolecular ion [MndashH]minusat 119898119911 659 (C33H39O14) The major fragment ions at 119898119911366 351 and 323 result from the concurrent loss of therhamnose chain the successive loss of a methyl group andCO respectively

As shownFigure 7M8 is themost abundantmetabolite oficariin By comparison with the reference standard it is iden-tified as icariside II Its MS spectrum shows a deprotonatedmolecular ion at 119898119911 513 (C27H29O10) And similar to the

MS119899 pattern of icariin the major fragment ions at 119898119911 366351 and 323 separately match the loss of rhamnose moietythe sequential loss of rhamnose and methyl groups and theloss of CO

M9 has a quasimolecular ion [MndashH]minus at 119898119911 383(C21H19O7) 146Da less than that of M5 (hydroxyl icarisideII) It is tentatively identified as hydroxyl icaritin Its MSMSspectrum shows themain fragments of119898119911 365 355 and 339respectively corresponding to the neutral loss of H2O CO

16 BioMed Research International

and CO2 MS3 spectrum the fragment ions at 119898119911 350 and321 individually result from the neutral loss of methyl radicaland CO2

The quasimolecular ion [MndashH]minus of M10 is at 119898119911 353146Da less than that of M3 (baohuoside II) The majorfragment ion in its MSMS spectrum is at119898119911 298 matchingthe loss of isobutenyl radical The fragment at 119898119911 219 ispossibly produced by the neutral loss of C8H6O2 (134Da) dueto the retro-Diels-Alder (RDA) fragmentation of the flavonersquosC ring Moreover the MS3 spectrum affords fragment ionsat 119898119911 279 269 253 and 164 resulting from the loss ofH2O CO CO2 and C8H6O2 (134Da) respectively Thus thismetabolite is reasonably identified as desmethyl icaritin

M11 was observed as anothermajormetabolite in rat fecesof both two administration groups (Figure 7) By comparisonwith the reference standard it is identified as icaritin Its MSspectrum yields a quasimolecular ion at119898119911 367 (C21H19O6)And themajor fragment ion at119898119911 352 is characterized as theloss of methyl group The MS3 fragment at 119898119911 309 resultsfrom the loss of methyl group and CO and ion at 119898119911 297is the outcome of the neutral loss of isopentene (70Da) fromthe deprotonated molecule

4 Conclusion

Themenopausal syndrome improvement and possible cancerpreventive activity of Herba Epimedii is receiving more andmore attention Information on the in vivo disposition ofHerba Epimedii components is important for better under-standing the biological effects of this herbal drug For thispurpose our investigation was designed to obtain informa-tion on the plasma pharmacokinetics and tissue distributionof icariin in rat and the excretion of icariin and itsmetabolitesIn this study we firstly established and validated a rapidsensitive and reliable LC-MS method for the determinationof icariin in various biological samples collected at differenttime points and then the method was applied to the plasmapharmacokinetics and tissue distribution study of icariin inrats after oral administration of the total flavonoid extractof Herba Epimedii In addition the differences of the tissuedistribution of icariin in between male and female rats werestudied after oral and intramuscular administration of icariinAnd the metabolic pattern of icariin was also investigatedThe data showed the plasma and tissue concentration oficariin were kept at a very low level at all collection pointswhich suggests icariin is poorly absorbed after oral adminis-trationThepeak concentration in plasma and collected tissuesamples appeared at 05ndash1 h and then quickly dropped at 4 hafter administration demonstrating that a small amount oficariin can be rapidly absorbed and eliminated and there isno accumulation in rat tissues Moreover the concentrationin liver lung and reproductive organs was much higherthan that in other tissues indicating that liver lung andreproductive organs are the main target sites of icariin in rats

The gender-related difference of icariin in tissue distribu-tion has not been reported in the past published literatureOur study demonstrated that the distribution features oficariin are similar in most of tissues between male andfemale rats However there exist a few remarkable gender

differences such that thewhole tissue concentration of icariinin male rats was much higher than female rats except in gen-ital organs (high in female and low in male) In China HerbaEpimedii is a famous tonic and aphrodisiac to invigoratekidney and strengthenYang therefore it is commonly used totreat male sexual functional decline and female menopausalsyndrome As one of the predominant constitutes in HerbaEpimedii icariin plays a very crucial role in exerting effectsso the relatively high icariin concentration in female genitalorgans actually supports the above-mentioned traditionalChinese medicine theory and clinical practice and makesthis compound a good future prospect for treating womenrsquosmenopausal related diseases

In the present study the complete metabolic profile oficariin has also been investigated The results illustrate thatfeces should be the major excretion way for both oral andintramuscular administration of icariin Based on LC-UVand LC-MS119899 analyses eleven metabolites were detected infecal samples and were further identified Among them10 metabolites appeared in rat feces of oral administrationgroup and 9 in the feces of intramuscular introduction groupThe major metabolic routes of clearance are proposed to behydrolysis demethylation oxidation and conjugation

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

Authorsrsquo Contributions

Shunjun Xu and Jiejing Yu contributed equally to this work

References

[1] X Wang Y He B Guo et al ldquoIn vivo screening for anti-osteo-porotic fraction from extract of herbal formula xianlinggubaoin ovariectomized micerdquo PLoS ONE vol 10 no 2 Article IDe0118184 2015

[2] R-H Liu X Kang L-P Xu et al ldquoEffects of the combinedextracts of Herba Epimedii and Fructus Ligustri Lucidi on bonemineral content and bone turnover in osteoporotic ratsrdquo BMCComplementary and Alternative Medicine vol 15 no 1 article112 2015

[3] B C L Chan H Y Lee W S Siu et al ldquoSuppression of mastcell activity contributes to the osteoprotective effect of an herbalformula containing Herba Epimedii Fructus Ligustri Lucidiand Fructus Psoraleaerdquo Journal of Pharmacy and Pharmacologyvol 66 no 3 pp 437ndash444 2014

[4] S CW Sze Y Tong T B Ng C L Y Cheng andH P CheungldquoHerba Epimedii anti-oxidative properties and its medicalimplicationsrdquoMolecules vol 15 no 11 pp 7861ndash7870 2010

[5] R Niu ldquoAction of the drug Herba Epimedii on testosterone ofthe mouse plasma and its accessory sexual organ before andafter processingrdquo China Journal of Chinese Materia Medica vol14 no 9 pp 530ndash574 1989

[6] F-X Liu S Sun and Z-Z Cui ldquoAnalysis of immunologi-cal enhancement of immunosuppressed chickens by Chineseherbal extractsrdquo Journal of Ethnopharmacology vol 127 no 2pp 251ndash256 2010

BioMed Research International 17

[7] J-H Kim Y-J Mun S-J Im J-H Han H-S Lee and W-HWoo ldquoEffects of the aqueous extract of Epimedii Herba on theantibody responses in micerdquo International Immunopharmacol-ogy vol 1 no 5 pp 935ndash944 2001

[8] X Yu Y Tong X-Q Han et al ldquoAnti-angiogenic activity ofHerba Epimedii on zebrafish embryos in vivo and HUVECs invitrordquo Phytotherapy Research vol 27 no 9 pp 1368ndash1375 2013

[9] D-C Zhang J-L Liu Y-B Ding J-G Xia and G-Y ChenldquoIcariin potentiates the antitumor activity of gemcitabine ingallbladder cancer by suppressingNF-120581BrdquoActa PharmacologicaSinica vol 34 no 2 pp 301ndash308 2013

[10] L Kong J Liu J Wang et al ldquoIcariin inhibits TNF-120572IFN-120574induced inflammatory response via inhibition of the substanceP and p38-MAPK signaling pathway in human keratinocytesrdquoInternational Immunopharmacology vol 29 no 2 pp 401ndash4072015

[11] H Shao J Shen M Wang et al ldquoIcariin protects against tita-nium particle-induced osteolysis and inflammatory response ina mouse calvarial modelrdquo Biomaterials vol 60 pp 92ndash99 2015

[12] Q Wei J Zhang G Hong et al ldquoIcariin promotes osteogenicdifferentiation of rat bone marrow stromal cells by activatingthe ER120572-Wnt120573-catenin signaling pathwayrdquo Biomedicine ampPharmacotherapy vol 84 pp 931ndash939 2016

[13] S-Q Zhang W-J Cai J-H Huang et al ldquoIcariin a naturalflavonol glycoside extends healthspan in micerdquo ExperimentalGerontology vol 69 pp 226ndash235 2015

[14] M N Makarova O N Pozharitskaya A N Shikov S VTesakova V G Makarov and V P Tikhonov ldquoEffect of lipid-based suspension of Epimedium koreanum Nakai extract onsexual behavior in ratsrdquo Journal of Ethnopharmacology vol 114no 3 pp 412ndash416 2007

[15] Y Chen J Wang X Jia X Tan and M Hu ldquoRole of intestinalhydrolase in the absorption of prenylated flavonoids present inyinyanghuordquoMolecules vol 16 no 2 pp 1336ndash1348 2011

[16] J J Yu Y J Liu S J Xu S Sun Y J Xu and L Yang ldquoSimulta-neous quantitative determination of four epimediumflavonoidsin rat urine by LC-MSMSrdquo Traditional Chinese Drug Researchamp Clinical Pharmacology vol 27 no 4 pp 537ndash541 2016

[17] Y Lu N Li Y Deng et al ldquoSimultaneous determination oficariin naringin and osthole in rat plasma by UPLC-MSMSand its application for pharmacokinetic study after oral admin-istration of Gushudan capsulesrdquo Journal of Chromatography Bvol 993-994 pp 75ndash80 2015

[18] N Wang X Huang X Wang Y Zhang R Wu and D ShouldquoPipette tip solid-phase extraction and high-performance liquidchromatography for the determination of flavonoids fromEpimedii herba in rat serum and application of the techniqueto pharmacokinetic studiesrdquo Journal of Chromatography B vol990 pp 64ndash72 2015

Submit your manuscripts athttpswwwhindawicom

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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

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ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

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Autoimmune Diseases

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Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

12 BioMed Research International

38218

36714 5293950114

0

20

40

60

80

100

Rela

tive a

bund

ance

400 500 600300mz

M5

MS2

36722

33916 382180

20

40

60

80

100

Rela

tive a

bund

ance

300 400 500200mz

M5

MS3

35217

36711

0

20

40

60

80

100

Rela

tive a

bund

ance

300 400 500200mz

M6

MS3

36724

35227 6452032309

0

20

40

60

80

100

Rela

tive a

bund

ance

400 600 800200mz

M6

MS2

36614

6592335118

0

20

40

60

80

100

Rela

tive a

bund

ance

400 600 800200mz

M7

MS2

35116

32317

31112

36607

0

20

40

60

80

100

Rela

tive a

bund

ance

300 400 500200mz

M7

MS3

35109

32308

31113

3660629520

0

20

40

60

80

100

Rela

tive a

bund

ance

300 400 500200mz

M8

MS3

36615

51313

400 600200mz

0

20

40

60

80

100

Rela

tive a

bund

ance M8

MS2

36514

35528 38328

33929

0

20

40

60

80

100

Rela

tive a

bund

ance

400300mz

M9

MS2

35019

36508

33724

0

20

40

60

80

100

Rela

tive a

bund

ance

400200mz

M9

MS3

Figure 7 Continued

BioMed Research International 13

25308

16310

27902

29817

0

20

40

60

80

100

Rela

tive a

bund

ance

400200mz

M10

MS3

29810

35312

21913

400200mz

0

20

40

60

80

100

Rela

tive a

bund

ance M10

MS2

35208

36723

309280

20

40

60

80

100

Rela

tive a

bund

ance

400 600200mz

M11

MS2

30911

2810435215

400200mz

0

20

40

60

80

100

Rela

tive a

bund

ance M11

MS3

Figure 7 Representative LC-MS119899 spectra of icariin and its metabolites M1ndashM11 in rat feces

necessary to characterize the MS119899 behavior of icariin toidentify and assign the metabolites of the constituent Thedeprotonated molecule [MndashH]minus of icariin appears at 119898119911675 which readily eliminates its glucosyl and rhamnosylgroups to produce an [MndashHndash308]minus ion at 119898119911 367 The[MndashHndash308]minus ion yields a fragment at 119898119911 352 by losing amethyl radical And of theMS4 spectrum of [MndashHndash308ndash15]minusion the fragment at 119898119911 323 can be reasonably attributed tothe loss of 3-COH of flavone skeletonrsquos C ring

M1 as aminormetabolite of icariin was only found in thefeces of the oral group Its quasimolecular ion occurs at 119898119911837 (C39H49O20) whose molecular mass is 162Da more thanthat of icariin And its major MS119899 fragment ions are the sameas those of icariinThemost abundantMS2 andMS3 fragmentions also appear at 119898119911 367 and 352 indicating the loss ofone rhamnosyl and two glucosyl moieties and the sequentialloss of methyl group respectively Although the molecularweight of M1 is equal to that of epimedin A there existsa significant difference on their chromatographic retentionbehavior Therefore M1 is tentatively identified as an isomerof epimedin A

Both M2 and M6 show the same quasimolecular ion[M minus H]minus at 119898119911 645 (C32H37O14) with the elution time at5044 and 5743min respectively M2 was only observed infeces after intramuscular introduction of icariin while M6was found in feces following both routes of administrationM6 is unambiguously identified as sagittatoside B incomparison with the reference standard Analogous to theMS119899 fragments of icariin (see Figure 7 and Table 6) M2also yielded the major fragment ions at 119898119911 352 323 309296 281 and 268 strongly suggesting that M2 should be

a conjunction metabolite of icariin Thus M2 is tentativelyassigned as sagittatoside B isomer

M3 was found in rat feces after both intramuscular andoral administration of icariin Its MS spectrum exhibits adeprotonated molecule [MndashH]minus at 119898119911 499 (C26H27O10)176Da less than that of icariin By comparison with thereference standard it is identified as baohuoside II TheMSMS spectrum shows the most abundant fragment ionat 119898119911 353 indicating the loss of rhamnose moiety TheMS3 spectrum displays the major fragment ions at 119898119911 325298 and 284 separately corresponding to the loss of COisobutenyl and isopentenyl radicals

M4 is a minor metabolite of icariin detected in the fecesof rats in the oral administration group Its deprotonatedmolecule (C33H37O16) is at 119898119911 689 And the major MSMSfragment at 119898119911 367 results from the loss of glucuronicacid and rhamnose moieties Its MS3 spectrum produces afragment at119898119911 352 (the loss of a methyl group) Thus M4 istentatively characterized as icariin glucuronide

M5ndashM11 of icariin metabolites were observed in rat fecesafter both intramuscular and oral administration of icariin

M5 has a deprotonated molecular ion at 119898119911 529(C27H29O11) in its MS spectrum The MS119899 spectra show themajor fragment ions at119898119911 501 (the loss of CO) 382 (the lossof rhamnosyl group) 367 (the loss of methyl radical from theion at119898119911 382) and 339 (the successive loss of methyl radicaland CO) Therefore M5 is tentatively identified as hydroxylicariside II

M7 is identified as 210158401015840-O-rhamnosyl icariside II based onthe reference standard and related mass spectral pattern The

14 BioMed Research International

Table6Ch

aracteriz

ationof

metaboliteso

ficariinin

ratfeces

Num

ber

Com

poun

dRT

Form

ula

Observedmass

RDB

Error

LCM

S119899data

[MndashH]minus

(ppm

)

PlowastIcariin

5519

C 33H39O15

67522888

145

0790

MS2[675]367(100)352(25)

MS3[675-367]352(100)

MS4[675-367-352]323(

30)309(100)296(45)281

(65)268

(50)255

(20)

M1

Epim

edin

Aiso

mer

a5038

C 39H49O20

8372

819

155

0824

MS2[837]790(50)367

(100)

MS3[837-367]352(100)297(20)

M2

Sagitta

tosid

eBiso

mer

b5044

C 32H37O14

6452186

145

1314

MS2[645]481(20)352(100)

MS3[645-352]323(40)309

(60)296

(90)281

(100)268(60)

M3

lowastBa

ohuo

sideIIa

b5278

C 26H27O10

4991

605

135

1295

MS2[499]353(100)

MS3[499-353]325(20)298

(40)284

(100)255(30)

M4

Icariin

glucuron

idea

5404

C 33H37O16

6892

084

155

1202

MS2[689]367(100)321(60)

MS3[689-367]352(100)

M5

Hydroxylicaris

ideIIa

b5412

C 27H29O11

5291

716

135

1132

MS2[529]501(20)382(100)367(40)

MS3[529-382]367(100)339(10)

M6

lowastSagitta

tosid

eBab

5743

C 32H37O14

6452184

145

1020

MS2[645]367(100)352(30)

MS3[645-367]352(100)

M7

lowast210158401015840-O

-Rhamno

syl-icaris

ideIIa

b5756

C 33H39O14

6592

344

145

1529

MS2[659]366(100)351(20)323(15)

MS3[659-366]351(100)323

(80)311(60)305

(20)

M8

lowastIcariside

IIab

6146

C 27H29O10

5131760

135

0851

MS2[513]366(100)

MS3[513-366]351(100)323

(80)311(50)305

(20)

M9

Hydroxylicaritin

ab6233

C 21H19O7

3831137

125

3160

MS2[383]365(100)355(60)339

(40)

MS3[383-365]350(100)321(20)230(40)

M10

Desmethylicaritin

ab6636

C 20H17O6

3531024

125

1119

MS2[353]298(100)219(10)

MS3[353-298]279(60)269

(40)253

(100)241(30)164(70)

M11

lowastIcaritinab

8265

C 21H19O6

3671183

125

1921

MS2[367]352(100)309(20)297

(20)

MS3[367-352]309(100)297(60)

lowastCom

paredwith

ther

eference

standardaObservedin

ratfeces

after

oraladministratio

nb O

bservedin

ratfeces

after

intram

usculara

dministratio

n

BioMed Research International 15

O

OO

O

O

O

OHOH

OH

OH

OH OHOH

OH

OHOH

OHOH

OH

OHOH OH

OH

OH

OH

OHOH

OH

OH

OH

OH

OH

OH

OH

OHOH

OH

OH

OH

OH

OHOH

OHOH

OHOH

OH

OH

OH

OH

OHOH

HO HO

HO

HO

HO

HOHO

HO

HO

HO

OH

O

OO

O

OOHOOC

O

OO

O

O

OO

OO

O

O

OO

OO

O

O

OO

O

O

OO

O

O

O

O

O

O

O

icariin

epimedin A isomer

minus rh

a

minus methyl

+ rhaOxidation

minus methyl

Icariin glucuronide

+ xyl

+ glcGlucuronidation

minus rha

Oxidation

minus rha

+ xyl

Sagittatoside B isomer

minus glc

M1

M4

M2

OCH3 OCH3

OCH3

OCH3

OCH3

OCH3

OCH3

OCH3

M7 2-O-rhamnosyl icariside II

M5 hydroxyl icariside II

M3 baohuoside II

M8 icariside II

M6 sagittatoside B

M9 hydroxyl icaritinM10 desmethyl icaritin

M11 icaritin

Figure 8 Proposed metabolic pathways for icariin in rat feces

MS spectrum of M7 shows a quasimolecular ion [MndashH]minusat 119898119911 659 (C33H39O14) The major fragment ions at 119898119911366 351 and 323 result from the concurrent loss of therhamnose chain the successive loss of a methyl group andCO respectively

As shownFigure 7M8 is themost abundantmetabolite oficariin By comparison with the reference standard it is iden-tified as icariside II Its MS spectrum shows a deprotonatedmolecular ion at 119898119911 513 (C27H29O10) And similar to the

MS119899 pattern of icariin the major fragment ions at 119898119911 366351 and 323 separately match the loss of rhamnose moietythe sequential loss of rhamnose and methyl groups and theloss of CO

M9 has a quasimolecular ion [MndashH]minus at 119898119911 383(C21H19O7) 146Da less than that of M5 (hydroxyl icarisideII) It is tentatively identified as hydroxyl icaritin Its MSMSspectrum shows themain fragments of119898119911 365 355 and 339respectively corresponding to the neutral loss of H2O CO

16 BioMed Research International

and CO2 MS3 spectrum the fragment ions at 119898119911 350 and321 individually result from the neutral loss of methyl radicaland CO2

The quasimolecular ion [MndashH]minus of M10 is at 119898119911 353146Da less than that of M3 (baohuoside II) The majorfragment ion in its MSMS spectrum is at119898119911 298 matchingthe loss of isobutenyl radical The fragment at 119898119911 219 ispossibly produced by the neutral loss of C8H6O2 (134Da) dueto the retro-Diels-Alder (RDA) fragmentation of the flavonersquosC ring Moreover the MS3 spectrum affords fragment ionsat 119898119911 279 269 253 and 164 resulting from the loss ofH2O CO CO2 and C8H6O2 (134Da) respectively Thus thismetabolite is reasonably identified as desmethyl icaritin

M11 was observed as anothermajormetabolite in rat fecesof both two administration groups (Figure 7) By comparisonwith the reference standard it is identified as icaritin Its MSspectrum yields a quasimolecular ion at119898119911 367 (C21H19O6)And themajor fragment ion at119898119911 352 is characterized as theloss of methyl group The MS3 fragment at 119898119911 309 resultsfrom the loss of methyl group and CO and ion at 119898119911 297is the outcome of the neutral loss of isopentene (70Da) fromthe deprotonated molecule

4 Conclusion

Themenopausal syndrome improvement and possible cancerpreventive activity of Herba Epimedii is receiving more andmore attention Information on the in vivo disposition ofHerba Epimedii components is important for better under-standing the biological effects of this herbal drug For thispurpose our investigation was designed to obtain informa-tion on the plasma pharmacokinetics and tissue distributionof icariin in rat and the excretion of icariin and itsmetabolitesIn this study we firstly established and validated a rapidsensitive and reliable LC-MS method for the determinationof icariin in various biological samples collected at differenttime points and then the method was applied to the plasmapharmacokinetics and tissue distribution study of icariin inrats after oral administration of the total flavonoid extractof Herba Epimedii In addition the differences of the tissuedistribution of icariin in between male and female rats werestudied after oral and intramuscular administration of icariinAnd the metabolic pattern of icariin was also investigatedThe data showed the plasma and tissue concentration oficariin were kept at a very low level at all collection pointswhich suggests icariin is poorly absorbed after oral adminis-trationThepeak concentration in plasma and collected tissuesamples appeared at 05ndash1 h and then quickly dropped at 4 hafter administration demonstrating that a small amount oficariin can be rapidly absorbed and eliminated and there isno accumulation in rat tissues Moreover the concentrationin liver lung and reproductive organs was much higherthan that in other tissues indicating that liver lung andreproductive organs are the main target sites of icariin in rats

The gender-related difference of icariin in tissue distribu-tion has not been reported in the past published literatureOur study demonstrated that the distribution features oficariin are similar in most of tissues between male andfemale rats However there exist a few remarkable gender

differences such that thewhole tissue concentration of icariinin male rats was much higher than female rats except in gen-ital organs (high in female and low in male) In China HerbaEpimedii is a famous tonic and aphrodisiac to invigoratekidney and strengthenYang therefore it is commonly used totreat male sexual functional decline and female menopausalsyndrome As one of the predominant constitutes in HerbaEpimedii icariin plays a very crucial role in exerting effectsso the relatively high icariin concentration in female genitalorgans actually supports the above-mentioned traditionalChinese medicine theory and clinical practice and makesthis compound a good future prospect for treating womenrsquosmenopausal related diseases

In the present study the complete metabolic profile oficariin has also been investigated The results illustrate thatfeces should be the major excretion way for both oral andintramuscular administration of icariin Based on LC-UVand LC-MS119899 analyses eleven metabolites were detected infecal samples and were further identified Among them10 metabolites appeared in rat feces of oral administrationgroup and 9 in the feces of intramuscular introduction groupThe major metabolic routes of clearance are proposed to behydrolysis demethylation oxidation and conjugation

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

Authorsrsquo Contributions

Shunjun Xu and Jiejing Yu contributed equally to this work

References

[1] X Wang Y He B Guo et al ldquoIn vivo screening for anti-osteo-porotic fraction from extract of herbal formula xianlinggubaoin ovariectomized micerdquo PLoS ONE vol 10 no 2 Article IDe0118184 2015

[2] R-H Liu X Kang L-P Xu et al ldquoEffects of the combinedextracts of Herba Epimedii and Fructus Ligustri Lucidi on bonemineral content and bone turnover in osteoporotic ratsrdquo BMCComplementary and Alternative Medicine vol 15 no 1 article112 2015

[3] B C L Chan H Y Lee W S Siu et al ldquoSuppression of mastcell activity contributes to the osteoprotective effect of an herbalformula containing Herba Epimedii Fructus Ligustri Lucidiand Fructus Psoraleaerdquo Journal of Pharmacy and Pharmacologyvol 66 no 3 pp 437ndash444 2014

[4] S CW Sze Y Tong T B Ng C L Y Cheng andH P CheungldquoHerba Epimedii anti-oxidative properties and its medicalimplicationsrdquoMolecules vol 15 no 11 pp 7861ndash7870 2010

[5] R Niu ldquoAction of the drug Herba Epimedii on testosterone ofthe mouse plasma and its accessory sexual organ before andafter processingrdquo China Journal of Chinese Materia Medica vol14 no 9 pp 530ndash574 1989

[6] F-X Liu S Sun and Z-Z Cui ldquoAnalysis of immunologi-cal enhancement of immunosuppressed chickens by Chineseherbal extractsrdquo Journal of Ethnopharmacology vol 127 no 2pp 251ndash256 2010

BioMed Research International 17

[7] J-H Kim Y-J Mun S-J Im J-H Han H-S Lee and W-HWoo ldquoEffects of the aqueous extract of Epimedii Herba on theantibody responses in micerdquo International Immunopharmacol-ogy vol 1 no 5 pp 935ndash944 2001

[8] X Yu Y Tong X-Q Han et al ldquoAnti-angiogenic activity ofHerba Epimedii on zebrafish embryos in vivo and HUVECs invitrordquo Phytotherapy Research vol 27 no 9 pp 1368ndash1375 2013

[9] D-C Zhang J-L Liu Y-B Ding J-G Xia and G-Y ChenldquoIcariin potentiates the antitumor activity of gemcitabine ingallbladder cancer by suppressingNF-120581BrdquoActa PharmacologicaSinica vol 34 no 2 pp 301ndash308 2013

[10] L Kong J Liu J Wang et al ldquoIcariin inhibits TNF-120572IFN-120574induced inflammatory response via inhibition of the substanceP and p38-MAPK signaling pathway in human keratinocytesrdquoInternational Immunopharmacology vol 29 no 2 pp 401ndash4072015

[11] H Shao J Shen M Wang et al ldquoIcariin protects against tita-nium particle-induced osteolysis and inflammatory response ina mouse calvarial modelrdquo Biomaterials vol 60 pp 92ndash99 2015

[12] Q Wei J Zhang G Hong et al ldquoIcariin promotes osteogenicdifferentiation of rat bone marrow stromal cells by activatingthe ER120572-Wnt120573-catenin signaling pathwayrdquo Biomedicine ampPharmacotherapy vol 84 pp 931ndash939 2016

[13] S-Q Zhang W-J Cai J-H Huang et al ldquoIcariin a naturalflavonol glycoside extends healthspan in micerdquo ExperimentalGerontology vol 69 pp 226ndash235 2015

[14] M N Makarova O N Pozharitskaya A N Shikov S VTesakova V G Makarov and V P Tikhonov ldquoEffect of lipid-based suspension of Epimedium koreanum Nakai extract onsexual behavior in ratsrdquo Journal of Ethnopharmacology vol 114no 3 pp 412ndash416 2007

[15] Y Chen J Wang X Jia X Tan and M Hu ldquoRole of intestinalhydrolase in the absorption of prenylated flavonoids present inyinyanghuordquoMolecules vol 16 no 2 pp 1336ndash1348 2011

[16] J J Yu Y J Liu S J Xu S Sun Y J Xu and L Yang ldquoSimulta-neous quantitative determination of four epimediumflavonoidsin rat urine by LC-MSMSrdquo Traditional Chinese Drug Researchamp Clinical Pharmacology vol 27 no 4 pp 537ndash541 2016

[17] Y Lu N Li Y Deng et al ldquoSimultaneous determination oficariin naringin and osthole in rat plasma by UPLC-MSMSand its application for pharmacokinetic study after oral admin-istration of Gushudan capsulesrdquo Journal of Chromatography Bvol 993-994 pp 75ndash80 2015

[18] N Wang X Huang X Wang Y Zhang R Wu and D ShouldquoPipette tip solid-phase extraction and high-performance liquidchromatography for the determination of flavonoids fromEpimedii herba in rat serum and application of the techniqueto pharmacokinetic studiesrdquo Journal of Chromatography B vol990 pp 64ndash72 2015

Submit your manuscripts athttpswwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

BioMed Research International 13

25308

16310

27902

29817

0

20

40

60

80

100

Rela

tive a

bund

ance

400200mz

M10

MS3

29810

35312

21913

400200mz

0

20

40

60

80

100

Rela

tive a

bund

ance M10

MS2

35208

36723

309280

20

40

60

80

100

Rela

tive a

bund

ance

400 600200mz

M11

MS2

30911

2810435215

400200mz

0

20

40

60

80

100

Rela

tive a

bund

ance M11

MS3

Figure 7 Representative LC-MS119899 spectra of icariin and its metabolites M1ndashM11 in rat feces

necessary to characterize the MS119899 behavior of icariin toidentify and assign the metabolites of the constituent Thedeprotonated molecule [MndashH]minus of icariin appears at 119898119911675 which readily eliminates its glucosyl and rhamnosylgroups to produce an [MndashHndash308]minus ion at 119898119911 367 The[MndashHndash308]minus ion yields a fragment at 119898119911 352 by losing amethyl radical And of theMS4 spectrum of [MndashHndash308ndash15]minusion the fragment at 119898119911 323 can be reasonably attributed tothe loss of 3-COH of flavone skeletonrsquos C ring

M1 as aminormetabolite of icariin was only found in thefeces of the oral group Its quasimolecular ion occurs at 119898119911837 (C39H49O20) whose molecular mass is 162Da more thanthat of icariin And its major MS119899 fragment ions are the sameas those of icariinThemost abundantMS2 andMS3 fragmentions also appear at 119898119911 367 and 352 indicating the loss ofone rhamnosyl and two glucosyl moieties and the sequentialloss of methyl group respectively Although the molecularweight of M1 is equal to that of epimedin A there existsa significant difference on their chromatographic retentionbehavior Therefore M1 is tentatively identified as an isomerof epimedin A

Both M2 and M6 show the same quasimolecular ion[M minus H]minus at 119898119911 645 (C32H37O14) with the elution time at5044 and 5743min respectively M2 was only observed infeces after intramuscular introduction of icariin while M6was found in feces following both routes of administrationM6 is unambiguously identified as sagittatoside B incomparison with the reference standard Analogous to theMS119899 fragments of icariin (see Figure 7 and Table 6) M2also yielded the major fragment ions at 119898119911 352 323 309296 281 and 268 strongly suggesting that M2 should be

a conjunction metabolite of icariin Thus M2 is tentativelyassigned as sagittatoside B isomer

M3 was found in rat feces after both intramuscular andoral administration of icariin Its MS spectrum exhibits adeprotonated molecule [MndashH]minus at 119898119911 499 (C26H27O10)176Da less than that of icariin By comparison with thereference standard it is identified as baohuoside II TheMSMS spectrum shows the most abundant fragment ionat 119898119911 353 indicating the loss of rhamnose moiety TheMS3 spectrum displays the major fragment ions at 119898119911 325298 and 284 separately corresponding to the loss of COisobutenyl and isopentenyl radicals

M4 is a minor metabolite of icariin detected in the fecesof rats in the oral administration group Its deprotonatedmolecule (C33H37O16) is at 119898119911 689 And the major MSMSfragment at 119898119911 367 results from the loss of glucuronicacid and rhamnose moieties Its MS3 spectrum produces afragment at119898119911 352 (the loss of a methyl group) Thus M4 istentatively characterized as icariin glucuronide

M5ndashM11 of icariin metabolites were observed in rat fecesafter both intramuscular and oral administration of icariin

M5 has a deprotonated molecular ion at 119898119911 529(C27H29O11) in its MS spectrum The MS119899 spectra show themajor fragment ions at119898119911 501 (the loss of CO) 382 (the lossof rhamnosyl group) 367 (the loss of methyl radical from theion at119898119911 382) and 339 (the successive loss of methyl radicaland CO) Therefore M5 is tentatively identified as hydroxylicariside II

M7 is identified as 210158401015840-O-rhamnosyl icariside II based onthe reference standard and related mass spectral pattern The

14 BioMed Research International

Table6Ch

aracteriz

ationof

metaboliteso

ficariinin

ratfeces

Num

ber

Com

poun

dRT

Form

ula

Observedmass

RDB

Error

LCM

S119899data

[MndashH]minus

(ppm

)

PlowastIcariin

5519

C 33H39O15

67522888

145

0790

MS2[675]367(100)352(25)

MS3[675-367]352(100)

MS4[675-367-352]323(

30)309(100)296(45)281

(65)268

(50)255

(20)

M1

Epim

edin

Aiso

mer

a5038

C 39H49O20

8372

819

155

0824

MS2[837]790(50)367

(100)

MS3[837-367]352(100)297(20)

M2

Sagitta

tosid

eBiso

mer

b5044

C 32H37O14

6452186

145

1314

MS2[645]481(20)352(100)

MS3[645-352]323(40)309

(60)296

(90)281

(100)268(60)

M3

lowastBa

ohuo

sideIIa

b5278

C 26H27O10

4991

605

135

1295

MS2[499]353(100)

MS3[499-353]325(20)298

(40)284

(100)255(30)

M4

Icariin

glucuron

idea

5404

C 33H37O16

6892

084

155

1202

MS2[689]367(100)321(60)

MS3[689-367]352(100)

M5

Hydroxylicaris

ideIIa

b5412

C 27H29O11

5291

716

135

1132

MS2[529]501(20)382(100)367(40)

MS3[529-382]367(100)339(10)

M6

lowastSagitta

tosid

eBab

5743

C 32H37O14

6452184

145

1020

MS2[645]367(100)352(30)

MS3[645-367]352(100)

M7

lowast210158401015840-O

-Rhamno

syl-icaris

ideIIa

b5756

C 33H39O14

6592

344

145

1529

MS2[659]366(100)351(20)323(15)

MS3[659-366]351(100)323

(80)311(60)305

(20)

M8

lowastIcariside

IIab

6146

C 27H29O10

5131760

135

0851

MS2[513]366(100)

MS3[513-366]351(100)323

(80)311(50)305

(20)

M9

Hydroxylicaritin

ab6233

C 21H19O7

3831137

125

3160

MS2[383]365(100)355(60)339

(40)

MS3[383-365]350(100)321(20)230(40)

M10

Desmethylicaritin

ab6636

C 20H17O6

3531024

125

1119

MS2[353]298(100)219(10)

MS3[353-298]279(60)269

(40)253

(100)241(30)164(70)

M11

lowastIcaritinab

8265

C 21H19O6

3671183

125

1921

MS2[367]352(100)309(20)297

(20)

MS3[367-352]309(100)297(60)

lowastCom

paredwith

ther

eference

standardaObservedin

ratfeces

after

oraladministratio

nb O

bservedin

ratfeces

after

intram

usculara

dministratio

n

BioMed Research International 15

O

OO

O

O

O

OHOH

OH

OH

OH OHOH

OH

OHOH

OHOH

OH

OHOH OH

OH

OH

OH

OHOH

OH

OH

OH

OH

OH

OH

OH

OHOH

OH

OH

OH

OH

OHOH

OHOH

OHOH

OH

OH

OH

OH

OHOH

HO HO

HO

HO

HO

HOHO

HO

HO

HO

OH

O

OO

O

OOHOOC

O

OO

O

O

OO

OO

O

O

OO

OO

O

O

OO

O

O

OO

O

O

O

O

O

O

O

icariin

epimedin A isomer

minus rh

a

minus methyl

+ rhaOxidation

minus methyl

Icariin glucuronide

+ xyl

+ glcGlucuronidation

minus rha

Oxidation

minus rha

+ xyl

Sagittatoside B isomer

minus glc

M1

M4

M2

OCH3 OCH3

OCH3

OCH3

OCH3

OCH3

OCH3

OCH3

M7 2-O-rhamnosyl icariside II

M5 hydroxyl icariside II

M3 baohuoside II

M8 icariside II

M6 sagittatoside B

M9 hydroxyl icaritinM10 desmethyl icaritin

M11 icaritin

Figure 8 Proposed metabolic pathways for icariin in rat feces

MS spectrum of M7 shows a quasimolecular ion [MndashH]minusat 119898119911 659 (C33H39O14) The major fragment ions at 119898119911366 351 and 323 result from the concurrent loss of therhamnose chain the successive loss of a methyl group andCO respectively

As shownFigure 7M8 is themost abundantmetabolite oficariin By comparison with the reference standard it is iden-tified as icariside II Its MS spectrum shows a deprotonatedmolecular ion at 119898119911 513 (C27H29O10) And similar to the

MS119899 pattern of icariin the major fragment ions at 119898119911 366351 and 323 separately match the loss of rhamnose moietythe sequential loss of rhamnose and methyl groups and theloss of CO

M9 has a quasimolecular ion [MndashH]minus at 119898119911 383(C21H19O7) 146Da less than that of M5 (hydroxyl icarisideII) It is tentatively identified as hydroxyl icaritin Its MSMSspectrum shows themain fragments of119898119911 365 355 and 339respectively corresponding to the neutral loss of H2O CO

16 BioMed Research International

and CO2 MS3 spectrum the fragment ions at 119898119911 350 and321 individually result from the neutral loss of methyl radicaland CO2

The quasimolecular ion [MndashH]minus of M10 is at 119898119911 353146Da less than that of M3 (baohuoside II) The majorfragment ion in its MSMS spectrum is at119898119911 298 matchingthe loss of isobutenyl radical The fragment at 119898119911 219 ispossibly produced by the neutral loss of C8H6O2 (134Da) dueto the retro-Diels-Alder (RDA) fragmentation of the flavonersquosC ring Moreover the MS3 spectrum affords fragment ionsat 119898119911 279 269 253 and 164 resulting from the loss ofH2O CO CO2 and C8H6O2 (134Da) respectively Thus thismetabolite is reasonably identified as desmethyl icaritin

M11 was observed as anothermajormetabolite in rat fecesof both two administration groups (Figure 7) By comparisonwith the reference standard it is identified as icaritin Its MSspectrum yields a quasimolecular ion at119898119911 367 (C21H19O6)And themajor fragment ion at119898119911 352 is characterized as theloss of methyl group The MS3 fragment at 119898119911 309 resultsfrom the loss of methyl group and CO and ion at 119898119911 297is the outcome of the neutral loss of isopentene (70Da) fromthe deprotonated molecule

4 Conclusion

Themenopausal syndrome improvement and possible cancerpreventive activity of Herba Epimedii is receiving more andmore attention Information on the in vivo disposition ofHerba Epimedii components is important for better under-standing the biological effects of this herbal drug For thispurpose our investigation was designed to obtain informa-tion on the plasma pharmacokinetics and tissue distributionof icariin in rat and the excretion of icariin and itsmetabolitesIn this study we firstly established and validated a rapidsensitive and reliable LC-MS method for the determinationof icariin in various biological samples collected at differenttime points and then the method was applied to the plasmapharmacokinetics and tissue distribution study of icariin inrats after oral administration of the total flavonoid extractof Herba Epimedii In addition the differences of the tissuedistribution of icariin in between male and female rats werestudied after oral and intramuscular administration of icariinAnd the metabolic pattern of icariin was also investigatedThe data showed the plasma and tissue concentration oficariin were kept at a very low level at all collection pointswhich suggests icariin is poorly absorbed after oral adminis-trationThepeak concentration in plasma and collected tissuesamples appeared at 05ndash1 h and then quickly dropped at 4 hafter administration demonstrating that a small amount oficariin can be rapidly absorbed and eliminated and there isno accumulation in rat tissues Moreover the concentrationin liver lung and reproductive organs was much higherthan that in other tissues indicating that liver lung andreproductive organs are the main target sites of icariin in rats

The gender-related difference of icariin in tissue distribu-tion has not been reported in the past published literatureOur study demonstrated that the distribution features oficariin are similar in most of tissues between male andfemale rats However there exist a few remarkable gender

differences such that thewhole tissue concentration of icariinin male rats was much higher than female rats except in gen-ital organs (high in female and low in male) In China HerbaEpimedii is a famous tonic and aphrodisiac to invigoratekidney and strengthenYang therefore it is commonly used totreat male sexual functional decline and female menopausalsyndrome As one of the predominant constitutes in HerbaEpimedii icariin plays a very crucial role in exerting effectsso the relatively high icariin concentration in female genitalorgans actually supports the above-mentioned traditionalChinese medicine theory and clinical practice and makesthis compound a good future prospect for treating womenrsquosmenopausal related diseases

In the present study the complete metabolic profile oficariin has also been investigated The results illustrate thatfeces should be the major excretion way for both oral andintramuscular administration of icariin Based on LC-UVand LC-MS119899 analyses eleven metabolites were detected infecal samples and were further identified Among them10 metabolites appeared in rat feces of oral administrationgroup and 9 in the feces of intramuscular introduction groupThe major metabolic routes of clearance are proposed to behydrolysis demethylation oxidation and conjugation

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

Authorsrsquo Contributions

Shunjun Xu and Jiejing Yu contributed equally to this work

References

[1] X Wang Y He B Guo et al ldquoIn vivo screening for anti-osteo-porotic fraction from extract of herbal formula xianlinggubaoin ovariectomized micerdquo PLoS ONE vol 10 no 2 Article IDe0118184 2015

[2] R-H Liu X Kang L-P Xu et al ldquoEffects of the combinedextracts of Herba Epimedii and Fructus Ligustri Lucidi on bonemineral content and bone turnover in osteoporotic ratsrdquo BMCComplementary and Alternative Medicine vol 15 no 1 article112 2015

[3] B C L Chan H Y Lee W S Siu et al ldquoSuppression of mastcell activity contributes to the osteoprotective effect of an herbalformula containing Herba Epimedii Fructus Ligustri Lucidiand Fructus Psoraleaerdquo Journal of Pharmacy and Pharmacologyvol 66 no 3 pp 437ndash444 2014

[4] S CW Sze Y Tong T B Ng C L Y Cheng andH P CheungldquoHerba Epimedii anti-oxidative properties and its medicalimplicationsrdquoMolecules vol 15 no 11 pp 7861ndash7870 2010

[5] R Niu ldquoAction of the drug Herba Epimedii on testosterone ofthe mouse plasma and its accessory sexual organ before andafter processingrdquo China Journal of Chinese Materia Medica vol14 no 9 pp 530ndash574 1989

[6] F-X Liu S Sun and Z-Z Cui ldquoAnalysis of immunologi-cal enhancement of immunosuppressed chickens by Chineseherbal extractsrdquo Journal of Ethnopharmacology vol 127 no 2pp 251ndash256 2010

BioMed Research International 17

[7] J-H Kim Y-J Mun S-J Im J-H Han H-S Lee and W-HWoo ldquoEffects of the aqueous extract of Epimedii Herba on theantibody responses in micerdquo International Immunopharmacol-ogy vol 1 no 5 pp 935ndash944 2001

[8] X Yu Y Tong X-Q Han et al ldquoAnti-angiogenic activity ofHerba Epimedii on zebrafish embryos in vivo and HUVECs invitrordquo Phytotherapy Research vol 27 no 9 pp 1368ndash1375 2013

[9] D-C Zhang J-L Liu Y-B Ding J-G Xia and G-Y ChenldquoIcariin potentiates the antitumor activity of gemcitabine ingallbladder cancer by suppressingNF-120581BrdquoActa PharmacologicaSinica vol 34 no 2 pp 301ndash308 2013

[10] L Kong J Liu J Wang et al ldquoIcariin inhibits TNF-120572IFN-120574induced inflammatory response via inhibition of the substanceP and p38-MAPK signaling pathway in human keratinocytesrdquoInternational Immunopharmacology vol 29 no 2 pp 401ndash4072015

[11] H Shao J Shen M Wang et al ldquoIcariin protects against tita-nium particle-induced osteolysis and inflammatory response ina mouse calvarial modelrdquo Biomaterials vol 60 pp 92ndash99 2015

[12] Q Wei J Zhang G Hong et al ldquoIcariin promotes osteogenicdifferentiation of rat bone marrow stromal cells by activatingthe ER120572-Wnt120573-catenin signaling pathwayrdquo Biomedicine ampPharmacotherapy vol 84 pp 931ndash939 2016

[13] S-Q Zhang W-J Cai J-H Huang et al ldquoIcariin a naturalflavonol glycoside extends healthspan in micerdquo ExperimentalGerontology vol 69 pp 226ndash235 2015

[14] M N Makarova O N Pozharitskaya A N Shikov S VTesakova V G Makarov and V P Tikhonov ldquoEffect of lipid-based suspension of Epimedium koreanum Nakai extract onsexual behavior in ratsrdquo Journal of Ethnopharmacology vol 114no 3 pp 412ndash416 2007

[15] Y Chen J Wang X Jia X Tan and M Hu ldquoRole of intestinalhydrolase in the absorption of prenylated flavonoids present inyinyanghuordquoMolecules vol 16 no 2 pp 1336ndash1348 2011

[16] J J Yu Y J Liu S J Xu S Sun Y J Xu and L Yang ldquoSimulta-neous quantitative determination of four epimediumflavonoidsin rat urine by LC-MSMSrdquo Traditional Chinese Drug Researchamp Clinical Pharmacology vol 27 no 4 pp 537ndash541 2016

[17] Y Lu N Li Y Deng et al ldquoSimultaneous determination oficariin naringin and osthole in rat plasma by UPLC-MSMSand its application for pharmacokinetic study after oral admin-istration of Gushudan capsulesrdquo Journal of Chromatography Bvol 993-994 pp 75ndash80 2015

[18] N Wang X Huang X Wang Y Zhang R Wu and D ShouldquoPipette tip solid-phase extraction and high-performance liquidchromatography for the determination of flavonoids fromEpimedii herba in rat serum and application of the techniqueto pharmacokinetic studiesrdquo Journal of Chromatography B vol990 pp 64ndash72 2015

Submit your manuscripts athttpswwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

14 BioMed Research International

Table6Ch

aracteriz

ationof

metaboliteso

ficariinin

ratfeces

Num

ber

Com

poun

dRT

Form

ula

Observedmass

RDB

Error

LCM

S119899data

[MndashH]minus

(ppm

)

PlowastIcariin

5519

C 33H39O15

67522888

145

0790

MS2[675]367(100)352(25)

MS3[675-367]352(100)

MS4[675-367-352]323(

30)309(100)296(45)281

(65)268

(50)255

(20)

M1

Epim

edin

Aiso

mer

a5038

C 39H49O20

8372

819

155

0824

MS2[837]790(50)367

(100)

MS3[837-367]352(100)297(20)

M2

Sagitta

tosid

eBiso

mer

b5044

C 32H37O14

6452186

145

1314

MS2[645]481(20)352(100)

MS3[645-352]323(40)309

(60)296

(90)281

(100)268(60)

M3

lowastBa

ohuo

sideIIa

b5278

C 26H27O10

4991

605

135

1295

MS2[499]353(100)

MS3[499-353]325(20)298

(40)284

(100)255(30)

M4

Icariin

glucuron

idea

5404

C 33H37O16

6892

084

155

1202

MS2[689]367(100)321(60)

MS3[689-367]352(100)

M5

Hydroxylicaris

ideIIa

b5412

C 27H29O11

5291

716

135

1132

MS2[529]501(20)382(100)367(40)

MS3[529-382]367(100)339(10)

M6

lowastSagitta

tosid

eBab

5743

C 32H37O14

6452184

145

1020

MS2[645]367(100)352(30)

MS3[645-367]352(100)

M7

lowast210158401015840-O

-Rhamno

syl-icaris

ideIIa

b5756

C 33H39O14

6592

344

145

1529

MS2[659]366(100)351(20)323(15)

MS3[659-366]351(100)323

(80)311(60)305

(20)

M8

lowastIcariside

IIab

6146

C 27H29O10

5131760

135

0851

MS2[513]366(100)

MS3[513-366]351(100)323

(80)311(50)305

(20)

M9

Hydroxylicaritin

ab6233

C 21H19O7

3831137

125

3160

MS2[383]365(100)355(60)339

(40)

MS3[383-365]350(100)321(20)230(40)

M10

Desmethylicaritin

ab6636

C 20H17O6

3531024

125

1119

MS2[353]298(100)219(10)

MS3[353-298]279(60)269

(40)253

(100)241(30)164(70)

M11

lowastIcaritinab

8265

C 21H19O6

3671183

125

1921

MS2[367]352(100)309(20)297

(20)

MS3[367-352]309(100)297(60)

lowastCom

paredwith

ther

eference

standardaObservedin

ratfeces

after

oraladministratio

nb O

bservedin

ratfeces

after

intram

usculara

dministratio

n

BioMed Research International 15

O

OO

O

O

O

OHOH

OH

OH

OH OHOH

OH

OHOH

OHOH

OH

OHOH OH

OH

OH

OH

OHOH

OH

OH

OH

OH

OH

OH

OH

OHOH

OH

OH

OH

OH

OHOH

OHOH

OHOH

OH

OH

OH

OH

OHOH

HO HO

HO

HO

HO

HOHO

HO

HO

HO

OH

O

OO

O

OOHOOC

O

OO

O

O

OO

OO

O

O

OO

OO

O

O

OO

O

O

OO

O

O

O

O

O

O

O

icariin

epimedin A isomer

minus rh

a

minus methyl

+ rhaOxidation

minus methyl

Icariin glucuronide

+ xyl

+ glcGlucuronidation

minus rha

Oxidation

minus rha

+ xyl

Sagittatoside B isomer

minus glc

M1

M4

M2

OCH3 OCH3

OCH3

OCH3

OCH3

OCH3

OCH3

OCH3

M7 2-O-rhamnosyl icariside II

M5 hydroxyl icariside II

M3 baohuoside II

M8 icariside II

M6 sagittatoside B

M9 hydroxyl icaritinM10 desmethyl icaritin

M11 icaritin

Figure 8 Proposed metabolic pathways for icariin in rat feces

MS spectrum of M7 shows a quasimolecular ion [MndashH]minusat 119898119911 659 (C33H39O14) The major fragment ions at 119898119911366 351 and 323 result from the concurrent loss of therhamnose chain the successive loss of a methyl group andCO respectively

As shownFigure 7M8 is themost abundantmetabolite oficariin By comparison with the reference standard it is iden-tified as icariside II Its MS spectrum shows a deprotonatedmolecular ion at 119898119911 513 (C27H29O10) And similar to the

MS119899 pattern of icariin the major fragment ions at 119898119911 366351 and 323 separately match the loss of rhamnose moietythe sequential loss of rhamnose and methyl groups and theloss of CO

M9 has a quasimolecular ion [MndashH]minus at 119898119911 383(C21H19O7) 146Da less than that of M5 (hydroxyl icarisideII) It is tentatively identified as hydroxyl icaritin Its MSMSspectrum shows themain fragments of119898119911 365 355 and 339respectively corresponding to the neutral loss of H2O CO

16 BioMed Research International

and CO2 MS3 spectrum the fragment ions at 119898119911 350 and321 individually result from the neutral loss of methyl radicaland CO2

The quasimolecular ion [MndashH]minus of M10 is at 119898119911 353146Da less than that of M3 (baohuoside II) The majorfragment ion in its MSMS spectrum is at119898119911 298 matchingthe loss of isobutenyl radical The fragment at 119898119911 219 ispossibly produced by the neutral loss of C8H6O2 (134Da) dueto the retro-Diels-Alder (RDA) fragmentation of the flavonersquosC ring Moreover the MS3 spectrum affords fragment ionsat 119898119911 279 269 253 and 164 resulting from the loss ofH2O CO CO2 and C8H6O2 (134Da) respectively Thus thismetabolite is reasonably identified as desmethyl icaritin

M11 was observed as anothermajormetabolite in rat fecesof both two administration groups (Figure 7) By comparisonwith the reference standard it is identified as icaritin Its MSspectrum yields a quasimolecular ion at119898119911 367 (C21H19O6)And themajor fragment ion at119898119911 352 is characterized as theloss of methyl group The MS3 fragment at 119898119911 309 resultsfrom the loss of methyl group and CO and ion at 119898119911 297is the outcome of the neutral loss of isopentene (70Da) fromthe deprotonated molecule

4 Conclusion

Themenopausal syndrome improvement and possible cancerpreventive activity of Herba Epimedii is receiving more andmore attention Information on the in vivo disposition ofHerba Epimedii components is important for better under-standing the biological effects of this herbal drug For thispurpose our investigation was designed to obtain informa-tion on the plasma pharmacokinetics and tissue distributionof icariin in rat and the excretion of icariin and itsmetabolitesIn this study we firstly established and validated a rapidsensitive and reliable LC-MS method for the determinationof icariin in various biological samples collected at differenttime points and then the method was applied to the plasmapharmacokinetics and tissue distribution study of icariin inrats after oral administration of the total flavonoid extractof Herba Epimedii In addition the differences of the tissuedistribution of icariin in between male and female rats werestudied after oral and intramuscular administration of icariinAnd the metabolic pattern of icariin was also investigatedThe data showed the plasma and tissue concentration oficariin were kept at a very low level at all collection pointswhich suggests icariin is poorly absorbed after oral adminis-trationThepeak concentration in plasma and collected tissuesamples appeared at 05ndash1 h and then quickly dropped at 4 hafter administration demonstrating that a small amount oficariin can be rapidly absorbed and eliminated and there isno accumulation in rat tissues Moreover the concentrationin liver lung and reproductive organs was much higherthan that in other tissues indicating that liver lung andreproductive organs are the main target sites of icariin in rats

The gender-related difference of icariin in tissue distribu-tion has not been reported in the past published literatureOur study demonstrated that the distribution features oficariin are similar in most of tissues between male andfemale rats However there exist a few remarkable gender

differences such that thewhole tissue concentration of icariinin male rats was much higher than female rats except in gen-ital organs (high in female and low in male) In China HerbaEpimedii is a famous tonic and aphrodisiac to invigoratekidney and strengthenYang therefore it is commonly used totreat male sexual functional decline and female menopausalsyndrome As one of the predominant constitutes in HerbaEpimedii icariin plays a very crucial role in exerting effectsso the relatively high icariin concentration in female genitalorgans actually supports the above-mentioned traditionalChinese medicine theory and clinical practice and makesthis compound a good future prospect for treating womenrsquosmenopausal related diseases

In the present study the complete metabolic profile oficariin has also been investigated The results illustrate thatfeces should be the major excretion way for both oral andintramuscular administration of icariin Based on LC-UVand LC-MS119899 analyses eleven metabolites were detected infecal samples and were further identified Among them10 metabolites appeared in rat feces of oral administrationgroup and 9 in the feces of intramuscular introduction groupThe major metabolic routes of clearance are proposed to behydrolysis demethylation oxidation and conjugation

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

Authorsrsquo Contributions

Shunjun Xu and Jiejing Yu contributed equally to this work

References

[1] X Wang Y He B Guo et al ldquoIn vivo screening for anti-osteo-porotic fraction from extract of herbal formula xianlinggubaoin ovariectomized micerdquo PLoS ONE vol 10 no 2 Article IDe0118184 2015

[2] R-H Liu X Kang L-P Xu et al ldquoEffects of the combinedextracts of Herba Epimedii and Fructus Ligustri Lucidi on bonemineral content and bone turnover in osteoporotic ratsrdquo BMCComplementary and Alternative Medicine vol 15 no 1 article112 2015

[3] B C L Chan H Y Lee W S Siu et al ldquoSuppression of mastcell activity contributes to the osteoprotective effect of an herbalformula containing Herba Epimedii Fructus Ligustri Lucidiand Fructus Psoraleaerdquo Journal of Pharmacy and Pharmacologyvol 66 no 3 pp 437ndash444 2014

[4] S CW Sze Y Tong T B Ng C L Y Cheng andH P CheungldquoHerba Epimedii anti-oxidative properties and its medicalimplicationsrdquoMolecules vol 15 no 11 pp 7861ndash7870 2010

[5] R Niu ldquoAction of the drug Herba Epimedii on testosterone ofthe mouse plasma and its accessory sexual organ before andafter processingrdquo China Journal of Chinese Materia Medica vol14 no 9 pp 530ndash574 1989

[6] F-X Liu S Sun and Z-Z Cui ldquoAnalysis of immunologi-cal enhancement of immunosuppressed chickens by Chineseherbal extractsrdquo Journal of Ethnopharmacology vol 127 no 2pp 251ndash256 2010

BioMed Research International 17

[7] J-H Kim Y-J Mun S-J Im J-H Han H-S Lee and W-HWoo ldquoEffects of the aqueous extract of Epimedii Herba on theantibody responses in micerdquo International Immunopharmacol-ogy vol 1 no 5 pp 935ndash944 2001

[8] X Yu Y Tong X-Q Han et al ldquoAnti-angiogenic activity ofHerba Epimedii on zebrafish embryos in vivo and HUVECs invitrordquo Phytotherapy Research vol 27 no 9 pp 1368ndash1375 2013

[9] D-C Zhang J-L Liu Y-B Ding J-G Xia and G-Y ChenldquoIcariin potentiates the antitumor activity of gemcitabine ingallbladder cancer by suppressingNF-120581BrdquoActa PharmacologicaSinica vol 34 no 2 pp 301ndash308 2013

[10] L Kong J Liu J Wang et al ldquoIcariin inhibits TNF-120572IFN-120574induced inflammatory response via inhibition of the substanceP and p38-MAPK signaling pathway in human keratinocytesrdquoInternational Immunopharmacology vol 29 no 2 pp 401ndash4072015

[11] H Shao J Shen M Wang et al ldquoIcariin protects against tita-nium particle-induced osteolysis and inflammatory response ina mouse calvarial modelrdquo Biomaterials vol 60 pp 92ndash99 2015

[12] Q Wei J Zhang G Hong et al ldquoIcariin promotes osteogenicdifferentiation of rat bone marrow stromal cells by activatingthe ER120572-Wnt120573-catenin signaling pathwayrdquo Biomedicine ampPharmacotherapy vol 84 pp 931ndash939 2016

[13] S-Q Zhang W-J Cai J-H Huang et al ldquoIcariin a naturalflavonol glycoside extends healthspan in micerdquo ExperimentalGerontology vol 69 pp 226ndash235 2015

[14] M N Makarova O N Pozharitskaya A N Shikov S VTesakova V G Makarov and V P Tikhonov ldquoEffect of lipid-based suspension of Epimedium koreanum Nakai extract onsexual behavior in ratsrdquo Journal of Ethnopharmacology vol 114no 3 pp 412ndash416 2007

[15] Y Chen J Wang X Jia X Tan and M Hu ldquoRole of intestinalhydrolase in the absorption of prenylated flavonoids present inyinyanghuordquoMolecules vol 16 no 2 pp 1336ndash1348 2011

[16] J J Yu Y J Liu S J Xu S Sun Y J Xu and L Yang ldquoSimulta-neous quantitative determination of four epimediumflavonoidsin rat urine by LC-MSMSrdquo Traditional Chinese Drug Researchamp Clinical Pharmacology vol 27 no 4 pp 537ndash541 2016

[17] Y Lu N Li Y Deng et al ldquoSimultaneous determination oficariin naringin and osthole in rat plasma by UPLC-MSMSand its application for pharmacokinetic study after oral admin-istration of Gushudan capsulesrdquo Journal of Chromatography Bvol 993-994 pp 75ndash80 2015

[18] N Wang X Huang X Wang Y Zhang R Wu and D ShouldquoPipette tip solid-phase extraction and high-performance liquidchromatography for the determination of flavonoids fromEpimedii herba in rat serum and application of the techniqueto pharmacokinetic studiesrdquo Journal of Chromatography B vol990 pp 64ndash72 2015

Submit your manuscripts athttpswwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

BioMed Research International 15

O

OO

O

O

O

OHOH

OH

OH

OH OHOH

OH

OHOH

OHOH

OH

OHOH OH

OH

OH

OH

OHOH

OH

OH

OH

OH

OH

OH

OH

OHOH

OH

OH

OH

OH

OHOH

OHOH

OHOH

OH

OH

OH

OH

OHOH

HO HO

HO

HO

HO

HOHO

HO

HO

HO

OH

O

OO

O

OOHOOC

O

OO

O

O

OO

OO

O

O

OO

OO

O

O

OO

O

O

OO

O

O

O

O

O

O

O

icariin

epimedin A isomer

minus rh

a

minus methyl

+ rhaOxidation

minus methyl

Icariin glucuronide

+ xyl

+ glcGlucuronidation

minus rha

Oxidation

minus rha

+ xyl

Sagittatoside B isomer

minus glc

M1

M4

M2

OCH3 OCH3

OCH3

OCH3

OCH3

OCH3

OCH3

OCH3

M7 2-O-rhamnosyl icariside II

M5 hydroxyl icariside II

M3 baohuoside II

M8 icariside II

M6 sagittatoside B

M9 hydroxyl icaritinM10 desmethyl icaritin

M11 icaritin

Figure 8 Proposed metabolic pathways for icariin in rat feces

MS spectrum of M7 shows a quasimolecular ion [MndashH]minusat 119898119911 659 (C33H39O14) The major fragment ions at 119898119911366 351 and 323 result from the concurrent loss of therhamnose chain the successive loss of a methyl group andCO respectively

As shownFigure 7M8 is themost abundantmetabolite oficariin By comparison with the reference standard it is iden-tified as icariside II Its MS spectrum shows a deprotonatedmolecular ion at 119898119911 513 (C27H29O10) And similar to the

MS119899 pattern of icariin the major fragment ions at 119898119911 366351 and 323 separately match the loss of rhamnose moietythe sequential loss of rhamnose and methyl groups and theloss of CO

M9 has a quasimolecular ion [MndashH]minus at 119898119911 383(C21H19O7) 146Da less than that of M5 (hydroxyl icarisideII) It is tentatively identified as hydroxyl icaritin Its MSMSspectrum shows themain fragments of119898119911 365 355 and 339respectively corresponding to the neutral loss of H2O CO

16 BioMed Research International

and CO2 MS3 spectrum the fragment ions at 119898119911 350 and321 individually result from the neutral loss of methyl radicaland CO2

The quasimolecular ion [MndashH]minus of M10 is at 119898119911 353146Da less than that of M3 (baohuoside II) The majorfragment ion in its MSMS spectrum is at119898119911 298 matchingthe loss of isobutenyl radical The fragment at 119898119911 219 ispossibly produced by the neutral loss of C8H6O2 (134Da) dueto the retro-Diels-Alder (RDA) fragmentation of the flavonersquosC ring Moreover the MS3 spectrum affords fragment ionsat 119898119911 279 269 253 and 164 resulting from the loss ofH2O CO CO2 and C8H6O2 (134Da) respectively Thus thismetabolite is reasonably identified as desmethyl icaritin

M11 was observed as anothermajormetabolite in rat fecesof both two administration groups (Figure 7) By comparisonwith the reference standard it is identified as icaritin Its MSspectrum yields a quasimolecular ion at119898119911 367 (C21H19O6)And themajor fragment ion at119898119911 352 is characterized as theloss of methyl group The MS3 fragment at 119898119911 309 resultsfrom the loss of methyl group and CO and ion at 119898119911 297is the outcome of the neutral loss of isopentene (70Da) fromthe deprotonated molecule

4 Conclusion

Themenopausal syndrome improvement and possible cancerpreventive activity of Herba Epimedii is receiving more andmore attention Information on the in vivo disposition ofHerba Epimedii components is important for better under-standing the biological effects of this herbal drug For thispurpose our investigation was designed to obtain informa-tion on the plasma pharmacokinetics and tissue distributionof icariin in rat and the excretion of icariin and itsmetabolitesIn this study we firstly established and validated a rapidsensitive and reliable LC-MS method for the determinationof icariin in various biological samples collected at differenttime points and then the method was applied to the plasmapharmacokinetics and tissue distribution study of icariin inrats after oral administration of the total flavonoid extractof Herba Epimedii In addition the differences of the tissuedistribution of icariin in between male and female rats werestudied after oral and intramuscular administration of icariinAnd the metabolic pattern of icariin was also investigatedThe data showed the plasma and tissue concentration oficariin were kept at a very low level at all collection pointswhich suggests icariin is poorly absorbed after oral adminis-trationThepeak concentration in plasma and collected tissuesamples appeared at 05ndash1 h and then quickly dropped at 4 hafter administration demonstrating that a small amount oficariin can be rapidly absorbed and eliminated and there isno accumulation in rat tissues Moreover the concentrationin liver lung and reproductive organs was much higherthan that in other tissues indicating that liver lung andreproductive organs are the main target sites of icariin in rats

The gender-related difference of icariin in tissue distribu-tion has not been reported in the past published literatureOur study demonstrated that the distribution features oficariin are similar in most of tissues between male andfemale rats However there exist a few remarkable gender

differences such that thewhole tissue concentration of icariinin male rats was much higher than female rats except in gen-ital organs (high in female and low in male) In China HerbaEpimedii is a famous tonic and aphrodisiac to invigoratekidney and strengthenYang therefore it is commonly used totreat male sexual functional decline and female menopausalsyndrome As one of the predominant constitutes in HerbaEpimedii icariin plays a very crucial role in exerting effectsso the relatively high icariin concentration in female genitalorgans actually supports the above-mentioned traditionalChinese medicine theory and clinical practice and makesthis compound a good future prospect for treating womenrsquosmenopausal related diseases

In the present study the complete metabolic profile oficariin has also been investigated The results illustrate thatfeces should be the major excretion way for both oral andintramuscular administration of icariin Based on LC-UVand LC-MS119899 analyses eleven metabolites were detected infecal samples and were further identified Among them10 metabolites appeared in rat feces of oral administrationgroup and 9 in the feces of intramuscular introduction groupThe major metabolic routes of clearance are proposed to behydrolysis demethylation oxidation and conjugation

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

Authorsrsquo Contributions

Shunjun Xu and Jiejing Yu contributed equally to this work

References

[1] X Wang Y He B Guo et al ldquoIn vivo screening for anti-osteo-porotic fraction from extract of herbal formula xianlinggubaoin ovariectomized micerdquo PLoS ONE vol 10 no 2 Article IDe0118184 2015

[2] R-H Liu X Kang L-P Xu et al ldquoEffects of the combinedextracts of Herba Epimedii and Fructus Ligustri Lucidi on bonemineral content and bone turnover in osteoporotic ratsrdquo BMCComplementary and Alternative Medicine vol 15 no 1 article112 2015

[3] B C L Chan H Y Lee W S Siu et al ldquoSuppression of mastcell activity contributes to the osteoprotective effect of an herbalformula containing Herba Epimedii Fructus Ligustri Lucidiand Fructus Psoraleaerdquo Journal of Pharmacy and Pharmacologyvol 66 no 3 pp 437ndash444 2014

[4] S CW Sze Y Tong T B Ng C L Y Cheng andH P CheungldquoHerba Epimedii anti-oxidative properties and its medicalimplicationsrdquoMolecules vol 15 no 11 pp 7861ndash7870 2010

[5] R Niu ldquoAction of the drug Herba Epimedii on testosterone ofthe mouse plasma and its accessory sexual organ before andafter processingrdquo China Journal of Chinese Materia Medica vol14 no 9 pp 530ndash574 1989

[6] F-X Liu S Sun and Z-Z Cui ldquoAnalysis of immunologi-cal enhancement of immunosuppressed chickens by Chineseherbal extractsrdquo Journal of Ethnopharmacology vol 127 no 2pp 251ndash256 2010

BioMed Research International 17

[7] J-H Kim Y-J Mun S-J Im J-H Han H-S Lee and W-HWoo ldquoEffects of the aqueous extract of Epimedii Herba on theantibody responses in micerdquo International Immunopharmacol-ogy vol 1 no 5 pp 935ndash944 2001

[8] X Yu Y Tong X-Q Han et al ldquoAnti-angiogenic activity ofHerba Epimedii on zebrafish embryos in vivo and HUVECs invitrordquo Phytotherapy Research vol 27 no 9 pp 1368ndash1375 2013

[9] D-C Zhang J-L Liu Y-B Ding J-G Xia and G-Y ChenldquoIcariin potentiates the antitumor activity of gemcitabine ingallbladder cancer by suppressingNF-120581BrdquoActa PharmacologicaSinica vol 34 no 2 pp 301ndash308 2013

[10] L Kong J Liu J Wang et al ldquoIcariin inhibits TNF-120572IFN-120574induced inflammatory response via inhibition of the substanceP and p38-MAPK signaling pathway in human keratinocytesrdquoInternational Immunopharmacology vol 29 no 2 pp 401ndash4072015

[11] H Shao J Shen M Wang et al ldquoIcariin protects against tita-nium particle-induced osteolysis and inflammatory response ina mouse calvarial modelrdquo Biomaterials vol 60 pp 92ndash99 2015

[12] Q Wei J Zhang G Hong et al ldquoIcariin promotes osteogenicdifferentiation of rat bone marrow stromal cells by activatingthe ER120572-Wnt120573-catenin signaling pathwayrdquo Biomedicine ampPharmacotherapy vol 84 pp 931ndash939 2016

[13] S-Q Zhang W-J Cai J-H Huang et al ldquoIcariin a naturalflavonol glycoside extends healthspan in micerdquo ExperimentalGerontology vol 69 pp 226ndash235 2015

[14] M N Makarova O N Pozharitskaya A N Shikov S VTesakova V G Makarov and V P Tikhonov ldquoEffect of lipid-based suspension of Epimedium koreanum Nakai extract onsexual behavior in ratsrdquo Journal of Ethnopharmacology vol 114no 3 pp 412ndash416 2007

[15] Y Chen J Wang X Jia X Tan and M Hu ldquoRole of intestinalhydrolase in the absorption of prenylated flavonoids present inyinyanghuordquoMolecules vol 16 no 2 pp 1336ndash1348 2011

[16] J J Yu Y J Liu S J Xu S Sun Y J Xu and L Yang ldquoSimulta-neous quantitative determination of four epimediumflavonoidsin rat urine by LC-MSMSrdquo Traditional Chinese Drug Researchamp Clinical Pharmacology vol 27 no 4 pp 537ndash541 2016

[17] Y Lu N Li Y Deng et al ldquoSimultaneous determination oficariin naringin and osthole in rat plasma by UPLC-MSMSand its application for pharmacokinetic study after oral admin-istration of Gushudan capsulesrdquo Journal of Chromatography Bvol 993-994 pp 75ndash80 2015

[18] N Wang X Huang X Wang Y Zhang R Wu and D ShouldquoPipette tip solid-phase extraction and high-performance liquidchromatography for the determination of flavonoids fromEpimedii herba in rat serum and application of the techniqueto pharmacokinetic studiesrdquo Journal of Chromatography B vol990 pp 64ndash72 2015

Submit your manuscripts athttpswwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

16 BioMed Research International

and CO2 MS3 spectrum the fragment ions at 119898119911 350 and321 individually result from the neutral loss of methyl radicaland CO2

The quasimolecular ion [MndashH]minus of M10 is at 119898119911 353146Da less than that of M3 (baohuoside II) The majorfragment ion in its MSMS spectrum is at119898119911 298 matchingthe loss of isobutenyl radical The fragment at 119898119911 219 ispossibly produced by the neutral loss of C8H6O2 (134Da) dueto the retro-Diels-Alder (RDA) fragmentation of the flavonersquosC ring Moreover the MS3 spectrum affords fragment ionsat 119898119911 279 269 253 and 164 resulting from the loss ofH2O CO CO2 and C8H6O2 (134Da) respectively Thus thismetabolite is reasonably identified as desmethyl icaritin

M11 was observed as anothermajormetabolite in rat fecesof both two administration groups (Figure 7) By comparisonwith the reference standard it is identified as icaritin Its MSspectrum yields a quasimolecular ion at119898119911 367 (C21H19O6)And themajor fragment ion at119898119911 352 is characterized as theloss of methyl group The MS3 fragment at 119898119911 309 resultsfrom the loss of methyl group and CO and ion at 119898119911 297is the outcome of the neutral loss of isopentene (70Da) fromthe deprotonated molecule

4 Conclusion

Themenopausal syndrome improvement and possible cancerpreventive activity of Herba Epimedii is receiving more andmore attention Information on the in vivo disposition ofHerba Epimedii components is important for better under-standing the biological effects of this herbal drug For thispurpose our investigation was designed to obtain informa-tion on the plasma pharmacokinetics and tissue distributionof icariin in rat and the excretion of icariin and itsmetabolitesIn this study we firstly established and validated a rapidsensitive and reliable LC-MS method for the determinationof icariin in various biological samples collected at differenttime points and then the method was applied to the plasmapharmacokinetics and tissue distribution study of icariin inrats after oral administration of the total flavonoid extractof Herba Epimedii In addition the differences of the tissuedistribution of icariin in between male and female rats werestudied after oral and intramuscular administration of icariinAnd the metabolic pattern of icariin was also investigatedThe data showed the plasma and tissue concentration oficariin were kept at a very low level at all collection pointswhich suggests icariin is poorly absorbed after oral adminis-trationThepeak concentration in plasma and collected tissuesamples appeared at 05ndash1 h and then quickly dropped at 4 hafter administration demonstrating that a small amount oficariin can be rapidly absorbed and eliminated and there isno accumulation in rat tissues Moreover the concentrationin liver lung and reproductive organs was much higherthan that in other tissues indicating that liver lung andreproductive organs are the main target sites of icariin in rats

The gender-related difference of icariin in tissue distribu-tion has not been reported in the past published literatureOur study demonstrated that the distribution features oficariin are similar in most of tissues between male andfemale rats However there exist a few remarkable gender

differences such that thewhole tissue concentration of icariinin male rats was much higher than female rats except in gen-ital organs (high in female and low in male) In China HerbaEpimedii is a famous tonic and aphrodisiac to invigoratekidney and strengthenYang therefore it is commonly used totreat male sexual functional decline and female menopausalsyndrome As one of the predominant constitutes in HerbaEpimedii icariin plays a very crucial role in exerting effectsso the relatively high icariin concentration in female genitalorgans actually supports the above-mentioned traditionalChinese medicine theory and clinical practice and makesthis compound a good future prospect for treating womenrsquosmenopausal related diseases

In the present study the complete metabolic profile oficariin has also been investigated The results illustrate thatfeces should be the major excretion way for both oral andintramuscular administration of icariin Based on LC-UVand LC-MS119899 analyses eleven metabolites were detected infecal samples and were further identified Among them10 metabolites appeared in rat feces of oral administrationgroup and 9 in the feces of intramuscular introduction groupThe major metabolic routes of clearance are proposed to behydrolysis demethylation oxidation and conjugation

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

Authorsrsquo Contributions

Shunjun Xu and Jiejing Yu contributed equally to this work

References

[1] X Wang Y He B Guo et al ldquoIn vivo screening for anti-osteo-porotic fraction from extract of herbal formula xianlinggubaoin ovariectomized micerdquo PLoS ONE vol 10 no 2 Article IDe0118184 2015

[2] R-H Liu X Kang L-P Xu et al ldquoEffects of the combinedextracts of Herba Epimedii and Fructus Ligustri Lucidi on bonemineral content and bone turnover in osteoporotic ratsrdquo BMCComplementary and Alternative Medicine vol 15 no 1 article112 2015

[3] B C L Chan H Y Lee W S Siu et al ldquoSuppression of mastcell activity contributes to the osteoprotective effect of an herbalformula containing Herba Epimedii Fructus Ligustri Lucidiand Fructus Psoraleaerdquo Journal of Pharmacy and Pharmacologyvol 66 no 3 pp 437ndash444 2014

[4] S CW Sze Y Tong T B Ng C L Y Cheng andH P CheungldquoHerba Epimedii anti-oxidative properties and its medicalimplicationsrdquoMolecules vol 15 no 11 pp 7861ndash7870 2010

[5] R Niu ldquoAction of the drug Herba Epimedii on testosterone ofthe mouse plasma and its accessory sexual organ before andafter processingrdquo China Journal of Chinese Materia Medica vol14 no 9 pp 530ndash574 1989

[6] F-X Liu S Sun and Z-Z Cui ldquoAnalysis of immunologi-cal enhancement of immunosuppressed chickens by Chineseherbal extractsrdquo Journal of Ethnopharmacology vol 127 no 2pp 251ndash256 2010

BioMed Research International 17

[7] J-H Kim Y-J Mun S-J Im J-H Han H-S Lee and W-HWoo ldquoEffects of the aqueous extract of Epimedii Herba on theantibody responses in micerdquo International Immunopharmacol-ogy vol 1 no 5 pp 935ndash944 2001

[8] X Yu Y Tong X-Q Han et al ldquoAnti-angiogenic activity ofHerba Epimedii on zebrafish embryos in vivo and HUVECs invitrordquo Phytotherapy Research vol 27 no 9 pp 1368ndash1375 2013

[9] D-C Zhang J-L Liu Y-B Ding J-G Xia and G-Y ChenldquoIcariin potentiates the antitumor activity of gemcitabine ingallbladder cancer by suppressingNF-120581BrdquoActa PharmacologicaSinica vol 34 no 2 pp 301ndash308 2013

[10] L Kong J Liu J Wang et al ldquoIcariin inhibits TNF-120572IFN-120574induced inflammatory response via inhibition of the substanceP and p38-MAPK signaling pathway in human keratinocytesrdquoInternational Immunopharmacology vol 29 no 2 pp 401ndash4072015

[11] H Shao J Shen M Wang et al ldquoIcariin protects against tita-nium particle-induced osteolysis and inflammatory response ina mouse calvarial modelrdquo Biomaterials vol 60 pp 92ndash99 2015

[12] Q Wei J Zhang G Hong et al ldquoIcariin promotes osteogenicdifferentiation of rat bone marrow stromal cells by activatingthe ER120572-Wnt120573-catenin signaling pathwayrdquo Biomedicine ampPharmacotherapy vol 84 pp 931ndash939 2016

[13] S-Q Zhang W-J Cai J-H Huang et al ldquoIcariin a naturalflavonol glycoside extends healthspan in micerdquo ExperimentalGerontology vol 69 pp 226ndash235 2015

[14] M N Makarova O N Pozharitskaya A N Shikov S VTesakova V G Makarov and V P Tikhonov ldquoEffect of lipid-based suspension of Epimedium koreanum Nakai extract onsexual behavior in ratsrdquo Journal of Ethnopharmacology vol 114no 3 pp 412ndash416 2007

[15] Y Chen J Wang X Jia X Tan and M Hu ldquoRole of intestinalhydrolase in the absorption of prenylated flavonoids present inyinyanghuordquoMolecules vol 16 no 2 pp 1336ndash1348 2011

[16] J J Yu Y J Liu S J Xu S Sun Y J Xu and L Yang ldquoSimulta-neous quantitative determination of four epimediumflavonoidsin rat urine by LC-MSMSrdquo Traditional Chinese Drug Researchamp Clinical Pharmacology vol 27 no 4 pp 537ndash541 2016

[17] Y Lu N Li Y Deng et al ldquoSimultaneous determination oficariin naringin and osthole in rat plasma by UPLC-MSMSand its application for pharmacokinetic study after oral admin-istration of Gushudan capsulesrdquo Journal of Chromatography Bvol 993-994 pp 75ndash80 2015

[18] N Wang X Huang X Wang Y Zhang R Wu and D ShouldquoPipette tip solid-phase extraction and high-performance liquidchromatography for the determination of flavonoids fromEpimedii herba in rat serum and application of the techniqueto pharmacokinetic studiesrdquo Journal of Chromatography B vol990 pp 64ndash72 2015

Submit your manuscripts athttpswwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

BioMed Research International 17

[7] J-H Kim Y-J Mun S-J Im J-H Han H-S Lee and W-HWoo ldquoEffects of the aqueous extract of Epimedii Herba on theantibody responses in micerdquo International Immunopharmacol-ogy vol 1 no 5 pp 935ndash944 2001

[8] X Yu Y Tong X-Q Han et al ldquoAnti-angiogenic activity ofHerba Epimedii on zebrafish embryos in vivo and HUVECs invitrordquo Phytotherapy Research vol 27 no 9 pp 1368ndash1375 2013

[9] D-C Zhang J-L Liu Y-B Ding J-G Xia and G-Y ChenldquoIcariin potentiates the antitumor activity of gemcitabine ingallbladder cancer by suppressingNF-120581BrdquoActa PharmacologicaSinica vol 34 no 2 pp 301ndash308 2013

[10] L Kong J Liu J Wang et al ldquoIcariin inhibits TNF-120572IFN-120574induced inflammatory response via inhibition of the substanceP and p38-MAPK signaling pathway in human keratinocytesrdquoInternational Immunopharmacology vol 29 no 2 pp 401ndash4072015

[11] H Shao J Shen M Wang et al ldquoIcariin protects against tita-nium particle-induced osteolysis and inflammatory response ina mouse calvarial modelrdquo Biomaterials vol 60 pp 92ndash99 2015

[12] Q Wei J Zhang G Hong et al ldquoIcariin promotes osteogenicdifferentiation of rat bone marrow stromal cells by activatingthe ER120572-Wnt120573-catenin signaling pathwayrdquo Biomedicine ampPharmacotherapy vol 84 pp 931ndash939 2016

[13] S-Q Zhang W-J Cai J-H Huang et al ldquoIcariin a naturalflavonol glycoside extends healthspan in micerdquo ExperimentalGerontology vol 69 pp 226ndash235 2015

[14] M N Makarova O N Pozharitskaya A N Shikov S VTesakova V G Makarov and V P Tikhonov ldquoEffect of lipid-based suspension of Epimedium koreanum Nakai extract onsexual behavior in ratsrdquo Journal of Ethnopharmacology vol 114no 3 pp 412ndash416 2007

[15] Y Chen J Wang X Jia X Tan and M Hu ldquoRole of intestinalhydrolase in the absorption of prenylated flavonoids present inyinyanghuordquoMolecules vol 16 no 2 pp 1336ndash1348 2011

[16] J J Yu Y J Liu S J Xu S Sun Y J Xu and L Yang ldquoSimulta-neous quantitative determination of four epimediumflavonoidsin rat urine by LC-MSMSrdquo Traditional Chinese Drug Researchamp Clinical Pharmacology vol 27 no 4 pp 537ndash541 2016

[17] Y Lu N Li Y Deng et al ldquoSimultaneous determination oficariin naringin and osthole in rat plasma by UPLC-MSMSand its application for pharmacokinetic study after oral admin-istration of Gushudan capsulesrdquo Journal of Chromatography Bvol 993-994 pp 75ndash80 2015

[18] N Wang X Huang X Wang Y Zhang R Wu and D ShouldquoPipette tip solid-phase extraction and high-performance liquidchromatography for the determination of flavonoids fromEpimedii herba in rat serum and application of the techniqueto pharmacokinetic studiesrdquo Journal of Chromatography B vol990 pp 64ndash72 2015

Submit your manuscripts athttpswwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Submit your manuscripts athttpswwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of