research article simultaneous determination of ...downloads.hindawi.com/archive/2013/786151.pdf ·...

Hindawi Publishing CorporationISRN Analytical ChemistryVolume 2013 Article ID 786151 7 pageshttpdxdoiorg1011552013786151

Research ArticleSimultaneous Determination of Glycyrrhizin and15 Flavonoids in Licorice and Blood by High PerformanceLiquid Chromatography with Ultraviolet Detector

Yongqian Zhang1 Jin Cao2 Yingping Wang3 and Shengyuan Xiao13

1 School of Life Science Beijing Institute of Technology Beijing 100081 China2 Institute for Food and Cosmetics Control National Institutes for Food and Drug Control Beijing 100050 China3 Institute of Special Wild Economic Animals and Plants Chinese Academic of Agricultural Science Jilin Jilin City 132109 China

Correspondence should be addressed to Shengyuan Xiao shyxiaogmailcom

Received 8 May 2013 Accepted 11 June 2013

Academic Editors P Campıns-Falco and A Taga

Copyright copy 2013 Yongqian Zhang et alThis is an open access article distributed under theCreativeCommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Licorice is the most frequently used herb in traditional Chinese medicine (TCM) with versatile functions It is also a popularnatural dietary supplement While the dosages is very important for there are some side effects caused by licoriceThe compositionof licorice its products should be well determined thereof A simple method for simultaneous determining sixteen compoundsin vary high dynamic range of content has been established This method based on the detection at the characteristic ultravioletspectra of different types of compounds in licorice Glycyrrhizin and fifteen flavonoids were well measured All of these compoundscan be precisely quantified at their characteristic wavelengthsThismethod has been successfully applied to the analyses of differentlicorices Sini Tang decoction and rat plasma after oral administration of Sini Tang decoction These compounds were found to beover 3000 times in content (from 001 120583gg to 345 120583gg) in some samples

1 Introduction

It is well known that herbal prescription is becoming increas-ingly popular as supplementary and alternative medicinein recent years The herbal medicines are believed to bemultitarget therapies in modern medicine [1ndash4] Herbaldrugs usually consist of a large number of ingredients [5]These ingredients might interact with each other during theprocess of decoction absorption metabolism or at physi-ological processes [6ndash9] Multiconstituent detection is veryimportant to uncover these interactions during the processof the herb application It is also important to summarize thecommonbehavior of some constituentswith similar structureduring somebioprocesses which is a crucialmethod to unveilthe bioactive mechanism of a herbal drug [10] Howeverthere are two kinds of obstacles that should be succeeded formultiple compounds detection The first is the large numberof compounds in the herb The second is the high dynamicrange of their contents High resolution and peak capacity areneeded to resolve the overlaid signals of these compounds

In order to estimate the different concentrations of themdetector with very long-range linearity and good selectivityof the detector response should be employed

Licorice is an important herb in oriental medicine It iswidely used to treat gastric ulcer hepatitis C and pulmonaryand skin diseases [11ndash15] Licorice is also a popular naturaldietary supplement [16] According to a statistic investigation[17] on Chinese herbal prescription licorice is the mostfrequently used herb in traditional Chinesemedicine (TCM)It has been proved that flavonoids and triterpenoids are themain bioactive ingredients in licorice whereas licorice wasproved to have side effects that is hypertension [18ndash22] saltand water retention [23] and low potassium levels [24 25] ifit is overdosed The major side effect of licorice is due to thecortisol-like structure of triterpenoids [7 26] The phenoliccompounds in licorice were also proved to play a dual role[27] Isoliquiritigenin has also been found to have dual effectin regulating gastrointestinalmotility It showed spasmogenicand spasmolytic effects in different dosages [27]The contentsof these compounds in licorice and its productions as well

2 ISRN Analytical Chemistry

their dosages should be well controlled thereof On the otherhand phenolic compounds interfere with the metabolismof triterpenoids Phenolic compounds in licorice extract candecrease the bioavailability of glycyrrhizin (G) [7 8] andincrease the excretion ofG into bile in a rat [7] Phenolic com-pounds in licorice can also decrease the hydrolyzation of G inintestine [6] Pure G and Glycyrrhetic acid (GA) have muchhigher risk of hypertension than the total extract of licorice[7] This is due to the metabolic interaction of phenoliccompounds and triterpenoids in the extract Determinationof the pharmacokinetic and metabolic interaction amongthese compounds is very important for the guidance oflicorice application Simultaneous quantification of phenoliccompounds and triterpenoids is therefore strongly neededfor researches on pharmaceutical mechanism of licorice andquality control of its preparations Several attempts havebeen made to satisfy this requirement These great jobshave been well summarized by Zhang and Ye [28] HPLC isusually the most popular tool for natural products analysisThree flavonoids and four triterpenoids in licorice have beendetermined by using a method of HPLC with UV detectionbut had very long run time (more than 140min for one run)[29] Mass spectrometer is always combined with HPLC forcomplex sample analysisThis kind of approach ismuchmoresensitive and selective 42 compounds have been successfullyidentified by a method of HPLC UV TOFMS but only threephenolic compounds and one triterpenoid among them wereidentified unambiguously [30] Eight flavonoids have beensuccessfully determined quantitatively by another HPLCMSMS method [31] HPLC MS is a relatively expensiveinstrument The response range of electrospray ionizationion source (ESI) and some mass analyzers are sometimesnot so broadened for quantification of multiple compoundswith long dynamic range in contents whereas in order tounderstand the mechanism of interactions among the ingre-dients of licorice during the process of decoction absorptionmetabolism or at the physiological process alternations of aseries of compounds with similar structure in these processeswere needed More efficient methods that can quantifymore compounds simultaneously are needed to satisfy thisrequirement

Herein a simple HPLC method based on the differentUV characters of triterpenoids flavones flavans and chal-cones for simultaneous determination of glycyrrhizin and 15flavonoidswas developed and validatedTheir contents in twowild licorices prepared licorice Sini Tang decoction and therat plasma after oral administration of Sini Tang decoctionwere estimated as a proof application

2 Material and Methods

21 Materials and Sample Preparation Two wild licoricesamples were collected fromdifferent environments in north-east of Inner Mongolia by Professor Lijun Han from JilinAgricultural University Changchun Jilin Province Both ofthem were identified to be the root of Glycyrrhiza uralensisFisch The wild licorice samples were dried under ambi-ent temperature Approximately 2 g powder of licorice wasimmersed in 20mL of methanol at ambient temperature

overnight before 30 minutes of treatment by an ultrasonicThe suspensions were filtered through 045 120583m pore sizemembrane and then 20120583L of filtration was finally applied tothe HPLC analysis

Honey-roasted licorice was bought from Beijing TongRen Tang Chinese Medicine Co Ltd It was identified to bethe root of G uralensis Fisch The slice of the licorice wassmashed to small piecesThe smashed honey-roasted-licoricewas then processed in the same procedure with wild licorice

Sini Tang decoction was composed and prepared accord-ing to the method described by Chinese Pharmacopoeia(2010 edition) [32] It consisted of honey-roasted-licoriceRadix Aconiti Carmichaeli and Rhizoma Zingiberis witha ratio of 3 3 2 Both aconite and ginger were boughtfrom China National Group Crop of Traditional and HerbalMedicine Licorice is the same source as the previous Thedecoction was made by three steps The first step is thedecoction of aconite and licorice together 300 g aconiteand 300 g licorice were mixed and immersed into 4000mLwater for 30 minutes Approximately 1500mL supernatantwas taken out from the mixture after 120 minutes of boilingThe same volume of water was added into the residue thenAbout 1600mL supernatant was taken out after the mixturewas boiled for another 90 minutes The second step is thedecoction of zinger 200 g dehydrated zinger was immersedinto 1200mL water for about 1 hour in a 2500mL flask Themixture was refluxed for 120 minutes to collect the volatileoil Approximately 500mL supernatant was taken out fromthe flask at the same time 500mL water was added into thezinger residue after that All supernatants were taken out afteranother 90 minutes of boiling The third step is Sini Tangdecoction composing All supernatants were mixed togetherexcept the volatile oil which was added in to the mixture afterit has been further treated The mixture was concentratedto a volume of about 400mL before 1200mL alcohol (95ethanol) was mixed into the slurry The precipitate wasremoved by filtration after about 24 hours Alcohol wasremoved by distillation under reduced pressure then Thevolatile oil was finally mixed into the extract and then itsvolume was adjusted to 800mL by water to make the finaldecoction 200120583L decoction was diluted to 5mL for analysisThe diluted solution was filtered through a 045 120583m pore sizemembrane 20120583L of filtration was applied to HPLC analysis

Plasma sample was kindly offered by Dr Honhxia LiuThe plasma was collected from male Wistar rats about 350 gweight The rats had been subjected to an overnight fast Theblood was collected after 6 hours [33] of oral administrationof Sini Tang decoction (10mLkg body weight equal to 10 gherb per kg body weight) 1000120583L of plasma was diluted with3000 120583LofmethanolTheprecipitate of proteins in the plasmawas removed by a centrifuge at a speed of 10000 rounds perminute (rpm) The supernatant was dried under a flow ofnitrogen at ambient temperature The residue was dissolvedin 120120583L methanol 20120583L of solution was applied to HPLCanalysis

22 Reagents and Standards Compounds used as referencesshown in Table 1 were isolated and purified by Dr Hong-xia Liu [34 35] The purities of them are all not less then

ISRN Analytical Chemistry 3

Table 1 Glycyrrhizin and 15 flavonoids their electrospray ionization mass and ultraviolet spectra data

Comp no Name 119872119908119877119905min MSMS data UV spectrum

1 Liquiritigenin 41015840-O-glucopyranyl-(1-2)-glucopyranoside 580 104 489 417 255 F2 Sophoraflavone B 416 142 415 253 C3 Liquiritin apioside 550 155 549 417 295 255 F4 Liquiritigenin 741015840-O-diglucopyranoside 580 158 417 255 F5 Liquiritin 418 162 255 135 119 F6 610158401015840-O-a-hydroxypropynoylliquiritin 490 184 417 399 327 255 135 F7 IsoLiquiritin apioside 550 196 549 429 417 357 327 297 255 D8 Isoliquiritin 418 207 297 255 135 119 D9 Kumatakenin B 254 218 253 C10 Medicarpin-3-O-glucoside 432 235 477 431 311 269 G11 Liquiritigenin 256 243 255 153 135 119 F12 Isoliquiritigenin 256 309 255 153 135 119 D13 Formononetin 268 317 267 252 B14 Glycyrrhizin 822 324 803 777 645 627 351 A15 Neoglycyrol 366 410 365 350 307 295 E16 Isoglycyrol 366 450 365 350 E

90 Acetonitrile and methanol (both HPLC grade) wereproduced by Fisher USA Analytical grade formic acid waspurchased from Beijing Chemical Factory

23 Chromatography and ESI MS Analysis HPLC UV ESIMSMS was carried on an Agilent 1100 series LC MSD Trapequipped with a diode array detector a binary pump adegasser anRheodyne valvemanual sampler and anESI TrapMS A gradient mobile phase was applied on a PhenomenexLuna C18 column (46 times 250mm 5 120583m) for separation Themobile phase consisted of water (with 01 formic acid A)and acetonitrile (with 01 formic acid B) The gradient was0 B at 0 to 3 minutes the ratio of B increased linearly to100 in 57 minutes then The flow rate was 08mLminHPLC chromatograms were recorded at 205 nm 254 nm280 nm 310 nm and 355 nm with a gate width of 16 nmsimultaneously in each run UV spectra were obtained from190 to 400 nm

ESI MSMS analysis was acquired in negative mode Thefluent was split about 200120583L of eluent was introduced intothe mass spectrometer per minute The pressure of nebulizergas was 021 mega-Pascal (MPa) The flow rate of dry gas was8 Lmin and its temperature was 325∘C The scan range ofMS was from 100 to 1200 atom mass unit (amu) The targetion accumulated in the trapwas 30000 countsThemaximumaccumulation time was 50millisecondsThe parameter of thetrap was set in a smart modeThe target ion was119898119911 350 thecompound stability was 100 the trap drive level was 80An auto MS2 mode was applied to obtain MSMS spectraof different compounds The MSMS parameter was set asfollows mass range of parent ion was from 100 to 1200 amu 1precursor was selected each cycle each precursor would beexcluded after 3 spectra the release time was 05 minutesthe fragmentation voltage was optimized automatically from03 to 20 electrical volt the fragmentation time was 20

Wavelength (nm)220 240 260 280 300 320 340 360 380

Abso

rban

ce (m

AU) (c)

(d)

(g)

(f)

(e)

(b)

(a)

400

Figure 1 Ultraviolet spectra for different types of compounds(a) Glycyrrhizin (b) Formononetin (c) Sophoraflavone B andKumatakenin B (d) Isoliquiritin apioside Isoliquiritin and Isoli-quiritigenin (e) Neoglycyrol and Isoglycyrol (f) 41015840-O-glucopyran-yl-(1-2)-Liquiritigenin-glucopyranoside Liquiritin apioside 741015840-O-Liquiritigenin-diglucopyranoside Liquiritin 610158401015840-O-a-hydroxypro-pynoylliquiritin and Liquiritigenin (g) Medicarpin-3-O-glucoside

milliseconds the activation width was 10 amu the cutoff fordaughter ion range was 27

3 Results and Discussion

31 UV Spectrum Characters of the Target Analytes All targetanalytes are shown in Table 1 These compounds can bedivided into three groups according to their characters ofUV spectroscopy Their representative of UV spectra is alsoshown in Table 1 The representative ultraviolet spectrum ofeach type is shown in Figure 1 The first group consists ofFormononetin (13) and Glycyrrhizin (14) (spectra A and B)Their absorption peaks are both at approximately 254 nmThe following seven compounds can be included in thesecond group Sophoraflavone B (2) and Kumatakenin B (9)


0 10 20 30 40 50 60

30 40 50 601

7

8

9

9

12

1315

16

25 30

1011

14

1 2

35

10 14 18

6

UV

(mAU

)

(a) 280 nm (b) 254 nm

(c) 255 nm

(d) 355 nm

T (min)

Figure 2 Representative chromatograms for licorice Compounds1 2 3 4 5 and 6were shown in a chromatogram recorded at 280 nmcompounds 7 8 9 12 13 14 and 16 were shown in a chromatogramrecorded at 355 nm compounds 10 11 and 14 were shown in achromatogram recorded at 254 nm all chromatograms shown wererecorded in the same run

Isoliquiritin apioside (7) Isoliquiritin (8) Isoliquiritigenin(12) Neoglycyrol (15) and Isoglycyrol (16) (2 and 9 spec-tra C 7 8 and 12 spectra D 15 and 16 spectra E) Themaximum UV absorption of these compounds ranged from330 nm to 370 nm at a much longer wavelength where thecompounds from the other two groups have no absorbanceAdditionally all compounds in the second group have anabsorption valley at about 280 nm The third group includesmainly liquiritigenin and its derivatives (1 3sim6 and 11) anda flavan (Medicarpin-3-O-glucoside 10) These compoundshave maximum a absorption at about 280 nm the absorptionvalley of compounds in group two as well as an absorptionvalley at approximately 254 nm the maximum absorptionof compounds in group one In view of this compoundsfrom different groups can be distinguished from each otherby detecting at different wavelengths 254 280 and 355 nmwhen they are coeluted

32 Separation and Selectivity Representative chromatogramof all 16 target analytes in licorice is shown in Figure 2All 16 compounds can be the baseline separated from thebackground constituents in licorice in a related short runtime (60min) when the detector response was recordedat a certain wavelength for each type of compound Theselectivity of the method was further proved by HPLC ESIMS analysis and purity analyses of HPLC UV peaks TheMSMS data of 16 target analytes is shown in Table 1 Peakpurities of all target analytes were larger than 950 at the rangeof their detection wavelengths Peak purities of compounds2 3 5 6 7 8 14 15 and 16 were also proved by the MSspectra obtained at the range of the peaks Compound 1 wascoeluted with a flavone-C-glycoside (according to its MSMSdata [36 37]) The ultraviolet spectrum of a flavone is Ctype (Figure 1) which may interfere with the detection ofcompound 1 (spectrum F) theoretically However the purityanalysis of chromatographic peak of compound 1 indicated

23 24 25

10

11

220 240 260 280 300 320 340 360 380

10

11

Wavelength (nm)

mAU

Figure 3 The representative chromatograms and their UV spectraforMedicarpin-3-O-glucoside (10) and Liquiritigenin (11) (a) Chro-matogram recorded at 205 nm (b) Overlay of UV spectra at 5 datapoints on each chromatographic peak at 205 nm for compounds 10and 11

that all spectra in the range of the peak were well overlappedThe purity of the chromatographic peak of compound 1 wastested at wave width that ranged from 200 to 400 nm witha purity factor of more than 950 The coeluted flavone-C-glycoside did not affect the detection This may be becausethe concentration of the coeluted flavone was very lowcompound 1 can be precisely determined in this distancecompounds 3 and 5 may interfere with the detection ofcompound 4 a little The UV spectrum of compound 4 isthe same as them so it is not able to be distinguished fromcompounds 3 and 5 by detecting at different wavelengths Butthey exhibited near the baseline separation The resolutionfactors of compounds 4 to 3 and 5 are 141sim152 and 243sim244 respectivelyThe chromatographic peaks of compounds12 and 13 were partly overlapped with that of compound 14(glycyrrhizin) Compounds 12 and 13 can be precisely quanti-tatively determined without interference from compound 14by detecting at 355 and 310 nm where compound 14 has noabsorbance Concentration of compound 14wasmuch higherin the samples than the other two the estimation of its contentwas not affected by coeluted compounds 12 and 13 Ionsrepresent glycyrrhizin type compounds (the production of adaughter ion 119898119911 351 which represents the structure of theglycoside chain [38]) that were recognized in the MS spectraof compounds 9 (119898119911 823 837 969 and 989) 10 (119898119911 809821 and 983) 11 (119898119911 821 and 983) but the detection of thesecompounds was not interfered by the coeluted constituentsby detecting at 355 nm 280 nm and 280 nm respectivelyHowever Medicarpin-3-O-glucoside (10) was estimated withits detector response at 205 nm because its detector responseat 280 nm is too weak to be recognized in real samples for itslow concentration The representative chromatogram for 10as well as its peak purity analysis is shown in Figure 3

33 Linearity Recovery and Precision The detector responseagainst compound mass perinjection was used to establishthe linearity of the method Due to the large dynamic contentrange of those compounds in licorice the linearity coveredranges for different compounds were quite a little differentfrom each other The linearity experiment for each analyte


Table 2 Linearity and recovery of all target analytes

No Linearity (119899 = 3) Recovery ( 119899 = 2) Wave length (nm)Slop Intercept 119877

2 Range (120583ginj)1 399310 plusmn 01047 minus59005 plusmn 14168 09964 plusmn 000107 0122sim9760 907 plusmn 14 2802 1000500 plusmn 01735 minus161570 plusmn 25922 09991 plusmn 000045 0135sim10800 924 plusmn 34 3103 3127770 plusmn 05550 minus1081800 plusmn 317830 09998 plusmn 000006 0550sim55000 977 plusmn 33 2804 1787300 plusmn 03786 minus824700 plusmn 76173 09934 plusmn 000038 0186sim18600 887 plusmn 38 2805 5132200 plusmn 05100 minus1405100 plusmn 150200 09995 plusmn 000006 0282sim28200 976 plusmn 18 2806 867590 plusmn 01000 minus348500 plusmn 15015 09896 plusmn 000010 0166sim16600 933 plusmn 10 2807 1217400 plusmn 00404 minus262100 plusmn 10551 09985 plusmn 000010 0240sim19200 918 plusmn 02 3558 5114600 plusmn 03550 416910 plusmn 116620 09964 plusmn 000000 0295sim23600 1003 plusmn 14 3559 753170 plusmn 01334 minus104140 plusmn 47618 09989 plusmn 000030 0338sim27040 968 plusmn 19 35510 643160 plusmn 00153 minus65371 plusmn 17859 09990 plusmn 000021 0263sim21040 949 plusmn 04 20511 1647600 plusmn 01429 minus188230 plusmn 41025 09988 plusmn 000015 0265sim21200 942 plusmn 02 28012 4054600 plusmn 00900 minus884210 plusmn 81045 09993 plusmn 000031 0260sim13000 967 plusmn 16 35513 739750 plusmn 00976 minus81110 plusmn 23016 09988 plusmn 000023 0251sim20080 948 plusmn 07 31014 8407600 plusmn 01600 minus451990 plusmn 20570 09977 plusmn 000000 0123sim12300 1053 plusmn 04 25415 1005700 plusmn 01375 minus109290 plusmn 28705 09988 plusmn 000025 0194sim15520 954 plusmn 14 35516 934020 plusmn 58669 minus25702 plusmn 08098 09988 plusmn 000020 0071sim5680 947 plusmn 11 355

10 15 20 25 30

30 3510 15 20 25

3

5

116

14

7

8

12

UV

(mAU

)

(a) 280 nm (b) 254 nm

(c) 355 nm

T (min)

Figure 4 Representative chromatograms for plasma from ratadministered Sini Tang decoction Compounds 3 5 6 and 11were shown in a chromatogram recorded at 280 nm compounds7 8 and 12 were shown in a chromatogram recorded at 355 nmcompound 14 was shown in a chromatogram recorded at 254 nmall chromatograms shown were recorded in the same run

was triplicate The fit of linearity for each compound wasgood The linearity correlation coefficients (1198772) of all 16target analytes varied from 09896 to 09998 with standardderivations ranging from 0000 to 0107 respectively(shown in Table 2) The slope of each compound was stable(RSD 0019sim6281 119899 = 3)

The recovery of the methanol extracting method wasestimated by adding known amounts of compounds into thepowder of licoriceThe difference between spiked licorice andnonspiked licorice was used to define the recovery of themethodThe recoveries of all 16 target analytes are also shown

in Table 2The average recoveries were quite close to 100 foreach compound at the detected amount

The relative standard derivation (RSD) of the three analy-ses of the mixture of 16 compounds was used for determiningthe precision of the method According to the results shownin Table 2 the precision of the method was good None ofthem was more than 63 when the injected amounts forcompounds 1 to 16 were 0122 0270 2750 0372 1410 01660480 1475 0135 0263 0265 1300 0100 2460 0097 and0028120583g per injection respectively

34 Determination of 16 Target Analytes in Licorices andSini Tang Decoction Figure 3 shows the representative chro-matograms of different licorices The separation and selec-tivity of the detection are stated in Section 32 The con-centrations of these compounds were determined by com-paring with the external references The contents of all 16compounds in two wild licorices prepared licorice and SiniTang decoction are shown in Table 3 The highest contentis glycyrrhizin in one of the wild licorices The minimumcontent detected is Formononetin in Sini Tang decoctionTheamount of glycyrrhizin in wild licorice was consistent withthe results in the literature [29 39]

35 Determination of Constituents from Licorice in RatPlasma The constituent from licorice in rat plasma whichwas orally administered Sini Tang decoction was also esti-mated in this work The experiment was carried out onlyone time Seven constituents including Liquiritin apioside(3) Liquiritin (5) Isoliquiritin apioside (7) Isoliquiritin (8)Liquiritigenin (11) Isoliquiritigenin (12) and Glycyrrhizin(14) were unambiguously detected (Figure 4) Their con-centrations were estimated by comparing to the externalreferences (shown in Table 3) Nine of 16 target analytes hadnot been detected in the plasmaThismay be because the dose


Table 3 Contents of all 16 compounds in different sample (119899 = 3)

No licorice 1(mgg)

licorice 2(mgg)

R-licorice(mgg)

gly-D(mgg) sini-Dlowast (mgg) Serum

(mgL)gly-Dgly-E

()sinigly-E

()1 3296 plusmn 0302 1536 plusmn 0100 0526 plusmn 0034 0352 plusmn 0032 0576 plusmn 0037 nd 669 plusmn 57 1095 plusmn 75

2 0238 plusmn 0251 0166 plusmn 0200 0580 plusmn 0076 0113 plusmn 0004 0077 plusmn 0010 nd 195 plusmn 07 132 plusmn 17

3 12382 plusmn 1110 13892 plusmn 0489 8587 plusmn 0302 1796 plusmn 0161 3843 plusmn 0135 1190 209 plusmn 19 448 plusmn 16



6 nd nd 0143 plusmn 0005 0207 plusmn 0014 0082 plusmn 0003 0127 1446 plusmn 71 573 plusmn 21




10 nd 2059 plusmn 0023 0026 plusmn 0000 0006 plusmn 0000 0041 plusmn 0000 nd 253 plusmn 50 1607 plusmn 50


12 0833 plusmn 0016 nd 0371 plusmn 0012 0125 plusmn 0002 0364 plusmn 0007 0357 337 plusmn 27 982 plusmn 27




16 nd nd 0087 plusmn 0015 0006 plusmn 0000 0004 plusmn 0000 nd 72 plusmn 26 51 plusmn 30

lowastContents were estimated based on the mass of licorice in the mixture R-licorice honey-roasted-licorice Sini-D decoction of Sini Tang

administered should be a little low for plasma metabolitesanalysis requirements according to the previous work [5 33]But this work provided a stable proof in which this methodcan be used for simultaneous determination of the abovementioned compounds in biosample

4 Conclusion

A simple and reproducible HPLC UV method was reportedfor simultaneous determination of sixteen constituents inlicorice and its products This method was proved to be fitfor biosample analysis It will provide a tool for chemical andpharmacological studies of licorice

Conflict of Interests

All herbal materials used in the current research werepurchased from local markets with no conflict of interestsrelated to the manufacturers

Authorsrsquo Contribution

Yongqian Zhang and Jin Cao had equal contribution in thispaper

Acknowledgments

This work was supported by the Beijing Institute of Technol-ogy Basic Research Foundation no 20091642014 and JilinProvince Science and Technology Development Foundation(20096040)

References

[1] VNandakumar T Singh and S K Katiyar ldquoMulti-targeted pre-vention and therapy of cancer by proanthocyanidinsrdquo CancerLetters vol 269 no 2 pp 378ndash387 2008

[2] A Goel S Jhurani and B B Aggarwal ldquoMulti-targeted therapyby curcumin how spicy is itrdquo Molecular Nutrition and FoodResearch vol 52 no 9 pp 1010ndash1030 2008

[3] H Wagner and G Ulrich-Merzenich ldquoSynergy researchapproaching a new generation of phytopharmaceuticalsrdquo Phy-tomedicine vol 16 no 2-3 pp 97ndash110 2009

[4] H Wagner ldquoSynergy research approaching a new generationof phytopharmaceuticalsrdquo Fitoterapia vol 82 no 1 pp 34ndash372011

[5] C Xiang X Qiao Q Wang et al ldquoFrom single compoundsto herbal extract a strategy to systematically characterize themetabolites of licorice in ratsrdquo Drug Metabolism and Disposi-tion vol 39 no 9 pp 1597ndash1608 2011

[6] Y-C Hou S-L Hsiu H Ching et al ldquoProfound differenceof metabolic pharmacokinetics between pure glycyrrhizin andglycyrrhizin in licorice decoctionrdquo Life Sciences vol 76 no 10pp 1167ndash1176 2005

[7] G Cantelli-Forti M A Raggi F Bugamelli F Maffei A Villariand N M Trieff ldquoToxicological assessment of liquorice biliaryexcretion in ratsrdquo Pharmacological Research vol 35 no 5 pp463ndash470 1997

[8] M A Raggi F Maffei F Bugamelli and G Cantelli FortildquoBioavailability of glycyrrhizin and licorice extract in rat andhuman plasma as detected by a HPLCmethodrdquoDie Pharmazievol 49 no 4 pp 269ndash272 1994

[9] S-P Lin S-Y Tsai Y-C Hou and P-D L Chao ldquoGly-cyrrhizin and licorice significantly affect the pharmacokineticsof methotrexate in ratsrdquo Journal of Agricultural and FoodChemistry vol 57 no 5 pp 1854ndash1859 2009


[10] S-Y Xiao H-X Liu W-H Lin and J-S Yang ldquoDiarylhep-tanoids in rhizoma zingiberis and their stereoslective reactionsduring the process of decoctingrdquo Chinese Journal of AnalyticalChemistry vol 35 no 9 pp 1295ndash1300 2007

[11] M V Palagina N S Dubnyak I N Dubnyak and P S ZorikovldquoCorrection of respiratory organ impairment with ural licoricepreparations in chronic skin diseasesrdquo Terapevticheskii Arkhivvol 75 no 1 pp 63ndash65 2003

[12] B Liu J Yang Q Wen and Y Li ldquoIsoliquiritigenin a flavonoidfrom licorice relaxes guinea-pig tracheal smooth muscle invitro and in vivo role of cGMPPKG pathwayrdquo EuropeanJournal of Pharmacology vol 587 no 1ndash3 pp 257ndash266 2008

[13] B Jayaprakasam S Doddaga R Wang D Holmes J Goldfarband X-M Li ldquoLicorice flavonoids inhibit eotaxin-1 secretion byhuman fetal lung fibroblasts in vitrordquo Journal of Agricultural andFood Chemistry vol 57 no 3 pp 820ndash825 2009

[14] Y-C Xie X-W Dong X-M Wu X-F Yan and Q-M XieldquoInhibitory effects of flavonoids extracted from licorice onlipopolysaccharide-induced acute pulmonary inflammation inmicerdquo International Immunopharmacology vol 9 no 2 pp 194ndash200 2009

[15] A Agarwal D Gupta G Yadav P Goyal P K Singh and USingh ldquoAn evaluation of the efficacy of licorice gargle for atten-uating postoperative sore throat a prospective randomizedsingle-blind studyrdquo Anesthesia and Analgesia vol 109 no 1 pp77ndash81 2009

[16] M Katayama T Fukuda T Okamura et al ldquoEffect of dietaryaddition of seaweed and licorice on the immune performanceof pigsrdquoAnimal Science Journal vol 82 no 2 pp 274ndash281 2011

[17] H Chang B-B Su Y-P Zhou and D-R He ldquoAssortativity andact degree distribution of some collaboration networksrdquoPhysicaA vol 383 no 2 pp 687ndash702 2007

[18] M Schambelan ldquoLicorice ingestion and blood pressure regulat-ing hormonesrdquo Steroids vol 59 no 2 pp 127ndash130 1994

[19] B R Walker and C R W Edwards ldquoLicorice-induced hyper-tension and syndromes of apparent mineralocorticoid excessrdquoEndocrinology andMetabolism Clinics of North America vol 23no 2 pp 359ndash377 1994

[20] C Schulze zur Wiesch N Sauer and J Aberle ldquoHyperten-sion and hypokalemiamdasha reninoma as the cause of suspectedliquorice-induced arterial hypertensionrdquo Deutsche Medizinis-che Wochenschrift vol 136 no 17 pp 882ndash884 2011

[21] H E Miettinen K Piippo T Hannila-Handelberg et alldquoLicorice-induced hypertension and common variants of genesregulating renal sodium reabsorptionrdquo Annals of Medicine vol42 no 6 pp 465ndash474 2010

[22] P Pant L Nadimpalli M Singh and J C Cheng ldquoA caseof severe hypokalemic paralysis and hypertension Licorice-induced hypokalemic paralysisrdquo American Journal of KidneyDiseases vol 55 no 6 pp A35ndashA37 2010

[23] A S Cooney and J T Fitzsimons ldquoIncreased sodium appetiteand thirst in rat induced by the ingredients of liquoriceglycyrrhizic acid and glycyrrhetinic acidrdquo Regulatory Peptidesvol 66 no 1-2 pp 127ndash133 1996

[24] N Mumoli and M Cei ldquoLicorice-induced hypokalemiardquo Inter-national Journal of Cardiology vol 124 no 3 pp e42ndashe44 2008

[25] C Noguchi J Yang K Sakamoto et al ldquoInhibitory effects ofisoliquiritigenin and licorice extract on voltage-dependent K+currents in H9c2 cellsrdquo Journal of Pharmacological Sciences vol108 no 4 pp 439ndash445 2008

[26] J Hukkanen O Ukkola and M J Savolainen ldquoEffects of low-dose liquorice alone or in combination with hydrochloroth-iazide on the plasma potassium in healthy volunteersrdquo BloodPressure vol 18 no 4 pp 192ndash195 2009

[27] G Chen L Zhu Y Liu Q Zhou H Chen and J YangldquoIsoliquiritigenin a flavonoid from licorice plays a dual rolein regulating gastrointestinal motility in vitro and in vivordquoPhytotherapy Research vol 23 no 4 pp 498ndash506 2009

[28] Q Zhang and M Ye ldquoChemical analysis of the Chinese herbalmedicine Gan-Cao (licorice)rdquo Journal of Chromatography Avol 1216 no 11 pp 1954ndash1969 2009

[29] Y-C Wang and Y-S Yang ldquoSimultaneous quantification offlavonoids and triterpenoids in licorice using HPLCrdquo Journalof Chromatography B vol 850 no 1-2 pp 392ndash399 2007

[30] G Tan Z Zhu H Zhang et al ldquoAnalysis of phenolic andtriterpenoid compounds in licorice and rat plasma by high-performance liquid chromatography diode-array detectiontime-of-flightmass spectrometry and quadrupole ion trapmassspectrometryrdquo Rapid Communications in Mass Spectrometryvol 24 no 2 pp 209ndash218 2010

[31] L Li S Liang F Du and C Li ldquoSimultaneous quantificationof multiple licorice flavonoids in rat plasmardquo Journal of theAmerican Society for Mass Spectrometry vol 18 no 4 pp 778ndash782 2007

[32] ldquoSini Tangrdquo in Chinese Pharmacopoeia Chinese Pharma-copoeia Committee Ed p 650 Chinese Press of Pharmaceuti-cal andMedicinal Science andTechnology Beijing China 2010

[33] Y Ozaki M Noguchi H Kamakura and M Harada ldquoStudieson concentration of glycyrrhizin in plasma and its absorptionafter oral administration of licorice extract and glycyrrhizinrdquoYakugaku Zasshi vol 110 no 1 pp 77ndash81 1990

[34] H-X Liu W-H Lin X-L Wang and J-S Yang ldquoFlavonoidsfrom preparation of traditional Chinese medicines named Sini-Tangrdquo Journal of Asian Natural Products Research vol 7 no 2pp 139ndash143 2005

[35] H-X Liu W-H Lin and J-S Yang ldquoStudies on chemicalconstituents of sini tangrdquo China Journal of Chinese MateriaMedica vol 29 no 5 pp 434ndash436 2004

[36] A P Rauter A Martins C Borges et al ldquoLiquidchromatography-diode array detection-electrospray ionisationmass spectrometrynuclear magnetic resonance analyses ofthe anti-hyperglycemic flavonoid extract of Genista tenerastructure elucidation of a flavonoid-C-glycosiderdquo Journal ofChromatography A vol 1089 no 1-2 pp 59ndash64 2005

[37] PWaridel J-L Wolfender K Ndjoko K R Hobby H J Majorand K Hostettmann ldquoEvaluation of quadrupole time-of-flighttandem mass spectrometry and ion-trap multiple-stage massspectrometry for the differentiation of C-glycosidic flavonoidisomersrdquo Journal of Chromatography A vol 926 no 1 pp 29ndash41 2001

[38] M Jin Y Yang B Su and Q Ren ldquoDetermination of soyas-aponins Ba andBb in human serumbyhigh-performance liquidchromatography coupled with electrospray ionization tandemmass spectrometryrdquo Journal of Chromatography B vol 846 no1-2 pp 169ndash175 2007

[39] S Tsukamoto M Aburatani T Yoshida Y Yamashita A A El-Beih and T Ohta ldquoCYP3A4 inhibitors isolated from licoricerdquoBiological and Pharmaceutical Bulletin vol 28 no 10 pp 2000ndash2002 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy


Carbohydrate Chemistry

International Journal of


Journal of

Chemistry


Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of


Chromatography Research International


Applied ChemistryJournal of



Theoretical ChemistryJournal of


Journal of

Spectroscopy

Analytical ChemistryInternational Journal of


Journal of


Quantum Chemistry


Organic Chemistry International

ElectrochemistryInternational Journal of



CatalystsJournal of


their dosages should be well controlled thereof On the otherhand phenolic compounds interfere with the metabolismof triterpenoids Phenolic compounds in licorice extract candecrease the bioavailability of glycyrrhizin (G) [7 8] andincrease the excretion ofG into bile in a rat [7] Phenolic com-pounds in licorice can also decrease the hydrolyzation of G inintestine [6] Pure G and Glycyrrhetic acid (GA) have muchhigher risk of hypertension than the total extract of licorice[7] This is due to the metabolic interaction of phenoliccompounds and triterpenoids in the extract Determinationof the pharmacokinetic and metabolic interaction amongthese compounds is very important for the guidance oflicorice application Simultaneous quantification of phenoliccompounds and triterpenoids is therefore strongly neededfor researches on pharmaceutical mechanism of licorice andquality control of its preparations Several attempts havebeen made to satisfy this requirement These great jobshave been well summarized by Zhang and Ye [28] HPLC isusually the most popular tool for natural products analysisThree flavonoids and four triterpenoids in licorice have beendetermined by using a method of HPLC with UV detectionbut had very long run time (more than 140min for one run)[29] Mass spectrometer is always combined with HPLC forcomplex sample analysisThis kind of approach ismuchmoresensitive and selective 42 compounds have been successfullyidentified by a method of HPLC UV TOFMS but only threephenolic compounds and one triterpenoid among them wereidentified unambiguously [30] Eight flavonoids have beensuccessfully determined quantitatively by another HPLCMSMS method [31] HPLC MS is a relatively expensiveinstrument The response range of electrospray ionizationion source (ESI) and some mass analyzers are sometimesnot so broadened for quantification of multiple compoundswith long dynamic range in contents whereas in order tounderstand the mechanism of interactions among the ingre-dients of licorice during the process of decoction absorptionmetabolism or at the physiological process alternations of aseries of compounds with similar structure in these processeswere needed More efficient methods that can quantifymore compounds simultaneously are needed to satisfy thisrequirement

Herein a simple HPLC method based on the differentUV characters of triterpenoids flavones flavans and chal-cones for simultaneous determination of glycyrrhizin and 15flavonoidswas developed and validatedTheir contents in twowild licorices prepared licorice Sini Tang decoction and therat plasma after oral administration of Sini Tang decoctionwere estimated as a proof application

2 Material and Methods

21 Materials and Sample Preparation Two wild licoricesamples were collected fromdifferent environments in north-east of Inner Mongolia by Professor Lijun Han from JilinAgricultural University Changchun Jilin Province Both ofthem were identified to be the root of Glycyrrhiza uralensisFisch The wild licorice samples were dried under ambi-ent temperature Approximately 2 g powder of licorice wasimmersed in 20mL of methanol at ambient temperature

overnight before 30 minutes of treatment by an ultrasonicThe suspensions were filtered through 045 120583m pore sizemembrane and then 20120583L of filtration was finally applied tothe HPLC analysis

Honey-roasted licorice was bought from Beijing TongRen Tang Chinese Medicine Co Ltd It was identified to bethe root of G uralensis Fisch The slice of the licorice wassmashed to small piecesThe smashed honey-roasted-licoricewas then processed in the same procedure with wild licorice

Sini Tang decoction was composed and prepared accord-ing to the method described by Chinese Pharmacopoeia(2010 edition) [32] It consisted of honey-roasted-licoriceRadix Aconiti Carmichaeli and Rhizoma Zingiberis witha ratio of 3 3 2 Both aconite and ginger were boughtfrom China National Group Crop of Traditional and HerbalMedicine Licorice is the same source as the previous Thedecoction was made by three steps The first step is thedecoction of aconite and licorice together 300 g aconiteand 300 g licorice were mixed and immersed into 4000mLwater for 30 minutes Approximately 1500mL supernatantwas taken out from the mixture after 120 minutes of boilingThe same volume of water was added into the residue thenAbout 1600mL supernatant was taken out after the mixturewas boiled for another 90 minutes The second step is thedecoction of zinger 200 g dehydrated zinger was immersedinto 1200mL water for about 1 hour in a 2500mL flask Themixture was refluxed for 120 minutes to collect the volatileoil Approximately 500mL supernatant was taken out fromthe flask at the same time 500mL water was added into thezinger residue after that All supernatants were taken out afteranother 90 minutes of boiling The third step is Sini Tangdecoction composing All supernatants were mixed togetherexcept the volatile oil which was added in to the mixture afterit has been further treated The mixture was concentratedto a volume of about 400mL before 1200mL alcohol (95ethanol) was mixed into the slurry The precipitate wasremoved by filtration after about 24 hours Alcohol wasremoved by distillation under reduced pressure then Thevolatile oil was finally mixed into the extract and then itsvolume was adjusted to 800mL by water to make the finaldecoction 200120583L decoction was diluted to 5mL for analysisThe diluted solution was filtered through a 045 120583m pore sizemembrane 20120583L of filtration was applied to HPLC analysis

Plasma sample was kindly offered by Dr Honhxia LiuThe plasma was collected from male Wistar rats about 350 gweight The rats had been subjected to an overnight fast Theblood was collected after 6 hours [33] of oral administrationof Sini Tang decoction (10mLkg body weight equal to 10 gherb per kg body weight) 1000120583L of plasma was diluted with3000 120583LofmethanolTheprecipitate of proteins in the plasmawas removed by a centrifuge at a speed of 10000 rounds perminute (rpm) The supernatant was dried under a flow ofnitrogen at ambient temperature The residue was dissolvedin 120120583L methanol 20120583L of solution was applied to HPLCanalysis

22 Reagents and Standards Compounds used as referencesshown in Table 1 were isolated and purified by Dr Hong-xia Liu [34 35] The purities of them are all not less then








Wavelength (nm)220 240 260 280 300 320 340 360 380

Abso

rban

ce (m

AU) (c)

(d)

(g)

(f)

(e)

(b)

(a)

400






0 10 20 30 40 50 60

30 40 50 601

7

8

9

9

12

1315

16

25 30

1011

14

1 2

35

10 14 18

6

UV

(mAU

)

(a) 280 nm (b) 254 nm

(c) 255 nm

(d) 355 nm

T (min)




23 24 25

10

11

220 240 260 280 300 320 340 360 380

10

11

Wavelength (nm)

mAU








10 15 20 25 30

30 3510 15 20 25

3

5

116

14

7

8

12

UV

(mAU

)

(a) 280 nm (b) 254 nm

(c) 355 nm

T (min)










No licorice 1(mgg)

licorice 2(mgg)

R-licorice(mgg)


(mgL)gly-Dgly-E

()sinigly-E



















4 Conclusion






Acknowledgments


References


















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of








Wavelength (nm)220 240 260 280 300 320 340 360 380

Abso

rban

ce (m

AU) (c)

(d)

(g)

(f)

(e)

(b)

(a)

400






0 10 20 30 40 50 60

30 40 50 601

7

8

9

9

12

1315

16

25 30

1011

14

1 2

35

10 14 18

6

UV

(mAU

)

(a) 280 nm (b) 254 nm

(c) 255 nm

(d) 355 nm

T (min)




23 24 25

10

11

220 240 260 280 300 320 340 360 380

10

11

Wavelength (nm)

mAU








10 15 20 25 30

30 3510 15 20 25

3

5

116

14

7

8

12

UV

(mAU

)

(a) 280 nm (b) 254 nm

(c) 355 nm

T (min)










No licorice 1(mgg)

licorice 2(mgg)

R-licorice(mgg)


(mgL)gly-Dgly-E

()sinigly-E



















4 Conclusion






Acknowledgments


References


















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


0 10 20 30 40 50 60

30 40 50 601

7

8

9

9

12

1315

16

25 30

1011

14

1 2

35

10 14 18

6

UV

(mAU

)

(a) 280 nm (b) 254 nm

(c) 255 nm

(d) 355 nm

T (min)




23 24 25

10

11

220 240 260 280 300 320 340 360 380

10

11

Wavelength (nm)

mAU








10 15 20 25 30

30 3510 15 20 25

3

5

116

14

7

8

12

UV

(mAU

)

(a) 280 nm (b) 254 nm

(c) 355 nm

T (min)










No licorice 1(mgg)

licorice 2(mgg)

R-licorice(mgg)


(mgL)gly-Dgly-E

()sinigly-E



















4 Conclusion






Acknowledgments


References


















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of





10 15 20 25 30

30 3510 15 20 25

3

5

116

14

7

8

12

UV

(mAU

)

(a) 280 nm (b) 254 nm

(c) 355 nm

T (min)










No licorice 1(mgg)

licorice 2(mgg)

R-licorice(mgg)


(mgL)gly-Dgly-E

()sinigly-E



















4 Conclusion






Acknowledgments


References


















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of



No licorice 1(mgg)

licorice 2(mgg)

R-licorice(mgg)


(mgL)gly-Dgly-E

()sinigly-E



















4 Conclusion






Acknowledgments


References


















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of









































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

research article simultaneous determination of ...downloads.hindawi.com/archive/2013/786151.pdf ·...

Documents