simultaneous qualitation and quantification of four phytoecdysones in radix achyranthis bidentatae...

Copyright © 2007 John Wiley & Sons, Ltd. Biomed. Chromatogr. 21: 823– 828 (2007)DOI: 10.1002/bmc

Simultaneous qualitation and quantification of four phytoecdysones 823ORIGINAL RESEARCHORIGINAL RESEARCH

Copyright © 2007 John Wiley & Sons, Ltd.

BIOMEDICAL CHROMATOGRAPHYBiomed. Chromatogr. 21: 823–828 (2007)Published online 12 April 2007 in Wiley InterScience(www.interscience.wiley.com) DOI: 10.1002/bmc.824

Simultaneous qualitation and quantification of fourphytoecdysones in Radix Achyranthis Bidentatae byhigh-performance liquid chromatography with diodearray detection

Juan Li, Hui-Jun Li,* Ping Li and Hui Qi

Key Laboratory of Modern Chinese Medicines, Ministry of Education and Department of Pharmacognosy, China Pharmaceutical University,Nanjing, People’s Republic of China

Received 8 December 2006; revised 23 January 2007; accepted 30 January 2007

ABSTRACT: A high-performance liquid chromatographic method with diode array detection was developed and validated tosimultaneously identify and quantify four phytoecdysones, i.e., polypodine B (1), ecdysterone (2), 25-R inokosterone (3) and 25-Sinokosterone (4), in Radix Achyranthis Bidentatae. The analysis was performed using a C18 column with 6% tetrahydrofuranaqueous and acetonitrile isocratic elution, and the detection was carried out by DAD at 242 nm. The identities of the analyteswere determined by comparing the retention time and UV spectrum with those of reference compounds. The validation of themethod included linearity, sensitivity, recovery and stability. All calibration curves of the four phytoecdysones showed good linearregression (r2 ≥ 0.9993). The limit of detection (S/N = 3) and limit of quantification (S/N = 10) were less than 7.5 and 12.3 ng,respectively. The method provided desirable repeatability with overall intra- and inter-day variations of less than 4.67%. Theobtained recoveries varied between 95.1 and 104.4% while the relative standard deviations were below 4.85% (n = 3). The analy-sis results indicated that the contents of the investigated phytoecdysones in Radix Achyranthis Bidentatae from different locationswere highly variant, and the genuine crude drug indigenous to Henan province did not possess the highest concentration ofphytoecdysones. Copyright © 2007 John Wiley & Sons, Ltd.

KEYWORDS: HPLC-DAD; phytoecdysones; Radix Achyranthis Bidentatae; quantification

INTRODUCTION

Radix Achyranthis Bidentatae (RAB), derived fromthe dried roots of Achyranthes bidentata Bl, is animportant traditional Chinese medicine (TCM) andis extensively used by TCM practitioners for thetreatment of osteodynia of the lumbar region andknees, spasm and flaccidity of limbs (PharmacopoeiaCommittee of China, 2005). Chemical investigations onRAB disclosed that triterpenoids, phytoecdysonesand polysaccharides existed in this herbal drug (Mengand Li, 2001). As a group of active components,Achyranthes phytoecdysones showed a number ofbiological activities, such as stimulation of RNA and

protein synthesis, effect on carbohydrate metabolismin blood, improvement of lipid metabolism, immuno-modulation and effects on central nervous system (Xuet al., 1997). Moreover, a pharmacological preparationfrom ecdysterone (a predominant Achyranthes phyto-ecdysone) is already available as a commercial pre-paration for its anabolic, tonic and other effects(Osipova et al., 2002; Slama et al., 1996).

Consequently, it is necessary to determine the Achy-ranthes phytoecdysones in RAB in order to ensure itseffective clinical use. Several methods, including ultra-violet spectrophotometry (Wang et al., 2005), thin-layerchromatography scanning (Zhang et al., 2000a), high-performance liquid chromatography (HPLC) (Ma et al.,2000; Zhang et al., 2000b; Zhang et al., 2001) and infra-red spectrometry (Bai et al., 2005), have been devel-oped to quantify ecdysterone considering its relativelyhigh content in RAB. However, due to the absence ofreference compounds, these analytical methods haveneglected other Achyranthes phytoecdysones.

In order to evaluate RAB more effectively, sufficientamounts of four main phytoecdysones, i.e. polypodineB (1), ecdysterone (2), 25-R inokosterone (3) and25-S inokosterone (4), were obtained by a series ofchromatographic techniques, and based on the above

*Correspondence to: Hui-Jun Li, Key Laboratory of Modern ChineseMedicines, Ministry of Education and Department ofPharmacognosy, China Pharmaceutical University, No. 24 TongjiaLane, Nanjing 210009, People’s Republic of China.E-mail: [email protected]

Abbreviations used: DAD, diode array detection; LOD, limit ofdetection; LOQ, limit of quantification; Pa, peak area; RAB, RadixAchyranthis Bidentatae; RSD, relative standard deviation; TCMs,traditional Chinese medicines; tR, retention time.

Contract/grant sponsor: Distinguished Young Scholars from NationalScience Foundation of China; Contract/grant number: 30325046.


824 J. Li et al.ORIGINAL RESEARCH

chemical investigation, we developed an HPLC coupledwith diode array detection (DAD) method for simulta-neous determination of four phytoecdysones in RAB,and report it here for the first time.

EXPERIMENTAL

Chemicals and reagents

Acetonitrile was of HPLC grade (Merck, Darmstadt, Ger-many). Ultra pure water was obtained in a Milli-Q systemfrom Millipore (Bedford, MA, USA). Tetrahydrofuran andother solvents used in extraction and isolation were of ana-lytical grade and purchased from Nanjing Chemical Factory(Nanjing, China). Column chromatography was performedwith silica gel (200–300 mesh, Qingdao Ocean ChemicalCooperation, China), Sephadex LH-20 (Pharmacia Biotech,Sweden) and pre-HPLC (Agilent Technologies, Palo Alto,CA, USA).

Apparatus

An Agilent 1100 series HPLC apparatus, equipped with aG-1312 binary pump, a G-1313 auto-sampler, a G-1316column oven and a G-1315 diode array detector, was used.This system was operated by HP Chemstation software(Agilent Technologies, Palo Alto, CA, USA).

Chromatographic conditions

Chromatography was performed on an Agilent Zorbax C18

column (250 × 4.6 mm, 5.0 µm) at a column temperature of20°C. The mobile phase consisted of (A) 6% tetrahydrofuranaqueous (v/v) and (B) acetonitrile. The flow rate was 0.8 mL/min. An isocratic elution of solvent A–solvent B (96:4, v/v)was used. The DAD detected analytes at 242 nm with refer-ence wavelength at 360 nm, bandwidth at 4 nm, with fullspectral scanning from 190 to 400 nm.

Material

Four cultivated samples of RAB were gathered from dif-ferent provinces of China: two from Henan province, onefrom Inner Mongolia Autonomous Region and one fromHebei province. The 10 commercial samples were purchasedfrom local markets all over the country. All samples wereauthenticated by Dr Ping Li, Professor of Pharmacognosy,China Pharmaceutical University, Nanjing, China. Thevoucher specimens were deposited in the Department ofPharmacognosy, China Pharmaceutical University.

Extraction and isolation of phytoecdysonesin RAB

The air-dried and powdered roots (65 kg) were extractedwith 70% MeOH under reflux for 4 h at 70°C for three times.The total filtrate was concentrated under reduced pressure togive an MeOH extraction (2.5 kg). An aqueous suspension ofthis extract was portioned successively with EtOAc (0.2 kg)

and n-BuOH (1.2 kg). The EtOAc extract was chromato-graphed over silica gel (5.0 × 150 cm) with a gradient systemof CHCl3–MeOH (100:1, 30:1, 10:1, 5:1) to give four mainsubfractions: E-1 (20 g), E-2 (14 g), E-3 (15 g) and E-4 (30 g).Subfraction E-3 (15 g) was further subjected to a silica gelcolumn (3.0 × 100 cm, CHCl3-MeOH, 10:1) to afford fractionsE-3-1 (0.1 g) and E-3-2 (2 g). Fraction E-3-1 (100 mg) wasrechromatographed over Sephadex LH-20 (1.5 × 120 cm) byMeOH, yielding polypodine B (1, 11 mg). Part of fractionE-3-2 (200 mg) was isolated and purified by pre-HPLC(pre-ODS, 15 × 250 mm, ACN–H2O, 15:85) to yield ecdy-sterone (2, 30 mg), 25-R inokosterone (3, 10 mg) and 25-Sinokosterone (4, 12 mg). Their structures were elucidatedby comparison of their spectral data (UV, MS, 1H NMR and13C NMR) with literature values (Zhu et al., 2004; Coll et al.,1994; Giranlt et al., 1988). The structures were shown inFig. 1. The purity of these reference compounds were deter-mined to be more than 98% by normalization of the peakareas detected by HPLC with DAD.

Preparation of sample solution

Samples of RAB were dried at 60°C to constant weight.Approximately 1.5 g of the pulverized samples (through 40mesh), accurately weighed, was added to a conical flask con-taining 40 mL of methanol and the mixture was ultrasoundedon a KH-5000B ultrasonic bath (Kunshan Factory, Kunshan,China) at 35°C for 1 h. The methanol extract was evaporatedto dryness with a rotary evaporator. The residue was dis-solved in 15 mL water, followed by extracting with 15 mL ofn-butanol three times; the n-butanol extract was combinedand evaporated; the residue was redissolved with methanolinto a 5 mL flask, and the volume measured. The resultantsolution was filtrated through a 0.45 µm syringe filter (TypeMillex-HA, Millipore, USA), and 10 µL of the filtrate wasinjected into the HPLC system.

Validation procedure

Calibration curve. A methanol stock solution, which con-tained four analytes, was prepared and diluted to appropriateconcentrations for the construction of calibration curves. Sixconcentrations of the mixed standard solution were injectedin triplicate, and the calibration curves were constructed byplotting the peak area vs the concentration of each analyte.The results are given in Table 1.

Limits of detection and quantification. The methanolstock solution containing four reference compounds wasfurther diluted to a series of appropriate concentrations withmethanol and an aliquot of 10 µL diluted solution wasinjected into HPLC for analysis. The limits of detection(LOD) and quantification (LOQ) under the present chro-matographic conditions were determined at a signal-to-noiseratio (S/N) of 3 and 10, respectively. The LOD and LOQ foreach compound are shown in Table 1.

Precision and accuracy. Intra-day and inter-day variationswere chosen to determine the precision of the developedassay. The intra-day variation was determined by analyzing in


Simultaneous qualitation and quantification of four phytoecdysones 825ORIGINAL RESEARCH

Figure 1. Chemical structures of four phytoecdysones from Radix AchyranthisBidentatae polypodine B (1), ecdysterone (2), 25-R inokosterone (3) and 25-Sinokosterone (4).

triplicate the same mixed standard methanol solution sixtimes within one day. For the inter-day variability test, thesolution was examined in triplicate on three consecutive days.The relative standard deviations (RSDs) of the retention time(tR) and peak area (Pa) were taken as the measures of pre-cision. The results are given in Table 2.

The recovery test was used to evaluate the accuracy ofthe method. Accurate amounts of four standards were addedto approximate 0.75 g of cultivated sample 1, and then ex-tracted and analyzed as described above. The mean recoverywas calculated on three assays for the standard. The resultsare given in Table 3.

Stability. Stability was tested with mixed standard solutionstored at room temperature and analyzed at 0, 2, 4, 8, 12, 24and 48 h within 2 days. The analytes showed very stable inmethanol solution over the tested period (RSD ≤ 4.1%).

RESULTS AND DISCUSSION

Optimization of chromatographic conditions

Different mobile phase compositions were tested. Even-tually, the use of a water–tetrahydrofuran–acetonitrile

Table 1. Calibration curves for four analytes

Analyte Calibration curve r2 Test range (µg/mL) LOD (ng) LOQ (ng)

1 y = 1089.0x + 27.828 0.9996 5.9–472 7.5 12.32 y = 1141.9x + 41.063 0.9999 10.6–848 6.0 11.83 y = 1362.5x − 2.8061 0.9999 4.2–336 5.9 9.54 y = 1390.9x + 12.439 0.9993 5.5–440 6.3 10.6

y, Peak area; x, concentration of standard (µg/mL); r2, correlation coefficient of the equation; LOD, limit of detection; LOQ, limit ofquantification.

Table 2. Precision of the four analytes for retention time (tR) and peak area (Pa), expressed as RSD%

Intra-day variability Inter-day variability

Analyte RSD of tR (%) RSD of Pa (%) RSD of tR (%) RSD of Pa (%)

1 0.26 1.69 0.89 4.552 0.15 1.70 0.99 4.413 0.03 1.55 0.21 4.674 0.02 1.62 0.17 4.11

tR, retention time; Pa, peak area.The concentrations of four reference compounds were 0.059, 0.106, 0.042 and 0.055 mg/mL, respectively.



Table 3. Recoveries of the four phytoecdysones in Radix Achyranthis Bidentatae

Analyte Contained (µg) Added (µg) Found (µg) Recovery (%) Average (%) RSD (%)

1 4.1 3.2 7.3 100.9 96.6 4.854.0 4.0 7.7 92.24.0 4.8 8.8 99.8

2 399.0 315.2 688.0 91.7 96.9 4.66397.0 394.0 790.2 99.8396.0 472.8 865.0 99.2

3 56.0 44.0 100.9 102.1 104.4 3.6755.0 55.0 111.2 102.255.0 66.0 126.8 108.8

4 121.0 95.2 212.8 96.4 95.1 3.06120.0 119.0 229.2 91.8119.0 142.8 257.8 97.2

Recovery (%) = (amount found − amount contained)/amount added × 100%.

Validation of the method

All calibration curves showed good linear regression (r2

≥ 0.9993) within the test ranges. As shown in Table 2,the overall intra- and inter-day variations were less than4.67% for all four analytes. As demonstrated in Table 3,the developed analytical method provided good accu-racy with the recoveries from 95.1 to 104.4% for theanalytes concerned. The LOD (S/N = 3) and the LOQ(S/N = 10) were less than 7.5 and 12.3 ng, respectively.Hence, this verified HPLC-DAD method was precise,accurate and sensitive enough for the quantitativeevaluation of major phytoecdysones in RAB.

Analysis of phytoecdysones in samples

The established analytical method was successfullyapplied for the determination of four phytoecdysonesin the samples of RAB. The individual amounts ofanalytes were calculated using the external standardmethod and the results are summarized in Table 4.

solvent system as mobile phase resulted in baselineseparation. Furthermore, other chromatographic vari-ables including the brand of analytical column, thecolumn temperature and the flow rate were alsooptimized. The optimal separation was achieved on anAgilent Zorbax C18 column (250 × 4.6 mm, 5.0 µm) at acolumn temperature of 20°C and the mobile phase wasdelivered with a flow rate of 0.8 mL/min.

The representative chromatograms of standardssolution and extraction solution of three cultivatedcrude drugs under specific conditions are shown inFig. 2, along with the on-line DAD-UV spectra ofthe peak from 190 to 400 nm. The maximum absorp-tion wavelengths of the four analytes were all at242 nm.

To guarantee the purity of each peak analyzed, threespectra from 190 to 400 nm were measured from theupslope, apex and downslope of the peaks correspond-ing to the standards in these chromatograms. Theycompletely overlapped each other, indicating that thepeaks were pure.

Table 4. Content of four phytoecdysones in samples of Radix Achyranthis Bidentatae

Content (mg/g)

Sample 1 2 3 4 Total

Cultivated sample 1 0.0053 0.5250 0.0742 0.1592 0.7638Cultivated sample 2 0.0027 0.4147 0.0730 0.1101 0.6005Cultivated sample 3 0.0108 0.7972 0.1602 0.2258 1.1941Cultivated sample 4 0.0024 0.5489 0.0884 0.1330 0.7727Commercial sample 1 0.0039 0.3874 0.0633 0.0954 0.5500Commercial sample 2 0.0035 0.5554 0.0932 0.1383 0.7904Commercial sample 3 0.0185 0.8330 0.1504 0.2312 1.2332Commercial sample 4 0.0059 0.6369 0.1079 0.1632 0.9138Commercial sample 5 0.0002 0.2683 0.0483 0.0689 0.3858Commercial sample 6 0.0009 0.6095 0.1102 0.1571 0.8777Commercial sample 7 0.0035 0.4452 0.0732 0.1064 0.6283Commercial sample 8 0.0026 0.4445 0.0767 0.1056 0.6294Commercial sample 9 0.0032 0.6755 0.1196 0.1660 0.9643Commercial sample 10 0.0043 0.4704 0.0789 0.1269 0.6805

Cultivated samples 1 and 2 were gathered at Henan Province, 3 at Inner Mongolia Autonomous Region and 4 at HebeiProvince. Commercial samples 1–10 were purchased from local markets in different provinces of China.


Simultaneous qualitation and quantification of four phytoecdysones 827ORIGINAL RESEARCH

RAB is one of the four genuine crude drugs indi-genous to Henan Province, China (the other threegenuine crude drugs are Rhizoma Dioscoreae, RadixRehmanniae and Flos Chrysanthemi). Thus the com-mercial RAB products from Henan province enjoy agood reputation and are believed to be of top quality.Previously, comparative research on the determinationof oleanolic acid and polysaccharides in RAB indicatedthat RAB from Henan Province surpassed those fromother places in quality (Zhang, 1998; Li et al., 2002;Liu and Jiao, 2000). Consequently, the original plantAchyranthese bidentata Bl was cultivated widely inHenan Province during the generalization of GoodAgricultural Practice for TCMs, while other varieties ofRAB declined sharply over the past five years. How-ever, our findings show that total phytoecdysones inRAB are lower in plants from Henan Province (0.7638and 0.6050 mg/g) than in those from the MongoliaAutonomous Region (1.1941 mg/g) and Hebei Province(0.7727 mg/g). The above evidence possibly reflectsthat the accumulative pathway of phytoecdysones maydiffer from other metabolites (triterpenoids andpolysaccharides) in RAB in specific habits. On theother hand, it also suggests that the determination of allcurative compounds, including triterpenoids, phytoec-dysones and polysaccharides, might be more helpfulfor the quality evaluation of this herbal drug due to itschemical and pharmacological complexity.

CONCLUSION

In conclusion, a newly validated HPLC-DAD methodhas been developed for the qualification and quanti-fication of four main phytoecdysones in RAB. Thismethod was successfully applied to quantification ofpolypodine B, ecdysterone, 25-R inokosterone and 25-Sinokosterone in commercial RAB samples with goodsensitivity, accuracy and repeatability.

Acknowledgments

This research was financially supported by Distin-guished Young Scholars from National Science Foun-dation of China (no.30325046).

REFERENCES

Bai Y, Yu ZX, Sun SQ, Zhu FY, Wang D and Chen ZH. Determina-tion of ecdysterone in Radix Achyranthis Bidentatae by near-infrared diffuse reflectance spectroscopy, Chinese Traditional andHerbal Drug (Zhongcaoyao) 2005; 36: 1391–1394.

Chinese Pharmacopoeia Committee. Pharmacopoeia of the People’sRepublic of China, Chemical Industry Press: Peking, 2005; 49.

Coll J, Reixach N, Sanchez Baeza F, Casas J and Camps F. Newecdysteroids from Polypodium vulgare. Tetrahedron 1994; 50:7247–7252.

Figure 2. Representative HPLC chromatograms of mixedstandards and ethanol extract of Radix AchyranthisBidentatae. (A) Mixed standards; (B) cultivated samplefrom Henan Province; (C) cultivated sample from HebeiProvince; and (D) cultivated sample from Inner MongoliaAutonomous Region. The concentrations of four refer-ence compounds were 0.472, 0.848, 0.336 and 0.44 mg/mL,respectively. This figure is available in colour online atwww.interscience.wiley.com/journal/bmc

In general, the overall patterns of phytoecdysones inall samples are not different from each other. Ecdys-terone (2) was present in the highest concentrationfollowed by 25-S inokosterone (4) and 25-R inoko-sterone (3). Polypodine B (1) was present at the lowestconcentration. In four cultivated samples, the totalphytoecdysones ranged from 0.6005 to 1.1941 mg/g.With respect to the 10 commercial samples, the totalphytoecdysones ranged from 0.3858 to 1.2332 mg/g, in-dicating a higher variation than the cultivated samples.



Giranlt JP, Lafont R, Varga E, Hajdu ZS, Herke I and Szendrei K.Ecdysteroids from Leuzea carthamoides. Phytochemistry 1988; 27:737–741.

Li GL, Du TX and Liang SW. The determination of polysaccharidesin Radix Achyranthis Bidentatae. Chinese Journal of ExperimentalTraditional Medical Formulae (Zhongguo Shiyanfangjixue Zazhi)2002; 8: 6–7.

Liu W and Jiao HJ. Determination of oleanolic acid in theroot of Achyranthes Bidentata Bl. from different places of pro-duction by TLC-scanning. Henan Journal of Traditional ChineseMedicine and Pharmacy (Henan Zhongyiyao Xuekan) 2000; 15:12–13.

Ma Y, Che ZT, Bi KS, Wang X and Wang W. Determinationof ecdysterone in the root of Achyranthes Bidentata BL. byRP-HPLC. Acta pharmaceutica Sinica (Yaoxue Xuebao) 2000; 35:313–315.

Meng DL and Li X. Progression of researches on chemical con-stituents and pharmaco-activity of Radix Achyranthis Bidentatae.Journal of Chinese Medicinal Chemistry (Zhongguo YaowuhuaxueZazhi) 2001; 11: 120–124.

Osipova SO, Islamova ZhI, Syrov VN, Badalova NS andKhushbaktova ZA. Ecdysterone in the treatment of giardiasis.Medicinskaa Parazitologia A (Mosk) 2002; 1: 29–33.

Slama K, Koudela K, Tenora J and Mathova A. Insect hormones invertebrates: anabolic effects of 20-hydroxyecdysone in Japanesequail. Experientia 1996; 52: 702–706.

Wang L, Xu XY, Zheng YM, Fu SQ and Yi ZH. Determination oftotal phytoecdysone from Achyranthes Bidentata by spectrometry.LiShiZhen Medicine and Medica Research (Shizhen Guoyiguoyao)2005; 16: 18–19.

Xu NJ, Guo YY and Li X. Advances in pharmacological researchon ecdysterone. Journal of Shenyang Pharmaceutical University(Shenyang Yaokedaxue Xuebao) 1997; 14: 300–302.

Zhang CY, Li ZG and Zhang ZL. Comparing content of ecdysteronein Achyranthes Bidentata BL. sitir-frying with three wines.Journal of Chinese Medicinal Material (Zhongyaocai) 2000b; 23:683–684.

Zhang CY, Liang SW and Zhang GQ. Determination of ecdysteronein Achyranthes Bidentata BL. from different locations. ChinesePharmaceutical Journal (Zhongguo Yaoxue Zazhi) 2001; 36: 699–700.

Zhang H, Zhang ZZ and Wei YJ. An approach to content ofecdysterone in Achyranthes Bidentata BL. collected in differentperiod. Journal of Chinese Medicinal Material (Zhongyaocai)2000a; 23: 734–735.

Zhang HH. Primary evaluation about quality of Radix AchyranthisBidentatae. LiShiZhen Medicine and Medica Research (ShizhenGuoyiguoyao) 1998; 9: 585–586.

Zhu TT, Liang H, Zhao YY and Wang B. Isolation and structureidentification of C-25 epimers of inokosterone from AchyranthesBidentata Blume. Acta Pharmaceutica Sinica (Yaoxue Xuebao)2004; 39: 913–916.

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