AJAC  Vol.2 No.6 , October 2011
Chemical Profiling and Quantification of Isoflavone Phytoestrogens in Kudzu Using LC/UV/MSD
Abstract: Method development for determination of isoflavones in kudzu was achieved by HPLC/UV/ESI-MSD. Us- ing three kudzu species of Pueraria lobata, P. thomsonii and P. edulis, and analyzing the isoflavones sepa- rately by species and from different plant tissues (roots, stems, leaves, flowers and fruits) in each species, a total of 25 isoflavones were identified by their molecular ions and characteristic fragment ion peaks using LC/MSD under MS and MS/MS mode, and in comparison with standard isoflavones. Two main chemical groups were identified: 1) 8-C-glycosyl isoflavone of puerarin and the analogues of 5-OH puerarin, 3’-OH puerarin, 3’-OMe puerarin, and their glycosides; and 2) daidzein, genistein, glycitein and their glycosyl and malonyl derivatives, which are similar to those known in soy. To accurately quantitate total isoflavones, acidic hydrolysis during extraction of kudzu samples was applied to convert the oxygen glycosides into their respective isoflavone aglycones of daidzein, genistein and glycitein, or non-hydrolyzed carbon glycosides of puerarin, 5-OH puerarin, 3’-OH puerarin and 3’-OMe puerarin. Under the multiple optimized conditions, all seven isoflavones in acidic hydrolyzed kudzu extracts were successfully separated within 30 min and quanti- fied individually with calycosin used as internal standard by both UV and MS detectors. For the quantitative study, several standards e.g. 5-OH puerarin, 3’-OH puerarin and 3’-OMe puerarin are not commercially available. Using polyamide, sephdex-LH20 chromatography and Prep-HPLC, we purified these three stan- dards from kudzu extracts and then elucidated their structures by UV, MS and NMR spectrometric methods. This is the first method to simultaneously quantitate all the isoflavones in kudzu.
Cite this paper: nullQ. Wu, Y. Yang and J. Simon, "Chemical Profiling and Quantification of Isoflavone Phytoestrogens in Kudzu Using LC/UV/MSD," American Journal of Analytical Chemistry, Vol. 2 No. 6, 2011, pp. 665-674. doi: 10.4236/ajac.2011.26076.

[1]   X. Wang, J. Wu, H. Chiba, K. Umegaki, K. Yamada and Y. Ishimi, “Puerariae Radix Prevents Bone Loss in Ova-riectomized Mice,” Journal of Bone and Mineral Meta-bolism, Vol. 21, No. 5, 2003, pp. 268-275. doi:10.1007/s00774-003-0420-z

[2]   Y. Lu, T. Zhang, J. S. Tao and L. Y. Xu, “Pharmacokinetics of Pueraria for Intranasal on Spray in Rabbits,” Journal of Chinese Medicinal Materials, Vol. 30, No. 9, 2007, pp. 1102-1105.

[3]   Y. M. Zheng, X. Y. Xu and S.Q. Fu, “Study on Data Analysis Method of Kudzu Root Total Flavone Finger- print,” China Journal of Chinese Materia Medica, Vol. 30, 2005, pp. 70-71.

[4]   Q. Chang, L. Sun, R. H. Zhao, M. S. Chow and Z. Zuo, “Simultaneous Determination of Ten Active Components in Traditional Chinese Medicinal Products Containing both Gegen (Pueraria lobata) and Danshen (Salvia miltiorrhiza) by High-Performance Liquid Chromatog- phy,” Phytochemical Analysis, Vol. 19, No. 4, 2008, pp. 368-375. doi:10.1002/pca.1063

[5]   D. M. Tham, C. D. Gardner and W. L. Haskell, “Clinical Review 97: Potential Health Benefits of Dietary Phytoe- trogens: A Review of the Clinical, Epidemiological, and Mechanistic Evidence,” Journal of Clinical Endocrino- logy and Metabolism, Vol. 83, No. 7, 1998, pp. 2223- 2235. doi:10.1210/jc.83.7.2223

[6]   K. D. R. Setchell and A. Cassidy, “Dietary Isoflavones: Biological Effects and Relevance to Human Health,” Journal of Nutrition, Vol. 129, No. 3758, 1999, pp. 758S- 767S

[7]   K. Yanagihara, A. Ito, T. Toge and M. Numoto, “Anti-proliferative Effects of Isoflavones on Human Cancer Cell Lines Established from the Gastrointestinal Tract,” Cancer Research, Vol. 53, No. 23, 1993, pp. 5815-5821.

[8]   K. K. Hintz and L. Ren, “Phytoestrogenic Isoflavones Daidzein and Genistein Reduce Glucose-Toxicity-Induced Cardiac Contractile Dysfunction in Ventricular Mycytes,” Endocrine Research, Vol. 30, 2004, pp. 215-223. doi:10.1081/ERC-120037730

[9]   X. P. Song, P. P. Chen and X. S. Chai, “Effects of Puera-rin on Blood Pressure and Plasma Renin Activity in Spontaneously Hypertensive Rats,” Acta Pharmacologica Sinica, Vol. 9, No. 1, 1988, pp. 55-58.

[10]   L. P. Yan, S. W. Chan, A. C. Chan, S. L. Chen, X. J. Ma and H. X. Xu, “Puerarin Decreases Serum Total Choles-terol and Enhances Thoracic Aorta Endothelial Nitric Oxide Synthase Expression in Diet-Induced Hypercho-lesterolemic Rats,” Life Sciences, Vol. 79, 2006, pp. 324-330. doi:10.1016/j.lfs.2006.01.016

[11]   W. M. Keung and B. L. Vallee, “Kudzu Root: An Ancient Chinese Source of Modern Antidipsotropic Agents,” Phytochemistry, Vol. 47, No. 4, 1998, pp. 499-506. doi:10.1016/S0031-9422(97)00723-1

[12]   D. H. Overstreet, J. E. Kralic, A. L. Morrow, Z. Z. Ma, Y. W. Zhang and D. Y. Lee, “NPI-031G (Puerarin) Reduces Anxiogenic Effects of Alcohol Withdrawal or Benzod- azepine Inverse or 5-HT2C Agonists,” Pharmacology Bio-chemistry and Behavior, Vol. 75, No. 3, 2003, pp. 619-625. doi:10.1016/S0091-3057(03)00114-X

[13]   R. C. Lin and T. K. Li, “Effects of Isoflavones on Alcohol Pharmacokinetics and Alcohol-Drinking Behavior in Rats,” American Journal of Clinical Nutrition, Vol. 68, No. 6, 1998, pp. 1512S- 1515S.

[14]   P. Delmonte and J. I. Rader, “Analysis of Isoflavones in Foods and Dietary Supplements,” Journal of AOAC International, Vol. 89, No. 4, 2006, pp. 1138-1146.

[15]   C. B. Fang, X. C. Wan, H. R. Tan and C. J. Jiang, “Iden-tification of Isoflavonoids in Several Kudzu Samples by High-Performance Liquid Chromatography Coupled with Electrospray Ionization Tandem Mass Spectrometry,” Journal of Chromatographic Science, Vol. 44, No. 2, 2006, pp. 57-63.

[16]   P. Delmonte, J. Perry and J. I. Rader, “Determination of Isoflavones in Dietary Supplements Containing Soy, Red Clover and Kudzu: Extraction Followed by Basic or Acid Hydrolysis,” Journal of Chromatography A, Vol. 1107, No. 1-2, 2006, pp. 59-69. doi:10.1016/j.chroma.2005.11.060

[17]   Y. Zhang, Q. Xu, X. Z. Zhang, J. P. Chen, X. M. Liang and A. Kettrup, “High-Performance Liquid Chromato-graphy-Tandem Mass Spectrometry for Identification of Isoflavones and Description of the Biotransformation of Kudzu Root,” Analytical and Bioanalytical Chemistry, Vol. 383, No. 5, 2005, pp. 787-796. doi:10.1007/s00216-005-0068-8

[18]   E. Benlhabib, J. I. Baker, D. E. Keyler and A. K. Singh, “Quantitative Analysis of Phytoestrogens in Kudzu-Root, Soy and Spiked Serum Samples by High-Pressure Liquid Chromatography,” Biomedical Chromatography, Vol. 18, No. 6, 2004, pp. 367-380. doi:10.1002/bmc.326

[19]   J. K. Prasain, K. Jones, M. Kirk, L. Wilson, M. Smith- Johnson, C. Weaver and S. Barnes,“Profiling and Quan-tification of Isoflavonoids in Kudzu Dietary Supplements by High-Performance Liquid Chromatography and Elec-trospray Ionization Tandem Mass Spectrometry,” Journal of Agricultural and Food Chemistry, Vol. 51, No. 15, 2003, pp. 4213-4218. doi:10.1021/jf030174a

[20]   Y. Niiho, Y. Nakajima, T. Yamazaki, M. Okamoto, R. Tsuchihashi, M. Kodera, J. Kinjo and T. Nohara, “Simul-taneous Analysis of Isoflavones and Saponins in Pueraria Flowers Using HPLC Coupled to an Evaporative Light Scattering Detector and Isolation of a New Isoflavone Diglucoside,” Journal of Natural Medicines, Vol. 64, No. 3, 2010, pp. 313-320. doi:10.1007/s11418-010-0411-z

[21]   J. G. Mun, M. D. Grannan, P. J. Lachcik, R. B. Rogers, G. G. Yousef, M. H. Grace, E. M. Janle, Q. L. Wu, J. E. Si-mon, C. W. Weaver and M. A. Lila, “Tracking Depos- tion of a 14C-Radiolabeled Kudzu Hairy Root-Derived Isoflavone-Rich Fraction into Bone,” Experimental Biol-ogy and Medicine (Maywood), Vol. 235, No. 10, 2010, pp. 1224-1235. doi:10.1258/ebm.2010.010134

[22]   Q. L. Wu, M. F. Wang and J. E. Simon, “Determination of Isoflavones in Red Clover and Related Species by High-performance Liquid Chromatography Combined with Ultraviolet and Mass Spectrometric Detection,” Journal of Chromatography A, Vol. 1016, No. 2, 2003, pp. 195-209. doi:10.1016/j.chroma.2003.08.001

[23]   Q. L. Wu, M. F. Wang, J. S. William and J. E. Simon, “LC/UV/ESI-MS Analysis of Isoflavones in Edamame and Tofu Soybeans,” Journal of Agricultural and Food Chemistry, Vol. 52, No. 10, 2004, pp. 2763-2769. doi:10.1021/jf035053p