Identification of Isoflavone Daidzein

Table of Contents

Title Page No. 1.0 Introduction 1 2.0 Sample Treatment 3 3.0 Extraction 4 4.1 Solid-Phase Extraction 4.0 Separation 6 5.2 High Performance Liquid Chromatography (HPLC) 5.3 Thin Layer Chromatography (TLC) 5.0 Detection and Identification 7 6.4 UV-Vis Spectrophotometry 6.5 Mass Spectrometry 6.6 Nuclear Magnetic Resonance Spectrometry 6.0 Health Benefits of Daidzein 18 7.7 Exposure to Daidzein 7.8 Anti-Tumour Effect of Isoflavone 7.9 Daidzein and Cardioovascular Disease 7.0 Conclusion 21 8.0 References 22

1.0 Introduction
Flavonoids are present in plants as mixtures and it is extremely rare for a plant tissue to only contain a single flavonoid component (Harborne 1998). They are a class of plant secondary metabolite, which means the survival of the plant does not depend on these compounds. The chemical structures of flavonoids are based on a C6-C3-C6 skeleton and they differ in the saturation of the heteroatomic ring C, in the placement of aromatic ring B at the positions C-2 or C-3 of ring C, and in the overall hydroxylation patterns (Grotewold 2008).

FIGURE 1.0 General molecular structure of flavonoid. [Adapted from Xu et al. 2007] Out of the many known classes of flavonoid, isoflavone is the class of interest. Isoflavones are colourless, often found in roots, and are only common in one family, Leguminosae (Harborne 1998). Members of the isoflavone class include genistein, glycitein, and the particular compound chosen for this assignment, daidzein. Daidzein, also known by its chemical name 4’,7-dihydroxyisoflavone, has a molecular formula of C15H10O4.

FIGURE 1.1 Structural formula of daidzein. [Adapted from Holder et al. 1999] Isoflavones can occur in the forms of aglycones (daidzein), glucosides (daidzin), acetylglucosides (acetyldaidzin), and

References: 1. Adlercreutz, H., 1990. Western diet and Western diseases: some hormonal and biochemical mechanisms and associations. Scandinavian Journal of Clinical Laboratory Investigation, 50, 3S-23S. 2. Albert, K., 1999. Liquid chromatography-nuclear resonance spectroscopy. Journal of Chromatography A, 856, 199-211. 3. Andersen, M., and Markham, K.R., 2006. Flavonoids: Chemistry, Biochemistry and Applications. Boca Raton: CRC Press, pg 20, 36, 84 and 104. 4. Anderson, J. W., Johnstone, B.M., and Cook-Newell, M.E., 1995. Meta-analysis of the Effects of Soy Protein Intake on Serum Lipids. The New England Journal of Medicine, 333, 276-282. 6. Barfi, B., Hadjmohammadi, R, M., and Kasaai, M, R., 2009. Determination of daidzein and genistein in soybean and its waste by matrix solid-phase dispersion extraction and HPLC. Monatsh chem 140, 1143-1148. 8. Brown, W.H., Foote, C.S., Iverson, B.L., and Anslyn, E.V., 2011. Organic Chemistry. 5th Ed. United States of America: Brooks/Cole Cengage Learning, pg 486-489. 9. Carroll, K. K. and Kurowska. E. M., 1995. Soy Consumption and Cholesterol Reduction: Review of Animal and Human Studies. Journal of Nutrition, 125, 594S-597S. 11. Chen, H., Zuo, Y.G., and Deng, Y.W., 2001. Separation and determination of flavonoids and other phenolic compounds in cranberry juice by high-performance liquid chromatography. Journal of Chromatography A, 913, 387–395. 15. Fair, J. D. and Kormos, C. M., 2008. Journal of Chromatography A, 1211(1-2), 49-54. 17. Franke, A.A., Custer, L.J., Cerna, C.M., and Narala, K.K., 1994. Quantitation of phytoestrogens in legumes by HPLC. Journal of Agricultural and Food Chemistry, 42(9), 1905-1913. 18. Golkhoo, S., Ahmadi, A.R., Hanachi, P., Barantalab, F., and Vaziri, M., 2008. Determination of Daidzein and Genistein in Soy Milk in Iran by Using HPLC Analysis Method. Journal of Biological Sciences, 11:2254-2258. 22. Grotewold, E., 2008. The Science of Flavonoids. United States of America: Springer, pg 47-48. 23. Harborne, J.B., 1994. The Flavanoids: Advances in Research Since 1986. London: Chapman and Hall, pg 19, 441. 24. Harborne, J.F., 1998. Phytochemical Methods:A Guide to Modern Techniques of Plant Analysis. 3rd Ed. United Kingdom: Chapman and Hall, pg 60-61. 25. He, J., Zhao, Z., Shi, Z., Zhao, M., Li, Y., and Chang, W., 2005. Analysis of isoflavone daidzein in Puerariae radix with micelle-mediated extraction and preconcentration. Journal of Agricultural and Food Chemistry, 53:518-523. 26. Hodgson, J.M., Puddey, I.B., Beilin, L.J., Mori, T.A., and Croft, K.D., 1998. Supplementation with Isoflavonoid Phytoestrogens Does Not Alter Serum Lipid Concentrations: A Randomized Controlled Trial in Humans. Journal of Nutrition, 128, 728-732. 27. Holder, C.L., Churchwell, M.I., and Doerge, D.R., 1999. Quantification of soy isoflavones, genistein and daidzein, and conjugates in rat blood using LC/ES-MS. Journal of Agricultural and Food Chemistry, 47:3764-3770. 28. Hurst, W. J., 2002. Methods of Analysis for Functional Foods and Nutraceuticals. 2nd Ed. Florida: CRC Press. 29. Jacobsen, N.E., 2007. NMR Spectroscopy Explained: Simplified Theory, Applications and Examples for Organic Chemistry and Structural Biology. New Jersey: John Wiley and Sons, Inc., pg 1-3. 30. Kale, M.S., and Ladda, K.S., 2013. Isolation, characterization and quantification of isoflavone in Momordica dioica Roxb. Ex wild (Cucurbitaceae) fruits. International Journal of Applied Research in Natural Products, 5(4), 28-36. 31. Kennedy, A., 1995. The Evidence for Soybean Products as Cancer Preventive Agents. Journal of Nutrition, 125, 733S-743S. 34. Klejdus. B., Trinacty, J., Hrdlicka, P., and Kuban, V., 2001. Comparison of sloid-Phase Extration procedures for isolation of plant isoflavones prior to liquid chromatographic determination. Research Institude of Animal Nutrition, Ltd., 23, 691. 35. Kuhnle, G.G.C., Dell Aquila, C., Low, Y.L., Kussmaul, M., and Bingham, S.A., 2007. Extraction and quantification of phytoestrogens in food using automated SPE and LC/MS/MS/. Analytical Chemistry, 79(23), 9234-9239. 36. Kulling, S.E., Honig, D.M., Simat, T.J., and Metzler, M., 2000. Oxidative in vitro metabolism of the soy phytoestrogens daidzein and genistein. Journal of Agricultural and Food Chemistry, 48, 4963-4972. 37. Li, W., Asada, Y., and Yoshikawa, T., 2000. Flavonoid constituents from Glycyrrhiza glabra hairy root cultures. Phytochemistry, 55, 447. 38. Lichtenstein, A.H., 1998. Soy Protein, Isoflavones and Cardiovascular Disease Risk. Journal of Nutrition, 128, 1589-1592. 39. Mabry, T.J., Markham, K.R., and Thomas, M., 1970. The systematic identification of flavonoids. New York: Springer-Verlag, pg 235. 40. Mattila, P., Astola, J., and Kumpulainen, J., 2000. Determination of flavonoids in plant material by HPLC with diode-array and electro-array detections. Journal of Agricultural and Food Chemistry, 48, 5834–5841. 41. Martin, P.M., Horwitz, K.B., Ryan, D.S., and McGuire, W.L., 1978. Phytoestrogen Interaction with Estrogen Receptors in Human Breast Cancer Cells. Endocrinology, 103, 1860-1867. 42. Mayo, B., and van Sinderen, D., 2010. Bifidobacteria: Genomics and Molecular Aspects. Great Britain: Caister Academic Press, pg 102. 43. Merken, H.M., and Beecher, G.R., 2000. Liquid chromatographic method for the separation and quantification of prominent flavonoid aglycones. Journal of Chromatography A, 897, 177–184. 45. Montgomery, R., Conway, T. W., Spector, A.A., and Chappell, D., 1996. Molecular Endocrinology: Hormones Active Inside the Cell. In Biochemistry: A Case- Oriented Approach. Missouri: Mosby-Year Book, Inc, pg 587-618. 46. Naim, M., Gestetner, B., Bondi, A., and Birk, Y., 1976. Antioxidative and Antihemolytic Activities of Soybean Isoflavones. Journal of Agricultural Food Chemistry, 24, 1174-1177. 47. Niessen, W.M.A., 2010. Liquid Chromatography-Mass Spectrometry. 3rd Ed. London: Taylor and Francis, pg 24. 48. Peterson, G., and Barnes., S., 1991. Genistein Inhibition Of The Growth Of Human Breast Cancer Cells: Independence From Estrogen Receptors And The Multi-Drug Resistance Gene. Biochemical and Biophysical Research Communications 179, 661-667. 49. Potter, S. M., 1996. Soy protein and serum lipids. Lipidology, 7, 260-264. 50. Sam, J.B., and Manuel, T.V., 2002. Beneficial role of dietary phytoestrogens in obesity and diabetes. Am J Clin Nutr December, 76(6), 1191-1201. 51. Tew, B.Y., Xu, X., Wang, H.J., Murphy, P.A., and Hendrich., S., 1996. A Diet High In Wheat Fiber Decreases the Bioavailability of Soybean Isoflavones in a Single Meal Fed to Women. Journal of Nutrition 126, 871-877. 52. Prasain, J.K., Wang, C.C., and Barnes, S., 2004. Mass spectrometric methods for the determination of flavonoids in biological samples. Free Radical Biology and Medicine, 37, 1324-1350. 53. Preedy, V.R., 2013. Isoflavones: Chemistry, Analysis, Function and Effects. Cambridge: RSC Publishing, pg 205. 54. Robards, K., 2003. Strategies for the determination of bioactive phenols in plants, fruits and vegetables. J. Chromatogr. A 1000, 657–691. 55. Rostagno, M.,A., Palma, M., and Barroso, C.G., 2005. Solid-phase extraction of soy isoflavones. Journal of Chromatography A, 1076, 110-117. 56. Sarker, S.D., and Nahar, L., 2007. Chemistry for Pharmacy Students: General, Organic and Natural Product Chemistry. Great Britain: Antony Rowe, pg 319. 57. Schneider, V., 1995. Evaluation of small amounts of flavonoid phenols in white wines by colorimetric assays. American Journal of Enology and Viticulture, 46, 274–277. 58. Setchell, K.D.R., 1998. Phytoestrogens: the biochemistry, physiology, and implications for human health of soy isoflavones. American Journal of Clinical Nutrition, 68, 1333S-46S. 59. Smith, J., and Charter, E., 2010. Functional Food Product Development. Singapore: Wiley-Blackwell, pg 111. 60. Ungar, Y., Osundahunsi, O.F., and Shimoni, E., 2003. Thermal stability of genistein and daidzein and its effect on their antioxidant activity. Journal of Agricultural and Food Chemistry, 51:4394-4399. 61. Waksmundzka-Hajnos, M., and Sherma, J., 2011. High Performance Liquid Chromatography in Phytochemical Analysis. Boca Raton: CRC Press, pg 340-341. 62. Wang, H.J., and Murphy, P.A., 1994. Isoflavone content in commercial soybean foods. Journal of Agricultural Food Chemistry, 42, 1666–1673. 63. Wei, F., Zhong, M., Ma, L., Jin, H., and Lin, R., 2007. Quality Control of Traditional Chinese Medicine. Chinese Herbal Drug Research Trends, 173-188. 64. Wei, H., Wei, L., Frenkel, K., Bowen, R., and Barnes, S., 1993. Inhibition of Tumor Promoter-Induced Hydrogen Peroxide Formation In Vitro and In Vivo by Genistein. Nutrition and Cancer , 20, 1-12. 65. Wilson, I.D., 2000. Multiple hyphenation of liquid chromatography with nuclear magnetic resonance spectroscopy, mass spectrometry and beyond, Journal of Chromatography A, 892, 315-327. 66. Wrolstad, R.E., 2003. Current Protocols in Food Analytical Chemistry. New York: John Wiley and Sons, Inc., pg 1. 67. Xu, Y.C., Leung, S.W.S., Yeung, D.K.Y., Hu, L.H., Chen, G.H., Che, C.M., and Man, R.Y.K., 2007. Structure–activity relationships of flavonoids for vascular relaxation in porcine coronary artery. Phytochemistry, 68(8), 1179-1188. 68. Yang, F., Ma, Y., and Ito, Y., 2001. Separation and purification of isoflavones from a crude soybean extract by high-speed counter-current chromatography. Journal of Chromatography A, 928, 163-170.