Development of Quantitative Analytical Method for Isoflavonoid Compounds from Fruits of Cudrania Tricuspidata

꾸지뽕나무 열매 isoflavonoid 화합물에 대한 정량분석법 개발

Yoon, Sun Young;Kim, Seok Ju;Sim, Su Jin;Lee, Hak-Ju

  • Received : 2016.03.02
  • Accepted : 2016.05.10
  • Published : 2016.05.25


In this study, an analytical method to evaluate the quality of isoflavonoid compounds purified and isolated from the fruits of Cudrania tricuspidata was developed and validated using Ultra Performance Liquid Chromatography (UPLC). The fruits of C. tricuspidata were extracted with methanol and further fractioned with n-hexane, ethyl acetate and water. The resulting ethyl acetate extract separated into four isoflavonoid compounds by a combination of silica gel and sephadex LH-20 column chromatography. The structures of the compounds were elucidated as alpinumisoflavone, 6,8-diprenyl genistein, 6,8-diprenyl orobol, 4'-O-methylalpinumisoflavone by various techniques such as UV-Vis, ESI-MS, $^1H\;NMR$ and $^{13}C\;NMR$ spectroscopy. Finally, a method to characterize the compounds was developed by using the UPLC equipped with a $C_{18}$ column and a gradient mobile phase system consisting of 2% acetic acid in water (solvent A) and 2% acetic acid in methanol (solvent B). The developed method was validated with the parameters such as selectivity, linearity, limit of detection, limit of quantitation, accuracy, and precision, which are defined by the ICH (International Conference on Harmonization). Using the validated method, the compounds in the fruits harvested in different months were also quantitatively analyzed. We propose this approach this approach can readily be utilized as an efficient evaluation method to quantify the extracts of C. tricuspidata.


Cudrania tricuspidata;alpinumisoflavone;6,8-diprenyl genistein;6,8-diprenyl orobol;4'-O-methyl-alpinumisoflavone;ultra performance liquid chromatography;validation


  1. Choi, S.R., You, D.H., Jang, I., Ahn, M.S., Song, E.J., Seo, S.Y., Choi, M.K., Kim, Y.S., Kim, M.K., Choi, D.G. 2012. Cytotoxicity of methanol extracts from Cudrania tricuspidata Bureau. Korean Journal of Medicinal Crop Science 20(3): 153-158.
  2. Choi, S.R., You, D.H., Kim, J.Y., Park, C.B., Kim, D.H., Ryu, J. 2009. Antioxidant activity of methanol extracts from Cudrania tricuspidata Bureau according to harvesting parts and time. Korean Journal of Medicinal Crop Science 17(2): 115-120.
  3. Collison, M.W. 2008. Total soy isoflavones in dietary supplements, supplement ingredients, and soy foods. Journal of AOAC International 91(3): 489-500.
  4. International Conference on Harmonisation (ICH). 2005. Validation of analytical procedures: text and methodology Q2 (R1).
  5. Jeong, C.H., Choi, G.N., Kim, J.H., Kwak, J.H., Jeong, H.R., Kim, D.O., Heo, H.J. 2010. Protective effects of aqueous extract from Cudrania tricuspidata on oxidative stress-induced neurotoxicity. Food Science and Biotechnology 19(4): 1119-1117.
  6. Jung, G.T., Ju, I.O., Choi, S.R., You, D.H., Noh, J.J. 2013. Food nutritional characteristics of fruit of Cudrania tricuspidata in its various maturation stages. The Korean Society of Food Preservation 20(3): 330-335.
  7. Kim, H.J. 2012. Antioxidant and antibacterial activity of extracts from Cudrania tricuspidata Bureau. MS Thesis, Chonnam National University, Korea.
  8. Kim, S.J., Lee, K.-T., Youe, W.-J., Lee, S.-S., Kim, Y.S. 2014. Structure analysis of water-soluble polysaccharides extracted from the unripe fruit of Cudrania tricuspidata. Journal of The Korean Wood Science and Technology 42(6): 740-746.
  9. Koh, B.S., Park, H.J., Kim, S.R., Moon, I.J., Leem, D.W., Park, K.H., Lee, J.H., Park, J.-W. 2014. Adverse drug reactions after taking the extract of Cudrania tricuspidata. Allergy Asthma & Respiratory Disease 2(5): 387-390.
  10. Korea Food & Drug Administration (KFDA). 2008. Guide on standardization of functional raw material for health functional food developer. KFDA. Korea. pp. 40-47.
  11. Korea Food & Drug Administration (KFDA). 2014. Health Functional Food Code. KFDA. Korea. pp. 340-347.
  12. Lee T.H., Kwon, J.Y., Lee, D.H., Mar, W.C. 2015. Effects of Cudrania tricuspidata fruit extract and its active compound, 5,7,3',4'-tetrahydroxy-6,8-diprenylisoflavone, on the high-affinity IgE receptor-mediated activation of Syk in mast cells. Journal of Agricultural and Food Chemisty 63: 5459-5467.
  13. Mauricio A.R., Miguel P., Carmelo G.B. 2003. Ultrasound-assisted extraction of soy isoflavones. Journal of Chromatography A 1012: 119-128.
  14. Mauricio A.R., Miguel P., Carmelo G.B. 2005. Solid-phase extraction of soy isoflavones. Journal of Chromatography A 1076: 110-117.
  15. Qizhen D., Zhonghua L., Yoichiro I. 2001. Preparative separation of isoflavone components in soybeans using high-speed conter-current chromatography. Journal of Chromatography A 923: 271-274.
  16. Radka M., Petr H., Pavel H., Vojtech A., Sona K., Ladislav H., Marie S. 2007. Determination of isoflavones using liquid chromatography with electrochemical detection. Acta Chimica Slovenica 54: 92-97.
  17. Seo, M.J., Kang, B.W., Park, J.U., Kim, M.J., Lee, H.H., Kim, N.H., Kim, K.H. Rhu, E.J., Jeong, Y.K. 2013. Effect of fermented Cudrania tricuspidata fruit extracts on the generation of the cytokines in mouse spleen cells. Journal of Life Science 23(5): 682-688.
  18. Seo, J.Y. 2012. Simultaneous analysis of main compounds from Cudrania tricuspidata stem by HPLC. MS Thesis, Chung-Ang University, Korea.
  19. Uddin, G.M., Lee, H.J., Jeon, J.S., Chung, D.H., Kim, C.Y. 2011. Isolation of prenylated isoflavonoids from Cudrania tricuspidata fruits that inhibit A2E photooxidantion. Natural Product Sciences 17(3): 206-211.
  20. USP Monograph. 2011. USP32-NF29; Soy isoflavones. The United States Pharmacopeial Convention pp. 1236.
  21. Xiang, H.H., Hong, S.S., Hwang, J.S., Heong, S.H., Hwang, J.H., Lee, M.H., Lee, M.K., Lee, D.H., Ro, J.S., Hwang, B.Y. 2005. Monoamine oxidase inhibitory constituents from the fruits of Cudrania tricuspidata. Archives of Pharmacal Research 28(12): 1324-1327.
  22. Xiang, H.H., Hong, S.S., Qinghao, J., Dayu, L., Kim, H.K., Lee, J., Kwon, S.H., Lee, D.H., Lee, C.K., Lee, M.K., Hwang, B.Y. 2009. Prenylated and benzylated flavonoids from the fruits of Cudrania tricuspidata, Journal of Natural Products 72(1): 164-167.
  23. Youn, K.S., Kim, J.W. 2012. Antioxidant and angiotensin converting enzyme I inhibitory activities of extracts from mulberry (Cudrania tricuspidata) fruit subjected to different drying methods. Journal of the Korea society of food science and nutrition 41(10): 1388-1394.


Grant : 목재이용 기반기술 연구

Supported by : 국립산림과학원