A simple and selective method for genistein (GNST) determination in rat liver and kidney was validated in order to study the phytoestrogenic effect of GNST in ovariectomised female Wistar rats. GNST was separated on a Kromasil 100-RP8 column, 150 mm x 4.6 mm, 5 mm equipped with a Kromasil RP 8 precolumn. The mobile phase was 55:45 (v / v) phosphoric acid, 15 mmol in water: methanol at a flow rate of 1.3 ml / min. Luteolin 20 μg / ml in methanol was used as internal standard (IS). The retention time of GNST was tR = 13.22 min and tR =11.60 min for the IS. Calibration curves in the range 40-400 μg GNST/100g liver and 20-200 μg GNST/100g kidney presented a coefficient of determination higher than 0.99. The method developed presented a good precision and accuracy at the lower limit of quantification LLOQ. 10 white Wistar female rats, 8 weeks of age were treated s.c. with 10 mg GNST/kg bw/day for 8 weeks, while a group of 10 animals were used as controls. The values obtained for GNST in the liver were 192.12 ± 53.46 μg/100g and 74.51 ± 12.77 μg/100g in kidney samples.
1. McCarty MF. Isoflavones made simple. Genistein’s agonist activity for the beta-type estrogen receptor mediates their health benefits. Med Hypotheses. 2006;66(6):1093-114. DOI: 10.1016/j. mehy.2004.11.046
2. Cederroth CR, Nef S. Soy, phytoestrogens and metabolism: A review. Molecular and Cellular Endocrinology. 2009 May 25;304(1-2):30-42. DOI: 10.1016/j. mce.2009.02.027
3. Newbold RR, Banks EP, Bullock B, Jefferson WN. Uterine adenocarcinoma in mice treated neonatally with genistein. Cancer Res. 2001 Jun 1;61(11):4325-8.
4. Weber KS, Setchell KD, Stocco DM, Lephart ED. Dietary soy-phytoestrogens decrease testosterone levels and prostate weight without altering LH, prostate 5alpha- reductase or testicular steroidogenic acute regulatory peptide levels in adult male Sprague-Dawley rats. J Endocrinol. 2001 Sep;170(3):591-9. DOI: 10.1677/ joe.0.1700591
5. Coldham NG, Sauer MJ. Pharmacokinetics of [14C] genistein in the rat: gender-related differences, potential mechanisms of biological action and implications for human health. Toxicol Appl Pharmacol. 2000;164(2):206-15. DOI: 10.1006/taap.2000.8902
6. Grace PB, Mistry NS, Carter MH, Leath AJ, Teale P. High throughput quantification of phytoestrogens in human urine and serum using liquid chromatography / tandem mass spectrometry (LC-MS/MS). J Chromatogr B. 2007;853(1-2):138-46. DOI: 10.1016/j. jchromb.2007.03.011
7. Tamames-Tabara C, Imbuluzqueta E, Campanero MA, Horcajada P, Blanco-Prietoa MJ. A simple and robust high-performance liquid chromatography coupled to a diode-array detector method for the analysis of genistein in mouse tissues. J Chromatogr B. 2013;935:47-53. DOI: 10.1016/j.jchromb.2013.07.020
8. Matthies A, Loh G, Blaut M, Braune A. Daidzein and Genistein Are Converted to Equol and 5-Hydroxy- Equol by Human Intestinal Slackia isoflavoniconvertens in Gnotobiotic Rats. J Nutr. 2012 Jan 1;142(1):40-6. DOI: 10.3945/jn.111.148247
9. Chen C-Y, Bakhiet RM. Age decreased steady-state concentrations of genistein in plasma, liver, and skeletal muscle in Sprague-Dawley rats. Mech Ageing Dev. 2006 Apr;127(4):344-8. DOI: 10.1016/j. mad.2005.12.003
10. Joshi JV, Vaidya RA, Pandey SN, Agashe S, Chandrasekharan S, Menon SK, et al. Plasma levels of genistein following a single dose of soy extract capsule in Indian women. Indian J Med Res. 2007 Apr;125(4):534-41.
11. Tero-Vescan A, Imre S, Vari CE, Dogaru MT. Determination of genistein in plasma by HPLC-UV. Timisoara Medical Journal. 2008;58(Supplement 2):10-4
12. *** International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for human use, Guidance on nonclinical safety studies for the conduct of human clinical trials and marketing authorization for pharmaceuticals, ICH Harmonised Tripartite Guideline M3(R2), 2009
13. *** ICH Common Technical Document (CTD), vol 2B - Notice to Applicants, Medicinal products for Human use (http://ec.europa.eu/health/files/eudralex/vol-2/b/update_200805/ctd_05-2008_en.pdf)
14. **** „Order no. 244/401/2005 of the Minister of Agriculture, Forestry and Rural Development and the Minister of Health of Romania”
15. Bursztyka J, Perdu E, Tulliez J, Debrauwer L, Delous G, Canlet C, et al. Comparison of genistein metabolism in rats and humans using liver microsomes and hepatocytes. Food and Chemical Toxicology. 2008 Mar;46(3):939-48. DOI: 10.1016/j.fct.2007.10.023
16. Moon YJ, Wang X, Morris ME. Dietary flavonoids: Effects on xenobiotic and carcinogen metabolism. Toxicology in Vitro. 2006 Mar;20(2):187-210. DOI: 10.1016/j.tiv.2005.06.048