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Estrogenic activity of griffonianone C, an isoflavone from the root bark of Millettia griffoniana: regulation of the expression of estrogen responsive genes in uterus and liver of ovariectomized rats.

ABSTRACT

Griffonianone C (Griff C) is an isoflavone produced by Millettia griffoniana (Bail) and exhibits estrogenic properties in in vitro reporter gene assays. In order to validate its estrogenic potency in vivo, we administered subcutaneously Griff C (2, 10, or 20 mg/kg/d BW), 17beta-estradiol (10 microg/kg/d BW) as positive control, and a vehicle control to ovariectomized Wistar rats. After three consecutive days of treatment animals were sacrificed 24 hours after receiving the last dose. The uteri and livers were excised, weighed and stored for mRNA expression analysis by real-time PCR. The uterine wet weight was not significantly increased by Griff C, although there was a trend towards an increase. In contrast, 17beta-estradiol increased uterine wet weight 4.5-fold in comparison to the vehicle control. However, as revealed by real-time PCR Griff C affected the expression of estrogen-responsive genes in uterus and liver of ovariectomized rats. E2 induced a 550-fold stimulation of uterine C3 mRNA expression. Griff C at the dose 20 mg/kg/d BW caused a 50-fold up-regulation of complement C3 mRNA compared to the control. A significant increase in calcium binding protein 9-kilodalton mRNA expression was observed in the uterus of ovariectomized rats treated with E (2) (41-fold versus control) or 20 mg/kg/d BW of Griff C (25-fold versus control). In contrast, the expression of clusterin and progesterone receptor in the uterus was strongly decreased by both E2 and Griff C at the highest dose. We also found a repression of clusterin mRNA in the liver while carbonic anhydrase 2 and major acute phase protein were slightly up-regulated. In conclusion, Griff C showed a clear estrogenic action on uterine and hepatic tissues of ovariectomized rats, although its effect was less than the effect of estradiol. This suggests that some of the biological effects attributed to Millettia griffoniana are probably related to estrogen-mediated function.