Studies were conducted using an ovariectomized rat model to determine the estrogenic and antiestrogenic activity of Trifolium pratense L. (red clover) extracts. A red clover extract, standardized to contain 15% isoflavones was administered by gavage [250, 500 and 750 mg/(kg x d)] to virgin, ovariectomized 50-d-old Sprague-Dawley rats, for 21 d in the presence and absence of 17beta-estradiol [50 microg/(kg x d)]. Estrogenic effects included an increase in uterine weight, vaginal cell cornification and mammary gland duct branching. Red clover produced a dose-dependent increase in uterine weight and differentiated vaginal cells at the two higher doses, but it did not stimulate cell proliferation in the mammary glands. Neither antiestrogenic nor additive estrogenic properties were observed in any of the tissues studied. These data suggest that red clover extract is weakly estrogenic in the ovariectomized rat model.

P-Glycoprotein (Pgp)-mediated drug efflux can yield a multidrug-resistance phenotype that is associated with poor response to cancer chemotherapy. Pervilleines B and C (PB and PC), two new tropane alkaloid aromatic esters obtained from a chloroform extract of the roots of Erythroxylum pervillei as the result of bioactivity-guided fractionation, were found to restore the vinblastine (VLB) sensitivity of cultured multidrug-resistant KB-V1 cells, with 50% inhibitory concentration values of 0.17 microM in each case. To explore the potential relevance of this response, KB-V1 cells were placed in hollow fibers and implanted into NCr nu/nu mice. Cell growth was not significantly inhibited when VLB or PB or PC were administered as single agents, but when used in combination with vinblastine inhibition of up to 77.7% was observed. Equimolar doses of verapamil were less effective. These data suggest that PB and PC are effective inhibitors of Pgp and should be further evaluated for clinical utility.

P-Glycoprotein-mediated drug efflux can yield a multidrug-resistance (MDR) phenotype that is associated with a poor response to cancer chemotherapy. Pervilleine A, a novel tropane alkaloid obtained from a chloroform extract of Erythroxylum pervillei as the result of bioactivity-guided fractionation, was found to restore the vinblastine sensitivity of cultured multidrug-resistant KB-V1 and CEM/VLB(100) cells, with IC(50) values of 0.36 and 0.02 microM, respectively. Similarly, the chemosensitivity of KB-8-5 cells to colchicine was restored with an IC(50) value of 0.61 microM. The mechanism of this response was evaluated with a number of model systems. First, incubation of multidrug-resistant KB-V1 and CEM/VLB(100) cells with up to 45 microM pervilleine A for 72 h did not significantly affect either the transcription of MDR1, as revealed by reverse transcriptional-PCR-based analysis of MDR1 mRNA, or levels of P-glycoprotein, as shown by Western blots. ATP-dependent binding of [(3)H]vinblastine observed with isolated multidrug-resistant KB-V1 cell membrane vesicles was inhibited by pervilleine A in a dose-dependent manner, and kinetic analysis indicted competitive inhibition with respect to vinblastine binding with a K(i) of 7.3 microM. Consistent with this effect, intracellular accumulation of [(3)H]vinblastine was increased from 0.18 pmol [(3)H]vinblastine/50 x 10(4) cells to approximately 5 pmol [(3)H]vinblastine/50 x 10(4) cells in the presence of 40 microM pervilleine A. To explore the potential relevance of these responses, KB-V1 or KB-8-5 cells were placed in hollow fibers and implanted into NCr nu/nu mice. Cell growth was not significantly inhibited when vinblastine or pervilleine A were administered as single agents, but when used in combination, inhibition of up to 75% was observed. Equimolar doses of verapamil were less effective. These data suggest that pervilleine A is an effective inhibitor of P-glycoprotein and should be further evaluated for clinical utility.