| Caragana sinica (Buc'hoz) Rehd. (Fabaceae) is widely distributed in China. Its dried roots (Chinese name: Jinquegen) have been used in China as a folk medicine for the treatment of asthenia syndrome, vascular hypertension, leukorrhagia, bruises and contused wounds. In our previous study, we found that the EtOAc extract of the roots contained many oligostilbenes which had multi-faceted bioactivities. Kobophenol A (1) is a stilbene tetramer, accounts for nearly 20% of the EtOAc extract. Using improved E-screen assay, we found that 1 is a phytoestrogen and has strong estrogenic activity. MTT assay showed that 1 is a phytoestrogen and can strongly stimulate the proliferation of osteoblasts cultured in vitro up to 25.6%. It can be likely developed as a new drug to cure and prevent osteoporosis. Based on this, we conducted studies on its metabolites in vivo and thereafter their mechanism so that uncovered its behavior in vivo. These studies might supply the preclinical research of 1 with sound theoretic foundation.Four main metabolites were isolated and purified from rats' feces orally administered with 1, koboquinone A-C (2, 3, 4) and carasinol B (5). 2. 3 and 4 are new compounds, phase I oxidative metabolites of 1. 5 is the isomeric metabolite of 1. During the isolation of metabolites, we also purified two phase II endogenous metabolites, indoxyl sulfate (IS, 6) and indole-3-acetylglycine (IAA-Gly, 7). from rats' urine. Compound 6 is a harmful uremic toxin while 7 is the phase II metabolite of indole-3-acetic acid (IAA. Auxin). Compound 7 has not been heard to be isolated from rats' urine, to our knowledge. Additionally, we isolated and purified the terminal metabolite of phytoestrogen daidzin, (-)-(S)-equol (8), from rats' feces and reported its X-ray single crystal analysis for the first time.To investigate the behavior of 1 and validate the rationality of the metabolites of 1 in animals, we conducted pharmacokinetic experiments of 1 in animals. The absolute rate of adding sample recovery of three concentrations (1, 5. 25 μg/ml) of 1 were between 78.7-98.6% which can meet needs of the pharmacokinetic and tissue distribution experiments; The serum protein binding rates of 1 at the concentrations of 5. 10, 25μg/ml were 66.0. 75.4 and 85.9%, respectively; After iv administration of 1 at the dose 4, 10, 20 mg/kg, the Drug Concentration-Time Curves were assayed by 3P87 software and best fit to a two-compartmental model (P<0.05). AUC and Dose relationship shows an excellent linearity (R~2 = 1); The pharmacokinetics are notdose-dependent, ti/2 values were 0.64^ 0.58 n 0.83 h, respectively; This shows quick absorption and distribution for 1 in rats. Kinetic process of 1 in rats after ig administration at dose of 250 mg/kg also was fitted to a two-compartmental model. Parameters values for 1 were Cmax 1.84 mg/L, Tmax 0.57 h, AUCo-c 22.41 h?mg?L'\ t1/2 5.78 h, respectivly; The absolute oral bioavailability of 1 was calculated to be 2.0%; There is a slightly second absorption peak on the Drug Concentration-Time Curve. This suggests a formation of a hepatoenteral circulation. 'Primary parent drug was eliminated with feces after iv and ig administration. Mice tissue distribution experiments show that 1 was prominently concentrated in livers. These pharmacokinetic parameters indicated that 1 be metabolized by gastrointestinal tract microflora and liver microsomes when orally administered. The former is primary.Liver is an important organ in which a majority of drug metabolism takes place. In order to validate the pathway of the metabolites of 1 in vivo, we incubated 1 with liver microsomes in vitro. Within the range 5-200 (xg/ml of 1 we tested, we found that 1 was conjugated with coenzyme I (J3-NADPH) and thereafter no metabolite was detected. To exclude the possibility of a high concentration of coenzyme I . we incubated 1 with hepatocyte and still we did not get the metabolite.So we began the study on the metabolism of 1 by intestinal bacteria. After 1 being incubated with human intestinal bacterium Escherichia coli, there were very slight differences between the HPLC chromatograms of incubation solution and the negative contrast. Fortunately, after 1 being incubated with human intestinal bacterium klebsiella pneumoniae. the HPLC peak of the primary metabolite 2 in the chromatogram was so obvious that we identified it by LC-ESI-MS/MS analysis. Results in this study indicated that oxidative metabolites 3 and 4 might also be the products of 1 by the action of intestinal bacteria.Since compound 5 is one of the isomers of 1, there is possibility that synthetic strategy can be used to find the relationship between compound 1 and its isomeric metabolite 5. 1 has two benzofuran moirties and one tetrahydrofuran. Provided the carbon atoms that connect to the oxygen atom were substituted by electron withdrawing group, they are ready to take the isomerization reaction when catalyzed by acid. To avoid protonation effect, lewis acid boron trifluoride etherate was used and the isomeric metabolite 5 was successfully synthesized from 1 in an anhydrous condition. Therefore, ingested 1 in rats might be transformed to 5 by the acidic gastric juice.
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