Metabolic Studies Of Evodiamine, Rutaecarpine And L-Tetrahydropalmatine In Microbial Organisms And Rats

Evodia rutaecarpa Juss. Benth is the most important component of famous Wu-Zhu-Yu decoction of China. Previous studies showed that Evodia is effective in the treatment of stomach diseases. Evodiamine and rutaecarpine are the two major active indole alkaloids in the title plant. In recent years, they have been found to exhibit cardiotonic, anti-inflammation and antitumor activities and received more and more attention from scientists of various fields. 1-Tetrahydropalmatine (1-THP), a naturally occurring neuroactive alkaloid from Stephania ainiaca Diels, has been widely used as analgetic medicine-Lu Tong Ding in China for many years. To find more effective compounds with low toxicity or to identify the in vivo metabolites, scientists have been searching for the approach to modify the structures by efficient methods.An important factor in the evaluation of safety and efficacy of any drug is the knowledge of how the drug is metabolized. Microbial metabolic studies have such advantages over chemical synthesis as high stereo- and regio-selectivity and are becoming a complementary tool in the study of drug metabolism in mammals for the reason that most families of human liver cytochrome P450 have been expressed in microorganisms as individual enzymes. For obtaining sizable amount of metabolites for pharmacological and toxicological studies, microbial metabolism is clearly useful.In efforts to study the chemical constituents responsible for antiemetic activity of Evodia and the metabolisms in microbial organism and rats of evodiamine and rutaecarpine, the chemical, pharmacological and metabolic studies were carried out. Metabolism of 1-THP in microbial organisms and rats were investigated at the same time.Two HPLC methods were set up to determinate the concentrations of evodiamine, rutaecarpine, rutaevine and evodin in Evodia, respectively, which get the results of 0.71%, 0.72%, 0.57% and 0.43%. Effects on stomach strips of different fractions of Evodia were evaluated. Fractions a, d and e showed obvious activity of antagoning contraction to stomach. Analgesic effect of different fractions of Evodia was also invesgated using acetic-acid induced twitching in mice and its analgesic potency was compared with ibuprofen. The fractions a, c, d and e all showed potential analgesic effect.Microbial transformations of evodiamine and rutaecarpine were carried out. Twenty strains of fungi were screened for their capabilities to transform the above two alkaloids. It was found that Mucor, Cunninghamella and Penicilliurn species could metabolize evodiamine and rutaecarpine to produce versatile products. Penicilliumjanthinellum AS 3.510 and Cunningharnella blakesleana AS 3.970 were found to be the most potent strain for evodiamine and rutaecarpine and were selected for the preparative-scale biotransformation. Eight products were isolated from the culture supernatant by silica gel column chromatography and preparative RP-HPLC. By means of extensive spectroscopic techniques, their structures were identified as 10-hydroxyevodiamine (J-3-1), 11- hydroxyevodiamine (J-1), 3-hydroxyevodiamine (J-3-2), p-hydroxybenzyl alcohol (J-2-1), p-hydroxyphenylethyl alcohol (J-2-2), 10-hydroxyrutaecarpine (C-1), 3-hydroxyrutaecarpine (C-2) and 3,10-dihydroxyrutaecarpine (C-6), respectively, among which products J-3-1, J-3-2, J-1, C-1, C-2 and C-6 are new compounds. Two substrates were converted into hydroxyl metabolites in 7 days of incubation with low efficiency and the possible biotransforrnation pathways are proposed to account for the formation of the observed products. The in vitro cytotoxicities of the biotransformed products were determined by MTT method. J-1, J-3-1, J-3-2, C-2 and C-6 were found to exhibit potent inhibitory activities against human cancer cell lines (BGC-823, Bel-7402, MCF-7 and HL-60), in which J-1 showed stronger inhibitory activities against four cell lines than evodiamine.A similar biotransformation process for 1-THP was carried out. Penicillium janthinellum AS 3.510 was selected as the strain for the preparative-scale biotransformation and 3 products were isolated. Their structures were identified as 1-corydalmine (T-1), 1-corypalmine (T-2) and 9-demethyl-1-THP (T-3), respectively. Time-course investigation revealed that Penicillium janthinellum AS 3.510 could slowly demethylate 1-THP at different -OCH₃ positions.LC/MS/MS analysis was used in the metabolism studies of evodiamine, rutaecarpine and 1-THP in rats. The structures of two metabolites (J-3-1 and J-3-2) of evodiamine and three metabolites of 1-THP (T-1, T-1 and T-3) in rat urine and feces were identified with the reference standards separated from biotransformation. Structures of three metabolites of 1-THP in rat feces were tentative deduced by MS and MS/MS analysis. The metabolisms of ebodiamine, rutaecarpine and 1-THP in microbial organisms and rats were compared.