Absorptive constituents and their metabolites in drug-containing urine samples from Wuzhishan miniature pigs orally administered with Buyang Huanwu decoction

Buyang Huanwu decoction (BYHWD), a famous traditional Chinese medicine prescription for the treatment of cerebrovascular diseases, is composed of seven commonly used Chinese herbs—Astragali Radix, Angelicae Sinensis Radix, Paeoniae Radix Rubra, Chuanxiong Rhizoma, Carthami Flos, Persicae Semen and Pheretima. To determine the main absorptive constituents and the metabolites of BYHWD in vivo, urine samples from Wuzhishan (WZS) miniature pigs orally administered with BYHWD (13.6 g crude drugs/kg) were collected to investigate the characteristic compounds. By comparing the high-performance liquid chromatography of a drug-containing urine sample with that of a drug-free sample, 17 characteristic compounds were isolated from the methanol extract of a drug-containing urine sample by column chromatography. Their structures, including 11 isoflavanoids, 2 pterocarpanoids and 4 isoflavonoids, were identified by spectroscopic means. Of the 17 compounds, 8 (1–8) were new compounds with the following structures: 3S-7,3′,4′-trihydroxyisoflavan-3′-O-β-d-glucuronide (1), 3S-7,3′,4′-trihydroxyisoflavan-4′-O-β-d-glucuronide (2), 3S-7,2′,4′-trihydroxyisoflavan-2′-O-β-d-glucuronide (3), 3R-7,2′-dihydroxy-3′,4′-dimethoxyisoflavan-2′-O-β-d-glucuronide (4), 3R-7,2′-dihydroxy-3′,4′-dimethoxyisoflavan-2′-O-β-d-glucuronide-6″-methyl ester (5), 3R-7,2′-dihydroxy-3′,4′-dimethoxyisoflavan-7-O-β-d-glucuronide-6″-methyl ester (6), 3R-7,2′,3′-trihydroxy-4′-methoxyisoflavan-3′-O-β-d-glucuronide-6″-methyl ester (7), and 3S-7,4′,5′-trihydroxy-2′,3′-dimethoxyisoflavan-5′-O-β-d-glucuronide (8). Based on the possible relationship and metabolic pathways of the 17 compounds in vivo, 3R-7,2′-dihydroxy-3′,4′-dimethoxyisoflavan (isomucronulatol, 11), 6aR,11aR-3-hydroxy-9,10-dimethoxypterocarpan (methylnissolin, astrapterocarpan, 13), 7,3′-dihydroxy-4′-methoxyisoflavone (calycosin, 16) and 7-hydroxy-4′-methoxyisoflavone (formononetin, 17) were thought to be the most important absorptive original isoflavonoid constituents of BYHWD in vivo, which underwent reactions of glucuronidation, hydroxylation, demethylation and reduction. The other 13 compounds were deduced to be their metabolites.