A sensitive and rapid high-performance liquid chromatography (HPLC) method with solid-phase extraction (SPE) to simultaneously determine albiflorin and paeoniflorin in rat serum was described. Serum samples were pretreated with solid-phase extraction using Extract-CleanTM cartridges, and the extracts were analyzed by HPLC on a reversed-phase C18 column and a mobile phase of acetonitrile-0.03% formic acid (17:83 (v/v)) with ultraviolet detection at 230nm. Pentoxifylline was used as the internal standard (IS). The linear ranges of the calibration curves were 29-1450ng/ml for albiflorin and 10-2000ng/ml for paeoniflorin. The intra-and inter-day precisions (R.S.D.) were≤10.49% for albiflorin and≤11.29% for paeoniflorin, respectively. Mean recovery was determined to be 89.75% for albiflorin and 85.82% for paeoniflorin. The limit of quantification was 29ng/ml for albiflorin and 10ng/ml for paeoniflorin, respectively. The validated method was applicable to pharmacokinetic studies of albiflorin and paeoniflorin from rat serum after oral administration of Si-Wu decoction. The pharmacokinetic study indicated that albiflorin and paeoniflorin had poor absorption and rapid elimination. This assay result was necessary for the pharmacokinetic evaluation of Si-Wu decoction.

Artemisinin (1) was transformed by Mucor polymorphosporus and Aspergillus niger. Five products were identified as 9β-hydroxyartemisinin (2), 3β-hydroxyartemisinin (3), deoxyartemisinin (4), 3β-hydroxydeoxyartemisinin (5), and 1α-hydroxydeoxyartemisinin (6). Products 2, 3, and 6 are new compounds.

Directional modifications of resibufogenin 1 by Mucor subtilissimus and Pseudomonas aeruginosa were carried out. The substrate was hydroxylated at C-12 by M. subtilissimus AS 3.2454, from which a major product 12-hydroxyresibufogenin 2 was obtained. Then product 2 was dehydrogenated by P. aeruginosa AS 1.860, which resulted in a new compound 12β-hydroxy-3-keto-resibufogenin 3.

The biotransformations of cinobufagin (1), an animal-originated bufadienolide, by cell suspension cultures of Catharanthus roseus and Platycodon grandiflorum were investigated. Incubation for 6 days of 1 with C. roseus yielded four products, desacetylcinobufotalin (2), 3-epi-desacetylcinobufagin (3), 1β-hydroxyl desacetylcinobufagin (4) and 3-epi-desacetylcinobufotalin (5), among which 4 is a new compound. Time course investigation revealed that the biotransformation rates of two major products, 2 and 3, reached their highest levels of 44.7 and 16.5%, respectively, on the third day after substrate administration. Compounds 2-5 showed more potent cytotoxic activities against HL-60 cell lines than the parent compound 1. A plausible biotransformation pathway is proposed to account for the formation of the observed products. From the culture supernatant of P. grandiflorum, 2 was isolated in 37.8% yield after 8 days of incubation with 1. Cinobufotalin (6) and desacetylcinobufagin (7) were also obtained as minor products. The two plant suspension cultures exhibited similar transformation patterns on compound 1.

Microbial transformation was used to prepare novel cytotoxic bufadienolides. Twelve products (3-14) were obtained from bufalin (1) by the fungus Mucor spinosus. Their structures were elucidated by high-resolution mass spectroscopy (HR-MS) and extensive NMR techniques, including 1H NMR, 13C NMR, DEPT, 1H-1H correlation spectroscopy (COSY), two dimensional nuclear Overhauser effect correlation spectroscopy (NOESY), heteronuclear multiple quantum coherence (HMQC), and heteronuclear multiple bond coherence (HMBC). Compounds 3, 4, 9 and 11-14 are new mono-or dihydroxylated derivatives of bufalin with novel oxyfunctionalities at C-1β, C-7β, C-11β, C-12β and C-16α positions. The in vitro cytotoxic activities against human cancer cell lines of 3-14, together with 16 biotransformed products derived from cinobufagin (15-30) were determined by the MTT method, and their structure-activity relationships (SAR) were discussed.