Abstract
To study the metabolism of dauricine in vivo and in vitro and identify the structure of its main metabolites, urine of rats after drug administration as the samples of dauricine metabolism in vivo was studied. Rat liver S9 fraction was prepared and the oxygenation metabolism system reconstituted to perform phase I reaction of dauricine in vitro. TLC, HPLC-DAD and MS were used to analyze and identify dauricine and its main phase I metabolites in the samples. The results showed that besides the untransformed dauricine, in the urine samples there was little product of X' which had the same features of TLC, HPLC-DAD and MS as those of N-desmethyl dauricine (N-ddau). Part of dauricine could be transformed to a main metabolite X after incubating with S9 fraction in appropriate conditions. The molecular ion peak of X was m/z 611. The full scan MS2 spectrum of m/z 611 peak from S9 sample were m/z 580, m/z 566, m/z 552, m/z 206, which were same as those of N-ddau. Liver is the major organ for dauricine metabolism and part of dauricine is biotransformed by liver. The major metabolite is considered to be N-ddau.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 253-256 |
| Number of pages | 4 |
| Journal | Journal of Tongji Medical University = Tong ji yi ke da xue xue bao |
| Volume | 20 |
| Issue number | 3 |
| DOIs | |
| State | Published - 2000 |
Bibliographical note
Funding Information:1.1 Chemicals Both Dau and N-desmethyl dau (N-ddau) were kindly donated by Dr. Pan XP, Department of Clinical Pharmacology, Tongji Medical University. Dau was a fair yellow powder with purity being 99.8 ~, MP being 98--100 "C and Mr. being 624.7. Methanol was HPLC grade. Other agents were analytical reagent quality and were distilled before use. NADPH was purchased from Sigma (USA). 1.2 Induction of Animals \[2\] Male Wistar rats, weighing 230-f-270 g, were provided by the Experimental Animal Center, Tongji Medical University. The rats received phenobarbital 80 mg/kg per day intraperitoneally on 3 consecutive days. The experiments were performed 24 h after the last injection of phenobarbital and the rats were CHEN Shujuan, female, born in 1973, Lecturer T his project was suported by a grant from the foundation of Youth Sciences and Technology Chenguang Planning of Wuhan (No. 20005004033) fasted for 16 h with free access to water before use. 1.3 Preparation of Liver $9 Supernatant ~ Rats were killed by decapitation, the livers excised rapidly and washed in ice-cold aqueous potassium chloride (KCI, 1.15 ~ w. v, 0. 154 mol/L). All subsequent procedures were carried out at g "C. The tissue was blotted dry, weighed and homogenized in 3 volumes of KC1 using a Potter-Elvehjem glass-Teflon homogenizer driven at 1200 r/rain. The suspension was centrifuged at 9000 for 20 min. The supernatant was diluted (1 : 1, v/v) with KCI (0. 154 tool/L) and used directly. All of the $9 supernatant was prepared freshly. 1.4 Incubation and Extraction of $9 Supernatant\[~.s\] $9 supernatant (0.78 g liver tissue per 10 ml reaction solution) was incubated with 0. 1 mg/ml dau (diluted to 1 retool/L) at 37'C for 1 h under aeration. The incubation was terminated by adding 20 % CCI~COOH as protein precipitation. After centrifu-gating, the supernatant was alkalized to pH 7.0--7. 5 by adding ammonia water. And then, the supernatant was extracted with chloroform 3 times. The combined extracts were evaporated to dryness and the dry residue was dissolved in chloroform for thin layer chromatography (TLC) analysis or in mobile phase for HPLC analysis. 1.5 Collection of Urine Samples and Pretreatment Rats were housed in metabolic cages. Urine was collected 24 h before the ora\[ administration. Dau was suspended in a 0. 5 ~ carboxymethyl cellulose (CMS) solution and was given at a dose of 150 rag/ kg. After dosing, 48-h urine was collected and stored at --20 C. The samples were extracted with chloroform 3 times. The combined extracts were dehydrated by adding sodium sulphate, decompressed and evaporated. The residue was dissolved in chloroform or mobile phase for TLC or HPLC analysis respectively.