Contraceptive and hormonal properties of the stem bark of Dysoxylum binectariferum in rat and docking analysis of rohitukine, the alkaloid isolated from active chloroform soluble fraction.

Background: This study was aimed to investigate the pregnancy interceptive activity of the stem bark of Dysoxylum binectariferum Hook. f. administered during the pre- and peri-implantation periods and immediately after implantation by oral route in adult female Sprague-Dawley rats.

Study Design: Ethanolic extract and its fractions were administered to female rats on Days 1-10, Days 1-7, Days 1-5 or Day 1 postcoitum by oral route. At autopsy on Day 12, the number and status of corpora lutea and implantations were recorded. For estrogenic activity, ovariectomized immature rats received the test extract or the vehicle once daily for 3 days and at autopsy on Day 4, uterine weight and status of vaginal opening and extent of vaginal cornification were recorded. For antiestrogenic activity, the extract was administered along with ethinyl estradiol. Docking analysis of rohitukine, the alkaloid isolated from active chloroform soluble fraction, to estrogen receptor (ERalpha) was conducted using AutoDock 3.0.5 on a Linux workstation.

Results: The ethanolic extract intercepted pregnancy in rats at a daily dose of 500 mg/kg on Days 1-7 postcoitum. On fractionation, the activity was localized in the chloroform fraction, which inhibited pregnancy in all females at the 35-mg/kg dose on Days 1-7, at the 50-mg/kg dose on Days 1-5 or at the single 300-mg/kg dose on Day 1 postcoitum. Chromatography of this fraction yielded an alkaloid, rohitukine, which prevented pregnancy at the 10-mg/kg dose administered on Days 1-7 but was partially (45%) effective at this dose when administered during the entire preimplantation period and ineffective even at 10 times this dose when administered only on Day 1 postcoitum, except that there was a significant reduction in implantation number in pregnant females. While the active chloroform soluble fraction was devoid of any estrogen agonistic or antagonistic properties, a mild uterotropic effect without induction of premature opening of vagina or cornification of vaginal epithelium was observed in rohitukine at the 10-mg/kg dose. Rohitukine, with an almost similar molecular size (mol. wt. 305) as 17beta-estradiol, fits ideally into the hydrophobic pocket of ER. While it does not appear to simultaneously interact with GLU353, ARG394 and HIS524 as estradiol to elicit frank estrogenic response, different conformations of the ligand or its metabolite(s) might acquire geometry with phenolic groups at C-3', C-5 and C-7 positions disposed in a fashion to interact with active site(s) of ER, which might be responsible for its contraceptive and/or weak uterotropic effects. The absence of a basic side chain directed toward the antiestrogen binding site (ASP351) on the receptor appears to be responsible for the lack of any estrogen antagonistic activity.

Conclusions: Findings demonstrate the antifertility activity of the ethanolic extract of D. binectariferum, its chloroform soluble fraction and rohitukine. Efforts are being made to enhance the anti-implantation activity of rohitukine by structural modifications.