Study On The Separation, Identification And The Mechanism Of Antifertility Bioactive Components From Castorbean (Ricinus Communis L.) Seed Extract In Female Rodents

Rodent pest is a worldwide calamity, which is one of leading disasters in the field of bio-agriculture. In China, tremendous economic losses in the fields of agriculture, forestry and animal husbandry are caused by Rodent pests. The effective control of rodents is of importance to ensure the agriculture sustainable development. Presently, the mainly rodents control method in the world is chemical control, most of the rodenticides mainly cause the increase of the mortality of mice and could not keep long effect, the low density of mice can maintain only one year, since the density of mice rise gradually and restore to the previous level. In addition, chemical rodenticides possess the side-effects on environment and living things. Due to the shortcomings of traditional rodenticides, the new rodenticide is urgently necessary for the efficacious and continuous reduction of mice: the population density of mice could be reduced by decreasing the reproduction rate of mice, namely, developing a new strategy of biological sterilization of rodents.The results of the investigation on the effect of sterility on the rodent reproduction and their population demonstrated that: Decreasing the reproductive ratio instead of increasing mortality had been suggested as a promising means for managing the impact of overabundant species. Sterilized resident females were thought to restrict reproduction on the population level if they maintained their territories and social status and prevented subordinates from breeding. Many botanic sterilants development in recently years acted on male rodents, but few acted on female. The reason was that the choice of guide for bioassay-guided fractionation of antifertility components in female rodents was difficult.Castorbean oil, an aether extract from the castorbean seeds(Ricinus communis L.)(AEC), has been known to possess medicinal property of antifertility activity on both female and male mammals. In order to developing new sterilants, the antifertility effect and the machinsm of castorbean oil in female rodents were investigated, the in vitro method for separating the contraceptive components of AEC was established; a colorless crystal which showed a significant inhibitory activity on rat decidual stormal cells was separated by means of bioassay-guided fractionation, and the colorless crystal was chemically analyzed. The results of the research not only supplied high-effective and safe botanic medicine for antifertility of rodents, but also established a new way of comprehensive utilization of castorbean resource of our country, which was beneficial to developing castorbean industry. It will helpful to the establishment of the substantial rationale for the developing of high-effective botanic antifertility medicine of our country possessing independent intellectual property rights, and also for the developing of the neotype botanic rodentcide. The main results are showed as follows. 1 Kunming mice were gavaged the AEC at doses of 0mg/kg/d, 20mg/kg/d, 40mg/kg/d, 80mg/kg/d and 160 mg/kg/d in specified time respectively. The amount of pregnancy mice and the embryos were added up for the research on the anti-early pregnancy and anti-implantation of AEC. The results were that the AEC at doses of 40mg/kg/d, 80mg/kg/d and 160 mg/kg/d possessed evident antifertility effects on mice. Their fertility inhibition ratio were 72.2%,88.2% and 90%(compared with control, p<0.01), respectively, the amount of foetus were reduced apparently. In addition, the fertility inhibition effect was found dose dependent as increase in concentration. The implantation ratio at doses of 40mg/kg/d,80mg/kg/d and 160 mg/kg/d were 3/8,1/6 and 1/6; The ratio of pregnancy at the dose of 40 mg/kg/d was reduced apparently. There were almost no changes in the index of ovary, spleen and drenal gland in the mice treated with AEC, but the index of thymus was increased at the dose of 40 mg/kg/d, moreover, the increase of the index of thymus was dose-dependent(p<0.01).2 Kunming mice were gavaged AEC at doses of 0mg/kg/d, 20mg/kg/d, 40mg/kg/d, 80mg/kg/d and 160 mg/kg/d for one week respectively. In order to investigate the antifertility mechanism of AEC, the mice were taken to study the sexual cycle, pregnancy-associated plasma protein A(PAPP-A) and the level of sexual hormone such as estradiol(E2) and progesterone(P), as well as the microstructure of ovaries, fallopian tube and uterus after the administration. The results were that the sexual cycles of mice treaded with 40mg/kg/d, 80mg/kg/d and 160 mg/kg/d were changed, mainly diestrus was erratically extended. From the 20 mg/kg/d, the PAPP-A of the treaded mice was lower than that of control, and from 40 mg/kg/d the decrease of PAPP-A was evident. There were no notable changes in the E2 level of treated mice, but the P level was increased in a certain extent. The 77.78% un-pregnant uteri suffused with colorless transparent fluid treated at a dose of 40mg/kg/d was evidently inflated. Histological observation showed that the height of epithelium in AEC treated un-pregnant uteri was significantly increased, and that the thickness of endometrium stromal layer, in which the glands were fewer and the muscle layer of which the cells ranged irregularly, was decreased.3 The morphology and the viability of the cells being taken as an index, the direct effects of AEC on the primary cultured rat decidual stormal cells(DSC) and rat luteal cells were observed. The results indicated that: AEC has inhibitory effects on rat decidual stomal cells(DSC) at a dose of 400μg/mL(P<0.05). In addition, the depressant effect was found dose dependent as increase in concentration increased the inhibition. When the concentration reached 80μg/mL, many DSC died(P^-0.001). The IC₅₀ of AEC on DSC was 568.6±5.3μMg/mL, r=+0.9790. But AEC has no inhibiting effects on rat luteal cells. The results suggested that the test on the viability of cultured rat decidual stormal cells could be used as a guide of bioassay-guided separation and isolation of AEC contraceptive activity components.4 AEC, which possessed antifertility activity and contraceptive efficacy in female rodents, were evaluated for their in vitro inhibitory activity in the primary cultured rat decidual stromal cells(DSC). A bioassay-guided fractionation technique was used to separate active components from crude extracts. A colorless crystal showed a significant inhibitory activity(IC₅₀=63.84±3.04μg/mL, r=+0.9478). The chemical analysis of the colorless crystal by capillary gas chromatography-mass spectrometry(GC-MS) revealed that the colorless crystal was a mixture of five components: four phytosterols which were ergost-5-en-3-ol(6.10%), stigmasterol(35.80%),γ-sitosterol(44.77%) and fucosterol(8.40%); and one probucol analog(4.93%). It was presumed thatγ-sitosterol may be the main component contributing to inhibit the viability of DSC, and the stigmasterol might act as an assistant.