A Study On Antifertility Effect And Chemical Analysis Of Extractions From Roots Of Rhynchosia Volubilis Lour

AIM To evaluate the antifertility activity of the solvent extract of Rhynchosia volubilis Lour in male mice and analyze their chemical composition.METHODS1 Solvent extractions Rhynchosia volubilis Lour was extracted by an ultrasound- assisted impregnation method and separated by columniation layer analysis, and the structures of the chemical composition in solvent extractions were determined by ¹HNMR, ¹³CNMR, IR spectrum.2 Antifertility2.1 The fertility was assessed by mating tests. The male mice were administrated by ig, and male and female mouse naturally mated one-to-one for 1 week. After 10 days, female mice were sacrificed by cervical dislocation to observe the situation of pregnancy and number of fetus, and to calculate the pregnant rate, inhibitory rate of pregnancy and corrected inhibitory rate of pregnancy.2.2 The number and motility of sperm in cauda epididymide were determined.2.3 Morphology of epididymis and testisEpididymis and testis were fixed in Bouin's solution[7], dehydrated in ethanol, embedded in paraffin wax. The sections cut at 5?m were stained with haematoxylin and eosin. Under light microscope, Morphology of epididymis and testis was observed. Under fluorescence microscope with immunohistochemistry, the distribution of the fucosidase in epididymis and testis was observed.Epididymis and testis were also fixed in 2.5% glutaraldehyde solution and 1% osmic acid. The sections cut at 1?m were stained with uranium and plumbum. Under transmission electron microscope, ultrastructure of epididymis and testis was observed.2.4 The antifertility of a few pure chemical substances was assessed in vitro fertilization experiment with ovarian stimulation in female mice.3 Antibacterial effect The antibacterial effect of solvent extract of Rhynchosia volubilis Lour roots was observed with the agar dilution method.RESULTS1 Four Kinds of Extractions from Roots of Rhynchosia volubilis Lour, i.e., ethanol extract of Rhynchosia volubilis Lour?EERVL? , ethyl acetate extract of Rhynchosia volubilis Lour?EAERVL? , n-butanol extraction of Rhynchosia volubilis Lour?BERVL? , and aqueous extract of Rhynchosia volubilis Lour?AERVL?, were obtained by an ultrasound-assisted impregnation method.2 Four kinds of extractions from root of Rhynchosia volubilis Lour, 100mg/d/kg, respectively, were administered orally for 11 weeks to adult male mice. The fertility was assessed by mating tests. The results showed that Four extractions, especially aqueous extract, from root of Rhynchosia volubilis Lour decreased the pregnant rate of mice, but the effect of BERVL was not obvious?P>0.05, compaired with NS? .3 After male mice were treated with NS, 10 mg·kg^-1·d^-1 Tripterygium wilfordii glycoside, 100 mg·kg^-1·d^-1EAERVL and 400 mg·kg^-1·d^-1EAERVL, the pregnant rates of female mice at the end of 3rd week were 80%, 15%, 40% and 20%, respectively , and that at the end of 11th week was 80%, 0%, 30% and 10%. The result of immunohistochemistry showed that the distribution of the fucosidase in epididymis was markedly decreased.4 Aqueous extract from root of Rhynchosia volubilis Lour gradually decreased the pregnant rate of mice since days14⁷0 of treatment with a decrease of the number of spermatozoa. The pregnant rates of female mice under the dosage of AERVL, that is, 25mg·kg^-1·d^-1, 100 mg·kg^-1·d^-1 and 400 mg·kg^-1·d^-1, at the end of 3rd week were 70%, 25% and 20%, respectively , and that at the end of 11th week was 50, 10% and 10%. The results, observed by transmission electron microscope, showed that in testis, the maturity of spermatids was inhibited and the arrangement of spermatogonia, spermatocytes and spermatids was in disturbance, and that in cauda epididymides, the membrane of acrosome was in the situation of irregular projections and the mitochondrion in epithelial cells was swelling.5 Four kinds of extracts of Rhynchosia volubilis Lour roots was applied in vitro spermicidal tests containing 10 mouse sperm samples and 10 human sperm samples. The results showed that the spermicidal effect of EERVL or AERVL was stronger than that of EAERVL or BERVL. But the difference among them was no significance.6 Four kinds of extracts of Rhynchosia volubilis Lour roots had the antibacterial effect on staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli. Among them, EERVL had a stronger antibacterial effect on standard or clinical staphylococcus aureus and clinical Pseudomonas aeruginosa, and BERVL had a stronger antibacterial effect on standard or clinical staphylococcus aureus, and all extracts, except AERVL, had a weaker antibacterial effect on standard or clinical Escherichia coli.7 Six kinds of pure compounds were separated from EAERVL, and they were identified as dihydro Isoflavone Derivatives , 3-D-glucose stigmast -[24]- ene,3-D-glucose kaempferol, 3-D-glucose-7-methoxy -ethyl-kaempferol, 3-D-glucose-7-sesquiterpenes kaempferol , and?-methyl-2,4- dimethoxy-L- pyranefucoside . Only one of pure compound was separated from AERVL, and they were identified as 5?, 14-dihydroxy-10?-carboxy-3?-cardiac glycoside-15 ?16?, 20 ?22?-diene .8 The rates of fertilization of ovum in vitro of five kinds of pure compounds were dihydro Isoflavone Derivatives 62.2%, 3-D-glucose kaempferol 43.0%,?-methyl-2,4-dimethoxy-L-pyranefucoside 38.9%, FucT-V ?antibody of fucosidase? 39.4%, and 5?, 14-dihydroxy-10?-carboxy-3?-cardiac glycoside-15 ?16?, 20 ?22?-diene 34.7%,respectively. 14-dihydroxy-10?-carboxy-3?-cardiac glycoside-15 ?16?, 20?22?-diene had the strongest inhibiting effect on fertilization in vitro.9 Compaired with NS, RVL did not cause any significant change in serum testosterone level and in Increment of the body weight of treated mice.CONCLUSIONSEERVL, EAERVL and AERVL have the antifertility on male mice, and the antifertility of AERVL is the strongest among them. Four kinds of extracts of Rhynchosia volubilis Lour roots had the antibacterial effect on staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli. There are Dihydro-Isoflavone Derivatives ?which content is rich?, and 3-D-glucose-7-sesquiterpenes kaempferol ?which chemical structure is novel? and?-methyl-2, 4-dimethoxy-L-pyranefucoside ?which active mechanism is especial? in EAERVL. In view of the quantity and a stronger antifertility of AEVRL, 5?, 14-dihydroxy-10?-carboxy-3?-cardiac glycoside-15 ?16?, 20?22?-diene deserves further research and development.