Research Of The Pharmacodynamics Of Kidney Invigorating And Miscarriage Prevention Effect Of Semen Cuscutae And Its Active Ingredients

ObjectiveTo extract the component of Modified Shoutai Wan(MSTW) recipe and its principal drug semen cuscutae, its active ingredients such as the total flavonoids and polysaccharide component. Purify and identify the extraction, prepare the drug contain serum of the high and low dosage solution of the different extract substances of semen cuscutae and establish human cytotrophoblast(CTB) cell model and identify the cell. Using the abortion animal models of corepus luteum inhibition due to renal deficiency rats and CTB cell model as research carriers, the extraction of MSTW and the active ingredients of semen cuscuta and their drug containing serum as study targets, to observe the abortion inhibition effect of the different dosage solution of the active ingredients for the abortion model rats and the effects of serum of high and low dosage solution of the different extraction on the cell proliferation, differentiation and apoptosis of the CTB cell, to screen the best dosage of kidney invigorating and miscarriage prevention effect of semen cuscutae and its active ingredients, and to explore its possible mechanism of action as well.Methods1. Extraction and ingredients identification of the drugs:Utilizing ultrasonic extraction method, Ethanol extraction plus water extraction craft to extract the MSTW recipe and semen cuscutae total extraction. Semen cuscutae water extract of the polysaccharide part were collected by alcohol precipitation, the filtrated liquid were passed through macroporous resin column, collecting the 30% and 60% ethanol part, merge and concentrate them to get the total flavonoids of cuscutae. Concentrate the water extract, adding high density ethanol, stirring and standing, then filter to derive the polysaccharides alcohol precipitation samples. The filtrate liquid was passed through macroporous resin column, collecting the liquid washing part and concentrating to derive the polysaccharide washed samples. Using UV spectrophotometry and phenol sulfuric acid method to detect the content of flavonoids and polysaccharides respectively, and identify the extracts with high performance liquid chromatography (HPLC).2. Pharmacodynamic research of the semen cuscutae extraction:Using abortion animal models of corepus luteum inhibition due to renal deficiency which were established by Hydroxy carbamide and mifepristone as the Pharmacodynamic tools, and allocating pregnant SPF SD rats into 13 groups:the control group, the model group, the positive group, the MSTW group, the high, medium and low dose of semen cuscutae total extract group (SCTEH group, SCTEM group, SCTEL group), the high, medium and low dose of semen cuscutae flavonoids group(SCFH group, SCFM group, SCFL group), the high, medium and low dose of semen cuscutae polysaccharide group(SCPH group, SCPM group, SCPL group), and 10 rats in each group. Observe the differences of the pregnancy rate, the average implantation number, weight of the uterus, ovary and a single embryo in each group. Check the differences of the serum progesterone(P) and estrodiol (E2) level among all the groups through Radioimmunoanalysis method. Meanwhile, use Real Time PCR method to observe the expression distinguishes of the mRNA level in the endometrium and ovary among the groups so as to explore possible mechanisms of the components.3. Preparation of the drug containing serum:SPF SD rats were allocated into 8 groups randomly:the blank control group(treated with saline), the MSTW group (with 1g/kg solution), the SCTEH and SCTEL group, the SCFH and SCFL group, the SCPH and SCPL,5 rats in each group. Intragastric administration two times for three days, anesthesia to take blood 1 hour after the last administration, centrifuge to get serum, prepare solution containing serum of the above groups and inactivate them for the following experiment.4. Effects of drug containing serum on the CTB cells:Take human chorionic villi tissues under sterile conditions to culture CTB cell, identify the cultured cell with immunocytochemistry and establish the CTB cell model. Using the prepared containing serum to interfere the cells, checking 0D data and cell cycle condition with thiazolyl blue (MTT) assay and flow cytometry methods respectively to observe the effect of serum on the proliferation potential and apoptosis of CTB cell.Results1. Successfully extracted the MSTW recipe and semen cuscutae total extract, and separated the active ingredients such as total flavonoids and total polysaccharides of semen cuscutae. The content of flavonoid is 55.938%, and the average content of polysaccharide is 13.891%. The extracted components were identified, and provided material foundation for the follow-up animal and cell experiments.2. The effects of different active ingredients of semen cuscutae on the abortion situation of the abortion model rats:2.1 Impact on the general situation of the rats:the model group rats start to appear the performance of Kidney yang deficiency symptoms, some rats in the positive group and the extract groups also appear similar performances, but the emergence time slightly delayed than the model group, and the symptoms gradually improved later in the experiment. There was no statistically significant differences among each groups for the weight gain of D5, D10, D11 (P>0.05).2.2 Impact on the embryo number and the abortion situation of the abortion model rats:The total embryo number in each group showed no significant statistical difference (P>0.05), while there are statistically significant differences in the number of live birth and abortions (P<0.05). Compared with the control group,number of live birth in the model group, the MSTW group, the SCTEL group and the SCPH group were significantly reduced (P<0.05), while that of the SCTEM group and SCFL group were significantly increased than the model group(P<0.05). As for the abortion number, the model group, the MSTW group, the SCTEH group, SCTEL group, the SCFM and the SCPH group were significantly higher than the control group(P<0.05), while that of the positive group, the SCTEM group, the SCFH, SCFM and SCFL group were significantly lower than the model group(P<0.05).As for the abortion ratio, the model group, the SCFM group, the SCPH, SCPM and SCPL group were significantly higher than the control group (P<0.05), while that of the positive group, the SCTEH, SCTEM, SCTEL group, the SCFH, SCFM and SCFL group, the SCPM and SCPL group were significantly lower than the model group(P<0.05 or P<0.01). The best dosage groups for the effects of increase live birth number and reduce abortion rate are the SCTEM group, the SCFL group and the SCPM group respectively.The comparison of the abortion symptoms, there was obvious difference between the control and model group (P<0.05), the model group and positive group and the SCFL group (P<0.05). As for the stasis condition of the uterus, model group greatly increased than the control and other treating groups, the stasis ratio of the SCTEH, SCTEL and SCPM group were much lower than the model group (P<0.05).2.3 Impact on the organ weight:There were no statistic differences for the weight of lateral uterus and ovary among each group (P>0.05). As for the single embryo weight, the model group, positive group and all treating groups were greatly lower than the control group (P<0.05 or P<0.01), while that of the positive group, the SCTEH, SCTEM group, the SCFM group, the SCPH and SCPL group were significantly heavier than the model group(P<0.05 or P<0.01).2.4 Impact on the serum hormone level:As for the P level, the positive group, the MSTW group, the SCTEH group, the SCFH and SCFM group were higher than the model group (P<0.05 or P<0.01). The best dosage group for the effects of improve P level are the SCTEH group, the SCFL group and the SCPH group respectively. As for serum E2 level, the model group and SCPL group were obviously lower than the control group (P<0.05), while that of the SCTEM, SCTEL group, SCFH, SCFM and SCFL group were obviously higher than the model group (P<0.05). The best dosage group of improve E2 level effect are the SCTEH group, the SCFL group and the SCPM group respectively.2.5 Impact on the expression of PR mRNA and ER mRNA of the uterus decidua and ovary:As for the expression of PR mRNA of the uterus decidua, the model group, SCTEL group, SCFL group, SCPH and SCPM group were obviously lower than the control group (P<0.05 or P<0.01), while that of the positive group, MSTW group, SCTEH, SCFH, SCFM group and the SCPL group were higher than the model group (P<0.05). There were no significant difference among the extract group and the positive group (P>0.05)Comparison of the expression of ER mRNA of the uterus decidua and ovary, the level of the model group was lower than the control group, but there were no obvious differences among the groups (P>0.05).3. Effects of serum of different active ingredients of the extract of Semen cuscutae on the proliferation activity and apoptosis of CTB cells: 3.1 Impact on the proliferation potential of the CTB cell:After treated with the high and low dose of drug containing serum for 48 hours, the optical density in 490 nm wave accelerated in a certain degree, especially for the concentration of 10% and 20% were significantly different from that of the blank control group (P<0.05 or P<0.01). As for the OD of different concentrations among the different groups, after treated with 5%,10% and 20% drug containing serum for 48h respectively, the OD of 490 nm wave accelerated gradually, which indicated that the proliferation potential increased with the concentration of serum dosage in an obvious dosage dependent tendency. The optimal dosage groups of increase proliferation potential effect are the SCTEL group, the SCFH group and the SCPH group respectively.3.2 After treated with 10% serum of different extracts for 24hs, the percentage of S-phase were obviously accelerated than the blank group (P<0.05 or <0.01), and the percentage of G2/M phase were obviously reduced than the blank group(P <0.05 or <0.01), there were no statistic difference for the percentage of the G1/G0 phase (P>0.05). Changes of 48hs treatment were accordance with the 24hs. Conclusion1. This study successfully optimized and determined the extract technology of the different active ingredients of Semen cuscutae, and detected the content and identified these ingredients as well. We also prepared drug containing serum of the extracts and established CTB cell model and using them to systematically study the pharmacodynamics and dose-effect relationship in the maintenance of pregnancy of the total flavonoids and total polysaccharides of Semen cuscutae.2. Using Kidney-Lutael abortion model rats as study carrier, the abortion number and ratio, serum P and E2 level as well as gene expression of PR and ER as pharmacodynamic parameters, the study confirmed that extract of the total extract, total flavonoids and total polysaccharides can reduce the abortion ratio and have kidney tonifying and miscarriage preventing effect. Multi-proxy analysis indicated the optimal dose of the efficacy were the SCTEM, the SCFL and the SCPM respectively.3. Using the CTB cell model as study carrier, we preliminarily confirmed that high and low concentrations of serum of the extracts can increase the proliferative activity of the CTB cells and reduce the apoptosis of the cells, the best dose were the SCTEL, the SCFH and the SCPH respectively.