Studies On The Fingerprints Of Radix Achyranthis Bidentatae And Extraction Of Achyranthes Bidentata Saponin Together With Its Pharmacological Action

Radix Achyranthis bidentata is one of commonly used Chinese medicinal herbs, which has the activities of removing silt, nutriting the liver and kidney, strengthening bones and tendons, diuretic, citing the effect of blood downlink, clinical congestion on block for many irregular menstruation, dysmenorrhea, amenorrhea, post-deposition resistance abdominal pain, bonesetter waist and knee, lowering limb weakness, urinating blood, urine disadvantage, urethral pain etc. Saponins is an important material in a class with ranges of biological activities. The study shows that there are uterus excitement and anti-fertility role; anti-tumor effects; cardiovascular pharmacological effects; the role of anti-inflammatory and analgesic action, etc.The present study was conducted to investigate the distinction of Achyranthes bidentata Bl. and Cyathula officinalis Kuao; to explore the toxicity and pharmacological effects of ABS; the determination of haemolytic index; the morphology and development of the zebrafish embryos; the proliferation of lymphocyte,peritoneal macrophage phagocytosis and metabolism in vitro; as well as the immune effects on broilers infected by coccidia.The dried root powder of Achyranthes bidentata Bl. and Cyathula officinalis Kuao were taken as the materials in this study. The ultraviolet-visible spectroscopy, infrared spectroscopy, X-ray diffraction and SDS-PAGE method were respectively used to identify the distinction between the two different species. Setting up two species fingerprints is to identify the distinction between two different species. The results showed that the ultraviolet spectroscopy, infrared spectroscopy and X-ray diffraction method could effectively distinguish the two, and the protein fingerprint is similar to the distinction between the two,which could not be used as the means.In this experiment, the method of extracting Achyranthes bidentata Bl. Saponin (ABS) was studied and the ABS content was determined. The optimal extraction process was selected with the orthogonal design, taking four main factors which effected the selection process: ethanol concentration, solvent volume, extraction time, extraction of the frequency of three levels of each factor to test. The optimal extracion parameters were: adding 80% alcohol into crude Archyranthes bidentata Bl.(8:1 in weight) and extracting 2 times for 3h each extraction. We also took the oleanolic acid as the reference substance, using vanillin-perchloric acid colorimetry to determine the content of total saponins of ABS. Its content was 60%.In order to observe the toxicity and pharmacological effects of ABS, we studied its hemolytic index, its toxicity effects on zebrafish embryos. The results showed that the hemolytic index of ABS was 5555, that is, HD50 is 180μg/ml, which noted the smaller hemolytic. The zebrafish embryonic hatching rates of low concentration of ABS was 100%, the death effect of high concentrations(20mg/ml)of ABS on zebrafish embryonic development is apparent.The bioactivities investigated in vitro by MTT method on lymphocyte proliferation rates were measured. The results suggested low concentration(1.56-25μg/ml) of ABS had no promoting effects on the lymphocyte proliferation, but T,B-lymphocyte proliferation were inhibited at 1600μg/ml concentration. Low concentration of ABS had no effects on abdominal macrophage phagocytosis function and metabolism. With the increasing of concentration(50-200μg/ml), ABS had promoting function. When the concentration was 500μg/ml, there was inhibitment effect on the macrophage metabolism.In order to observe the effects of ABS on the immune effects of broilers effected by coccidia, we examined the lymphocyte transformation rates by MTT method, and flow cytometry was used to detect the dynamic changes of CD4+ and CD8+ T lymphocyte cells in peripheral blood of chickens, while the weight gains and spleen indexs at different times were also evaluated. The results suggested that ABS could increase the lymphocyte transformation rate of the chickens immunized by oocysts and promoted the ratio of CD4+/CD8+ T lymphocytessubsets, which indicated that the injection of ABS could promote the immunity state of chickens immunized with E.tenella. In addition,comparing with the control group, there was no significant difference in body weight gains with aging.Taken together, these results indicated that the ultraviolet spectroscopy, infrared spectroscopy and X-ray diffraction method could effectively distinguish the two; the best extraction method of ABS was adding 80% ethanol into crude Archyranthes bidentata Bl.(8:1 in weight) and extracting 2 times for 3h each extraction, its content was 60%. ABS had the hemolytic index; the high-quality saponin significantly hindered the development of zebrafish embryos, which had obvious death effects; promoting the lymphocyte proliferation and the phagocytosis and metabolism of the peritoneal macrophages; promoting the immunity state of chickens immunized with E.tenella., through the injection of ABS.