Study On The Quality Analysis Of Index Components And Blood-Activating Active Fractions Of Cyathula Officinalis Kuan

The main content of this topic is studying the quality control of index components of Cyathula officinalis Kuan and evaluation of the blood-activating efficacy of active fraction of Cyathula officinalis Kuan.The main research of contents and results in this study are as follows:1.Research of the quality control of Cyathula officinalis Kuan(1)Establish the high performance liquid chromatography(HPLC)fingerprints of Cyathula officinalis KuanThrough the optimization of mobile phase,elution gradient,flow rate,column temperature,preparation of test materials and other conditions,the HPLC fingerprints of Cyathula officinalis Kuan and Achyranthes bidentata Blume are successfully established.After adjustment of fingerprint chromatogram peaks,27 common peaks were found in the fingerprints of Cyathula offcinalis Kuan and 16 common peaks in the fingerprints of Achyranthes bidentata Blume.Through the comparative analysis between the fingerprints of Cyathula officinalis Kuan and Achyranthes bidentata Blume,it was found that they have some differences on the fingerprints.Therefore,the study of index components that can control the quality of them needs to be further researched.(2)The HPLC-ESI-MS analysis of Cyathula officinalis Kuan and Achyranthes bidentata BlumeIn this part,we are using the technology of HPLC-MS and comparison with the standards to identify the compounds in Cyathula officinalis Kuan and Achyranthes bidentata Blume.There are 64 compounds detected from Cyathula officinalis Kuan,including 11 phytoecdysteroids and 26 triterpenoid saponins.There are 39 compounds detected from Achyranthes bidentata Blume,including 12 phytoecdysteroids and 17 triterpenoid saponins.The triterpenoid saponins of Cyathula officinalis Kuan are similar with Achyranthes bidentata Blume,but the retention time of phytoecdysteroids with high contents in Cyathua officinalis Kuan and Achyranthes bidentata Blume have significant differences.Phytoecdysteroids and triterpenoid saponins may play an important role in the quality of them.(3)Combining the chromatographic fingerprints and chemometrics methods to analysis the markers of Cyathula officinalis KuanThe fingerprints of twenty-two batches Cyathula officinalis Kuan samples were established.Then differences among the whole samples batches and chemical markers are found using similarity analysis(SA),cluster analysis(CA)and principal component analysis(PCA).It is concluded that compounds 24-Hydroxycyasterone,Cyasterone,Isocyasterone,Precyasterone,Chikusetsusaponin Ⅳ,Chikusetsusaponins IVa,Zingibroside R1 and 3-O-Rha-(1→3/4)-Glc-28-O-Glc hederagenin affect the quality of Cyathula officinalis Kuan samples,while compounds β-Ecdysone,25R-Inokosterone,25S-Inokosterone,Chikusetsusaponin IV,Chikusetsusaponins Ⅳa and 3-O-β-D-Glucopyranosyl oleanolic acid affect the quality of Achyranthes bidentata Blume samples.The differences between Cyathula officinalis Kuan and Achyranthes bidentata Blume on the chemical markers provided the basis for the specification application and revision of quality standards of them.2.Evaluation of the efficacy of Cyathula officinalis Kuan(1)Study on antioxidant effects of Cyathula officinalis Kuan and its different solvents extraction in vitroUV was used to evaluate antioxidant effects of various fractions of Cyathula officinalis Kuan extracts by 2,2-Diphenyl-1-picryl-hydrazy(DPPH-),hydroxyl free radical(·OH),superoxide radical(O2-)scavenging activities and ferric reducing power.Results indicated that the total extract of Cyathula officinalis Kuan and the n-butanol fraction as well as 80%ethanol precipitation fraction have stronger antioxidant activities with significant dose-effect relationship.(2)Effects of Cyathula officinalis Kuan and its active fraction on acute blood stasis model ratsAcute blood stasis rat model was established by subcutaneous injection of adrenaline hydrochloride and ice water bath.Whole blood viscosity,plasma viscosity and the levels of interleukin-6(IL-6),nitric oxide(NO),tumor necrosis factor-α(TNF-α)and cyclooxygenase-2(COX-2)in plasma were measured.Results indicated that the total extract of Cyathula officinalis Kuan and n-butanol fraction as well as 80%ethanol precipitation fraction significantly reduced whole blood viscosity,plasma viscosity and levels of IL-6,NO,TNF-α and COX-2 in vivo.(3)The chemical constituents of the n-butanol fractionBy using HPLC-QTOF/MS/MS to analyse the components of the n-butanol fraction,40 compounds were identified,mainly as phytoecdysteroids and triterpenoid saponins.3.Evaluation of the efficacy of Cyasterone and Chikusetsusaponin IVThe properties of two representative components(Cyasterone and Chikusetsusaponin IV)from n-butanol fraction on thrombin-induced human umbilical vein endothelial cells(HUVECs)damage model were also assessed by levels of thromboxaneA2(TXA2),endothelin(ET),malonaldehyde(MDA),COX-2,endothelial nitric oxide synthase(eNOS)and superoxide dismutase(SOD).Results indicated that Cyasterone and Chikusetsusaponin IV have significant protective effect on HUVECs damage model by inhibiting levels of TXA2,ET,MDA,and COX-2 and promoting the activities of eNOS and SOD.Especially,the inhibitions of COX-2,ET and promotion of SOD in Cyasterone(100 μM)were better than Aspirin group(100μM).