Study On Three Chemical Components And Bioactivities In Euphorbia Humifusa Willd

Objectives:1.There are ten batches samples of Euphorbia humifusa Willd.from different areas to be studied by analysing their quality and quantity,and biological activities with active components.2.To study the effects of Euphorbia humifusa Willd.on the activity of Streptococcus mutans.3.To investigate autophagy and apoptosis in NRK cells and Hela cells induced by Euphorbia humifusa Willd.as a basis for cervical cancer therapy or other related applications.Methods:Identification and quantification of three substances from ten batches samples were analyzed by thin-layer chromatography（TLC）and high performance thin-layer chromatography（HPTLC）.The antioxidant compounds and antioxidant activity in Euphorbia humifusa Willd.were screened by TLC-DPPH.We also determined Euphorbia humifusa Willd.using high performance liquid chromatography（HPLC）.The inhibitory effects on the activity of Streptococcus mutans was investigated by the 96 pore plate method,and then used laser scanning confocal microscopy（LSCM）for observing morphological and antibacterial effect;the antiproliferation activity against Hela cells were evaluated by MTT assay;the changes of cell morphology,cell autophagy and apoptosis were observed by LSCM;Cell apoptosis was detected by Hoechst 33342staining and flow cytometry（FCM）;the autophagic vacuoles and other ultrastructure of cells were observed under Transmission electron microscopy（TEM）;the expression levels of autophagy-related protein was subjected to western blot.Results:The results of preliminary study showed that analysis of TLC was performed on silica gel GF254 plates with chloroform-ethyl acetate-formic acid with 5.2:4.4:1 proportion as the mobile phase,which are optimized conditions to determination ten batches of E.humifusa Willd..There are some differences in the contents of GA、KA and QU in E.humifusa Willd.from ten batches.And the sample from Jiangxi,Changji and Kashi have highest content of GA、QU and KA,especially.The antioxidant activities of E.humifusa Willd.with ten batches were tested by TLC-DPPH,and GA、KA and QU show their antioxidant activity with a little differences in ten batches.The contents of GA,KA,and QU determined by HPLC were measured 2.3342-4.1443,0.4636-1.5922,0.3831-3.1500 mg/g,respectively.The inhibitory effect of E.humifusa Willd.on Streptococcus mutans growth and its biofilm growth had been tested.Hela cells treated with different concentrations of E.humifusa Willd.extracts and bioactive compounds showed an growth being inhibited in a time-and dose-dependent manner which were observed by MTT and FC.Autophagy induced by extracts and gallic acid was more obvious after observation of LSCM and Hoechst 33342staining.The expression level of autophagy-associated marker protein LC₃-Ⅱincreased with increase of drug concentrations,and the expression level of P62 protein was opposite,which are consistent with above experiments.Conclusions:In this paper,HPTLC and TLC-DPPH,a kind of drug screening method withseparating compounds and determining activities,were performed to analyze and screen the antioxidant activity and components.And then TLCS method was combined with above methods to provide a reliable basis for analyzing chemicals,screening activity and selecting medicines,etc.The contents of gallic acid,quercetin andkaempferol in E.humifusa Willd.were determined by HPLC,which provided a scientific basis for the control of medicinal quality and the development and utilization of medicinal resources.At the same time,we studied the inhibitory effect of E.humifusa Willd.on Streptococcus mutans to lay a theoretical foundation for related oral diseases treatment.In this study,we also studied the extracts and active ingredients had an inhibitory activity on cervical cancer cells by inducing autophagy and apoptosis,which may play an important role in cervical cancer therapy or otherrelated applications.