Isolation Of Chemical Compositions From Vernonia Amygdalina By DOSY Technology,Antioxidant Activity And Anti-breast Cancer Activity Of Vernonia Amygd Alin A

Vernonia amygdalina?family of compositae?is a valuable vegetable and medicinal plant that is widespread in East and West Africa.This plant contains complex active components that are pharmacologically useful.It is known as bitter leaf and may be used as anticancer agent,anti-bacteria,anti-malaria,and anti-parasites.In addition to being used medicinally by local people,it weas also eaten as vegetables,making the incidence of breast cancer in local women lower than normal,indicating that it had a very good anti-breast cancer activity.In this study,in vitro anti-breast cancer activity test was used as a verification method.The potential anti-breast cancer active components?steroidal saponins?in V.amygdalina were tracked with DOSY technology,and isolation and purification of steroidal saponins were guided by DOSY technology,which laid the foundation for the application of DOSY technology in the separation and purification of natural products.At the same time,the anti-breast and anti-oxidant activities of the components were evaluated to provide important data for further research in the future.In this study,three different methods were used to extract V.amygdalina.Petroleum ether,ethyl acetate,85%ethanol and deionized water were used as solvents in the extraction of V.amygdalina obtaining ten extractions?VA1-10?.The antioxidant activities of four different polarity extractions?VA4,VA5,VA6 and VA7?were investigated using various in vitro assays?DPPH,ABTS and hydroxyl free radical scavenging assay?.What's more,in order to determine the active site against breast cancer and the optimal extraction method for anti-breast cancer active sites,ethyl acetate extracts?VA2,VA6 and VA9?and 85%ethanol extracts?VA3,VA7 and VA8?obtaining with different conditions against human breast cancer lines?MCF-7?were investigated by the CCK-8 assay.The DOSY spectrum of ethyl acetate extract was tested to determine whether it had steroidal saponins.Ethyl acetate extract?A1?and its six crude fractions?A2-7?were evaluated for anti-breast cancer activity and DOSY spectra.In addition,A6,an active component,was isolated and purified rapidly by DOSY technology,obtaining five compounds?F1-5?.The structure of F5?vernonioside D1 and vernonioside D₂?was identified by NMR,FT-IR,UV,HPLC,and MS.The results showed that F5 was a mixture of two steroidal saponins?vernonioside D1 and vernonioside D₂?.F5 was evaluated for anti-breast cancer activity.The results of antioxidant assay showed that:?1?VA4,VA6,and VA7 all possessed excellent abilities on scavenging DPPH and ABTS free radical while VA5 had moderate activity during the concentration 0.025-1.0 mg·mL-1.?2?In the concentration range of 0.1-5.0 mg·mL-1,VA4,VA6 and VA7 all possessed excellent abilities on scavenging hydroxyl free radical while VA5 had moderate activity.Generally,VA7 had the strongest scavenging ability on three kinds of free radicals and VA5 had the weakest scavenging ability at the same concentration.The results of DOSY assay showed that:The steroidal saponins in ethyl acetate extract from V.amygdalina were tracked by DOSY technology.A6 and A7,crude fractions of ethyl acetate extract,also have steroidal saponins.The steroidal saponins?F5?were isolated and purified from active component A6 using DOSY technology.It suggested that natural products with specific functional groups could be isolated and purified rapidly by DOSY technology.The results of CCK-8 assay showed that:?1?pre-experiment:The results indicated the ethyl acetate extractions from V.amygdalina could inhibit proliferation of human breast cancer cells MCF-7 in vitro with the concentration-effect relationship.VA6 had the best anti-breast cancer activity at the concentration of 50 ?g mL-1 with the inhibition rate of 85.43%.It suggested the optimal extraction conditions for active ingredients against breast cancer was extracted with ethyl acetate at 50 ?.?2?After extensive extraction:A5,A6,and A7 all showed good inhibitory effects on MCF-7 breast cancer cells at the concentration of 50 ?g mL-1,with the inhibition rates of 64.08%,81.75%and 78.57%,respectively,and the inhibition rates were higher than A1.It suggested A5,A6 and A7 were the active sites in A1,and A6 had the best anti-breast cancer activity?3?F5:The anti-breast cancer activity of F5 was evaluated by CCK-8 method.The inhibitory rate of F5 was 77.53%at the concentration of 100 ?g·mnL-1.It was demonstrated that F5 had a good inhibitory effect on MCF-7 breast cancer cells,suggesting steroidal saponins was one of the anti-breast cancer active substances in V.amygdalina.However,the inhibitory rate of F5 was lower than A1 and A6,suggesting a possible synergistic effect of steroidal saponins and any other anti-breast cancer constituents in ethyl acetate extractions.