Study On Extraction,purification And Biological Activity Of Flavonoids From Veronicastrum Latifolium(Hemsl.) Yamazaki

Veronicastrum latifolium(Hemsl.)Yamazaki is a perennial herb of Veronicastrum Heist,ex Farbic of Scrophulariaceae.Veronicastrum latifolium(Hemsl.)Yamazaki could be used as a whole herb to remedy cirrhotic ascites,nephritic edema,bruises,burns,and furuncle swollen disease.Veronicastrum latifolium(Hemsl.)Yamazaki is rich in flavonoids,which have multiple biological activities such as antioxidation,antibiosis,anti-inflammatory,enhancement of the immunity,lower blood pressure and blood lipids,protection of the cardiovascular and cerebrovascular diseases,etc.This study takes Veronicastrum latifolium(Hemsl.)Yamazaki as the research object,the flavonoids were analyzed and identified by HPLC-MS/MS.It was the first time to establish HPLC method for the quantitative analysis of flavonoids.On the basis of the quantitative analysis,the conditions in the extraction process were optimized,and the macroporous adsorption resins were used to enrich and purify the flavonoids.The antioxidant,antibacterial,and anti-inflammatory activities of the crude extract and purified product in the Veronicaslrum latifolium(Hemsl.)Yamazaki were discussed for the first time.The results indicate that:1.The eight flavonoids in Veronicastrum latifolium(Hemsl.)Yamazaki were identified by LC-MS/MS and HPLC qualitative analysis mainly as vitexin,luteolin,wogonoside,apigenin,hispidulin,chrysoeriol,acacetin,and pectolinarigenin.The developed HPLC chromatographic conditions:chromatographic column:Diamonsil C18 column,(250×4.6mm i.d..5?m),the mobile phase was constituted of the 0.5%phosphoric acid(H3PO4)in aqueous solution(A)and acetonitrile(B),the gradient of the mobile phase was as follows:0-6 min,10-15%B,6-7 min,15-18%B,7-30 min,18-25%B,30-65 min,25-40%B,65-80 min,40-52%1B.The column temperature was maintained at 35?,and the flow rate was 0.8 mL/min.The detection wavelength was 350 nm,and the injection volume was set as 20 ?L.The column temperature was maintained at 35?,and the flow rate was 0.8 mL/min.It was confirmed that this method can be successfully used for the qualitative and quantitative analysis of the main flavonoids in Veronicastrum latifolium(Hemsl.)Yamazaki.2.The process conditions of microwave-assisted extract vitexin,luteolin,wogonoside,apigenin,hispidulin,chrysoeriol,acacetin,and pectolinarigenin in the Veronicastrum latifolium(Hemsl.)Yamazaki were optimized based on the single factor experiment and the response surface methods,and determined the optimum process conditions:70%ethanol was used as solvent,the microwave temperature was 59?,the microwave time was 18 min,and the solid-liquid ratio was 1:48(g/mL).Under the optimal extraction conditions,the extraction yields of the vitexin,lutcolin,wogonoside.apigenin,hispidulin,chrysoeriol,acacetin,and pectolinarigenin in the Veronicastrum latifolium(Hemsl.)Yamazaki were 0.284 mg/g,0.298 mg/g,5.59 mg/g,0.161 mg/g,0.297 mg/g,0.263 mg/g,0.285 mg/g,and 0.781 mg/g,respectively.It was confirmed that this method has a high rate of extraction of flavonoids from Veronicastrum latifolium(Hemsl.)Yamazaki,and the extraction process is completely feasible.D101 resin had high adsorption rate,desorption rate,high purity,and recovery rate for the flavonoids in the Veronicastrum latifolium(Hemsl.)Yamazaki,and the values were 83.7%,81.2%,88.5%,and 41.4%,respectively.D101 resins were suitable for enrichment and purification of the flavonoids from the crude extract in the Veronicastrum latifolium(Hemsl.)Yamazaki.The purity of the flavonoids in Veronicastrum latifolium(Hemsl.)Yamazaki was 9.36 times higher compared with crude extract after purification through D101 resin.3.The DPPH free radical scavenging experiment,ABTS free radical scavenging experiment,and FRAP total antioxidant capacity experiment were carried out on crude extract and purified product of the Veronicasrrum latifolium(Hemsl.)Yamazaki in this study,and selecting Vc as control.Both the crude extract and purified product had antioxidant effects in the three antioxidant experiments,and the purified product had stronger antioxidant capacity compared with the crude extract.The gram-positive bacteria(Staphylococcus aureus,Bacillus subtilis,Candida albicans)and gram negative bacteria(Escherichia coli,Pseudomonas aeruginosa,Acinetobacter haumannii)were selected to study antibacterial activity for the crude and purified product of the Veronicastrum latifolium(Heinsl.)Yamazaki.Both the crude extract and purified product showed some inhibition on the six tested strains,but the inhibitory effect on the gram-positive bacteria was stronger compared with the gram-negative bacteria.The antibacterial effect of the purified product on the six tested strains was stronger compared with the crude extract,and the purified product had the strongest antibacterial activity against(Candida albicans.The effect of the crude extract and purified product in the range of 25-500 ?g/mL on the cell viability of the RAW 264.7 cells was determined by MTT assay.This study found that the crude extract and purified product in the concentration range of 25-300 ?g/mL has no significant effect on cell viability.The secretion of NO in the RAW 264.7 cells induced by LPS was detected by Griess method.It was found that the crude extract and purified product of the Veronicastrum latifolium(Hemsl.)Yamazaki inhibited the secretion of NO,and the inhibitory ability of the purified product was stronger compared with the crude extract.It was verified that the crude extract and purified product of the Veronicastrum latifolium(Hemsl.)Yamazaki had anti-inflammatory effects,and the anti-inflammatory effects of the purified product was stronger compared with the crude extract.