| Objective:In this study,optimization of extraction technology of total flavonoids from Corepsis tinctoria Nutt.,expect to obtain higher content,establish a method for the determination of chemical composition and content of total flavonoids by macroporous adsorptive resin to purificate,identify the inhibition capacity of DPPH radical scavenging,ABTS radical scavenging,superoxide anion radical scavenging and anti-lipid peroxidation from the active ingredient of total flavonoids,provide the basic data for drug discovery of Corepsis tinctoria Nutt.and the research of drugs pharmacokinetics in vivo.Methods:(1)Response surface methodology was used to optimize the parameters of ultrasonic extraction of total flavonoids with Box-Behnken central composition design principle,The adsorption and desorption capacities of 10 kinds of macroporous adsorption resins were investigated such as XDA-1,NKA-10 and AB-8,which was selected to purify and enrich the effective part of macroporous resin.(2)Uesd UPLC-PDA to qualitative analysis of total flavonoids from Corepsis tinctoria Nutt.after purification of macroporous resin,The sample was analyzed on an ACQUITY UPLC?HSS T3(1.8 ?m,2.1×100 mm)column at 30 temperature,with mobile phase consisting of methanol and 0.5% formic acid by gradient eluteion at the flow rate of 0.2 ml·min-1,volume of sample was 1 ?l,the wavelength was at 280 nm.establish a determination method of chemical components of total flavonoids before and after.(3)Uesd the TLCbioautography,establishment the method of free radical scavenging activity and experimental method for inhibition of glycosidase activity in vitro.Results:(1)Optimized concentration of ethanol,liquid-solid ratio 21:1,the ultrasonic time was 60%,30 min.To obtain stable quality,high content of total flavonoids extract which was 4.65%.Selected the AB-8macroporous resin optimum technology conditions were as follows :sample concentration was 0.54mg·ml-1,sample volume was 2.1 BV,adsorption equilibrium 4 h,2-3 BV flushing fluid,eluted by 2.2 BV50% ethanol.(2)Determine the 10 components used UPLC method,just as cyanidin-3-O-glucoside,chlorogenic acid,favanomarein,quercetagitin-7-O-?-D-glucopyranoside,rutin,flavanokanin,marein,okanin,luteolin,quercetin.The linear ranges of the 10 components are 10.04~100.4 ?g·ml-1 ?2.51~40.16 ?g·ml-1?49.80~298.80 ?g·ml-1?49.80~298.80 ?g·ml-1?2.51~40.16 ?g·ml-1?50.00~300.00?g·ml-1?64.00~384.00 ?g·ml-1?50.20~301.20 ?g·ml-1?2.48~39.68 ?g·ml-1?2.50~40.00 ?g·ml-1,above have good inter relationships within the concentration range(average r>0.999),sample recovery between 98%and 100%.(3)TLC-Bio assay was used to identify the active components with antioxidant and?-glucosidase inhibitory effects,total flavonoids were eluted with methyl benzene-acetic ether-formic acid(8:7.4:3)on the silica gel plate,and the antioxidant components were identified after the coloration with the1,1-diphenyl-2-picryl-hydrazyl(DPPH)and 3-ethylbenz-thiazoline-6-sulphonic acid(ABTS)methanol solution.The total flavonoids were eluted with the same method,incubated for 1 huor in ?-glucosidase solution which was sprayed on the silica gel plate and were colored with the solution of p-Nitrophanyl-?-D-Gluopyranoside and fast blue B salt(1:1)as the color developing agent,thus?-glucosidase inhibitors were obtained and identified by resolution in visible light;The antioxidant activities in vitro of total flavonoids were measured by the methods of DPPH radical scavenging capacity,ABTS radical scavenging capacity radical scavenging capacity and anti-lipid peroxidation capacity.The value of IC50were(58.75±2.549),(23.13±0.0004)and(19.74±1.09)?g·ml-1;p NPG was used to determine ?-glucosidase and ?-glucosidase of rat small intestine inhibiting activity.The value of IC50were(46.79±9.024)and(0.771±7.735)mg·ml-1.Conclusion:(1)Response surface methodology was used to optimize of ultrasonic extraction of total flavonoids,which can obtained the higher content of total flavonoids.The AB-8 macroporous resin has better purification effect for total flavonoids.(2)The flavanokanin,marein,flavanomarein,okanin of total flavonoids have good effect for DPPH radical scavenging,ABTS radical scavenging and inhibition of ?-glucosidase;(3)The total flavonoids has strong antioxidant activity,which can be used as a new free radical scavenging scavenger and natural antioxidants;total flavonoids have medicinal development values as a new ?-glucosidase inhibitor and antioxidant. |
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