| There are many flavonoids in Apocynum venetum,and flavonoids have antioxidant activity,lipid-lowering and anti-fatigue effects.However,the purity of flavonoids in crude extracts is not high,and there are few studies on the purification of flavonoids,which limits their further application.In this paper,flavonoids were extracted,separated and purificated from Apocynum venetum(Kashgar,Xinjiang province),The structures of the purified products were identified,and their antioxidant activities were studied.These researches provide theoretical support for the comprehensive utilization and development of Apocynum venetum.Orthogonal experiment was used to study the effects of ethanol concentration,temperature,extraction time,feed-liquid ratio and ultrasonic power on the extraction rate of total flavonoids.The best conditions were as follows:ethanol concentration,extraction temperature,extraction time,solid-liquid ratio and ultrasonic power were selected at 55%,50°C,25min,1:35(g/mL)and 400W,respectively.Under this condition,the extraction rate reached 2.033%.The content of total flavonoids in different parts of Apocynum venetum was:leaf(20.33mg/g)>flower(8.57mg/g)>stem(3.62mg/g)>root(0.59mg/g).The macroporous resin was used to separate and purify the crude solution of Apocynum venetum.The adsorption and desorption rates of flavonoids in the crude solution were compared among the six resins(AB-8,D101,S-8,X-5,HPD-500,and NKA-9).And the HPD-500 resin exhibited the optimal separation and purification efficiency.The influence of concentration,p H and flow rate of the sample solution on the purification effect were investigated.The optimal conditions for purification of total flavonoids with HPD-500 macroporous resin column chromatography were as follows:sample solution of 1.2mg/mL(total flavonoids),p H5.0,flow rate of2.0mL/min.70%ethanol solution,2.0mL/min to dynamically elution,and elution rate reached 91.35%.Under this condition,the purity of flavonoids increased from7.8%to 57.3%,and the effect of purification was obvious.The structures of the purified product were analyzed by UV,IR and LC-MS.And four major flavonoids were identified:aesculin,quercetin-3-O-rutinoside(rut in),quercetin-3-O-?-D-glucoside,quercetin-3-O-B-D-glucoside.Ascorbic acid(VC)was used as a reference to study the scavenging effect of crude extraction,ethyl acetate purification,macroporous resin purification against DPPH(diphenylpicrylhydrazide)radical,hydroxyl radical,superoxide anion radical,ABTS~+·radical.The results showed that:(1)When the concentration was 1.2 mg/mL,the clearance rates of the crude extraction to the four types of free radicals were54.86%,78.45%,56.24%,and 40.24%.The macroporous resin purification to DPPH radical(93.87%)and ABTS~+·radical(94.52%)was significantly improved,and the scavenging ability of superoxide anion radical(50.68%)was slightly decreased.(2)The macroporous resin purification to four free radicals(93.87%,89.27%,50.68%,94.52%)was significantly better than ethyl acetate purification(68.29%,83.68%,43.57%,63.75%).(3)The antioxidant activity was ranked as follows:VC>macroporous resin purification>ethyl acetate purification>crude extraction.When the concentration was 1.2mg/mL,the antioxidant activity of macroporous resin purification was close to VC,and the antioxidant acitivity of ethyl acetate purification and crude extraction was significantly poor than VC. |
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