Study On Extraction,isolation And Bioactivity Of Total Flavonoids From Poplar Bark

In this paper,the optimum extraction conditions of total flavonoids from poplar bark by ethanol extraction and enzyme-ultrasound-assisted extraction were systematically studied,making a systematic study and analysis about isolation and purification,structure identification,antioxidant activity in vitro,hypoglycemic activity in vitro,antitumor activity and antifungal activity to provide basic data for the development and utilization of poplar bark.1.Qualitative analysis of flavonoids from poplar bark was carried out,and 11 methods were used to extract flavonoids from poplar bark.The best extraction method was enzyme-ultrasound assisted extraction technology.The results of single factor experiment and response surface experiment showed that the optimum extraction technology of total flavonoids from poplar bark was as follows:poplar bark 1 g,weight of cellulase 37 mg per gram of poplar bark,p H value 4.6,enzymolysis time 1.75 h,enzymolysis temperature 56℃,ultrasonic temperature 40℃,ultrasonic time 30 min and ultrasonic power 160 W.Under these conditions,the yield of flavonoids was4.56%.The optimum extraction conditions of total flavonoids from poplar bark by traditional ethanol method were as follows:ethanol concentration 40%,solid-liquid ratio 1:19（g:m L）,extraction temperature 42℃,extraction time 84 min,the yield of flavonoids was 3.85%.Thus,the yield of total flavonoids by enzyme-ultrasound assisted extraction technology was 18.4%higher than that by traditional ethanol extraction method.2.Five extractive components including petroleum ether phase,dichloromethane phase,ethyl acetate phase,n-butanol phase and water phase were separated from extract by extraction in turn.The ethyl acetate phase with the highest flavonoid content was eluted by AB-8 macroporous resin,and the components Fr1 to Fr8 were obtained.The component Fr7 was purified by Sephadex LH-20 and preparative high performance liquid chromatograph,and the compounds A,B,C and D were obtained.The structures of compounds A,B,C and D were analyzed by ultraviolet and visible spectrum（UV）,infrared spectrum（IR）,high performance liquid chromatography（HPLC）,high performance liquid chromatography-mass spectrometry（HPLC-MS）,¹H-NMR,¹³C-NMR,¹H-¹H COSY,HSQC,HMBC and NOESY;The identification results of substances A,C and D were5,7,4’,5’-tetrahydroxy-3’-methoxyflavanonol,myricetin and 7-O-methyltaxifolin,substance B is4’-hydroxyflavonol or 7,4’-dihydroxyflavonol.3.The antioxidant and hypoglycemic activities in vitro of different extracts from poplar bark were studied.The results showed that the scavenging capacity of ethyl acetate phase and dichloromethane phase to DPPH free radicals at 0.08 mg/m L was similar to that of L（+）-ascorbic acid phase.The IC₅₀ values of the two phases were 0.025 mg/m L and 0.026 mg/m L,respectively.The scavenging ability of ethyl acetate phase and dichloromethane phase to hydroxyl radicals was stronger than that of other extraction phases,but still weaker than that of L（+）-ascorbic acid.IC₅₀values of them were 3.407mg/m L and 7.401mg/m L,respectively.Petroleum ether phase can promote oxidation process.In hypoglycemic activities,the inhibitory effect of extractive components on alpha-amylase was studied.The inhibition rate of five extractive components on alpha-amylase was much lower than that of Acarbose,which indicated that the hypoglycemic effect of five extractive components was not obvious.4.The antitumor activity in vitro of flavonoids isolated from poplar bark was studied by MTT method.Compound D has a certain inhibitory effect on human hepatocellular carcinoma cell line（Hep G2）and human breast cancer cell line（MCF7）.IC₅₀ values of it were 47.1μmol/L,96.9μmol/L.Compound A has a certain inhibitory effect on human breast cancer cell line（MCF7）with IC₅₀value 97.9μmol/L.Compound C and D had no significant inhibitory effect on this two cancer cells.In antibacterial activities,compound A began to inhibit the growth of Fusarium oxysporum at the concentration of 125μg/m L,compound B,C and D showed no inhibitory activity against the four fungi tested.