Extraction,Purification,and Identification Of Flavonoids From Moringa Oleifera Lam.Leaves And Their Bioactivity Research

Moringa oleifera Lam.belonging to Moringaceae family,is a perennial deciduous tropical tree,and native to the south of Himalayas in Northwestern India.Moringa oleifera Lam.was called as Miracle Tree due to its edible and medicinal value.In recent years,Moringa oleifera Lam.is widely cultivated in the southwestern provinces of China,such as Guangdong,Guangxi,Yunnan.Moringa oleifera Lam.leaf was announced as a new resource food by Chinese health authorities in 2012,and flavonoids are one of its important functional components.The flavonoids from Moringa oleifera Lam.leaves?FML?have many physiological activities,such as anti-oxidation,antibacterial,anti-cancer and hypoglycemic,and can be developed into functional food,clinical drugs,and natural preservatives.The extraction and purification process,the structure and biological activities of FML were studied in this research.The main content and conclusions of this study are as follows:1.The conditions for microwave-assisted extraction of FML was optimized by response surface methodology.On the basis of single-factor experiments,a set of 4-factor and 3-level experiments were designed by Box-Behnken experimental design,with ethanol concentration,ratio of material to liquid,extraction time,extracting power as influencing factor and the extraction yield of flavonoids as response value.The results of 29 groups of experiments were fitted and performed with statistical analysis.The optimal extraction conditions were:ethanol concentration is 75%,solid-liquid ratio is 1:52 g/mL,extraction time is 308 s,extraction power is 302 W.Under this optimized condition,the extraction yield of flavonoids was 5.53%,which was very close to the model predicted value of 5.69%.2.The FML were separated and purified by macroporous resin column and polyamide column chromatography.The AB-8 macroporous resin was selected to purify FML,and its static adsorption dynamics and thermodynamics were studied.The conditions for separation and purification process of FML were optimized.The optimal conditions for AB-8 macroporous resin column were:the concentration of sample is 7 mg/mL,flow rate of sample is 2 BV/h,volume of the sample is 1 BV,eluent is 70%ethanol solution,eluent flow rate was 2 BV/h,volume of eluent is 2.5 BV;the optimal conditions for polyamide resin column were:concentration of the sample is 4 mg/mL,flow rate of sample is 1.5 BV/h,volume of the sample is 1 BV,eluent is 70%ethanol solution,eluent flow rate was 1.5 BV/h,volume of eluent is 3 BV.The flavonoids content was increased from 13.38%to 76.45%after twice purification,and the rutin content of FML was determined by HPLC.3.The structure of purified FML was analyzed and identified by UPLC-Q-Orbitrap MS technology.Based on the chromatogram information,mass spectrometry information and relevant literature,11 flavonoids and 2 phenolic compounds were identified.Including:Multiflorin B,Quercetin-3-rutinoside,Apigenin-8-C-glucoside,Quercetin-3-O-glucoside,Quercetin-3-O-?6-malonylgluc-oside?,Quercetin-3-O--hydroxy methylglutaroyl galactoside,Isorhamnetin-3-o-rutinoside,Quercetin-3-O-acetyl glucoside,Kaempferol-3-O-hydroxy methylgluta--royl hexose,Kaempferol-3-O-malonyl hexose,Kaempferol-3-O--glucoside,3-caffeoylquinic acid and 4-caffeoylquinic acid.4.The antioxidation ability of FML was evaluated by DPPH,hydroxyl radical scavenging ability and reducing power,and their antibacterial activity was studied by measuring the diameter of inhibition zone and the minimum inhibitory concentration.The EC₅₀ values of the unpurified and purified FML for DPPH were 0.03 and 0.438 mg/mL,respectively,and the EC₅₀ for hydroxyl radicals were 0.054 mg/mL and 0.874 mg/mL,respectively.0.075 mg/mL purified FML and 0.048 mg/mL Vc have the similar reduction ability.The unpurified FML have an inhibitory effect on E.coli,B.subtilis and M.luteus,and the diameter of inhibition zone is 16.71,10.66 and 15.77 mm at 20 mg/mL,and the MIC is 10,5,10 mg/mL,no inhibitory effect on S.aureus and P.aeruginosa.The purified FML have a better inhibitory effect on E.coli,S.aureus,B.subtilis and M.luteus,the diameter of inhibition zone is 21.59,17.80,20.91 and 23.94 mm at 20 mg/mL,and the rMIC is 1.250,2.500,0.625 and 1.250 mg/mL,no inhibitory effect on P.aeruginosa.5.The inhibitory effects of FML on pancreatic lipase and a-glucosidase were determined by PNPP and PNPG method.The type of inhibitory was determined by Lineweaver-Burk double reciprocal method.The results showed that the purified FML had a good inhibitory effect on pancreatic lipase,with a IC₅₀ of 0.86 mg/mL,and the type of inhibitory was non-competitive inhibition.The purified FML also had inhibitory effect on a-glucosidase,with a IC₅₀ of 4.18 mg/mL,and the type of inhibitory was competitive inhibition.