Extraction,Isolation And Identification Of Polyphenols From Moringa Oleifera Lam. Leaves And Their Antioxidant Activities

Moringa oleifera Lam.,belonging to Moringaceae family,is a perennial plant and native to the southern Himalayas in northern India.Since the introduction and cultivation of M.oleifera in Yunnan Province in the 1960s,the promotion of planting in China has gradually increased and the Moringa industry has developed rapidly.M.oleifera leaves are rich in polyphenolic active substances and have antioxidant,anti-tumor,hypoglycemic,hypolipidemic,anti-bacterial and anti-inflammatory activities.They have broad prospects for development in the health care products and pharmaceutical industries.At present,the research on the polyphenols from M.oleifera leaves is mostly focused on the extraction method and its drug value.However,the use of polyphenols from M.oleifera leaves as a lipid antioxidant has rarely been reported.It needs further development and research.This paper studied the extraction technology of polyphenols from M.oleifera leaves.And the polyphenols from M.oleifera leaves were isolated,purified and the components were identified.The antioxidant activity in vitro and the anti-oxidation effect on soybean oil were determined.Different extraction methods of polyphenols from M.oleifera leaves were optimized and studied.The optimal extraction conditions for ultrasonic-assisted extraction method were:ethanol concentration 70%,solid-liquid ratio 1:30,extraction temperature 50°C,and extraction time 42 min.Under these conditions,the total flavonoid yield was 4.83±0.07%,the total phenol yield was 2.44±0.10%.The optimal extraction conditions for pressurized solvent extraction method were:ethanol concentration 70%,solid-liquid ratio 1:40,extraction temperature 110°C,extraction time 30 min.Under these conditions,the total flavonoid yield was 4.66±0.13%,the total phenol yield was 2.42±0.05%.The total flavonoid yield and total phenol yield of M.oleifera leaves obtained by ultrasonic-assisted extraction were higher than those by pressurized solvent extraction.The components of the polyphenol crude extracts from M.oleifera leaves obtained by ultrasonic-assisted extraction and pressuried solvent extraction method was identified by high performance liquid chromatography-mass spectrometry(HPLC-ESI-MS).The results showed that there were 23 polyphenols in total.The 21 compounds that have been initially identified were:3-caffeoyl quinic acid,coumaroylquinic acid isomer 1,coumaroylquinic acid isomer 2,caffeoylquinic acid isomer 1,4-caffeoylquinic acid,4-p-coumaroylquinic acid,caffeoylquinic acid isomer 2,coumaroylquinic acid Isomer 3,6,8-di-C-glucosylapigenin,quercetin triacetylgalactoside,isolariciresinol glycoside,apigenin glucoside isomer 1,apigenin glucoside isomer 2,Quercetin-3-O-β-D-glucoside,quercetin-acetyl-glucoside isomer 1,quercetin-acetyl-glucoside isomer 2,kaempferol-3-O-glucoside,quercetin-acetyl-glucoside isomer 3,kaempferol-acetyl-glucoside isomer 1,kaempferol-acetyl-glucoside isomer 2,kaempferol-acetyl-glucoside isomer 3.There were two other compounds that were unknown and still needed further analysis.Ultrasonic-assisted extraction method and pressurized solvent extraction method have different species of polyphenols from M.oleifera leaves.Pressurized solvent extraction method under the same temperature conditions,pure water,pure ethanol and ethanol/water mixed system compounds have obvious differences,but different proportions of ethanol/water mixed systems have the same type of extraction compounds,the same solvent at 140°C,the extracted compounds and their relative contents were less than those at 110°C.The crude extracts of polyphenols from M.oleifera leaves was isolated and purified by organic solvent extraction and polyamide column chromatography.The results showed that the purity of the total flavonoids(76.58±0.65%)eluted by the 30%ethanol in n-butanol phase after polyamide column chromatography was the highest,which was 4.33 times higher than that before purification.The purified polyphenol extracts from M.oleifera leaves can be used to further study the anti-oxidation of oils.This purified product was further separated by Sephadex LH-20 column chromatography and preparative liquid phase to obtain a single compound quercetin-3-O-glucoside.The antioxidant activity of polyphenol crude extracts of M.oleifera leaves and their isolates was evaluated in vitro by the determination of DPPH free radicals,ABTS free radicals,hydroxyl radical scavenging ability and total reducing power.The results showed that the antioxidant activity of polyphenol crude extracts of M.oleifera leaves obtained by ultrasonic-assisted extraction method was higher than that of pressurized solvent extraction method.Among four organic extraction sites,the DPPH radical,ABTS radical,hydroxyl radical scavenging ability and total reducing power of n-butanol phase were higher than ethyl acetate phase,petroleum ether phase and water phase,slightly lower than that of V_C at the same concentration.The n-butanol phase has a certain separation effect on polyphenols.Different ethanol elution phases showed good antioxidant effects,of which,30%ethanol eluting phase had the best active ingredient enrichment effect and oxidation resistance.The DPPH radical and ABTS radical scavenging ability of the monomeric compound quercetin-3-O-glucoside was higher than that of V_C.When the sample concentration was 0.1mg/m L,its DPPH radical and ABTS radical scavenging were 96.33%and 97.98%,respectively,and there was no significant difference with V_C at the same mass concentration.It had strong antioxidant activity.The antioxidant effect of polyphenols from M.oleifera leaves on soybean oil was studied by oven heating experiments.The results showed that the antioxidant effect of polyphenols from M.oleifera leaves on soybean oil increased with the increase of the addition amount at 60°C.The addition of 0.1%polyphenols from M.oleifera leaves had the best anti-oxidant effect on soybean oil,which could effectively inhibit the increase of soybean oil peroxidation value,anisidine value and total oxidation value,and the inhibition rate of saturated fatty acid production reached 61.29%;0.1%polyphenols from M.oleifera leaves can prolong the shelf life of soybean oil at 20°C for 30.24 days.At 60°C,ascorbic acid and citric acid had good synergistic effects on the antioxidant activity of polyphenols from M.oleifera leaves in soybean oil,and the synergistic effect of ascorbic acid was stronger than that of citric acid.The heating temperature had a significant effect on the antioxidant effect of polyphenols from M.oleifera leaves.With the increase of temperature,the oxidation inhibition effect of polyphenols from M.oleifera leaves on soybean oil was weaker.