Separation,Purifying,Structural Analysis Of Antioxidative Substances From Melaleuca Bracteata Leaves

The antioxidative substances of M.bracteata leaves were researched systematically in this study.The main results were as follows:1 The study of the chemical compositions of Melaleuca bracteata leavesThe chemical constituents of Melaleuca bracteata leaves were pre-tested by preliminary systematic test,the results showed that the leaves probably contained amino acids,peptides,proteins,carbohydrates,glycosides,phenols,tannins,resorcino,phloroglucinol compounds,organic acids,flavonoids,coumarins,lactones,cardiac glycosides,volatile oils,grease,terpenes,sterols et al,but without saponins,alkaloids,anthraquinones,or contained only a little.In addition,The contents of total phenolic,total flavonoids and total polysaccharide of freeze-dried leaves from M bracteata were 43.56 mg GA/g?11.34 mg RT/g and 16.13 mg GC/g respectively.2 Extraction of antioxidative substances from M.bracteata leavesThe reducing power and scavenging capacities of DPPH free radicals and ABTS+free radicals of M.bracteata leaves extracts of water,75%ethanol,75%methanol,75%acetone were determined.The results showed 75%methanol extract had the most efficient and comprehensive antioxidant capacity.Then the extraction process of methanol extract from Melaleuca bracteata leaves was optimized by response surface methodology,and the extraction yield was 25.451%under the conditions of 26 h extraction by 65%methanol with the material/solvent ratio of 1:30.Based on the analysis,the primary and secondary order of three main factors which influenced the extraction yield was extraction time>methanol concentration>ratio of material to solvent.3 Separation,purification and structural analysis of antioxidative substances extract from M.bracteata leavesAntioxidative substances of M.bracteata leaves(methanol extract,ME)was successively partitioned with petroleum ether,ethyl acetate and normal-butyl alcohol and obtaining the fraction in petroleum ether fraction(PF),ethyl acetate fraction(EF),n-butyl alcohol fraction(BF)and the aqueous fraction(AF)respectively.The antioxidant activity of ME and fractions extracted with different polarity solvents were determined in 5 antioxidant systems,and BF showed the strongest reducing power and scavenging capacity of DPPH free radicals,ABTS+ free radicals,superoxide anion free radical and nitrogen oxide radical,and there was a positive correlation between the antioxidant activity and phenolics content of different extracts,except for the scavenging capacity of nitrogen oxide radical.Further more,12 different components(B1?B12)and a monomer compound named MB1 were obtained by the column chromatography of C18 from BF.The molecular weight of MB1 is about 462,and speculated that MB1 had the structure of phenoxy ethyl ketone and another which is similar to furan.4 The activity of antioxidant and antibacterial of different separated fractionsThe in vitro antioxidant activities of B1?B12 and MB1 were determined by different assays in this study.Compared with BE,the oxidation resistance of B3?B7 were obviously improved at different degrees,and B4 displayed best activities in the DPPH free radical scavenging activity,ABTS+free radicals scavenging activity and reducing power,B5 came second place.B4 and B5 were more effective than Vc in the 3 assays,and there were no significant differences between B6 and Vc.B4?B6 all have broad-spectrum and efficient antioxidant activity,and were expected to be developed as natural antioxidant.On the other hand,Staphylococcus aureus,Bacillus subtilis,Pseudomonas aeruginosa,Escherichia coli,Serratia marcescens H30 and Serratia marcescens MG1 were selected as tested bacteria to evaluate the antibacterial activity of ME,four different polar fractions of ME,B1?B12 and MB1 on the concentration of 50 mg/mL.The results revealed that not all compound possessed the antibacterial activity,such as MB1,which displayed no antibacterial effect against the 6 tested bacteria on the concentration of 50 mg/mL.B4 possess the strongest antibacterial activity to all six tested bacteria,especially to Staphylococcus aureus and Escherichia coli,the inhibition zone diameters of the two bacteria were all over 20 mm,which showed the higher antimicrobial activity of B4,and the antibacterial activity of B4 against Staphylococcus aureus was equivalent to kanamycin(2 mg/mL)without significant differences.