Study On Antibacterial Effect And Separation Active Components Of Flavonoids From Pinus Massoniana Needles

In this paper, food spoilage bacteria and plant pathogenic fungi as experimental funguswere employed to study the activity of bacteriostasis and effects of abiotic factors on theflavonoids which were extracted with water,75%ethanol and75%acetone from Pinusmassoniana needles. The structures of the antibacterial components separated by solventextraction and purified by thin layer chromatography were characterized using spectroscopiesand their activities on bacteriostasis were also tested. The results of research were asfollowing:1.Though the choice of the solvents for total，the extraction rates and yields indicatedthat the solubility of flavonoids from Pinus massoniana needles in acetone and ethanol washigher than be in water. It was found that three extracts had strong antibacterial effects onfood spoilage bacteria and also inhibited the growth of some plant pathogenic fungus. Thecomponents abstracted by75%acetone had significant bacteriostatic effects. Therefore,75%acetone was the perferrable solvent for flavonoids extraction.2. The effects of various factors such as pH, storage time, saccharides, metal ion andfood additives on the antibacterial activity and stability of flavonoids against Staphylococcusaureus Rosenbach, Salmonella paratyphi B Castellani et Chalmers, Bacillus subtilis(Ehrenberg) Cohn were investigated. The results showed that the flavonoid extracts were verystable to temperature and ultraviolet heating．High activities on bacteriostasis of the extractswere showed at acidic condition, but the activities were decreased while abjusting the pH toalkaline. The antibacterial activity of flavonoids increased with the addition of metal ions. Themetal ions (Na+, K+, Ca2+and Mg2+) showed little effects on the antimicrobial activity ofextracts except for Fe2+. The addition of Fe2+could enhance the antibacterial activity offlavonoids on S.aureus and S.paratyphi B, but not on B.subtilis. The activity of flavonoidsagainst food spoilage bacteria was enhanced by Na2SO3, Vc, EDTA-2Na and sodiumdihydrogen phosphate, among them the effect of EDTA-2Na was more significant. However,the antibacterial activity of flavonoids was partially suppressed by CMC-Na and citric acid.3. Investigation on the types and proportions of expansion agent on separating andpurifying components of flavonoids was carried out. The antibacterial ingredients mainlysolublized in acetic ether phase according to the results of bacterium experiments. Theflavonoids in ethyl acetate extraction phase were analyzed by TLC, and a mixed solvent ofmethylene dichloride: methanol: acetic acid (8.5:1.5:0.05) was employed as the mobile phase.Under the mobile phase, nine clear and focused bands were isolated from the acetate ethyl acetate extraction phase from Pinus massoniana needles, in which five components had Rfranging from0.3to0.8. They were collected and numbered C, D, E, F, and G.4. The bacteriostasis activities of four fractions were studied. The result of MIC testsshowed component C, D and E had the prominent antibacterial activities against the modelbacteria, but component F and G had no antibacterial activity. In addition, the antibacterialactivity of component C, D and E were as good as benzoate and potassium sorbate. IR andNMR were employed to analyze the structures of components. The three antibacterialcompounds were identified as (C) dihydromyricetin,(D) diihydroquercetin,(E) quercetin,respectively.