| The petroleum ether、chloroform、ethyl acetate、acetone、ethanol and water extracts from Pinus massoniana needles were examined for the antimicrobial activities against eight bacteria by the inhibition zone diameters、MIC and MBC. Systematic solvents were examined for the antimicrobial activities against eight bacteria. This thesis focus on the antibacterial activity of crude extracts of Pinus massoniana needles against bacteria in vitro, chemical composition and stability of the aqueous extract were diseussed by the numbers,The screening for active part, isolation and purification for active component were studied by a bioassay-directed fractionation and its inhibiting against Gram-positive bacteria. The results are shown as follows:(1) Study on the antibacterial activity of different polarity solvent extracts of Pinus massoniana needles against bacteria. It was found that all extracts of Pinus massoniana needles have strong antibacterial effects and the antibacterial activity of different solvent extracts decreased in the order:ethanol> acetone> water> ethyl acetate> chloroform> petroleum ether.The MIC and MBC values also showed that The ethanol extract showed better antibacterial activity than other extracts. The ethanol extract had resemble antibacterial action to Staphylococcus aureus compared with sodium benzoate, because of MIC was3.125mg/mL. Stability study made done with ethanol extract had shown that it was stabile to temperature. When the pH value was less than7,the antimicrobial activities were weaker with pH value increasing. The ethanol extract was stable under ultraviolet light and long term storage.(2) The extract from Pinus massoniana needles by systematic solvents (petroleum ether, chloroform, ethyl acetate, n-butanol, ethanol, water) were examined for the antimicrobial activities against eight bacteria.The results showed that the rates of petroleum ether, chloroform, ethyl acetate, n-butanol, ethanol, water extracts were18.4%,9.8%,14.6%,5.1%,52.1%。All extracts showed inhibitory effect against tested bacteria. And it was observed that the antibacterial activity of ethyl acetate extracts was significantly higher than other extracts. The lowest MIC of the ethyl acetate extracts against Staphylococcus aureus Rosenbach was2.5mg/mL.Chemical composition of Pinus massoniana needles ethyl acetate extracts were tested by the method of test tube and thin-layer chromatography.The result showed that the extract contained organic acid, flavones, phenols, acids, it may contain alkaloids, saponin, anthraq, lactones, coumarins, have no cardic glycosides. Determination of Flavonoid proved that the extracts contain flavones.(3) Bioassay-directed fractionation of ethyl acetate extracts of Pinus massoniana needles was conducted by the thin-layer chromatography technology. Qualitative analysis by TLC, a mixed solvent of chloroform-methyl alcohol-acetic acid(4.5:1:0.05) was employed as the mobile phase. Four fractions were collected and numbered C、D、E、F. The bacteriostasis activity of four fractions were studied. The result showed fractions D and F had the prominent antibacterial activities against the tested bacteria,fraction C had no antibacterial activitie,fraction E had a unsignificance antibacterial activity. Fractions D and F was identified by1H NMR and13C NMR, fractions F was identified as flavone.(4) The antimicrobial activity of Pinus massoniana needles against three species of Gram-positive bacteria (S.aureus、B.subtilis and B.cereus) cells were investigated. As the results showed, the ethyl acetate extracts from Pinus massoniana needles had antimicrobial ratio against three species of Gram-positive bacteria with the increase of and time. The electric conductivity had an increase of12%,and total sugar concentration of microbial broth were increased by0.27mg/100mL at least compared with the control. After the treatment with the extract for12hours, the consumption of phosphorous and oxygen ware gradually reduced till completely ceased. The facts indicated that extract could damage the structure of cell membrane, which resulted in the increase of permeability of cell membrane and release of cell components. |
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