Study On Fat-soluble Antibacterial Components Of Periplanet A Americana L. And Blaps Rhynchopetera Fairmaire Larvae

It has always been one of the main ways of new drug research, that search goodactivity and low side effects of natural products from plants, microorganisms, insects and other creatures. After a long evolution from400million years ago, insect adaptes tothe environment and becomes one of the most successful organisms on the earth. Asone of the important natural resources in the world, insect has not been sufficient development and utilization. As the research and developement of insect breeding techology,chemical components and pharmacological effects of insect, people pay more and more attention to insect resources. The aboundant of insect resources and diversities of chemical components provide a broad prospect for searching new nature products as medicine from insect.In this dissertation, extraction, purification and identification of fat-soluble antimicrobial chemical components from Periplaneta americana L. and the larvae of Blaps rhynchoptera Fairmaire, were systematically studied. Firstly, two insects powders were extracted for four times by petroleum ether and ethyl acetate. The extractions were tested their antimicrobal activities against five species of bacteria based on bioassay-guided.The strains for testing antimicrobial activity were Staphyloccocus aureus, Micrococcus luteus, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa. Then the fat-soluble antimicrobial compounds or components were obtained from activity extractions of two insects by silica gel column chromatography and Sephadex LH-20column gel chromatography.Lastly, NMR, MS and GC-MS had been applied to identify the antimicrobial fat-soluble compounds. The main research results are listed as follows:（1） Antimicrobial activity of crude extractions of the two medicinal insectsDifferent polarity fat-soluble substances of two insects were extracted by using petroleum ether and ethyl acetate. Antibacterial activity was tested under1.0g·L^-1.Petroleum ether extraction of P. americana L. only showed antibacterial activity to B. subtilis,and the average inhibition zone diameter was2.58mm. Ethyl acetate extraction of P. americana L. showed some inhibition effects to all tested gram-positive bacteria, and thebest inhibition effect was against B. subtilis. But the effect was a little weaker than that of petroleum ether extraction. The average inhibition zone diameter was2.00mm.Petroleum ether extraction had no antimicrobial activity from the larvae of B. rhynchopt era, but ethyl acetate extraction showed antibacterial activity to S. aureus and M. luteus, the average inhibition zone diameter were3.78and1.06mm respectively.（2） Antibacterial activity of fractionsEthyl acetate extractions of two insects were separated by silica gel column chromatography. Their antimicrobial activities were detected under0.1g·L^-1. Nine fractions which were obtained after silica gel column chromatography from ethyl acetate extractionof P. americana L., showed different degrees of antibacterial activity. Among them, FrZ3showed some inhibition effects to all tested gram-positive bacteria. The best inhibition effect was against B. subtilis, and the average inhibition zone diameter was5.10mm. FrZ7showed inhibition effects to all tested bacteria, and the antibacterial activity togram-positive bacteria was better than that to gram-negative bacteria. The inhibition zone diameters were4.00and3.94mm for M. luteus and B. subtilis. Eight fractionswhich were obtained after silica gel column chromatography from ethyl acetate extraction of the larvae of B. rhynchoptera, showed different degrees of antibacterial activity. Among them, FrP3showed especially antibacterial activity to M. luteus. The average inhibition zone diameter was8.38mm. FrP4^FrP7showed some inhibition effects to alltested bacteria, and the antibacterial activity to gram-positive bacteria was better than that to gram-negative bacteria.（3） Antibacterial activity of compoundsThirty compounds were obtained from FrZ6, FrZ7and FrZ8of P. americana L..Fifteen compounds which quantities were more than15mg were selected to test theirantibacterial activities against five bacteria under5mg·L^-1. Thirteen compounds showeddifferent degrees of antibacterial activity and antibacterial activity to gram-positive bacteria was better than that to gram-negative bacteria. Seven compounds showed antibacterial activity to all test bacteria. Among them, antibacterial activity of compund25was the best to S. aureus, and the average inhibition zone diameter was3.53mm. Antibacterial activity of compund18was the best to M. luteus, and the average inhibition zonediameter was19.70mm. Antibacterial activity of compund21was the best to B. subtilis, and the average inhibition zone diameter was6.00mm. Antibacterial activity of compunds18and21were the best to E. coli, and the average inhibition zone diameterwas1.34mm. Antibacterial activity of compunds25and26was the best to P. aeruginosa, and the average inhibition zone diameter was2.20mm.（4） Compound structure analysis By NMR, MS and GC-MS methods, compund16,18,21,22,23,25,26, were determined as high polar unsaturated fatty acids, which were relatively stable and had relatively good antibacterial activity. Compound16identified as oleic acid-1-glyceride. Compound23identified as linoleic acid-1-glyceride.In short, Antibacterial activity substances of P. americana L. were contained in petroleum ether and ethyl acetate extractions, and antibacterial spectrum of ethyl acetate extraction was larger than that of petroleum ether extraction. Dozens of fat-soluble antibacterial chemical components were obtained from ethyl acetate extraction of P. americana L.. The unsaturated fatty acids were one of the main active components. Two unsaturated fatty acids identified as oleic acid-1-glyceride and linoleic acid-1-glyceride. Antibacterial active substances of the larvae of B. rhynchoptera were concentration in largerpolarity fractions FrP3^FrP7from ethyl acetate extraction.