Separation And Mechanism Of Insecticidal Ingredient From Buddleja Albiflora Hemsl

Buddleja albiflora Hemsl belongs to Buddleja family. So far, more than 100 species have been found in Buddleja family around the world, and half of them can be found in China. The ethanol extract and the essential oil of B. albiflora H. Showed biological activities in previous studies. The ethanol extract had excellent antifeedant activity against several kinds of insects. Further sepearation lead to the active fraction, ethyl acetate extraction. The essential oil exhibited certain fumigation effect against several kind of insects. In order to clarify the antifeedant components of B. albiflora H. ethyl acetate fraction were carried out further separation. The major constituents of essential oil were identified by GC-MS, among which, p-cymene and methyl benzoate(MeBA) were main active components based on the biological activity assay. The action mechnism of p-cymene was studied. Main results were as follows:(1) Six substances were isolated from ethyl acetate extracts of 95% ethanol extract of B. albiflora H., including three flavonoids and three sterols. And they were indentified as 1,3-dihydroxyxanthone(Eb), luteolin(Ib), apigenin(Fd), ergosterol(Ea), stigmasterol(Fa) and ?-Sitosterol(Ia). 1,3-dihydroxyxanthone, which was isolated from Buddleja for the first time, showed antifeedant activity against 3rd-instar of Mythima Separata Walker. The antifeedant rate, > 80% against 3rd-instar of M. separata W. during 24 h, and the rates was more than 60% during 48 h and 72 h under the concentration of 0.2mg/ml.(2) The chemical constituents of essential oil from B. albiflora H. were analyzed by GC/MS, 20 compounds were identified. The main components were alkanes and fatty acids. The most abundant alkane component was Heptacosane with content of 16.17%, followed by Hentriacontane with content of 16.05%; The content of Tetracosanoic acid was the most among fatty acids, which was 2.82%. The main low boiling point components were p-cymene and methyl benzoate(MeBA), The content were 13.58% and 3.47%.(3) MeBA and p-cymene had fumigation and contact activities against several kinds of insect. The fumigating LC50 of MeBA against 3-day old adults of Culex pipiens was 1.05 uL/L during 4h, 10.92 uL/L against 5rd-instar of M.separata W. during 24 h, and 6.80 uL/L against 4-day old adults of Musca domestica during 6h. The fumigating LC50 of p-cymene against 5rd-instar and 6rd-instar of M. separata W. were 4.67 uL/ and 7.31 L uL/L during 24 h, while 5.89 uL/L against 3-day old adults of C. pipiens was during 4h. The toxicities of MeBA and p-cymene against Tetranychina hani and 3rd-instar of C. pipiens pallens were also tested, the LC50 were 151.84 uL/L and 296.60 uL/L against T. hani, the LC50 were 16.87 mg/L and 158.69 mg/L against 3rd-instar of C. pipiens pallens. The contact LD50 of MeBA and p-cymene against Rhopalosiphum maidis were 179.31 ng/larvae and 570.10 ng/ larvae. Besides, The contact activities of p-cymene against M.separata W. and Helicoverpa armigera Hubner were also tested, the LD50 were 128.55 ug/larvae and 418.22 ug/larvae.(4) The poisoning symptoms caused by p-cymene could be divided into 4 stages against M.separata W., that is, excitation, convulsion, paralysis and death. In vivo, the activity of AchE and Na+,K+-ATPase in brain of 5rd-instar larvae tested was strongly inhibited; the specific activities of AchE were 0.86, 0.77 and 0.69 times of the control. and the specific activities of Na+,K+-ATPase were 0.88, 0.76 and 0.66 times of the control, however the specific activities of GPT had no significant effect. In vitro, the specific activities of AchE and Na+,K+-ATPase were also tested. No significant inhibiting effects were found on AchE, while Na+,K+-ATPase was activated to some extent. In addition, the cAMP production showed increasing tendency along with the deepening of poisoning symptom, 1.85, 2.83, and 3.27 times of the control during excitation stage, convulsion stage, and paralysis stage.(5) The activities of several detoxification enzymes, including carboxylesterase(Car E), glutathione S-transferase(GSTs), P450 O-demethylase, acid phosphatase(ACP), alkaline phosphatase(AKP), were determined at all poisoning stages. The CarE in Midgut of 6rd-instar larvae for test was obviously activated, While ACP and AKP induced an insignificant change. Besides, GSTs and P450 O-demethylase in Midgut of 6rd-instar larvae tested were distinctly inhibited, the inhibition ratio was more than 40% in paralysis stage.In conclusion, the insecticidal substances of B. albiflora H. were structural complex, and action mode diverse. Further work should be focused both on the higher active components of B. albiflora H. and their action mechanism.