Isolation And Identification Of Active Components From Vitex Negundo Linn. And The Biological Effectiveness Against Aphis Gossypii Glover And Pieris Rapae Linn.

Vitex negundo Linn. belongs to Vitex Linn., Verbenaceae. It distributes widely in China, and is abundant in the natural resources of Mountain Tai, Shandong province. To explore the insecticidal plant, we isolated and identified the insecticidal active constituents of V. negundo weed utilizing silica gel column chromatography, thin layer chromatography, GC-MS technology and biological activity tracking. We also investigated the action modes and virulence of insecticidal active constituents and total alkaloids from V. negundo weed against A. gossypii and P. rapae, two important agricultural pests, and speculated its possible action mechanisms. It has great theoretical and practical significance for the further development and utilizing of this insecticidal plant and development of new environmentally friendly botanical insecticides. The main results were as follows:1. Tested with A. gossypii, we got one high active insecticidal component from V. negundo weed dichloromethane extract and identified its main constituents.β-caryophyllene,α-pinene and 1-methylnaphthalene were isolated and identified from V. negundo weed dichloromethane extract by silica gel column chromatography repeatedly, which accounted for 7.68%, 5.45% and 35.67%, respectively. Total alkaloids were extracted from V. negundo seed methanol extract utilizing acid water.2. We investigated the virulence and action mechanisms ofβ-caryophyllene andα-pinene against A. gossypii and P. rapae using bioassay and biochemical analysis. The results showed that:(1) Action modes ofβ-caryophyllene andα-pinene were variety. The contact toxicities and repellent effects ofβ-caryophyllene andα-pinene against A. gossypii were strong and the higher one wasβ-caryophyllene. At the dose of LD30, they had significantly adverse effects on the fertility, honeydew excretion frequency and honeydew production. In vitro and vivo conditions,β-caryophyllene andα-pinene distinctly inhibited the activities of A. gossypii acetylcholine esterase, polyphenol oxidase, carboxylesterase and glutathione S-tramsferases.(2)β-caryophyllene andα-pinene had excellent antifeedant activity and contact toxicity against P. rapae. The LC50 values of contact activity at 72 h were 642.54 and 729.69 mg/L, respectively.β-caryophyllene also showed obvious stomach toxicity. At the dosage of LC30 of contact toxicity at 72 h,β-caryophyllene andα-pinene inhibited the larva growth of P. rapae distinctly. The developmental time and pupa time were prolonged compared with the control over the same period. Body weight, male pupal weight and female pupal weight of P. rapae larval were decreased significantly. PPO was the key target ofβ-caryophyllene to P. rapae, and the IC₅₀ was 1.09×10³ mg/L. However, AChE was the key target ofα-pinene to P. rapae, and IC₅₀ was 1.32×10³ mg/L.β-Caryophyllene andα-pinene could activate the activity of P. rapae GST.β-Caryophyllene also could inhibit the activity of protease and amylase of the midgut in P. rapae.3. The inhibitory effect on the activity of A. gossypii AChE and P. rapae PPO byβ-caryophyllene was determined, and the possible mechanism of the inhibitor was discussed also. The results showed that:β-Caryophyllene was reversible competitive inhibitor on A. gossypii AChE and the IC₅₀ was 1.76×10³ mg/L. The inhibitory constant (Ki) was also determined to be 665.50 mg/L. The inhibitory mechanism ofβ-caryophyllene against P. rapae PPO was mixed inhibition, the IC₅₀ was 1.09×10³ mg/L and the inhibitory constant (Ki) was 1.00×10^-4 mg/L, the Kis was 2.00×10^-6 mg/L.4. The main action modes and the action mechanisms of total alkaloids from V. negundo weed against A. gossypii and P. rapae showed that:(1) The contact toxicity of total alkaloids from V. negundo weed against A. gossypii was obviously and the LD50 value was 0.73×10^-1μg individual. At the dose of LD30, total alkaloids from V. negundo weed could shorten the survival time of A. gossypii for 7 d compared with the control. The number of fertility of single female aphid was reduced by 4.9 heads per female. Total alkaloids from V. negundo weed distinctly inhibited the activities of AChE and CarE in A. gossypii and IC₅₀ were 1.90×10³ and 777.81 mg/L, while it activated the activity of A. gossypii PPO.(2) Total alkaloids from V. negundo weed showed obvious contact toxicity, antifeedant activity and stomach toxicity. 72 h after treated, LC₅₀ of contact toxicity was 2346.61 mg/L, AFC50 of antifeedant activity was 2.13×10³ mg/L and LC₅₀ of stomach toxicity was 2.99×10³ mg/L, respectively. It inhibited the activity of P. rapae AChE and CarE.5. The main action modes and the action mechanisms of ursolic acid against A. gossypii and P. rapae showed that:(1) The contact toxicity of ursolic acid against A. gossypii was the main action mode and LD50 value was 0.71×10^-1μg individual. AChE was the key target of ursolic acid against A. gossypii. Ursolic acid was reversible competitive inhibitor on A. gossypii AChE, and the IC₅₀ were 2.23×10³ mg/L. The inhibitory constant (Ki) was also determined to be 489.40 mg/L.(2) Ursolic acid showed significant antifeedant activity and contact toxicity against P. rapae. AFC50 of antifeedant activity and LC₅₀ of contact toxicity were 829.27 and 1745.12 mg/L. Ursolic acid inhibited the activities of AChE and PPO in P. rapae, and the IC₅₀ were 1.60×10³ and 2.68×10³ mg/L, respectively. It was reversible competitive inhibitor on P. rapae AChE.