Separation And Preparation Of Polyclonal Antibody Of Periplocoside T

One of the effective pathways to develop new insecticides is to screen novel bioactiveleading compounds from natural products in order to use the bioactive compounds as probesto find new target sites. Based on this idea, insecticidal components of Periploca sepium werestudied systematacially by bioassay-guided fractionation. And polyclonal antibody ofPeriplocoside T was prepared, as Periplocoside T has the highest insecticidal activity. whichcould elucidate insecticide mechanism of Periplocosides. The main research findings arelisted as the following:1. Six compounds were isolated from the root barks of Periploca sepium Bge by silicagel chromatograph, high pressure liquid chromatograph (HPLC) and bioassay-guidedfractionation. Their structures have been identified by physicochemical evidences andspectral methods (MS,2D-NMR and1D-TOCSY). Three compounds were new pregnaneglycosides (Periplocosides T, P and U) while the other three were reported compoundsPeriplocosides D, E and F.Bioactivity assay were operated to test the insecticidal activities of six compounds,which indicated that Periplocosides D, F, P and T had insecticidal activities againstPseudaletia separata and Plutella xylostella. LC50values of48h against3rdP. separata were0.39,0.34,0.11and0.07mg/mL respectively while those against3rdP. xylostella were1.21,1.39,0.89and0.35mg/mL. As periplocosides had the same toxic Symptoms of Mythimnaseparata to Periplocoside NW, it is estimated that digestive system might be the main targetof Periplocosides, the same to the target site of Periplocoside NW.2. Derivative of Periplocoside T (hapten) was synthesized by combining PeriplocosideT and succinic anhydride and the hapten was identified by HPLC-ELSD, MS and NMR. Thenthe hapten was conjugated to bovine serum albumin (BSA) and ovalbumin (OVA)respectively to form the complete antigens by carbodiimide method, which were identified byultraviolet spectrum (UV) and infrared spectrum (IR). The ratios of hapten-BSA andhapten-OVA were5:1and7:1respectively, which were confirmed by2,4,6-trinitrobenzenesulfonicacid method (TNBS).3. Balb/c mices were immunized by hapten-BSA. ELISA was applied for testing titer of antibody and primary optimization for condition of ELISA were operated. Periplocoside P andPeriplocoside E were chosen for cross-reactivity tests, as these two compounds had similarstructure to Periplocoside T. Results from ELISA indicated that the best concentration ofcoating antigen was10μg/mL, the titer of antisera was1600. Competitive inhibition test ofantisera demonstrated that artificial antibody had a high affinity to Periplocoside T and IC50was12.4294μg/mL. The cross-reactivity ratios between antibody and Periplocoside T,Periplocoside E were25.6%and8.6%respectively.