Studies On The Immunochemistry For Residue Analysis And Safety Assessment Of Botanical Podophyllotoxin

Research aiming at the development of new agents for pest control based on natural products is increasingly important because it has implications in pesticide risk reduce in agriculture, and interest in the use of botanical (plant-derived) pesticides was driven primarily by concerns over the longer-lasting synthetic pesticides'environmental impacts and their residues on food. Based on the extensively screening from flora, extracts of Sabina vulgris, belonging to Cupressaceae family, were found to show insecticidal activities. From extract, an insecticidal compound, podophyllotoxin was isolated by bioassay-guided fractionation. In bioassays, podophyllotoxin showed anti-feeding and growth inhibition activities against several insects. Therefore, a podophyllotoxin-based insecticide (0.75%, EC) from S. vulgris, was developed by R & D Center of Biorational Pesticide, Norwest A & F University. This dissertation focused particularly on studies on the immunochemistry for residue analysis and safety assessment of the podophyllotoxin-based insecticide from S. vulgris. 1. From Chapter 2 to 4, an indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) for podophyllotoxin was developed by using polyclonal antibody, and its suitability for the determination of this analyte in spiked water samples was studied. To avoid antibody production to the linker, the succinoyl-podophyllotoxin (hapten) mimicking the analyte, podophyllotoxin, was synthesized and conjugated with the carrier proteins bovine serum albumin (BSA) and ovalbumin (OVA) by mixed anhydride reaction (MAR) and active ester method (AEM). Polyclonal antibodies raised against Hapten-BSA1 synthesized by MAR and Hapten-BSA2 by AEM were screened and selected for the IC-ELISA. One set of antibodies from the rabbit M4440 immunized with Hapten-BSA1showed an I50 value of 2.21μg/mL with a detection limit of 0.12 μg/mL, and the other set from the rabbit M4469 immunized with Hapten-BSA2 had an I50 value of 0.7897 μg/mL with a detection limit of 0.0056 μg/mL. This assay showed the cross-reactivities with the structurally closely related compounds, including epipodophyllotoxin, 4-acetyl-epipodophyllotoxin and deoxypodophyllotoxin. 2. In Chapter 5, a high performance liquid chromatography (HPLC) was established for determination of residues of podophyllotoxin in tape water, soil, and emulsifiable concentrates preparation. When the spiked concentration of podophyllotoxin in tape water and soil was from 0.1-10μg/mL,podophyllotoxin recoveries determined by HPLC were in the range of 86.32%-101.67%and 81.50%-103.60%, respectively. 3. Based on results from Chapter 2 to5, recoveries from the podophyllotoxin-fortified water determined by IC-ELISA and HPLC were in the range of 72.00-115.00% and 80.50-102.12%, respectively. A good correlation between podophyllotoxin concentration measured by the ELISA and HPLC (R2=0.9924, Y=1.195X-0.257) was obtained from linear regression analysis. These results indicate that the ELISA could be a convenient and supplemental analytical tool for monitoring podophyllotoxin and its analogues in waters without previous extraction or cleanup. 4. The environmental fate of podophyllotoxin under laboratory conditions was conducted by IC-ELISA and HPLC from Chapter 6 to 8. Abiotic hydrolysis of podophyllotoxin in neutral and acidic sterile buffer solutions with half-lives of approximately 145 and 10 days, respectively. In pH 9 solutions the half-life was approximately 12 hours. Podophyllotoxin degradation in soils was biphasic, showing an initial faster degradation followed by a slower rate. The half-lives of podophyllotoxin in aerobic soils ranged from 10-71 days in 3 soils ranging in texture from loamy sand to clay. Podophyllotoxin was less persistent in soils with a higher pH. This decrease in stability with increasing water and soil pH were observed. Podophyllotoxin degraded in sterile water with a half-life of 75.33 hours in sunlight irradiated pH 5 aqueous solutions; the half-life in the dark controls was 147.45 days. Photolytic half-lives of podophyllotoxin in acetone, methanol and n-hexone under high-pressure mercury lamp or sunlight were in the range of 43.86-3523.8min. Photodegradation appears to be an important route of dissipation for podophyllotoxin. The studies on environmental fate of podophyllotoxin illuminate that podophyllotoxin appears to degrade fast in soil and water under both aerobic and anaerobic conditions, and it is lower persistent in environmental media. 5. Chapter 9 has adequate ecological toxicity data to assess the hazards of technical grade podophyllotoxin and the podophyllotoxin product to nontarget organisms for dietary exposures. Podophyllotoxin was toxicity free to quail (Coturnix chinensis), with acute LD50 1337.92 and 1133.09 mg/kg for technical grade podophyllotoxin and the product, respectively. Acute contact LD50 values for technical grade podophyllotoxin and the product for honeybee (Apis mellifera L.) were 333.98 and 196.19 μg/bee, respectively. Podophyllotoxin had acute contact LC50 was more than 4000mg/L and AFC50 was lessthan 50mg/L for silkworm (Bombyxmori Linnaeus). The contact LC50s for earthworm (Eisenia fetida) with technical grade podophyllotoxin and the product were 1445.70 and 116.75μg /cm2, respectively. The podophyllotoxin product was highly toxic to beneficial insect (Serangium japonicum) with a LC50 of 5.12mg/L, but technical grade podophyllotoxin had a much higher LC50 of 550.87 mg/L. Acceptable and supplemental acute 96-hour toxicity tests indicate that the podophyllotoxin product was toxic to fish (Carassius auratus) and tadpole (Rana limnochris), with LC50 values of 8.15 and 5.60mg/L, respectively. Technical grade podophyllotoxin was low toxic against fish and tadpole, with LC50 values of 161.64 and 33.4 mg/L, respectively. All results show that podophyllotoxin and podophyllotoxin-based product have a low toxicity to nontarget organisms. 6. The effect of podophyllotoxin on photosynthetic rate (Pn), the anti-oxidative enzyme system and the content of chlorophyll, soluble protein, soluble sugar, malondialdehyde (MDA) in Brassica rapa L. was investigated in Chapter 10. The activities of superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), and glutathione peroxidase (GSHPx), were measured with NBT assay, Na2S2O3 titration method, ABTS method, and DTNB method, respectively. The thiobarbituric assay (TBA) was used to analyze MDA. The results showed that activities of SOD, CAT, POD and GSHPx and contents of soluble protein, soluble sugar and MDA were increased by the podophyllotoxin product with a dosage of 50X (9.6mL/L). The lower concentrations (250X and 500X) of the product had little effect on the anti-oxidative enzyme system and the content of MDA. All level treatment with podophyllotoxin increased the content of chlorophyll. This well indicates that the low concentration of pesticides applied on the vegetable may not severely affect on the growth of the plant because of the regulation of anti-oxidative enzyme system and MDA.