Isolamento, purificação e caracterização de uma lectina das sementes de crotalaria stipularia (desv., 1814) (farbales: fabaceae) e sua avaliação inseticida contra cerconota anonella (sepp., 1830) (lepidoptera: oecophoridae)

Abstract

The fruit borer, Cerconota anonella (Sepp., 1830) (Lepidoptera: Oecophoridae), is an important pest of sugar apple and graviola crops, causing significant damage to these crops. To control this pest, pesticides are used mainly. However, the resistance acquired by insects and the high toxicity of these compounds on the environment and non-target organisms led to an increase in the search for natural products free of toxicity and effective in controlling insect pests. Extracts and lectins (proteins or glycoproteins that recognize carbohydrates and agglutinate cells) from plants have shown insecticidal activity and, for this reason, have been extensively studied. Among them, Crotalaria stipularia, belonging to the Fabaceae family, is widely distributed in Atlantic Forest environments and is important in cattle feeding. Thus, the present study aimed to isolate, purify and characterize the lectin from Crotalaria stipularia seeds and to evaluate the insecticidal activity of this extract protein and the seed protein protein against adults of Cerconota anonella. For this purpose, the crude extract of the plant seeds was prepared in 50 mM Tris-HCl, pH 8.0, followed by saline precipitation with ammonium sulfate and SDS-PAGE electrophoresis. Subsequently, extract, collection and were quantified by the Bradford method and evaluated for their hemagglutinating activity. The fraction with the highest hemagglutinating activity was then maintained for affinity chromatography on a chitin matrix. Then, solutions of different concentrations were prepared for the extract (90, 45, 30 µg of protein/mL of diet), for absorption (30, 15, 10 µg of protein/mL of diet) and for lectin (10, 5, 3 µg of protein/ml of diet). Then, the insecticidal activity of the extract, extraction and lectin, the food deterrence, the nutritional parameters of the treated insects, and the effects of the extract, extraction and lectin on the biochemical profile presented by the insects at the end of the 7 days of trial were evaluated. In addition, the secondary metabolites presented in the extract were qualitatively determined through phenolic composition analysis and High Performance Liquid Chromatography (HPLC). The extract was also submitted to an ecotoxicological evaluation against Lactuca sativa. The lectin of C. stipularia was isolated and its purity visualized in gel electrophoresis (SDS-PAGE). It was presented thermally maintaining the HA at 50ºC and maintaining up to 50% of its HA up to 100 ºC, it maintained its HA in the face of pHs susceptible to neutral and did not vary its HA by EDTA and divalent ions (Mn2+ and Ca2+ cations). The phytochemical analysis showed that the phenolic content in the aqueous extract of dry seeds of C. stipularia was 7.05 µg EAG/g of the crude sample, while through high performance liquid chromatography (HPLC) it was possible to identify the compound chlorogenic acid (2,8642 mg/kg) and syringaldehyde (0,2763 mg/kg). The extract was also submitted to an ecotoxicity analysis against L.sativa (lettuce) where its non-toxicity was proven. The results with the bioassays show that all treatments (extract, rotation and lectin from the seeds of C. stipularia) present insecticidal activity against C. anonella and both affected in the nutritional parameters (relative rate of biomass gain, relative consumption rate and efficiency of conversion of the ingested food) of the insects tested and equivalent, the rates of proteins, glucose, cholesterol and triglycerides were also transformed, revealing that the treatments (both, extract, and lectin) are in the metabolism of the insects. Thus, C. stipularia seeds are a biomaterial with potential to be used in the control of Cerconota anonella.