CaracterizaÃÃo BioquÃmica Parcial do LÃtex de Cryptostegia grandiflora R. Br. e AÃÃo Contra o Vetor da Dengue. / Biochemical characterization of latex of Cryptostegia grandiflora and larvicidal activity against Aedes aegypti

AUTOR(ES)
FONTE

IBICT - Instituto Brasileiro de Informação em Ciência e Tecnologia

DATA DE PUBLICAÇÃO

25/02/2010

RESUMO

Cryptostegia grandiflora R. Br. is a shurb belonging to Apocynaceae family popularly known as âunha de bruxaâ. There are few studies available about the latex from this species and preliminary assays had shown its action against Dengue vector. Thus, this study aimed to characterize the biochemical properties of Cr. grandiflora latex and to evaluate the action of its proteins against Aedes aegypti. After collected the latex was separated into two fractions one consisting of insoluble rubber (97%) and another containing soluble proteins (3%). Electrophoretic analysis showed the presence of proteins of several molecular weights whose profile has changed completely after treatment with reducing agent and predominance of acidic proteins with pI less than 5.0 (59%). Proteins from soluble fraction were also detected in the rubber fraction after extraction with acidic, basic and saline solutions. In the protein fraction were detected glycoproteins, chitinolytic activity (58.68 Â 4.45 nKat/Âg) and antioxidant enzymes such as catalase (0.254 Â 0.03 ÂMol H2O2/min g), superoxide dismutase (886.36 Â 0.35 UA/g) and ascorbate peroxidase (0.389 Â 0.03 ÂMol H2O2/min g). Latex proteins also showed strong proteolytic activity at pH 5.0 (5.43 UA/μgP) which was increased to 6.13 UA/μgP after activation by the reducing agent DTT. The strong proteolytic activity with BANA (20.95 UA/μgP) and inhibition of the same in the presence of specific inhibitors E-64 and IAA showed the predominance of cysteine proteases in latex. Serine, aspartic and metalloproteases were not detected. The proteolytic activity was resistant to high temperatures, remaining at 50% after 30 min ate 75. Papain and trypsin inhibitors seem to occur in the latex as the activity of these enzymes was virtually eliminated when previously incubated with protein fraction devoid of endogenous proteases. When fractioned by ion exchange chromatography (DEAE-Sepharose Fast-flow; pH 5.0) the protein fraction produced two peaks. The proteolytic activity was concentrated in non-retained proteins and residual in retained proteins eluted with 1M NaCl. Biological assays showed that the protein fraction was not able to inhibit egg hatching until 24h, however, it was able to interefere upon larval development (60% of mortality after 72h). Larval mortality caused by protein fraction (96.66%) increased after treatment with DTT (100%) and was reduced after treatment with cysteine protease inhibitors E-64 (36.66%) and IAA (21.42%). The larvicidal activity was detected only in the chromatographic peak which concentrated proteolytic activity. Purified non-specific (pronase) and serine proteases (trypsin and chymotrypsin) were not toxic to the larvae even at concentrations equal to those of the cysteine protease papain that showed toxic action. Bromelain also had no larvicidal activity. Photomicrography of larvae subjected to various treatments did not evidence any morphological damage although hypertrophy of abdominal segments was observed. There were alterations in the electrophoretic profile of proteins larval incubated with protein fraction of latex and purified proteases. Larvae exposed to FITC-PL exhibited fluorescence throughout the body and only discrete differences were documented. Latex proteins did not exhibit inhibitory activity against Dengue virus type 1, 2 and 3. The acute toxic action in mice was not evident at doses up to 100 mg / kg body weight, however, the dose of 1000 mg / kg showed 33.33% mortality within 24h. This study showed that proteins from Cr. grandiflora latex have strong larvicidal effect against Ae. aegypti and proteolytic activity of cysteine type seems to be correlated with the deleterious effects observed, however, in addition to proteases other proteins may be involved in toxicity upon larvae.

ASSUNTO(S)

bioquimica lÃtex cryptostegia grandiflora protease aedes aegypti latex, protease, cryptostegia grandiflora, aedes aegypti

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