Abstract:We genetically modified Eclipta alba using Agrobacterium rhizogenes LBA 9402, with the aim of producing secondary metabolites with pharmacological properties against phospholipase A 2 and the myotoxic activities of snake venom. Extracts from in natura aerial parts and roots, both native and genetically modified ( in vitro ), were prepared and analysed by high-performance liquid chromatography. In natura materials showed the coumestan wedelolactone at higher concentration in the aerial parts, while demethylwedelolactone appeared at higher concentration in roots. Among the modified roots, clone 19 showed higher concentrations of these coumestans. Our results show that the in natura extracts of plants collected from Botucatu and Ribeirão Preto were efficient in inhibiting snake venom phospholipase A 2 activity. Regarding in vitro material, the best effect against Crotalus durissus terrificus venom was that of clone 19. Clone 19 and isolated coumestans (wedelolactone and demethylwedelolactone) inhibited the myotoxic activity induced by basic phospholipases A 2 isolated from the venoms of Crotalus durissus terrificus (CB) and Bothrops jararacussu (BthTX-I and II). The search for antivenom is justified by the need of finding active principles that are more efficient in neutralizing snake venoms and also as an attempt to complement serum therapy.Eclipta alba (L.) Hassk. (syn. Eclipta prostrata ), popularly known as dye weed and belonging to the Asteraceae family, is an annual herbaceous species [1]. Native to Brazil and other tropical and subtropical regions, this plant is a source of several secondary metabolites, such as polypeptides, polyacethylenes and triterpenes [2], flavonoids, phytosterols and coumestans [3].Coumestans represent an important class of natural oxygenated aromatic products, including wedelolactone and its demethylated form, demethylwedelolactone, both responsible for the main medicinal effects of Eclipta , such as its anti-hepatotoxic, anti-hypertensive, antitumor, antiphospholipase A 2 and antidote activities against snake venoms [2,4 -7].With the development of in vitro plant tissue cultures, it is possible to produce viable, economically valuable compounds [8]. Among the procedures for tissue culture, those for genetically modified plants have developed significantly, and the obtainment of transgenic plants is made by indirect modifications with a biological vector, such as Agrobacterium [9].Hundreds of plants have been related with anti-snake venom action, but only a few have been scientifically investigated [5][6][7] [11]. More recently, the potential antivenom property of wedelolactone against Calloselasma rhodostoma venom (by neutralizing isolated phospholipases A 2 and venom lethality) has been observed [12]. In this study, E. alba was genetically modified, aiming at the production of secondary metabolites of pharmacological interest, which were assayed against phospholipase A 2 and myotoxic activities of snake venoms and isolated toxins.
Materials and MethodsAnimals, venoms and t...