Miriam Verginia Lourenço

Medicinal Plants with Inhibitory Properties Against Snake Venoms

Envenomations due to snake bites are commonly treated by parenteral administration of horse or sheep-derived polyclonal antivenoms aimed at the neutralization of toxins. However, despite the widespread success of this therapy, it is still important to search for different venom inhibitors, either synthetic or natural, that could complement or substitute for the action of antivenoms. Several plants have been utilized in folk medicine as antiophidian. However, only a few species have been scientifically investigated and still less had their active components isolated and characterized both structurally and functionally. This article presents a review of plants showing neutralizing properties against snake venoms which were assayed in research laboratories, correlating them with ethnopharmacological studies, as (i) the part of the plant used as antidote, (ii) its respective genus and family and (iii) inhibition of the main pharmacological, toxic and enzymatic activities of snake venoms and isolated toxins. Protective activity of many of these plants against the lethal action of snake venoms has been confirmed by biological assays. Compounds in all of them belong to chemical classes capable of interacting with macromolecular targets (enzymes or receptors). Popular culture can often help to guide scientific studies. In addition, biotechnological application of these inhibitors, as helpful alternative or supplemental treatments to serum therapy, and also as important models for synthesis of new drugs of medical interest, needs to be better oriented and scientifically explored.

Inhibition of Snake Venoms and Phospholipases A2 by Extracts from Native and Genetically Modified Eclipta alba: Isolation of Active Coumestans

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.

Micropropagation of Pothomorphe umbellata via direct organogenesis from leaf explants

The establishment of a micropropagation protocol for Pothomorphe umbellata was carried out using leaf segments cultured on 1/4 strength Murashige and Skoog medium supplemented with 0.5 mg l-1 6-benzyladenine, 0.1 mg l-1 gibberelic acid added with 10 g l-1 sucrose. Rooting was achieved using MS medium devoid of growth regulators. An anatomical study confirmed shoot regeneration via direct organogenesis.

Anti-snake venom activities of extracts and fractions from callus cultures of Sapindus saponaria

Context: Sapindus saponaria L. (Sapindaceae) bark, root, and fruits are used as sedatives and to treat gastric ulcer and also demonstrate diuretic and expectorant effects. Objective: The anti-snake venom properties of callus of S. saponaria are investigated here for the first time. Materials and methods: In vitro cultivated callus of Sapindus saponaria were lyophilized, and the extracts were prepared with different solvents, before submitting to phytochemical studies and evaluation of the anti-ophidian activity. Crude extracts were fractionated by liquid–liquid partition and the fractions were monitored by thin layer chromatography (TLC). Subsequently, anti-ophidian activities were analyzed toward Bothrops jararacussu Lacerda (Viperidae), B. moojeni Hoge (Viperidae), B. alternates Duméril (Viperidea) and Crotalus durissus terrificus Lineu (Viperidae) venoms and isolated myotoxins and phospholipase A2 (PLA2). Results: Fractions A1, A2 and the extract in MeOH:H2O (9:1) significantly inhibited the toxic and pharmacological activities induced by snake venoms and toxins, when compared to other extracts and fractions. The lethal, clotting, phospholipase, edema-inducing, hemorrhagic and myotoxic activities were partially inhibited by the different extracts and fractions. TLC profiles of the crude extracts (B and C) and fractions (A1 and A2) showed β-sitosterol and stigmasterol as their main compounds. Stigmasterol exhibited inhibitory effects on enzymatic and myotoxic activities of PLA2. Discussion and conclusion: Sapindus saponaria extracts and fractions presented anti-ophidian activity and could be used as an adjuvant to serum therapy or for its supplementation, and in addition, as a rich source of potential inhibitors of enzymes involved in several pathophysiological human and animal diseases.

Enhanced triterpene production in Tabernaemontana catharinensis cell suspension cultures in response to biotic elicitors

Cell suspension cultures of Tabernaemontana catharinensis were treated with autoclaved homogenates of Candida albicans, Fusarium oxysporum, Penicillium avelanium and Saccharomyces cerevisiae. The effects caused by the concentration, exposure time and the type of elicitor on the accumulation of pentacyclic triterpenes were monitored. When exposed to biotic elicitors for longer periods, some cell lines redoubled the production of those triterpenes. Saccharomyces cerevisiae homogenate was the best elicitor of triterpenes in all cell lines investigated.

Atividade fitorreguladora de jasmonatos produzidos por Botryosphaeria rhodina

O acido jasmonico e seus derivados jasmonatos sao reguladores vegetais endogenos produzidos por varias especies vegetais, que atuam no mecanismo de defesa das plantas e agem como sinalizadores de estresse. O objetivo deste trabalho foi comparar o potencial fitorregulador de um fermentado, obtido pela fermentacao do fungo filamentoso Botryosphaeria rhodina, contendo jasmonatos, comparado a acao do acido jasmonico (AJ) usado como padrao. Sementes selecionadas de Capsicum frutescens (pimenta malagueta) e Physalis angulata (camapu) foram plantadas em terra contendo fertilizantes orgânicos e minerais. Plantas apos 37 dias de germinacao foram tratadas via pulverizacao foliar com duas diferentes concentracoes de AJ (25,0 e 50,0 mg L-1) e com fermentado contendo jasmonatos (correspondendo a 50 mg L-1 de AJ) e como controle foi utilizado agua. Os experimentos foram realizados em casa de vegetacao, em blocos casualizados, com tres repeticoes de cinco plantas por tratamentos, com os tratamentos dispostos em fatorial de 4x1 (1 controle; 2 doses de AJ e 1 dose do filtrado). A avaliacao das plantas foi realizada 80 dias apos a aplicacao de AJ e o fermentado. Plantas controle de Capsicum produziram 6,4 frutos/planta enquanto que plantas tratadas com 25,0 e 50,0 mg L-1 de AJ produziram 7,2 frutos/planta e 9,2 frutos/planta, respectivamente e plantas tratadas com o fermentado produziram 11,7 frutos/planta. Adicionalmente, plantas controle de Physalis produziram 10,5 botoes/planta e quando tratadas com 25,0 e 50,0 mg L-1 de AJ produziram 15,5 e 16,0 botoes/planta, respectivamente. Plantas tratadas com o fermentado dobraram a producao de botoes/planta (23,0) quando comparados ao controle. Resultados obtidos validam o potencial fitorregulador do AJ e do fermentado contendo jasmonatos, comprovado pelo aumento na producao de frutos e botoes florais pelas especies vegetais testadas, evidenciando assim o potencial de uso deste fermentado como alternativa de menor custo ao AJ obtido comercialmente.

Optimization of jasmonates bioproduction

Several studies have shown that jasmonates (jasmonic acid, (+)-7-isojasmonic acid and methyl jasmonate) exhibit potential anticancer activity, with the advantage of displaying selective cytotoxicity to cancer cells that spares normal lymphocytes to this function [1]. This class of compounds is present in several families of plants, being also produced by certain microorganisms, including Gibberela fujikuroi and Botryosphaeria rhodina. Bioprocess studies based on the use of strains of B. rhodina have shown the potential of this microorganism to produce jasmonates under controlled conditions [2]. There are several factors that affect the productivity of a fermentative process, including the strain of the microorganism, the inoculum, and the morphological and rheological properties of the broth [3]. Once selected the most producing strains, it becomes necessary to define the conditions of the fermentation process, and the standardization of the inoculum is of fundamental importance when seeking increased productivity. In view of the morphology of filamentous fungus B. rhodina and the difficulty to induce sporulation, the standardization of the inoculum is a major challenge. In this context, the aim of this work was to conduct the standardization of B. rhodina inoculum preparation for jasmonate bioproduction.

Influence of linolenic acid in the production of jasmonate

Background Research shows that jasmonic acid (JA) and Methyl Jasmonate (MJ), compounds that participate in the metabolism’s control, development and protection of plant processes [1], also possess anticancer activities [2]. In studies, researchers found that Jasmonate were able to inhibit the growth of human cancer cells, and MJ induced death in lung carcinoma cells [3], breast, prostate, melanoma, lymphoma and leukemic cells [4], without reaching normal lymphocytes. The predominant route of obtaining these compounds is the vegetal extraction, however it takes about 800 kg of flowers of Jasminum grandiflorum to produce 1 kg of jasmone containing only 0.25% of AJ. However, the jasmonatos can also be produced by the microorganisms [5]. A microorganism which shows promising results in the production of Jasmonate is the fungus Botryosphaeria rhodina, making it possible to produce systems which can control the parameters involved in the fermentation process, including the use of precursors in this process.

Evaluation of process parameters in the production of jasmonic acid

The term jasmonates is used to describe lipid derivatives synthesized via octadecanoid pathway and are mainly represented by jasmonic acid (JA) and its ester methyl jasmonate (MJ). In plants, jasmonates act in the defense mechanism [1], and recent reports have attributed to JA and MJ the ability to inhibit the growth of human cancer cells, yet having cytotoxic selectivity [2]. Due to the diverse biological activities related to jasmonates, studies have been conducted to optimize the bioproduction route of these molecules, and the use of strains of Botryosphaeria rhodina seems to be a viable alternative when compared to the extraction from plants [3]. In order to optimize the production process, some of the important parameters must be standardized and controlled, such as the microorganism strain, the culture medium composition and the operational features of the process [4]. This study aimed to evaluate the influence of the inoculum size, medium supplementation with tryptone, the fermentation time and the interaction among these factors on the jasmonates production by the fungus Botryosphaeria rhodina.

Bioproduction and extraction of jasmonates

Jasmonates are a growing class of compounds of vegetable origin which present phytoregulator activity and toxicity against some types of cancer cells. Additionally the jasmonates have also shown activity against some types of nematodes [1]. Jasmonates available on the market are of plant origin or synthetic, present costs are still high. Like plants, some microorganisms have the potential to produce jasmonates. Some microorganisms have the potential to produce this class of compounds, including filamentous fungus Botryosphaeria rhodina has shown to be the most promising. The choice of the strain and optimization of process steps are fundamental premises for increasing the scale of production [2]. Furthermore, the optimization of the steps of extraction and purification of the product in the fermentation broth are essential to ensure the viability of the process. Currently, studies of recovery of jasmonates produced by Botryosphaeria rhodina are based on organic solvents. In this work, an alternative recovery route is proposed, based on adsorption, and the determination of the adsorption kinetics of jasmonates present in fermented using ion exchange resins Amberlite ® is presented.