Partial least squares model and design of experiments toward the analysis of the metabolome of Jatropha gossypifolia leaves: Extraction and chromatographic fingerprint optimization.

A major challenge in metabolomic studies is how to extract and analyze an entire metabolome. So far, no single method was able to clearly complete this task in an efficient and reproducible way. In this work we proposed a sequential strategy for the extraction and chromatographic separation of metabolites from leaves Jatropha gossypifolia using a design of experiments and partial least square model. The effect of 14 different solvents on extraction process was evaluated and an optimized separation condition on liquid chromatography was estimated considering mobile phase composition and analysis time. The initial conditions of extraction using methanol and separation in 30 min between 5 and 100% water/methanol (1:1 v/v) with 0.1% of acetic acid, 20 µL sample volume, 3.0 mL min(-1) flow rate and 25°C column temperature led to 107 chromatographic peaks. After the optimization strategy using i-propanol/chloroform (1:1 v/v) for extraction, linear gradient elution of 60 min between 5 and 100% water/(acetonitrile/methanol 68:32 v/v with 0.1% of acetic acid), 30 µL sample volume, 2.0 mL min(-1) flow rate, and 30°C column temperature, we detected 140 chromatographic peaks, 30.84% more peaks compared to initial method. This is a reliable strategy using a limited number of experiments for metabolomics protocols.

EFFECTS OF SINGLE, BINARY AND TERTIARY COMBINATIONS WITH Jatropha gossypifolia AND OTHER PLANT-DERIVED MOLLUSCICIDES ON REPRODUCTION AND SURVIVAL OF THE SNAIL Lymnaea acuminata / Efeitos de combinações unitárias, binárias e terciárias de Jatropha gossypifolia e outros muluscicidas derivados de plantas na reprodução e sobrevivência do caramujo Lymnaea acuminata

The effect of sub-lethal doses (40% and 80% of LC50/24h) of plant derived molluscicides of singly, binary (1:1) and tertiary (1:1:1) combinations of the Rutin, Ellagic acid, Betulin and taraxerol with J. gossypifolia latex, leaf and stem bark powder extracts and their active component on the reproduction of freshwater snail Lymnaea acuminata have been studied. It was observed that the J. gossypifolia latex, stem bark, individual leaf and their combinations with other plant derived active molluscicidal components caused a significant reduction in fecundity, hatchability and survival of young snails. It is believed that sub-lethal exposure of these molluscicides on snail reproduction is a complex process involving more than one factor in reducing the reproductive capacity.

O efeito de doses sub-letais (40% e 80% de LC50/24h) de moluscicidas derivados de plantas com combinações unitárias, binárias (1:1) e terciárias (1:1:1) de Rutin, ácido Elágico, Betulin e taraxerol com látex da J. gossypifolia, folhas e extrato em pó de casca de caule e seus componentes ativos foram estudados na reprodução do caramujo de água fresca Lymnaea acuminata. Foi observado que o látex da J. gossypifolia, casca do caule, folhas individualmente e suas combinações com componentes moluscicidas ativos derivados de outras plantas causaram redução significante na fecundidade, incubação e sobrevivência dos caramujos jovens. Acredita-se que a exposição sub-letal destes moluscicidas sobre a reprodução dos caramujos é processo complexo envolvendo mais de um fator na redução da capacidade reprodutiva.

Establishment of cell suspension cultures of two Costa Rican Jatropha species (Euphorbiaceae) / Establecimiento de suspensiones celulares de dos especies Jatropha (Euphorbiaceae) de Costa Rica

J. curcas has been studied in different countries and some interesting agronomic, pharmacological and industrial properties have been reported. More recently, it has been considered an important alternative source for biofuel production. The objective of this study was to establish a long-term method for the maintenance of calli and cell suspension cultures of the local species J. curcas and J. gossypifolia, in order to allow future studies for novel compounds with pharmaceutical or industrial applications. For this, friable calli were successfully induced from hypocotyl segments of J. curcas and J. gossypifolia that were cultured in semisolid MS media supplemented with 1.5mg/L, and 0.5mg/L of 2,4-D, respectively. Cell suspension cultures of J. curcas were established using 1g of 35 and 60-day calli, in 50mL of liquid MS media supplied with 1.5mg/L of 2,4-D; sucrose and maltose were additionally evaluated as carbon sources. After 35 days, cell suspension cultures initiated with 35-day calli, showed greater cell growth with a maximum biomass of 194.9g/L fresh weight, 6.59g/L dry weight and 17.3% packed volume. The exponential phase ended at day 35 for cultures initiated with 35-day calli, and at day 21 for cultures initiated with 60-day calli. Higher biomass production was obtained with sucrose. Cell cultures were established with 35-day calli in MS media with the same 2,4-D concentration used for calli induction and 30g/L sucrose. This medium was considered optimum for the maintenance and growth of cell suspensions for both species, with sub-cultures every 20 days. The biotechnological potential for the production of bioactive compounds in these species for pharmacological, agricultural and industrial applications is being evaluated.

J. curcas es un importante recurso alternativo de biocombustible. Por otro lado, propiedades de interés agronómico, farmacológico e industrial han sido reportadas para esta especie. El objetivo de este estudio fue el establecimiento y mantenimiento a largo plazo de callos y cultivos celulares en suspensión de J. curcas y J. gossypifolia, con el objetivo de permitir futuros estudios para nuevos compuestos con aplicaciones farmaceúticas e industriales. Los callos friables fueron exitosamente inducidos a partir de segmentos de hipocótilos J. curcas and J. gossypifolia cultivados en medio MS semisólido suplementado con 1.5mg/L y 0.5mg/L of 2,4-D, respectivamente. Los cultivos celulares en suspensión de J. curcas fueron establecidos utilizando 1g de callos de 35 y 60 días de edad en 50mL de medio MS líquido adicionado con 1.5mg/L de 2,4-D. Después de 35 días, los cultivos en suspensión celular iniciados con callos de 35 días, mostraron mayor crecimiento celular con una biomasa máxima de 194.9g/L de peso fresco y 6.59g/L de peso seco y 17.3% de volumen empacado. La fase exponencial finalizó al día 35 en los cultivos iniciados con callos de 35 días, y al día 21 en los cultivos iniciados con callos de 60 días. Dos fuentes de carbono fueron evaluadas: sacarosa y maltosa. La producción de mayor biomasa fue obtenida con sacarosa. Los cultivos celulares se establecieron con callos de 35 días cultivados en medio MS con la misma concentración de 2,4-D utilizada para la inducción de callos y 30g/L de sacarosa. Este medio fue considerado el óptimo para el mantenimiento y crecimiento de suspensiones celulares en ambas especies con subcultivos cada 20 días. El potencial biotecnológico para la producción de compuestos bioactivos en estas especies, para aplicaciones farmacológicas, agrícolas e industriales está siendo evaluado.

Intoxicação experimental com as folhas de Jatropha gossypifolia (Euphorbiaceae) em ovinos / Experimental poisoning by the leaves of Jatropha gossypifolia (Euphorbiaceae) in sheep

As folhas recém-colhidas de Jatropha gossypifolia (Euphorbiaceae) foram letais para ovinos em administrações únicas de 40g/kg. A dose de 5g/kg não causou sintomas de intoxicação; as doses intermediárias provocaram a morte de parte dos animais. A evolução da intoxicação foi de 6 a 22 dias. O quadro clínico-patológico nos ovinos experimentais era caracterizado por perturbações digestivas, pulmonares, cardíacas e ainda alterações regressivas leves somente evidenciadas através de exames histológicos, hepáticas e renais. Esses achados são semelhantes aos observados em experimentos com as sementes de Jatropha curcas em caprinos, ovinos e bezerros, e com os com os frutos e as folhas de Jatropha glauca e Jatropha aceroides em caprinos, realizados por outros autores. Estas comparações indicam que, independentemente das espécies, as folhas de Jatropha spp. devem conter compostos semelhantes aos encontrados nas sementes.

Fresh green leaves of Jatropha gossypifolia (Euphorbiaceae) were lethal for sheep in single administrations of 40g/kg. The dose of 5g/kg did not cause poisoning, but intermediate doses caused death in part of the animals. The clinical course of poisoning was 6 to 22 days. The clinic and pathological picture in the experimental sheep was characterized by digestive, lung and heart disturbances, and also by slight microscopic liver and renal regressive alterations. These findings are similar to those observed in experiments with the seeds of Jatropha curcas in goats, sheep and calves, and with the fruits and leaves of Jatropha glauca and Jatropha aceroides in goats, performed by other authors. A comparison indicates that, independently of the plant species, the leaves of Jatropha spp. contain toxic compounds similar to those found in the seeds.