Characterization of different genotypes of Jatropha curcas L.

Jatropha curcas L. (Euphorbiaceae family) is a perennial plant, which is widely distributed in tropical areas. Different parts of this plant have been traditionally used for various purposes. In particular, the oil from seeds has been used for soap production, for treatment of skin diseases, etc. The leaves have been used against coughs, as antiseptics and diuretics. Recently, J. curcas achieved significant importance for biodiesel production (green energy) from its seed oil (Thomas et al., 2008). Various active substances have been isolated from all parts of the plant, including: apigenin and vitexin (from leaves); alkaloids (from latex); other substances such as phorbol esters, responsible for the toxicity of J. curcas seeds. However, different genotypes from the same species could have different composition and, as a consequence, different biological activities. Aim of this study was to characterize genotypes of J.curcas at genomic, transcriptomic, phytochemical and biological levels. The genotypes were obtained by specific breeding program; to perform the study two divergent genotypes (A and B) were used. Genomic analysis were applied to estimate genetic distance into the segregant population. From each genotype leaves and petiole, at equal physiological phase, were collected and extracted using solvents with increasing polarity. Transcriptomic analysis (Milella et al., 2006) were carried out to isolate differentially expressed genome fraction. The sequence analysis of the differential fragments was obtained using FASTA and BLAST algorithm, and comparing with EMBL databases. Phytochemical studies were performed by chromatography (CC, TLC), spectroscopy (NMR). The biological activity (cell proliferation and cytotoxicity) of J. curcas was assessed in a wide range of concentrations (6-0.05 mg/ml of methanolic extract) on BRL-3A hepatic cell line, by MTT test, Trypan blue exclusion test and Neutral Red assay (Vitalone et al., 2003; Ganbold et al., 2010). The genomic analysis have shown sufficient differences between genotype A and B structures. This aspect was confirmed by transcriptomic analysis. Moreover, sequences analysis highlighted differences in enzyme activities and transcription factors among the genotypes. In vitro study indicated that petiole of two different genotypes of J. curcas did not affect BRL-3A cell proliferation. On the other hand, leaves inhibited cell proliferation in a concentration dependent manner. Genotype B was more potent than Genotype A (IC50= 0.12 mg/ml and 0.47 mg/ml, respectively in MTT test), and more cytotoxic at the higher concentrations tested (at 6 mg/ml, percentage of dead cells were 65% and 45% for Genotype B and A, respectively in Trypan blue test). Lysosomes appears less sensitive than mitochondria to genotype B effect (neutral red test), indicating that mitochondrial dysfunction occurs without the lysosomal permeabilization. A further characterization of the more potent Genotype B indicated that nonpolar fraction contained mainly polyunsaturated fatty acids, whereas several flavonoids were detected in the medium polarity fraction in their glycosides forms. Even if mitochondrial impairment without lysosomes destabilization, on hepatocytes, is generally due to fatty acids (Li et al., 2008) also flavonoids could contribute to this effect. In summary, this study provided evidence that two divergent genotypes of J. curcas have large differences at genomic and transcriptomic levels; this leads to a difference in the biological activity, with genotype B as more potent in the inhibition of cell proliferation. Further studies will additional characterize phytochemistry and other biological effects of J. curcas