## 4 Discussion and Conclusions  
_J. curcas_ is a multiple-use plant and, as shown in our study, the assessment of its phytochemical composition and biological characteristics needs a multistep procedure of analysis. First of all, the leaves often represent the waste material of the plant; however, taking into account the variability of the herbal material and the in vitro effects, they could be considered in the research for other biological activities. In our study, the leaf extracts of the two genotypes of the same genus and species of _J. curcas_ showeddifferent phytochemical composition and biological activity. One genotype (JB)was richer in lipid compounds and flavonoid glycosides than the other one. Indeed, the organic fraction of JB, analyzed by NMR analysis, was characterized by unsaturated linear fatty acids (linoleic acid type) and methyl esters. Both genotypes inhibited cell proliferation in a concentration-dependent manner, but in our biological assays, JB was more potent in inhibiting hepatic cells proliferation than JA, with this effect being mainly observed in mitochondria. Indeed, lysosomes appeared less sensitive than mitochondria to the genotype effect of _J. curcas_, indicating that mitochondrial dysfunction occurs without lysosomal permeabilization. Lysosomal damage can probably occur a second time--when the membrane is damaged, becomes permeable, and the lysosomal enzymatic content is poured into the cytoplasm. Only when the cell is compromised does it undergo autolysis. This type of cytotoxicity was highlighted in our study at the highest concentrations tested (through the trypan blue exclusion and chromatin condensation tests). [PERSON] et al. [7] observed a high cytotoxic effect on human hepatocytes (Chang liver cells) of methanolic extracts from leaves of _J. curcas_. This effect has also been confirmed by our study. [PERSON] and colleagues [28] analyzed the anti-metastatic activity of methanolic fraction of _J. curcas_ (MFJC), and found that MFJC reduced the formation of pulmonary metastasis of B16F10 melanoma cells. In an in vitro cytotoxicity test, an assay on MFJC showed a significant reduction in melanoma cells proliferation, with an IC\({}_{50}\) of 24.8 \(\mu\)g/mL [28]. In our study, the antiproliferative effect of _J. curcas_ had a stronger result (IC\({}_{50}\) of about 11 \(\mu\)g/mL), probably because we used a normal hepatic cell line as a model for the screening of cytotoxicity. Our results showed that the biological activity of JB is not due to the single chemical components here identified, suggesting that other secondary metabolites of the extract are involved in the effect on cell proliferation. The presence of unsaturated fatty acids (such oleic and linoleic acids) could probably explain the lack of inhibition of proliferation of BRL-3A cells in the MTT test. The lack of effect on  
Figure 10: Hoechst 33258 staining in BRL-3A hepatic cells after 6 h (**A**), 24 h (**B**), and 48 h (**C**) of treatment with two concentrations of _J. curcas_ genotype B. Figures are representative of five replicates for each sample. Original magnification: \(\times 20\). cell proliferation of the flavonoid glycoside, isolated from the polar fraction of JB, is reported in the literature [29; 30]. Another very important issue of this study is that the extracts of the leaves of _J. curcas_ do not contain phorol esters, which are present in other plants. These data agree with previous studies that found that _J. curcas_ from Guatem (even if in the presence of high genetic variation) did not contain toxins [31] or phorol esters [32]. Therefore, even if current strategies for the detoxification of _J. curcas_ seeds from phorol esters are continuously studied [33], there is the possibility that there are specific phenotypes that actually do not contain them. The use of plant species \"phorol ester-free\" allows for a great saving of costs in the choice/application of their removal methods (i.e., chemical, physical, biological, or combined processes) [33].