## 3 Results
### Antiproliferative Activity  
#### 3.3.1 MTT Assay and Trypan Blue Exclusion Test  
The extracts of leaves of the two different genotypes of _J. curcas_ inhibited cell proliferation in a concentration-dependent manner. In the MTT reduction test, JB was more potent than JA (IC\({}_{50}\) were 0.12 mg/mL and 0.46 mg/mL, respectively) after 24 h of treatment (Figure 7). These data were confirmed after 48 h exposure when the IC\({}_{50}\) values were 0.18 mg/mL and 0.47 mg/mL for JB and JA, respectively (data not shown).  
Figure 6: NMR spectra of nonpolar (**A**) and polar (**B**) fractions of _J. curcas_ (genotype B) leaves. Spectrum A shows the main constituent, an unsaturated lipid-alcohol methylester. At higher fields, the signals of the aliphatic protons are present, whereas at 4.08 ppm the signals of methyl groups are visible and at 5.32 ppm those of the double bond proton are visible. In spectrum B, the presence of a glucosyl-3-methylether of kaempherd is indicated. At lower fields the signals of the flavonoid aglycone are present, as AA’BB’ system for protons of ring B and as two doublets for proton H-6 and H-8 of ring A; visible in the middle of the spectrum are the signals of the sugar moiety with, in particular, the H-3′′ of the glucose with an ethoxy group.  
Figure 7: Effect of _J. curcas_ extracts of JA and JB on proliferation of Buffalo normal rat liver cell line (BRL-3A) cells measured by MTT test, after 24 h treatment. Data represent the mean (\(\pm\)SE) of two independent experiments done in triplicate. * _p_\(<\) 0.05 vs. control (Dunnett’s test).  
In the trypan blue exclusion test, both genotypes were cytotoxic at the higher concentration tested; at 1.5 mg/mL, the percentage of dead cells were 45% and 65% for JA and JB, respectively, after 24 h of treatment (Figure 8A,B). These data were confirmed after 48 h exposure (data not shown).  
Given the greater potency of genotype B, itwas chosen for further investigation and was divided into fractions and single compounds. MTT test on JB fractions showed that the whole extract was more active than its (nonpolar and polar) fractions and its single compounds, after 24 h (not shown) and after 48 h of treatment (Figure 9A). In particular, the IC\({}_{50}\) of the non-polar and polar fractions after 24 h of exposure were 11.70 \(\mu\)g/mL and 10.36 \(\mu\)g/mL, respectively (corresponding to 0.15 and 0.13 mg/mL of the total extract). After 48 h of treatment, the polar fraction (IC\({}_{50}\) of 34.32 \(\mu\)g/mL) presented a very weak antiproliferative activity compared to the non-polar one (IC\({}_{50}\) of 12.23 \(\mu\)g/mL) (Figure 9B), indicating a reversible cytotoxic effect. The pure substances isolated from the non-polar and polar fractions showed activity inhibiting cell proliferation only at the maximum concentration tested. In any case, for all single compounds, it was not possible to determine the IC\({}_{50}\) because it was certainly higher than the maximum concentration tested (Figure 9A,B).  
Figure 8: Cytotoxic effect of _J. curcas_ extracts JA (**A**) and JB (**B**) on BRL-3A cells assayed by Trypan blue exclusion assay, after 24 h of treatment. All data are statistically different from control (* \(p\) < 0.05).