Chemical Characterization and DNA Fingerprinting of Griffonia simplicifolia Baill.

BACKGROUND: Griffonia simplicifolia Baill. (Caesalpiniaceae) is a medicinal plant whose seeds are widely used in traditional medicine for their high content of 5-hydroxy-l-tryptophan (5-HTP), a direct precursor and enhancer of the activity of the brain hormone serotonin (5-HT). The plant extracts are used in dietary supplements aimed to alleviate serotonin-related disorders. METHODS: In order to characterize the chemical components of G. simplicifolia seeds and their identity, we used a combined methodology by using HPLC-DAD-ESI-MS/MS for the qualitative and quantitative determination of the N-containing compounds, GC-FID and GC-MS for the characterization of the major fatty acids, and DNA fingerprinting based on PCR⁻RFLP for the unequivocal identification of the plant. RESULTS: 5-HTP was the most representative compound, followed by lower percentages of the ß-carboline alkaloid derivative griffonine and other alkaloids. Fatty acids were dominated by the unsaturated fatty acids linoleic acid and oleic acid, followed by the saturated fatty acids stearic and palmitic acids. PCR analysis of the internal transcribed spacer amplified sequence showed a major band at about 758 bp, whereas the PCR⁻RFLP analysis of this sequence using three different restriction enzymes (MspI, HhaI, and HaeIII) generated a specific fingerprinting useful for the plant identification. CONCLUSIONS: The combined chemical and molecular analysis of G. simplicifolia provided an interesting integrated approach for the unequivocal identification of commercial G. simplicifolia seeds.

Calcium modulates protease resistance and carbohydrate binding of a plant defense legume lectin, Griffonia simplicifolia lectin II (GSII).

Site-directed mutagenesis previously identified the residues responsible for the biological activity of the plant defense legume lectin, Griffonia simplicifolia lectin II (GSII) [Proc. Natl. Acad. Sci. USA 95, (1998) 15123-15128]. However, these results were inconclusive as to whether these residues function as direct defense determinants through carbohydrate binding, or whether substantial changes of the protein structure had occurred in mutated proteins, with this structural disruption actually causing the loss of biochemical and biological functions. Evidence shown here supports the former explanation: circular dichroism and fluorescence spectra showed that mutations at carbohydrate-binding residues of GSII do not render it dysfunctional because of substantial secondary or tertiary structure modifications; and trypsin treatment confirmed that rGSII structural integrity is retained in these mutants. Reduced biochemical stability was observed through papain digestion and urea denaturation in mutant versions that had lost carbohydrate-binding ability, and this was correlated with lower Ca(2+) content. Accordingly, the re-addition of Ca(2+) to demetalized proteins could recover resistance to papain in the carbohydrate-binding mutant, but not in the non-binding mutant. Thus, both carbohydrate binding (presumably to targets in the insect gut) and biochemical stability to proteolytic degradation in situ indeed contribute to anti-insect activity, and these activities are Ca(2+)-dependent.