UPLC-MS-QTOF analysis and antifungal activity of Cumaru (Amburana cearensis).

This study was aimed at investigating the phytochemical constituents, antifungal properties and antibiotic-modifying activity of the aqueous crude extract and fractions of Amburana cearensis seeds (CEFAC). The CEFAC were chemically characterized by LC-MS/MS-QTOF. In addition, the antifungal activity was assayed by the microdilution method against strains of Candida albicans. The phytochemical profile of CEFAC exhibited phenolic compounds, organic acids, and polyphenols. The results of the assessment of antifungal activity reveled an IC50 ranging from 45.6 to 2048 µg/mL. Interestingly, when CEFAC was associated with Fluconazole, we evidenced a decreased IC50 (1.81-11.9 µg/mL), suggesting a synergism with antibiotic. It was possible to identify in the crude extract and fractions several phenolic compounds, organic acids, and some polyphenols in positive ionization mode. These results suggest that CEFAC may present compounds with the ability to interact and act synergistically with antimicrobial drugs, highlighting its potential as an alternative source for the development of new antimicrobial agents.

Biochemical characterisation of a Kunitz-type inhibitor from Tamarindus indica L. seeds and its efficacy in reducing plasma leptin in an experimental model of obesity.

A trypsin inhibitor isolated from tamarind seed (TTI) has satietogenic effects in animals, increasing the cholecystokinin (CCK) in eutrophy and reducing leptin in obesity. We purified TTI (pTTI), characterised, and observed its effect upon CCK and leptin in obese Wistar rats. By HPLC, and after amplification of resolution, two protein fractions were observed: Fr1 and Fr2, with average mass of [M + 14H]+ = 19,594,690 Da and [M + 13H]+ = 19,578,266 Da, respectively. The protein fractions showed 54 and 53 amino acid residues with the same sequence. pTTI presented resistance to temperature and pH variations; IC50 was 2.7 × 10-10 mol.L-1 and Ki was 2.9 × 10-11 mol.L-1. The 2-DE revealed spots with isoelectric points between pH 5 and 6, and one near pH 8. pTTI action on leptin decrease was confirmed. We conclude that pTTI is a Kunitz trypsin inhibitor with possible biotechnological health-related application.

Expression in Escherichia coli of cysteine protease inhibitors from cowpea (Vigna unguiculata): The crystal structure of a single-domain cystatin gives insights on its thermal and pH stability.

Two cysteine proteinase inhibitors from cowpea, VuCys1 and VuCys2, were produced in E. coli ArcticExpress (DE3). The recombinant products strongly inhibited papain and chymopapain as well as the midgut proteases from Callosobruchus maculatus larvae, a bruchid that uses cysteine proteases as major digestive enzymes. Heat treatment at 100°C for up to 60min or incubation at various pH values caused little reduction in the papain inhibitory activity of both inhibitors. Moreover, minor conformational variations, as probed by circular dichroism spectroscopy, were observed after VuCys1 and VuCys2 were subjected to these treatments. The crystal structure of VuCys1 was determined at a resolution of 1.95Å, revealing a domain-swapped dimer in the asymmetric unit. However, the two lobes of the domain-swapped dimer are positioned closer to each other in VuCys1 in comparison to other similar cystatin structures. Moreover, some polar residues from opposite lobes recruit water molecules, forming a hydrogen bond network that mediates contacts between the lobes, thus generating an extended open interface. Due to the closer distance between the lobes, a small hydrophobic core is also formed, further stabilizing the folded domain-swapped dimer. These structural features might account for the extraordinary thermal and pH stability of VuCys1.

Trypsin inhibitor from tamarindus indica L. seeds reduces weight gain and food consumption and increases plasmatic cholecystokinin levels

OBJECTIVES: Seeds are excellent sources of proteinase inhibitors, some of which may have satietogenic and slimming actions. We evaluated the effect of a trypsin inhibitor from Tamarindus indica L. seeds on weight gain, food consumption and cholecystokinin levels in Wistar rats. METHODS: A trypsin inhibitor from Tamarindus was isolated using ammonium sulfate (30-60%) following precipitation with acetone and was further isolated with Trypsin-Sepharose affinity chromatography. Analyses were conducted to assess the in vivo digestibility, food intake, body weight evolution and cholecystokinin levels in Wistar rats. Histological analyses of organs and biochemical analyses of sera were performed. RESULTS: The trypsin inhibitor from Tamarindus reduced food consumption, thereby reducing weight gain. The in vivo true digestibility was not significantly different between the control and Tamarindus trypsin inhibitor-treated groups. The trypsin inhibitor from Tamarindus did not cause alterations in biochemical parameters or liver, stomach, intestine or pancreas histology. Rats treated with the trypsin inhibitor showed significantly elevated cholecystokinin levels compared with animals receiving casein or water. CONCLUSION: The results indicate that the isolated trypsin inhibitor from Tamarindus reduces weight gain by reducing food consumption, an effect that may be mediated by increased cholecystokinin. Thus, the potential use of this trypsin inhibitor in obesity prevention and/or treatment should be evaluated. .