Antifungal activity of a trypsin inhibitor from Salvia hispanica L. (chia) seeds against fluconazole-resistant strains of Candida spp. and evaluation of its toxicity in vitro.

The incidence of Candida species resistant to traditional antifungal drugs is increasing globally. This issue significantly impacts patients' lives and increases healthcare expenses, confirming the need to develop novel therapeutic strategies. Recently, a thermostable trypsin inhibitor named ShTI (11.558 kDa), which has antibacterial effects on Staphylococcus aureus, was isolated from Salvia hispanica L. (chia) seeds. This study aimed to assess the antifungal effect of ShTI against Candida species and its synergism with fluconazole and to evaluate its mode of action. Preliminary toxicological studies on mouse fibroblasts were also performed. ShTI exhibited antifungal effects against C. parapsilosis (ATCC® 22,019), C. krusei (ATCC® 6258), and six clinical fluconazole-resistant strains of C. albicans (2), C. parapsilosis (2), and C. tropicalis (2). The minimum inhibitory concentration (MIC) values were 4.1 µM (inhibiting 50% of the isolates) and 8.2 µM (inhibiting 100% of the isolates). Additionally, when combined with fluconazole, ShTI had a synergistic effect on C. albicans, altering the morphological structure of the yeast. The mode of action of ShTI against C. krusei (ATCC® 6258) and C. albicans involves cell membrane permeabilization, the overproduction of reactive oxygen species, the formation of pseudohyphae, pore formation, and consequently, cell death. In addition, ShTI (8.65 and 17.3 µM) had noncytotoxic and nongenotoxic effects on L929 mouse fibroblasts. These findings suggest that ShTI could be a promising antimicrobial candidate, but further research is necessary to advance its application as a novel antifungal agent.

Chia (Salvia hispanica L.) Seeds Contain a Highly Stable Trypsin Inhibitor with Potential for Bacterial Management Alone or in Drug Combination Therapy with Oxacillin.

The emergence of antibiotic resistance poses a serious and challenging threat to healthcare systems, making it imperative to discover novel therapeutic options. This work reports the isolation and characterization of a thermostable trypsin inhibitor from chia (Salvia hispanica L.) seeds, with antibacterial activity against Staphylococcus aureus sensitive and resistant to methicillin. The trypsin inhibitor ShTI was purified from chia seeds through crude extract heat treatment, followed by affinity and reversed-phase chromatography. Tricine-SDS-PAGE revealed a single glycoprotein band of ~ 11 kDa under nonreducing conditions, confirmed by mass spectrometry analysis (11.558 kDa). ShTI was remarkably stable under high temperatures (100 °C; 120 min) and a broad pH range (2-10; 30 min). Upon exposure to DTT (0.1 M; 120 min), ShTI antitrypsin activity was partially lost (~ 38%), indicating the participation of disulfide bridges in its structure. ShTI is a competitive inhibitor (Ki = 1.79 × 10-8 M; IC50 = 1.74 × 10-8 M) that forms a 1:1 stoichiometry ratio for the ShTI:trypsin complex. ShTI displayed antibacterial activity alone (MICs range from 15.83 to 19.03 µM) and in combination with oxacillin (FICI range from 0.20 to 0.33) against strains of S. aureus, including methicillin-resistant strains. Overproduction of reactive oxygen species and plasma membrane pore formation are involved in the antibacterial action mode of ShTI. Overall, ShTI represents a novel candidate for use as a therapeutic agent for the bacterial management of S. aureus infections.

The Essential Oil of Hyptis crenata Inhibits the Increase in Secretion of Inflammatory Mediators.

Background: Hyptis crenata is a plant of great ethnopharmacological importance widely distributed in South American countries. In Northeast Brazil, teas or infusions of its aerial parts are used in folk medicine to treat several acute and chronic inflammatory diseases. In a previous work we have demonstrated that the essential oil of H. crenata (EOHc) has an antiedematogenic effect. The aim of this work was to evaluate the effect of EOHc on cytokines secretion and cellular infiltration. Methods: Peritonitis and paw edema models induced by carrageenan were used to determine leucocyte count, myeloperoxidase (MPO) activity, nitrite, and cytokines secretion. Results: EOHc (10−300 mg/kg) significantly inhibited leucocyte migration and reduced the neutrophil count (control: 1.46 × 103 ± 0.031 × 103/mL) of the total leucocytes population in extracellular exudate (control: 2.14 × 103 ± 0.149 × 103/mL) by 15.00%, 43.29%, 65.52%, and 72.83% for the doses of 10, 30, 100, and 300 mg/kg EOHc, respectively (EC50: 24.15 mg/kg). EOHc (100 mg/kg) inhibited the increase in myeloperoxidase activity and completely blocked the increase in nitrite concentration induced by carrageenan. EOHc markedly reduced the pro-inflammatory cytokines (IL-6, MCP-1, IFN-γ, TNF-α, and IL-12p70) and increased IL-10, an anti-inflammatory cytokine (compared to control group, p < 0.05). Conclusions: This study demonstrates that EOHc has a long-lasting anti-inflammatory effect mediated through interference on MPO activity, and nitrite, and cytokines secretion. This effect, coupled with low EOHc toxicity, as far as results obtained in mice could be translated to humans, suggests that EOHc has great potentiality as a therapeutic agent.

Therapeutic effects of a lipid transfer protein isolated from Morinda citrifolia L. (noni) seeds on irinotecan-induced intestinal mucositis in mice.

This work aimed to evaluate the activity of a lipid transfer protein isolated from Morinda citrifolia L. seeds, McLTP1, on the development of intestinal mucositis following irinotecan administration. McLTP1 (0.5, 2, and 8 mg/kg, i.v.) was injected into mice 1h before irinotecan administration (75 mg/kg, i.p.; 4 days), and then for additional 6 days. Seven days after the first dose of irinotecan, diarrhea was assessed, and the intestine was removed for histological evaluation, assessment of intestinal over-contractility, measurement of myeloperoxidase (MPO), proinflammatory cytokines and chemokine (IL-1, IL-6, and KC levels - a murine homolog of human IL-8 chemokine), analysis of cyclooxygenase 2 (COX-2), nuclear factor kappa B (NF-κB), and nitric oxide synthase (iNOS) expression. At the two highest doses, McLTP1 administration decreased mortality and diarrhea. McLTP1 (8 mg/kg, i.v.) significantly prevented irinotecan-induced intestinal damage and led to a reduction in over-contractility of the intestinal muscle (p < 0.05). Moreover, McLTP1 decreased the MPO, IL-1ß, IL-6, and KC levels by 74.7%, 42%, 92.9%, and 95.9%, respectively. Also, the expression of COX-2, NF-κB, and iNOS was reduced. Our study provides a potential new therapeutic for preventing irinotecan-induced mucositis, improved clinical parameters, and reduced inflammation.

Noni (Morinda citrifolia L.) fruit as a new source of milk-clotting cysteine proteases.

This work reports the characterisation of caseinolytic and milk-clotting activities of proteases extracted from ripe fruits of Morinda citrifolia L., as a potential of their use in cheese production. Noni puree extract (NPE) was obtained by homogenising the fresh puree in 150 mM NaCl/50 mM sodium phosphate buffer (pH 7.0). The resulting protein concentration was of 0.367 ±â€¯0.006 mg/mL, and an electrophoretic profile of the extract revealed protein bands ranging from 14 to 55 kDa. The proteolytic activity of NPE was higher when the extract had been previously incubated at pH 6.0 (8.859 ±â€¯0.216 U/mg), whereas the optimum caseinolytic activity was observed at 50 °C. Noni puree proteases were strongly (98%) inhibited by iodoacetamide and E-64, suggesting the presence of only cysteine proteases in the crude extract. NPE proteases showed a milk-clotting activity (MCA) of 238.80 ±â€¯5.29 U/mL, a specific milk-clotting activity (SMCA) of 9950.17 ±â€¯220.74 U/mg, and an SMCA/PA ratio of 1124.31 ±â€¯24.94, this last being comparable to those of commercial calf rennet. The cheese manufactured using NPE presented brittle and soft texture, high humidity, and showed sanitary conditions compatible with current Brazilian regulations. The product showed a slightly bitter taste, but still good acceptability, rating between 6 and 7 in the hedonic scale for flavour, texture, and overall acceptance. Lastly, there was 60% of positive purchase intent, demonstrating that noni fruit is a promising source of milk-clotting enzymes for the dairy industry.

Purification of a chitin-binding protein from Moringa oleifera seeds with potential to relieve pain and inflammation.

Moringa oleifera Lam. is a perennial multipurpose tree that has been successfully used in folk medicine to cure several inflammatory processes. The aim of this study was to purify and characterize a chitin-binding protein from Moringa oleifera seeds, named Mo-CBP4, and evaluate its antinociceptive and anti-inflammatory effects in vivo. The protein was purified by affinity chromatography on chitin followed by ion exchange chromatography. Acetic acid-induced abdominal constrictions assay was used for the antinociceptive and anti-inflammatory activity assessments. Mo-CBP4 is a glycoprotein (2.9% neutral carbohydrate) composed of two protein subunits with apparent molecular masses of 28 and 18 kDa (9 kDa in the presence of reducing agent). The intraperitoneal injection of Mo-CBP4 (3.5 and 10 mg/kg) into mice 30 min before acetic acid administration potently and significantly reduced the occurrence of abdominal writhing in a dose dependent manner by 44.7% and 100%, respectively. In addition, the oral administration of the protein (10 mg/kg) resulted in 18% and 52.8% reductions in abdominal writhing when given 30 and 60 min prior to acetic acid administration, respectively. Mo-CBP4, when administered by intraperitoneal route, also caused a significant and dose-dependent inhibition of peritoneal capillary permeability induced by acid acetic and significantly inhibited leukocyte accumulation in the peritoneal cavity. In conclusion, this pioneering study describes that the chitin-binding protein Mo-CBP4, from M. oleifera seeds, exhibits anti-inflammatory and antinociceptive properties and scientifically supports the use of this multipurpose tree in folk medicine.