Antifungal, Antimycobacterial, Protease and α‒Amylase Inhibitory Activities of a Novel Serine Bifunctional Protease Inhibitor from Adenanthera pavonina L. Seeds.

Antifungal resistance poses a significant challenge to disease management, necessitating the development of novel drugs. Antimicrobial peptides offer potential solutions. This study focused on extraction and characterization of peptides from Adenanthera pavonina seeds with activity against Candida species, Mycobacterium tuberculosis, proteases, and α-amylases. Peptides were extracted in phosphate buffer and heated at 90°C for 10 min to create a peptide rich heated fraction (PRHF). After confirming antimicrobial activity and the presence of peptides, the PRHF underwent ion exchange chromatography, yielding retained and non-retained fractions. These fractions were evaluated for antimicrobial activity and cytotoxicity against murine macrophages. The least toxic and most active fraction underwent reversed-phase chromatography, resulting in ten fractions. These fractions were tested for peptides and antimicrobial activity. The most active fraction was rechromatographed on a reversed-phase column, resulting in two fractions that were assessed for antimicrobial activity. The most active fraction revealed a single band of approximately 6 kDa and was tested for inhibitory effects on proteases and α-amylases. Thermal stability experiments were conducted on the 6 kDa peptide at different temperatures followed by reassessment of antifungal activity and circular dichroism. The 6 kDa peptide inhibited yeasts, M. tuberculosis, human salivary and Tenebrio molitor larvae intestine α-amylases, and proteolytic activity from fungal extracts, and thus named ApPI. Remarkably, ApPI retained antifungal activity and conformation after heating and is primarily composed of α-helices. ApPI is a thermally stable serine protease/α-amylase inhibitor from A. pavonina seeds, offering promise as a foundational molecule for innovative therapeutic agents against fungal infections and tuberculosis.

Neutrophils regulate the expression of cytokines, chemokines and nitric oxide synthase/nitric oxide in mice injected with Bothrops atrox venom.

Bothrops atrox crude venom injected intraperitoneal (i.p.) into BALB/c mice induced local afflux of inflammatory cells, one neutrophil-rich peak after 6h and another macrophage-rich peak after 48 h. A similar pattern of local cell afflux plus edema, Delta lesions of some skeletal muscle cells, and hemorrhage were observed in mice intramuscular (i.m.) injected with the venom. Measurement of serum cytokines in neutrophil-depleted (by anti-mouse rat monoclonal antibody (mAb) RB6-8C5) and non-depleted BALB/c mice was performed by ELISA. With the exception of IL-1beta (78 pg/ml), higher levels of IL-6 (1348 pg/ml), MIP-1beta (437 pg/ml) and MIP-2 (904 pg/ml) were observed in neutrophil-depleted mice, in comparison to the values found in non-neutrophil depleted mice: IL-1beta (437 pg/ml), IL-6 (750 pg/ml), MIP-1beta (165 pg/ml) and MIP-2 (90 pg/ml). TNF-alpha was not detected. NO was detected (18 microM) 24h after venom injection in neutrophil-depleted mice. RT-PCR using representative primers detected expression of mRNA in cells from BALB/c mice injected with B. atrox venom: (a) for IL-1beta, IL-6, inducible nitric oxide synthase (iNOS), CXCR2, MIP-2 and RANTES in cells from mice that were neutrophil-depleted or not; (b) for CCR1, CCR5 and MIP-1beta in cells from neutrophil-depleted mice; (c) for MIP-1alpha in cells from non-neutrophil-depleted mice; (d) TNF-alpha and TGF-beta were not detected in either of the mice. These results indicate that neutrophils play a role in regulating the production of some cytokines and chemokines as well as locally expressed or liberated iNOS/NO in tissues injected with B. atrox crude venom.