STUDY OF ANTI-INFLAMMATORY POTENTIAL OF WHOLE CELL EXRACTS OF CYANOBACTERIA BY MEMBRANE STABILIZATION & TRYPSIN INHIBITION ASSAY

Inflammation is a complex mechanism in response to any infection, injury, or irritation. The prolonged inflammation leads to tissue damage and loss of function result in various disease conditions like osteoarthritis, and autoimmune diseases like lupus, rheumatoid arthritis, asthma and Crohn’s disease. Anti-inflammatory drugs are used to control inflammatory response and prevent tissue damage .Cyanobacteria are rich sources of phytochemicals like phenolics, flavonoids are known to have anti-inflammatory activity. Because of ease of cultivation and faster growth rate than plants, they are preferred candidates over plants. This study focuses on screening of cyanobacterial isolates for their anti-inflammatory activity. The human erythrocytes (HRBC) membrane stabilization assay indicated the potential of whole cell extracts of cyanobacteria to stabilize lysosomal membrane and thereby prevent tissue damage by lysosomal chemokines and enzymes. The trypsin inhibition assay is an indicator for potential to inhibit proteinases and decelerate tissue damage. The whole cell extracts of 10 cyanobacterial isolates of different genus namely Weistellopsis, Pseudophormidium, Oscillatoria, Nostoc, Phormidium, Chlorella, Hapalosiphon under study showed 85%-94% membrane stabilization in hypotonicity induced hemolysis and 92% to 97% membrane stabilization in heat induced hemolysis. The test extracts also showed 48% to 52% inhibition of trypsin. Thus, the isolates under study have application as anti-inflammatory agent.

Bacterial Protease Inhibitors as Antibacterial agents to prevent Bacterial Infections Associated with Biofilms

Isolation of protease inhibitor producing bacteria from microbial mat and investigating its anti-biofilm potential against biofilm producing organism was selected as the main objective of the present study. Protease inhibitor (PI) was produced from bacterial isolates and purified using ammonium sulphate precipitation methods. Primary and secondary protease inhibitor assay was carried out separately to confirm the inhibition of protease enzyme activity both qualitatively and quantitatively. Antibacterial activity and anti-biofilm assay was performed to determine the biofilm prevention capabilities of PI. Three isolates (B1PI, B2PI and B3PI) were screened and B2PI bacterial culture was selected based on the results of primary and secondary protease inhibitor assay. Maximum trypsin inhibition of 77.5±0.25% was recorded for the isolate B2PI. Antibacterial activity of the B2PI protease inhibitor fractions exhibited inhibitory zones of 22.3±1.04mm and 20.2±0.25mm against Escherichia coli and Staphylococcus aureus respectively. Anti-biofilm assay of protease inhibitor fractions expressed 31.2μl/ml of MBIC against Escherichia coli and Staphylococcus aureus. The results conclude that, the protease inhibitor from the microbial mat isolate will be an effective alternative to the commercial antibiotics either alone or in combination with other drugs synergistically which shall be studied elaborately in future.

A novel approach to trypsin inhibition by flavonoids

Trypsin is a key protease related to digestion and absorption of proteins, which its inhibition must be studied when natural compounds, such as flavonoids, are used as part of alternative treatments for obesity and diabetes mellitus type 2, since trypsin and together with other pancreatic enzymes worked at small intestine. Considering that flavonoids are good lipase and amylase inhibitors, trypsin-flavonoids interactions were analyzed through UV-Vis, intrinsic and extrinsic fluorescence spectroscopies, circular dichroism, and molecular docking. The interaction between porcine pancreas trypsin and five flavonoids: hesperetin (HES), luteolin (LUT), quercetin (QUE), catechin (CAT), and rutin (RUT) was evaluated. Most of them exhibited a mixed-type mode. LUT was the best trypsin inhibitor (e.g., lower IC50, 45.20±1.00 µM). All flavonoids-trypsin complexes showed static quenching, and QUE and LUT exhibited higher affinity (higher Ka values, 0.90±0.10 and 1.60±0.20·10-1 mM-1, respectively). Hydrophobic interactions between trypsin and flavonoids were predominant.

Angiotensin Converting Enzyme and Trypsin Inhibition by Garcinia Cambogia Seeds Extract

In this project work, Garcinia cambogia seeds extract was prepared and analysed for inhibitory studies. G. cambogia or the Malabar tamarind, commonly used as a food preservative, flavouring agent or food-bulking agent, and as a traditional remedy to treat constipation, piles, rheumatism, edema, irregular menstruation and intestinal parasites in many Asian countries. G. cambogia seeds solvents (Methanol, ethylacetate, petroleum ether and acetone) extract were prepared and pharmacological activities were analyzed such as antioxidant activity, ACE inhibitory and trypsin inhibitory activities. Among the extracts tested, methanolic extract of G. cambogia has shown potent antioxidant, angiotensin converting enzyme and trypsin inhibitory activities.

Identification and Target-Modification of SL-BBI: A Novel Bowman–Birk Type Trypsin Inhibitor from Sylvirana latouchii

The peptides from the ranacyclin family share similar active disulphide loop with plant-derived Bowman–Birk type inhibitors, some of which have the dual activities of trypsin inhibition and antimicrobial. Herein, a novel Bowman–Birk type trypsin inhibitor of the ranacyclin family was identified from the skin secretion of broad-folded frog (Sylvirana latouchii) by molecular cloning method and named as SL-BBI. After chemical synthesis, it was proved to be a potent inhibitor of trypsin with a Ki value of 230.5 nM and showed weak antimicrobial activity against tested microorganisms. Modified analogue K-SL maintains the original inhibitory activity with a Ki value of 77.27 nM while enhancing the antimicrobial activity. After the substitution of active P1 site to phenylalanine and P2′ site to isoleucine, F-SL regenerated its inhibitory activity on chymotrypsin with a Ki value of 309.3 nM and exhibited antiproliferative effects on PC-3, MCF-7 and a series of non-small cell lung cancer cell lines without cell membrane damage. The affinity of F-SL for the β subunits in the yeast 20S proteasome showed by molecular docking simulations enriched the understanding of the possible action mode of Bowman–Birk type inhibitors. Further mechanistic studies have shown that F-SL can activate caspase 3/7 in H157 cells and induce apoptosis, which means it has the potential to become an anticancer agent.

Reduction of Allergenic Potential in Bread Wheat RNAi Transgenic Lines Silenced for CM3, CM16 and 0.28 ATI Genes

Although wheat is used worldwide as a staple food, it can give rise to adverse reactions, for which the triggering factors have not been identified yet. These reactions can be caused mainly by kernel proteins, both gluten and non-gluten proteins. Among these latter proteins, α-amylase/trypsin inhibitors (ATI) are involved in baker’s asthma and realistically in Non Celiac Wheat Sensitivity (NCWS). In this paper, we report characterization of three transgenic lines obtained from the bread wheat cultivar Bobwhite silenced by RNAi in the three ATI genes CM3, CM16 and 0.28. We have obtained transgenic lines showing an effective decrease in the activity of target genes that, although showing a higher trypsin inhibition as a pleiotropic effect, generate a lower reaction when tested with sera of patients allergic to wheat, accounting for the important role of the three target proteins in wheat allergies. Finally, these lines show unintended differences in high molecular weight glutenin subunits (HMW-GS) accumulation, involved in technological performances, but do not show differences in terms of yield. The development of new genotypes accumulating a lower amount of proteins potentially or effectively involved in allergies to wheat and NCWS, not only offers the possibility to use them as a basis for the production of varieties with a lower impact on adverse reaction, but also to test if these proteins are actually implicated in those pathologies for which the triggering factor has not been established yet.