In vitro model using cytokine cocktail to evaluate apoptosis in Min6 pancreatic beta cells.

INTRODUCTION: Development of therapy options for treatment of type 1 diabetes mellitus is hampered by non-availability of appropriate experimental models that can exactly mimic the in vivo situation. Apoptosis of beta cells by T cells and cytokine action leads to loss of beta cells. We propose a simple and elegant model using cytokine cocktail of TNF-α, IFN-γ and IL-1ß, the major cytokines responsible for apoptosis in Min6 beta cell line. METHODS: A cocktail of TNF-α, IFN-γ and IL-1ß was used to induce apoptosis in Min6 beta cell line. Apoptosis was assessed by flow cytometry using CytoFLEX (Beckman Coulter). The destruction of beta cells is through production of nitric oxide (NO), oxidative stress and change in mitochondrial membrane permeability. NO was measured using Griess reagent. Oxidative stress was assessed using 2',7'-dichlorofluorescein diacetate, a cell-permeable fluorogenic dye and mitochondrial membrane potential was determined on the basis of retention of rhodamine 123 using flow cytometer. RESULTS AND DISCUSSION: Very low concentration of the cocktail viz. TNF-α 25 ng/ml, IFN-γ 25 ng/ml and IL-1ß 50 ng/ml has demonstrated effective early and late apoptosis in as short a time period as 6 h. The experimental model used demonstrated 1.5 fold higher production of NO, 1.2 fold increased oxidative stress and lower mitochondrial membrane potential as compared to the positive control used. Hence the above model can be easily used for assessment and screening of drugs that can prevent apoptosis of beta cells and stop progression of type 1 diabetes.

Biofilm inhibition mechanism from extract of Hymenocallis littoralis leaves.

ETHNOPHARMACOLOGICAL RELEVANCE: Hymenocallis littoralis (Jacq.) Salisb. has been referred as beach spider lily and commonly known for its rich phytochemical diversity. Phytochemicals such as alkaloids, volatile constituents, phenols, flavonoids, flavonols extracted from different parts of these plants like bulbs, flowers, leaf, stem and root had been used in folk medicines from ancient times because of their excellent antimicrobial and antioxidant properties. The leaf and bulb extract of H. littoralis plant was traditionally used for wound healing. Alkaloids extracted from bulb of this plant possess anti-viral, anti-neoplastic and cytotoxic properties. However, these phytochemicals have also shown antibiofilm activity, which is considered as one of the important factor accountable for the drug resistance in microorganisms. Thus, the investigation of medicinal properties of H. littoralis could be useful to control biofilm producing pathogens. AIM OF THE STUDY: Explore antimicrobial, antibiofilm and antioxidant potentials of H. littoralis against pathogenic microorganisms using experimental and computational biology approach. MATERIALS AND METHODS: Phytochemical extraction from dried powder of H. littoralis leaves was done by solvent extraction using methanol. Antimicrobial and antibiofilm activities of leaves extract were carried out using agar well diffusion method, growth curve, minimum inhibitory concentration (MIC) and Scanning Electron Microscopy (SEM). Liquid Chromatography and Mass Spectroscopy (LCMS) technique was used for the identification of phytochemicals. Molecular docking studies of antibiofilm agents with adhesin proteins were performed using Autodock 4.2. Antioxidant activity of extract was carried out by FRAP assay. The noxious effect of extract was investigated by histological studies on rat skin. RESULTS: The preliminary phytochemical analysis of methanolic leaves extract revealed the presence of alkaloids, flavonoids, terpenoid, glycosides, terpene, terpenoids and phenolics. The various phytochemicals such as Apigenin 7-(4'', 6'' diacetylalloside)-4'- alloside, Catechin 7-O- apiofuranoside, Emodic acid, Epicatechin 3-O- ß-D-glucopyranoside, 4 - Methylesculetin, Methylisoeugenol, Quercetin 5,7,3',4'-tetramethyl ether 3-rutinoside, 4 - Methylumbelliferyl ß-D- glucuronide were extracted, characterized and recognized from the leaves extract of H. littoralis. The identification of these phytochemicals was performed using LC-MS. The antimicrobial property of H. littoralis leaf extract was investigated against different pathogenic microorganisms. Out of these tested microorganisms, promising antibiofilm and antimicrobial activities were confirmed against S. aureus NCIM 2654 and C. albicans NCIM 3466 by using growth curve and SEM analysis. MIC of this leaf extract was identified as 45 µg/ml and 70 µg/ml for S. aureus NCIM 2654 and C. albicans NCIM 3466 respectively. The leaves extract also showed good antioxidant activity due to presence of phenols and flavonoids. Molecular docking of these identified antibiofilm components interacts with the active site residues of adhesin proteins, Sortase A and Als3 from S. aureus and C. albicans respectively. Histological studies of extracted phytochemicals revealed non-noxious effects on rat skin. CONCLUSION: Thus, the present study revealed that the leaves extract of H. littoralis contains various phytochemicals having good extent of antimicrobial, antibiofilm and antioxidant properties. The in-vitro and in-silico results would be useful to design new lead compounds against biofilm producing pathogenic microorganisms.