Production optimization and antioxidant potential of exopolysaccharide produced by a moderately halophilic bacterium Virgibacillus dokdonensis VITP14.

This study aimed to enhance the extracellular polymeric substances (EPS) production of Virgibacillus dokdonensis VITP14 and explore its antioxidant potential. EPS and biomass production by VITP14 strain were studied under different culture parameters and media compositions using one factor at a time method. Among different nutrient sources, glucose and peptone were identified as suitable carbon and nitrogen sources. Furthermore, the maximum EPS production was observed at 5% of inoculum size, 5 g/L of NaCl, and 96 h of fermentation. Response surface methodology was employed to augment EPS production and investigate the optimal levels of nutrient sources with their interaction. The strain was observed to produce actual maximum EPS of about 26.4 g/L for finalized optimum medium containing glucose 20 g/L, peptone 10 g/L, and NaCl 50 g/L while the predicted maximum EPS was 26.5 g/L. There was a nine fold increase in EPS production after optimization study. Additionally, EPS has exhibited significant scavenging, reducing, and chelating potential (>85%) at their higher concentration. This study imparts valuable insights into optimizing moderately halophilic bacterial EPS production and evaluating its natural antioxidant properties. According to findings, V. dokdonensis VITP14 was a promising isolate that will provide significant benefits to biopolymer producing industries.

Inhibition of the Naja naja venom toxicity by polymeric nanoparticles loaded with Leucas aspera methanolic extract.

Background: Snakebite is a neglected tropical disease that affects millions of people worldwide. Developing effective treatments can make a significant contribution to global health efforts and public health initiatives. To reduce mortality due to snakebite, there is an immediate need to explore novel and effective treatment methodologies. In that context, nanoparticle-based drug delivery is gaining a lot of attention. Hydrophilic nanoparticles are suitable for the delivery of therapeutic peptides, proteins, and antigens. Methods: The present investigation is aimed at evaluating the anti-ophidian potential of the methanolic extract of the ethno-medicinal herb Leucas aspera (Willd.) loaded within chitosan nanoparticles (CNP-LA), against the Indian cobra (Naja naja) venom enzymes. For this purpose, nanoparticles were prepared using the ionic gelation method to enhance the efficacy of the extract. The physicochemical and structural features of nanoparticles were investigated using dynamic light scattering (DLS), Fourier-transform Infrared (FTIR), field emission scanning electron microscopy (FE-SEM), and X-ray diffraction (XRD) techniques. Results: It was found that CNP-LA has an average size of 260 nm with a polydispersity index of 0.132 (PDI) and zeta potential of 34.7 mV, with an encapsulation efficiency of 92.46%. The in vitro release study was performed at pH 5.0 and 7.4. Furthermore, in vitro studies indicated that CNP-LA inhibited the phospholipase A2, hemolytic, and caseinolytic activities of Naja naja venom with the percentage inhibition of 92.5%, 83.9%, and 94.5%, respectively. Conclusion: This is the first report on the application of herbal methanolic extract loaded within chitosan nanoparticles for neutralizing snake venom enzymes with increased efficiency.

Molecular Characterization and Biocompatibility of Exopolysaccharide Produced by Moderately Halophilic Bacterium Virgibacillus dokdonensis from the Saltern of Kumta Coast.

The use of natural polysaccharides as biomaterials is gaining importance in tissue engineering due to their inherent biocompatibility. In this direction, the present study aims to explore the structure and biocompatibility of the EPS produced by Virgibacillus dokdonensis VITP14. This marine bacterium produces 17.3 g/L of EPS at 96 h of fermentation. The EPS was purified using ion exchange and gel permeation chromatographic methods. The porous web-like structure and elemental composition (C, O, Na, Mg, P, S) of the EPS were inferred from SEM and EDX analysis. AFM analysis revealed spike-like lumps with a surface roughness of 84.85 nm. The zeta potential value of −10 mV indicates the anionic nature of the EPS. Initial molecular characterization showed that the EPS is a heteropolysaccharide composed of glucose (25.8%), ribose (18.6%), fructose (31.5%), and xylose (24%), which are the monosaccharide units in the HPLC analysis. The FTIR spectrum indicates the presence of functional groups/bonds typical of EPSs (O-H, C-H, C-O-H, C-O, S=O, and P=O). The polymer has an average molecular weight of 555 kDa. Further, NMR analysis revealed the monomer composition, the existence of two α- and six ß-glycosidic linkages, and the branched repeating unit as → 1)[α-D-Xylp-(1 → 2)-α-D-Glcp-(1 → 6)-ß-D-Glcp-(1 → 5)]-ß-D-Frup-(2 → 2)[ß-D-Xylp-(1 → 4)]-ß-D-Xylp-(1 → 6)-ß-D-Fruf-(2 → 4)-ß-D-Ribp-(1 →. The EPS is thermally stable till 251.4 °C. X-ray diffraction analysis confirmed the semicrystalline (54.2%) nature of the EPS. Further, the EPS exhibits significant water solubility (76.5%), water-holding capacity (266.8%), emulsifying index (66.8%), hemocompatibility (erythrocyte protection > 87%), and cytocompatibility (cell viability > 80% on RAW264.7 and keratinocyte HaCaT cells) at higher concentrations and prolongs coagulation time in APTT and PT tests. Our research unveils the significant biocompatibility of VITP14 EPS for synthesizing a variety of biomaterials.

Virgibacillus dokdonensis VITP14 produces α-amylase and protease with a broader operational range but with differential thermodynamic stability.

Extracellular α-amylase and protease were coproduced from halo tolerant Virgibacillus dokdonensis VITP14 with banana peels (2% w/v) as substrate. The pH optima for α-amylase and protease were 6.5 and 7.0, respectively. The temperature optima of α-amylase and protease were 30 and 50 °C, respectively. Both the enzymes were active in the presence of various metal ions (1 mM of Ni2+ , Ca2+ , Ba2+ , Sr2+ , and Mg2+ ), detergents (Tween 20, Tween 80, Triton X-100), and other additives (2-mercaptoethanol and urea). Both the enzymes followed Michaelis-Menten type enzyme kinetics with Vmax of 121.40 and 4.17 µmol Min-1 mL-1 and Km of 0.59 and 0.28 mg mL-1 for amylase and protease, respectively. Amylase showed higher activation energy for inactivation (75.55 kJ mol-1 compared to 59.70 kJ mol-1 for protease) and higher thermal stability (reflected by longer half-life 53.23 Min compared to 0.11 Min for protease) at 60 °C. The coexistence of amylase and protease could be attributed to the difference in the optimum temperatures of activity and thermal stability of the two enzymes.

Metabolites from halophilic bacterial isolates Bacillus VITPS16 are cytotoxic against HeLa cells.

The present study was aimed at evaluating the cytotoxic potential of selected halophilic bacterial metabolites. The use of the metabolomics approach in identifying the unexplored bioactive metabolites from halophilic bacterial isolate reduces time and complex experiments. In our study, we used UV/Visible spectroscopy, LC-MS/MS, and NMR to identify the metabolites present in the methanolic extract of the halophilic bacterium Bacillus VITPS16. MTT assay revealed that metabolite fractions (S1-79.61% and S2-85.74%) possess cytotoxic activity. Colonogenic assay confirmed the cytotoxic potential of the fractions and apoptosis assays showed that 83.37% of the cells undergo apoptosis at 10 mg/mL concentration (MF-S2). The DNA binding studies revealed the metabolite fraction interacts with DNA resulting in cytotoxicity. The study states that MF- S2 induced an antiproliferative effect that led to apoptosis through DNA binding as one of the possible pathways. The toxicity analysis using zebrafish indicated that the metabolite fractions are non-toxic even at 10 mg/mL concentration. Fraction MF-S2 is found to contain phosphoethanolamines, glycerophospholipids, sphingolipids, apocarotenoid, enigmol and its analogue, ankaflavin and flavonoid type of metabolites, which have been previously reported to have anti-cancer activity. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02724-9.

Marine sulfated polysaccharides as potential antiviral drug candidates to treat Corona Virus disease (COVID-19).

The viral infection caused by SARS-CoV-2 has increased the mortality rate and engaged several adverse effects on the affected individuals. Currently available antiviral drugs have found to be unsuccessful in the treatment of COVID-19 patients. The demand for efficient antiviral drugs has created a huge burden on physicians and health workers. Plasma therapy seems to be less accomplishable due to insufficient donors to donate plasma and low recovery rate from viral infection. Repurposing of antivirals has been evolved as a suitable strategy in the current treatment and preventive measures. The concept of drug repurposing represents new experimental approaches for effective therapeutic benefits. Besides, SARS-CoV-2 exhibits several complications such as lung damage, blood clot formation, respiratory illness and organ failures in most of the patients. Based on the accumulation of data, sulfated marine polysaccharides have exerted successful inhibition of virus entry, attachment and replication with known or unknown possible mechanisms against deadly animal and human viruses so far. Since the virus entry into the host cells is the key process, the prevention of such entry mechanism makes any antiviral strategy effective. Enveloped viruses are more sensitive to polyanions than non-enveloped viruses. Besides, the viral infection caused by RNA virus types embarks severe oxidative stress in the human body that leads to malfunction of tissues and organs. In this context, polysaccharides play a very significant role in providing shielding effect against the virus due to their polyanionic rich features and a molecular weight that hinders their reactive surface glycoproteins. Significantly the functional groups especially sulfate, sulfate pattern and addition, uronic acids, monosaccharides, glycosidic linkage and high molecular weight have greater influence in the antiviral activity. Moreover, they are very good antioxidants that can reduce the free radical generation and provokes intracellular antioxidant enzymes. Additionally, polysaccharides enable a host-virus immune response, activate phagocytosis and stimulate interferon systems. Therefore, polysaccharides can be used as candidate drugs, adjuvants in vaccines or combination with other antivirals, antioxidants and immune-activating nutritional supplements and antiviral materials in healthcare products to prevent SARS-CoV-2 infection.

Optimization of extracellular lipase production by halotolerant Bacillus sp. VITL8 using factorial design and applicability of enzyme in pretreatment of food industry effluents.

The effect of macro and micronutrients of media components on lipase production by Bacillus sp. VITL8 was investigated using classical as well as statistical methods. Initially, the carbon source, nitrogen source, inducer and metal ions that affect lipase production were selected using the classical approach. Subsequently, selected nutrients along with other key factors (such as pH, agitation rate, gum acacia and tween 80) were investigated using Placket Burman design. Finally, three significant factors, viz., olive oil, peptone and tween 80 were studied using a 22 full factorial central composite design. Under optimized condition [6% (v/v) of olive oil, 0.7% peptone, 0.9% tween 80 and 25 h of incubation], the enzyme production was found to be 2.2 times higher with an overall enzyme production of 325.0 ± 1.4 U mL-1. Laboratory scale experiment proved that the enzyme could be utilized for pretreatment of food industry effluent rich in fat and oil (dairy, bakery and poultry). The enzyme was capable of hydrolyzing more than 50% of the initial fat present in all these effluents and enabled the reduction in the levels of biological oxygen demand (BOD) and chemical oxygen demand (COD) of the effluent samples. The study thus reveals the utility of the lipase produced by the halotolerant bacterium Bacillus sp. VITL8 in the pretreatment of industrial effluents contaminated with oil and fat.HighlightsLipase production was enhanced by 2.2-fold using statistical methodsOne of the few reports on lipase production by a halotolerant bacterium, especially by Bacillus sp.Production of 325.0 ± 1.4 U mL-1 lipase within 25 h by a halotolerant bacteriumPretreatment of food industry effluents using Bacillus sp. VITL8 lipaseImprovement in effluent quality within 8 h of treatment.

Nε-Acetyl L-α Lysine Improves Activity and Stability of α-Amylase at Acidic Conditions: A Comparative Study with other Osmolytes.

BACKGROUND: Nε-acetyl L-α lysine is an unusual acetylated di-amino acid synthesized and accumulated by certain halophiles under osmotic stress. Osmolytes are generally known to protect proteins and other cellular components under various stress conditions. OBJECTIVE: The structural and functional stability imparted by Nε-acetyl L-lysine on proteins were unknown and hence was studied and compared to other commonly known bacterial osmolytes - ectoine, proline, glycine betaine, trehalose and sucrose. METHODS: Effects of osmolytes on the temperature and pH profiles, pH stability and thermodynamic stability of the model enzyme, α-amylase were analyzed. RESULTS: At physiological pH, all the osmolytes under study increased the optimal temperature for enzyme activity and improved the thermodynamic stability of the enzyme. At acidic conditions (pH 3.0), Nε-acetyl L-α lysine and ectoine improved both the catalytic and thermodynamic stability of the enzyme; it was reflected in the increase in residual enzyme activity after incubation of the enzyme at pH 3.0 for 15 min by 60% and 63.5% and the midpoint temperature of unfolding transition by 11°C and 10°C respectively. CONCLUSION: Such significant protective effects on both activity and stability of α-amylase imparted by addition of Nε-acetyl L-α lysine and ectoine at acidic conditions make these osmolytes interesting candidates for biotechnological applications.

Structural features of microbial exopolysaccharides in relation to their antioxidant activity.

Oxidative damage caused by free radicals is an inevitable and pervasive phenomenon that leads to cell damage and the emergence of diseases including ageing, cancer, diabetes, cardiovascular disease and neurodegenerative disorders. In this context, antioxidants play a significant role in encountering free radicals by delaying or reducing the oxidative damage of cells. Evidence suggests that synthetic antioxidants are double-edged swords wherefore the requirement for natural antioxidants is increasing globally. Exploring non-toxic, biodegradable and compatible natural molecules like exopolysaccharides can favour the current antioxidant limitations. Microbial exopolysaccharides represent a structurally diverse class of carbohydrate molecules secreted at the cell wall. Recently, bioprospecting exopolysaccharides for their astounding physiochemical properties and the reliable structure-activity relationship have motivated more research towards the investigation of their antioxidant properties. Here we propose that structural features of exopolysaccharides such as monosaccharide residues, branching, molecular weight, glycosidic linkage, functional groups, protein, selenium, and chemical modifications are likely to influence their antioxidant activity. To support this hypothesis we review the interdependence of structural features of exopolysaccharides to the observed antioxidant activity. In light of its importance, this review focuses on the understanding of the elimination of free radicals by microbial exopolysaccharides derived from marine and nonmarine sources during the last six years.

Calcium ion induced thermodynamic stability, bisubstrate specificity, and differential organic solvent tolerance of a predominantly ß-sheet serine protease from Bacillus aquimaris VITP4.

The present study was aimed to get insights on the role of calcium ions on the thermodynamic stability, substrate specificity, and organic solvent compatibility of the extracellular protease produced by Bacillus aquimaris VITP4. Presence of Ca2+ enhanced the activity of the enzyme in the temperature range of 30-60 °C and increased the half-life from 164 to 234 Min. Circular dichroism experiments indicated that the temperature of half-denaturation (Tm ) of the protease increased from 76 to 86 °C. As judged by fluorescence emission profiles, the overall fold of the enzyme around the tryptophan residues could be similar. Further, thermal inactivation experiments revealed that the enzyme followed first order kinetics, with increase in energy for inactivation (Eai ) by 24.2 ± 1.2 kJ mol -1 in the presence of Ca2+ . Studies with synthetic peptides as well as with bovine serum albumin signified preferential hydrolysis of the peptide bonds at the C-terminus of alanine residues (with a kcat /KM of 141,400 M-1  Sec-1 ) and at the C-terminus of arginine residues with a lower specificity (72,400 M-1  Sec-1 ), indicating bisubstrate specificity of the enzyme. The enzyme was found to be compatible with organic solvents (50%, v/v) such as acetonitrile and butanol, indicating possible application under demanding nonaqueous conditions.

Evaluation of the anti-oxidant property and cytotoxic potential of the metabolites extracted from the bacterial isolates from mangrove Forest and saltern regions of South India.

The objective was to evaluate the anticancer and antioxidant activities of the methanolic extracts of halophilic bacteria, isolated from soil samples of Marakkanam saltern and Pichavaram mangrove forest, India. Radical Scavenging activity, reducing power, and metal ion chelation ability was used to evaluate the antioxidant potential of the metabolic extracts, whereas cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The methanolic extract of Bacillus VITPS7 exhibited significant antioxidant property. Bacillus VITPS14 and Bacillus VITPS16 extracts were cytotoxic against HeLa cell lines but not to A549 cell lines. Colorimetric assays for the presence of specific metabolites including, total flavonoid and ß carotene content were performed. The presence of these specific classes of metabolites was confirmed by UV-Visible spectrophotometry, Nuclear Magnetic Resonance (NMR) spectroscopy and Gas Chromatography-Mass Spectrometry (GC-MS). Specific NMR signals revealed the presence of aromatic and unsaturated metabolites whereas GC-MS analysis indicated the presence of metabolites such as squalene and methyl hexadeconate. The present study thus reports for the first time the presence of squalene in Bacillus VITPS12 and Planococcus maritimus VITP21, in addition to other metabolites that contribute to the observed antioxidant or/and cytotoxicity, thus revealing the therapeutic potential of these selected halophilic bacterial isolates.

In vitro inhibition potential of mono-n-octyl phthalate on Mycobacterium tuberculosis H37Ra: Possibility of binding to mycobacterial PknB-An in silico approach.

Fatty acids of specific chain lengths have been shown to inhibit the growth of Mycobacterium tuberculosis. In the present study, specific synthetic aromatic derivatives of n-octyl esters were investigated for their property to inhibit the growth of M. tuberculosis H37Ra. Agar well diffusion assay indicated that the crude synthetic derivatives obtained by the esterification of phthalic acid (PA) and n-octanol exhibited antimycobacterial activity. Further, the activity was authenticated with the Miroplate Alamar Blue Assay (MABA). Subsequently, the active component was purified by bioactivity guided chromatographic fractionation. The structure of the synthetic derivative was deduced by UV-Vis, FT-IR, LC-MS, GC-mass spectrometry, and NMR spectroscopy. Molecular docking and molecular dynamic simulation (MDS) were performed with Autodock 4.0 and GROMACS 5.1.2 softwares, respectively. It was found that mono-n-octyl phthalate (MOP) exhibited antimycobacterial activity with a MIC of 20 µg/mL, and not by any other related compounds, including di-n-octyl phthalate, PA, phthalic anhydride, and n-octanol. Binding of MOP with protein kinase B can participate in the binding cavity region, which was previously reported. Subsequently, we authenticate the stability with MDS. This is first report on the inhibition of M. tuberculosis growth by MOP.

Structural insights into the binding mode and conformational changes of BSA induced by bixin and crocin.

Bixin and crocin are natural apocarotenoids utilized as food colorants and additives in food industries worldwide. For safety assessment, it is necessary to understand the biological interaction of food colorants. In our present study, we report the interaction of two apocarotenoids with bovine serum albumin (BSA) at physiological pH using spectroscopic techniques and in silico tools. The binding constant and the mode of binding sites have been studied. The enthalpic and entropic contribution to the intermolecular binding event was analyzed and it was found that the contribution of hydrogen bonding and hydrophobic interactions was dominant. The adverse temperature dependence in the unusual static quenching is found to be a reasonable consequence of the large activation energy requirement in the binding process, which is required to overcome the fundamental block and is a direct result of the unique microstructure of the binding sites. To confirm the experimental analysis, we investigated the binding patterns using different in silico tools. A combination of molecular docking, molecular dynamics, and toxicity analysis was performed, and the obtained results revealed that both the apocarotenoids had high binding affinity with a binding energy of -5.44 and -5.93 kcal/mol for bixin and crocin, respectively, with no toxic effects and are in accordance with our experimental analysis. The results directly revealed the flexibility of the protein toward bixin and crocin which has a great impact on the interaction. Thus bixin and crocin can guardedly be used as food colorants in food industries.

Quercetin-3-O-rhamnoside from Euphorbia hirta protects against snake Venom induced toxicity.

BACKGROUND: The plant Euphorbia hirta is widely used against snake envenomations in rural areas and it was proved to be effective in animal models. Therefore, the scientific validation of its phytoconstituents for their antiophidian activity is aimed in the present study. METHODS: E. hirta extract was subjected to bioactivity guided fractionation and the fractions that inhibited different enzyme activities of Naja naja venom in vitro was structurally characterized using UV, FT-IR, LC-MS and NMR spectroscopy. Edema, hemorrhage and lethality inhibition activity of the compound were studied in mice model. In addition, molecular docking and molecular dynamic simulations were also performed in silico. RESULTS: The bioactive fraction was identified as Quercetin-3-O-α-rhamnoside (QR, 448.38 Da). In vitro experiments indicated that protease, phospholipase-A(2), hemolytic activity and hemorrhage inducing activity of the venom were inhibited completely at a ratio of 1:20 (venom: QR) w/w. At the same concentration, the edema ratio was drastically reduced from 187% to 107%. Significant inhibition (93%) of hyaluronidase activity was also observed at a slightly higher concentration of QR (1:50). Further, in in vivo analysis, QR significantly prolonged the survival time of mice injected with snake venom. CONCLUSION: For the first time Quercetin-3-O-α-rhamnoside, isolated from E. hirta, has been shown to exhibit anti-snake venom activity against Naja naja venom induced toxicity. GENERAL SIGNIFICANCE: Exploring such multifunctional lead molecules with anti-venom activity would help in developing complementary medicine for snakebite treatments especially in rural areas where anti-snake venom is not readily available.

Biochemical changes induced by salt stress in halotolerant bacterial isolates are media dependent as well as species specific.

Halophilic bacteria respond to salt stress by regulating the cytosolic pools of organic solutes to achieve osmotic equilibrium. In order to understand the metabolic regulation of these organic solutes, for the first time, we have investigated the effect of salt on growth and biochemical changes in four major moderately halophilic bacterial strains isolated from a saltern region of the Kumta coast, India. The strains under study were Halomonas hydrothermalis VITP9, Bacillus aquimaris VITP4, Planococcus maritimus VITP21, and Virgibacillus dokdonensis VITP14, which exhibited similar salt tolerance (0% to 10% w/v NaCl) with optimal growth at 5% w/v NaCl. Biochemical analysis showed that the total intracellular organic solutes increased significantly with increasing NaCl concentration in the growth medium, and the compositions of the solutes were dependent on the type of strain and also on the nutrient richness of the growth medium. Glutamic acid levels increased in all the strains under salt stress, indicating the significance of glutamic acid as the anionic counterpart of K(+)/Na(+) ions and precursor for other synthesized nitrogenous osmolytes. Though initial studies were performed with thin-layer chromatography, mass spectrometry was used to identify the major solutes accumulated by the strains under salt stress, such as proline (VITP4), ectoine (VITP14 and VITP9), and sugars (VITP21) under minimal medium and glycine betaine (by all the strains under study) under complex growth medium conditions. Such comparative study on the stress-dependent metabolic differences of different microbes, under identical experimental condition, helps to identify possible bacterial sources for the production of industrially important solutes.

Optimization of Parameters that Affect the Activity of the Alkaline Protease from Halotolerant Bacterium, Bacillus acquimaris VITP4, by the Application of Response Surface Methodology and Evaluation of the Storage Stability of the Enzyme.

BACKGROUND: It was previously shown that the activity of a serine protease from a moderately halotolerant Bacillus aquimaris VITP4 strain is active in a wide range of pH and temperatures and could be modulated by the presence of the divalent metal ions. OBJECTIVES: In the present study, a quantitative analysis was done in order to explore the parameters that are contributing to the protease activity. MATERIALS AND METHODS: Changes in the secondary structure of the enzyme was determined by circular dichroism analysis. The conditions for the optimal activity was investigated by Response Surface Methodology. Stability of the enzyme was determined by thermal inactivation experiments. RESULTS: The initial one-factor-at-a-time experiments have indicated that the activity of the enzyme could be enhanced not only by the presence of low concentrations of NaCl but also by divalent metal ions, such as Ca2+, Mn2+ and Cu2+. A clear dependence of the activity to the secondary structure of the enzyme could be established using circular dichroism spectroscopy. In the next level of optimization, four factors; viz. pH, temperature, concentration of Ca2+, and Mn2+ were used to optimize the conditions required for the maximal activity of the enzyme by Response Surface Methodology, and the data could be explained using quadratic model. Under optimal condition of 43°C, pH 8.0, 8.2 mM Ca2+, and 4.3 mM Mn2+ a 1.5 times enhancement in the enzyme activity could be achieved. The storage stability of the enzyme under these selected conditions has indicated a non-linear relation between the conditions for the enzymatic activity as well as stability. However, the condition for the maximal stability (267±18 min) has corresponded to that of the optimal conditions for the maximal activity. CONCLUSIONS: This study, for the first time, has explored the possibility of using statistical methods for identifying the optimal conditions for alkaline protease activity isolated from the halotolerant Bacillus aquimaris VITP4.

Protective effect of Euphorbia hirta and its components against snake venom induced lethality.

ETHNOPHARMACOLOGICAL RELEVANCE: Despite the use of snake anti-venom therapy, herbal medicine is still in practice to treat snakebites. Euphorbia hirta is traditionally used as antidote for snakebites and also for numerous other ailments. However, the scientific evidence for its anti-snake venom property is still lacking. MATERIALS AND METHODS: Methanolic extract of E. hirta was evaluated for anti-venom activity under in vitro and ex vivo conditions. Histopathological changes in the vital organs of the mice were also monitored. UHPLC-SRM/MS was used to estimate the phenolic constituents whereas GC-MS analysis was performed to analyze the volatile metabolites present. The major compound was further evaluated for its contribution to the overall inhibitory potential of the extract. RESULTS: Methanolic extract of E. hirta completely inhibited the venom enzymes under in vitro and reduced the edema ratio. The extract increased the survival time (>24h) of mice which was further evidenced by histopathological analysis of vital organs. Phytochemical analysis revealed higher content of phenolic (144 mg/g extract) compounds in the extract. UHPLC-SRM/MS demonstrated that ellagic acid, gallic acid and quinic acid are the major phenolics whereas GC-MS analysis revealed pyrogallol as the major constituent (60.07%) among the volatile components of the extract. It was also shown that pyrogallol has the ability to differentially inhibit venom protease but not phospholipase A2. CONCLUSION: The present study confirmed that E. hirta methanolic extract was able to completely inhibit Naja naja venom induced toxicity under in vitro as well as ex vivo conditions, thus providing scientific evidence to its traditional use.

Biophysical characterization and molecular docking studies of imidazolium based polyelectrolytes-DNA complexes: role of hydrophobicity.

Nonviral gene delivery vectors are acquiring greater attention in the field of gene therapy by replacing the biological viral vectors. DNA-cationic polymer complexes are one of the most promising systems to find application in gene therapy. Hence, a complete insight of their biophysical characterization and binding energy profile is important in understanding the mechanism involved in nonviral gene therapy. In this investigation, the interaction between calf thymus DNA (ctDNA) and imidazolium-based poly(ionic liquids) (PILs) also known as polyelectrolytes with three different alkyl side chains (ethyl, butyl, and hexyl) in physiological conditions using various spectroscopic experiments with constant DNA concentration and varying polyelectrolyte concentrations is reported. UV-visible absorption, fluorescence quenching studies, gel electrophoresis, circular dichroism (CD), and Fourier transform infrared spectroscopy (FTIR) have confirmed the binding of polyelectrolytes with DNA. UV-vis absorption measurements and fluorescence quenching revealed that the binding between DNA and the polyelectrolyte is dominated by electrostatic interactions. Additionally, CD and FTIR results indicated that the DNA retained its B-form with minor perturbation in the phosphate backbone without significant change in the conformation of its base pairs. Preference for alkyl side chains (K(PIL-Ethyl Br) < K(PIL-Butyl Br) < K(PIL-Hexyl Br)) toward efficient binding between the polyelectrolyte and DNA was inferred from the binding and quenching constants calculated from the absorption and emission spectra, respectively. Further, in silico molecular docking studies not only validated the observed binding trend but also provided insight into the binding mode of the polyelectrolyte-DNA complex.

Formulation and characterisation of antibody-conjugated soy protein nanoparticles--implications for neutralisation of snake venom with improved efficiency.

The present study reports the formulation of soy protein nanoparticles and its conjugation to antivenom. The conditions for nanoparticle formation were optimised by considering particle size, count rate, stability and zeta potential. The smallest particle size of 70.9 ± 0.9 nm with a zeta potential of -28.0 ± 1.4 mV was obtained at pH 6.2, with NaOH 5.4 % and 28 µg/mg glutaraldehyde. The nanoparticle was conjugated with antisnake venom immunoglobulins (F(ab')2 fragments) using 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide. TEM analysis indicated the increased size of particle to 600 nm after conjugation to antivenom. Further, in vitro studies indicated that conjugated antibodies inhibited the activity of protease, phospholipase and hyaluronidase enzymes of Bungarus caeruleus venom more efficiently than the free antivenom. This is the first report on the use of protein nanoparticles for conjugating snake venom antibodies and their implications for neutralising snake venom enzymes with increased efficiency.

Metal ion activated lipase from halotolerant Bacillus sp. VITL8 displays broader operational range.

Lipase producing halo tolerant Bacillus sp. VITL8 was isolated from oil contaminated areas of Vellore. The identity of the organism was established by 16S rDNA sequence, in addition to the morphological and biochemical characterization. The purified enzyme (22kDa, 8680U/mg) exhibited optimal activity at pH 7.0 and 40°C and retained more than 50% of its activity in the NaCl concentration range of 0-3.0M, pH 6.0-10.0 and 10-60°C. Secondary structure analysis, using circular dichroism, revealed that the enzyme is composed of 38% α-helix and 29% ß-turns. The lipase activity significantly increased in the presence of (1mM) Mn(2+) (139%), Ca(2+) (134%) and Mg(2+) (130%). Organic solvents such as butanol and acetonitrile (25%, v/v) enhanced the activity whereas DMSO (25% v/v) retained the activity. The Km of enzyme-p-Nitrophenyl palmitate complex was determined to be 191µM with a Vmax of 68µM/mg/min. Though halotolerant Bacillus sp. has been explored for hydrocarbon degradation, to our knowledge this is the first report on the lipase activity of the isolate. The characteristics of the enzyme presented in this report, imply broader operational range of the enzyme and therefore could be suitable for many of the industrial chemical processes.