Bio prospecting of Riboflavin producing bacteria from different riboflavin enriched food sources

Riboflavin is an essential, water-soluble vitamin (B2) and a component of basic cellular metabolism. The aim of the present study is to isolate and characterize riboflavin producing bacteria from different food sources. Ten different riboflavin enriched food sources were collected from Vellore district. Totally 72 bacterial strains were isolated and cultured on nutrient agar plates. Out of these, 43 strains were identified as riboflavin producers. Isolated bacterial strains HDS27, HDS07, HDS14, HDS18, HDS38 and HDS54 isolated from milk, mushroom, spinach, lamb kidney, beef liver and mackerel fish were found to be potent riboflavin producers. Based on morphological, biochemical and molecular characterization, the potent strains were identified as Lactobacillus plantarum (HDS27), Bacillus cereus (HDS07), Delftia tsuruhatensis (HDS14), Citrobacter freundii (HDS18), Enterobacter cloacae (HDS38) and Bacillus cereus (HDS54). The selected potent isolates HDS27 from milk and HDS07 from mushroom showed a maximum riboflavin production of 3.69 mg/L and 2.9mg/L respectively. The present study explores the riboflavin producing novel bacteria from different food sources. This is the first report that the Enterobacter cloacae isolated from beef liver, Delftia tsuruhatensis from spinach and Citrobacter freundii from lamb kidney has the ability to produce riboflavin. These potent strains could be a better starter for substituting the conventional bacteria for large scale production of riboflavin in industry.

Fibrinolytic and ACE Inhibitory Activity of Nattokinase Extracted from Bacillus subtilis VITMS 2: A Strain Isolated from Fermented Milk of Vigna unguiculata.

The current research work aims at optimization, production, purification and evaluation of fibrinolytic extracellular protease from Bacillus subtilis VITMS2 isolated from fermented milk of Vigna unguiculata. The optimal production was achieved at 4.0% inoculum, pH7.0, 30 °C with (1% w/v) sucrose, (2% w/v) soya bean meal and (2% w/v) malt extract and 10 mM of CaCl2, MgSO4, Na2HPO4 and K2HPO4. The clear cell-free supernatant was purified using conventional ammonium sulphate salt fractionation (75%), ultrafiltration, ion-exchange (DEAE Sepharose FF) and gel filtration (Sephadex G-50). The molecular mass was determined to be 29 kDa using SDS-PAGE analysis. The purified enzyme showed strong fibrinolytic activity with a specific activity of 2418.85 U/mg and has a yield of 12.01%. The enzyme was highly stable up to 60 °C and a pH range of 10.0 until 72 h of incubation. The purified enzyme showed 97.4% in vitro thrombolytic activity. The Km and Vmax values of the enzyme was determined to be 0.0114 mM and 147.8 µmol min-1 using the chromogenic substrate S-7388. IC50 of ace inhibition was assessed to be 0.06 mg/mL suggesting anti-hypertensive property of the fibrinolytic enzyme. The above-obtained ace-inhibition results was supported by in silico molecular docking studies which revealed better binding affinity of nattokinase with a HADDOCK score of - 22.0 ± 8.5 confirms affinity towards angiotensin converting enzyme.

A review on interleukins: The key manipulators in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease associated with weakening of bones and joint pain. It primarily involves autoimmunity, matrix destruction, osteoclastogenesis, inflammation, and angiogenesis. Numerous cellular and humoral components of the immune system are involved in the etiology of diseases; however, the cardinal part is played by the inter-cellular signaling messengers called cytokines. Interleukins is a vaguely defined sub-class of cytokines that are abundantly found in the RA patients. The multifariousness and diversity in the function of the interleukins make them very likely to be associated with the pathogenesis in multiple ways. Nonetheless, the variety in opinions of researchers globally has led to contentious inferences. Ergo, in this review we have amalgamated the views of researchers from the past two decades till date to provide a comprehensive report about the role of interleukins in rheumatoid arthritis.

In vitro Thrombolytic Potential of Actinoprotease from Marine Streptomyces violaceus VITYGM.

Back ground: One of the most prevailing diseases that required effective drug as therapeutic purposes is cardiac related illness includes myocardial infarction Current scenario makes enzyme to raises their sector towards therapeutic as effective active component for such diseases. OBJECTIVES: The main aim of the study was to isolate, screen, characterize and produce an extracellular thrombolytic protease from marine actinomycetes. METHODS: Marine actinomycete was isolated and characterized on the basis of morphological, biochemical, and molecular characterization. The primary screening for protease activity was done by casein hydrolysis method followed by radial caseinolytic assay. The actinoprotease was partially purified using ammonium sulfate precipitation technique followed by dialysis and Sephadex G-50 gel permeation chromatography. RESULTS: 16srDNA sequencing and BLAST search analysis of the sequence revealed close affiliation with Streptomyces genera and identified as Streptomyces violaceus VITYGM with 99% similarity. The specific activity of purified protease was found to be 1437 units/mg along with purification fold up to 1.5 times. The blood clot lysis activity was compared with the standard and found to lyse the blood clot with 97.43%. Till now very less evidences have been reported on actinoprotease. A single peak at retention time 0.9 min observed on high performance liquid chromatography (HPLC) confirmed the homogeneity of the preparation. CONCLUSION: This is the first study to report on actinoprotease from Streptomyces violaceus VITYGM. This study emphasizes the potency of novel actinoproteases as active compound in drugs for the treatment of cardiovascular diseases.

In vitro thrombolytic potential of bioactive compounds from marine Streptomyces sp. VITJS4 / Potencial trombolítico in vitro de compostos bioativos de Streptomyces marinhos sp. VITJS4

The most practical approach to reduce morbidity and mortality of coronary heart disease (CHD) is to delay the process of thrombus by usage of clot-dissolving agents. The necessities of such safer compounds are to be critically examined for thrombolytic activity especially, from marine sources. Thrombolytic agents have been investigated as a possible treatment for thrombus. The aim of this study was to investigate the in vitro thrombolytic potential of Streptomyces sp.VITJS4 (NCIM No. 5574); (ACC No: JQ234978.1) active compounds. The fibrin degradation revealed a clear transparent zone of clearance with 500µg/mL concentration showing 24mm hydrolysis. The thrombolytic effect of Streptomyces sp.VITJS4 compounds was also demonstrated in vitro clot lysis assay where The percent of thrombolysis by the crude extract showed 90±1.7% at the concentration of 1000µg/mL, whereas percent of thrombolysis by streptokinase was found 100± 00%%. The bioactive compounds were further studied for spectrophotometric analysis. The UV-VIS profile showed different peaks ranging from 400-700 nm with different absorption respectively. The data confirmed the presence of both analogues with absorption maxima at 210 and 310 nm. A sensitive method using LC-MS technique was optimized for the separation and identification of bioactive metabolites which was indicated by the fingerprints. The results of the LC-MS analysis provided different peaks determining the presence of compounds with different therapeutic activities. The current study refers the bioactive compound as impressive thrombolytic agent for further laboratory study. Further studies should be conducted to ensure the efficacy and safety of different concentration of bioactive compounds for drug development. Hence the results reported perhaps useful for the discovery of novel thrombolytic drugs from marine origin.

A abordagem mais prática para reduzir a morbidade e a mortalidade da doença arterial coronariana (CHD, do inglês coronary heart disease) consiste em retardar o processo de trombo através da utilização de agentes de dissolução de coágulos. As necessidades de tais compostos mais seguros devem ser criticamente examinadas para a atividade trombolítica, especialmente de fontes marinhas. Agentes trombolíticos tem sido estudados como um possível tratamento para o trombo. O objetivo deste estudo foi investigar o potencial trombolítico in vitro dos compostos ativos do Streptomyces sp.VITJS4 (NCIM No. 5574); (ACC No: JQ234978.1). A degradação da fibrina revelou um clara zona livre transparente com concentração de 500µg/mL mostrando uma hidrólise de 24mm. O efeito trombolítico dos compostos de Streptomyces sp.VITJS4 também foi demonstrado no ensaio in vitro de lise dos coágulos em que a percentagem de trombólise pelo extrato bruto mostrou 90±1.7% a uma concentração de 1000µg/mL, enquanto que a percentagem de trombólise pela estreptoquinase foi de 100± 00%. Os compostos bioativos foram estudados posteriormente através da análise espectrofotométrica. O perfil ultra violeta visível (UV-VIS profile, em inglês) mostrou diferentes picos variando entre 400-700 nm com diferentes absorções respectivamente. Os dados confirmaram a presença de ambos os análogos com absorção máxima em 210 e 300 nm. Um método sensível usando a técnica LC-MS (Liquid chromatography­mass spectrometry) foi otimizado para a separação e identificação metabólitos bioativos que foram indicados pelas impressões digitais (?). Os resultados da análise LC-MS forneceram diferentes picos determinando a presença de compostos com diferentes atividades terapêuticas. O estudo atual refere-se ao composto bioativo como um agente trombolítico impressionante para futuros estudos em laboratório. Estudos futuros devem ser conduzidos para assegurar a eficácia e segurança de diferentes concentrações dos compostos bioativos para o desenvolvimento de drogas. Assim, os resultados reportados talvez sejam úteis para a descoberta de novas drogas trombolíticas de origem marinha.

Binding and molecular dynamic studies of sesquiterpenes (2R-acetoxymethyl-1,3,3-trimethyl-4t-(3-methyl-2-buten-1-yl)-1t-cyclohexanol) derived from marine Streptomyces sp. VITJS8 as potential anticancer agent.

The main aim of the current study is to explore the bioactive potential of Streptomyces sp. VITJS8 isolated from the marine saltern. The cultural, biochemical, and morphological studies were performed to acquire the characteristic features of the potent isolate VITJS8. The 16Sr DNA sequencing was performed to investigate the phylogenetic relationship between the Streptomyces genera. The structure of the compound was elucidated by gas chromatography-mass spectrometry (GC-MS), infra-red (IR), and ultra-violet (UV) spectroscopic data analysis. The GC-MS showed the retention time at 22.39 with a single peak indicating the purity of the active compound, and the molecular formula was established as C14H9ONCl2 based on the peak at m/z 277 [M](+). Furthermore, separated by high-performance liquid chromatography (HPLC), their retention time (t r) 2.761 was observed with the absorption maxima at 310 nm. The active compound showed effective inhibitory potential against four clinical pathogens at 500 µg/mL. The antioxidant activity was found effective at the IC50 value of 500 µg/mL with 90 % inhibition. The 3-(4,5-dimethylthiazol-2-yl)-2,5-ditetrazolium bromide (MTT) assay revealed the cytotoxicity against HepG2 cells at IC50 of 250 µg/mL. The progression of apoptosis was evidenced by morphological changes by nuclear staining. The DNA fragmentation pattern was observed at 250 µg/mL concentration. Based on flow cytometric analysis, it was evident that the compound was effective in inhibiting the sub-G0/G1 phase of cell cycle. The in vitro findings were also supported by the binding mode molecular docking studies. The active compound revealed minimum binding energy of -7.84 and showed good affinity towards the active region of topoisomerase-2α that could be considered as a suitable inhibitor. Lastly, we performed 30 ns molecular dynamic simulation analysis using GROMACS to aid in better designing of anticancer drugs. Simulation result of root mean square deviation (RMSD) analysis showed that protein-ligand complex reaches equilibration state around 10 ns that illustrates the docked complex is stable. We propose the possible mechanism of sesquiterpenes to play a significant role in antitumor cascade. Hence, our studies open up a new facet for a potent drug as an anticancer agent.

NGA- A Novel Hypothetical Drug Model: Combinatorial Approach to Multifactorial Alzheimer's Disease.

Alzheimer's disease (AD) is supposed to stanch from inappropriate waving in the brain sections related to memory and perception. The incidence of AD in distressed person associated with an upsurge in the accumulation of amyloid plaque-rich senile plaques and neurofibrillary tangles in the brain. We hypothesize that a combination therapy provides a new treatment for AD. We propose that an anti-AD drug, NGA, a combination of NSAIDS, Galanthamine and ACS-40 may be useful in preventing the formation of amyloid plaques from ß-amyloid. Being a widespread incurable disease, the treatment for Alzheimer's has been at the forefront of the medical research work. We propose a novel drug-like NGA will allow for the effective control and treatment of the progression of AD by preventing acetylcholinesterase activity and reducing plaque formation that forms the distinctive symptom for the identification of the onset of AD. A combinatory use of NSAID with a natural neurotransmitter will allow for an efficient control of amyloid beta toxicity and will open doors for the treatment of a myriad of other neurodegenerative diseases.

Fibrinolytic Actinokinase--A Short Review.

The prevalence of cardiovascular disease is one of the major causes of overall mortality. It kills almost 18-19 million individuals annually. There are a number of synthetic drug departures but the major effects are hemorrhagic impact, immunogenicity, and high price, due to restricted applications. Actinomycetes are the most economically and biotechnologically valuable prokaryotes. They are known to be responsible for the production and successful exploitation as a source of secondary metabolites, and are found to be abundant and active in marine sediments. Natural thrombolytic drugs are increasingly reported as safer, more fascinating and less costly. Actinokinase is a serine protease which cleaves α-chain, ß-chains and γ-chains of fibrinogen. Hence, such mechanistic property makes actinokinase an interesting feature. These microbial fibrinolytic proteases are used for therapeutic approach of medical interest and have biotechnological applications to treat cardiovascular diseases.

Exploring the Anticancer Activity of Grape Seed Extract on Skin Cancer Cell Lines A431

In this study, grape seeds were extracted using ethyl acetate and petroleum ether by solvent-solvent extraction method. The phytochemical tests were performed to identify different phytochemical compounds present in the grape seed extract (GSE). Antibacterial activity of the GSE was determined using agar diffusion method against Gram- positive and Gram-negative bacteria. Gas chromatography-mass spectrometry (GC-MS) and Fourier transform infrared spectroscopy (FTIR) analysis was done to identify the presence of bioactive compounds and their functional groups. The GC-MS results revealed a total of four compounds, known to have potent activity against cancer cells, viz, squalene, the most potent compound found in ethyl acetate extract and diethyl phthalate, ethyl-9- cis -11- trans octadecadienoate and (R)-(-)-14,-methyl-8-Hexadecyn-1-ol in petroleum ether extract. Cytotoxic activity of the GSE was observed against skin cancer cell lines A4321 using 3-(4, 5-dimethylthiazol-2-yl)-2-5-diphenyl tetrazolium bromide) MTT assay. The IC50 value of the GSE against A431 skin cancer cell line was 480 µg/mL. This is first such report against A4321 cell lines. The study gives the overall perception about importance of GSE in medicine and nutraceuticals purposes.

Production and partial purification of antifungal chitinase from Bacillus cereus VITSD3

The current work was designed to isolate and characterize chitin degrading bacteria. Among the 55 bacterial colonies isolated from 7 different soil samples, 4 isolates were capable of degrading chitinase, among which one strain VITSD3 was found to be potent. Based on the morphological, biochemical and molecular characterization of VITSD3 the isolate was confirmed as Bacillus cereus (Genbank accession number: KC961638), designated as Bacillus cereus VITSD3. The crude enzyme had a total activity of 220 U, precipitated with 44.8 U and 22.5 U for dialysed sample. The hydrolysed product NAG (N-Acetyl Glucosamine) from chitin was analysed by high-pressure liquid chromatography (HPLC).The molecular weight of the chitinase was determined using SDS PAGE and found to be 55 kDa. The partially purified chitinase produced from Bacillus cereus VITSD3 showed antifungal activity against Aspergillus fumigatus (18 mm), Aspergillus niger (6 mm) and Aspergillus flavus (15 mm). Hence the investigation suggests a potential benefit of partially purified chitinase extracted from Bacillus cereus VITSD3 which will serve as an excellent antifungal potential with therapeutic use.

O presente trabalho atual foi delineado para isolar e caracterizar bactérias degradadoras de quitina. Entre as 55 colónias bacterianas isoladas a partir de 7 amostras de solo diferentes, quatro isolados foram capazes de degradar quitinase, entre os quais uma estirpe, VITSD3, mostrou-se potente. Com base na caracterização morfológica, bioquímica e molecular de VIT D3 a soluto foi confirmada como Bacillus cereus (número de acesso Genbank: KC961638), designada como Bacillus cereus VITSD3. A enzima bruta tinha uma actividade total de 220 L, precipitou-se com 44,8 L e 22,5 L de amostra dialisada. O produto hidrolisado NAG (N-acetil-glucosamina) a partir de quitina foi analisado por cromatografia líquida de alta pressão (HPLC) .O peso molecular da quitinase foi determinado, utilizando-se SDS-PAGE e verificou-se ser 55 kDa. A quitinase parcialmente purificada produzida a partir de Bacillus cereus VITSD3 mostrou actividade antifúngica contra Aspergillus fumigatus (18 mm), Aspergillus niger (6 mm) e Aspergillus flavus (15 mm). Por isso, a investigação sugere um potencial benefício de quitinase parcialmente purificada extraída de Bacillus cereus VITSD3 o que poderá servir como um excelente potencial antifúngico para uso terapêutico.

Antimicrobial, Antioxidant and Cytotoxic Activity of Marine Streptomyces parvulus VITJS11 Crude Extract

The main aim of the study was to evaluate the bioactive properties of ethyl acetate crude extract of Streptomyces parvulus VITJS11 with a view to assess their therapeutic potential. The biological activity of ethyl acetate extract was tested against fungal and bacterial pathogens. The free radical scavenging potential of the crude extract was determined by DPPH assay. The chemo preventive properties of S. parvulus VITJS11 ethyl acetate extract was examined by MTT assay on HepG2 cells. The morphological, physiological and the biochemical properties of the strain S. parvulus VITJS11 was confirmed by conventional methods. Genotypic characterization was done using 16S r-DNA partial gene amplification and sequencing. The authenticity of the crude chemical constitutes were determined by the GC-MS. The ethyl acetate extract of VITJS11 showed maximum antifungal activity against three Aspergillus species and prominent antibacterial activity against two Gram positive and Gram negative bacteria at 20 mg/mL. The antioxidant potential of the crude extract exhibited strong reducing power activity at 5mg/ mL with 85% inhibition and the cytotoxic effect was found with IC50 of 500µg/ mL on HepG2 cell lines. The GC-MS analysis and the chromatogram patterns revealed 16 peaks, indicating the presence of bioactive constituents, which included several important organic compounds, namely 9-(2',2'-dimethylpropanoilhydrazono)-3,6-dichloro-2,7-Bis-[2-(diethylamino)-ethoxy]fluorine (23.1) Dotriacontylpentafluoropropionate,(25.0) Octadecanoic acid, (20.0); Trans-2-methyl-4-n-butylthiane, S, S-dioxide.(19.0). The results showed the benefit of ethyl acetate extract from S. parvulus VITJS11 in treating microbial infections and indicated their broad spectrum of activity with beneficial virtues for therapeutic use.

Larvicidal and repellent properties of Streptomyces sp. VITJS4 crude extract against Anopheles stephensi, Aedes aegypti and Culex quinquefasciatus (Diptera: Culicidae).

The aim of the present study was to assess the larvicidal and repellent properties of marine Streptomyces sp. VITJS4 crude extracts. The marine soil samples were collected from the Puducherry coast, Tamil Nadu, India. The isolate Streptomyces sp. VITJS4 was taxonomically characterized and identified. The ethyl acetate crude extract tested for larvicidal property showed 100% mortality for all the 3 species after 24 h exposure against the early fourth instar larvae of malarial vector--Anopheles stephensi at 50% and 90% lethal concentration (LC50 = 132.86, LC90 396.14 ppm); dengue vector--Aedes aegypti (LC50 = 112.78, LC90 336.42 ppm) and filariasis vector--Culex quinquefasciatus (LC50 = 156.53, LC90 468.37 ppm). The Streptomyces sp. VITJS4 solvent extracts of hexane, ethyl acetate, benzene, chloroform and methanol were tested for repellent activity against A. stephensi, A. aegypti and C. quinquefasciatus. The ethyl acetate extract showed complete protection for 210 min at 6 mg/cm2 against these mosquito bites. The crude extract was analyzed further for Fourier Transform-infrared spectroscopy (FT-IR) analysis. In addition to the importance of bioactive compounds, the utilization of Streptomyces sp. VITJS4 crude extracts revealed effective larvicidal and repellent activity against the vectors, which perhaps represents a promising tool in the management of mosquito control.

Evaluation on clot buster activity of staphylokinase from Staphylococcus sp.

Thrombolytic agents play a major role in the treatment of cardiovascular diseases. Streptokinase is the prominently commercialized thrombolytic drug used for the treatment of cardiovascular diseases. The later studies on staphylokinase (SaK) showed promising results as an alternative fibrinolytic drug. The present study explores the isolation, production and purification of SaK producing Staphylococcus sp. from milk samples. The potent isolate MSA4 of Staphylococcus sp. was selected for production and purification of SaK. The total activity and specific activity of purified staphylokinase was found to be 1266 IU mL(-1) and 815.5 IU mg(-1), respectively. The partially purified enzyme was lysed the euglobulin clot completely within 18 h of incubation and the purified enzyme showed 79% of blood clot lysis activity.