PURIFICATION AND CHARACTERIZATION OF L-ASPARAGINASE BY PSEUDONOCARDIA ENDOPHYTICA VUK-10 ISOLATED FROM NIZAMPATNAM MANGROVE ECOSYSTEM

Objective: L-asparaginase has been a promising therapeutic agent in the treatment of acute lymphoblastic leukaemia. In the present study a rare actino bacterial strain Pseudonocardia endophytica VUK-10 isolated from Nizampatnam mangrove ecosystem was explored for the production of L-asparaginase.Methods: The extracellular L-asparaginase enzyme was purified to homogeneity from the P. endophytica VUK-10 strain. The crude culture filtrate was subjected to different purification steps including ammonium sulphate fractionation followed by separation on Sephadex G-100 gel filtration and CM-Sephadex C-50 ion exchange chromatography to obtain a pure enzyme preparation.Results: The enzyme was purified 96 fold and showed a final specific activity of 702.04 IU/mg with a 61% yield. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified enzyme revealed its nature as single peptide chain with molecular weight of 120 kDa. This is the first report on production and purification of L-asparaginase from P. endophytica of mangrove origin.Conclusion: The extracellular L-asparaginase of the P. endophytica may be effectively used as potential chemotherapeutic agent.Keywords: Mangrove ecosystem, Pseudonocardia endophytica, L-asparaginase, Purification, SDS-PAG

ANTIMICROBIAL POTENTIAL OF STREPTOMYCES CHEONANENSIS VUK-A FROM MANGROVE ORIGIN

Objectives: The aim of the present study was to isolate, characterize and evaluate the activity of compounds produced by Streptomyces cheonanensis VUK-A.Methods: Chemical examination of the secondary metabolites of the strain Streptomyces cheonanensis VUK-A has led to the segregation of one bioactive compound (1) and a partially purified fraction (2). The strain was isolated from the sediment samples of mangrove ecosystem of Coringa, south coastal Andhra Pradesh, India. The chemical structure of the active compound 1 was established on the basis of spectroscopic analysis including 1H NMR, 13C NMR spectroscopy, FTIR and EIMS. The partially purified sub-fraction (2) subjected to Gas Chromatography-Mass spectroscopy. The antimicrobial activity of the bioactive compounds produced by the strain was expressed in terms of minimum inhibitory concentration.Results: The compound 1 was isolated from the fermentation broth was characterized as benzoic acid (1) based on spectroscopic analysis. The partially purified sub-fraction (2) subjected to Gas Chromatography-Mass spectroscopy contained nine analogues: 1-tetradecene, tetradecane, 1-hexadecene, hexadecane, 5-octadecene, octadecane, 5-eicosene, 1-nonadecene and cyclo tetracosane. The compounds recorded moderate to significant antimicrobial activity against medicinally and agriculturally important bacteria and fungi. This is the first report of six partially purified compounds 1-tetradecene, tetradecane, hexadecane, octadecane, 5-eicosene and cyclo tetracosane from the genus Streptomyces.Conclusion: The results of the present study showed that the metabolites of Streptomyces cheonanensis VUK-A exhibited antibacterial and antifungal activities. The study also supports that Coringa, a promising mangrove ecosystem remained to be explored for new bioactive compounds.Keywords: Streptomyces cheonanensis, Mangrove Ecosystem, Natural Products, Antimicrobial activity

Isolation, structure elucidation and bioactivity of secondary metabolites produced by marine derived Streptomonospora arabica VSM-25

73-93The strain VSM-25 with an exhilarating bioactive potential isolated during our systematic screening of marine actinomycetes was identified as Streptomonospora arabica based on polyphasic taxonomy. The ethyl acetate extract of culture filtrate was purified by silica gel column chromatography. The chemical structure of active compounds was determined by NMR, FTIR, and ESIMS and were established as Indole-3-carboxaldehyde (C1), 2, 3-dihydroxy benzoic acid (C2), Vanillic acid (C3), Daidzein (C4), and 3, 4-Dihydroxy benzaldehyde (C5). The antimicrobial activities of the compounds were tested against medicinally and agriculturally significant bacteria and fungi. C1 displayed a high inhibitory effect against bacteria and fungi to that of the other compounds tested. C5 exerted the strongest scavenging activity of free radicals such as DPPH and NO at a concentration of 400 μg/mL. C1 inhibited alpha-amylase effectively at 400 μg/mL although it was less potent than acarbose. C3 and C4 exerted significant anti-inflammatory and anti-arthritic activities at 400 μg/mL. The anti-inflammatory activity of compound C3 was found to be more potent than Diclofenac sodium, the reference drug. MTT assays of five compounds against MDA-MB-231 and MCF-7 cell lines using taxol as standard documented cytotoxicity. C4 showed highest activity of 67.81% and 54.33% (IC50 -1 μg/mL) against MDA-MB-231 and MCF-7. The cytotoxicity of five compounds was also evaluated by soft agar colony forming assay to determine the ability of MDA-MB-231 cells to proliferate while cell cycle arrest at sub G1 and induction of apoptosis was documented with MDA-MB-231 cells after treatment with C1, C2, C3, C4, and C5

PREVALENCE OF KRAS CODON 12 MUTATION IN PATIENTS WITH ORAL SQUAMOUS CELL CARCINOMA (OSCC) FROM SOUTH INDIAN POPULATION

Background: The RAS gene family is the most studied and best characterized of the known cancer-related genes KRAS is the most frequently altered gene, with mutations occurring in 17%–25% of all cancers. This study was designed to investigate the impact of Kras12 mutation in oral squamous cell Carcinoma Method: We genotyped 100 cases with oral squamous cell carcinoma (OSCC)and 100 controls, using a Polymerase Chain Reaction-Restriction Fragment Length Polymorphism method. Results: We found 13 cases harboring K-RAS G12D mutations & 9 cases harboring G12V among 100 OSCC cases. K-RAS codon 12 mutations was significantly different between OSCC patients and controls (P = 0.0007 & P=0.31) (Table 2). The G12D & G12V mutations were predominantly more in patients with chewing as the lone risk factor Conclusion: Prevalence of K-RAS codon 12 mutations G12D and G12V was 13% & 9% in the study group. The frequency of KRAS mutations appears higher in the south Indian population indicating that this testing is very crucial for targeted therapy management. Further studies on K-RAS mutation associations among south Indian OSCC patients are needed

Production of pullulan using jaggery as substrate by Aureobasidium pullulans MTCC 2195

Shake-flask fermentation, under batch cultivation, was investigated for the production of fungal exopolysaccharide, pullulan using jaggery (a traditional concentrated sugar cane juice) as a carbon substrate by Aureobasidium pullulans MTCC 2195. Change in the initial pH (from 3.0 to 7.0) of media containing jaggery was varied to study the effect of pH in the fermentation and maximum pullulan yield was obtained at a pH of 5.0. An increase in the initial concentrations (50, 75, 100 g/L) of jaggery in the media produced the maximum pullulan content as 21.6, 19.7 and 18.6 g per 100 g of jaggery, respectively, used. A sucrose based defined media were also used for comparison purposes. Fourier Transform InfraRed (FTIR) spectroscopic analysis was done to confirm the functional groups of synthesized pullulan and compared with that of commercial pullulan.Shake-flask fermentation, under batch cultivation, was investigated for the production of fungal exopolysaccharide, pullulan using jaggery (a traditional concentrated sugar cane juice) as a carbon substrate by Aureobasidium pullulans MTCC 2195. Change in the initial pH (from 3.0 to 7.0) of media containing jaggery was varied to study the effect of pH in the fermentation and maximum pullulan yield was obtained at a pH of 5.0. An increase in the initial concentrations (50, 75, 100 g/L) of jaggery in the media produced the maximum pullulan content as 21.6, 19.7 and 18.6 g per 100 g of jaggery, respectively, used. A sucrose based defined media were also used for comparison purposes. Fourier Transform InfraRed (FTIR) spectroscopic analysis was done to confirm the functional groups of synthesized pullulan and compared with that of commercial pullulan

Anticancer potential of Solanum lycopersicum L. extract in human lung epithelial cancer cells A549

The study aimed to reveal the phytochemical profile, free radical scavenging potential, and anticancer activity of Solanum lycopersicum L. leaf extract (SLLE). According to the study, SLLE contains plant secondary metabolites that are beneficial for health, like phenolics, flavonoids, ascorbic acid, alkaloids, and terpenoids. The SLLE has shown potential free radical scavenging potential in DPPH and ABTS free radical scavenging analysis and its EC50 values (concentration required to inhibit 50% of free radicals) were determined as 481.29 ± 33.82 and 527.56 ± 20.34 µg/mL, respectively. The SLLE has the ability to scavenge free radicals and could be used to treat illnesses brought on by oxidative stress. The anticancer activity of SLLE was assessed by MTT, LDH, micro-morphological, live/dead dual staining, and caspase-3 analysis. In the MTT assay, the IC50 value (concentration required to inhibit 50% of cell viability) of SLLE was determined as 190.41 ± 4.77 µg/mL. Furthermore, SLLE has shown potential anticancer activity by adversely affecting the plasma membrane integrity and escalating the caspase-3 levels. In the biomedical field, SLLE could be highly useful to treat cancer

Application of Syzygium aromaticum, Ocimum sanctum, and Cananga odorata essential oils for management of Ochratoxin A content by Aspergillus ochraceus and Penicillium verrucosum: An in vitro assessment in maize grains

172-182The study is directed to establish the minimizing effects of Syzygium aromaticum, Ocimum sanctum, and Cananga odorata essential oils on the growth and ochratoxin A (OTA) level of Aspergillus ochraceus and Penicillium verrucosum in maize grains. S. aromaticum essential oil (SAEO), O. sanctum essential oil (OSEO), and C. odorata essential oil (COEO) were extracted by hydro-distillation technique, and a total of 50, 44, and 48 chemical constituents were identified by gas chromatography-mass spectrometry (GC-MS), respectively.The SAEO and OSEO belong to the chemotype of eugenol, whereas, COEO was found to be the chemotype of thymol, limonene, and α-ylangene. The antifungal activity of essential oils (EOs) was determined by the micro-well dilution technique. The SAEO showed superior antifungal activity compared to OSEO, COEO, and synthetic antifungal agent nystatin, and its minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) values against A. ochraceous and P. verrucosum were noticed as 1251 ± 42.32 and 1878 ± 28.47 μg/mL, and 0815 ± 22.69 and 1146 ± 51.19 μg/mL, respectively.The antifungal mechanism of EOs was unveiled by assessing the intracellular reactive oxygen species (ROS), ergosterol content, and membrane integrity. The antifungal investigations found that EOs caused fungal mortality by increasing the intracellular ROS, depleting ergosterol synthesis, and distracting membrane integrity. Finally, antifungal and antimycotoxin activity of EOs was demonstrated in maize grains. The SAEO, OSEO, and COEO have reduced the complete fungal growth and OTA level of A. ochraceous and P.verrucosum correspondingly at 2500 and 2500, 3500 and 2500, and 3500 and 3500 μg/g in maize. The EOs could act asnatural antifungal agents; protect foodstuffs from fungal infection and mycotoxins during storage

Development of Monoclonal Antibodies Against Cry1Ac/Ab Protein for Designing of Sandwich ELISA to Detect BT Toxin from Cotton Seeds and Leaves

The design of the study is to develop monoclonal antibodies against Cry1Ac/Ab protein for designing os sandwich ELISA(hybridoma technology). Hybridoma technology was invented by Cesar Milstein and Georges J.F Kohler in the year 1975 and is an unique method used to produce identical antibodies in maximum quantities. Monoclonal antibodies were developed by immunization of Balb/C mice with Cry1Ac/Ab Protein. Titer values of mice tail bleeds were checked and the best mice with higher titer value was used for fusion. Immunized mice spleen cells were fused with Myeloma cells (SP2-O), using polyethylene glycol (PEG) and the fused cells were incubated with HAT medium for 12 days and initially 400 positive hybridoma clones were obtained, of which 13 potential clones were selected using indirect ELISA against Cry1Ac/Ab recombinant antigen. Cross reactivity was ruled out using indriet ELSA against cry proteins such as Cry2A, Cry1F and CP4EPSPS using. Cloning was carried out twice for all 13 clones by limiting dilution factor and pure single clones were selected. The class IgG/IgM/IgA and sub classes IgG1, IgG2, IgG3 antibodies are determined by isotyping. Determination of class and subclass of an antibody is very important for selecting proper purification methods. Commercially available rapid isotyping kits were used for isotyping which provides the information of 1) IgG, IgM, IgA, IgG2a, IgG2b or IgG3 2) Light chain identification as either kappa or lambda. All pure clones were preserved in Liquid Nitrogen for future use to develop immunological kits for detection of Cry1Ac/Ab present in the plant tissue.The design of the study is to develop monoclonal antibodies against Cry1Ac/Ab protein for designing os sandwich ELISA(hybridoma technology). Hybridoma technology was invented by Cesar Milstein and Georges J.F Kohler in the year 1975 and is an unique method used to produce identical antibodies in maximum quantities. Monoclonal antibodies were developed by immunization of Balb/C mice with Cry1Ac/Ab Protein. Titer values of mice tail bleeds were checked and the best mice with higher titer value was used for fusion. Immunized mice spleen cells were fused with Myeloma cells (SP2-O), using polyethylene glycol (PEG) and the fused cells were incubated with HAT medium for 12 days and initially 400 positive hybridoma clones were obtained, of which 13 potential clones were selected using indirect ELISA against Cry1Ac/Ab recombinant antigen. Cross reactivity was ruled out using indriet ELSA against cry proteins such as Cry2A, Cry1F and CP4EPSPS using. Cloning was carried out twice for all 13 clones by limiting dilution factor and pure single clones were selected. The class IgG/IgM/IgA and sub classes IgG1, IgG2, IgG3 antibodies are determined by isotyping. Determination of class and subclass of an antibody is very important for selecting proper purification methods. Commercially available rapid isotyping kits were used for isotyping which provides the information of 1) IgG, IgM, IgA, IgG2a, IgG2b or IgG3 2) Light chain identification as either kappa or lambda. All pure clones were preserved in Liquid Nitrogen for future use to develop immunological kits for detection of Cry1Ac/Ab present in the plant tissue

Hibiscus tiliaceus mediated phytochemical reduction of zinc oxide nanoparticles and demonstration of their antibacterial, anticancer, and dye degradation capabilities

565-574The present research focused on the green, non-toxic, low-cost synthesis of zinc oxide nanoparticles (ZnO NPs) using aqueous extract of Hibiscus tiliaceus leaves as a reducing and stabilizing agent. Thus, synthesized ZnO NPs were characterized by nanotechnological applications, i.e., ultraviolet-visible spectroscopy (UV-vis), dynamic light scattering (DLS), zeta potential, X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), and high-resolution transmission electron microscopy (HR-TEM). The nanotechnological applications showed that as-synthesized ZnO NPs have bandgap energy of 2.97 eV, zeta potential of 1.2 mV, crystalline in nature (JCPDS data card no-89-1397), and an average size of 30 to 60 nm. The FTIR showed that ZnO NPs have coated with plant secondary metabolites and assisted in the process of green synthesis. The ZnO NPs exhibited broad-spectrum antibacterial activity on Gram-positive and Gramnegative bacteria. The ZnO NPs showed potential anticancer activity against human breast cancer cells MCF-7 and determined the IC50 value as 65.83 ± 2.57 μg/mL by MTT assay. Furthermore, ZnO NPs were used as nano-catalyst for dye degradation of methylene blue, methyl orange, and malachite green with NABH4 as a reducing agent. The ZnO NPs exhibited potent dye degradation capability and followed pseudo-first order kinetics. The study concluded that ZnO NPs could be highly useful as anticancer and antibacterial agents in the biomedical field, and as an environmental cleaning agent for dye degradation in textile industries

Isolation, structure elucidation and bioactivity of secondary metabolites produced by marine derived Streptomonospora arabica VSM-25

The strain VSM-25 with an exhilarating bioactive potential isolated during our systematic screening of marine actinomycetes was identified as Streptomonospora arabica based on polyphasic taxonomy. The ethyl acetate extract of culture filtrate was purified by silica gel column chromatography. The chemical structure of active compounds was determined by NMR, FTIR, and ESIMS and were established as Indole-3-carboxaldehyde (C1), 2, 3-dihydroxy benzoic acid (C2), Vanillic acid (C3), Daidzein (C4), and 3, 4-Dihydroxy benzaldehyde (C5). The antimicrobial activities of the compounds were tested against medicinally and agriculturally significant bacteria and fungi. C1 displayed a high inhibitory effect against bacteria and fungi to that of the other compounds tested. C5 exerted the strongest scavenging activity of free radicals such as DPPH and NO at a concentration of 400 µg/mL. C1 inhibited alpha-amylase effectively at 400 µg/mL although it was less potent than acarbose. C3 and C4 exerted significant anti-inflammatory and anti-arthritic activities at 400 µg/mL. The anti-inflammatory activity of compound C3 was found to be more potent than Diclofenac sodium, the reference drug. MTT assays of five compounds against MDA-MB-231 and MCF-7 cell lines using taxol as standard documented cytotoxicity. C4 showed highest activity of 67.81% and 54.33% (IC50 -1 µg/mL) against MDA-MB-231 and MCF-7. The cytotoxicity of five compounds was also evaluated by soft agar colony forming assay to determine the ability of MDA-MB-231 cells to proliferate while cell cycle arrest at sub G1 and induction of apoptosis was documented with MDA-MB-231 cells after treatment with C1, C2, C3, C4, and C5