S. Nishanth Kumar

Identification of antimicrobial compound, diketopiperazines, from a Bacillus sp. N strain associated with a rhabditid entomopathogenic nematode against major plant pathogenic fungi

To purify and characterize antimicrobial compounds from Bacillus sp. strain N associated with rhabditid entomopathogenic nematode (EPN).

Cyclo(d‐Tyr‐d‐Phe): a new antibacterial, anticancer, and antioxidant cyclic dipeptide from Bacillus sp. N strain associated with a rhabditid entomopathogenic nematode

A new microbial cyclic dipeptide (diketopiperazine), cyclo(d‐Tyr‐d‐Phe) was isolated for the first time from the ethyl acetate extract of fermented modified nutrient broth of Bacillus sp. N strain associated with rhabditid Entomopathogenic nematode. Antibacterial activity of the compound was determined by minimum inhibitory concentration and agar disc diffusion method against medically important bacteria and the compound recorded significant antibacterial against test bacteria. Highest activity was recorded against Staphylococcus epidermis (1 µg/ml) followed by Proteus mirabilis (2 µg/ml). The activity of cyclo(d‐Tyr‐d‐Phe) against S. epidermis is better than chloramphenicol, the standard antibiotics. Cyclo(d‐Tyr‐d‐Phe) recorded significant antitumor activity against A549 cells (IC50 value: 10 μM) and this compound recorded no cytotoxicity against factor signaling normal fibroblast cells up to 100 μM. Cyclo(d‐Tyr‐d‐Phe) induced significant morphological changes and DNA fragmentation associated with apoptosis in A549 cells. Acridine orange/ethidium bromide stained cells indicated apoptosis induction by cyclo(d‐Tyr‐d‐Phe). Flow cytometry analysis showed that the cyclo(d‐Tyr‐d‐Phe) did not induce cell cycle arrest. Effector molecule of apoptosis such as caspase‐3 was found activated in treated cells, suggesting apoptosis as the main mode of cell death. Antioxidant activity was evaluated by free radical scavenging and reducing power activity, and the compound recorded significant antioxidant activity. The free radical scavenging activity of cyclo(d‐Tyr‐d‐Phe) is almost equal to that of butylated hydroxyanisole, the standard antioxidant agent. We also compared the biological activity of natural cyclo(d‐Tyr‐d‐Phe) with synthetic cyclo(d‐Tyr‐d‐Phe) and cyclo(l‐Tyr‐l‐Phe). Natural and synthetic cyclo(d‐Tyr‐d‐Phe) recorded similar pattern of activity. Although synthetic cyclo(l‐Tyr‐l‐Phe) recorded lower activity. But in the case of reducing power activity, synthetic cyclo(l‐Tyr‐l‐Phe) recorded significant activity than natural and synthetic cyclo(d‐Tyr‐d‐Phe). The results of the present study reveals that cyclo(d‐Tyr‐d‐Phe) is more bioactive than cyclo(l‐Tyr‐l‐Phe). To the best of our knowledge, this is the first time that cyclo(d‐Tyr‐d‐Phe) has been isolated from microbial natural source and also the antibacterial, anticancer, and antioxidant activity of cyclo(d‐Tyr‐d‐Phe) is also reported for the first time. Copyright

Purification of an antifungal compound, cyclo(l-Pro-d-Leu) for cereals produced by Bacillus cereus subsp. thuringiensis associated with entomopathogenic nematode

Mold spoilage is the main cause of substantial economic loss in cereals and might also cause public health problems due to the production of mycotoxins. The aim of this study was to separate and purify and to identify antifungal compounds of bacterium associated with novel entomopathogenic nematode and check the antifungal property of identified compound in particular food model systems. The antifungal compound was purified using silica gel column chromatography, TLC and HPLC and its structure was elucidated using NMR (¹H NMR, ¹³C NMR, ¹H-¹H COSY, ¹H-¹³C HMBC), HRMS and Marfeys method. Based on the spectral data, the active compounds were identified as diketopiperazine [cyclo(l-Pro-d-Leu)]. The antifungal activity of cyclo(l-Pro-d-Leu) was studied by MIC and paper disk assay against Aspergillus flavus MTCC 277 and Aspergillus niger MTCC 282 and best MIC value of 8μg/ml was recorded against A. flavus. Cyclo(l-Pro-d-Leu) strongly inhibit mycelia growth of fungus and thereby affecting aflatoxin production. To investigate the potential application of the cyclo(l-Pro-d-Leu) and to eliminate fungal spoilage in food and feed, soybean and peanut were used as models. White mycelia and dark/pale green spores of A. flavus were observed in the control soybeans after 2-day incubation. However the fungal growth was not observed in soybeans treated with cyclo(l-Pro-d-Leu). Almost the same result was observed for peanuts treated with cyclo(l-Pro-d-Leu) for A. niger. The cyclo(l-Pro-d-Leu) was nontoxic to two normal human cell lines (FS normal fibroblast and L231 lung epithelial) up to 200μg/ml. Thus the diketopiperazine derivative identified in the study may be a promising alternative to chemical preservatives as a potential biopreservative which prevent fungal growth and mycotoxin formation in food and feed.

Purification, structural elucidation and bioactivity of tryptophan containing diketopiperazines, from Comamonas testosteroni associated with a rhabditid entomopathogenic nematode against major human-pathogenic bacteria

The cell free culture filtrate of a Comamonas testosteroni associated with an Entomopathogenic nematode (EPN), Rhabditis (Oscheius) sp. exhibited promising antimicrobial activity. The ethyl acetate extract of the bacterial culture filtrate was purified by silica gel column chromatography to obtain five diketopiperazines or cyclic dipeptides (DKP 1-5). The structure and absolute stereochemistry of the compounds were determined based on extensive spectroscopic analyses (HR-MS, (1)HNMR, (13)CNMR, (1)H-(1)H COSY, (1)H-(13)C HMBC) and Marfeys method. Based on the spectral data the compounds were identified as Cyclo-(L-Trp-L-Pro) (1), Cyclo-(L-Trp-L-Tyr) (2), Cyclo-(L-Trp-L-Ile) (3), Cyclo-(L-Trp-L-Leu) (4) and Cyclo-(L-Trp-L-Phe) (5), respectively. Three diketopiperazines (DKP 2, 3 and 5) were active against all the ten bacteria tested. The highest activity of 0.5μg/ml by Cyclo-(L-Trp-L-Phe) was recorded against Vibrio cholerae followed by Salmonella typhi (1 μg/ml) a human pathogen responsible for life threatening diseases like profuse watery diarrhea and typhoid or enteric fever. The activity of this compound against V. cholerae and S. typhi is more effective than ciprofloxacin and ampicillin, the standard antibiotics. Cyclo-(L-Trp-L-Phe) recorded significant antibacterial activity against all the test bacteria when compared to other compounds. Five diketopiperazines were active against all the test fungi and are more effective than bavistin the standard fungicide. Diketopiperazines recorded no cytotoxicity to FS normal fibroblast and VERO cells (African green monkey kidney) except DKP 3 and 4. To our best knowledge this is the first report of antimicrobial activity of the tryptophan containing diketopiperazines against the human pathogenic microbes. The production of cyclic dipeptides by C. testosteroni is also reported here for the first time. We conclude that the C. testosteroni is promising sources of natural bioactive secondary metabolites against human pathogenic bacteria which may receive great benefit in the field of human medicine in near future.

Purification and characterization of antifungal phenazines from a fluorescent Pseudomonas strain FPO4 against medically important fungi

The strain FPO4 was isolated from the rhizoplane of rice plant root and identified as a fluorescent Pseudomonas aeruginosa on the basis of 16S rDNA sequences and BLAST analysis. The extracellular metabolites produced by this strain were purified by silica gel column chromatography and isolated four pure compounds. Based on the spectral data the four compounds were identified as phenazin-1-ol, phenazine-1-carboxylic acid (PCA), 2-heptyl-3-hydroxyl-4(1H)-quinolone (PQS), and phenazine-1-carboxamide (PCN), respectively. Phenazin-1-ol and PCA were active against all the eight fungi tested. The highest activity of 4 μg/mL by PCA was recorded against Trichophyton rubrum, a human pathogen responsible for causing athletes foot, jock itch, ringworm and fingernail fungus infections, followed by Candida albicans and Candida tropicalis. The activity of phenazin-1-ol, PCA against Candida spp. was found to be better than the standard antifungal agent amphotericin B. Furthermore, the present study reports the antimicrobial activity of the purified phenazines on major human pathogen, T. rubrum for the first time.

Synergistic activity of phenazines isolated from Pseudomonas aeruginosa in combination with azoles against Candida species

Candidiasis infections are caused by yeasts from the genus Candida. The types of infection range from superficial to systemic. Treatment often requires antifungals such as the azoles; however, increased use of these drugs has led to the generation of yeasts with increased resistance to these drugs. Here, we describe the synergistic anticandidal activity of three phenazines-phenazine-1-ol, phenazine-1-carboxylic acid, and phenazine-1-carboxamide. These phenazines were purified from Pseudomonas aeruginosa in combination with three clinically used azoles-fluconazole, itraconazole, and clotrimazole. The synergistic anticandidal activities of phenazines and azoles were assessed using the checkerboard microdilution and time-kill methods. Study results show that the combined effects of phenazines and azoles were predominantly synergistic activity (fractional inhibitory concentration index <0.5). The time-kill study, which included a combination of the minimum inhibitory concentration of phenazines and azoles, showed growth of Candida species that was completely attenuated after 0-6 h of treatment. These results, which suggest that the activity of phenazines and azoles may be beneficial, have potential implications in delaying the development of resistance, as the anticandidal effect is achieved with lower concentrations of both agents (phenazines and azoles). The cytotoxicity of phenazines was also tested against a normal human cell line (foreskin normal fibroblast). No cytotoxicity was recorded at concentrations up to 200 μg/ml. The in vitro synergistic activity of phenazines and azoles against Candida species is reported here for the first time.

Reactive oxygen species (ROS) mediated enhanced anti-candidal activity of ZnS–ZnO nanocomposites with low inhibitory concentrations

Enhanced antifungal activity against the yeast species Candida albicans, Candida tropicalis and Saccharomyces cerevisiae was displayed by ZnS–ZnO nanocomposites prepared by a simple precipitation technique. The antifungal activity was significantly more in the presence of indoor light than under dark conditions and was a clear confirmation of the inhibitory role of reactive oxygen species (ROS) generated in situ by the photocatalytic nanocomposites. The generation of ROS was further evidenced by flow cytometry results and membrane permeabilisation studies. Time kill assay and growth curve analysis indicated diminished antifungal activity under dark conditions due primarily to Zn2+ efflux in solution.

Characterization of three depside compounds from a Western Ghat lichen Parmelia erumpens Kurok with special reference to antimicrobial and anticancer activity

The aim of this study was to investigate the major chemical compositions of the chloroform extract of lichen Parmelia erumpens from Western Ghats, Kerala, India and its antimicrobial and anticancer activities. Chloroform extract was purified by silica gel column chromatography to obtain three major compounds and their chemical structures were characterized by 1H-NMR, 13C-NMR, UV and HR-MS spectroscopic methods as atranorin (1), (+)-usnic acid (2) and 2-hydroxy-4-methoxy-3,6-dimethylbenzoic acid (3). The minimal inhibitory concentration (MIC) by the broth micro dilution and agar disc diffusion methods was used to record the antimicrobial activity. Out of three compounds tested, 2-hydroxy-4-methoxy-3,6-dimethylbenzoic acid recorded excellent antimicrobial activity especially against medically important bacteria and fungi and the MIC values ranged from 0.06 to 4 μg ml−1 against test bacteria and 0.12 to 16 μg ml−1 against test fungi. The best MIC of 0.06 μg ml−1 by 2-hydroxy-4-methoxy-3,6-dimethylbenzoic acid was recorded against Vibrio cholera, a human pathogenic bacterium responsible for causing life threatening diseases like profuse watery diarrhea. Anti cancer activity was initially screened by MTT assay in A549, B16F10, Caski and HepG2 cell lines. MTT assay results showed that the growth of cancer cells was suppressed by 2-hydroxy-4-methoxy-3,6-dimethylbenzoic acid in both dose- and time-dependent manners. A549, B16F10 and Caski cells treated with 2-hydroxy-4-methoxy-3,6-dimethylbenzoic acid showed typical apoptotic morphology when stained with acridine orange–ethidium bromide and hoechst staining. Cell cycle analysis clearly indicated that cell death was due to apoptosis. Enhancement in the proliferation of lymphocytes suggested immunomodulatory activity of this compound. To our best knowledge anticancer activity of 2-hydroxy-4-methoxy-3,6-dimethylbenzoic acid was reported here for the first time. Thus the results of the present study suggest that 2-hydroxy-4-methoxy-3,6-dimethylbenzoic acid has a strong potential to be developed as an antimicrobial and anticancer drug target after further clinical evaluation.

Silanated nano ZnO hybrid embedded PMMA polymer coatings on cotton fabrics for near-IR reflective, antifungal cool-textiles

Textile surfaces engineered with infrared/ultraviolet energy shielding coatings are an emerging technology in the processing of solar heat protective cool-textiles. In this work, such multifunctional coatings were prepared using silane treated nano ZnO hybrid embedded PMMA colloids on a model black colored cotton textile. Black cotton fabrics were selected for the study because they exhibit a high heat-build up tendency due to the inherent black body radiation effect. 3-(Aminopropyl)trimethoxy silane modified nano ZnO hybrids (APZO NHs) were first prepared and transformed into a stable colloidal dispersion in a PMMA medium to obtain an APZO NHs/PMMA sol. This hybrid–polymer colloidal sol was dip-coated on the fabric surface to form multilayer coatings. This surface engineered black cotton was subjected to studies of its phase analysis, chemical interaction, and morphological features in addition to its NIR reflectance, UV shielding efficiency and antifungal properties. The NIR/UV reflectance performance was analyzed with respect to the number of coatings and compared with the standard white cotton fabric which has roughly 50% NIR reflectance. When silane treated ZnO hybrid particulates are embedded in the PMMA matrix, the polymer coatings offer mechanically stable, UV/NIR radiation shielding textile surfaces in the wavelength region between 360 to 1600 nm that eventually turns the black cotton to cool-black textiles. The coatings also offer hydrophobic functionality as well as strong protection against the growth of Aspergillus flavus and Aspergillus niger fungi species resulting in biosafe textiles.

Pseudopyronine B: A Potent Antimicrobial and Anticancer Molecule Isolated from a Pseudomonas mosselii.

In continuation of our search for new bioactive compounds from soil microbes, a fluorescent Pseudomonas strain isolated from paddy field soil of Kuttanad, Kerala, India was screened for the production of bioactive secondary metabolites. This strain was identified as Pseudomonas mosselii through 16S rDNA gene sequencing followed by BLAST analysis and the bioactive metabolites produced were purified by column chromatography (silica gel) and a pure bioactive secondary metabolite was isolated. This bioactive compound was identified as Pseudopyronine B by NMR and HR-ESI-MS. Pseudopyronine B recorded significant antimicrobial activity especially against Gram-positive bacteria and agriculturally important fungi. MTT assay was used for finding cell proliferation inhibition, and Pseudopyronine B recorded significant antitumor activity against non-small cell lung cancer cell (A549), and mouse melanoma cell (B16F10). The preliminary MTT assay results revealed that Pseudopyronine B recorded both dose- and time-dependent inhibition of the growth of test cancer cell lines. Pseudopyronine B induced apoptotic cell death in cancer cells as evidenced by Acridine orange/ethidium bromide and Hoechst staining, and this was further confirmed by flow cytometry analysis using Annexin V. Cell cycle analysis also supports apoptosis by inducing G2/M accumulation in both A549 and B16F10 cells. Pseudopyronine B treated cells recorded significant up-regulation of caspase 3 activity. Moreover, this compound recorded immunomodulatory activity by enhancing the proliferation of lymphocytes. The production of Pseudopyronine B by P. mosselii and its anticancer activity in A549 and B16F10 cell lines is reported here for the first time. The present study has a substantial influence on the information of Pseudopyronine B from P. mosselii as potential sources of novel drug molecule for the pharmaceutical companies, especially as potent antimicrobial and anticancer agent.