C. Mohandas

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.

Bioactive stilbenes from a Bacillus sp. N strain associated with a novel rhabditid entomopathogenic nematode.

Aims:  The aim of the present study was to purify and characterize a natural antimicrobial compound from Bacillus sp. strain N associated with a novel rhabditid entomopathogenic nematode.

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.

Protocetraric acid: an excellent broad spectrum compound from the lichen Usnea albopunctata against medically important microbes

The aim of this study was to investigate the antimicrobial property of the compounds present in the lichen Usnea albopunctata. Ethyl acetate extract of the lichen was purified by column chromatography to yield a major compound which was characterised by spectroscopic methods as protocetraric acid. In this study, protocetraric acid recorded significant broad spectrum antimicrobial property against medically important human pathogenic microbes. The prominent antibacterial activity was recorded against Salmonella typhi (0.5 μg/mL). Significant antifungal activity was recorded against Trichophyton rubrum (1 μg/mL), which is significantly better that the standard antifungal agent. Protocetraric acid is reported here for the first time from U. albopunctata. Thus the results of this study suggest that protocetraric acid has significant antimicrobial activities and has a strong potential to be developed as an antimicrobial drug against pathogenic microbes.

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.

Molecular characterization and amplified ribosomal DNA restriction analysis of entomopathogenic bacteria associated with Rhabditis (Oscheius) spp.

Bacterial strains associated with entomopathogenic nematodes (EPNs) Rhabditis (Oscheius) spp. were isolated from infected cadavers of Galleria mellonella. The obtained 18 isolates were subdivided into nine phylogenetically different genera based on comparative sequence analysis of their 16S rRNA genes. The isolates were affiliated to three different class namely γ-proteobacteria (Enterobacter, Proteus, Providencia, Pseudomonas, Stenotrophomonas), β-proteobacteria (Alcaligenes) and Bacilli (Bacillus, Enterococcus, Lysinibacillus). It was observed that Gram-positive strains (Bacilli) were more frequently associated with the EPN, whereas Gram-negative isolates were affiliated to six different genera with more genotypic diversity. Subsequently, all bacterial isolates used in this study were analyzed by amplified ribosomal DNA restriction analysis (ARDRA). Eight restriction endonucleases (CfoI, HinfI, RsaI, DdeI, Sau3AI, AluI, HaeIII, and MspI) were examined and a total of 15 different genotypes were obtained, forming two heterogenous main clusters after analysis by un-weighted pair-group method using arithmetic averages.

Purification and identification of two antifungal cyclic dipeptides from Bacillus cereus subsp. thuringiensis associated with a rhabditid entomopathogenic nematode especially against Fusarium oxysporum

Abstract The cell-free culture filtrate of Bacillus cereus subsp. thuringiensis associated with an entomopathogenic nematode (EPN), Rhabditis (Oscheius) sp., exhibited strong antimicrobial activity. The ethyl acetate extract of the bacterial culture filtrate was purified by silica gel column chromatography to obtain two cyclic dipeptides (CDPs). The structure and absolute stereochemistry of this compound were determined based on extensive spectroscopic analyses (FABMS, 1H NMR, 13C NMR, 1H--1H COSY, 1H--13C HMBC) and Marfey’s method. The compounds were identified as cyclo(D-Pro-L-Met) and cyclo(D-Pro-D-Tyr). CDPs showed significantly higher activity than the standard fungicide bavistin against agriculturally important fungi, viz., Fusarium oxysporum, Rhizoctonia solani and Penicillium expansum. The highest activity of 2 µg/ml by cyclo(D-Pro-D-Tyr) was recorded against F. oxysporum, a plant pathogen responsible for causing fusarium wilt followed by R. solani, a pathogen that causes root rot and P. expansum. To our knowledge, this is the first report on the isolation of these compounds from Rhabditis EPN bacterial strain Bacillus cereus subsp. thuringiensis.

Antimycobacterial activity of cyclic dipeptides isolated from Bacillus sp. N strain associated with entomopathogenic nematode

Abstract Context: Tuberculosis (TB) is one of the leading causes of morbidity and mortality with a global mortality rate of two million deaths per year; one-third of the world’s population is infected with Mycobacterium tuberculosis. Objective: The aim of this study was to determine the antimycobacterial activity of six diketopiperazines (DKPs) purified from a Bacillus sp. N strain associated with entomopathogenic nematode Rhabditis (Oscheius) sp. Materials and methods: The minimum inhibitory concentration (MIC) and minimum bactericidal concentration of DKPs were determined using the broth dilution method on Middlebrook 7H11 against M. tuberculosis H37Rv. Time-kill assay was used to determine the rate of killing of M. tuberculosis H37Rv by DKPs. The cytotoxicity of the DKPs was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay against the VERO cell line. Results: Out of six DKP-tested cyclo-(d-Pro-l-Leu), cyclo-(l-Pro-l-Met) and cyclo-(d-Pro-l-Phe) recorded antimycobacterial activity, the cyclo-(l-Pro-l-Met) showed the highest activity and MIC values of 4 μg/ml for M. tuberculosis H37Rv. The MIC value for rifampicin was 0.06 μg/ml. Growth curve study by the MIC concentration of cyclic dipeptides recorded significant inhibition when compared with control. Time-kill curve showed maximum reduction of colony count was between 3 and 5 weeks. The DKPs are nontoxic to the VERO cell line up to 200 µg/ml. The antimycobacterial activity of cyclo-(d-Pro-l-Leu), cyclo-(l-Pro-l-Met) and cyclo-(d-Pro-l-Phe) is reported in this study for the first time. Discussion and conclusion: In conclusion, the potency, low cytotoxicity and selectivity of these compounds make them valid lead compounds for treatment against TB.