Insights into diversity and L-asparaginase activity of fungal endophytes associated with medicinal plant Grewia hirsuta.

L-asparaginase is used as one of the prime chemotherapeutic agents to treat acute lymphoblastic leukemia. The present work aimed to study the endophytic fungal diversity of Grewia hirsuta and their ability to produce L-asparaginase. A total of 1575 culturable fungal endophytes belonging to four classes, Agaricomycetes, Dothideomycetes, Eurotiomycetes, and Sordariomycetes, were isolated. The isolates were grouped into twenty-one morphotypes based on their morphological characteristics. Representative species from each group were identified based on their microscopic characteristics and evaluation of the ITS and LSU rDNA sequences. Most of the fungal endophytes were recovered from the leaves compared to other plant parts. Diaporthe sp. was the predominant genus with a colonization frequency of 8.62%. Shannon-Wiener index for diversity ranged from 2.74 to 2.88. All the plant parts showed similar Simpson's index values, indicating a uniform species diversity. Among the sixty-three fungal endophytes screened, thirty-two were identified as L-asparaginase-producing isolates. The enzyme activities of fungal endophytes estimated by the nesslerization method were found to be in the range of 4.65-0.27 IU/mL with Fusarium foetens showing maximum enzyme activity of 4.65 IU/mL. This study for the first time advocates the production of L-asparaginase from Fusarium foetens along with the endophytic fungal community composition of Grewia hirsuta. The results indicate that the fungal endophyte Fusarium foetens isolated in the present study could be a potent source of L-asparaginase.

Endophytic Fungi as a Promising Source of Anticancer L-Asparaginase: A Review.

L-asparaginase is a tetrameric enzyme from the amidohydrolases family, that catalyzes the breakdown of L-asparagine into L-aspartic acid and ammonia. Since its discovery as an anticancer drug, it is used as one of the prime chemotherapeutic agents to treat acute lymphoblastic leukemia. Apart from its use in the biopharmaceutical industry, it is also used to reduce the formation of a carcinogenic substance called acrylamide in fried, baked, and roasted foods. L-asparaginase is derived from many organisms including plants, bacteria, fungi, and actinomycetes. Currently, L-asparaginase preparations from Escherichia coli and Erwinia chrysanthemi are used in the clinical treatment of acute lymphoblastic leukemia. However, they are associated with low yield and immunogenicity problems. At this juncture, endophytic fungi from medicinal plants have gained much attention as they have several advantages over the available bacterial preparations. Many medicinal plants have been screened for L-asparaginase producing endophytic fungi and several studies have reported potent L-asparaginase producing strains. This review provides insights into fungal endophytes from medicinal plants and their significance as probable alternatives for bacterial L-asparaginase.

Antimicrobial metabolite profiling of Nigrospora sphaerica from Adiantum philippense L.

BACKGROUND: Endophyte bestows beneficial aspects to its inhabiting host, along with a contribution to diverse structural attributes with biological potential. In this regard, antimicrobial profiling of fungal endophytes from medicinal plant Adiantum philippense revealed bioactive Nigrospora sphaerica from the leaf segment. Chemical and biological profiling through TLC-bioautography and hyphenated spectroscopic techniques confirmed the presence of phomalactone as an antimicrobial metabolite. RESULTS: The chemical investigation of the broth extract by bioassay-guided fractionation confirmed phomalactone as a bioactive antimicrobial secondary metabolite. The antimicrobial activity of phomalactone was found to be highest against Escherichia coli by disc diffusion assay. The MIC was found to be significant against both Escherichia coli and Xanthomonas campestris in the case of bacteria and dermatophyte Candida albicans at 150 µg/ml, respectively. CONCLUSIONS: Overall, the results highlighted the antimicrobial potential of phomalactone from the endophyte Nigrospora sphaerica exhibiting a broad spectrum of antimicrobial activity against human and phytopathogenic bacteria and fungi. This work is the first report regarding the antibacterial activity of phomalactone.

Application of Optimized and Validated Agar Overlay TLC-Bioautography Assay for Detecting the Antimicrobial Metabolites of Pharmaceutical Interest.

The agar overlay TLC-bioautography is one of the crucial methods for simultaneous in situ detection and separation of antimicrobial metabolites of pharmaceutical interest. The main focus of this research relies on the dereplication of an antimicrobial metabolite coriloxin derived from mycoendophytic Xylaria sp. NBRTSB-20 with a validation of agar overlay TLC-bioautography technique. This polyketide metabolite coriloxin was purified by column chromatography, and its purity was assessed by HPLC, UPLC-ESI-QTOF-MS, FT-IR and NMR spectral analysis. The antimicrobial capability of ethyl acetate extract and the purified compound coriloxin was determined by disc diffusion, minimal inhibitory concentration and agar overlay TLC-bioautography assay. The visible LOD of coriloxin antimicrobial activity was found at 10 µg for Escherichia coli and 20 µg for both Staphylococcus aureus and Fusarium oxysporum. Inter- and intra-day precision was determined as the relative standard deviation is less than 6.56%, which proved that this method was precise. The accuracy was expressed as recovery, and the values were found ranging from 91.18 to 108.73% with RSD values 0.94-2.30%, respectively. The overall findings of this investigation suggest that agar overlay TLC-bioautography assay is a suitable and acceptable method for the in situ determination of antimicrobial pharmaceuticals.

Chemogenomics driven discovery of endogenous polyketide anti-infective compounds from endosymbiotic Emericella variecolor CLB38 and their RNA secondary structure analysis.

In the postgenomic era, a new strategy for chemical dereplication of polyketide anti-infective drugs requires novel genomics and chromatographic strategies. An endosymbiotic fungal strain CLB38 was isolated from the root tissue of Combretum latifolium Blume (Combretaceae) which was collected from the Western Ghats of India. The isolate CLB38 was then identified as Emericella variecolor by its characteristic stellate ascospores culture morphology and molecular analysis of ITS nuclear rDNA and intervening 5.8S rRNA gene sequence. ITS2 RNA secondary structure modeling clearly distinguished fungal endosymbiont E. variecolor CLB38 with other lifestyles in the same monophyletic clade. Ethyl acetate fraction of CLB38 explored a broad spectrum of antimicrobial activity against multidrug resistant pathogens. Biosynthetic PKS type-I gene and chromatographic approach afford two polyketide antimicrobial compounds which identified as evariquinone and isoindolones derivative emerimidine A. MIC of purified compounds against test microorganisms ranged between 3.12 µg/ml and 12.5 µg/ml. This research highlights the utility of E. variecolor CLB38 as an anticipate source for anti-infective polyketide metabolites evariquinone and emerimidine A to combat multidrug resistant microorganisms. Here we demonstrates a chemogenomics strategy via the feasibility of PKS type-I gene and chromatographic approach as a proficient method for the rapid prediction and discovery of new polyketides compounds from fungal endosymbionts.

Corrigendum to "Mycosynthesis of silver nanoparticles bearing antibacterial activity" [Saudi Pharm. J. 24 (2016) 140-146].

[This corrects the article DOI: 10.1016/j.jsps.2015.01.008.].

Biomimetic Synthesis of Silver Nanoparticles Using Endosymbiotic Bacterium Inhabiting Euphorbia hirta L. and Their Bactericidal Potential.

The present investigation aims to evaluate biomimetic synthesis of silver nanoparticles using endophytic bacterium EH 419 inhabiting Euphorbia hirta L. The synthesized nanoparticles were initially confirmed with change in color from the reaction mixture to brown indicating the synthesis of nanoparticles. Further confirmation was achieved with the characteristic absorption peak at 440 nm using UV-Visible spectroscopy. The synthesized silver nanoparticles were subjected to biophysical characterization using hyphenated techniques. The possible role of biomolecules in mediating the synthesis was depicted with FTIR analysis. Further crystalline nature of synthesized nanoparticles was confirmed using X-ray diffraction (XRD) with prominent diffraction peaks at 2θ which can be indexed to the (111), (200), (220), and (311) reflections of face centered cubic structure (fcc) of metallic silver. Transmission electron microscopy (TEM) revealed morphological characteristics of synthesized silver nanoparticles to be polydisperse in nature with size ranging from 10 to 60 nm and different morphological characteristics such as spherical, oval, hexagonal, and cubic shapes. Further silver nanoparticles exhibited bactericidal activity against panel of significant pathogenic bacteria among which Pseudomonas aeruginosa was most sensitive compared to other pathogens. To the best of our knowledge, present study forms first report of bacterial endophyte inhabiting Euphorbia hirta L. in mediating synthesizing silver nanoparticles.

Implication of PKS type I gene and chromatographic strategy for the biodiscovery of antimicrobial polyketide metabolites from endosymbiotic Nocardiopsis prasina CLA68.

Advanced approach in probing for polyketide antimicrobials requires novel genomics and chromatographic strategies. An endophytic strain CLA68 was isolated from the root of Combretum latifolium Blume (Combretaceae) collected from the Western Ghats of Southern India. Strain CLA68 was then identified as Nocardiopsis prasina by its characteristic culture morphology and analysis of 16S rRNA gene sequence. Biosynthetic polyketide synthase genes were investigated using two pairs of degenerate primers. Ethyl acetate extract of CLA68 exhibited broad spectrum activity against a panel of test human pathogens. PKS type-I gene detection and chromatographic strategy yielded a robust polyketide antimicrobial compound which identified as nocapyrone E. Minimum inhibitory concentration of the purified compound against MRSA and other human pathogens ranged between 25 and 100 µg/ml. The present work highlights the utility of N. prasina CLA68 as potential source for antimicrobial polyketide nocapyrone E which could help to combat multidrug-resistant pathogens. This study demonstrates feasibility of PKS type-I gene-based molecular approach and chemical investigation by chromatographic approach is the best method for prediction and rapid discovery of novel polyketides from endosymbiotic actinomycetes. The sequence data of this endosymbiotic actinomycete is deposited in GenBank under the accession no. KP269077.

Mycosynthesis of silver nanoparticles bearing antibacterial activity.

Mycosynthesis of silver nanoparticles was achieved by endophytic Colletotrichum sp. ALF2-6 inhabiting Andrographis paniculata. Well dispersed nanoparticles were characterized using UV-Visible spectrometry with maximum absorption conferring at 420 nm. FTIR analysis revealed possible biomolecules reducing the metal salt and stabilization of nanoparticles. XRD analysis depicted the diffraction intensities exhibiting between 20 and 80 °C at 2theta angle thus conferring the crystalline nature of nanoparticles. Morphological characteristic using TEM revealed the polydispersity of nanoparticles with size ranging from 20 to 50 nm. Synthesized nanoparticles exhibited bactericidal activity against selected human pathogens. Nanoparticles mode of action was carried out to reveal DNA damage activity. Thus the present investigation reports facile fabrication of silver nanoparticles from endophytic fungi.

Molecular characterization of an endophytic Phomopsisliquidambaris CBR-15 from Cryptolepis buchanani Roem. and impact of culture media on biosynthesis of antimicrobial metabolites.

An endophytic fungus Phomopsis liquidambaris CBR-15, was isolated from Cryptolepis buchanani Roem. (Asclepiadaceae) and identified by its characteristic culture morphology and molecular analysis of the ITS region of rDNA and intervening 5.8S rRNA gene. The impact of different culture media on biosynthesis of antimicrobial metabolites was tested by disc diffusion assay. Polyketide synthase gene (PKS) of the endophytic fungus was investigated using three pairs of degenerate primers LC1-LC2c, LC3-LC5c and KS3-KS4c by PCR. TLC-bioautography method was employed to detect the antimicrobial metabolites. Antimicrobial metabolites fractionated with ethyl acetate extract showed significant antimicrobial activity against the test bacteria and fungi. Biosynthesis of antimicrobial metabolites was optimum as depicted by zone of inhibition from ethyl acetate extract cultured in potato dextrose broth. Strain CBR-15 was identified as Phomopsisliquidambaris and PKS genes of the fungus were amplified with LC3-LC5c and KS3-KS4c sets of degenerate primers. These findings suggest that endophytic P.liquidambaris CBR-15 harbor iterative type I fungal PKS gene domain which indicates the biosynthetic potential of endophytic fungi as producers of natural antimicrobial metabolites. The study also demonstrates the utilization and optimization of different culture media which best supports for the biosynthesis of the antimicrobial metabolites from P.liquidambaris.

Molecular profiling and antimicrobial potential of endophytic Gliomastix polychroma CLB32 inhabiting Combretum latifolium Blume.

Fungal endophytes as a source of bioactive metabolites have led to the development of pharmaceutical products finding new applications. In a survey of endophytic fungal biodiversity, an antimicrobial endophytic strain CLB32 was isolated from the leaf of Combretum latifolium Blume (Combretaceae) from the Western Ghats of Southern India. CLB32 was then identified as Gliomastix polychroma (KR704576) by morphological and phylogenetic analysis based on internal transcribed spacer (ITS) nuclear rDNA and intervening 5.8S rRNA gene. CLB32 here constituted the first report on incidence of endophytic fungi from C. latifolium Blume. Ethyl acetate fraction of strain CLB32 was evaluated for antimicrobial activity by disc diffusion assay. Secondary metabolites produced effectively inhibited methicillin-resistant Staphylococcus aureus (18.33 ± 0.33 mm), Pseudomonas aeruginosa (14.66 ± 0.33 mm) and Candida albicans (14.00 ± 0.57 mm). Biosynthesis of these antimicrobial compounds was detected by analytical TLC-bioautography method as depicted by zone of inhibition on intensive the band. These findings suggest that G. polychroma CLB32, as a producer of natural antimicrobial drugs, could help to combat against multidrug-resistant infections and also provide baseline information for industrial applications.

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Plant mediated nanoparticles' synthesis has led to a remarkable progress via unfolding a green synthesis protocol towards nanoparticles' synthesis. It seems to have drawn quite an unequivocal attention with a view of reformulating the novel strategies as alternatives for popular conventional methods. Hence, the present review summarizes the literature reported thus far and envisions towards plants as emerging sources of nanofactories.