Bioprospecting the antimicrobial, antibiofilm and antiproliferative activity of Symplocos racemosa Roxb. Bark phytoconstituents along with their biosafety evaluation and detection of antimicrobial components by GC-MS.

BACKGROUND: Plants provide a ray of hope to combat the ever increasing antibiotic resistance and Symplocos racemosa is a valuable medicinal plant. The study focused on highlighting the importance of this plant's phytoconstituents as potential source of novel antimicrobials against planktonic as well as biofilm forming microorganisms, along with their antiproliferative activity. The biosafety of the phytoconstituents was also established, followed by detection of probable antimicrobial components. METHODS: The best organic extractant and major groups of phytoconstituents were tested for their antimicrobial activity against reference microbial strains and drug-resistant clinical isolates. The anti-proliferative potential of the most active group of phytoconstituents was evaluated against cancerous cell lines. The in vitro biosafety of phytoconstituents was evaluated by Ames and MTT assay, while in vivo biosafety of the most active phytoconstituents, i.e., flavonoids was determined by acute oral toxicity. Further, the probable antimicrobial components in the flavonoids were detected by TLC and GC-MS. RESULTS: Ethyl acetate extract was the most effective among various organic extracts, whereas phytoconstituents such as flavonoids, cardiac glycosides, saponins, tannins, triterpenes and phytosterols were the major groups present, with flavonoids being the most potent antimicrobials. The phytoconstituents displayed a significant antibiofilm potential, as exhibited by inhibition of initial cell attachment, disruption of the pre-formed biofilms and reduced metabolic activity of biofilms. The phytoconstituents were significantly active against the drug-resistant strains of E.coli, MRSA and Salmonella spp. Further, flavonoids showed significant cytotoxic effect against the cancerous cell lines but were non-cytotoxic against Vero (normal) cell line. All the test preparations were biosafe, as depicted by the Ames test and MTT assay. Also, flavonoids did not induce any abnormality in body weight, clinical signs, biochemical parameters and organs' histopathology of the Swiss albino mice during in vivo acute oral toxicity studies. The flavonoids were resolved into 4 bands (S1-S4), where S3 was the most active and its GC-MS analysis revealed the presence of a number of compounds, where Bicyclo [2.2.1]heptan-2-one,1,7,7-trimethyl-, (1S)- was the most abundant. CONCLUSIONS: These findings suggest that the phytoconstituents from Symplocos racemosa bark could act as potential source of antimicrobial as well as antiproliferative metabolites.

Bioactive potential of endophytic fungus Chaetomium globosum and GC-MS analysis of its responsible components.

The recent exploration of various medicinal plants for bioactive potential has led to the growing interest to explore their endophytes for such bioactive potential which may turn out to be better option than the plants. In the present study, Chaetomium globosum, an endophytic fungus isolated from Moringa oleifera Lam has been explored for its various biological activities. The chloroformic extract of C. globosum showed good antimutagenicity against the reactive carcinogenic mutagen, 2-aminofluorene (2-AF) in Ames test. The antiproliferative activity against various cell lines such as HCT-15, HeLa and U87-MG was found to be dose dependent and the viability reduced to 9.26%, 15.7% and 16.3%, respectively. Further, the chloroformic fungal extract was investigated for free radical scavenging activity using 2, 2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethyl-benzthiazolin-6-sulfonic acid) assay which showed the IC50 value of 45.16 µg/ml and 50.55 µg/ml, respectively. The fungal extract also showed good ferric reducing power. Total phenolic and flavonoid content was found to be in linear relationship with the antioxidant potential of the fungal extract. High performance liquid chromatography showed the presence of phenolics which may help to combat the free radicals. The presence of various bioactive compounds was analysed by GC-MS which endorsed Chaetomium globosum to be a promising candidate for drug development.

Antibiofilm, antiproliferative, antioxidant and antimutagenic activities of an endophytic fungus Aspergillus fumigatus from Moringa oleifera.

An endophytic fungus Aspergillus fumigatus isolated from Moringa oleifera has been evaluated for its various bioactivities. The chloroformic fungal extract exhibited a good antimicrobial as well as antibiofilm activity against various pathogenic microorganisms. It also demonstrated a good antimutagenicity against the reactive carcinogenic ester generating mutagen, 2-aminofluorene (2-AF) with IC50 values of 0.52 mg ml-1 and 0.36 mg ml-1 in case of co-incubation and pre-incubation, respectively. The antiprolifertive activity against different cancer cell lines; such as HCT-15, HeLa A549 and U87-MG showed the IC50 values of 0.061, 0.065 and 0.072 mg ml-1, respectively. The antioxidant activity of fungal extract has been assessed by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethyl-benzthiazolin-6-sulfonicacid) (ABTS) methods with IC50 values of 40.07 µg and 54.28 µg, respectively. Total phenolics and flavonoid contents have been also determined. Ultra-high performance liquid chromatography (UPLC) of fungal extract revealed the presence of various phenolic compounds (caffeic acid, rutin, ellagic acid, quercetin and kaempferol). Further an attempt has been made to purify the bioactive compounds by column chromatography and GC-MS analysis. The above studies demonstrated a good bioactive potential of endophytic fungus Aspergillus fumigatus and shows the pharmacological importance of an endophytic fungus and justify the need to carry out further studies.

Antioxidant compounds from microbial sources: A review.

Free radicals are one or more unpaired electrons containing reactive molecules, which can damage nucleic acids, proteins, carbohydrates, and lipids, leading to several diseases including early aging, cancer and atherosclerosis. Antioxidants can scavenge these free radicals to prevent cellular damage by ultimately reducing the oxidative stress and thus have a beneficial effect on human health. Epidemiological studies have already revealed that higher intake of antioxidants as food supplements results in reduced risk of many diseases. Exploring natural antioxidants and its role in human health & nutrition is an emerging field. Several biological sources like medicinal plants, vegetables, spices and fruits have been evaluated as sources of potentially safe natural antioxidants. Beside plants, microorganisms are the potential source of novel bioactive compounds to be used in medical, agricultural, and industrial sectors. As compared to plants, microbes can be grown under controlled conditions at a faster rate, which make them a potential source of natural bioactive molecules for food and nutraceutical applications. This review summarizes the potential of different microorganisms including actinomycetes, bacteria, blue green algae, fungi, lichens and mushrooms to be explored as the source of such bioactive compounds.

Major Phytoconstituents of Prunus cerasoides Responsible for Antimicrobial and Antibiofilm Potential Against Some Reference Strains of Pathogenic Bacteria and Clinical Isolates of MRSA.

Prunus cerasoides is a traditionally well known for human health in various ways and particularly its bark is reported to possess high therapeutic applications in wound healing, foot and mouth disease, and indigestion etc. But there is scanty literature available on its systematic studies and phytoconstituents responsible for antimicrobial activity so the work is proposed. The main aim of this study is to reveal the phytoconstituents responsible for antimicrobial and antibiofilm action to demonstrate the effectiveness of such compounds by extrapolating the data using clinical isolates of pathogenic bacteria. In the present study, evaluation of P. cerasoides organic extract and phytoconstituents for their antimicrobial and antibiofilm potential against reference microbial strains was carried out. Antimicrobial potential was carried out using agar diffusion assay and biosafety of organic extract and its phytoconstituents was evaluated by MTT and Ames mutagenicity assay. Ethyl acetate was found to be the best organic extractant, where Klebsiella pneumoniae 1 (39.5 mm) and Staphylococcus aureus (22.5 mm) were the most sensitive microorganisms, respectively. Among the major phytoconstituents, flavonoids (14.5-33.5mm), diterpenes (14-28.7 mm), and cardiac glycosides (11.5-20.5mm) exhibited broad-spectrum antimicrobial activity. Ethyl acetate extract showed better potency with lowest minimum inhibitory concentration (0.1-10 mg/ml) than the most active partially purified phytoconstituents (0.5-10 mg/ml). Total activity potency for ethyl acetate extract ranged from 26.66-2666 ml/g and for flavonoids, it was 41-410 ml/g, thus considered as highly potent and bactericidal in nature as evidenced from VCC study. The major bioactive compounds were found to be biosafe. The most active phytoconstituents were found to have antibiofilm potential, as well as effective against clinical isolates of MRSA, thus, the findings indicate that P. cerasoides stem bark could be a potential source for development of broad-spectrum drugs against multidrug-resistant bugs.

In vitro antimicrobial potential of extracts and phytoconstituents from Gymnema sylvestre R.Br. leaves and their biosafety evaluation.

The in vitro antimicrobial screening of Gymnema sylvestre leaves against 13 test pathogens established its broad spectrum activity with average inhibition zone ranging from 14 to 23 mm. The antimicrobial activity of the classically- optimized aqueous extract was enhanced up to 1.45 folds, when subjected to statistical optimization using Response Surface Methodology (RSM) and was thermostable. Ethyl acetate was found to be the best organic extractant with Klebsiella pneumoniae 1 (31.5 mm) and Staphylococcus epidermidis (25.5 mm) being the most sensitive among Gram negative and Gram positive bacteria, respectively. Among the major group of phytoconstituents detected, tannins were the most abundant followed by flavonoids and phytosterols, while triterpenes were absent. Flavonoids and cardiac glycosides exhibited a broad range of antimicrobial potential, with inhibition zone ranging from 13 to 35 mm, where Candida albicans was the most sensitive organism. Ethyl acetate extract showed better potency with lowest Minimum inhibitory concentration (0.1-1 mg ml-1) than the aqueous extract (1-3 mg ml-1) and all partially purified phytoconstituents (0.1-10 mg ml-1). The ethyl acetate extract and flavonoids were highly potent, as they exhibited a total activity potency ranging from 41.4 to 1045 ml g-1. Time kill studies revealed their microbicidal action, where ethyl acetate extract had a kill time from 0 to 12 h. However, among phytoconstituents, flavonoids were the most effective (0-8 h). The MIC and time kill study was also compared to that of standard antibiotics. These findings indicate that Gymnema sylvestre can be a potential source for development of leading metabolites against pathogens of clinical importance like Pseudomonas aeruginosa, Candida albicans, Escherichia coli, Staphylococcus aureus etc. They were neither mutagenic nor cytotoxic, as revealed by Ames and MTT assay.

Antimicrobial Potential of Callistemon lanceolatus Seed Extract and its Statistical Optimization.

Plants have always been eminent source of medicinal products. Screening of the aqueous seeds extract of Callistemon lanceolatus (bottle brush) revealed its broad spectrum antimicrobial potential with an inhibition zone ranging from 13 to 28 mm against various pathogenic microorganisms. While optimizing the different parameters the antimicrobial activity was better expressed at 15 % concentration, prepared by extracting the material at 60 °C for 20 min. The extract was filtered through muslin cloth and gave best results at its natural pH. Statistical optimization by Response surface methodology enhanced the antimicrobial activity up to 1.6-fold. Minimum inhibitory concentration values of the aqueous extract of seeds of C. lanceolatus against different organisms ranged from 1-5 mg/ml. The viable cell count studies indicated a bactericidal effect against most of the pathogens. The aqueous extract was found to be relatively thermostable at 100 °C. When treated for shelf life at ambient conditions and refrigeration temperature (2-8 °C), the latter only showed a 28 % loss in antimicrobial activity. The aqueous extract was found to be biosafe when evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide reagent (MTT toxicity) assay and Ames mutagenicity assay.

Bioprocessing of wheat and paddy straw for their nutritional up-gradation.

Solid-state bioprocessing of agricultural residues seems to be an emerging and effective method for the production of high quality animal feed. Seven strains of white-rot fungi were selected to degrade wheat and paddy straw (PS) under solid-state conditions. Degradation of different components, i.e., hemicellulose, cellulose and lignin was evaluated along with nutritional parameters including; in vitro digestibility, crude protein, amino acids, total phenolic contents (TPC) etc. Effect of nitrogen-rich supplements on degradation of lignocellulosics was evaluated using two best selected fungal strains (Phlebia brevispora and Phlebia floridensis). The best selected conditions were used to upscale the process up to 200 g batches of wheat and PS. Lignin was selectively degraded up to 30 % with a limited loss of 11-12 % in total organic matter. Finally, the degraded agro-residues demonstrated 50-62 % enhancement in their digestibility. Two-threefold enhancement in other nutritional quality (amino acids, TPCs and antioxidant activity) fortifies the process. Thus the method is quite helpful to design an effective solid-state fermentation system to improve the nutritive quality of agricultural residues by simultaneous production of lignocellulolytic enzyme production and antioxidants.

Production of lignocellulolytic enzymes and enhancement of in vitro digestibility during solid state fermentation of wheat straw by Phlebia floridensis.

Degradation by white rot fungi has the potential to increase digestibility of wheat straw and thus improve its value as animal feed. To optimize conditions for production of lignocellulolytic enzymes by Phlebia floridensis during solid state fermentation of wheat straw along with enhancement of in vitro digestibility, a response surface methodology (RSM) based experiment was designed. Effect of moisture content, inorganic nitrogen source (NH(4)Cl) and malt extract on lignocellulolytic enzymes, changes in chemical constituents and digestibility of wheat straw was evaluated. With increase in moisture content, laccase production increased up to 34-fold, while Manganese peroxidase was optimally produced in the presence of almost equal amount (50-55 mg/g of WS) of NH(4)Cl and malt extract. These supplements also significantly (p<0.05) enhanced the production of CMCase and xylanase. In vitro digestibility was increased by almost 50% with a loss of 27.6% and 14.6% in lignin and total organic matter, respectively. The present findings revealed P. floridensis to be an efficient organism for lignocellulolytic enzymes production and simultaneous enhancement in in vitro digestibility of wheat straw.

Comparative production of ligninolytic enzymes by Phanerochaete chrysosporium and Polyporus sanguineus.

The aim of the present study was to compare the effect of a wide range of culture conditions on production of ligninolytic enzymes by Polyporus sanguineus and Phanerochaete chrysosporium. Lignin peroxidase production by P. sanguineus was comparable with that of P. chrysosporium, although the culture conditions giving the highest yield varied greatly between the two fungi. Highest yield of manganese peroxidase by P. sanguineus obtained in 0.5% malt extract medium and peptone or malt extract supplemented mineral salts broth could not be surpassed by P. chrysosporium in any of the optimization experiments. In addition to lignin peroxidase and manganese peroxidase, P. sanguineus also produced laccase, which was best expressed in malt extract medium supplemented with sugarcane bagasse.

Decolourisation of diverse industrial dyes by some Phlebia spp. and their comparison with Phanerochaete chrysosporium.

Three species of Phlebia, viz. P. brevispora, P. fascicularia and P. floridensis have been evaluated for their potential to decolourise eight industrial dyes including; reactive yellow, reactive orange, reactive red, rathidol scarlet, coracryl black, coracryl pink, coracryl violet and coracryl red. The cultures used for the present study were pre adapted by growing these on yeast glucose agar medium supplemented with Poly-R 478, a reference dye. The fungal cultures were grown in mineral salts broth and harvested after different incubation periods to obtain their cell free enzyme extracts which were then used to assess their ability to decolourise the above mentioned dyes. The extracts obtained from the cultures grown for six days significantly decolourised the tested dyes. The study revealed Phlebia spp. to be better dye decolourisers than Phanerochaete chrysosporium.