INTRODUCTION:

Development of resistance to the wide range of antibiotics by the pathogenic microorganisms leads to severe problem in treatment of infectious diseases.Bioactive compounds derived from natural sources have been extensively utilized in modern medicine as antimicrobial and anti tumour agents because of high efficacy and best biocompatibility [1,2].Among the various sources, microorganism are the major candidates of bioactive producers particularly fungi. Fungi are the major group of microorganism present in diverse environment [3,4].

An endophyte is a microorganism (bacteria, actinomycetes, fungi) which present and colonizing inter- and/or intra cellular healthy tissues of the host plant, typically causing no apparent symptoms of disease.[5] Endophytes are being accepted as an important source of novel bioactive secondary metabolites that can be excellent new starting points for the development of novel pharmaceuticals and/or agrochemicals [6]. Endophytes are well known as producer of antibiotics and other biologically active substances of higher commercial value, such as vitamins, alkaloids, plant growth factors, enzymes and enzyme inhibitors. They play major role in physiological activities of host plants influencing enhancement of stress, insect, nematode and disease resistance [7,8]. Many plants are known to harbour endophytic fungi that are believed to be associated with the production of pharmaceutical products [9]. Fungal endophytes have been recognized as a repository of novel secondary metabolites, some of which have beneficial biological activities [10].A recent comprehensive study has indicated that 51% of bioactive substances isolated from endophytic fungi [11,12].These studies revealed the biological activities of endophytic fungi mediated metabolites as an effective preventive agents of infection and cancer. In the present study, antibacterial activity of ethyl acetate extract of endophytic fungi isolated from weed plant against human pathogenic bacteria

MATERIALS AND METHODS:

Isolation of endophytic fungi

P. europhyrena was isolated from leaflets of Clerodendron by surface washing method [13]. Collected leaves were washed with distilled water to remove surface debrices and the washed leaves were cut into 1cm ² using sterile blade. Cut pieces were washed by 0.1 % mercuric chloride solution followed by successive washing with sterile distilled water. Washed leaflets were placed on sterile molten potato dextrose agar plates supplemented with chloroamphenicol antibacterial antibiotic to prevent bacterial growth. Seeded plates were incubated at 28^oC for seven days. Plates were observed daily to record the fungal growth. Fungal colonies grown on the leaflets were transferred to the PDA media and slant for identification .Respective fungal organism was identified based on cultural characteristics and microscopic observation of fungal spores by lactophenol cotton blue.

Extraction of crude metabolites

Inoculum preparation

Inoculum for the crude extraction of metabolites was obtained from slant culture of fungi. Spore suspension was derived from seven days slant culture by flooding with sterile distilled water containing 0.1 % tween 80. Collected suspension thus obtained was filtered through cheese cloth and the collected was stored in sterile screw cap vial used for further studies.

Extraction

For the metabolites production, the fungal spores suspension (1ml) was inoculated into 250 ml of sterile potato dextrose broth. Seeded flasks were incubated at 30ºC for ten days kept at shaking incubator (Remi, India).After the incubation period, the flasks were filtered through the filter paper ,the filtrate thus obtained was extracted with double the volume of ethyl acetate. Extracts were concentrated on rotator vacuum evaporator under optimum condition. Concentrated extracts was collected and used for antibacterial studies.

Antibacterial activity

Anti bacterial activity of crude metabolites was studied against human pathogenic bacterial strains Pseudomonas aeuroginosa and Staphylococcus aureus by well diffusion assay. Both the strains were received from Microbial type culture collection (MTCC) and maintained on Tryptic soy agar (TSA) slants. Inocula of the respective bacterial strain was prepared in tryptic soy broth under standard condition. The respective broth culture was uniformly spread with sterile cotton swabs on sterile Mueller Hinton (MH)Agar Media (Hi-media, India). The wells were made using cork borer and different concentration of ethyl acetate extracts (10, 25, 50, 75 and 100 µg/ml was loaded into the wells. The plates were incubated at 37ºC for 24 hours. Plates were monitored for the zone of inhibition after the incubation period.

Determination of Minimum Inhibition Concentration (MIC)

Turbidometric method [14] was used to determine MIC and MBC of tested extracts against both the bacterial strains. The MIC was determined as the minimum concentration at which there is no visible change in the turbidity of the medium. The minimum bactericidal concentration (MBC), defined as the lowest concentration of sample that kills 99.9% or more of the initial inoculum, was determined in those test samples after the MIC test showed no growth.

Biofilm inhibition study

^Biofilm^inhibition was studied by the micrototre plate spectrophotometric assay [15]. Known volume (100 μl) of respective bacterial cell suspension and the respective concentration of metabolites was added into the wells of a 96-well PVC microtiter plate..The microtiter plates were covered and sealed before incubation under stationary conditions at 37 °C for 48 hours. After the incubation time, the content was discarded and the plates thoroughly washed with water. 100 μl of 0.1% aqueous solution of crystal violet was added and incubated at room temperature for 30 minutes followed by washing with water the remaining stain was solubilized with 200 μL of 95% ethanol. Biofilm inhibition was studied by determination of the absorbance of the ethanol solubilised mixture at 540 nm in an UV spectrophotometer. Control (without bacteria only crystal violet),three replicates were maintained for each treatment [16]

RESULT AND DISCUSSION:

Due to the increasing pattern of antibiotics resistance, treatment of many of the infectious disease is highly complicated. Effective and biocompatible anti microbial compounds have been isolated from biological sources specially from microorganism [8], Endophytic fungi are the unique group of organism that present in live tissues of plants known to exhibit a wide range of bioactive compounds. In the present study, endophytic fungi isolated from the leaflets of garden weed was evaluated against human pathogenic strains. A total of 16 isolates belong to 3 species were recorded in leaflets of tested weed. Fungal organisms were identified based on cultural and morphological characteristics by the standard methods. Identified fungi were Aspergillus niger, Phoma europhyrena and Penicillium sp. Among the fungal isolates, antibacterial activity was studied with metabolited derived from P. europhyrena. The active principle of the antibacterial activity of the fungal culture filtrate was extracted into ethyl acetate solvent. Metabolites thus obtained were reconstituted in solvent at different concentration and evaluated against human pathogenic bacterial strains.

Table 1. Minimum inhibition concentration (MIC) and minimum bacteriacidal concentration of ethyl acetate extract of P.europhyrena against tested bacteria

Tested Bacteria MIC ((mg/ml) MBC (mg/ml)

P.aeruginosa 21.3 41.2

Staph.aureus 32.2 51.2

Mean value of three replication

Figure 1. Zone of inhibition (mm) of ethyl acetate extract of P. europhyrena against tested bacteria

Figure 2.Biofilm inhibition (%) of ethyl acetate extract of P. europhyrena against tested bacteria

Well diffusion assay, turbidometric assay and biofilm inhibition assay was employed to study the antibacterial activity. Both the tested strains were susceptible to the all the tested concentration of fungal extracts as dose dependent manner. An increase in zone of inhibition was observed in high concentration (figure 1). Turibidometric assay used to study determination of MIC and MBC of the fungal extracts.MIC and MBC values were presented in table 1.It can be seen that ethyl acetate extract showed maximum inhibition at least concentration which revealed high antibacterial efficacy of the extracts against the both tested bacterial strains. Further confirmation of antibacterial activity was carried out by biofilm inhibition assay using crystal violet microtitre plat assay. Results were represented as inhibition percentage of biofilm development by the earlier studies [15]. Biofilm inhibition was observed as dose dependent manner (figure 2).Maximum inhibition was recorded at high concentration which can be seen in both the tested bacterial strains. Antibacterial activity of the endophytic fungal metabolites have been reported in previous works would support the present findings. Potential antibacterial and antifungal activity of endophytic fungal metabolites isolated from Quercus variabilis reported by Wang et al [13].Effective inhibition of bacterial strains is due to the presence of bioactive compounds in the ethyl acetate extract of P. europhyrena and the future study will helpful to find out the possible bioactive compounds in the extract would helpful to use as an effective antibacterial agent.

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Received on 20.03.2016 Modified on 20.04.2016

Research J. Pharm. and Tech. 9(4): April, 2016; Page 437-439

DOI: 10.5958/0974-360X.2016.00080.9