Endophytic fungi mediated silver nanoparticles as effective of antibacterial agents

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1 World Journal of Pharmaceutical Sciences ISSN (Print): ; ISSN (Online): Published by Atom and Cell Publishers All Rights Reserved Available online at: Original Article Endophytic fungi mediated silver nanoparticles as effective of antibacterial agents T. Thaslimmunisha, R. Bharathidasan, L. Prince PG & Research Department of Microbiology, Marudupandiyar College, Vallam , Thanjavur Dist, Tamilnadu, India ABSTRACT Received: / Revised: / Accepted: / Published: In the present study of fifteen endophytic fungi isolated from Andrographis paniculata and Carica papaya plant leaf. Among, six genera and fifteen species was identified by lactophenol cotton blue mounting techniques. To induce the biosynthesize of silver nanoparticles () using Aspergillus flavus, Aspergillus terreus and Penicillium janthinellum and evaluate their antibacterial potential activity. The characterization of silver nanoparticles on the fungi by FT-IR, UV-VIS spectroscopy and scanning electron microscopic (SEM) analysis were performed to study the structural morphology of the biosynthesized silver nanoparticles. Antibacterial activity was performed using agar well diffusion method against Escherichia coli, Klebsiella pneumoniae, Streptococcus pyogens, Staphylococcus aureus and Pseudomonas aeruginosa. Further, investigations in the field can lead to the improvement of the medicinal methods for the treatment of microbial infections. Keywords: Silver nanoparticles synthesizing Aspergillus flavus, Aspergillus terreus and Penicillium janthinellum, FT-IR, UV-VIS spectroscopy, Scanning electron microscopy (SEM), Antibacterial agents INTRODUCTION Endophytic microorganisms are recognized as one of the most chemically promising groups of microorganisms in terms of diversity and pharmaceutical potential. These are microorganisms that grow in the intercellular spaces of higher plants without causing visible damage to their hosts and comprise especially fungi and bacteria [1].These microorganisms, in some way contribute to the wellbeing of the plant and being associated with living tissues they are not considered as saprophytes. There are reports indicating the endophytic organisms to be the chemical synthesizers inside the host plant [2]. The biosynthesized chemicals include bioactive compounds used by the host as a defense against pathogens. Some of these bioactive compounds have been proven to be a source for normal drug which are reported to be useful as agro-chemical, antibiotics, immunosuppressant, antimicrobial, anti-parasitic, antioxidant, anticancer agents [3]. Many natural products associated with endophytic fungi have been to be potential as antifungal, antioxidant,anticancer, anti-inflammatory and antimicrobial agents [4]. Nanotechnology is an emerging field of science which involves synthesis and development of various nanomaterials. At present, different types of metal nanomaterials are being produced by copper, zinc, titanium, magnesium, gold, alginate and silver. These nanomaterials were used in various fields such as optical devices, catalytic, bactericidal, electronic, sensor technology, biological labeling and treatment of some cancers. Nanotechnology involves the production, manipulation and use of materials ranging in size from less than a micron to that of individual atoms [5]. One of the most important criteria of nanotechnology is that of the development of clean, nontoxic and eco-friendly green chemistry producers [6]. Silver nanoparticles have found potential application in many fields such as antibacterial effect, biological sensors, drug delivery, textile and filters [7]. Nanoparticles can be synthesized by physical, chemical and biological methods [8]. Biological methods for nanoparticle synthesis would help circumvent many of the detrimental features by enabling synthesis at mild ph, pressure and temperature and at a substantially lower cost. A number of microorganisms such as bacteria, fungus, yeast and plants either intra or extracellular [9] which are of higher production yields and with low expenses have been found to be capable of *Corresponding Author Address: Thaslimmunisha T., Marudupandiyar College, Thanjavur, Tamilnadu, India: thaslimmunisha@gmail.com

2 synthesizing nanoparticles. Fungi are ideal candidates in the synthesis of metal nanoparticles, because of their ability to secrete large amount of enzymes [8]. Silver nanoparticles, having a long history of general use as an antiseptic and disinfectant, are able to interact with disulfide bonds of the glycoprotein/protein contents of microorganisms such as viruses, bacteria [10,11] and fungi [12]. Both silver nanoparticles and silver ions can change the three dimensional structure of proteins by interfering with S-S bonds and block the functional operations of the microorganism [13,14]. MATERIALS AND METHODS Sample collection: The healthy leaf samples were collected during day time from vallam employing sterile polythene bags. The fresh cut ends of plant samples were placed in zip-lock plastic bags and stored less than 72hrs in a refrigerator prior to isolation of endophytic fungi. Samples were cleaned under running tap water and then air dried. Materials 70% ethanol (70 ml of ethanol in 30 ml distilled water. 0.1% Mercuric chloride (0.1g of mercuric chloride in 100ml distilled water). Potato Dextrose Agar (PDA) : Potato -200g, Dextrose 20g, Agar 18g, Distilled water 1000ml. Streptomycin (purchased from Himedia Laboratories Pvt, Ltd, India). Potato Dextrose Broth: Potato-200g, Dextrose- 20g. Potato was boiled and dextrose was added to the potato extract. 1Mm (0.2 g of was dissolved in 100ml of deionized water). Isolation of the endophytic fungi: Leaf samples of Andrographis paniculata and Carica papaya were cleaned under running tap water to remove debris and then air dried and processed within 5hrs of collection. From each leaf sample, 4 segments of 1cm length were separated and treated as replicates. Surface sterilization was carried out by submerging them in 70% ethanol for 2 min. The explants were further sterilized sequentially in 5.3% sodium hypochlorite (NaOCl) for 5 min and 70% ethanol for 0.5 min [15]. Samples were allowed to dry on paper towel in a laminar air flow chamber. Four segments per plant were placed horizontally on separate Petri dishes containing Potato Dextrose Agar (PDA). After incubation at 28 o C for three days, the endophytic fungi was collected placed onto PDA and incubated for 3 days and checked for culture purity. Eventually, pure cultures were Thaslimmunisha et al., World J Pharm Sci 2016; 4(4): transferred to PDA slant tubes and subcultured regularly. Morphological features: Microscopically, the endophytic fungal isolates were identified on the basis of their hyphal features, arrangement of spores and reproductive structures using lactophenol cotton blue mounting techniques [16]. Production of biomass and synthesis of Silver Nanoparticles: The fungi obtained were grown aerobically in liquid broth containing potato dextrose broth. The culture flasks were incubated on room temperature at 28 o C.The biomass was harvested after7 days of growth by sieving through a plastic sieve followed by extensive washing with sterile double distilled water to remove any medium components from the biomass. Typically 25g of biomass (wet weight) were brought into contact with 100 ml sterile double-distilled water for 72 hours at 28 o C in an Erlenmeyer flask and agitated at 150 rpm. After incubation the cell filtrate was obtained by filtering using Whatman filter paper No ml of cell filtrate is challenged 1mm silver nitrate and incubated under dark conditions [17]. Characterization of silver nanoparticles UV-VISIBLE SPECTROSCOPY: The formation of silver nanoparticles was monitored by visual observation of color change from pale white to reddish brown and was further confirmed by sharp peaks given by silver nanoparticles in the visible region from UV-visible spectrum of the reaction solution using double beam UV visible spectrophotometer [18]. FT-IR Data: Silver nanoparticles solution was purified by centrifugation at 10,000 rpm for 15 min, and then the pellets were resuspended in sterile distilled water and again centrifuged at 10,000 rpm for 10 min. The collected pellets were air dried at room temperature for IR analysis. The probable biomolecules involved in the synthesis and stabilization of nanoparticles was recorded by FT-IR spectrum [18] SEM ANALYSIS: The silver nanoparticle synthesized using Fungi were allowed to dry completely by fixing the fungal mat at various percentage of acetone. Finally the fungal samples were fixed in 100% acetone for SEM analysis. Since the specimen is at high vacuum, living cells and tissues and whole, soft bodied organisms usually require chemical fixation to preserve and stabilize. Fixation is usually performed by incubation in a solution of a buffered chemical fixative, such as glutraldehyde. The fixed tissue is then dehydrated [19].

3 ANTIBACTERIAL ACTIVITY OF SILVER NANOPARTICLES: Biosynthesis of silver nanoparticles was studied for antibacterial activity against pathogenic bacteria (clinical isolates) using agar well diffusion method [20,21]. The test organisms used were Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus aureus and Streptococcus pyogens. The bacterial test organisms were grown in nutrient broth for 18 hrs. Lawns of pathogenic bacteria were prepared on nutrient agar plates using swabs. Agar wells were made on nutrient plates using sterile steel cork borer and each well was loaded with 200µl silver nanoparticle solution. The plates containing bacterial and silver nanoparticles were incubated at 37 o C. The plates were examined for the zone of inhibition, which appeared as clear area around the wells. Inhibition zone diameter was observed. RESULT AND DISCUSSION Isolation of endophytic fungi: From the surface sterilized leaf segment of Andrographis paniculata and Carica papaya, the endophytic fungi was grown from from the cut ends of the leaves after 48 hrs and luxuriant growth after 72 hrs. Subculturing was done on PDA, The microscopic images and morphological characteristic features study revealed that the fungal isolate is six genera and fifteen species Aspergillus flavus, Aspergillus terreus and Penicillium janthinellum. Extracellular synthesis of silver nanoparticles: Enzyme filtrate was treated with equal volume of 1mM silver nitrate solution, the color change from pale white to reddish brown was observed after 24rh, indicating the formation of silver nanoparticles with the reduction of silver ions. Characterization of silver nanoparticles Antibacterial Activity of Silver Nanoparticles: Antibacterial activity of biosynthesized silver nanoparticles were studied against pathogenic bacteria (clinical isolates) using agar well diffusion Thaslimmunisha et al., World J Pharm Sci 2016; 4(4): method and zone of inhibition were depicted. Wells were loaded loaded with same concentration 200µl of silver nanoparticles. Maximum zone of inhibition (29mm) was observed with Klebsiella penumoniae at 200µl of in Penicillium janthinellum. Minimum zone of inhibition (8mm) was observed with Escherichia coli Verma et al., [24] reported the antibacterial properties of silver nanoparticles produced by endophytic fungi, Aspergillus clavatus which revealed the zone of inhibition of 16mm in case of Pseudomonas sp and 10mm in case of E.coli. Similartly, reports of swetha sunkar and vallinachiyar [25] regarding antibacterial activity of. Fig:1 Antibacterial activity of silver nanoparticles with Aspergillus terreus 78

4 Thaslimmunisha et al., World J Pharm Sci 2016; 4(4): Fig:2 Antibacterial activity of silver nanoparticles with Aspergillus flavus Fig:3 Antibacterial activity of silver nanoparticles with Penicillium janthinellum TABLE: 1 Zone of inhibition AgNO3 produced by the endophytic fungi against pathogenic bacteria Name of the organism A.terrus With A.terreus Without A.flavus with A.flavus without P.janthinellum with Escherichia coli 13mm _ 9mm _ 8mm _ P.janthinellum Without Klebsiella pneumoniae Streptococcus pyogens Staphylococcus aureus Pseudomonas aeruginosa 24mm _ 16mm _ 29mm _ 17mm _ 15mm _ 15 mm _ 17mm _ 17mm _ 10mm _ 16mm _ 18 mm 14 mm 79

5 UV-VISIBLE SPECTROSCOPY: Silver nanoparticle synthesized, initially observed by color change from pale white to reddish brown was further confirmed by UV- visible spectroscopy. The color change occurs due to the excitation of surface Plasmon resonance in the silver metal nanoparticle. Silver nanoparticles from endophytic fungi, Pencillium janthinellum maximum absorbance at 445 nm after 24h of incubation, Thaslimmunisha et al., World J Pharm Sci 2016; 4(4): implying that the bioreduction of.surface Plasmon peaks were also located at 410nm as reported by Shivaraj et al., [21] using Aspergillus flavus. Whereas, Afreen et al., [22] reported peak at 422nm with Rhizopus stolonifer. Maliszewska et al., [23] reported the absorption spectrum of spherical silver nanoparticles produced Penicillium janthinellum presents a maximum peak between nm. Fig:4 UV visible spectroscopy of AgNo 3 of endophytic fungi Penicillium janthinellum FOURIER TRANSFORM INFRARED SPECTROSCOPY (FT-IR) FTIR spectroscopic analysis is carried out to determine the possible interaction between silver and bioactive molecules which are responsible for the synthesis and stabilization of silver nanoparticles. FT-IR spectrum revealed that the silver nanoparticles synthesized from endophytic fungi,penicillium janthinellum. The representative spectra of nanoparticles obtained manifest absorption peak located at about cm -1 (- NH group of amines), cm -1 (-OH group of phenols), cm -1 (-NHCO of amide), and cm -1 (C-Cl). [26]. SEM ANALYSIS: The size and shape of the nanoparticles that plays a significant role in their function is identified by SEM analysis. The SEM micrographs recorded showed comparatively spherical nanoparticles that were observed to be uniformly distributed. Acknowledgement The authors wish to acknowledge the staffs and students of the Department of Microbiology, Marudupandiyar College, Thanjavur, for their cooperation and use of the department s laboratory facilities. 80

6 Thaslimmunisha et al., World J Pharm Sci 2016; 4(4): Fig:5 FTIR spectrum showing the presence of proteins as capping agents for AgNO3, synthesized by endophytic fungi Penicillium janthinellum Fig:6 SEM image show silver nanoparticles, synthesized by endophytic fungi Penicillium janthinellum REFERENCE 1. Petrini O. Fungal endophytes of tree leaves. In: Andrews JH, Hirano SS (Ed) Microbial Ecology of the Leaves. Springer- Verlag, NewYork. 1991; pp. 2. Owen NL, Hundley N. Biodiversity of Marine derived fungi and identification of their metabolites. Sci. Prog. 2004; 87: Gunatilaka AAL. Natural products from plant associated microorganisms: Distribution, Structural diversity, bioactivity and implications of their occurrence. J. Nat. Prod. 2006; 69: Verma VC et al. Chemical and functional diversity of natural products from plant associated endophytic fungi.nat. Prod. Commun.2009; 4: Narayanan KB Sakthivel N. Biological synthesis of metal nanoparticles by microbes. Adv. Colloid Interface Sci. 2010; 156: Sharma VK,Yangard RA. Green synthesis and antimicrobial activities. J. Colloid Interface Sci. 2009; 9: Eleechiguerra J et al.interaction of silver nanoparticles with HIV-I. Nanobiotechnol. 2005; 03: 06 81

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