Document Type : Original Research Paper
School of Biosciences, Mahatma Gandhi University, Kottayam, Kerala, India
The endophytic fungi Fusarium solani from the remedial plant Withania somnifera (L.) is used for development of nano material (Bio- AgNP).
The Bio AgNP was very much portrayed by UV –spec, TEM, SEM, XRD and AFM investigation and is of 10-50 nm in size.
It demonstrated antibacterial action at a concentration of 50 µg ml-1 with a RBC lysis rate of 3.906 % and about 69% cell viability in MTT assay.
The preliminary studies thus emphasise that the synthesized Bio- AgNP are safe to use as medicine.
In the area of nanotechnology evolution of dependable and green collar process for construction of nanoparticles is of great importance. Nanotechnology deals with the materials which exhibits remarkable physical, chemical and biological properties because of their nanoscaled size . Nanoparticles are particles with a size range of 1-100 nm  . A wide variety of physical and chemical methods to synthesize nanoparticles are in practice but their inherent flaws that include contamination from precursor chemicals, use of toxic solvents and generation of hazardous by products makes their usage inappropriate in biological systems. These disadvantages insisted the use of novel and well refined methods that opened the doors to explore benign and green routes for synthesizing high – yielding, low cost, non toxic and environment friendly nanoparticles . Microorganisms have been explored as potential bio factory for synthesis of nanoparticles such as cadmium sulphide, gold and silver . Sastry et al. studied that fungus Verticillium sp. and Fusarium oxysporum when exposed to gold and silver ions, reduced the metal ion fairly into metal nanoparticles. Biological synthesis of silver nanoparticles using microorganisms has received profound interest because of their potential to synthesize nanoparticles of various size, shape and morphology either extracellularly or intracellularly [5; 6; 7]. Chauhan et al., reported the extracellular biosynthesis of silver and zinc oxide nanoparticles from extracellular components of Pichia fermentans JA2.Fungi are more versatile in growth and metal tolerance in contrast to bacterial population. Hence they have been used as bio factories for synthesis of nanoparticles and offer environment friendly solutions for different problems. Bio fabricated silver nanoparticles produced by various fungi via different approaches [9; 10; 11]. Intracellular synthesized nanoparticles require additional downstream processing steps to release the particles from cells. The extracellular synthesis is economical and downstream processing is simple .
Endophytes are one such organism that are under explored and can be exploited in the biogenic synthesis of silver nanoparticles. According to Baker et al. endophytes are microbes that colonize living internal tissue of plants without causing any immediate, overt negative effects. Owing to the fact that the endophytic fungi provide a broad variety of bioactive secondary metabolites with unique structures, they could be explored for their ability to biosynthesis of silver nanoparticles to develop an efficient environment friendly process. An attempt was made to investigate endophytic fungi associated with an ethno medicinal plant Potentilla fulgens L. for their ability to synthesize silver nanoparticles and the ultra structure and size distribution of the particles were studied using electron microscopy techniques . Plant endophytic fungi are significant and unique source of natural bioactive composites with their possible applications in medicine, food and agriculture. Some of the endophytic fungi are associated with the production of anti-tumor drug taxol (paclitaxel)[14; 15; 16]. So endophytic fungi can be used as an encouraging optional unit in mycofabrication of nanoparticles with a progressive employment for potential upcoming drug .
Withania somnifera(L.) Dunal (aswaganda) is regularly used in ayurvedic medicine. It holds anti-inflammatory, antitumor, antistress, antioxidant, immunomodulatory, hematopoietic, and rejuvenating characteristics. It also influences the endocrine, cardiovascular and pulmonary system. Introductory research shows that diverse components of aswaganda display a range of remedial effects. It nurture with large population of endophytes and they contributes to the medicament compounds bring out by the plant . Hence in the current study we have adopted Withania somnifera for the isolation of endophytes and are then used for production of silver nanoparticles. The antimicrobial, preliminary cytotoxicity studies and MTT assay were carried out with the biogenic nanoparticles. The accurate component which silver nanoparticles utilize to bring about antimicrobial impact is not unmistakably known and is a wrangled about subject. There are however different speculations on the activity of silver nanoparticles on organisms to bring about the microbicidal impact. Silver nanoparticles can grapple to the bacterial cellwall and in this way enter it, consequently bringing about auxiliary changes in the cell membrane like the porousness of the cell layer and passing of the cell. There is development of “pits” on the cell surface, and there is gathering of the nanoparticles on the cell surface. The silver Nanoparticles also interact with respiratory enzymes.
Principle and extreme objective of this work is to study in point of interest the procedure variables like concentration of silver nitrate , response pH, temperature and time of response on the bio mimetic amalgamation of silver Nanoparticles. In the current study we evaluated the antimicrobial and cytotoxicity patterns of Bio- AgNP. The reports are exceptionally encouraging.Further in vitro and in vivo studies are required to establish the medicinal exploitation of the particles.
MATERIALS AND METHODS
All chemicals used in the experiment were collected from sigma (Sigma, Bangalore, India) and Medias from Himedia (Himedia Labs, India).
Isolation of Endophytic Fungi
The healthy leaf samples of Withania somnifera (L.) (ashwaganda) were collected and identified with the snippets of Herbarium of Department of Botany at St. Berchmans college, Changanacherry (RHK 6350). The samples were then cleansed in the running tap water for 5- 8 min (4 – 5 times). Then leaf samples were cut in to small fragments of 1 cm length and surface sterilized with 70% ethanol for 2 minutes proceeded by soaking in Sodium hypochlorite (NaOCl 1-13%) for 3-10 minutes and washed in 70% alcohol for 1 minute. The segments were then washed 3 – 4 times in sterile distilled water and dried by using sterile blotting paper. The dried pieces (4-5) were placed aseptically on sterile Potato Dextrose Agar plate (PDA) plates augmented with streptomycin (100 μg ml-1) and incubated at room temperature for 3 – 5 days .The potency of surface sterilization was established by spread plating the water sample used for last wash on the PDA plates and offered as control. The fungi that rise out from the portions were regularly isolated and identified by dispensing the hyphal tips to fresh PDA plates without antibiotics.
Screening of Silver Nanoparticles Produced by Isolated Endophytic Fungi
The acquired fungal isolates were cultured in liquid Potato Dextrose medium and incubated at room temperature in rotary shaker for 5 days. The biomass obtained was accumulated by a sterile sieve and cleansed with sterile distilled water to remove components of medium. It was then centrifuged at 7000 g at 4ºC for 10 minutes and the pellets were collected. Generally 20 g of biomass were carried in contact with 100 ml sterile double distilled water for 72 hours at room temperature in rotary shaker. Later the cell filtrate was obtained by filtering with sterile sieve and then centrifuged at 7000 g at 4º C for 10 minutes to remove cell debris. The collected supernatant was treated with 1mM silver nitrate (AgNO3) and incubated at dark condition. It was then observed for colour change. AgNO3without supernatant was served as control.
Identification of Fungi
The potential fungal isolates which showed colour change from colourless to yellowish brown were selected for further studies. The preliminary identification was carried out by Lacto Phenol Cotton Blue staining. Cultural characteristics were also studied. The Fungi was then identified by molecular characterization using 18 S rRNA amplification. Pure culture pellet was used for genomic DNA extraction. D1/D2 region was amplified by PCR from fungal genomic DNA using universal primers DF- 5’-ACCCGCTGAACTTAAGC-3’ and DR - 5’-GGTCCGTGTTTCAAGACGG-3’.The amplified DNA was sequenced at Sci Genomics (Sci Genomics, Cochin). The sequences were submitted to NCBI gene bank for accession code.
Purification of Silver Nanoparticles
Suspension containing Bio- AgNP was centrifuged at 5000 g at 4º C for 10 minutes. The pellets were collected and resuspended in Milli Q water and again centrifuged at 5000 g at 4º C for 10 minutes. The cleansing step was replicated three to four times to remove any impurities present in it. Ultrasound sonication was carried out with the pellets and then again subjected to centrifugation. Finally the purified nanoparticles were weighed and put back into suspension with Milli Q water. This purified nanoparticles were stored at 4º C for further characterization .
Characterization of Biosynthetic Silver Nanoparticles
Formation of Bio- AgNP was proved by UV Vis spectrophotometer (Hitachi U5100) in the visible region from 350 – 800 nm. The morphology, size, shape and distribution of nanoparticles were studied by Scanning Electron Microscope (SEM). SEM analysis of dried samples was performed by mounting nanoparticles on specimen stubs with double adhesive tape and coated with platinum in a sputter coater and examined under JEOL 6390 SEM JSM at 10 kV. Transmission Electron Microscope (TEM) analysis of drop-coated solution of nanoparticles onto the carbon-coated copper grid was carried out for better evaluation of the size and shape of the nanoparticles using JOEL-JEM-2011. The possible bimolecules associated with the synthesis and stabilization of nanoparticles was listed using Fourier Transform Infrared (FTIR) Spectroscopy analysis (Nicolet, USA) in 400 to 4000 ranges. Atomic force microscopy (AFM (APER-A-100 SPM, Italy)) was carried out for the sample to know their size and three dimensional arrangements. The purified powdered Bio- AgNP were analyzed using powder X-Ray Diffractometer (BRUKER, Germany) in a range of 20º -80º 2 Ɵ angles.
Optimization of Silver Nanoparticle Production
Prominent yield of nanoparticles were obtained by optimizing the conditions of production. Optimization studies were carried out for the crucial parameters such as temperature, pH, concentration of silver nitrate, and days of incubation. At first concentration of silver nitrate was optimized and that value was used in addition for optimization of pH. Then both optimized values were used for temperature optimization and then respectively for days of incubation. Optical density was measured at 430 nm in UV – spectral analysis for evaluating the production rate.
Antimicrobial Activities of Silver Nanoparticles
To analyse the antibacterial activity of the synthesized Bio- AgNP, antimicrobial sensitivity assay was performed by agar well diffusion in Muller Hinton Agar (MHA) plates. The pathogens such as Escherichia coli (MTCC723) Pseudomonas sp (MTCC 121), salmonella typhi (MTCC 734), Klebsiella pneumoniae (MTCC 109) and Staphylococcus aureus (MTCC 734) were used for the assay. MTCC strains of bacterial pathogens were grown in Muller Hinton broth at 37 0C for 18–24 h. A confluent lawn of bacterial growth was prepared with the pathogens and wells were cut using sterile cork borer. In to the wells AgNP was applied at concentration of 50, 100, 150 µg/ml and incubated at 37 º C. The plates were examined for appearance of zone of inhibition and the diameter of zones were measured in millimetres and compared with standard antibiotic Ciprofloxacin (Hi Media, India).
Cytotoxicity Assay-RBC Lysis Test for AgNp
In vitro erythrocyte lysis test was performed as a rudimentary cytotoxicity assay. The analysis was by quantifying the release of haemoglobin due to membrane disruption by the AgNP. Fresh blood drawn from a healthy donor was collected in anticoagulant solution (EDTA) and was centrifuged at 1000g for 10 minutes at 4º C. The washed RBC was diluted (50%) with 20mM PBS buffer. To study haemolysis RBC suspension was mixed with diverse concentrations of Bio- AgNP (50 to 150µg/ml) and incubated at 37 º C for 1 hour . It was again centrifuged at 1500 g after incubation and supernatant was interpreted by measuring the absorbance at 576 nm in UV – visible spectroscopy. The percentage haemolysis was calculated by equation (1);
Where Abs (T) is the absorbance of test and Abs(c) is absorbance for control (normal saline). Complete lysis in 1% triton X 100 act as Abs (100%).
Determination of in vitro Cytotoxic Effect of Bio- AgNp on Cultured L929 Cellsl929 (Fibroblast Cells)
Cell lines were purchased from NCCS Pune was maintained in Dulbecco’s modified eagles media (Hi Media) supplemented with 10% FBS (Invitrogen) and grown to confluency at 370C in 5 % CO2 in a humidified atmosphere in a CO2 incubator (NBS, EPPENDORF, GERMANY). The cells were trypsinized (500µl of 0.025% Trypsin in PBS/0.5 mM EDTA solution (Himedia)) for 2 minutes and passaged to T flasks in complete aseptic conditions. Extract was added to grown cells at a final concentration of 6.25, 12.5,25,50 and 100 µg/ml from a stock of 1mg/ml and incubated for 24 hours. The % difference in viability was determined by standard MTT assay after 24 hours of incubation.
MTT Assay 
MTT is a colorimetric assay that measures the reduction of yellow 3-(4, 5dimethythiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) by mitochondrial succinate dehydrogenase. The MTT enters the cells and passes into the mitochondria where it is reduced to an insoluble, coloured (dark purple) formazan product. The cells are then solubilised with an organic solvent Dimethyl sulfoxide (Himedia) and the released, solubilised formazan product was measured at 540nm. Since reduction of MTT can only occur in metabolically active cells the level of activity is a measure of the viability of the cells as in Equation (2).
The cells was washed with 1x PBS and then added 30 µl of MTT solution to the culture (MTT -5mg/ml dissolved in PBS). It was then incubated at 370C for 3 hours. MTT was removed by washing and centrifuged at top speed for 2 minutes to precipitate cell debris. Optical density was read at 540 nm using DMSO as blank in a microplate reader (ELISA SCAN, ERBA).
Primary Screening for Biosynthesis of Silver Nanoparticles
From the sterilized leaf segments of Withania somnifera fungal colonies were grown out after 5 days of incubation. The fungal colonies were then subcultured on PDA without antibiotics and maintained as pure cultures. The fungal strains were screened for their efficiency for the extracelllar production of Bio- AgNP. Cell free supernatant of one strain showed production of silver nanoparticles with a visible colour change from colour less to dark yellowish brown when exposed to silver nitrate solution Fig. 1. The potential isolate was selected and used for further studies.
Fig 1 Biosynthesis of silver nanoparticles by Fusarium solani showing colour change from colourless to brown colour
Identification of Fungal Isolate
The isolate grown on PDA showed white cottony smooth appearance and on reverse it showed a pigmentation of dull white colour to pale yellow . Hyaline crescent shaped macro conidia and small micro conidia were observed on Lacto Phenol Cotton Blue staining. Hence the isolate was preliminarily identified as Fusarium sp. Fig. 2 and was later identified by molecular characterization with 18S r RNA. Amplification of 18 S r RNA genes was carried out and sequenced. The accession code: KJ 193849 was obtained for the sequence from NCBI gene bank and the organism was identified as Fusarium solani.
Fig 2 Cultivation of Fusarium solani on the PDA and Microscopic observation showing micro and macro conidia by Lacto phenol cotton blue staining.
Characterization of Biosynthetic Silver Nanoparticles
The colour change from colourless to brownish yellow perceived was later established by UV – Vis spectroscopy analysis. To analyse the particles they were purified and were stored at 4º C for further studies. The purified nanoparticles were weighed and the total yield was calculated as 62 mg/100ml.
The initial characterization was carried out with UV –Vis spectroscopy analysis. The formation of distinguishing brown colour is due to Surface Plasmon Resonance (SPR). A distinctive strong absorption peak was observed at 424 nm Fig. 3. It indicates the reduction of silver nitrate to silver nanoparticles. In the blank solution there was no characteristic absorption spectral response.
SEM analysis of Bio - AgNP on drop coated films was recorded (Fig. 4). It confirmed the formation of AgNP. SEM micrographs showed well dispersed spherical nanoparticles with size ranging from10 to 50 nm. SEM images indicate the uniform size, distribution and spherical shape of stabilized nonagglomerated nanoparticles.
TEM micrographs Figs. 5(a) and 5(b) showed that the Bio - AgNP are spherical in nature with measurements ranging from 10 nm to 50 nm. The crystalline nature of AgNP was also analyzed and confirmed by TEM using Selected Area Electron Diffraction analysis (SAED). The SAED pattern showed well defined diffraction lattice in the form of rings in the silver region which confirmed the nanocrystalline nature of the particles.
AFM surface topology analysis of bio fabricated silver nanoparticles thin film on mica sheet showed more insight into the size, shape and distribution of nanoparticles. Spherical particles of varying size below 50 nm Fig. 6 were observed on AFM analysis. It also showed uniform distribution without agglomeration.
Additional studies were carried out using XRD to validate the crystalline nature of nanoparticles. The pattern displayed the powerful peaks in a 2Ɵ value ranging from 20 to 80. X- Ray diffractometer showed characteristic peaks at 32.3670, 38.2520 and 46.3370 Fig. 7.
These values corresponds with the plane of silver 111(38.2520 ) and 200 (46.3370 ) when compared with the standard powder diffraction card of Joint Committee on Powder Diffraction Standards (JCPDS), silver file No. 04–0783. Two strong Bragg reflections were found which correspond to pure silver metal. The particles size can be calculated by using the Equation (3):
The calculated particle sizes (Table 1) for the particles are 16.98 and 51 nm respectively.
FTIR analysis of silver nanoparticle samples was carried out in order to obtain information about functional groups involved in reduction of silver nitrate Fig. 8. Compared to silver nitrate certain new prominent peaks at band width 2918.30, 1340.55, 1195.87, 1159.22, 1134.14, 943.19 and 916.19 were observed in treated sample, which represents C-H stretch of alkenes, N-O symmetric stretch of nitro compounds, C-H wag of alkyl halides, C-N stretch of aliphatic amines and O-H bend of carboxylic acid respectively. These representative peaks contributed by the supernatant manifest the possible role of biomolecules mainly proteins in reduction of silver nitrate to nanoparticles.
Table 1 The particle size of Bio – Ag NP calculated by Debye – Scherrer formula
Fig 3 UV – Vis spectroscopy of biosynthetic silver nanoparticles showing peak at 424 nm
Fig 4 SEM micrograph showing well dispersed spherical silver Nanoparticles