INTRODUCTION:

Nanotechnology is the newest and one of the most promising areas of research in modern medical sciences. Nanoparticles exhibit new and improved properties based on size, distribution and morphology than larger particles of the bulk materials from which the nanoparticles are made. Metal nanoparticles which have a high specific surface area and a high fraction of surface atoms have been studied extensively because of their unique physicochemical characteristics including catalytic activity, optical properties, electronic properties, antibacterial properties and magnetic properties [1]. Synthesis of noble nanoparticles for the applications such as electronics, environmental and biotechnology is an area of constant interest [2]. Generally metal nanoparticles are synthesized and stabilized by using chemical methods such as chemical reduction [3], electrochemical techniques [4] and now a days via green chemistry route [5]. Metal nanomaterials like copper, zinc, titanium [6], magnesium, gold [7], alginate [8] and silver have come up but silver nanoparticles have proved to be most effective as it has good antimicrobial efficacy against bacteria. Of these, silver nanoparticles plays a major role in the field of nanotechnology and nanomedicine [9, 10].

Use of plants in synthesis of nanoparticles is quite novel leading to truly green chemistry which provide advancement over chemical and physical method as it is cost effective and environment friendly easily scaled up for large scale synthesis and in this method there is no need to use high pressure, energy, temperature and toxic chemicals [11]. An important branch of biosynthesis of nanoparticles is the application of plant extract to the biosynthesis reaction. Synthesis of quasi spherical silver nanoparticles using purified apiin compound, extracted from henna leaf at ambient conditions [12]. Using green tea, Camellia sinensis extract as reducing and stabilizing agents produced gold nanoparticles and silver nanostructures in aqueous solution at ambient conditions [13]. Plant extracts from live Alfalfa, the broths of lemongrass, geranium leaves and others have served as green reactants in silver nanoparticles synthesis [14]. The reaction of aqueous silver nitrate with an aqueous extract of leaves of a common ornamental geranium plant, Pelargonium graveolens gave silver nanoparticles after 24 h [15]. A vegetable, Capsicumannum L., was also used to synthesize silver nanoparticles [16].

Cocculus hirsutus (L.) is a widely growing plant found in the plains of India in dry localities and is used medicinally by the Indian tribes for a wide range of ailments, including constipation and kidney problems [17, 18, 19]. The aqueous extract of this palnts are used for the treatment of rheumatism, fever and also as anti-inflammatory and analgesic [20, 21]. In the present investigation, we report the easy synthesis of silver nanoparticles by an environmental friendly procedure involving the insitu reduction of Ag by Cocculus hirsutus leaf extracts and the evaluation of their antimicrobial activity against various human pathogenic bacteria.

MATERIALS AND METHODS:

Plant material and preparation of the extract

Fresh and healthy Cocculus hirsutus leaves were collected from the botanical garden of Periyar university, leaves were washed thoroughly with distilled water, incised into small pieces and air dried. About 25g of those finely cut Cocculus hirsutus leaves were weighed and transferred into 500ml beaker containing 100ml distilled water, mixed well and boiled for 25min. The extract obtained was filtered through Whatman No.1 filter paper and the filtrate was collected in a 250ml Erlenmeyer flask and stored in refrigerator for further use.

Biosynthesis of silver nanoparticles

Silver nitrate used in this study was obtained from Himedia Laboratories Pvt. Ltd., Mumbai, India. 1.5g of the Cocculus hirsutus leaves extract were boiled in 100ml of deionized water. 2.5ml of ammonium solution was added to 5ml of 1mM silver nitrate solution, followed by addition of plants extract 1-10ml and the final volume was adjusted to 50ml by adding the appropriate amount of deionized water. For silver nanoparticles, the solution turned from yellowish to bright yellow and then to dark brown. The Erlenmeyer flasks were incubated at 37°C under agitation (200 rpm) for 5h [12].

Characterization of Silver nanoparticles

UV-Visible spectroscopy analysis

In order to study the formation of silver nanoparticles, the UV-Visible (UV-vis) adsorption spectrophotometer was used. UV-vis spectral analysis was performed by using Systronics 2202 double beam model spectrophotometer. The UV-vis spectra were recorded at room temperature using the periodic sampling of quartz cuvette with a UV-vis spectra wavelength range 300-700 nm.

Fourier Transform infrared spectroscopy (FTIR)

FTIR measurements were carried out using spectrum RXI MODEL FTIR spectroscopy. After complete reduction of silver nitrate ions by Cocculus hirsutus leaf extract, the solution was centrifuged at 8000 rpm for 10 minutes to isolate silver nanoparticles free from protein or other bioorganic compounds present in the solution. The silver nanoparticles pellet obtained after centrifugation was redispersed in water and washed with distilled water for three times. The purified suspension was air dried to obtain fine powder. Finally, the dried nanoparticles were analysed by FTIR.

X- Ray Diffraction spectroscopy (XRD)

The crystalline metallic silver structure and composition were analysed by Shimadzu MODEL XRD 6000. The silver nanoparticles solution obtained was purified by repeated centrifugation at 8000 rpm for 10 minutes followed by redispersion of the pellet of silver nanoparticles in sterile distilled water. The pellet was air dried. After air drying the purified pellet was collected for analyzing crystalline nature of silver nanoparticles.

Scanning electron microscopy (SEM)

Scanning electron microscopy analysis was carried out using JEOL-MODEL-6390 SEM machine. SEM was used to record the micrograph images of synthesized silver nanoparticles. Thin films of the sample were prepared on a carbon coated copper grid by just dropping a very small amount of the sample on the grid, extra solution was removed using a blotting paper and then the film on the SEM grid were allowed to dry by keeping it under a mercury lamp for 5 min.

Antimicrobial activity by well diffusion method

The silver nanoparticles synthesized from Cocculus hirsutus were tested for their antimicrobial activity by well diffusion method against pathogenic organisms like Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Bacillus subtilis. The pure cultures of organisms were subcultured on Muller Hinton broth at 35°C on rotary shaker at 200 rpm. Each strain was swabbed uniformly on the individual plates using sterile cotton swab. Wells of size 6mm have been made on Muller Hinton agar plates using gel puncture. Using micropipette 50μl of the sample of nanoparticles solution were poured into wells on all plates. After incubation at 35°C for 18h, the different levels of zone of inhibition were measured in millimetre.

RESULTS AND DISCUSSION:

The current study which shows that the aqueous silver ions were reduced to silver nanoparticles when added to natural plant extract of Cocculus hirsutus. It was observed that the color of the solution turned from yellow to bright yellow and then to dark brown after 5h of the reaction, which indicated the formation of silver nanoparticles. Absorption spectra of silver nanoparticles formed in the reaction media has absorbance peak at 360nm-380nm and the broadening of peaks indicated that the particles are polydispersed were shown in the figure 1. Almost all similar results were observed in leaf extracts of Clerodendrum inerme, Euphorbia hirta and Argimone maxicana [22, 23, 24].

FTIR analysis was used for the characterization of the extract and the resulting nanoparticles. FTIR absorption spectra of water soluble extract before and after reduction of Ag ions were shown in the figure 2. Absorbance bands in figure 2 are observed in the region are 3429.11cm^-1, 2361.49cm^-1, 1628.91cm^-1, 1383.48cm^-1, 1271.02cm^-1, 1020.89cm^-1, 669.46 cm^-1. The peak produced at 3429.11cm^-1 represents the stretching and H-bonded (phenols and alcohol), 2361.49cm^-1 represents the H-C=C-H stretching (aldehydes), 1628.91cm^-1 denotes the N-H (primary amines), 1383.48cm^-1 represents C-H (alkanes), 1271.02cm^-1 adenotes the C-N stretching (aromatic amines), 1020.89cm^-1 represents the C-N stretching (aliphatic amines), 669.46cm^-1 denotes the N-H (primary, secondary amines). These chemical groups are functional groups of many compounds and previously proved to have potential reducing agents in the synthesis of silver nanoparticles. Analysis of FTIR studies were confirmed that the carbonyl group from the amino acid residues and proteins has the stronger ability to bind metal indicating that the proteins could possibly from the metal nanoparticles (i.e., capping of silver nanoparticles) to prevent agglomeration and thereby stabilize the medium. This suggests that the biological molecules could possibly perform dual functions of formation and stabilization of silver nanoparticles in the aqueous medium [25].

Figure 1. UV-vis spectra of Silver nanoparticles synthesized from Cocculus hirsutus

Figure 2. FTIR spectra of Silver nanoparticles from the Cocculus hirsutus.

Figure 3. X-ray diffraction pattern of Silver nanoparticles synthesized by Cocculus hirsutus

Figure 4. SEM image of Silver nanoparticles formed by Cocculus hirsutus

Figure 5. Antimicrobial activity of Silver nanoparticles from Cocculus hirsutus

XRD clearly indicates the silver nanoparticles formed by reduction of silver ions by Cocculus hirsutus were shown in the figure 3. The Braggs reflections were observed in the XRD pattern at 2θ values of 27.9, 32.3, 38, 47 and 54. These Braggs reflections clearly indicated the presence of (111), (220) (226), (264) and (311) sets of lattice planes and further on the basis that they can be indexed as face-centered-cubic (FCC) structure of silver. Hence XRD pattern thus clearly illustrated that the silver nanoparticles formed in this present synthesis are crystalline in nature. No additional diffraction peaks were observed other than the characteristic peak of the silver structure that reflects the purity of synthesized silver nanoparticles, which is comparable with the Join Committee on Power Diffraction Standard (JCPDS) value. The silver nanoparticles synthesized with the help of Cocculus hirsutus extract were scanned using SEM from which we can conclude that the average mean size of silver nanoparticles was 139nm and it seems to be spherical in morphology were shown in the figure 4.

The antimicrobial activity of silver nanoparticles synthesized by natural plants extract was investigated against various pathogenic organisms such as Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli and Bacillus subtilis using well diffusion method. The diameter of inhibition zones (mm) around each well with silver nanoparticles solution were shown in the figure 5. The silver nanoparticles synthesized by Cocculus hirsutus were found to have highest antimicrobial activity against Pseudomonas aeruginosa (29 mm) respectively and the lesser antimicrobial activity of silver nanoparticles synthesized by Cocculus hirsutus extract was found against Staphylococcus aureus (19mm), Escherichia coli (23mm) and Bacillus subtilis (16mm). According to Morones et al., However these silver nanoparticles get attached to the cell membrane and also penetrated inside the bacteria. The bacterial membrane contains sulfur containing proteins and the silver nanoparticles interact with these proteins in the cell as well as with the phosphorus containing compounds like DNA.When silver nanoparticles enter the bacterial cell it forms a low molecular weight region in the center of the bacteria to which the bacteria conglomerates thus, protecting the DNA from the silver ions. The nanoparticles preferably attack the respiratory chain, cell division finally leading to cell death. The nanoparticles release silver ions in the bacterial cells, which enhance their bactericidal activity [26, 27].

CONCLUSION:

The silver nanoparticles have been produced by Cocculus hirsutus, which is an economical, efficient and eco-friendly process. UV-vis spectrophotometer, FTIR, XRD, and SEM techniques have confirmed the reduction of silver nitrate to silver nanoparticles. The zones of inhibition were formed in the antimicrobial screening test indicated, that the silver nanoparticles synthesized in this process has the efficient antimicrobial activity against pathogenic bacteria. The biologically synthesized silver nanoparticles could be of immense use in medical field for their efficient antimicrobial function.

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Received on 29.06.2013 Accepted on 07.07.2013

Asian J. Pharm. Tech. 2013; Vol. 3: Issue 3, Pg 93-97