Plant Mediated Synthesis of Biomedical Silver Nanoparticles by Using Leaf Extract of Citrullus colocynthis

MATERIALS AND METHODS

Plant material and preparation of the extract: Fresh Citrullus colocynthis leaves were collected from the Southeast coast of Parangipettai (Tamil Nadu) India. The specimen was certified by Botanical Survey of India (BSI) Coimbatore and documented in the Herbaria of C.A.S. in Marine Biology, Annamalai University, India, during 2010. Greenish-leaves of Citrullus colocynthis were collected and used to make the aqueous extract. The leaves weighing 25 g were thoroughly washed in distilled water, dried, cut into fine pieces and were crushed into 100 mL sterile distilled water and filtered through what man No.1 filter paper. The filtrate was further filtered through 0.6 μm sized filters. They were stored at 4°C and used within a week. The experimental chemicals were purchased from Sigma Chemicals (Mumbai).

Synthesis of silver nanoparticles and purification: Ten milliliter suspension of leaf extract was added to 90 mL aqueous solution of silver nitrate (1 mM) solution separately for reduction in to Ag⁺ ions and incubated at room temperature (35°C) for about 24 h. The primary detection of synthesized silver nanoparticles was carried out in the reaction mixture by observing the colour change of the medium from greenish to dark brown. After 5 h of incubation the silver nanoparticles were isolated and concentrated by repeated (4-5 times) centrifugation of the reaction mixture at 10,000xg for 10 min. The supernatant was replaced by distilled water each time and suspension stored as lyophilized powder for the optical measurements.

CHARACTERIZATION OF SILVER NANOPARTICLES

Ultra violet -visible spectroscope analysis: After 5 h of incubation the silver nanoparticles were isolated and concentrated by repeated (4-5 times) centrifugation of the reaction mixture at 10,000xg for 10 min. The supernatant was replaced by distilled water each time and subjected for the optical measurements which was carried out by using UV-Vis spectrophotometer (UV- 2450 (Shimadzu) and scanning the spectra between 200-700 nm at the resolution of 1 nm.

Atomic force microscope: Purified SNP in suspension was also characterized their morphology using a VEeco diNanoscope 3D AFM (Atomic Force Microscope). A small volume of sample was spread on a well-cleaned glass cover slip surface mounted on the AFM stub and was dried with nitrogen flow at room temperature. Images were obtained in tapping mode using a silicon probe cantilever of 125 μm length, resonance frequency 209-286 kHz, spring constant 20-80 nm^-1 minimum of five images for each sample were obtained with AFM and analyzed to ensure reproducible results.

Fourier transform infra red spectroscope: To identify silver nanoparticles associated biomolecules, the Fourier transform infra red spectra of washed and purified silver nanoparticles powder were recorded on the Nicolet Avatar 660 FT-IR Spectroscopy (Nicolet, USA) using KBr pellets. To obtain good signal to noise ratio, 256 scans of silver nanoparticles were taken in the range of 400-4000 cm^-1 and the resolution was kept as 4 cm^-1

Antibacterial assay: Antibacterial activity of leaf extract derived silver nanoparticles was assessed using the Standard agar diffusion method with 6mm diameter Whatmann No.1 filter paper discs (Becerro et al., 1994). In this 50 μL of silver nanoparticles prepared from leaf extract was mixed in 1 mL add distilled water and applied to sterile paper discs of 6 mm diameter and standard antibiotic disc (ampicillin and tetracycline) used for control. Zobell marine agar was used for antimicrobial test. The bacteria such as E.coli, Bacillus subtillus, Proteus mirabilis, Salmonella enteritids, Staphylococcus aureus, Streptococcus pyogens and Pseudomonas aeruginosa were inoculated into Zobell marine agar plates and incubated at 27°C for 24 h. Inhibition of zone was measured after 24-48 h of incubation.

RESULTS AND DISCUSSION

The leaf extract was used for the synthesis of silver nanoparticles. The reaction started with in first hour of the incubation with silver nitrate (1 mM). This was confirmed by the appearance of brown colour in the reaction mixture. The reaction rate was maximum after 25 h of incubation as indicated by the formation of silver nanoparticles. Present findings showed resemblance to the results already reported by in the case of extract of Capsicum annum (Li et al., 2007) and in case of extract of Aloe vera (Chandran et al., 2006). They reported that when the extracts of their respective test plants were challenged with silver nitrate (1 mM). They turned brown and the intensity of colour was increased with the time of incubation. In order to verify the synthesis of silver nanopartices, the test samples were subjected to the UV-Vis spectrophotometer analysis after 5 h of incubation. A peak specific for the synthesis of silver nanoparticles was obtained at 340 nm (Fig. 1). The detailed shape and size characterization of SNP was carried with AFM for leaf extract was spherical in shape and 31 nm as evident by AFM (Fig. 2), respectively. The larger size of the nanoparticles might be due to the capping of nanoparticles by polyphenols with aromatic ring and bound amide as confirmed from FT-IR analysis.

FT-IR spectra were obtained with Avatar-660 FT-IR spectroscopy using KBr pelleting. Earlier FT-IR measurements were carried out to identify the possible biomolecules responsible for the reduction of the Ag⁺ ions and capping of the bioreduced silver nanoparticles synthesized by fungal filtrate (Sastry et al., 2010).

Fig. 1:	Uv-visible absorpation spectra at different wave length for silver nano particals synthesized from leaf extract

Fig. 2:	AFM image of the silver nano particals formed by the reaction 1 mM AgNO3 with leafbroth

Representative spectrum of the nanoparticles obtained in the present study is presented in Fig. 3. Among them, the absorption bands are observed in the region of 500-4000 cm^-1 is 536.70, 1026.87, 1243.84, 1383.9, 1452.05, 1643.25, 2860.27, 2924.82, 3403.72, 3490.41 and 3463.01 cm^-1. These peaks corresponding to amide II and III aromatic rings, ether and polyphenols were present in nanoparticles synthesized by leaf extract. Result suggests that molecules attached with silver nanoparticles have free and bound amide group. These amide groups may also be in the aromatic rings.

Fig. 3:	FI-IR spectrum for leaf extract after treatment with 1mM silver nitrate

This concludes that the compounds attached with silver nanoparticles could be polyphenols with aromatic ring and bound amide region. The amount of polyphenols could be one of the crucial parameters determining the size and distribution of silver nano particles in Syzygium cumini (Sastry et al., 2010).

The antimicrobial properties of silver compounds and silver ions had been historically recognized and applied in the wide range of applications from disinfecting medical devices and home appliances to water treatment (Chou et al., 2005). Silver ion and silver based compounds are highly toxic to micro organisms, showing strong biocidal effect against microbial species. The silver nanoparticles produced by plant extracts was showed the maximum activity against E. coli, Bacillus subtillus, Pseudomonas aeruginosa and Streptococcus pyogens and also observed that it showed no activity against Proteus mirabilis, Salmonella enteritids and Staphylococcus aureus. The bioavailability of nano copper coated antidiabetic drug may help to reduce the dose of the drug and frequency of administration (Poovi et al., 2011).

The nanoparticles synthesized using the plant system have application in the field of medicines, cancer treatment, drug delivery, commercial appliances and sensors etc (Mude et al., 2009). As compared to other biological systems the plant system shows rapid and easy biosynthesis of nanoparticles. The synthesis of silver nanoparticles by the extract of Citrullus colocynthis may therefore, serve as a green simple, cheap and eco-friendly approach.

CONCLUSIONS

Present investigation reveals the bioreduction of aqueous Ag⁺ ions by the leaf extract of the Citrullus colocynthis has been demonstrated. The reduction of the metal ions through leaf extracts leading to the formation of silver nanoparticles of fairly well-defined dimensions. This green chemistry approach toward the synthesis of silver nanoparticles has many advantages such as, ease with which the process can be scaled up, economic viability etc. applications of such eco-friendly nanoparticles in bactericidal, wound healing and other medical and electronic applications make this method potentially exciting for the large-scale synthesis of other inorganic materials (nanoparticles). Though there is a report describing synthesis of silver nanoparticles using papaya callus but the present used leaf of Citrullus colocynthis as a source which is easily available and economic unlike callus which need extensive tissue culture facilities and expertise.

ACKNOWLEDGMENTS

The authors are gratefully acknowledge to the Dean, Faculty of Marine Sciences, Annamalai University, Parangipettai, Tamil Nadu, India for providing all support during the study period.