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Sunlight-induced rapid and efficient biogenic synthesis of silver nanoparticles using aqueous leaf extract of Ocimum sanctum Linn. with enhanced antibacterial activity
© Brahmachari et al.; licensee Springer. 2014
- Received: 4 September 2014
- Accepted: 27 November 2014
- Published: 29 December 2014
Nanotechnology is now regarded as a distinct field of research in modern science and technology with multifaceted areas including biomedical applications. Among the various approaches currently available for the generation of metallic nanoparticles, biogenic synthesis is of increasing demand for the purpose of green nanotechnology. Among various natural sources, plant materials are the most readily available template-directing matrix offering cost-effectiveness, eco-friendliness, and easy handling. Moreover, the inherent pharmacological potentials of these medicinal plant extracts offer added biomedical implementations of the synthesized metal nanoparticles.
A robust practical method for eco-friendly synthesis of silver nanoparticles using aqueous leaf extract of Ocimum sanctum (Tulsi) as both reducing and capping agent, under the influence of direct sunlight has been developed without applying any other chemical additives. The nanoparticles were characterized with the help of UV-visible spectrophotometer and transmission electron microscopy (TEM). The prepared silver nanoparticles exhibited considerable antibacterial activity. The effects were more pronounced on non-endospore-forming Gram-positive bacteria viz., Staphylococcus aureus, Staphylococcus epidermidis, and Listeria monocytogenes than endospore-forming species Bacillus subtilis. The nanoparticles also showed prominent activity on Gram-negative human pathogenic Salmonella typhimurium, Escherichia coli, Pseudomonas aeruginosa, and plant pathogenic Pantoea ananatis. A bactericidal mode of action was observed for both Gram-positive and Gram-negative bacteria by the nanoparticles.
- Silver nanoparticles
- Ocimum sanctum
- Antibacterial activity
- Mode of action
Nowadays, nanotechnology is regarded as a distinct field of research in modern science and technology with multidirectional applications -. Useful application of nanotechnology in medicinal purposes is currently one of the most fascinating areas of research. Metallic nanoparticles (NPs) have also been receiving considerable interest in biomedical applications ,; silver nanoparticles (AgNPs), in particular, are finding applications to the researchers as tools for antibacterial and antifungal , anti-inflammatory , wound healing , radio-imaging, retinal neovascularization ,, antiviral and antioxidant  agents, and also as novel cancer therapeutics, capitalizing on their unique properties to enhance potential therapeutic efficacy ,. In an in vivo experiment, silver oxide nanoparticles were found to exhibit notable antitumor efficacies in transplanted Pliss lymphosarcoma tumor models when administered by intravenous injection in the form of aqueous dispersions . Interest in the clinical uses of silver nanoparticles has been facilitated due to their rapid availability as well. The diameters of AgNPs are normally smaller than 100 nm and contain 20 to 15,000 silver atoms per particle ,. Thus, when cells or tissues get exposed to AgNPs, the active surface area of the nanoparticles become significantly large compared to that of the ordinary silver compounds. Such enhanced surface area of metallic nanoparticles is supposed to be responsible for exhibiting remarkably unusual physicochemical properties and biological activities . It is also assumed that promising inhibitory function of AgNPs originates from their interactions with sulfur-containing proteins as well as with phosphorus-containing compounds like DNA inside the microbial membranes . Besides, silver-embedded fabrics are now used in textile industry in manufacturing sporting equipments .
Among the various approaches currently available for the generation of metallic nanoparticles, biogenic synthesis that avoids the use of toxic and hazardous chemicals is of increasing demand for the purpose of green nanotechnology. It is well-established that biological efficacies of synthesized nanoparticles largely depend on the nature and concentration of capping agent(s) used for stabilizing the nanoparticles. Several matrixes for the biogenic synthesis of such nanoparticles are reported so far, and they include microorganisms such as bacteria , fungi , enzymes , and useful medicinal plant extracts ,,,. Among these natural sources, plant materials are the most readily available template-directing matrix offering cost-effectiveness, eco-friendliness, and easy handling much suitable for scaling up processes. Uses of plant materials in generating metallic nanoparticles in both micro- and macroscales are, thus, more advantageous over the microorganism-based methods involving complicated and sensitive cell culture processes. Moreover, the inherent pharmacological potentials of these medicinal plant extracts offer added biomedical implementations of the synthesized metal nanoparticles -.
In compliance with this view, varying extracts of a huge number of medicinal plants such as Azadirachta indica, Boswellia ovalifoliolata, Carica papaya, Catharanthus roseus, Cinnamomum camphora, Citrus aurantium, Datura metel, Jatropha curcas, Medicago sativa, Nelumbo nucifera (lotus) , Pelargonium graveolens, Solanum melongena, Tridax procumbens, etc. have already been used to synthesize and stabilize metallic nanoparticles, very particularly silver (Ag) and gold (Au) nanoparticles ,. However, a little has been carried out on engineering approaches, viz. rapid nanoparticles synthesis using plant extracts and size control of the synthesized nanoparticles ,. Besides, the uses of edible plants are in tremendous demand for the biomedical applications of AgNPs.
In ancient Ayurveda, ‘Tulsi’ (Ocimum sanctum Linn., family: Limiaceae) is known as the elixir of life since it promotes longevity and is used in many formulations for the prevention and cure of various ailments . All parts of the plant such as fresh leaves, juice, seeds, and volatile oil are very beneficial to us. The O. sanctum plant finds wide applications in the treatment of cough, coryza, hay asthma, bronchial infections, bowel complaints, worm infestations, and kidney stones in traditional systems of medicine ,. O. sanctum possesses diverse pharmacological properties that include antioxidant , antibiotic, antidiabetic, antiatherogenic, immunomodulatory ,, anti-inflammatory ,, analgesic, antiulcer , and chemo-preventive and antipyretic properties . Tulsi leaf extract reduces blood glucose and cholesterol and promotes immune system function , and one of the constituents, β-elemene, has been reported to have potent anticancer property . The major phytochemicals present in O. sanctum plant belong to terpenoid, phenolic, tannin, steroid, alkaloid, and saponin class of compounds .
That is why O. sanctum plant has recently drawn an attention for its possible uses in the biogenic synthesis and stabilization of metal nanoparticles ,,,; however, these reported methods suffer from certain shortcomings such as the use of other chemical additives and heating conditions. Hence, development of environmentally more benign, cost-effective, and efficient methodology for the rapid biogenic synthesis of nano-sized metal particles under mild reaction conditions using medicinally significant and edible O. sanctum plant as both metal ion reducing and good capping agent is still warranted. We, herein, wish to report for the first time a simple and efficient one-step protocol for the rapid synthesis of AgNPs (7 to 11 nm) by reducing Ag+ ions in aqueous silver nitrate solution using aqueous fresh leaf extract of O. sanctum in the absence of any chemical additive under direct sunlight irradiation with excellent stability. We compared the efficacy of O. sanctum with another two medicinal plants, Citrus limon Linn. (family: Rutaceae) and Justicia adhatoda Linn. (family: Acanthaceae) under the same conditions. Besides, the enhanced antimicrobial activity of the AgNPs plant leaf extract (PLE) against some known pathogenic strains is also evaluated.
General experimental procedures
Fresh leaves of three medicinal plants Tulsi (O. sanctum Linn.), C. limon L., and J. adhatoda L. were collected in October 2013 at and around Santiniketan, West Bengal, India, and identified by Dr. H. R. Chowdhury (Botany Department, Visva-Bharati University). Voucher specimens are preserved in the Laboratory of Natural Products and Organic Synthesis of this university. The water used as the solvent was previously subjected to deionization, followed by double distillation (first time in alkaline KMnO4). Fresh leaves of the three plants were washed thoroughly with double-distilled water for several times to make it free from dust and were then cut into small pieces. Silver nitrate (AgNO3) (Sigma-Aldrich, Bangalore, India) were used as the source of Ag(I) ion required for the synthesis of Ag nanoparticles. UV-vis absorption spectra were recorded on a Thermo Scientific Spectrascan UV 2700 1 nm double beam spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Sample for transmission electron microscopy (TEM) was prepared by drop-coating the Ag nanoparticles solution onto carbon-coated copper grid. The film on the grid was allowed to dry prior to the TEM measurement in a JEOL TEM-2010 instrument (JEOL Ltd., Akishima-shi, Japan). Solution pH values were measured by Mettler Toledo's pH meter (Mettler-Toledo Inc., Columbus, OH, USA).
Preparation of plant extracts
Fresh and healthy leaves of Tulsi (O. sanctum), C. limon, and J. adhatoda were collected, washed thoroughly with double-distilled water, and were then cut into small pieces. A 10 g of finely cut pieces of leaves of each plant were then transferred into three different 250-mL beakers containing 100 mL distilled water each and boiled for 10 min. After cooling, the aqueous leaf extract obtained from the three different plants were filtered through ordinary filter paper, the filtrates were collected in three separate 100-mL volumetric flasks, and these 10% broth solutions were stored in a refrigerator for further use. On the dilution of the respective mother extract (10%) with requisite amount of distilled water, aqueous extracts of varying concentrations (7%, 5%, and 3%) were used in the present work.
Synthesis of AgNPs and evaluation of reducing potential of the extracts
Analysis of bioreduced silver nanoparticles
To observe the optical property of biosynthesized silver nanoparticles, samples were periodically analyzed with UV-vis spectroscopic studies (Thermo Scientific Spectrascan UV 2700) at room temperature operated at a resolution of 1 nm between 200 and 600 nm ranges.
Transmission electron microscopy
TEM was performed for characterizing the size and shape of biosynthesized silver nanoparticles using JEOL TEM-2010 operated at an accelerating voltage of 300 kV. Prior to analysis, AgNPs were sonicated for 5 min, and a drop of appropriately diluted sample was placed onto carbon-coated copper grid. The liquid fraction was allowed to evaporate at room temperature.
The bacterial strains used in this study were procured from Microbial Type Culture Collection (MTCC), Institute of microbial technology, Chandigarh, India. The bacterial strains used belonged to both Gram-positive and Gram-negative categories. Bacillus subtilis MTCC121 was an endospore former; Staphylococcus aureus MTCC96, Staphylococcus epidermidis MTCC2639, and Listeria monocytogenes MTCC657 were Gram-positive bacteria; Salmonella typhimurium MTCC98, Escherichia coli MTCC1667, and Pseudomonas aeruginosa MTCC741 were human pathogenic Gram-negative bacteria; and Pantoea ananatis MTCC2307 was a plant pathogenic Gram-negative bacteria.
Antimicrobial spectra for each concentration (5%, 7%, and 10%) of aqueous leaf extracts of O. sanctum as well as silver nano formed by these extracts upon sunlight induction were studied against four Gram-positive and four Gram-negative bacteria described above. Initially, 50 μL of each of the above samples were tested by agar well diffusion for antimicrobial screening . Later, it was confirmed by colony-forming unit CFU counting method after serial dilution of the bacterial cultures under different treatments.
Antimicrobial mode of action
Antimicrobial mode of action of the silver nano formed by 7% leaf extracts was studied against one Gram-positive S. aureus and one Gram-negative P. aeruginosa. The study was performed by applying 7% silver nano to the actively growing cultures of the bacteria followed by counting their CFU at regular intervals.
UV-vis absorbance studies
Bioreduction and stabilization of silver nanoparticles by O. sanctum fresh aqueous leaf extract: chemical perspective
Count of colony forming units of different pathogenic bacteria and the AgNPs
AgNPs formed by 5% PLE
AgNPs formed by 7% PLE
AgNPs formed by 10% PLE
1.2 × 109
0.5 × 102
1.4 × 102
0.01 × 102
1.7 × 102
0.7 × 102
0.02 × 102
6.0 × 108
1.0 × 103
5.2 × 103
0.5 × 102
4.8 × 103
0.3 × 102
3.5 × 103
0.3 × 102
7.0 × 108
2.0 × 102
1.2 × 103
0.1 × 102
9.0 × 102
0.09 × 102
5.6 × 102
1.0 × 102
1.5 × 109
2.0 × 102
5.0 × 102
0.8 × 102
3.7 × 102
0.05 × 102
3.0 × 102
0.08 × 102
8.0 × 108
1.5 × 103
6.0 × 103
0.8 × 102
5.5 × 103
0.5 × 102
4.5 × 103
0.7 × 102
8.5 × 108
6.0 × 102
7.2 × 103
0.4 × 102
7.0 × 103
0.04 × 102
6.9 × 103
0.4 × 102
8.0 × 108
1.3 × 103
2.0 × 103
1.2 × 102
1.5 × 103
0.8 × 102
8 × 102
0.9 × 102
8.5 × 108
7.0 × 102
4.0 × 103
0.06 × 102
1.7 × 103
0.04 × 102
9 × 102
0.05 × 102
In conclusion, we have developed a very simple, efficient, and robust practical method for the synthesis of silver nanoparticles using aqueous leaf extract of O. sanctum (Tulsi) as both reducing and capping agent, under the influence of direct sunlight. The biosynthesis of silver nanoparticles making use of such a traditionally important medicinal plant without applying any other chemical additives, thus offers a cost-effective and environmentally benign route for their large-scale commercial production. The nanoparticles formed are very effective in killing a number of bacteria in a bactericidal mode that include endospore formers, food spoilage pathogens, human as well as plant pathogenic members thus indicating their importance in controlling the growth of such microorganisms.
The authors are thankful to the UGC, New Delhi for providing financial assistance under the SAP.
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