Microwave-assisted synthesis of silver nanoparticles from Origanum majorana and Citrus sinensis leaf and their antibacterial activity: a green chemistry approach
© Singh et al. 2016
Received: 1 December 2015
Accepted: 2 March 2016
Published: 16 March 2016
Silver nanoparticles (SNPs) play important role in the field of optics and electronics and also as a novel antibacterial agents. Here, we report a simple and green method for the biosynthesis of SNPs using aqueous leaf extract of Origanum majorana and Citrus sinensis as a novel bio source of cost-effective, non-hazardous reducing, and stabilizing agents. A 3 mM solution of silver nitrate was prepared. Five milliliter aqueous leaf extract was slowly added to 20 ml silver salt solution (3 mM) with constant stirring. No noticeable color change was observed. The solution was then heated in domestic microwave for variable time intervals. The intense brown colored solution was obtained on 1 min heating with O. majorana and 5 min heating with C. sinensis extract. The intense brown color indicated the formation of SNPs. The antibacterial activity of synthesized SNPs was investigated.
SNPs were rapidly synthesized using aqueous leaf extract of O. majorana and C. sinensis on microwave irradiation. Formation of SNPs was confirmed by the change in color from yellowish green to brown and absorption maximum around ~420 and 410 nm due to surface plasmon resonance of SNPs. They were also characterized by other physical–chemical techniques like Fourier transform infrared spectroscopy (FT-IR), scanning electron microscope coupled with X-ray energy dispersive spectroscopy, and high-resolution transmission electron microscopy. TEM analysis showed the presence of feather-shaped NPs in O. majorana and spherical as well as cubical-shaped NPs in C. sinensis-mediated SNPs. The synthesized SNPs showed significance antibacterial activity against two human pathogenic strains.
The SNPs were synthesized using leaf extract of plants. This synthesis method is nontoxic, eco-friendly, and a low-cost technology for the large-scale production. The SNPs can be used as a new generation of antibacterial agents.
In this article, we report a simple, robust, and eco-friendly method for the biosynthesis of SNPs using an aqueous leaf extract of “O. majorana” and “C. sinensis” as a bio-reductant and stabilizer. O. majorana is also called sweet marjoram. This plant is native to North Africa, Turkey, and SW Asia, extensively cultivated in India. Dried marjoram is extremely useful in industrial food processing and is used, together with thyme, in spice mixtures for the production of sausages. It is commonly called marwa in Hindi language. Its leaf contains protein, terpineol, sabinene hydrate (cis and trans) linalool oil, and pentosans. C. sinensis commonly called orange has high levels of glucose and ascorbic acid (vitamin C). Besides this, it also contains aldehyde such as n-octanal, 2,6-dimethyl-2,6-octadiene-1,8-dial, 4-isopropenyl-1-methyl-1,2-cyclohexanedial and alcohols like 1-octanol, β-linalool, and 1-nonanol. Further these biologically synthesized SNPs were found highly toxic against different pathogenic bacteria.
Silver nitrate (AgNO3, 99.995 %) was purchased from Merck, India. NaCl, NaOH, yeast, and Tryptone were purchased from Spectrochem, India. All the reagents were of analytical grade. The O. majorana and C. sinensis leaf were collected from botanical garden of Daulat Ram College, University of Delhi, India. These were washed with deionized water before use. All glassware was washed and rinsed with deionized water, followed by subsequent drying.
Preparation of plant leaf extract
The collected leaf were washed several times with deionized water to remove the dust particles and then sun-dried to remove the residual moisture. The dried leaf were cut into small pieces and boiled in 100 ml deionized water for 10–15 min. After boiling, the color of the aqueous solution changed from colorless to yellowish green color. The aqueous leaf extract was separated by filtration with in Whatman No. 1 filter paper and then centrifuged at 1200 rpm for 5 min to remove heavy biomaterials. The prepared leaf extract was stored at room temperature to be used for biosynthesis of SNPs from silver nitrate.
Synthesis of silver nanoparticles
The bioreduction of Ag+ ion in aqueous solution was monitored with the help of UV-2600 series Shimadzu spectrophotometer. Fourier transform infrared (FT-IR) spectra for plant leaf powder and SNPs were obtained in the range 4000–400 cm−1 with a Shimadzu FT-IR spectrophotometer using KBr pellet method. Scanning electron microscopy (SEM) analysis coupled with energy dispersive spectroscopy (EDAX) of synthesized SNPs was done using a model-JEOL-5800-LV 16. All TEM images were obtained using a JEOL model 1200 EX instrument with an accelerating voltage of 120 kV. TEM samples were prepared by drop casting of nanoparticles dispersion onto carbon-coated copper TEM grid, followed by air drying at ambient conditions. TEM samples were stored in a desiccator and imaged shortly after collection.
The antibacterial activity of SNPs was carried out on human pathogenic Escherichia coli and Bacillus subtilis by standard disk diffusion method. LB broth/agar medium was used to cultivate bacteria. Fresh overnight inoculum (100 μl) of each culture was spread on to LB agar plates. Sterile paper disks of 5 mm diameter containing various concentrations (µg/ml) of SNPs were placed in each plate. Each plate also contained a disk loaded with standard antibiotic as a reference. The plates containing bacteria and SNPs were incubated at 37 °C. The plates were then examined for zones of inhibition. The clear area around the disk is known as zone of inhibition. The diameters of such zones were measured using meter ruler and expressed in millimeter.
Results and discussions
UV–Vis spectral analysis
High-resolution transmission electron microscopy (HRTEM)
Antibacterial activity of Origanum majorana and Citrus sinensis-mediated SNPs
Zone of inhibition (mm)
Origanum majorana-mediated SNPs
Citrus sinensis-mediated SNPs
O. majorana-mediated SNPs showed better result against E. coli at the minimum concentration of 25 µg/ml, whereas -mediated SNPs were good against B. subtilis. The antibacterial activity of SNPs depends upon the concentration of NPs used. As evident from table, inhibition against bacteria increased with increasing the concentration of SNPs. The nanoparticles bind to the cell membrane and also pierced inside the bacteria. SNPs interact with sulfur present in the proteins as well as with the phosphorus containing compounds like DNA (Morones et al. 2005). When SNPs 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 (Lok 2006).
The present article reports the biosynthesis of SNPs from O. majorana and C. sinensis leaf for the first time. O. majorana/C. sinensis leaf extract is capable of reduction and stabilization of SNPs. Moreover, microwave radiation and its mode of heating make the synthesis of the metallic nanoparticles fast, uniform, and reproducible. SNPs showed superior antibacterial activity toward E. coli and B. subtilis pathogens. It is an environmental friendly process for the production of SNPs and completely free from toxic solvents and chemicals. So, it is one of the effective recycling processes to utilize the O. majorana and C. sinensis for the production SNPs. Therefore, biogenic synthesized SNPs can be used for waste water treatment, food and water storage, and manufacturing medicinal supplies such as beds.
All authors have been involved in the writing and interpretation of results during preparation of the manuscript. All authors read and approved the final manuscript.
The authors are grateful to the Daulat Ram College, University of Delhi for carrying out experimental work. I express my sincere thanks to the director, USIC, University of Delhi, for providing instrumentation technique which is necessary this research work.
The authors declare that they have no competing interests.
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