Green synthesis of silver nanoparticles with antimicrobial and azo dye (Congo red) degradation properties using Amaranthus gangeticus Linn leaf extract
© Kolya et al. 2015
Received: 19 September 2015
Accepted: 5 November 2015
Published: 12 November 2015
The present paper describes a less time-consuming and eco-friendly method for the synthesis of silver nanoparticles (AgNPs) using an aqueous solution of silver nitrate and Amaranthus gangeticus Linn (Chinese spinach) leaf extract. The synthesized AgNPs which are to be used as an antimicrobial and Congo red dye is to be used as a toxic-degrading agent.
AgNP was prepared by the reduction of silver nitrate solution by the leaf extract of Amarranthus Gangeticus Linn leaf extract in aqueous medium on heating for about 15 mins at 80 °C in presence of one drop 0.05 (M) NaOH.
The size of the synthesized silver nanoparticles (AgNPs) using Amaranthus gangeticus Linn leaf extract and aqueous solution of silver nitrate (10−3 M) are formed at their stable condition within the range of 11–15 nm. AgNPs are obtained by this process within a couple of minutes of reaction without using reducing and stabilizing agents or harsh conditions. High-resolution transmission electron microscope (HR-TEM), selected area electron diffraction (SAED), ultraviolet-visible (UV-VIS) spectroscopy, and Fourier transform infrared spectroscopy (FTIR) are used to characterize the prepared AgNPs which show that the nanoparticles are globular in shape and polycrystalline. The synthesized silver nanoparticles showed inhibitory activity towards Gram positive, Gram negative bacteria and fungus and also showed good Congo red dye-degrading agents.
The overall outcome of this study suggests that green synthesis AgNPs hold promise as a potent antibacterial and antifungal agent. The particles obtained were also found to degrade toxic Congo red dye.
KeywordsSilver nanoparticles Antibacterial activity Chinese red spinach Congo red
Over the past few years, synthesis of metal nanoparticles is one of the upcoming areas of research in the field of material science owing to their wide variety of applications in the field of catalysis (Kalidindi and Jadirdar 2012), photonics (Shen et al. 2000), chemical and bio-sensing (Zayats et al. 2003), medicine (Jotterand and Alexander 2011; Etheridge et al. 2013) etc. Nano crystalline silver is well known for possessing an inhibitory effect towards many bacterial stains and microorganisms (Zhang et al. 2012; Prabhu and Poulose 2012). Silver nanoparticles were synthesized using various chemical and biological approaches (Ericka et al. 2013; Shankar et al. 2003; Bar et al. 2009; Sivaram et al. 2009; Ahmad et al. 2010; and Dubey et al. 2010). Even though silver nanoparticles are considered bio-compatible, chemical synthesis methods may lead to the presence of some toxic chemicals absorbed on the metallic surface that may have adverse effects in medical applications (Bhattcharya and Mukherjee 2008). Therefore, there is an increasing demand of green procedure for synthesizing metal nanoparticles which are free from the use of toxic chemicals.
Congo red, methyl alcohol, and potassium bromide were procured from Loba chemie, Mumbai, India. Bacillus subtilis-11774, Shigella flexneri-2022 were collected from American Type Culture Collection (ATCC), USA. Fungus, collected from Amaranthus gangeticus Linn plants (Chinese red spinach), were procured from the local market of Midnapore town, Paschim Medinipur, West Bengal, India. All the solutions were prepared by double-distilled water.
Preparation of leaf extract
The freshly collected leaves of Amaranthus gangeticus Linn plant were cleaned several times using distilled water. The washed leaves were chopped into small sizes. Thereafter, the small-sized leaves were compactly packed into a thimble and then the solvent extraction (methanol, water = 1:3) was performed using a Soxhlet apparatus. Such solvent extraction leads to remove desirable compounds from the leaves and make them soluble in the used solvent having a definite polarity.
Preparation of silver nanoparticles
The 5 ml of prepared 10−3 M AgNO3 solution was taken in a freshly washed 50-ml beaker and then it was heated for about 5 min at 80 °C. Thereafter, 1.5 ml of plant leaf extract was added followed by one drop of 0.05 M NaOH. The solution was further heated up to 20 min in 80 °C at which the colour of the solution changed to a brownish yellow colour which had indicated the formation of silver nanoparticles (AgNPs) (Nalvothula et al. 2014). The concentration of AgNO3 solution and leaf extract was also varied at 4 to 6 mM of AgNO3 and 1 to 2 ml, respectively, keeping other parameters constant. Ultraviolet-visible (UV-VIS) spectra showed a strong absorption peak (SPR) band at 416 nm thus indicating the formation of silver nanoparticles. The synthesized AgNPs (hydrosol) were centrifuged at 12,000 rpm for 20 min. Thereafter, the AgNPs were redispersed in sterile distilled water for further use.
Study of antibacterial activity
The antibacterial activity of synthesized AgNPs was investigated by standard agar-well diffusion method (Bauer et al. 1966; Awhad et al. 2013) into one Gram positive (Bacillus subtilis) and one Gram negative (Shigella flexneri) bacteria. Solidified nutrient agar was cast onto petriplates, and the plates containing nutrient medium were evenly inoculated with 100 μg (108 CFU/ml) separately. The wells are prepared on the agar plate with the help of cork borer (0.6 cm diameter). Levofloxacin, 5 μg/disc, which was used as a standard, was placed in the well of each plate. The synthesized and redispersed hydrosol (30 mg/ml) was loaded onto the wells of each plate. The plates were then incubated 24 h at 37 °C, and the antibacterial activity was determined by measuring the diameter of the inhibition zone and expressed in millimeter.
Study of antifungal activity
Actively growing fungal plant Sclerotinia sp. pathogens were aseptically transformed on to midpoint of sterile standard potato dextrose agar (PDA) plates and incubated at 25 °C for 2 days. After 2 days, three wells (5 mm) were prepared by using a sterile cork borer at equal distance around the mycelia growth of pathogenic fungi. The prepared AgNPs at different concentrations ( 0.4 μg/ml,  0.2 μg/ml,  0.1 μg/ml) were loaded onto each well separately and allowed to grow for 3 days. The inhibition of growth of plant pathogenic fungi around the well refers to antifungal activity of the antimicrobial sample (here AgNPs). Antifungal activity test was done against one plant pathogenic fungi Sclerotinia sp.
Congo red dye degradation
Aqueous solution of Congo red (10−3 M) and 0.5 M ethanolic solution of sodium borohydride were prepared. Thereafter, a solution was prepared by adding 5 ml 10−3 M Congo red solution with 1 ml of ethanolic borohydride solution. From this solution, 1.5 ml was taken in an ultraviolet (UV) quartz cuvette. UV-VIS absorption study was done to record the change in absorbance at a time interval 1.5 min until the solution became completely colourless.
Characterization of AgNPs
UV-VIS adsorption spectra were measured in a 1-cm quartz cuvette using Shimadzu-1800 (Japan) spectrophotometer. Morphology and size of AgNPs were investigated using JEOL-JEM-2100 high-resolution transmission electron microscope (HR-TEM). Sample of HR-TEM study was prepared by placing a drop of redispersed silver sol onto a carbon film, supported on a copper grid followed by solvent evaporation. FTIR spectrum of the AgNPs was taken in a Perkin Elmer (L16000300 Spectrum Two LiTa, Llantrisant, UK) spectrophotometer, and the potassium bromide (KBr) pellet method was applied for spectral analysis.
Results and discussion
Fourier transform infrared spectroscopy
Degradation of Congo red dye
An eco-friendly and convenient green method for the synthesis of silver nanoparticles from silver nitrate solution using Amaranthus gangeticus Linn leaf extract was developed. Formation of globular-shaped and well-dispersed AgNPs with an average particle size of 11–15 nm was confirmed by UV-VIS, FTIR, HR-TEM, and SAED. FTIR spectroscopic study confirmed that the amino acids present in the leaf extract reduces the Ag (I) to Ag (0) in the nanoscale. Silver nanoparticles prepared by the present method have promising applications as an activity against both Gram negative and Gram positive bacteria and fungi. The prepared AgNPs also showed efficient catalytic activity towards degradation of Congo red dye thus having potential for industrial application.
The financial assistance of Midnapore College (Autonomous) is gratefully acknowledged.
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