Common Duckweed (Lemna minor) Assisted Green Synthesis of Silver Nanoparticles as Potent Anti-Fungal Nanomaterial
B.S. Naveen Prasad*1 and TVN. Padmesh2
1Department of Chemical Engineering, Sathyabama University, Chennai, India
2Department of Chemical Engineering, Manipal International University, Nilai, Malaysia
*Corresponding Author E-mail: bsnaveenprasad@gmail.com
ABSTRACT:
A simple and eco-friendly green synthesis of silver nanoparticles using Common duckweed (Lemna minor) as the reducing and stabilizing agent is reported. Lemna minor (Lm) extract was mixed with silver nitrate (AgNO3) solution for the production of silver nanoparticles (AgNPs). The reaction process was simple and the formation of highly stable silver nanoparticles at room temperature were observed. The morphology and crystalline phase of the Lm-AgNPs were determined using UV-Visible spectroscopy (UV-vis), Transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD) spectra. TEM studies showed that the average particle size of silver nanoparticles were in the range of 10 to 20 nm. The antifungal activity of Lm-AgNPs against Aspergillus flavus was also investigated and showed that the synthesized Lm-AgNPs has a remarkable potential as an antifungal agent in treating fungal diseases.
KEYWORDS: Common duckweed (Lemna minor); synthesis; silver nanoparticles; antifungal activity.
INTRODUCTION:
Nanomaterials are considered highly efficient and are used in the field of medicine for various therapeutic and drug delivery applications. The use of polymeric nanoparticles in targeted drug delivery systems is one of the milestones in medicine. The absence of some optical properties in polymeric nanoparticles made researchers to synthesize metal nanoparticles for applications like antimicrobial activity. Metal nanoparticles have wide applications ranging from photovoltaic to biological and chemical sensors.1 The availability of larger surface area for activity is one of the important reasons for incorporating nanomaterials into various application systems.2 Though different modes of synthesis of metal nanoparticles have been known, synthesis using green approach is preferable and most widely used since it is environment friendly. The mechanism involved in size controlled synthesis of the metal nanoparticles is not clear. However, shape controlled synthesis of spherical silver nanoparticles (AgNPs) has been achieved3 and a size dependant activity of silver nanoparticles is also demonstrated.4
Depending upon the source used for the synthesis of metal nanoparticles, they are scaled up for various applications like non-linear optics, selective coating for solar energy absorption and intercalation materials for electrical batteries, as optical receptors, catalysis in chemical reaction and biolabelling.5 The property of metal nanomaterials can be improved by tuning the surface plasmon resonance by altering the concentration and volume of stabilizing agents used generally for detection of nucleic acids.6 The use of the AgNPs for antimicrobial activity is considered as one of the most important properties for silver as they have the tendency to form free radicals which kills the microbes. In our previous work, we have demonstrated the antibacterial activity of the AgNPs synthesized from the marine alga Sargassum wightii Grevillie.7 AgNPs have the capability of forming free radicals that inhibit the growth of microbial species present in the vicinity. Interestingly, microbes have also been employed for the synthesis of silver nanoparticles since using chemical method is time consuming and the use of organic solvents remains as a threat to environment. Lm, a type of weed usually present in lakes and fresh water ponds where there is slow moving water have been employed for the synthesis of silver nanoparticles. In the present study, AgNPs were synthesized using Lm and its antimicrobial potential was investigated. The present study aims to develop a novel AgNPs using biological method and its activity against microbes was also tested.
MATERIALS AND METHODS:
Collection of Lemna minor (Duckweed) and synthesis of AgNPs
Silver nitrate was obtained from Sigma-Aldrich and used as received. All other chemicals used were of analytical grade. Lm was collected from Palar River, Vellore district, Tamilnadu, India and was washed with distilled water to remove the impurities present. 1 g of fresh Lm was mixed with 100 mL of 1mM silver nitrate (AgNO3) solution and kept at room temperature.
Characterization of Lm-AgNPs:
The biosynthesized Lm-AgNPs were characterized using various spectral and microscopic analyses. UV-vis spectra were used to know the absorbance of Lm-AgNPs which were recorded using Shimadzu UV-1800. Fourier Transform Infrared Spectroscopy (FTIR) was used to identify the possible groups involved in the reaction. The FTIR spectrum was recorded by employing KBr pellet technique at a resolution of 4 cm−1 in the range of 4000-450 cm−1 using Perkin Elmer model-983/G detector double beam spectrophotometer. X-Ray Diffraction (XRD) pattern were obtained using Rich Seifert P3000 instrument operated at a voltage of 40 kV and a current of 30 mA with Cu Kα1 radiations to determine the particle nature and approximate size of silver nanoparticles. High Resolution Transmission Electron Microscopy (HRTEM-JEOL 3010) was used to study the size and shape of the Lm-AgNPs.
Antifungal activity of Lm-AgNPs by agar well diffusion method:
Lm-AgNPs were tested for its antifungal activity against clinically isolated fungus Aspergillus flavus by agar well diffusion method. The pure cultures of pathogenic organisms were sub cultured. Each pathogen was swabbed uniformly onto individual plates using sterile cotton swabs. Wells of 10 mm diameter were made on culture plates using gel puncture and each well was filled with different concentrations of silver nanoparticles (10, 50 and 100 μL). The fungal plate was incubated at 28oC for 48 h. After incubation, the plate was observed for zone of inhibition around each well.
Studies on the interaction of silver nanoparticles with microbes:
Log phased cells of Aspergillus flavus (105) was cultured in specific culture medium. The cultured cells were harvested and Lm-AgNPs were incubated with Aspergillus flavus for 5 h. TEM was used to examine the thin fungal sections treated with silver nanoparticles using standard procedures for fixing.
RESULTS AND DISCUSSION:
Figure 1(a) shows the UV-spectrum of Lm-AgNPs, the peak at 433 nm. The reaction was rapid and there was an immediate colour change from colorless to yellowish brown colour which indicates the formation of Lm-AgNPs. Figure. 1(b) shows the FTIR spectrum of Lm-AgNPs. The prominent peak at 3449 cm-1 can be assigned to H–bonded hydroxyl groups7 and the peak at 1638 cm-1 corresponds to C=O stretching of the carboxylic acid group.8 In the spectrum, no other peaks were observed. Hence, these functional groups were suspected to involve in the stabilization of Lm-AgNPs.
(a)
(b)
Figure. 1. (a) UV-spectrum and (b) FTIR spectrum of Lm-AgNPs
XRD pattern (Figure. 2) shows the crystalline nature of Lm-AgNPs. The pattern shows three diffraction peaks at 2θ in the range of 10ş to 70ş which was indexed as (111), (200) and (220) appeared to be crystalline face centered cubic phase (JCPDS File No. 04-0783). No other peaks were seen and this indicates that the synthesized silver nanoparticles were in crystalline nature. The size of synthesized silver nanoparticles was calculated by Debye-Scherrer equation.9
D = K λ / β cosθ
Where D is the average crystal size, K is the Scherrer constant, λ is the wavelength of the X-ray source and θ is the diffraction angle. The particle size calculated from the above equation was found to be 18 nm which was found to be in agreement with the size of the nanoparticle from TEM analysis. The size and shape of Lm-AgNPs were also investigated by TEM analysis (Figure. 3). From the TEM analysis, the synthesized Lm-AgNPs showed polydispersed spherical shaped particles with size ranging from 10-20 nm.
Further, the antifungal activity of Lm-AgNPs against Aspergillus flavus was investigated. The fungus inoculated plates were supplemented with a positive control (100 µL aqueous AgNO3), a negative control (100 µL Lm-extract) and Lm-AgNPs at different concentrations (10 µL, 50 µL and 100 µL). There was a pronounced activity with increase in volume of Lm-AgNPs. The zones of inhibition were found to be 6.1, 13.2 and 17.4 mm for 10, 50 and 100 µL Lm-AgNPs respectively (Table .1). The positive control showed a zone of inhibition of 13.9 mm and the negative control showed a diameter of 1.2 mm. From this, it can be concluded that the synthesized Lm-AgNPs has a remarkable potential as an antifungal agent in treating fungal diseases. TEM studies were also performed to elucidate the physiological changes of Aspergillus flavus when treated with Lm-AgNPs. Figure. 4 shows moderate disruption of cell wall and disintegrity of cell components. Cells after treatment with AgNPs usually get ruptured by the action of silver particles. In general, electron dense particles which cause damage to the cells will be translucent to that of the undamaged cells.10 The ability of silver based materials have been tested for antimicrobial studies as recently silver chloride nanoparticles have been tested against gram negative bacteria E. coli and microbes have been analyzed using electron micrograph for studying the damage.11 Similarly, marine algae derived AgNPs have also been studied for efficiency in controlling microbes.12 Hence, Lm mediated synthesis of AgNPs was found to have a greater potential against the fungus A. flavus which can serve as a good candidate for the treatment of fungal diseases. It has also been reported that nano-Ag exhibited remarkable antifungal effects through destruction of membrane integrity of the fungal cells and hence deserving further investigation for clinical applications.13
Figure. 4. Transmission electron micrograph of Aspergillus flavus after treatment with Lm-AgNPs
Table 1. Antifungal activity of Lm-AgNPs
|
S. No |
Test Organism |
Zone of inhibition (mm) |
||||
|
Lm- extract (100 μl) |
Aqueous AgNO3 (100 μl) |
Lm-AgNPs (10 μl) |
Lm-AgNPs (50 μl) |
Lm-AgNPs (100 μl) |
||
|
1. |
Aspergillus flavus |
1.2 ± 0.3 |
13.9 ± 0.5 |
6.1 ± 0.4 |
13.2 ± 0.2 |
17.4 ± 0.2 |
Lm - Lemna minor; AgNPs - Silver nanoparticles; Lm-AgNPs - Lemna minor synthesized silver nanoparticles
CONCLUSION:
Green synthesis of silver nanoparticles using Common duckweed as reducing and stabilizing agent at room temperature were discussed. AgNPs have been characterized using various spectroscopic and microscopic analyses. The Lm-AgNPs synthesized in the present study exhibits potential antifungal activity, suggesting its prospective applications in antifungal formulations.
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Received on 01.08.2014 Modified on 10.08.2014
Accepted on 19.08.2014 © RJPT All right reserved
Research J. Pharm. and Tech. 7(9): Sept. 2014 Page 955-958