Silver Nanoparticles Synthesis of Mentha arvensis Extracts and Evaluation of Antioxidant Properties
T. SivaKumar*, T. Rathimeena, V. Thangapandian, T. Shankar
Department of Microbiology, Ayya Nadar Janaki Ammal College (Autonomous), Sivakasi, Tamilnadu, India
Abstract
Silver nanoparticle synthesis of selected plant extract were confirmed by Ultra violet visible and Fourier transform infrared spectroscopy The Mentha arvensis leaf extract mediated nanoparticles showed absorbance peaks at 340 nm region in the spectral analysis. Fourier transform infrared spectroscopy analysis of the silver nanoparticles showed absorption peaks of reduced silver at1650.95 cm−1. The total antioxidant of AgNO3) shows a maximum activity of 40% was observed at 600μg/ml. 1-Dibhenyl-2-Picrylhydrazlradical in Mentha arvensis mediated silver nanoparticles showed a maximum activity of 25% was observed at 600μg/ml. Hydrogen peroxide scavenging assay in Mentha arvensis mediated silver nanoparticles showed a maximum activity of 10% was observed at 600μg/ml. Reducing power of Mentha arvensis silver nanoparticles exhibited a higher activity of 19% in 600μg/ml. The selected plant exhibits better antioxidant properties.
Keywords
Mentha arvensis UV, FTIR, Antioxidant
Received: April 1, 2015
Accepted: April 18, 2015
Published online: May 27, 2015
@ 2015 The Authors. Published by American Institute of Science. This Open Access article is under the CC BY-NC license. http://creativecommons.org/licenses/by-nc/4.0/
1. Introduction
Numerous methods for the synthesis of silver nanoparticles have been reported which includes biological reduction (Lee and Meisel, 1982), warm air decomposition (Yang et al., 2007), laser ablation (Simakin et al., 2004), and Sonochemical synthesis (Salkar et al., 1999). Among these, chemical reduction method and laser ablation method are the most commonly employed synthetic routes. The natural reduction method involves the reduction of metal salt like silver nitrate in an appropriate medium using various reducing agents like citrate, borohydride, etc. to produce colloidal suspensions integrated by nanoparticles (Evanoffand Chumanov, 2005).In recent years there has been growing interest in the preparation and study of silver nanoparticles (AgNPs), because those nanoparticles have been found to exhibit interesting antibacterial activities. (Shahverdi et al., 2007) Production of nanosized metallic silver particles with different morphologies and sizes using different routes has been reported (Patel et al., 2007). Along with methods, the simple process involving a reduction of silver salts has already been well developed (Chen et al., 2007).
The exact antibacterial action of AgNPs is not completely understood. There are reports in the literature that show that electrostatic attraction between negatively charged bacterial cellsand positively charged nanoparticles is crucial for the activity of nanoparticles as bactericidal materials.(Sondi et al., 2004).AgNPs were effective against these pathogens. The anti-bacterial activity of the ethanolic extracts of leap of M. arvensis L. was studied against Gram-positive & Gram-negative bacteria .The ethanolic extract of M. arvensis exhibited a significant anti-bacterial activity. The anti-bacterial activity of Staphylococcus aureus was higher than the other bacteria. The inhibition zone diameter of S. aureus was 20 mm at 10 % concentration and it was 7 mm at 0.3 % concentration.
The antibacterial activity of P. aeruginosa was the lowest and the inhibition zone ranging from 7 mm to 12 mm at 0.6 % to 10 % of concentration. The inhibition zone of E. coli, K. pneumoniae and S. flexineri was ranging from 7 mm to 14 mm at 0.3 % to 10 % of concentration. The plant extract was found to have a moderate antibacterial activity against these three bacteria. M. arvensi Leaf extract was classified as very active against Staphylococcus aureus, against E. coli, K. pneumonia and Shigella flexineri, and partially active against P. aeruginosa. (Rachel Madhuri Sugandhi and Meera Bai, 2011) Ag NPs release silver ions, which make an additional contribution to the bactericidal effect.(Feng et al., 2000) In fact, showed that Ag NPs (where silver is present in the Ag0 form) also contain micromolar concentrations of Ag+, and they have shown that Ag+ and Ag0 both contribute to the antibacterial activity. The mechanism of inhibition by silver ions on microorganisms is partially known. It is believed that DNA loses its replication ability and cellular proteins become inactivated on silver ion treatment.(Gupta et al., 2008) Higher concentrations of Ag+ ions have been shown to interact with cytoplasmic components and nucleic acids.(Lim et al., 2006) The antibacterial effect of Ag NPs determined in this study was found to be similar to that described in the earlier reports.(Shrivastava et al., 2007) The particle size has an effect on microbes; the effect increased with smaller particle size.
The antibacterial properties of silver nanoparticles are associated with its slow oxidation and liberation of Ag+ ions to the environment making it an ideal biocidal agent. Moreover, the small size of these particles facilitates the penetration of these particles through cell membranes to affect intracellular processes from inside. Additionally, the excellent antibacterial properties exhibited by the nanoparticles are due to their well-developed surface which provides maximum contact with the environment (Krutyakov et al., 2008). A better understanding of the bactericidal action of silver would require a proper examination of the membrane-bound and intracellular nanoparticles. Silver nanoparticles were found to penetrate into the bacterial cell causing membrane damage and ultimately the death of the organism.
According to the reports of silver nanoparticles exhibited excellent antifungal activity on Candida albicans by disrupting the cell membrane and inhibiting the normal budding process (Kim et al., 2009). In order to compare the antifungal effects of silver nanoparticles, amphotericin B, an antifungal agent used to treat serious systemic infections was used as a positive control (Hartsel and Bolard, 1996). It showed remarkable antifungal activity against Trichophyton mentagrophytes and Candida species. Remarkably, these particles exhibited similar activity with amphotericin B, but more potent activity than fluconazole toward all the fungal strains examined. The present investigation was mainly focused on the fabrication of silver nanoparticles synthesis of Mentha arvensis plant and its antioxidant properties were evaluated.
2. Materials and Methods
2.1. Collection of Plant Materials
Mentha arvensis are evergreen tree, native to tropical northern South America, southern Caribbean and also India. Its flowers are orange, scarlet and pink in colour and form large bunches. leaf of Mentha arvensis were collected from Sorimuthu Ayyanar Koil, Pabanasam, Thirunelveli District, Tamil Nadu, India (8º 39’ N and 77 º 20’ E) with elevation of 1500 m above sea level, The voucher specimen was identified and deposited in the herbarium of the Ayya Nadar Janaki Ammal College (Autonomous) Sivakasi, Tamil Nadu. The samples were washed, air-dried and powdered.
2.2. Plant Extract Preparation
Mentha arvensis, leafs were broken with help of marton pestle and flowers broken by scissors The fruit pulp (white in color which converts into blue to brown within minutes) was collected for the synthesis of nanoparticles. The plant extracts (broth solutions) were prepared by using 5g of washed and cut leaves flowers petals and fruit pulp in a 250 ml Erlenmeyer flask with 50 ml of sterile distilled water and then boiling the mixture for 5min. The herbal aqueous extract was collected in separate conical flasks by standard filtration method and stored at 4°C in a refrigerator (Gardea-Torresdey et al., 2003).
2.3. Preparation of 1mm Aqueous Solution of Silver Nitrate
17mg of Silver nitrate (AgNO3) was added to the 100 ml of distilled water and the solution was stirred well continuously until the silver nitrate is dissolved. This 1mMSilver nitrate solution was stored in brown bottle at 4°C for further use for the synthesis of Silver nanoparticles from Mentha arvensis (Gardea-Torresdey et al., 2003).
2.4. Synthesis of AgNPs
1mM aqueous solution of silver nitrate (Himedia, Mumbai) was prepared for synthesis of silver nanoparticles. For the synthesis of AgNPs, two boiling tubes were taken, one containing 10ml of 1MmAgNO3 solution as control and the second containing 9ml of 1mM silver nitrate solution and 1ml of plant leaf extract as test solution. These were incubated at room temperature for 1-2 hours. The color change of the leaf extracts from pale yellow to dark brown was checked. The brown color formation indicates that the silver nanoparticles were synthesized from the plant extracts and they were centrifuged at 5000 rpm for 15 minutes in order to obtain the pellet which is used for further study. Supernatant is discarded and the pellet is dissolved in deionized water. The silver nanoparticles were confirmed by color changes and qualitatively characterized by UV-Visible spectrophotometer.
2.5. Characterization of Silver Nanoparticles
A) UV-visible spectroscopy
Synthesis of silver nanoparticles by reducing, the respective metal ion solution with leaves extract may be easily observed by UV- Vis spectroscopy. The absorption spectra of leaves extract quantities and metal concentration was measured using a spectrophotometer in 300-1000 nm range. The formation and completion of silver nanoparticles was characterized by UV-visible spectroscopy using a double beam spectrophotometer
B) FT-IR chemical analysis
Interaction of NPs obtained with PEG and gluconic acid products by reduction of sugar compound were confirmed by FT-IR spectra. (Geethalakshmi and Sarada, 2012).
2.6. Antioxidant Properties
2.6.1. Determination of Total Antioxidant Capacity (TAC)
Briefly, 0.3 ml of sample will be mixed with 3.0 ml reagent solution (0.6 M sulphuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate). Reaction mixture will be incubated at 95ºC for 90 minutes under water bath. Absorbance of all the sample mixtures will be measured at 695 nm after 15 min. Ascorbic acid will be used as standard (Prieto et al., 1999)
2.6.2. DPPH Radical Scavenging Assay
The free radical scavenging activity was measured by the 1-1-Diphenyl-2-picryl-hydrazyl (DPPH) following the method by Blois, (1958). DPPH will be used as a reagent which evidently offers a convenient and accurate method for titrating the oxidizable groups of natural (or) synthetic antioxidants. 0.1 mM solution of DPPH in methanol will be prepared and 1ml of this solution will be added to 3ml of seaweed extracts of different concentration (100, 250, 500, 750 and 1000μg). After 10 minutes, absorbance will be measured at 517 nm. The percentage scavenging activity values will be calculated using the following formula
Percentage of Scavenging = ((Ao-A1) / Ao) x 100
Where, Aois absorbance of control and A1 is absorbance of sample turbidity factor.
2.6.3. Hydrogen Peroxide Scavenging Assay
The free radical scavenging activity was determined by hydrogen peroxide assay (Gulcin et al., 2004).Hydrogen peroxide (10mM) solution will be prepared in phosphate buffered saline (0.1M, pH 7.4). 1ml of the extract containing samples of different concentration (100, 250, 500, 750 and 1000μg) will be rapidly mixed with 2ml of hydrogen peroxide solution. The absorbance will be measured at 230 nm in the UV spectrophotometer after 10 minutes of incubation at 37°C against a blank (without hydrogen peroxide). The percentage of scavenging of hydrogen peroxide will be calculated using the formula
Percentage scavenging (H2O2) = ((Ao-A1) / Ao) x 100
Ao - Absorbance of control; A1 - Absorbance of sample
2.6.4. Determination of Reducing Power
Reducing power was determined by the following method (Yamaguchi et al., 1998). Briefly, 4 ml of reaction mixture, containing samples of different concentration in phosphate buffer (0.2 M, pH 6.6) will be incubated with potassium ferricyanide (1% w/v) at 50°Cfor 20 min. The reaction will be terminated by TCA solution (10% w/v). The solution will be then mixed with distilled water and ferric chloride (0.1% w/v) solution and the absorbance will be measured at 700 nm.
3. Results
The Mentha arvensisflower extract mediated nanoparticles showed absorbance peaks at 370 nm region in the spectral analysis shown in Fig.1. The peaks were stable with time duration also.It indicates that the synthesis of silver nano particles reqires the reduction of α-NADPH to α- NADP+ and the hydroxy quinoline probably acts as electron shuttle tranforming the electron generated during the reduction of nitrate to Ag+ ions converting them to Ag0
Fourier transform infrared spectroscopy analysis of the silver nanoparticles showed absorption peaks of reduced silver at 1650.95 cm−1 (Fig.2). The stretching vibration of C=C obtained at 1625.88 cm−1 and the single absorbance peak located at 1108.99cm−1 is assigned to C-O Polyols, while 3379.05 and 3355.91 cm−1 corresponds to O-H and N-H stretching vibration.
3.1. Total Antioxidant Properties
Free radical scavenging activity of the silver nanoparticles was assessed by DPPH solution exhibited a deep purple colour with a maximum absorbance at 517nm. The disappearance of purple colour on adding synthesized silver nanoparticles might due to presence of antioxidant in the medium (Fig.3).
3.2. Dpph
Free radical scavenging activity of the silver nanoparticles was assessed by DPPH solution exhibited a deep purple colour with a maximum absorbance at 517nm. The disappearance of purple colour on adding synthesized silver nanoparticles might due to presence of antioxidant in the medium. Mentha arvensis plant extract of silver nanoparticles exhibit higher activity at 35% in600μg/ml, likewise lower activity observed at 200 μg/ml with 19% followed by the plant extract showing minimum activity at 200μg/ml with 30 % and also maximum activity observed at 600 μg/ml with 80%(Fig.4).
3.3. Hydrogen Peroxide Scavenging Assay
Mentha arvensis, Hydrogen peroxide scavenging activity of AgNO3showing a minimum activity observed 2% at 200μg/ml and maximum activity observed 10% at 600μg/ml followed by the plant extract showing a minimum activity at 30% at 200μg/ml and maximum activity observed 82% at 600μg/ml (Fig.5).
3.4. Reducing Sugar
Reducing power of Mentha arvensis silver nanoparticles exhibit higher activity at 19% in600μg/ml, minimum activity at200μg/ml with 35 % and also maximum activity observed at 600 μg/ml with 85% (Fig.6).
4. Discussion
In the present examination, Mentha arvensis were studied for antioxidant properties. DPPH is a stable and well characterized synthetic solid radical for evaluation of antioxidant potential of compounds. The DPPH will be reduced by accepting the hydrogen or electron, the DPPH reducing ability of silver nanoparticles was quantified spectrophotometrically by changing the DPPH color from purple to yellow. Inhibition was found to be high in silver nanoparticles, when compared with gold nanoparticles, which may be due to the facts that silver act as a good oxidant can easily lose electrons. The results obtained in the DPPH assay showed effective free radical inhibition by both AgNPs. The average percentage inhibition of synthesized AgNPs was observed in the range from 10%-40 as compared at different concentrations used in this study and the activity increased with increasing concentrations of AgNPs and plant extract also. Similar observations with enhanced DPPH scavenging activity by selenium, platinum, silver nanoparticles (Gao et al., 2002; Huang et al., 2003; Watanabe et al., 2009; Saikia et al., 2010 ) and by torolex and chitosan coated gold nanoparticles (Nie et al., 1997; Raghunandan et al., 2010) have been reported.
In the present examination, Mentha arvensis shows the superoxide scavenging activity of both the plant extract and AgNPs as determined by the PMS-NBT reduction system. Superoxide (O2 −) radicals easily react with DNA and protein which necessitate their immediate clearance in living systems. The superoxide radical quenching activity of plant extract and AgNPs was found to be increased with increasing concentrations and the average inhibition was about 40%. Similarly, The superoxide radical inhibition has been reported for platinum and selenium nanoparticles (Makari et al., 2008).The potential superoxide scavenging activity of gold and silver nanoparticles reported earlier (Ramamurthy et al., 2013) support our findings in the present study. In the same way (Huie et al., 1993; Pacher et al., 2007) explored that the role of nitric oxide radicals in carcinomas and inflammatory process is well established. The toxic effects of nitric oxide will increase when reacts with superoxide radicals that lead to vascular system damage and results in conditions including inflammation, juvenile diabetes and multiple sclerosis Because of the less stability of nitric oxide ions, they accept electrons from silver nanoparticles and form formazan when treated with Griess reagent that can be detected spectrophotometrically.
Mentha arvensis of H2O2 scavenging activity of Plant extract and AgNPs are active in quenching H2O2 radicals and the average inhibition was found to be 80 % By the same token, PLAgNPs were as effective as PLFE in quenching H2O2 radicals and the average inhibition was found to be 96% as compared to PLFE. In this study, it could be noted that the superoxide radical quenching activity and NO quenching activity of PLAgNPs was 60% and 70% respectively as compared to PLFE which can be explained on the fact that the concentration of phytocompounds responsible for the scavenging activities was higher in the extract than adhered to the nanoparticles. On the other hand, the observed increase in H2O2 scavenging activity of PLAgNPs (96%) may be because of the plant condensed tannins present in the extract that are involved in confirmation of nanoparticles (Subramanian et al.,2013).
5. Conclusion
Nanoparticles synthesis of Mentha arvensis exhibited high antioxidant properties.
Acknowledgements
The facilities provided by the Department of Microbiology, AyyaNadarJanakiAmmal College, Sivakasi, Tamilnadu are gratefully acknowledged.
References