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1 Available online at Nanoscience and Nanotechnology: An International Journal Universal Research Publications. All rights reserved ISSN: Original Article BIOLOGICAL REDUCTION OF SILVER NANOPARTICLES USING CASSIA AURICULATA FLOWER EXTRACT AND EVALUATION OF THEIR IN VITRO ANTIOXIDANT ACTIVITIES Velavan S*, Arivoli P and Mahadevan K 1. Department of Biochemistry, Marudupandiyar College, Thanjavur, Tamil Nadu, India 1 Harman Research Centre, Thanjavur, Tamil Nadu, India *Corresponding author: mayavelvan@gmail.com Accepted 26 December 2012 Abstract There is an increasing commercial demand for nanoparticles due to their wide applicability in various areas such as electronics, catalysis, chemistry, energy, and medicine. In this work deals with the synthesis and characterization of silver nanoparticles using Cassia auriculata flower and evaluation of their antioxidant activity. Synthesized nanoparticles were characterized by using UV Vis absorption spectroscopy and SEM analysis. The synthesized particles were evaluated its in vitro antioxidant activity. The reaction mixture turned to brownish gray color after 5 hrs of incubation and exhibits an absorbance peak around 450 nm characteristic of Ag nanoparticle. Scanning electron microscopy (SEM) analysis showed silver nanoparticles was pure and polydispersed and the size were ranging from nm. Biosynthesized silver nanoparticles exhibited strong antioxidant activity. The approach of green synthesis seems to be cost efficient, eco-friendly and easy alternative to conventional methods of silver nanoparticles synthesis. The powerful bioactivity demonstrated by the synthesized silver nanoparticles leads towards the antioxidant agent Universal Research Publications. All rights reserved Key words: Silver nanoparticles, Cassia auriculata, Biosynthesis, SEM, In vitro antioxidant activity 1. Introduction The field of nanotechnology is one of the most active areas of research in modern materials science. Nanoparticles exhibit completely new or improved properties based on specific characteristics such as size, distribution and morphology [1]. New applications of nanoparticles and nanomaterials are emerging rapidly. Nanocrystalline silver particles have found tremendous applications in the field of high sensitivity biomolecular detection and diagnostics, antimicrobials and therapeutics, Catalysis and microelectronics. However, there is still need for economic, commercially viable as well environmentally clean synthesis route to synthesize silver nanoparticles [2]. A number of approaches are available for the synthesis of silver nanoparticles for example, reduction in solutions, chemical and photochemical reactions in reverse micelles, thermal decomposition of silver compounds, radiation assisted, electrochemical, sonochemical, microwave assisted process and recently via green chemistry route [3-4]. The use of environmentally benign materials like plant leaf extract, bacteria, fungi and enzymes for the synthesis of silver nanoparticles offers numerous benefits of ecofriendliness and compatibility for pharmaceutical and other biomedical applications as they do not use toxic chemicals for the synthesis protocol [5]. Chemical synthesis methods lead to presence of some toxic chemical absorbed on the surface that may have adverse effect in the medical applications. Green synthesis provides advancement over chemical and physical method as it is cost effective, environment friendly, easily scaled up for large scale synthesis and in this method there is no need to use high pressure, energy, temperature and toxic chemicals [6-7]. Therefore, the objectives of the present study to investigate the size of reduced silver nanoparticles by Scanning Electron Microscope (SEM) and the in vitro antioxidant activity of Cassia auriculata flower through the iron reducing power, hydrogen peroxide and total antioxidant activity. 2. Material and Methods 2.1. Chemicals Materials used for the synthesis of silver nanoparticles are AR grade silver nitrate (AgNO 3 ), purchased from Merck, India, Nitro blue tetrazolium (NBT), ethylene diamine tetra acetic acid (EDTA), sodium nitroprusside (SNP), trichloro 30

2 acetic acid (TCA), thiobarbituric acid (TBA), Potassium hexacyano ferrate [K 3 Fe (CN) 6], and L-ascorbic acid were purchased from SISCO Research Laboratories Pvt. Ltd., India. All other chemicals and solvents used were of analytical grade available commercially Collection of plant materials The dried flowers of the Cassia auriculata were collected from Kantharvakottai, Thanjavur District, Tamil Nadu, and India. The flowers were rinsed with water thrice followed by distilled water to remove the fine dust materials and then, the flowers were dried under direct sun light for 1 week to completely remove the moisture Preparation of flower extract The dried flowers were pulverized well with mortar and pestle to make a powder. Twenty grams of powder sample was mixed into 100 ml of deionized water and the mixture was boiled for 10 min. After cooling the flower extract was filtered with Whatman No. 1 filter paper. The filtrate was stored at 4 C for further use Synthesis of Ag nanoparticles using flower extracts For the Ag nanoparticles synthesis, 5 ml of Cassia auriculata flower extract was added to 45 ml of 1 mm aqueous AgNO 3 solution in a 250 ml Erlenmeyer flask. The flask was then incubated in the dark at 5hrs (to minimize the photo activation of silver nitrate), at room temperature. A control setup was also maintained without flower extract. The Ag nanoparticle solution thus obtained was purified by repeated centrifugation at 10,000 rpm for 15 min followed by re-dispersion of the pellet in de-ionized water. Then the Ag nanoparticles were freeze dried using SEM analysis [7] UV-Vis Spectra analysis The reduction of pure Ag+ ions was monitored by measuring the UV-Vis spectrum of the reaction medium at 5 hours after diluting a small aliquot of the sample into distilled water. UV-Vis spectral analysis was done by using UV-Vis spectrophotometer UV-2450 (Shimadzu) SEM analysis of silver nanoparticles Scanning electron microscopic (SEM) analysis was done using VEGA3 LMU machine, Japan. Thin films of the sample were prepared on a carbon coated copper grid by just dropping a very small amount of the sample on the grid. Extra solution was removed using a blotting paper and then the films on the SEM grid were allowed to dry by putting it under a mercury lamp for 5 min. 2.7 Free radical scavenging activity of Cassia auriculata Determination of total antioxidant capacity The antioxidant activity of the extracts was evaluated by the phosphomolybdenum method according to the procedure of Prieto et al., [8]. The assay is based on the reduction of Mo(VI) Mo(V) by the extract and subsequent formation of a green phosphate/mo(v) complex at acid ph. 0.3 ml extract was combined with 3ml of reagent solution (0.6M sulfuric acid, 28mM sodium phosphate and 4mM ammonium molybdate). The tubes containing the reaction solution were incubated at 95 C for 90 min. Then the absorbance of the solution was measured at 695 nm using a spectrophotometer against blank after cooling to room temperature. Methanol (0.3 ml) in the place of extract is used as the blank. For standard, L-Ascorbic acid was used as a control and prepared by dissolving 2mg of L-ascorbic acid in 10ml. The antioxidant activity of sample was expressed as % Iron reducing power assay The Fe 3+ reducing power of the Cassia auriculata was determined by the method of Oyaizu [9] with slight modifications. The Cassia auriculata (0.75ml) at various concentrations was mixed with 0.75ml of phosphate buffer (0.2mole, ph 6.6) and 0.75ml of potassium hexacyanoferrate [K 3 Fe (CN) 6] (1%, w/v), followed by incubating at 50 o C in a water bath for 20min. The reaction was stopped by adding 0.75ml of trichloro acetic acid (TCA) solution (10%) and then centrifuged at 3000r/min for 10min. 1.5ml of the supernatant was mixed with 1.5ml of distilled water and 0.1ml of ferric chloride (FeCl 3 ) solution (0.1%, w/v) for 10min. The absorbance at 700nm was measured as the reducing power. Higher absorbance of the reaction mixture indicated greater reducing power Hydrogen peroxide scavenging activity assay: Hydrogen peroxide scavenging activity of the Cassia auriculata was estimated by replacement titration [10]. Aliquot of 1.0ml of 0.1mmole of H 2 O 2 and 1.0ml of various concentrations of Cassia auriculata were mixed, followed by 2drops of 3% ammonium molybdate, 10ml of 2mole of H 2 SO 4 and 7.0ml of 1.8mole KI. The mixed solution was titrated with 5.09mmole of NaS 2 O 3 until yellow color disappeared. Percentage of scavenging of hydrogen peroxide was calculated as: % Inhibition = (V 0 - V 1 ) / V Where V 0 was volume of NaS 2 O 3 solution used to titrate the control sample in the presence of hydrogen peroxide (without Cassia auriculata), V 1 was the volume of NaS 2 O 3 solution used in the presence of the Cassia auriculata Statistical analysis Tests were carried out in triplicate for 3 5 separate experiments. The free radical scavenging activity of Cassia auriculata expressed in percentage (%). Fig.1. Color changes after (AgNPs) the process of reduction of Ag + to Ag nanoparticles (A) and control (AgNO 3 ) (B) (A) = AgNPs (1 mm AgNO 3 with C. auriculata extract after 5 hrs of incubation (Brown colour) (B) = 1mM AgNO 3 without C. auriculata extract. 31

3 3. Results 3.1 Synthesis and characterization of AgNPs Addition of plant broth resulted in the gradual change of the colour of AgNO 3 solution from colourless to brown (Fig 1), indicating the synthesis of AgNPs. The color intensity also increased with the duration of incubation. The flower extracts without AgNO3 did not show any change in color and AgNO3 solution has white color. The plant-broths, yield of AgNPs as revealed by spectral analysis was best by broth of C. auriculata flower that exhibited the highest peak. The absorbance was monitored at a wave length in the range of nm and the resulting absorption spectrum showed the peak at 450 nm (Fig 2) that was characteristic of AgNPs. SEM image and size distribution of AgNP-P indicated that they were more or less spherical in shape with an average diameter of 20 ± 10 nm, distributed in the range of nm (Fig. 3and 4). reveals that the antioxidant activity of the extract is in the increasing trend with the increasing concentration of the plant extract and AgNPs. The observed scavenging effect of plant extract, AgNPs and standard on the total antioxidant activity decreases in the following order: AgNPs >L ascorbic acid > plant extract. Among this AgNPs possess potential antioxidant activity as compared with ascorbic acid. Fig. 4. High resolution scanning electron microscopic (SEM) image of individual silver nanoparticles (AgNPs). Fig. 2. UV-Vis absorption spectrum of silver nanoparticles synthesized by treating 1mM aqueous AgNO 3 solution with Cassia auriculata flower extract after 5 hrs. Fig 4a shows the schematic representation of synthesis of silver nanoparticles from Cassia auriculata flowers. Fig. 3. High resolution scanning electron microscopic (SEM) image of silver nanoparticles (AgNPs). Polydispersed AgNPs ranged between 10 40nm. 3.2 In vitro antioxidant activity The yield of the methanol extract of the plant extract and AgNPs and its total antioxidant capacity are given in Figure 5. Total antioxidant capacity of C. auriculata is expressed as the number of equivalents of ascorbic acid. The study Fig. 5. Total antioxidant assay activity of auriculata extract at different concentrations 32 Cassia Positive Hydrogen peroxide tests demonstrated that plant extract, AgNPs and ascorbic acid are free radical scavengers. The Hydrogen peroxide scavenging assay exhibited effective inhibition activity of AgNPs, plant extract and ascorbic acid. The Hydrogen peroxide activity of the nanoparticles was found to increase in a dosedependent manner. The observed scavenging effect of plant

4 extract, AgNPs and standard on the Hydrogen peroxide scavenging activity decreases in the following order: AgNPs >L ascorbic acid > plant extract. However, the AgNPs exhibited more inhibition with more than 57% scavenging activity of Hydrogen peroxide than ascorbic acid (Figure 6). Fig. 6. Hydrogen peroxide scavenging activity of auriculata extract at different concentrations Cassia The iron reducing power activity of C. auriculata and AgNPs were increased markedly with the increase of concentrations. The observed scavenging effect of plant extract, AgNPs and standard on the iron reducing power activity decreases in the following order: AgNPs >L ascorbic acid > plant extract. The iron reducing power capability is high in AgNPs. These results suggested that AgNPs had superior iron reducing power effect. The iron reducing power assay of AgNPs, plant extract and ascorbic acid represented in Figure 7. Fig. 7. Reducing power assay of Cassia auriculata extract at different concentrations 4. Discussion 4.1. Synthesis of silver nanoparticles The green synthesis of silver nanoparticles through plant extracts were carried out. Silver nitrate is used as reducing agent as silver has distinctive properties such as good conductivity, catalytic and chemical stability. Applications of such eco-friendly nanoparticles in bactericidal, wound healing and other medical and electronic applications, makes this method potentially exciting for the large-scale synthesis of other inorganic materials (nanomaterials). The aqueous silver ions when exposed to herbal extracts were reduced in solution, there by leading to the formation of silver hydrosol. The time duration of change in colour varies from plant to plant. The phytochemicals present in the flower extract were considered responsible for the reduction of silver ions. It is well known that silver nanoparticles exhibit yellowish - brown colour in aqueous solution due to excitation of surface plasmon vibrations in silver nanoparticles The appearances of yellowish-brown colour in the reaction vessels suggest the formation of silver nanoparticles (SNPs) [11]. It is generally recognized that UV Vis spectroscopy could be used to examine size- and shape-controlled nanoparticles in aqueous suspensions. Absorption spectra of silver nanoparticles formed in the reaction media has absorbance peak at 450 nm, broadening of peak indicated that the particles are polydispersed. Silver nanoparticles are being extensively synthesized using many different biological sources including fungi, bacteria and plants [12, 13]. Among them the plant mediated nanoparticles synthesis is getting more popular because of the high reactivity of plant extract and easy availability of plant materials [14]. This method of nanoparticles synthesis involves no toxic chemicals and termed as green chemistry procedure. In this present study, C. auriculata extract was used for the synthesis of silver nanoparticles. The aqueous AgNO 3 solution turned to brown colour in 30 min with the addition of flower extract, indicating the formation of AgNPs in the reaction solution probably as a result of the excitation of surface plasmon resonance (SPR) bands [15, 16]. The control tubes (AgNO 3 ) showed no change in colour when incubated in a similar condition. SEM analysis was carried out to understand the topology and the size of the Ag-NPs, which showed the synthesis of higher density polydispersed spherical Ag-NPs of various sizes. The SEM image showing the high density silver nanoparticles synthesized by the flower extract further confirmed the development of silver nanostructures. Most of the nanoparticles were scattered, as observed under SEM. The SEM analysis showed the particle size between 10-40nm as well the cubic, face-centred cubic structure of the nanoparticles Antioxidant activity The identification of antioxidant is beneficial to biological system against ROS ravage. Recently importance has been given for in vitro antioxidant study to understand the pharmacological role of medicinal plant and its isolate. In vitro techniques have been used for detection of antioxidants, which are based on the ability of compounds to scavenge peroxy radicals [17] Total antioxidant activity Total antioxidant capacity of Cassia auriculata is expressed as the number of equivalents of ascorbic acid. The phosphomolybdenum method was based on the reduction of Mo (VI) to Mo (V) by the antioxidant compound and the formation of a green phosphate/ Mo (V) complex with a maximal absorption at 695 nm. The assay is successfully used to quantify vitamin E in seeds and, being simple and independent of other antioxidant measurements commonly employed, it was decided to extend its application to plant extract [8]. Moreover, it is a quantitative one, since the antioxidant activity is expressed 33

5 as the number of equivalents of ascorbic acid. The study reveals that the antioxidant activity of the extract is in the increasing trend with the increasing concentration of the plant extract, ascorbic acid and AgNPs. Among this AgNPs possess potential antioxidant activity as compared with plant extract (Fig 5) Hydrogen peroxide scavenging activity Hydrogen peroxide is a weak oxidizing agent and can inactivate a few enzymes directly, usually by oxidation of essential thiol (-SH) groups. Hydrogen peroxide can cross cell membranes rapidly, once inside the cell, H 2 O 2 can probably react with Fe 2+, and possibly Cu 2+ ions to form hydroxyl radical and this may be the origin of many of its toxic effects [18]. It is therefore biologically advantageous for cells to control the amount of hydrogen peroxide that is allowed to accumulate. As shown in Fig 6. Cassia auriculata demonstrated hydrogen peroxide scavenging activity in a concentration dependent manner. The study reveals that hydrogen peroxide scavenging activity of the extract is in the increasing trend with the increasing concentration of the plant extract, ascorbic acid and AgNPs. Among this AgNPs possess hydrogen peroxide scavenging activity as compared with plant extract Iron reducing power activity For the measurements of the reducing ability, the Fe 3+ Fe 2+ transformation was investigated in the presence of Cassia auriculata. The reducing capacity of a compound may serve as a significant indicator of its potential antioxidant activity. However, the activity of antioxidants has been assigned to various mechanisms such as prevention of chain initiation, binding of transition-metal ion catalysts, decomposition of peroxides, reductive capacity and radical scavenging [19, 20]. Fig 7 depicts the reductive effect of Cassia auriculata. Similar to the antioxidant activity, the reducing power of Cassia auriculata increased with increasing dosage. All the doses showed significantly higher activities than the control exhibited greater reducing power, indicating that Cassia auriculata having the greater reducing power. The study reveals that reducing power activity of the extract is in the increasing trend with the increasing concentration of the plant extract, ascorbic acid and AgNPs. Among this AgNPs possess potential reducing activity as compared with plant extract. The present study concluded that the bio-reduction of silver ions through C. auriculata flower extract and testing for their in vitro antioxidant activity. The bio-reduction of aqueous Ag+ ions by the flower extract of the cassia auriculata has been demonstrated. The aqueous silver ions exposed to the extract, the synthesis of silver nanoparticles were confirmed by the change of colour of plant extract. Absorption spectra of silver nanoparticles formed in the reaction media has absorbance peak at 450 nm also confirmed. These environmentally benign silver nanoparticles were further confirmed by using SEM. The SEM analysis showed the particle size between 10-40nm as well the cubic structure of the nanoparticles. On the basis of the results of this study, it clearly indicates that C. auriculata, ascorbic acid and AgNPs possessing antioxidant activity against various in vitro antioxidant systems. The free radical scavenging activity of AgNPs was found to be higher than that standard confirmed in the present investigation. From the above assays, the possible mechanism of antioxidant activity of AgNPs includes reductive ability, electron donating ability and scavengers of radicals. These obtained silver nanoparticles are advantageous in medical and pharmaceutical purposes. It also has potential applications in the biomedical field and can be produced commercially at large scale. 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