PHOTO CATALYTIC REDUCTION OF METHYLENE BLUE DYE USING BIOGENIC SILVER NANOPARTICLES FROM THE AQUEOUS CLADODE EXTRACT OF Casuarina equisetifolia
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1 Page4562 Indo American Journal of Pharmaceutical Research, 2016 ISSN NO: PHOTO CATALYTIC REDUCTION OF METHYLENE BLUE DYE USING BIOGENIC SILVER NANOPARTICLES FROM THE AQUEOUS CLADODE EXTRACT OF Casuarina equisetifolia Saranya.V.T.K *, S. Uma Gowrie Department of Plant Biology and Plant Biotechnology, Ethiraj College for Women, Chennai ARTICLE INFO Article history Received 15/02/2016 Available online 05/03/2016 Keywords Casuarina Equisetifolia, Cladode, Silver Nanoparticles, Methylene Blue Dye, Degradation. Corresponding author Saranya.V.T.K. Depratment of Plant Biology and Plant Biotechnology, Ethiraj college for Women, Tamilnadu, Chennai-8. ABSTRACT There is an increasing demand commercially for the bio synthesis of metallic nanoparticles, due to its catalytic property and wider application in medicine, pharmaceutical, agriculture, defense etc. Casuarina equisetifolia is a tree species, with high calorific value which is widely cultivated to improve the soil nutrient, but its evaluation in the field of pharmacology is still under explored. The present study is a novel work where we report the synthesis of Silver nanoparticles (AgNPs) using Casuarina equisetifolia leaf extract as a potential reducing agent, with the help of 0.1mM of silver nitrate solution. Various optimization parameters like temperature, ph and time for synthesis were optimized. Thus the particles synthesized under this optimized condition were characterized using UV-Vis spectrophotometer, Fourier transform-infrared (FT-IR), X-ray diffraction (XRD) and Transmission electron microscope (TEM). Surface Plasma resonance peak of the synthesized nanoparticles was observed at 418nm, using UV-Vis spectrophotometer. FTIR spectrum was analyzed to identify the effective functional molecules, which are responsible for the reduction and stabilization of silver nanoparticles synthesized by leaf extract. XRD results confirmed the crystalline structure of the synthesized AgNO 3. The photocatalytic activity of the synthesized silver nanoparticles was observed to have potential efficacy to degrade methylene blue dye under sunlight irradiation. This was confirmed by the decrease in maximum absorbance of methylene blue dye with respect to time using UV-Vis spectrophotometer. The bio synthesized silver nanoparticles, effectively degraded nearly 35.13% of methylene blue dye at 5 hours of exposure time. The results clearly figure out that the biogenic silver nanoparticles synthesized from Casuarina equisetifolia can find its applications in textile industries, water treatment plants and also in pharmaceutical industry to prevent the adverse effect caused by methylene blue, like painful micturition, methymoglobinemia. Please cite this article in press as Saranya.V.T.K. et al. Photo Catalytic Reduction of Methylene Blue Dye Using Biogenic Silver Nanoparticles From The Aqueous Cladode Extract of Casuarina Equisetifolia. Indo American Journal of Pharmaceutical Research.2016:6(02). Copy right 2016 This is an Open Access article distributed under the terms of the Indo American journal of Pharmaceutical Research, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
2 Page4563 INTRODUCTION The term nanoparticles describes a particle with size in the range of 1nm-100nm, at least in one of the three possible dimensions. In this size range, the physical, chemical and biological properties of the nanoparticles changes in fundamental ways from the properties of both individual atoms/molecules and of the corresponding bulk materials. Silver nanoparticles have received considerable attention owing to their attractive physicochemical properties. Silver nanoparticles have its application in the field of bio labeling, sensor, antimicrobial, catalysis, electronic and other medical application such as drug delivery [1]. Recent studies suggest that metallic nanoparticle serves as an effective catalyst, to degrade complex structured organic dyes under effect natural and artificial illumination. Natural products, such as plants extract, either as pure compounds or as standardized extracts, provide unlimited opportunities for new drug discoveries because of the unmatched availability of chemical diversity [2]. Synthesis of silver nanoparticles using plant resources has so many advantages over conventional methods like chemical synthesis, due to its environmental friendly activity and low cost for efficient production [3]. Casuarina equisetifolia is a predominant, non-leguminous, nitrogen fixing tree species, in association with actinomycetes Frankia. Extracts of C. equisetifolia, shows significant anthehelmintic activities [4], anticancer [5], antifungal [6] and hypoglycemic activity. Aqueous extract of cladode proves to have potential phytochemicals, that efficacy to reduce silver nitrate to biosynthesized silver nanoparticles. Commercially available dyes are sources of environmentally hazardous organic compounds that affect both the aquatic life and human health. Biological degradation of these dyes seems to be ineffective due to its varied composition of organic compounds. Methylene blue (MB) (3,7bis(Dimethylamino)phenothiazin5iumchloride) is a thiazine dye, that causes difficulties in breathing, when consumed it results in vomiting, diarrhea and nausea [7]. The study was carried on to investigate the role of aqueous cladode extract of Casuarina equisetifolia in the synthesis of Silver nanoparticles. The synthesized nanoparticles were optimized for various parameter like temperature, time and ph. The optimization was followed by characterization; the silver nanoparticles were taken for UV-Vis Spectroscopy analysis, FTIR spectrum study and TEM micrograph, to characterize the synthesized particles. The study also aims to evaluate the catalytic property of Silver nanoparticles synthesized from Casuarina equisetifolia, on the reduction of Methylene Blue dye. Thus the objective of current study, aims to understand the synergistic interaction of active phytocompounds with the Silver nitrate solution for the formation of silver nanoparticles that serves as a potential source in degrading dye. MATERIALS AND METHOD PLANT MATERIAL AND PREPARATION OF EXTRACT: Fresh and healthy cladodes of Casuarina equisetifolia (Fig 1a) were collected from Pudhucherry. The cladodes were washed thoroughly using distilled water, incised into small pieces, shade dried for a week and was ground into fine powder. About 25g of powdered leaves sample was extracted with distilled water at 100 C for 25 mins [8]. The extract obtained was filtered through Whatman No1 filter paper and stored at 4 C for further use. SYNTHESIS OF SILVER NANOPARTICLES: 1mM of silver nitrate solution was used to synthesis silver nanoparticles, from the aqueous cladode extract of Casuarina equisetifolia. To synthesis silver nanoparticles, 1mM silver nitrate solution was mixed with aqueous cladode extract of Casuarina equisetifolia. The solution was kept in dark overnight. The reduction of Ag + was analysed visually by colour change (Fig 1b) and was also monitored by measuring the UV-Vis spectrum of the reaction mixture. 1 1b Fig 1a - Casuarina equisetifolia plantation ; Fig 1b Green synthesis of silver nanoparticles using aqueous cladode extract. OPTIMISATION: TEMPRATURE: Temperature serves as one of the important factor for the synthesis of silver nanoparticles. So it is very important to standardize an optimum temperature for perfect synthesis of nanoparticles. The various temperature of the reaction was set as 20 C, 40 C, 60 C, 80 C, 100 C using water bath. At the end the absorbance was observed using Uv-Visible spectrophotometer.
3 Page4564 ph: The reaction mixture ph was maintained at various ranges, such as 4.0, 7.0 and 9.0 individually. The ph was adjusted using 0.1N HCl and 0.1 NaOH. Absorbance was recorded using Uv-Visible spectrophotometer. TIME: The optimization of the time was done in order to check the stability of the synthesized Silver nanoparticles. For this purpose different reaction periods (10 mins, 20 mins, 30 mins) was chosen. Absorbance of the resulting reaction mixture was measured spectrophototmetrically. CHARACTERISATION: UV-Vis spectroscopy is widely used for characterization of silver nanoparticles. The fixed range for spectral analysis was 300nm to 500nm. Thus at this range the reaction mixture was scanned for absorption maxima. The freeze dried powder of the synthesized nanoparticles, was used for spectrum analysis with the help of Shimazdu IR prestrige 21 FTIR instrument with diffuse reflectance mode (DRS- 8000), to detect the functional molecules responsible for capping. X-ray diffraction (XRD) measurements were carried out with a Bruker D8 A vance diffract meter using Ni-filtered Cu Kα radiation (λ = nm). XRD patterns were recorded in the 2θ range at a scan speed of 10 min -1 at room temperature. TEM micrographs help to interpret details regarding external morphology and size of the synthesized nanoparticles. Micrographs were taken with the help of Transmission Electron Microscope Techai 10 Philips and photographed. PHOTOCATALYTIC DEGRADATION OF DYE: Methylene blue dye solution was prepared by dissolving 0.1mg of the methylene blue dye in 100 ml of water. To this about 10mg of silver nanoparticles synthesized from the aqueous cladode extract was added and mixed well for 20 minutes using magnetic stirrer. Control was also maintained without addition of the silver nanoparticles. This was subjected to exposure of sunlight and at the end of each hour, 3ml of suspension was taken to analyse the dye degradation percentage. This was done by measuring the absorbance spectrum at 660nm,specific for methylene blue dye, using UV-Vis Spectrophotometer. Percentage of dye degradation was calculated using the formula: ( 0 ) %Decolorisation = X where 0 is the initial concentration of dye solution and is the concentration of dye solution after photocatalytic degradation. RESULT AND DISCUSSION: TEMPRATURE: On addition of aqueous cladode extract of Casuarina equisetifolia, to silver nitrate, there was a change in colour to brown. Experimental temperature range was set as 20 C, 40 C, 60 C. 80 C and 100 C. Absorbance increased from 20 C to 80 C and then there was a drop in absorbance value at higher temperature. AgNPs, synthesis by Casuarina equisetifolia cladode extract, was optimized at temperature of 80 C, with maximum absorption. Similar results were obtained during the synthesis of silver nanoparticles using Hippophae rhamnoides Linn. leaves aqueous extract [8]. (Fig 2) Fig 2 - Uv-Visible Spectra of AgNP s showing effect of different reaction temperature. TIME: Silver nanoparticles are not stable in nature. So it is always necessary to optimize the duration, because Silver nanoparticles agglomerate after the optimum duration resulting in larger particle size. Increase in absorbance was observed with the increase in the incubation period [9]. The optimum time required for the completion of reaction was 30 mins, (Fig 3) for the stable synthesis of silver nanoparticles using cladode extract of Casuarina.equisetifolia.
4 Page4565 Fig 3 - Uv-Visible Spectra of AgNP s showing effect of different reaction time. ph: When the ph is low, larger nanoparticles are formed, whereas at higher ph agglomeration of silver nanoparticles take place. The optimum ph was found to be 7 (Fig 4), that is neutral ph ( temperature 80 C, time 30mins). It was observed that there was a decrease in absorbance with increase in ph [10]. Fig 4 - Uv-Visible Spectra of AgNP s showing effect of different reaction time. UV-Vis SPECTROPHOTOMETER: UV-Vis spectrophotometer serves as a valuable tool for characterizing AgNP s, with the help of the optical absorption spectra and surface plasmon resonances produced by the metal nanoparticles and the capping agents [11]. Synthesis of silver nanoparticles using the extracts was confirmed by determining the absorption peak maxima at regular time intervals using UV vis spectrophotometer in the range of nm. Throughout the experiment, during synthesis and optimization of the silver nanoparticles synthesized using cladode extract of Casuarina equisetifolia, lies between the range 425 nm to 440nm. FTIR ANALYSIS: FTIR measurements were carried out to identify the biomolecules for capping and efficient stabilization of the metal nanoparticles synthesized by the aqueous cladode extract of Casuarina equisetifolia.(fig 8) The band at cm -1, cm -1 corresponds to dimeric OH stretch of alchol and hydroxyl compounds, with strong and broad peak description. The peak at cm -1 corresponds to internally bonded OH stretch. The peak correspond to cm -1, cm -1 corresponds to NH bend of secondary amine. The peak corresponding to cm -1, cm -1, cm -1, cm -1, corresponds to aliphatic organo halogen frequency group. The presence of aromatic ring corresponds to the peak cm -1, cm -1 (aromatic C-H out of plane bend). The C-O ester group, secondary amine CN stretch, Phenol stretch peaks found in FTIR analysis of aqueous extract of cladode of Casuarina equisetifolia, was found to reduce and stabilize the nanoparticles formed. Thus the phytochemicals present in the cladode extract proves to serve as powerful reducing and stabilizing agents, which may be responsible for reduction of Ag +.
5 Page4566 Fig 5 - FTIR spectrum of the Silver nanoparticles synthesized using aqueous root extract of Casuarina equisetifolia. POWDER XRD ANALYSIS: To confirm and identify the crystalline nature of SNPs using X-ray diffraction pattern were recorded from the 2θ upto 10 to 70 degrees. The fig 6 shows, the characteristic 2θ values are 19.64º, 21.72º and 29.67º. The average crystalline size was found to be 40 Å using using Scherrer s formula, d = 0.9 λ / B cosθ. Fig 6 XRD pattern of the biosynthesized AgNP s. TRANSMISSION ELECTRON MICROSCOPE: TEM micrographs were used to analyze the shape and structure of the nanoparticles formed. From fig 7 it is evident that AgNPs were spherical in shape. The smallest nanoparticles size observed was 9nm and the largest was 22 nm. Few nanoparticles showed tendency to aggregate. Similar results were reported during the synthesis of Silver Nanoparticles using extracts of Ipomoea indica flowers [12]. Fig 7- TEM image of the biosynthesized AgNP s.
6 Page4567 PHOTOCATALYTIC DEGRADATION OF DYE: Degradation of dye was visually observed by the change in colour from deep blue to light blue at the end of 5 th hour. The control exhibited no colour change. With the increase in the time of exposure, the absorption peak at 660nm, specific for methylene blue decreased gradually, followed by the increase in absorption band of silver nanoparticles at 420nm. This clearly indicates the photocatalytic degradation of methylene blue dye, mediated by silver nanoparticles synthesized from aqueous cladode extract of Casuarina equisetifolia. The photocatylytic degradation was found to be more effective irradiation technique for degradation of dye [13, 14]. Fig8 Photo catalytic degradation of dye. CONCLUSION This plant based green synthesis of nanomaterial is the progress of eco-friendly method, for the fabrication of metal based nanoparticles. This study method was free from poisonous and dangerous solvents and waste. The overall optimized reaction condition was: Temperature = 80 C; Time = 30 mins; ph =7. TEM results shows that the shape of nanoparticle was spherical. FTIR result clearly marks the functional group, responsible for the stabilization of silver nanoparticles. Thus the optimized and synthesized silver nanoparticles from the aqueous extract of cladode, are further used for catalyzing the reduction of methylene blue dye. The nanocatalysed degradation of MB dye was found to be much effective compared to that of the control. This proves that it could be a cost effective way to treat textile industry effluents. In future, these biogenic fabricated nanoparticles could be employed in the development of drug, to prevent the adverse effect of methylene blue dye like mental confusion, micturition, methemoglobinemia. ACKNOWLEDGEMENNT The authors acknowledge the Principal and Head of the department for their constant support and encouragement. We would also thank Mrs.Kalaiselvi of Instrumentation Centre, Ethiraj college for Women, for her timely help. LIST OF ABBREVATIONS % -Percentage mm -Millimolar g -Gram AgNP s -Silver Nanoparticles UV-Vis -Ultra Violet Visible FTIR -Fourier transform-infrared XRD -X-ray diffraction TEM - Transmission Electron Microscope AgNO 3 - Silver Nitrate HCl -Hydrochloric acid NaOH -Sodium Hydroxide
7 Page4568 REFERENCE 1. Jong WHD., Borm PJA., Drug delivery and nanoparticles: Applications and hazards, International Jounnal Nanomedicine (2): Paul Cos., Arnold J. Vlietinck., Dirk Vanden Berghe., Louis Maes., Review Anti-infective potential of natural products: How to develop a stronger in vitro proof-of-concept, Journal of Ethnopharmacology 2006; 10: Sharma KV., Yngard AR., Lin Y., Silver nanoparticle: Green synthesis and their antimicrobial activities, Advances in Colloid and Interface Science 2009; 145: Aher AN., Pal SC., Patil UK., Yadav SK., Evaluation of anthelmintic activity of Casuarina equisetifolia Frost (Casuarinaceae), Planta Indica 2006; 2: Cock IE., Antibacterial Activity of Selected Australian Native Plant Extracts, Internet J. Microbiol. 2008; 4:2. 6. Han ST (1998). Medicinal Plants in South Pacific. WHO Regional Publications, Geneva, Switzerland. 7. Kavitha, D., Namasivayam, C., Experimental and kinetic studies on methylene blue adsorption by coir pith carbon, Bioresour. Technol. 2007; Bashir Ahmed., Javid Ali., Shumalia Bashir.,Optimisation and effects of different reaction conditions for the bioinspired synthesis of silver nano particles using Hippophae rhamnoides Linn. Leaves aqueous extract., World Applied Sciences Journal 2013; 22 (6): K.Allikarjuna, G.Narasimha., Green synthesis of silver nanoparticles using Ocimum leaf extract and their characterization, Digest journal of Nanomaterials and Biostructures, 2011; Vol 6, No 1, Ravichandran.V., Z.X.Tiah., G.Subashini., F.W.X.Terence,, F.C.Y. Eddy., J.Nelson., A.D.Sokkalingam, Biosynthesis of silver nanoparticles using Mangsteen leaf extract and evaluation of their antimicrobial activies. Journal of Saudi chemical Society, 2011; 15: Brause. R., Moeltgen.H., Kleinermanns. K., Characterization of laser ablatedand chemically reduced silver colloids in aqueous solution by UV/vis spec-troscopy and STM/SEM microscopy. Appl. Phys. B: Lasers Opt. 2002; 75, K.V.Pavani., Gayathramma.K., Aparajitha Banerjee., Shah Suresh., Phyto-syntheis of Silver Nanoparticles using Extracts of Ipomoea indica Flowers., American journal of Nanomaterial, 2013;Vol 1 No 1, Kansal SK., Singh M., Sudo D (2008) Studies on TiO2/ZnO photocatalysed degradation of lignin., J Hazard Mater, 2008; 153: Kumar P., Govindaraju M., Senthamilselvi S., Premkumar K., Photocatalytic degradation of methyl orange dye using silver nanoparticles synthesized from Ulva lactuca. Colloids Surf B: Biointerfaces 2013; 103:
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