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Micro and Nanosystems, 2012, 4, 192-198

Green Synthesis of Well Dispersed Nanoparticles using Leaf Extract of Medicinally useful Adhatoda Vasica Nees
V. Karthicka, V. Ganesh Kumara,*, T. Maiyalaganb, R. Deepaa, K. Govindarajua, A. Rajeswaria and T. Stalin Dhasa
a b

Nanoscience Division, Centre for Ocean Research, Sathyabama University,Chennai - 600 119, India

School of Chemical and Biomedical Engineering, Nanyang Technological University, 70 Nanyang Drive Singapore 639798
Abstract: Development of reliable method for the green synthesis of gold nanoparticles (AuNPs) using medicinally valued Adhatoda vasica Nees has been studied here. The color change and the Surface Plasmon Resonance (SPR) confirmed the formation of AuNPs. The biosynthesized AuNPs were characterized using UV-visible Spectroscopy (UV-vis), Fourier Transform Infrared Spectroscopy (FT-IR), X-Ray Diffraction (XRD), Scanning Electron Microscopy (SEM), Energy Dispersive Spectroscopy (EDAX) and Transmission Electron Microscopy (TEM) analysis. The nanoparticles synthesized were predominantly monodisperse, stable spherical in nature with well-defined dimensions of size ranging from 22 to 47 nm. The crystalline nature of the synthesized particles was also evident by the X-ray diffraction analysis.

Keywords: Adhatoda vasica, Gold nanoparticles, Biosynthesis, Electron Microscopy, Diffraction. INTRODUCTION Nanobiotechnology combines biological principles with physical and chemical procedures to generate nano-sized particles with well defined functions. Particles of interestingly small size make the field of drug delivery more interesting and effective. Synthesizing gold particles (AuNPs) with medicinal applications is the recent trend in the field of nanobiotechnology. The surface availability of nanoparticles for binding/reactivity of other species on them is an important function as it is synthesized in different structures like nanorods, spheres, prims and hexagons. The controlled growth of AuNPs of different morphologies and the various chemical mechanisms involved in the anisotropic growth were studied using different chemical procedures [1]. The low toxicity effects of green synthesized AuNPs on biological systems made researchers to synthesize it by biological method rather by chemical means. Extensive studies were done on AuNPs and its binding affinity towards nucleic acids and proteins in biological systems [2]. AuNPs have been synthesized from various sources like plants [3], microbes [4], seaweeds [5] and microalgae [6]. Sastry et al., 2007 have synthesized AuNPs using chitosan and showed good control on postprandial hyperglycemia which when loaded with insulin [7] proved AuNPs to be an effective drug carrier. AuNPs can be used for the detection of nucleic acids as Chandirasekar and co-worker have, 2011 synthesized AuNPs using bile salts and particles with different geometry was achieved by varying the reducing agent concentration [8]. AuNPs have also been employed in the imaging of cancer cells like AR42J pancreatic tumor cells by synthesizing it using laser ablation technique and reported that size of AuNPs plays a role in the intracellular uptake [9].
*Address correspondence to this author at the Nanoscience Division, Centre for Ocean Research, Sathyabama University,Chennai - 600 119, India; Tel: +91 44 24500646; Fax: +91 44 24503308; E-mail: ganesv@gmail.com

Adhatoda vasica Nees, a malabar nut tree belonging to the family Acanthaceae is native of South Asia and is exclusively studied for its active components like quinazoline alkaloids, vasicine, vasicinone, deoxyvasicine, oxyvasicinine, maiontone and other essential oils. The plant parts have been traditionally used for curing diseases like stomatitis, asthma and bronchititis [10]. The compound 73/602 an alkaloid isolated from the leaves and roots of A. vasica is a structural analogue of vasicinone which shows appreciable antiallergic activity in mice, rats and guinea pigs [11]. Synthesis of AuNPs using medicinally useful plants [12, 13] is gaining more importance in therapeutic applications, where the reducing agent responsible for the reduction of chloroauric acid will have its own effect on biological systems. In our previous work, we have demonstrated the use of antidiabetic potent plant Cassia auriculata for the synthesis of AuNPs [14]. Herein, we have used an antiallergic potent plant A. vasica for the synthesis of AuNPs which may possess antiallergic effect on animals and that will be a welcoming outcome in the field of drug delivery. MATERIALS USED Chloroauric acid (HAuCl43H2O) was obtained from Loba Chemie, India was used as received. All other reagents used in the reaction were of analytical grade with maximum purity. A. vasica Nees leaves were collected from forest area of Vellore, Tamilnadu, India and was cleaned with tap water and double distilled water twice to remove the dust. Further, it is shade dried for a week at room temperature and powdered for use. SYNTHESIS OF GOLD NANOPARTICLES For the preparation of A. vasica leaf extract, 4 g of powdered leaves is taken in a conical flask along with 40 mL of distilled water. The flask is then placed in an orbital shaker for 4 h and then the extract is taken by filtering the
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Fig. (1A). Aqueous extract of A. vasica (B ) Chloroauric acid solution (C) Ruby red colour indicating the formation of gold nanoparticles.

Fig. (2). UV-vis spectrum of the gold nanoparticles recorded immediately after synthesis (after 5 min).

whole content using whatman No.1 filter paper. The filtrate is then stored in refrigerator for further use. The reaction is carried out using different concentrations of plant extract and finally optimized to a ratio of 1:10 (Extract: Chloroauric acid solution). Further 3 mL of extract is added to 30 mL aqueous solution of chloroauric acid (10-3 M) and kept in an orbital shaker at room temperature. Formation of AuNPs takes place within 4 min, evident from the development of a stable ruby red color (Fig. 1) in the solution indicates the reduction of Au3+ to Au0 with no color change further. The experiment was repeated thrice to check the formation of AuNPs. The synthesized AuNPs are stable at a pH range of 3.6-9.1 showed no precipitation and color change for a period of 45 days. CHARACTERIZATION OF GOLD NANOPARTICLES The UV-vis spectra were recorded using Thermo Scientific Spectrascan UV 2700 with samples in quartz cuvette. FT-IR of Perkin Elmer spectrophotometer was used to identify the possible groups responsible for the reduction of chloroauric acid at a resolution of 4 cm-1 in the range of 4000-450 cm-1 and the FT-IR spectrum was recorded by employing KBr pellet technique using Perkin Elmer model-

983/G detector double beam spectrophotometer. XRD pattern of the A. vasica reduced AuNPs was carried out using a Rich Seifert P3000 instrument operated at a voltage of 40 kV with Cu K radiations. SEM-EDAX was studied to check the surface morphology and elemental analysis of the AuNPs using XL30 FESEM, Philips. TEM studies were carried out by drop coating AuNPs onto carbon-coated TEM grids using Philips Technai-10. RESULTS AND DISCUSSION: The formation of AuNPs is noted down by the color change after the addition of aqueous extract to chloroauric acid solution as illustrated in (Fig. 1). The formation and stability of metal nanoparticles in aqueous solution is studied using UV-vis spectrophotometer. The surface plasmon resonance (SPR) arising due to the oscillation of free conduction electrons induced by the absorption of electromagnetic field [15] as a absorption band with a maximum at 532 nm has been observed in the spectrum (Fig. 2). To identify the possible functional groups responsible for the reduction of chloroauric acid the FTIR spectrum is taken and the interaction of biomolecules have

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Fig. (3). FT-IR spectra of dried powder of (A) A. vasica extract (B) gold nanoparticles.

been studied (Fig 3). The spectrum show a clear difference in the functional group shifts. The peak seen at 3358 cm-1 corresponds to the OH or COOH group which has shifted to higher wavelength 3687 cm-1 thus, implying that the OH

or COOH group might have stabilized the AuNPs. The stretching of C=O can be observed in the spectrum indicating the red shift from 1625 to 1675 cm-1. The peak 1406 cm-1 corresponds to the COO- also shows a shift

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Fig. (4). Diffraction pattern of gold nanoparticles synthesized using A. vasica.

Fig. (5A). SEM image shows the presence of bioorganic compounds involved in the reduction (B) EDAX showing strong signals for gold nanoparticles.

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Fig. (6A&B). TEM image taken at various magnifications showing gold nanospheres.

which confirms the contribution of carboxylate groups in the reduction. In the spectrum aromatic resonances have not been identified indicating the absence of aromatic groups in the stabilizing compounds. Thus by using FT-IR, it is concluded that OH or COOH, -C=O, -COO- groups has involved in the stabilization of AuNPs. The crystalline nature of AuNPs was examined using XRD where three diffraction peaks were observed in the 2 range of 10 to 70 which can be indexed as (111), (200) & (220) reflections of fcc (face centered cubic) matches with Joint Committee on Powder Diffraction Standards (JCPDS No: 04-0784) revealing that synthesized AuNPs are composed of pure crystalline gold as there is no other peak found (Fig. 4). The XRD patterns obtained were similar to the results reported earlier on AuNPs [16]. The particle size of the AuNPs formed were calculated using Debye-Scherrer equation D = K / cos Where D is the average crystalline domain size perpendicular to the reflecting planes, K the Scherrer

constant with value from 0.9 to 1, is the wavelength of the X-ray source, is the full width at half maximum (FWHM) and is the Diffraction angle. From this equation, the particle size calculated was found to be 39 nm which is similar to the size observed in TEM image of the AuNPs. The surface morphology and the scale in which the size of the nanoparticle synthesized can be studied using the SEM. The presence of bioactive components responsible for the reduction of the chloroauric acid to AuNPs was revealed using SEM imaging (Fig. 5A). The image confirms the presence bioorganic compounds which has stabilized the AuNPs. The EDAX profile has showed strong signal for gold atoms (Fig. 5B) and weak signals for chlorine, oxygen, sodium and magnesium which implies that these signals might be from the biomolecules present in the aqueous extract. The morphology of the synthesized nanoparticles were determined by TEM image and shown in (Fig. 6A&B). For the analysis the image is taken in a random place on the grid and the morphology is observed carefully. The particles as seen in TEM image are triangular and spherical in shape

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Fig. (7). Possible mechanism for the reduction of chloroauric acid by bioactive compounds.

with an average size of 38 nm with many similar sized particles except a few. The uniformity in size is a welcoming result in the green synthesis as in most of the cases the particle size varies with greater range which is a concern when applied in a targeted drug for therapeutic applications. The particle shape usually observed in most of the green synthesis is triangles and spheres and sometimes hexagon [14]. The synthesis of gold nanoprisms has been demonstrated [17] using a plant Cymbopogon flexuosus and a very clear image of prism structures have been observed in TEM. The possible mechanism for the stabilization of AuNPs is illustrated in (Fig. 7). However, finding the exact chemistry involved in reduction and elucidating the capping agent need further substantiation. CONCLUSION In the present work, a simple and more rapid method to procure gold nanospheres of monodisperse nature been displayed and its characterization has been discussed. In future, such rapid and eco-friendly method may help researchers to synthesize AuNPs using green synthesis than chemical methods. The formation of AuNPs in the reaction indicates the presence of bioactive compounds in the plants which are present on the surface of the AuNPs. Such compound rich AuNPs can be further used in therapeutic applications and it may have antiallergeic activity if tested on a suitable animal model. CONFLICT OF INTEREST The author(s) confirm that this article content has no conflicts of interest. ACKNOWLEDGEMENT We thank DST-Nanomission, Government of India for its financial support for the project (SR/NM/NS-06/2009) and the management of Sathyabama University, Chennai for its stanch support in research activities.

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Received: April 02, 2012

Revised: May 18, 2012

Accepted: May 18, 2012