1. 192 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
Nanoscience Division, Centre for Ocean Research, Sathyabama University,Chennai - 600 119, India
b
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 Adhatoda vasica Nees, a malabar nut tree belonging to
the family Acanthaceae is native of South Asia and is
Nanobiotechnology combines biological principles with exclusively studied for its active components like
physical and chemical procedures to generate nano-sized quinazoline alkaloids, vasicine, vasicinone, deoxyvasicine,
particles with well defined functions. Particles of oxyvasicinine, maiontone and other essential oils. The plant
interestingly small size make the field of drug delivery more parts have been traditionally used for curing diseases like
interesting and effective. Synthesizing gold particles stomatitis, asthma and bronchititis [10]. The compound
(AuNPs) with medicinal applications is the recent trend in 73/602 an alkaloid isolated from the leaves and roots of A.
the field of nanobiotechnology. The surface availability of vasica is a structural analogue of vasicinone which shows
nanoparticles for binding/reactivity of other species on them appreciable antiallergic activity in mice, rats and guinea pigs
is an important function as it is synthesized in different [11]. Synthesis of AuNPs using medicinally useful plants
structures like nanorods, spheres, prims and hexagons. The [12, 13] is gaining more importance in therapeutic
controlled growth of AuNPs of different morphologies and applications, where the reducing agent responsible for the
the various chemical mechanisms involved in the anisotropic reduction of chloroauric acid will have its own effect on
growth were studied using different chemical procedures [1]. biological systems. In our previous work, we have
The low toxicity effects of green synthesized AuNPs on demonstrated the use of antidiabetic potent plant Cassia
biological systems made researchers to synthesize it by auriculata for the synthesis of AuNPs [14]. Herein, we have
biological method rather by chemical means. Extensive used an antiallergic potent plant A. vasica for the synthesis of
studies were done on AuNPs and its binding affinity towards AuNPs which may possess antiallergic effect on animals and
nucleic acids and proteins in biological systems [2]. AuNPs that will be a welcoming outcome in the field of drug
have been synthesized from various sources like plants [3], delivery.
microbes [4], seaweeds [5] and microalgae [6]”. Sastry et al.,
2007 have synthesized AuNPs using chitosan and showed MATERIALS USED
good control on postprandial hyperglycemia which when
Chloroauric acid (HAuCl4·3H2O) was obtained from
loaded with insulin [7] proved AuNPs to be an effective drug
Loba Chemie, India was used as received. All other reagents
carrier. AuNPs can be used for the detection of nucleic acids
used in the reaction were of analytical grade with maximum
as Chandirasekar and co-worker have, 2011 synthesized
purity. A. vasica Nees leaves were collected from forest area
AuNPs using bile salts and particles with different geometry
of Vellore, Tamilnadu, India and was cleaned with tap water
was achieved by varying the reducing agent concentration
and double distilled water twice to remove the dust. Further,
[8]. AuNPs have also been employed in the imaging of
it is shade dried for a week at room temperature and
cancer cells like AR42J pancreatic tumor cells by
powdered for use.
synthesizing it using laser ablation technique and reported
that size of AuNPs plays a role in the intracellular uptake [9]. SYNTHESIS OF GOLD NANOPARTICLES
For the preparation of A. vasica leaf extract, 4 g of
*Address correspondence to this author at the Nanoscience Division, Centre
for Ocean Research, Sathyabama University,Chennai - 600 119, India; powdered leaves is taken in a conical flask along with 40 mL
Tel: +91 44 24500646; Fax: +91 44 24503308; E-mail: ganesv@gmail.com of distilled water. The flask is then placed in an orbital
shaker for 4 h and then the extract is taken by filtering the
1876-4037/12 $58.00+.00 © 2012 Bentham Science Publishers

2. Green Synthesis of Well Dispersed Nanoparticles Micro and Nanosystems, 2012, Vol. 4, No. 3 193
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 983/G detector double beam spectrophotometer. XRD
is then stored in refrigerator for further use. The reaction is pattern of the A. vasica reduced AuNPs was carried out using
carried out using different concentrations of plant extract and a Rich Seifert P3000 instrument operated at a voltage of 40
finally optimized to a ratio of 1:10 (Extract: Chloroauric acid kV with Cu K radiations. SEM-EDAX was studied to check
solution). Further 3 mL of extract is added to 30 mL aqueous the surface morphology and elemental analysis of the AuNPs
solution of chloroauric acid (10-3 M) and kept in an orbital using XL30 FESEM, Philips. TEM studies were carried out
shaker at room temperature. Formation of AuNPs takes place by drop coating AuNPs onto carbon-coated TEM grids using
within 4 min, evident from the development of a stable ruby Philips Technai-10.
red color (Fig. 1) in the solution indicates the reduction of
Au3+ to Au0 with no color change further. The experiment was RESULTS AND DISCUSSION:
repeated thrice to check the formation of AuNPs. The The formation of AuNPs is noted down by the color
synthesized AuNPs are stable at a pH range of 3.6-9.1 showed change after the addition of aqueous extract to chloroauric
no precipitation and color change for a period of 45 days. acid solution as illustrated in (Fig. 1). The formation and
stability of metal nanoparticles in aqueous solution is studied
CHARACTERIZATION OF GOLD NANOPARTICLES
using UV-vis spectrophotometer. The surface plasmon
The UV-vis spectra were recorded using Thermo resonance (SPR) arising due to the oscillation of free
Scientific Spectrascan UV 2700 with samples in quartz conduction electrons induced by the absorption of
cuvette. FT-IR of Perkin Elmer spectrophotometer was used electromagnetic field [15] as a absorption band with a
to identify the possible groups responsible for the reduction maximum at 532 nm has been observed in the spectrum
of chloroauric acid at a resolution of 4 cm-1 in the range of (Fig. 2). To identify the possible functional groups
4000-450 cm-1 and the FT-IR spectrum was recorded by responsible for the reduction of chloroauric acid the FT-
employing KBr pellet technique using Perkin Elmer model- IR spectrum is taken and the interaction of biomolecules have

3. 194 Micro and Nanosystems, 2012, Vol. 4, No. 3 Karthick et al.
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 or –COOH group might have stabilized the AuNPs.
the functional group shifts. The peak seen at 3358 cm-1 The stretching of C=O can be observed in the spectrum
corresponds to the –OH or –COOH group which has shifted indicating the red shift from 1625 to 1675 cm-1. The
to higher wavelength 3687 cm-1 thus, implying that the –OH peak 1406 cm-1 corresponds to the –COO- also shows a shift

4. Green Synthesis of Well Dispersed Nanoparticles Micro and Nanosystems, 2012, Vol. 4, No. 3 195
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.

5. 196 Micro and Nanosystems, 2012, Vol. 4, No. 3 Karthick et al.
Fig. (6A&B). TEM image taken at various magnifications showing gold nanospheres.
which confirms the contribution of carboxylate groups in the constant with value from 0.9 to 1, is the wavelength of the
reduction. In the spectrum aromatic resonances have not X-ray source, is the full width at half maximum (FWHM)
been identified indicating the absence of aromatic groups in and is the Diffraction angle. From this equation, the
the stabilizing compounds. Thus by using FT-IR, it is particle size calculated was found to be 39 nm which is
concluded that –OH or –COOH, -C=O, -COO- groups has similar to the size observed in TEM image of the AuNPs.
involved in the stabilization of AuNPs. The crystalline nature The surface morphology and the scale in which the size of
of AuNPs was examined using XRD where three diffraction the nanoparticle synthesized can be studied using the SEM.
peaks were observed in the 2 range of 10º to 70º which can The presence of bioactive components responsible for the
be indexed as (111), (200) & (220) reflections of fcc (face reduction of the chloroauric acid to AuNPs was revealed
centered cubic) matches with Joint Committee on Powder using SEM imaging (Fig. 5A). The image confirms the
Diffraction Standards (JCPDS No: 04-0784) revealing that presence bioorganic compounds which has stabilized the
synthesized AuNPs are composed of pure crystalline gold as AuNPs. The EDAX profile has showed strong signal for
there is no other peak found (Fig. 4). The XRD patterns gold atoms (Fig. 5B) and weak signals for chlorine, oxygen,
obtained were similar to the results reported earlier on sodium and magnesium which implies that these signals
AuNPs [16]. The particle size of the AuNPs formed were might be from the biomolecules present in the aqueous
calculated using Debye-Scherrer equation extract. The morphology of the synthesized nanoparticles
were determined by TEM image and shown in (Fig. 6A&B).
D = K / cos For the analysis the image is taken in a random place on the
Where D is the average crystalline domain size grid and the morphology is observed carefully. The particles
perpendicular to the reflecting planes, K the Scherrer as seen in TEM image are triangular and spherical in shape

6. Green Synthesis of Well Dispersed Nanoparticles Micro and Nanosystems, 2012, Vol. 4, No. 3 197
Fig. (7). Possible mechanism for the reduction of chloroauric acid by bioactive compounds.
with an average size of 38 nm with many similar sized REFERENCES
particles except a few. The uniformity in size is a welcoming
[1] Grzelczak, M.; Juste, J.P.; Mulvaney, P.; Marza, L.M.L. Shape
result in the green synthesis as in most of the cases the control in gold nanoparticle synthesis, Chem. Soc. Rev., 2008, 37,
particle size varies with greater range which is a concern 1783-1791.
when applied in a targeted drug for therapeutic applications. [2] Niemeyer, C.M. Nanoparticles, proteins, and nucleic acids:
The particle shape usually observed in most of the green Biotechnology meets materials, Science, Angew. Chem. Int. Ed.,
2001, 40, 4128-4158.
synthesis is triangles and spheres and sometimes hexagon [3] Sharma, N.C.; Sahi, S.V.; Nath, S.; Parsons, J.G.; Torresdey,
[14]. The synthesis of gold nanoprisms has been J.L.G.; Pal, T. Synthesis of plant-mediated gold nanoparticles and
demonstrated [17] using a plant Cymbopogon flexuosus and catalytic role of biomatrix-embedded nanomaterials, Environ. Sci.
a very clear image of prism structures have been observed in Technol., 2007, 41, 5137-5142.
TEM. The possible mechanism for the stabilization of [4] Shankar, S.S.; Ahmad, A.; Pasricha, R.; Sastry, M. Bioreduction of
chloroaurate ions by geranium leaves and its endophytic fungus
AuNPs is illustrated in (Fig. 7). However, finding the exact yields gold nanoparticles of different shapes, J. Mater. Chem.,
chemistry involved in reduction and elucidating the capping 2003, 13, 1822-1826.
agent need further substantiation. [5] Singaravelu, G.; Arockimary, J.S.; Kumar, V.G.; Govindaraju, K.
A novel extracellular synthesis of monodisperse gold nanoparticles
CONCLUSION using marine alga, Sargassum wightii Greville, Colloids Surf., B
2007, 57, 97-101.
In the present work, a simple and more rapid method to [6] Govindaraju, K.; Basha, S.K.; Kumar, V.G.; Singaravelu, G. Silver,
procure gold nanospheres of monodisperse nature been gold and bimetallic nanoparticles production using single-cell
protein (Spirulina platensis) Geitler, J. Mat. Sci., 2008, 43, 5115-
displayed and its characterization has been discussed. In 5122.
future, such rapid and eco-friendly method may help [7] Bhumkar, D.R.; Joshi, H.M.; Sastry, M.; Pokharkar, V.B. Chitosan
researchers to synthesize AuNPs using green synthesis than reduced gold nanoparticles as novel carriers for transmucosal
chemical methods. The formation of AuNPs in the reaction delivery of insulin, Pharm. Res., 2007, 24, 1415-1426.
[8] Chandirasekar S.; Dharanivasan, G.; Kasthuri, J.; Kathiravan, K.;
indicates the presence of bioactive compounds in the plants Rajendiran, N. Facile synthesis of bile salt encapsulated gold
which are present on the surface of the AuNPs. Such nanoparticles and its use in colorimetric detection of DNA, J. Phys.
compound rich AuNPs can be further used in therapeutic Chem. C, 2011, 115, 15266-15273.
[9] Sobhan, M.A.; Sreenivasan, V.K.A.; Withford M.J.; Goldys, E.M.
applications and it may have antiallergeic activity if tested on Non-specific internalization of laser ablated pure gold
a suitable animal model. nanoparticles in pancreatic tumor cell, Colloids Surf., B, 2012, 92,
190-195.
CONFLICT OF INTEREST [10] Srivastava, S.; Verma, R.K.; Subhash, M.M.G.; Singh, C.; Kumar,
S. HPLC determination of vasicine and vasicinone in Adhatoda
The author(s) confirm that this article content has no vasica with photo diode array detection, J. Liq. Chrom. & Rel.
conflicts of interest. Technol., 2001, 24, 153-159.
[11] Paliwa, J.K.; Dwivedi, A.K.; Singh, S.; Gutpa, R.C.
ACKNOWLEDGEMENT Pharmacokinetics and in-situ absorption studies of a new
antiallergic compound 73/602 in rats, Int. J. Pharm., 2000, 197,
We thank DST-Nanomission, Government of India for its 213- 220.
financial support for the project (SR/NM/NS-06/2009) and [12] Philip, D.; Unni C. Extracellular biosynthesis of gold and silver
the management of Sathyabama University, Chennai for its nanoparticles using Krishna tulsi (Ocimum sanctum) leaf, Physica
stanch support in research activities. E, 2011, 43, 1318-1322.

7. 198 Micro and Nanosystems, 2012, Vol. 4, No. 3 Karthick et al.
[13] Kumar, K.P.; Paul, W.; Sharma, C.P. Green synthesis of gold [15] Mulvaney, P. Surface plasmon spectroscopy of nanosized metal
nanoparticles with Zingiber officinale extract: Characterization and particles, Langmuir 1996, 12, 788-800.
blood compatibility, Proc. Biochem., 2011, 46, 2007-2013. [16] Long, N.N.; Vu, L.V.; Kiem, C.D.; Doanh, S.C.; Nguyet, C.T.;
[14] Kumar V.G.; Gokavarapu, S.D.; Rajeswari, A.; Dhas, T.S.; Hang, P.T.; Thien, N.D.; Quynh, L.M. Synthesis and optical
Karthick, V.; Kapadia, Z.; Shrestha, T.; Barathy, I.A.; Roy, A.; properties of colloidal gold nanoparticles, J. Phy.: Conf. Ser., 2009,
Sinha, S.; Facile green synthesis of gold nanoparticles using leaf 187, 012026.
extract of antidiabetic potent Cassia auriculata, Colloids Surf. B, [17] Shankar, S.S.; Rai, A.; Ankamwar, B.; Singh, A.; Ahmad, A.;
2011, 87, 159-163. Sastry, M. Biological synthesis of triangular gold nanoprisms, Nat.
Mater., 2004, 3, 482-488.
Received: April 02, 2012 Revised: May 18, 2012 Accepted: May 18, 2012