International Journal of Physics

and Research (IJPR)

ISSN(P): 2250-0030; ISSN(E): 2319-4499
Vol. 5, Issue 6, Dec 2015, 39-48
TJPRC Pvt. Ltd.

SYNTHESIS AND CHARACTERIZATION OF SILVER NANOPARTICLES

USING ANDROGRAPHIS PANICULATA AND ITS ANTI-INFLAMMATORY
EFFECTS ON HUMAN BLOOD CELLS
P. ANITHA1 & P. SAKTHIVEL2*
1

Assistant Professor, Department of Physics, Roever College of Engineering and

Technology, Perambalur, Tamil Nadu, India

2*

Associate Professor, Department of Physics, Urumu Dhanalakshmi College (Autonomous),

Trichy, Tamil Nadu, India

ABSTRACT
Various researchers are interested in the synthesis and characterization of nanoparticles, due to its wide
applications in the field of medicine. In the present study the extract of Andrographis paniculata leaf (Family:
Acanthaceae) is synthesized as silver nanoparticles. The leaf extract is mixed with AgNO 3, and then it is incubated. The
as nanoparticles by color transformation. The characterization of silver nanoparticles was studied by UVVis
spectroscopy, FTIR, XRD&TEM. The silver nanoparticles synthesized were generally found in size 1-100 nm. The
results showed that the leaf extract is ideal for the synthesis of silver nanoparticles and it is also known to have the ability
to constrain the growth of various pathogenic microorganisms. The average size of synthesized silver nanoparticles is
found to be 15.59nm using XRD data by Scherrers formula, which is approximately similar as the size obtained in TEM
analysis (15.38nm). On the whole, the AgNPs prepared are safe to be discharged in the atmosphere and possibly utilized

Original Article

extract is kept in microwave oven for exposure of heat, dried and powdered. The synthesized dried powder is confirmed

in processes of pollution remediation. AgNPs may also be competently consumed in Anti-inflammatory activity of
Pharmaceutical research to obtain better result of plant as shown by our study. The Anti-inflammatory activity of silver
nanoparticles was tested on human blood cells which confirms that the plant mediated synthesis of silver nanoparticles
have a significant Anti-inflammatory effect on human blood cells.
KEYWORDS: Silver Nanoparticles, UVVis Spectroscopy, FTIR, TEM, XRD, Anti-Inflammatory, Human Blood Cells,
etc.,

Received: Oct 31, 2015; Accepted: Nov 05, 2015; Published: Dec 11, 2015; Paper Id.: IJPRDEC201506

INTRODUCTION
The field of Nanoscience has flourished over the last twenty years and the need for nanotechnology will
only increase as miniaturization becomes more important in areas such as computing, sensors, and biomedical
applications. Advances in this field largely depend on the ability to synthesize nanoparticles of various nano
materials, based on their sizes, and shapes, as well as their efficiency to assemble them into complex
architectures (David D et al., 2005)1.Nanotechnology provides the ability to engineer the properties of materials
by controlling their size, and this has been driven research toward a multitude of potential uses for Nanomaterial
(Saifuddin N et al., 2009)2.
Nanoparticle synthesis and characterization are fundamental importance in the advancement of recent
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P. Anitha & P. Sakthivel

research. It is found that the properties of metal nanoparticles also depend on their size, shape and chemical surroundings
(Das R et al., 2009)3.
Size of the Silver nanoparticles ranges from 1 - 100 nm. It is generally recognized that silver nanoparticles may
attach to the cell wall, thus disturbing cell-wall permeability and cellular respiration. Normally, 'silver' is composed of a
large percentage of silver oxide due to their large ratio of surface-to-bulk silver atoms (Prashant S et al., 2011)4.
Andrographis paniculata is an herbaceous plant of Acanthaceae family which is cultivated all over in India. It is used as
antibiotics and to treat some infectious diseases. Mostly the leaves ofAndrographis paniculata is used in traditional
Siddha and Ayurvedic systems of medicine as well as a tribal medicine in India and some other countries for several
clinical applications (X. Zhang2004)5.
Andrographis paniculata is also reported to possess anti-hepatotoxic, antibiotic, antimalarial, anti-hepatitic, antithrombogenic, anti-inflammatory, anti-snake venom, and antipyretic properties to mention a few, besides its general use
as an immune stimulant agent (Rahman NNNA et al., 1991)6. Synthesis of metal nanoparticles and their characterization
has been an emerging field of nanotechnology since the past few decades because of their exclusive properties and
potential application in the fields of physics, chemistry, biology and medicine 7.Widely, nanoparticles are synthesized by
different routes. However, the synthesis of nanoparticles by chemical methods is not eco-friendly. Therefore, the synthesis
of nanoparticles by biological route (using microorganisms, enzymes and plant extract) is the suggested alternative to the
eco-friendly methods8.
The bio reduction of Ag+ to silver nanoparticles involves plant extracts and microorganisms
shown that variety of plant extracts served as green reactants in silver nanoparticles synthesis

11, 12, 13, 14

9, 10

.It has been

. A recent study has

demonstrated that the synthesized silver nanoparticles using leaf extract of A. paniculata displayed good anti-plasmodial
activity15.Though there are some reports for synthesis and characterization of nanoparticles using Andrographis
paniculata, in this present study in silver nanoparticles are synthesized using aqueous extract of Andrographis paniculata
and was confirmed by colour transformation. The size of nanoparticles obtained was very small compared to previous
report of TEM analysis. The stability of nanoparticles was studied by UV, FTIR, XRD, TEM and Anti-inflammatory
activity on human blood cell was also analyzed.

MATERIALS AND METHODS

Collection of Leaf
Fresh leaf of Andrographis paniculata were collected from Perambalur, during the month of May and identified
by Dr.JohnBritto, The Director, Rabinat Herbarium and Center for Molecular Systematics, St.Josephs College (Campus),
Trichirappalli-2, Tamil Nadu. India. (Plant authentication no: PN004)
Preparation of Leaf Extract
The fresh and young leaf samples of Andrographis paniculata was collected washed thoroughly with sterile
double distilled water (DDW). Twenty gram of sterilized leaf samples were taken and cut into small pieces. Finely cut
leaves were placed in a 500 ml Erlenmeyer flask containing 100 ml of sterile DDW. After that the mixture was boiled for
5 min and filtered. The extract was stored in 4 0C.
Synthesis of Silver Nanoparticles
Silver nitrate was used as precursor in the synthesis of silver nanoparticles. 100 ml of Andrographis paniculata
Impact Factor (JCC): 2.3529

Index Copernicus Value (ICV): 3.0

Synthesis and Characterization of Silver nanoparticles using Andrographis paniculata

and its Anti-inflammatory effects on human blood cells

41

leaf extract was added to 100 ml of 0.1N AgNO3 aqueous solution in conical flask of 250 ml content at room temperature.
The flask was thereafter put into shaker (100 rpm) at 500 C and reaction was carried out for a period of 12 hrs. Then the
mixture is kept in microwave oven for exposure of heat. The mixture was completely dried after a period of 20 minutes
and hence nanoparticles in forms of powders were obtained.

Figure 1: Optical photograph of Andrographis paniculata- 0.1 N AgNO3 Solution B- Leaf extract
C- Leaf extract + AgNO3 D- Leaf extract + AgNO3 (After 30mins) E- Leaf extract + AgNO3 (After 1 hr)
F- Leaf extract + AgNO3 (After 2 hrs) G- Leaf extract + AgNO3 (After 24 hrs)
UV-Visible Spectroscopy Analysis
The colour change in reaction mixture (metal ion solution + leaf extract) was recorded through optical
observation. The bio reduction of silver ions in aqueous solution was monitored by periodic sampling of solid and
subsequently measuring UV-visible spectra of the solid sample. UV-visible spectra of sample were monitored as a
function of time of reaction on the UV-visible spectroscopy. The investigations were carried out using PERKIN ELEMER
(Lambda 35 model) spectrometer in the range of 190 nm to 1100 nm.
FT-IR Measurement
The Fourier transform infrared (FTIR) investigations were carried out using PERKIN ELEMER (Spectrum RXI)
spectrometer in the range of 400 cm-1 to 4000 cm-1. The functional groups were identified using the peak assignments.
XRD Measurement
The sample was drop-coated onto Nickel plate by just dropping a small amount of sample on the plate
frequently, allowed to dry and finally thick coat of sample was prepared. The particle size and nature of the silver
nanoparticle was determined using X-ray diffraction (XRD). This was carried out using Rigaku miniflex-3 model with
40kv, 15mA with Cuk radians at 2 angle.
TEM Analysis
Sample is dispersed with acetone and exposed in ultrasonics for 5 minutes. Take a drop of a solution from the
samples and drop it on the grid, leave it until it dries. After drying the samples is inserted into TEM instruments using
model is Tecnai T20Making in FEI, Netherlands operating at 200KeV Tungsten Filament.

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P. Anitha & P. Sakthivel

ANTI-INFLAMMATORY ACTIVITY
The Human Red Blood Cell (HRBC) Membrane Stabilization Method
The method as prescribed (Gopalkrishnan et al., 2009; Sakat et al., 2010) was adopted with some modifications.
The blood was collected from healthy human volunteer who had not taken any NSAIDS for 2 weeks prior to the
experiment and mixed with equal volume of Alsever solution (2 % dextrose, 0.8 % sodium citrate, 0.5 % citric acid and
0.42 % NaCl) and centrifuged at 3,000 rpm. The packed cells were washed with isosaline and a 10 % suspension was
made. Various concentrations of extracts were prepared in mg/ml using distilled water and to each concentration, 1 ml of
phosphate buffer, 2 ml hypo saline and 0.5 ml of HRBC suspension were added. It was incubated at 37 0C for 30 minutes
and centrifuged at 3,000 rpm for 20 minutes and the hemoglobin content of the supernatant solution was estimated
spectrophotometrically at 560 nm. Diclofenac (100 Jg/ml) was used as reference standard and a control was prepared by
omitting the extracts. The experiments were performed in triplicates and mean values of the three were considered. The
percentage (%) of HRBC membrane stabilization or protection calculated using the following formula,
Percentage of Protection (%) = (100- OD of Drug Treated Sample/OD of Control) X 100
Albumin Denaturation Method
The method as prescribed (Sakat et al., 2010) was followed with modifications. The reaction mixture was
consisting of test extracts and 1% solution of bovine albumin fraction. pH of the reaction mixture was adjusted using
small amount of HCl. The sample extracts were incubated at 37C for 20 minutes and then heated to 51C for 20 minutes.
After cooling the samples the turbidity was measured spectrophotometrically at 660 nm. Diclofenac sodium was taken as
a standard drug. The experiment was performed in triplicates and the mean value of the three was considered. Percent
inhibition of protein denaturation was calculated as follows,
Percentage of Inhibition (%) =(OD of Control- OD of Sample/ OD of Control) X 100

RESULTS
UV-Visible Spectroscopy Analysis
UV-Vis spectroscopy analysis showed that absorbance band of silver nanoparticles synthesized using
Andrographis paniculata leaf extract absorption band at 230.62nm as characteristic poly-unsaturated and aromatic
compound present (retinol) (Advanced strategies in food analysis ,UV-VIS spectrometry by Richard Koplk).

Figure 2: UV-visible spectrum of synthesized silver

nanoparticles using leaf extracts of Andrographis paniculata

Impact Factor (JCC): 2.3529

Index Copernicus Value (ICV): 3.0

Synthesis and Characterization of Silver nanoparticles using Andrographis paniculata

and its Anti-inflammatory effects on human blood cells

43

FT-IR Measurement
The Andrographis paniculata related functional groups were identified using the peak assignments. A strong
peak at 3920.80 cm-1,3792.82 cm-1and 3729.01 cm-1 was assigned to the OH stretching in Phenol group, The medium
peak at 3352.32 cm-1 was assigned to N-H stretching primary, secondary amines and amides group, The strong peak at
3210.11cm-1 was assigned to C-H stretching in alkynyl or broad and strong OH stretching in alcohol, Phenol group,
strong peak at 2923.34 cm-1 was assigned to OH and C-H stretching in carboxylic acids and alkanes group,The medium
peak at 2822.70 cm-1 was assigned to O-H stretching in carboxylic acids, The strong peak at 2357.59 cm-1 was assigned
to C-H stretching in alkynyl group, The very strong peak at 2254.81 cm-1 and 2171.34 cm-1was assigned to C-triple bond
N stretching in nitriles and C stretching in alkynes , The medium peak at 1599.28 cm-1 was assigned to C-C stretching in
aromatic group, , The strong peak at 1381.27 cm-1 was assigned to N-O stretching in nitro group, The medium peak at
1034.74 cm-1 was assigned to C-N stretching in aliphatic amines, , The small peak at 867.87 cm-1 was assigned to CH stretching in opparomatic, The medium peaks at 824.34 cm-1 and 769.70 cm-1
-1

was assigned to C-Cl stretching in

-1

alkyl halides, and also the medium peaks at 609.75 cm and 565.71 cm was assigned to C-Br stretching in alkyl halides
are observed.

Figure 3: FT-IR spectrum of synthesized silver nanoparticles using leaf extracts of Andrographis paniculata

XRD MEASUREMENT
Determination of Crystalline Size
Average crystallite size of silver was calculated using the Scherrers formula,
D = k / cos
D- Average crystallite size: K- Constant: - X- ray Wavelength: - Angular FWHM of the XRD peak at the
diffraction angle: - Diffraction angle.
By using Scherrers formula in XRD data, the size of the particle is approximately found to be 15.59nm.

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P. Anitha & P. Sakthivel

Figure 4: XRD spectrum of synthesized silver nanoparticles using leaf extracts of Andrographis paniculata
TEM Analysis
The figure shows the TEM image obtained by the reaction of Andrographis paniculata leaf extract with 0.1N
silver nitrate solution. This Andrographis paniculata Ag-NPs was found to be 15.38nm.

Figure 5: TEM image of synthesized silver nanoparticles using leaf extracts of Andrographis paniculata
Anti-Inflammatory Activity
Anti-inflammatory study like human red blood cell (HRBC), membrane stabilization, inhibition of albumin de
nutrition indicated that anti-inflammatory activity .The medical use of Andrographis paniculata has a good antiinflammatory activity.As the concentration of the sample increases, the percentage of inhibition also increases.
Table 1: Anti-inflammatory activity of human red blood cell (HRBC)
by using AgNPs of Andrographis paniculata
% of inhibition
S. No Concentration (g/Ml)
Membrane Stabilization MeanS. E. M
1
100
33.97 0.18
2
200
37.92 0.11
3
400
45.34 0.73
4
600
46.73 0.42
5
800
52.62 0.20

Impact Factor (JCC): 2.3529

Index Copernicus Value (ICV): 3.0

Synthesis and Characterization of Silver nanoparticles using Andrographis paniculata

and its Anti-inflammatory effects on human blood cells

45

Figure 6: Graphical representation of anti-inflammatory activity of human

red blood cell (HRBC) by using AgNPs of Andrographis paniculata

Table 2: Anti-inflammatory activity of albumin denaturation method by

using AgNPs of Andrographis paniculata
S. No

Concentration (g/Ml)

1
2
3
4
5

100
200
400
600
800

% of inhibition
Membrane Stabilization Mean S. E. M
30.18 0.39
34.27 0.71
42.22 0.98
45.30 0.32
48.47 0.12

Figure 7: Graphical representation of anti-inflammatory activity of albumin

denaturation method by using AgNPs of Andrographis paniculata

DISCUSSIONS
The present research work used A. paniculata for the biosynthesis of silver nanoparticles and studied their effect
on microbial growth. The whole plant material was dried and ground to fine powder before subjecting to crude
phytochemical extraction. The active phytochemical components are expected to be more concentrated in dry preparation
than in fresh plant material 16.The reduction of silver ions to silver nanoparticles by A. paniculata extract was measured
using UV-visible spectrophotometry. It was observed that the initial colour of silver nitrate treated with A. paniculata
extract turned from light to dark brown after 24 hrs of the reaction, which indicates the formation of silver nanoparticles.
The colour transformation of A. paniculata extract treated silver nitrate might be due to vibrations in surface plasmon of
silver17. The strong broad peak located at 430 nm indicates the reduction of Ag+ ions which further confirmed the
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P. Anitha & P. Sakthivel

formation of silver nanoparticles. It is corroborated to the findings of Vilchis-Nestor et al. 18, who have reported that the
noble metal silver displays characteristic absorbance at around 430 nm.
The green synthesis of noble nanoparticles using plant or fruit extracts and bio organisms leads to the formation
of crystalline nanoparticles with variety of shapes and sizes ranging from 1 -100 nm. Interestingly, the size of A.
paniculata reduced silver nanoparticles was found to range from 14-80 nm under SEM and TEM observation. The
functional groups of compounds adsorbed on the silver nanoparticles were identified using FTIR studies. The plant
extract of A. paniculata shows a strong peak at 3306 cm1, 684 cm-1 indicating the presence of hydroxyl group and C-S
group respectively. The peaks at 2364 cm1 and 2345 cm1 indicating the presence of aliphatic cyanide/ Nitrile group
(CN) in aliphatic /aromatic compounds19.Transmission Electron Micrograph of Silver nanoparticles finds the shape and
size of the nanoparticles. Result of TEM micrograph of silver nanoparticles at 200nm shows that synthesized silver
nanoparticles present in size between 11-22nm and spherical in shape 20.Transmission electron microscopy (TEM) is
characterised for analysing the nanoparticles morphology. The results confirmed the reduction of silver nitrate to silver
nanoparticles with high stability and without any impurity. Comparison of experimental results showed that the average
size of synthesized silver nanoparticles was about 55 nm21.

CONCLUSIONS
In conclusion, the bio-reduction of aqueous silver ions by the leaf extract of the Andrographis paniculata has
been demonstrated. The reduction of the metal ions through leaf extract leading to the formation of silver nanoparticles
and the synthesized nanoparticles are quite stable in solution. The size of silver nanoparticles was determined by using
XRD &TEM analysis. Transmission Electron Micrograph of Silver nanoparticles finds the size of the nanoparticles.
Result of TEM micrograph of silver nanoparticles at 50nm shows that, the average size of synthesized silver
nanoparticles is found to be 15.59nm using XRD data by Scherrers formula(Figure 4), which is approximately similar as
the size obtained in TEM analysis (15.38nm)(Figure 5). In addition to that anti-inflammatory study like human red blood
cell (HRBC), membrane stabilization, and inhibition of albumin de- nutrition indicated the anti-inflammatory activity.
The medical use of Andrographis paniculata has a good anti-inflammatory activity. As the concentration of the sample
increases, the percentage of inhibition also increases (Table 1 & 2). The synthetic method based on naturally occurring
biomaterials provides an alternative means for obtaining the nanoparticles. Use of plants in synthesis of nanoparticles is
quite novel leading to truly green Environment route. This green environment approach towards the synthesis of
nanoparticles has many advantages such as, process scaling up, economic viability and safe way to produce nanoparticles.
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