Industrial Crops and Products 60 (2014) 212–216

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Industrial Crops and Products

journal homepage: www.elsevier.com/locate/indcrop

Coriandrum sativum seed extract assisted in situ green synthesis of

silver nanoparticle and its anti-microbial activity
G.M. Nazeruddin a , N.R. Prasad a,b,∗ , S.R. Prasad d , Y.I. Shaikh a ,
S.R. Waghmare b , Parag Adhyapak c
a
Poona College of Arts, Commerce & Science, Pune, India
b
Solid State Research Laboratory, Department of Chemistry, Shivaji University, Kolhapur, Maharashtra 416004, India
c
Material Research Laboratory, Centre for Material for Electronic Technology, Pune, India
d
DKTE Society’s Textile and Engineering Institute, Ichalkaranji, India

a r t i c l e i n f o a b s t r a c t

Article history: Extracellular in situ synthesis of metal nanoparticles using extracts of plants like Azadirachta indica
Received 17 December 2013 (Neem), and Zingiber officinale (Ginger) has been successfully carried out earlier. In this study we have
Received in revised form 13 May 2014 developed a novel method to synthesize silver nanoparticles by the process of bio-chemical reaction
Accepted 26 May 2014
between silver salt solution with seed extract of Coriandrum sativum without using any surfactant or
external energy. By this method physiologically stable, bio-compatible Ag nanoparticles (NPs) were
Keywords:
formed. These functionalized AgNPs could be used for targeted drug delivery with enhanced therapeutic
AgNPs
efficacy and minimal side effects. It is well known that on treating the metallic salt solution with some
Green synthesis
C. Sativum
plant extracts, a rapid reduction occur leading to the formation of highly stable metal nanoparticles. This
SEM method resulted in rapid synthesis of nanoparticles, i.e. reaction time was 1–2 h as compared to 2–4 days
Antimicrobial required by microorganisms. These nanoparticles were analysed by various characterization techniques
to reveal their morphology and chemical composition. TEM image of these NPs indicated the formation
of spherical, non-uniform, poly dispersed nanoparticles of mean size of 13.09 nm. A detailed study of
anti-microbial activity of nanoparticles and its synergetic effectiveness with common narrow spectrum
antibiotic has been successfully carried out.
© 2014 Published by Elsevier B.V.

1. Introduction the gap between bulk materials and atomic and molecular level
(Thakkar et al., 2010). Large spectrum of research has been focused
Nanomaterials have a long list of applicability in improving to synthesize nanomaterials with complex morphologies and to
human life and its environment. It has been found that 5000 years control the size and shape which is crucial in tuning their properties
old Indian System of Medicine Ayurveda had some knowledge of (Khan et al., 2012; Vijaymohan et al., 2012). Several approaches are
nano-scale fabrication used for medicinal purposes (Prasad and in practice to generate the metallic nanoparticles such as electro-
Elumalai, 2011). The process of formation of Bhasma (a special chemical, sono-chemical and microwave assisted process, but most
kind of ayurvedic medicine) is well described in ancient book Ras- of these strategies suffer from utilization of high energy, hazardous
Ratnakar by Nagarjuna, 50 BC. A bulk material has constant physical chemicals and difficulty in purification (Vijaykumar et al., 2013). To
properties regardless of its size, but at the nano-scale often this switchover these technical hitches biological principles have been
is not true. Several well-characterized bulk materials have been established recently (Castro et al., 2011).
found to possess most interesting properties when studies in the DNA can be used as an ideal template for the synthesis of
nanoscale. There are many reasons for this including the fact that nanoparticles due to some key features such as inherent nanoscale
nanoparticles possess a very high aspect ratio, i.e. surface to volume dimensions, high specificity exerted towards inorganic materials
ratio. Nanoparticles are of great scientific interest as they bridge and structural flexibility to build NPs of different size and shape
(Aland, 2006).
Silver nanoparticles can be produced either intra or extra-
∗ Corresponding author at: Department of Nanoscience and Technology, Shivaji cellularly by using biological routes such as algae, fungi, bacteria
University, Kolhapur, India. Tel.: +91 9404265521. and plants, etc. There are some species of algae which are
E-mail address: neeraj prasad21@rediffmail.com (N.R. Prasad). used as bio-factory for the synthesis of metallic nanoparticles.

http://dx.doi.org/10.1016/j.indcrop.2014.05.040
0926-6690/© 2014 Published by Elsevier B.V.
 G.M. Nazeruddin et al. / Industrial Crops and Products 60 (2014) 212–216 213

While micro-organisms continue to be investigated for reduc- (Shankar, 2005). Due to the development of antibiotic resistance in
tion of metals to synthesize nanoparticles, the use of plant pathogenic bacteria the pharmaceutical companies and researchers
extracts is an exciting possibility and is relatively unexplored and are searching for new antibacterial agents and the AgNPs are
under-exploited (Mittal et al., 2013). promising candidate for the same. The reduction of Ag+ ions leads
Plants have been known to bio-mineralize calcium carbonate, to the formation of silver atoms (Ag) which is followed by agglom-
silica and even magnetite internally. Certain plants are known to eration into oligomeric clusters. These clusters eventually lead to
hyper-accumulate these heavy metals or can even be induced to the formation of colloidal AgNPs. When the colloidal particles are
hyper-accumulate within different parts of plants. The internal much smaller than the wavelength of visible light, the solution
accumulation of metal in plants can occur both via complexation of possess characteristic yellow colour with an intense band in the
the metal ion with a suitable bio-ligand in its native oxidation state 380–430 nm range and other less intense bands at longer wave-
or after its reduction to a lower oxidation state. The possibility of length in the UV–visible absorption spectrum.
reduction of metal ions by plants and the presence of metal com-
plexing agents in them entices a materials scientist to use plants for 2. Materials and methods
the goal of synthesizing nanoparticles and controlling their size and
shape and to experiment on the likelihood of forming nanoparti- Nanoparticles of Ag were prepared by using C. sativum seed
cles of low reduction potential metals (Anilkumar, 2007). Solution extract. C. sativum seeds have been purchased from local market
temperature, concentration of metal salt and reducing agent and Jaysingpur, India. 50 g of seeds have been taken for the prepara-
reaction time all influence the shape and size of particle. The possi- tion of extract. The obtained seeds were sieved and thoroughly
bility of synthesizing nanoparticles of different compositions using washed in running water and finally rinsed with deionized water
plants would offer an environmentally friendly alternative to the until no foreign material remained behind. After washing the col-
existing potentially toxic chemical and physical methods of prepa- lected seeds were kept in 100 ml of deionized water over night and
rations. This would also help stave off the growing apprehensions macerated. Then it was finely crushed in grinder mixer and filtered
related to environmental degradation and biological hazards apart by using cotton and collected in stoppered glass bottle. The extract
from being a cost effective process with potential to scale up for is diluted to 250 ml using deionized water and the solution was
large-scale synthesis. kept in refrigerator at 4 ◦ C. The AgNO3 used in reaction was analyt-
Shiv Shankar has reported the synthesis of silver and gold ical grade chemical obtained from Sigma Aldrich and used without
nanoparticles using decoction solution of Azadirachata indica. How- further purification.
ever, the nanoparticles synthesized by him are in the range of 100 ml of 0.1 N AgNO3 solution was taken in 500 ml beaker.
50–100 nm and are not well separated from each other and tend to Then 50 ml of C. sativum seed extract solution was taken in burette
form agglomerated structures. By reducing silver nitrate in solution and drop wise added at room temperature with constant stir-
of tea extract or epicatechin of varying concentrations, spherical ring. As soon as the seed extract solution was added dark brown
silver nanoparticles were formed that had controllable size dis- coloured precipitate appeared in the solution. After complete addi-
tribution depending upon the concentration of tea extract and tion of 50 ml of solution a sufficient amount of the precipitate
epicatechin in the samples (Kharisov et al., 2013). was observed and it was separated by high speed centrifugation
In this research work an attempt has been made to extend these at about 8000 rpm. The formation of AgNPs requires about 60 min
green chemistry approaches for synthesizing nano-materials from of time and that is determined using spectro-photometer. The sep-
lower class of organisms to higher forms of plants. It leads to green arated solid mass was washed with alcohol for few times to remove
chemistry route which is more advantageous because it does not organic impurities soluble in alcohol. After complete washing the
require elaborate process such as intra cellular synthesis, multi- solid mass was kept in oven for drying. The complete drying of
ple purification steps, and maintenance of microbial cell culture. this solid mass resulted in a black coloured material which was
In green chemistry approach of synthesis the reaction is normally powdered in mortar and sampled for characterization purpose.
carried out at room temperature although in some processes a lit-
tle heating below 100 ◦ C was applied. High amount of phenolic
compounds in the plant extract are generally supposed to influ- 3. Experimental results and discussion
ence the reduction process and stabilize nanoparticles preventing
agglomeration. These nanoparticles are studied for their antibac- Biosynthesis of AgNPs was indicated by colour developed in
terial properties and future work can be extended to study their solution during the course of reaction. The intensity of colour
antioxidant, and anti-tumour properties (Rao et al., 2013). depends upon degree of bio-reduction of silver particles. Forma-
Coriandrum sativum is widely available plant in tropical coun- tion of AgNPs was confirmed by using different characterization
try like India. In the present research work C. sativum seed extract techniques.
have been used as a reducing agent for AgNPs biosynthesis. The
same extract also acts as a capping agent. This plant is described to 3.1. UV spectroscopic graph showing absorbance of silver
possess diuretic, purgative and appetizer property in Ayurveda. It is nanoparticles
also used as a medicine against griping pain, piles, leucorrhoea and
in eye diseases. It is reported in the literature that high consump- The blackish brown coloured sample powder was dissolved in
tion of C. sativum leads to impotency in males and suppression of deionized water and sonicated. Then this solution was taken in
menstrual cycles in female. cuvette and exposed to UV–visible radiation and the absorbance
of the solution was recorded. Because of the surface plasmon reso-
1.1. Silver nanoparticles nance phenomena resonant peak occurs at different wavelength for
different nanoparticles solution and as per the theory of resonance
AgNPs can be used in antimicrobial agents, textile indus- maximum wavelength is absorbed at resonant wavelength. It is
tries, water treatment, sunscreen lotions, etc. Silver nanoparticles reported in the literature that a typical AgNPs shows the character-
exhibit tremendous applications in various areas such as biomed- istic SPR at the wavelength in the range of 400–480 nm. Fig. 1 shows
ical field, spectral selective coatings for solar energy absorption, SPR for the sample solution to occur at the wavelength of 421 nm
optical receptors, bio-labelling, intercalation materials for elec- which confirms the presence of silver nanoparticles in the pre-
trical batteries, filters, anti-microbial agents and sensors, etc. pared solution. The SPR absorbance is sensitive to the nature, size
214 G.M. Nazeruddin et al. / Industrial Crops and Products 60 (2014) 212–216

Fig. 3. Graph showing particle size distribution for Ag nanoparticles.

Fig. 1. UV spectroscopic graph showing absorbance of silver nanoparticles. found the different particle size of AgNPs by changing the char-
acterization techniques and the particle size order is reported to
and shape of particles present in the solution and also it depends be DLS > AFM > TEM > XRD and our results are consistent with this
upon their inner particle distance and the surrounding media. The finding. The MATLAB analysis of this particle size distribution image
MATLAB analysis of this UV image shows that the image format is shows the bit-depth of 24 bits and image resolution of 1024 × 768.
JPEG with bit-depth of 24 bits and image resolution is found to be Also the image file-size was 124.9 kB and image format JPEG with
492 × 492 and the file-size is 13.7 kB. Also it is seen that the image image class as uint8. The particle size distribution graph is taken
class is uint8. The UV characterization has been done at Department from Dr D Y Patil University, Kolhapur, India.
of Chemistry, Shivaji University, Kolhapur.
3.4. SEM image of silver nanoparticles
3.2. XRD graph showing silver nanoparticles
The SEM image from Fig. 4 reveals the formation of cluster of
XRD patterns of synthesized AgNPs are shown in Fig. 2 which spherical beadlike structure of silver nanoparticles with nonuni-
is an absorbance vs 2␪ graph. The graph shows eight major peaks form distribution. The MATLAB analysis gives the pixel depth of the
and the peak position explains about the translational symmetry image equal to 8 bits and the image resolution of 1280 × 960. Also
namely size and shape of the unit cell, whereas the peak intensities the image file-size was found to be 1.23 MB and the image format
give details about the electron density inside the unit cell. The syn- as TIFF. The SEM image has been taken with JSM-6360 instrument
thesized nanoparticles are crystalline in nature. These peaks are which uses accelerating voltage of 20 kV. The SEM Image has been
of cubic structure which is in agreement with the JCPDS file No. taken at Department of Physics, Shivaji University, Kolhapur India.
00-004-0783.
3.5. TEM image of silver nanoparticles
3.3. Particle size distribution
TEM images from Fig. 5 reveal that there was poly-disperse
Particle size distribution histogram from Fig. 3 indicates that spherical particles with nonuniform distribution of nanoparticles
10.07% particles size is around 113 nm with a standard deviation in the prepared sample. For TEM measurements, a drop of solution
of 16.48% size while the remaining particles are around 633.5 nm containing the particle was deposited on a copper grid covered with
with a standard deviation of 20.58%. The difference in size as amorphous carbon. After allowing the film to stand for 2 min the
observed from TEM and PDS may be due to the presence of bio-
active molecules of C. sativum on AgNPs surface. Prathna et al.

Fig. 2. XRD graph showing silver nanoparticles. Fig. 4. SEM image of silver nanoparticles.
 G.M. Nazeruddin et al. / Industrial Crops and Products 60 (2014) 212–216 215

Fig. 5. TEM image for silver showing size of nanoparticles.

extract solution was removed by means of blotting paper and the

grid allowed drying before the measurement. It was observed that Fig. 6. Image of culture plate showing antimicrobial activity of silver nanoparticles.

the nanoparticles formed were of different sizes and particle size

can be used in antimicrobial coatings in medical instruments and
was found to be 9.94, 13.94, 15.91 and 12.59 nm and the mean size
in textiles. The image was captured by Sony Cybershot camera and
of about 13.09 nm which lies in the nano range. The TEM measure-
shown in the picture. The MATLAB analysis of this antimicrobial
ment was done with JEOL model 1200Ex instrument operated at
culture image shows that the image format is JPEG with bit-depth
an accelerating voltage of 80 kV. The TEM image was taken with
of 24 bits, image class is uint8 and image resolution 4000 × 3000
very high resolution and MATLAB analysis gives the pixel depth of
and the file-size is 1.55 MB.
the image equal to 24 bits and the image format as JPEG. The TEM
images have been taken from National Chemical Laboratory, Pune,
India. 3.7. Possible mode of action of silver nanoparticles as
antibacterial agent
3.6. Antimicrobial activity of silver nanoparticles
There are different theories behind the mode of action of AgNPs
The antimicrobial activity of AgNPs was evaluated against a as antibacterial agent. One of the theory says that silver is a soft
Gram-positive and a Gram-negative pathogenic bacteria as per acid, and there is a natural tendency of an acid to react with a
below mentioned agar diffusion method. In this method first the base, in that case soft acid reacts with soft base. The cells are
bacterial cultures were developed on nutrient agar (NA) slants mainly made up of sulfur and phosphorus which are soft bases.
which contained different ingredients such as peptones (5.0 g), agar Thus the reaction between nanoparticles (soft acid) and cells (soft
(15.0 g), meat extract (q.s.), sodium chloride (q.s.), and yeast extract base) takes place which results in the formation of salt, i.e. degen-
(q.s.) per litre of distilled water. After solidification of prepared cul- eration and death of cells takes place. The interaction of the silver
ture medium the stainless steel cylinder were placed on the surface nanoparticles with the sulfur and phosphorus of DNA can lead to
and holes were punched in the medium. Freshly prepared AgNPs problems in the DNA replications of bacteria and thus terminate
were added into the wells that were made in the culture medium the microbes.
plates. The samples were initially incubated for 15 min at 4 ◦ C tem- However as per another theory positive charge on silver ion
perature and later were incubated for 24 h at 37 ◦ C temperature. may be responsible for its antimicrobial activity. It is possible
Positive test results were scored when a zone of inhibition was that the antibacterial activity is derived through the electrostatic
observed around the well after the incubation process. attraction between negatively charged cell membrane of micro-
The antimicrobial activity has been tested against Gram-positive organism and positively charged nanoparticles. In the present
and Gram-negative bacteria. A Gram-positive bacterium used for scenario, AgNPs as antimicrobial agent have come up as promis-
experimentation was Bacillus subtilis NCIM 2635. The antimicro- ing candidate in the medical field. But still the exact mechanism
bial study given in Fig. 6 showed the well size to be 8 mm and of the antibacterial effect of silver ions is not clearly under-
zone of inhibition of about 13 mm. Also the antimicrobial activity stood.
of common narrow spectrum drug Penicillin is seen and is found
to be 13 mm, but the synergetic study with 1:1 mixture of AgNPs 4. Discussion
and Penicillin shows a significant increase in antimicrobial activ-
ity and the zone of inhibition is found to be 17 mm. Thus it can It is well known that few medicinal plants exhibit anti-oxidant
be claimed that these AgNPs are exhibiting antibacterial property property. Thus they can act as biological source of reducing agent.
against Bacillus subtilis NCIM 2635 also the antimicrobial activity On this belief choice of plants for this purpose were those carrying
of Penicillin has increased in the presence of AgNPs. Thus AgNPs medicinal and aromatic properties.
216 G.M. Nazeruddin et al. / Industrial Crops and Products 60 (2014) 212–216

5. Conclusion

The present green synthetic method is an easy, economical,

rapid and eco-friendly way to synthesize metallic nanoparticles
and capable of synthesizing AgNPs at room temperature. There
is no need of any external capping agent. This method produced
poly-dispersed, spherical nanoparticles which are well separated
from each other and no aggregation was observed. The method
discussed here involves a single pot synthesis. Based on the exper-
imentation, it can be concluded that this approach can be used
for industrial scale fabrication of nanomaterials. The experimenta-
tion showed that AgNPs possess significant antimicrobial activity
against Gram-positive bacteria Bacillus subtilus. The synergistic
study of AgNPs and common narrow spectrum antibiotics indicates
that the potency of common antibiotics can be enhanced in the
presence of Ag nanoparticles. Thus there is a future platform for
preparing nano-medicines, and targeted drug delivery, etc. Lastly,
it can be concluded that the green synthesis of nanoparticles is
an emerging and exciting area of nanotechnology and may have
significant impact on further advances in biotechnology.

Acknowledgements

The authors are thankful to Assistant Professor Hasan Osman

for all kind support in the progress of research work. They are
thankful to Dr. Prakash Sane for helping in TEM images and Vish-
wajeet Khot for helping in particle size distribution They are also
grateful to Dr. Sonavane, Department of Biotechnology, Ms. Sonali
Fig. 7. Possible reaction mechanism for reduction of metal ions using plant.
Kalake, Ms. Dhanshree Mali, and Mr. Jaykumar Khot, Department
of Nanoscience and Technology, Shivaji University, Kolhapur, India
A thorough study on literature of C. sativum reveals that the
for analysis of antimicrobial activity.
major components of the plant are coriandrol-C10 H18 O, maleic acid
(about 0.25%), volatile oil, sucrose, tannin, fatty acids and minerals,
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