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Research Article CODEN: IJPRNK ISSN: 2277-8713 M Ramar, IJPRBS, 2015; Volume 4(1): 151-164 IJPRBS
Available Online at www.ijprbs.com 151
SYNTHESIS OF SILVER NANOPARTICLES USING NATURAL PRODUCTS FROM ACALYPHA INDICA (KUPPAIMENI) AND CURCUMA LONGA (TURMERIC) ON
ANTIMICROBIAL ACTIVITIES P. MANONMANI1 , M. RAMAR2, N. GEETHA1, M. VALAN ARASU4 , R. RASKIN ERUSAN3, R.
MARISELVAM5 , J. JERLIN SOWMIYA1 1. Department of Biotechnology, Mother Teresa Women’s University, Kodaikanal-624101, India 2. Entomology Research Institute, Loyola College, Chennai-600034, Tamil Nadu, India. 3. Department of Genetics, Dr. A. L. M. Post Graduate Institute of Basic Medical, Sciences, University of Madras, Chennai-600113, India 4. Department of Botany and Microbiology, College of Science, King Saud University, Riyadh, 11451, Saudi Arabia. 5. Department of Chemistry, Sri Paramakalyani College, Alwarkurichi, Tirunelveli, Tamilnadu, India
Accepted Date: 01/01/2015; Published Date: 27/02/2015
Abstract: In the present study, a total of four extracts of two species namely Acalypha indica, Curcuma longa and combination of both
species in two solvents (aqueous and methanol extract) were evaluated for their antibacterial activity. The antibacterial was measured by agar
disc diffusion method. All the extracts showed antibacterial activity all the test bacterial isolates. Aqueous extracts of A. indica did not exhibit
antibacterial activity against Salmonella sp., Klebsiella sp., Pseudomonas sp., Staphylococus sp., but it showed a very good inhibitory effect for
Bacillus sp., and E. coli with a zone of inhibition of 1.8 cm and 1.5 cm respectively. The methonolic extract of A. indica showed very good
activity for all the tested organisms with maximum zone of inhibition i.e 2.5 cm dia for Bacillus sp., and minimum in Pseudomonas sp., of 1.8
cm dia. Curcuma longa (Turmeric) showed a very good inhibitory activity against Staphylococus aureus with zone of inhibition 1.7 and 1.3 cm
for methanolic and aqueous extract respectively. For other species the methanolic extract of turmeric showed the zone inhibition 2.2, 2.1,
2.0, 1.7 and 1.6 cm dia for E.coli, Bacillus sp., Pseudomonas sp., Staphylococus sp., and Salmonella sp. respectively. The synergistic effect of
methanolic extract of both species (Acalypha indica and turmeric) also showed very good inhibitory effect. From the above results it was
inferred that among the two plant species methanolic extract of turmeric was found to be more effective (i.e showed maximum zone of
inhibition against all the bacterial species tested. The methanolic extract of turmeric was compared with the synthesized turmeric silver
nanoparticles. Among these, the silver nano particles showed better results when compared to the methanolic extract of turmeric and
antibiotic. In E. coli the zone of inhibition was maximum i.e 2.5 and 2.2 cm dia for silver nano particles and methanolic extract of turmeric
respectively. Silver nanoparticles of turmeric showed a inhibition zone of 2.3, 2.2, 2.4 and 2.4 cm dia for Salmonella sp., Pseudomonas sp.,
Bacillus sp., and Staphylococus sp., respectively. Findings of the present investigation supports the use of Acalypha indica and turmeric in
traditional medicine for the treatment of various bacterial infections.
.Keywords: Antibacterial, Antibiotics, Silver nanoparticles, Acalypha indica. Curcuma longa, Aqueous, Methanol
INTERNATIONAL JOURNAL OF
PHARMACEUTICAL RESEARCH AND BIO-SCIENCE
PAPER-QR CODE
Corresponding Author: DR. MARIMUTHU RAMAR
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How to Cite This Article:
M Ramar, IJPRBS, 2015; Volume 4(1): 151-164
Research Article CODEN: IJPRNK ISSN: 2277-8713 M Ramar, IJPRBS, 2015; Volume 4(1): 151-164 IJPRBS
Available Online at www.ijprbs.com 152
INTRODUCTION
Plants produce hundreds to thousands of diverse chemical compounds with different biological
activities (Hoareau and DaSilva, 1999). Thus, they have been used in the treatment of various
human diseases for thousands of years all over the world. Herbal medicine has been practicing
in rural areas for different diseases. Implication of the knowledge of those indigenous people
into practice helped the researchers to develop the present formulation into a better approach
with more therapeutic effect. Development of bacterial resistance to the available antibiotics
and increasing popularity of traditional medicine has led the researchers to investigate the
antibacterial compounds in plants.
Acalypha indica Linn. is commonly known as copper leaf (Indian Nettle) belongs to the family
Euphorbiaceae and is seen in many parts of Asia including India, Pakistan, Yemen, Sri Lanka and
throughout Tropical Africa and South America (Ramachandran, 2008). It is an annual herb,
about 80 cm high and commonly found in waste places or fields (Burkill, 1985). It is locally
known as “kucing galak” or “rumput lis-lis”, “kuppaimeni” in India. The plant grows widely on
the backyards of houses and through out the plains of India. It is a small erect herb grows up to
60 cms. Tribes of Kerala, Rajasthan and Madhya Pradesh use fruits: in asthma, cough, bronchitis
and ear ache; plant and fruit: as an expectorant, laxative, pneumonia and rheumatism; leaf: in
skin diseases like scabies. This plant is used as diuretic, antihelmintic and for respiratory
problems such as bronchitis, asthma and pneumonia (Varier, 1996). The roots of A. indica is
used as laxative and leaves for scabies and other cutaneous diseases (Perry, 1980). Major
phytochemicals identified from A. indica are acalyphine, cyanogenic glycoside, inositol, resin,
triacetomamine and volatile oils (Winter and Griffith, 1998). This plant has been used
extensively in herbal medicine in many tropical and sub tropical countries (Kirtikar and Basu,
1975; Ramachandran, 2008). Rahman et al. (2010) has reported that A. indica having analgesic
and anti-inflammatory effects. In Malaysia, A. indica is used for generations for the treatment
of superficial fungal and several other bacterial infections (Abdul Rahman, 1996). Previous
studies on A. indica revealed that this plant has antibacterial activity against several gram
positive bacteria (Govindarajan et al., 2008; Krishnaraj et al., 2010).
Curcuma longa is a medicinal plant that is related to Zingiberaceae family (Chattopadhyay et al.,
2004). C. longa, commonly known as ‘turmeric’, is widely used as a spice and colouring agent,
and is well known for its medicinal properties (Luthra et al., 2001). Components of turmeric are
named curcuminoids, which include mainly curcumin (diferuloyl methane),
demethoxycurcumin, and bisdemethoxycurcumin (Chainani-Wu, 2003). Curcumin is the most
important fraction which is responsible for the biological activities of turmeric. The melting
Research Article CODEN: IJPRNK ISSN: 2277-8713 M Ramar, IJPRBS, 2015; Volume 4(1): 151-164 IJPRBS
Available Online at www.ijprbs.com 153
point of curcumin, C2H2OO6, is 184º C. It is soluble in ethanol and acetone, but insoluble in
water (Joe et al., 2004). Curcumin is a potent antioxidant is believed to be the most bioactive
compound, and soothing portion of the herb turmeric and posses the properties like
antioxidant, anti-inflammatory, anti-platelet activity, lowering of cholesterol etc. Keeping this in
view the important role of A. indica and turmeric in inhibition of different cultures of bacteria
and its role as antioxidant and antibacterial, the present study was conducted to compare the
antibacterial activity extracts of A. indica, C. longa and synthesized turmeric silver nanoparticles
on some bacteria. This study also supports the use of turmeric in traditional medicines for the
treatment of bacterial infections.
Microbes are unlikely to develop resistance against silver as they do against for conventional
target antibiotics, because the metal attacks a broad range of targets in the organisms, which
means that they need to develop a range of mutations simultaneously to protect themselves
(Pal et al., 2007). As a result, Ag-NPs have been applied to a wide range of products, the most
important current use is as antimicrobial agents to prevent infection, such as in burn and
traumatic wound dressings, diabetic ulcers, coating of catheters, dental works, scaffold, and
medical devices (Kim and Kim, 2006; Silver et al., 2006; Kim et al., 2007; Thomas et al., 2007;
Law et al., 2008 and Rai et al., 2009). Ag-NPs are also used in hygienic products including water
purification systems, linings of washing machine, dishwashers, refrigerators, and toilet seats
(Silver et al., 2006 and Rai et al., 2009).
Thus, the objective of this present study was to evaluate the antibacterial activities of aqueous,
methanol extracts of A. indica and Curcuma longa (turmeric) and to compare it with a standard
antibiotic and synthesized turmeric silver nano particles.
MATERIALS AND METHODS
Collection of plant parts
Leaves of mature A. indica plants (1 kg wet weight) were collected from Madurai, Tamil Nadu,
India and identified. Like wise, dried plant parts (Rhizome) of turmeric (Curcuma longa) were
purchased in local supermarket from Madurai and evaluated for their antibacterial activity
against six bacteria.
Research Article CODEN: IJPRNK ISSN: 2277-8713 M Ramar, IJPRBS, 2015; Volume 4(1): 151-164 IJPRBS
Available Online at www.ijprbs.com 154
Fig.1. Showing the leaves and powder form of A. indica
Fig.2. Showing the dried rhizome and powder form of Curcuma longa
Protocol for phytochemical extraction
Leaves of A. indica were washed, oven dried at 45°C overnight, then grounded into powder
form and extracted using Soxhlet apparatus with either Aqueous (distilled water) or methanol
as solvent for 12 h. The solvent was concentrated under vacuum using a rotary evaporator. The
yields were, 3.8 and 7.9 % respectively. The solid residues were stored at -20°C prior to use. The
dried rhizomes of turmeric were grated in a blender and ground into fine powder. It was
extracted using Soxhlet apparatus with either Aqueous (distilled water) or methanol as solvent
for 12 h. The solvent was concentrated under vacuum using a rotary evaporator. The yields
were 4.2 and 8.6 % respectively. The solid residues were stored at -20°C prior to use.
Synthesis of silver nanoparticles
Research Article CODEN: IJPRNK ISSN: 2277-8713 M Ramar, IJPRBS, 2015; Volume 4(1): 151-164 IJPRBS
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The fresh turmeric were purchased, dried at C in hot air oven and powdered and then used
for extraction. About 2 grams of dried powder were ground to fine paste with 20 ml of
distilled water using mortar and pestle. It was centrifuged at 10,000 RPM for 10 minutes and
supernatant was taken for further processing. Ten ml of the extract was added to 90 ml of
aqueous solution of 5 mM Silver nitrate solution (AgNO3 for reduc on of silver nitrate into g
ions and maintained at room temperature C) in the incubator in static condition and the
completion of the reaction was carried out for a period of 2 h. The colourless silver nitrate
solution is changed from pale yellow to ruby red and finally dark brown colour which indicates
the formation of silver nanoparticles.
UV-visible spectroscopy analysis
The colour change in reaction mixture (Silver nitrate solution + leaf extract) was recorded
through visual observation. The bio reduced silver nanoparticle solution was filtered through
Whatmann No.1 filter paper and the filtrate was measured using UV-Visible absorbance. The
bioreduction of silver ions in aqueous solution was monitored by periodic sampling of aliquots
(1 ml) and subsequently measuring UV-vis spectra of the solution using Double beam UV-vis
spectrophotometer (model 2201) operated at a resolution of 1 nm.
Evaluation of plants for their antibacteral activity
Agar disc diffusion method
The antibacterial activity of 6 crude extracts (aqueous, methanolic and combined effect of two
plant parts (Acalypha indica and turmeric, combination of two) and synthesised silver nano
particles of turmeric) against six bacterial isolates was evaluated by using the agar disc diffusion
method (Ahmad and Beg, 2001; Srinivasan et al., 2001 and Somchit et al., 2004) and
experiments were conducted three separate times. Sterilised Muller Hinton agar petriplates
were inoculated with 1 μl 1x1 5 CFU/ml) of each bacterium (in triplicates) and spread with
sterile cotton swabs. Bacteria (Salmonella sp, Klebsiella sp., Pseudomonas sp., Bacillus sp.,
Escherichia coli and Staphylococcus aureus) used in this study were from clinical isolates and
identified at the Department of Pathology and Microbiology, Meenakshi Mission Hospital
Research Centre, Madurai, Tamil Nadu. Commercial antibiotics disc which consists of amphicilin
(10 mg/ml) were used as reference.
Sterile 10.0 mm diameter blank discs (Himedia, India) were used to impregnate two different
dilutions of the extracts. 1 μl volume of the plant extract was poured into a sterile disc and
evaporated. Discs were stored at –5° C prior to use. These discs were placed on the agar plates.
The plates thus prepared were left at room temperature for ten minutes allowing the diffusion
of the extract into the agar (Rios et al., 1988). After incubation for 24 h at 37oC, the plates were
Research Article CODEN: IJPRNK ISSN: 2277-8713 M Ramar, IJPRBS, 2015; Volume 4(1): 151-164 IJPRBS
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observed. If antibacterial activity was present on the plates, it was indicated by an inhibition
zone surrounding the disc containing the plant extract. The zone of inhibition was measured
and expressed in centimeters. Antibacterial activity was recorded if the zone of inhibition was
greater than 8 mm (Hammer et al., 1999). The antibacterial activity results were expressed in
term of the diameter of zone of inhibition and < 9 mm zone was considered as inactive; 9 – 12
mm as partially active; while 13 -18 mm as active and >18mm as very active (Junior and Zanil,
2000). The mean and standard deviation of the diameter of inhibition zones were calculated.
RESULTS
The results of antibacterial activity of both aqueous and methanolic extract of A. indica,
Curcuma longa and combined effect is presented in the Table 1 and represented in fig.2 , 3 & 4.
In the present study, the aqueous extracts of A. indica did not exhibit antibacterial activity
against Salmonella sp., Klebsiella sp., Pseudomonas sp., Staphylococus sp., but it showed a very
good inhibitory effect for Bacillus sp. and E. coli with a zone of inhibition of 1.8 cm and 1.5 cm
respectively. Turmeric showed a very good inhibitory activity against Staphylococus aureus with
zone of inhibition 1.7 and 1.3 cm for methanolic and aqueous extract respectively. For other
species the methanolic extract of turmeric showed the zone inhibition 2.2, 2.1, 2.0, 1.7 and 1.6
cm dia for E.coli, Bacillus sp., Pseudomonas sp., Staphylococus sp., and Salmonella sp.
respectively. The synergistic effect of methanolic extract of both species (Acalypha indica and
turmeric) also showed very good inhibitory effect. These results are on par with methanolic
extract of Turmeric. There were no much variations in the zone of inhibition and is shown in
Table 1.
The methanolic extract of turmeric was also compared with the synthesized turmeric silver
nanoparticles (Laboratory preparation) and represented in Table 2 and represented in fig.5.
Among these, the silver nano particles showed better results when compared to the methanolic
extract of turmeric and antibiotic. In E. coli the zone of inhibition was maximum i.e 2.5 and 2.2
cm dia for silver nano particles and methanolic extract of turmeric respectively. Silver
nanoparticles of turmeric showed a inhibition zone of 2.3, 2.2, 2.4 and 2.4 cm dia for Salmonella
sp., Pseudomonas sp., Bacillus sp., and Staphylococus sp., respectively.
DISCUSSION
Although, the primary purpose of herbs and spices is to impart flavour and piquancy to food.
Medicinal, antimicrobial and antioxidant properties of spices have also been exploited ((Uraih,
2004 and Souza et al., 2005). Naturally occurring water-soluble components in most plant
materials include various anionic components such as thiocynate, nitrate, chlorides and
sulphates, starches and tannins, saponins, terpenoids, polypeptides and lectins (Darout et al.,
2000). In the present study, the Acalypha indica, and turmeric extracts exhibited antibacterial
Research Article CODEN: IJPRNK ISSN: 2277-8713 M Ramar, IJPRBS, 2015; Volume 4(1): 151-164 IJPRBS
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activity in methanolic extracts. Phytochemicals having solubility in ethanol and methanol
include tannins, polyphenols, polyacetylenes, flavonol, sterols and alkaloids (Ivanovska et al.,
1996). Cowan (1999) examined a variety of extracts for their ability to solubilise antibacterial
from plants as well as other factors such as their relative ranking as biohazards and the ease of
removal of solvent from the fraction and ranked them in the order: methylene dichloride >
methanol > ethanol > water. Accordingly, in the present study, two solvents namely water and
methanol was selected for the plant extraction.
In the methanolic extract of A. indica showed very good activity for all the tested organisms
with maximum zone of inhibition i.e 2.5 cm dia for Bacillus sp., and minimum in Pseudomonas
sp., of 1.8 cm dia. There are many reports of plants in the family Euphorbiaceae possessing
anti-microbial activity (Perez et al., 1997; Awoyinka et al., 2007; Falodun et al., 2008). From the
analysis it was concluded that the preliminary phytochemical analysis that phenols and tannins
detected in the extracts may contribute to the antimicrobial effect. This may be the reason why
A. indica also showed similar anti-microbial activity. Indeed, previous study on A. indica
revealed this plant has antibacterial property against other bacteria (Govindarajan et al., 2008).
From the above results it was inferred that among the two plant species methanolic extract of
turmeric was found to be more effective (i.e showed maximum zone of inhibition against all the
bacterial species tested. The methanolic extract of turmeric being strongly active against E. coli
(2.4 cm) and Bacillus (2.6 cm) isolates while aqueous extracts strongly active against S. aureus
isolates (1.2 cm). Chandrana et al. (2005) who studied antimicrobial activity of turmeric
reported that it was effective against E. coli, B. subtilis and S. aureus and suggested that the
activity is due to the presence of curcuminoid, a phenolic compound. Gur et al. (2006) who
reported that the methanolic extract of turmeric was effective in extraction of antimicrobially
active substances as compared to water and hexane. Negi et al. (1999) demonstrated that
turmerone and curlone components of turmeric possess excellent antibacterial action against a
wide range of microbes such as B. cereus, B. coagulans, B. subtilis, S. aureus. E. coli and P.
aeruginosa. The antimicrobial property of turmeric has been attributed to the presence of
essential oil, an alkaloid, curcumins and other curcuminoids, turmeric oil, turmerol and veleric
acid (Cikrikci et al., 2008). The methanolic extract of turmeric was also compared with the
synthesized turmeric silver nanoparticles and among these, the silver nano particles showed
better results when compared to the methanolic extract of turmeric and antibiotic. In E. coli
the zone of inhibition was maximum i.e 2.5 and 2.2 cm dia for silver nano particles and
methanolic extract of turmeric respectively. Silver nanoparticles of turmeric showed a inhibition
zone of 2.3, 2.2, 2.4 and 2.4 cm dia for Salmonella sp., Pseudomonas sp., Bacillus sp., and
Staphylococus sp., respectively.
Research Article CODEN: IJPRNK ISSN: 2277-8713 M Ramar, IJPRBS, 2015; Volume 4(1): 151-164 IJPRBS
Available Online at www.ijprbs.com 158
Table 1. Antibacterial activity of Methanolic and aqueous extracts of selected plants (zone of
inhibition in cm)
Values are mean ± SD (cm) of 3 separate experiments.
S.No Name of Organisms Acalypha indica Curcuma longa
(Turmeric)
Combined effect
M
cm in dia
A
cm in
dia
M
cm in dia
A
cm in
dia
M
cm in dia
A
1. Salmonella sp., 2.1 ± 0.46 .
.
- 1.8 ± 0.34 - 1.9 ± 0.64 -
2. Klebsiella sp., 1.8 ± 0.36 - - - - -
3. Pseudomonas sp., 1.4 ± 0.54 - 2.2 ± 0.26 - 2.3 ± 0.38 -
4. Bacillus sp., 2.5 ± 0.65 1.8 ± 2.6 ± 0.52 - 2.1 ± 0.26 -
5. E.coli 2.1 ± 0.34 1.5 ± 2.4 ± 0.28 - 1.8 ± 0.47 -
6. Staphylococcus sp., - - 1.6 ± 0.48 1.2 2.0 ± 0.65 1.8 ± 0.53
Table 2. Antibacterial activity of Methanolic extract of Curcuma longa and Turmeric nano
particles (zone of inhibition in cm)
S.No Name of Organisms Antibiotics
Control
Turmeric methanol extract
(TM)
Turmeric Nano particle
(TNP)
cm in dia cm in dia Cm in dia
1. Salmonella sp., 0.7 ± 0.32 1.6 ± 0.35 2.3 ± 0.46
2. Pseudomonas sp., 1.0 ± 0.23 2.0 ± 0.54 2.2 ± 0.64
3. Bacillus sp., 0.9 ± 0.27 2.2 ± 0.37 2.3 ± 0.54
4. E. coli 1.2 ± 0.63 2.6 ± 0.54 2.1 ± 0.32
5. Staphylococcus sp., 0.8 ± 0.53 2.4 ± 0.26 1.8 ± 0.54
Values are mean ± SD (cm) of 3 separate experiments.
Research Article CODEN: IJPRNK ISSN: 2277-8713 M Ramar, IJPRBS, 2015; Volume 4(1): 151-164 IJPRBS
Available Online at www.ijprbs.com 159
Fig.3. Photograph of Antibacterial activity of Methanolic and aqueous extracts of Acalypha
indica
Fig.4. Photograph of Antibacterial activity of Methanolic and aqueous extracts of Curcuma
longa (Turmeric)
Research Article CODEN: IJPRNK ISSN: 2277-8713 M Ramar, IJPRBS, 2015; Volume 4(1): 151-164 IJPRBS
Available Online at www.ijprbs.com 160
Fig. 5. Photograph of Antibacterial activity of Methanolic and aqueous extracts of
combination Acalypha indica and Curcuma longa
Fig. 6. Photograph of Antibacterial activity of Methanolic extracts Curcuma longa and
synthesised silver nano particles
Research Article CODEN: IJPRNK ISSN: 2277-8713 M Ramar, IJPRBS, 2015; Volume 4(1): 151-164 IJPRBS
Available Online at www.ijprbs.com 161
Findings of the present investigation supports the use of Acalypha indica and Curcuma longa
(turmeric) in traditional medicine for the treatment of various bacterial infections.
CONCLUSION
It may be concluded from the present studies that both plant species of the methanolic extracts
can be used as a potential source of natural antimicrobial compound. Further research is
required for the identification of bioactive molecule present in the two extracts. Hence, they
can be used in the treatment of infectious diseases caused by tested strains and potential
antimicrobial agents may be developed. However, further studies have to be done to identify
the specific principles responsible for the antimicrobial activity of A. indica and Curcuma longa.
Silver nanoparticles of turmeric was found to be more effective and the zone of inhibiton found
to be higher when to compared to methanolic extract of turmeric,.
ACKNOWLEDGEMENT
We the authors acknowledge the Mother Teresa Women;s University for providing necessary
facilities. We extend our heartfelt gratitude to DST-CURIE for funding to carry out this project
successfully.
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