TERMINALIA CEBULA FRUIT EXTRACT MEDIATED SYNTHESIS OF
SILVER NANOPARTICLES AND THEIR ANTIMICROBIAL EFFECT
Yadav Shiv Kumar1*, Deepika1, Khar Roop K1, Mohd. Mujeeb2, Ansari S.H.2
1*
2
B. S. Anangpuria Institute of Pharmacy, Alampur, Faridabad
Department of Pharmacognosy, Faculty of Pharmacy, Jamia Hamdard University, Delhi
Address For correspondence
Dr. Shiv Kumar Yadav
Assistant Professor,
B. S. Anangpuria Institute of Pharmacy,
Alampur, Faridabad-121004 (Haryana)
Email: shivbsaip@gmail.com
ABSTRACT
There were numerous work have been produced based on the plant and its extract mediated
synthesis of nanoparticles, in this present study we tried to explore this novel approaches for the
biosynthesis of silver nanoparticles using plant fruit bodies. The plant, Terminalia Cebula L.
fruit bodies are used in this study, where the dried fruit body extract were prepared by using
different solvents and they were mixed with silver nitrate in order to synthesis of silver
nanoparticles. The active phytochemicals present in the plant were responsible for the quick
reduction of silver ion (Ag(+)) to metallic silver nanoparticles (Ag(0)). The reduced silver
nanoparticles were characterized by UV-vis spectroscopy. The spherical shaped silver
nanoparticles were observed. The silver nanoparticles formed showed a similar behavior with
maximum absorption peaks ranging between 410-430 nm due to characteristic surface plasmon
absorption. The antibacterial property of synthesized nanoparticles was observed by Agar Well
Diffusion method against different clinically isolated multi-drug resistant gram +ve and gram –
ve bacteria. The plant materials mediated synthesis of silver nanoparticles have comparatively
rapid and less expensive and wide application to antibacterial therapy in modern medicine.
KEYWORDS: Nanoparticles, Silver, Nanotechnology, Biotechnology, Antibacterial
1. INTRODUCTION
The emergence of nanotechnology is likely to have a significant impact in various fields
including the pharmaceutical sciences. Nanoparticles possess unique electrial, optical as well as
biological properties and are thus applied in catalysis, biosensing, imaging, drug delivery and
medicine1,2,3. Currently, there is a constant need to develop eco-friendly processes for the
synthesis of nanoparticles. The focus for synthesis of nanoparticles has shifted from chemical
towards green chemistry4. In recent years the term metal nanoparticles has arisen and attracted
great attention specifically silver nanoparticles because of their strong cytotoxicity towards a
broad range of microorganisms and moreover its use as antimicrobial agent is well known5,6.
As per literature survey there are numerous techniques available for synthesis of silver
nanoparticles like thermal decomposition7, microwave assisted synthesis8, electrochemical9 and
chemical reduction10. Almost all the above mentioned methods are extermely expensive and
involve uses of toxic, hazardous chemicals which can cause impending biological risks.
Biological technique for synthesizing silver nanoparticles using microbes11, enzymes or plant
extract12,13 has been considered as valuable alternative method to chemical or physical methods
as it does not require any elaborative processes or chemicals.
Nowadays multiple drug resistance has developed due to the indiscriminate use of commercial
antimicrobial drugs commonly used in the treatment of infectious disease. In addition to this
problem, antibiotics are sometimes associated with adverse effects on the host including
hypersensitivity, immune-suppression and allergic reactions. These situations forced scientists to
search for new antimicrobial substances or modified form of the existing antimicrobial drugs.
Given the alarming incidence of antibiotic resistance in bacteria of clinical importance, there is a
constant need for new and effective therapeutic agents. Therefore, there is a need to develop
modified form of antimicrobial drugs for the treatment of infectious diseases from medicinal
plants. Several screening studies are been carried out in different parts of the world for screening
of plant mediated synthesis of silver nanoparticle for their antimicrobial effect. Hence in present
study we have taken different extracts of fruits of T.chebula for synthesis of silver nanoparticle
and evaluated them for their antibacterial activity.
Terminalia chebula Retz.(Combretaceae) is native to Indian subcontinent and the adjacent areas
such as Pakistan, Nepal and the South-West of China stretching as far south as Kerala or even Sri
lanka14. Terminalia chebula Retz. is a medium to large deciduous tree, attaining a height of up to
30 m, with widely spreading branches and a broad roundish crown. Its wood is hard and bulky. It
occurs scattered in teak forest, mixed deciduous forest, extending into forests of comparatively
dry types15-19. According to Unani system of medicine, unripe fruit is an astringent and aperient.
The ripe fruit has purgative action, it is a tonic and has good in piles. It is reported to be
stimulant and tonic for brain, eyes and gums. In Ayurveda, fruits are used as- antimicrobial20,
Antioxidant21, anti-inflammatory, wound healing, stomachic, anthelminitic, antidysentric,
scorpion sting, burns, skin disorders22 and a variety of many other diseases.
2 MATERIALS AND METHODS
2.1. MATERIALS
The fruits of T.chebula were purchased from gobal herbs, Delhi. The voucher specimen was kept
in herbarium of Institute for further references. Analytical grade Silver nitrate used in this
experiment was obtained from Merck (India).
2.2. SYNTHESIS OF SILVER NANOPARTICLES
Plant fruit extract was prepared by using successive hot continuous extraction with pet. ether
(60-800), chloroform, methanol and chloroform water as solvents. The extracts were filtered and
concentrated on water bath. Ten milliliters of each extracts were taken in different Erlenmeyer
flask. For the reduction of silver (Ag+) ions 90 ml of 1mM aqueous solution of silver nitrate was
added in each Erlenmeyer flask respectively. Then each Erlenmeyer flask was incubated at 37 0 C
with periodic stirring for 48 hrs. The synthesis of silver nanoparticles was confirmed by color
change from yellowish orange to dark brown.
2.3. CHARACTERIZATION OF THE SYNTHESIZED SILVER NANOPARTICLE
Synthesis of silver nanoparticles solution with fruit extract may be easily observed by ultraviolet
–visible spectroscopy. The UV –VIS spectra were carried out as a function of time of the
reaction at room temperature at an interval of 1 hr, 24 hr, 48 hr operated in 300 to 540 nm range
at a resolution of 1 nm.
2.4. ANTIBACTERIAL ACTIVITY
Antibacterial activity of silver nanoparticles synthesized using fruit extracts of T.chebula was
tested against different gram +ve and gram –ve bacteria i.e. Bacillus cerius, Staphylococcus
aureus, Salmonella typhi, Pseudomonas aerogenus, Escherichia coli and Klebsiella pneumoniae
using the Agar Well Diffusion method as per Indian Pharmacopeia. The sterilized nutrient agar
media containing 0.5 ml of inoculum per 100 ml of media was added to the conical flask and
then flasks were shaken slowly avoiding formation of air bubbles and then transferred into
petridishes so as to obtain 6 mm thickness of medium. The medium in the plate was allowed to
solidify at room temperature. The sterile borer was used to prepare four cups of 8 mm diameter
in the medium of each Petri - dish. An accurately measured 0.1 ml of solution of each
concentration of silver nanoparticle and standard samples were added to the cups with the help of
micropipette. All the plates were kept at room temperature for effecting diffusion of drug
extracts and standards. Later, they were incubated at 370C for 24 hrs. The plates were examined
for evidence of zone of inhibition, which apperared as a clear area around the well. The diameter
of such zone of inhibition was measured using a meter ruler and the mean value for each
organism was recorded and expressed in millimeter.
3. RESULTS
3.1. UV-VIS SPECTRA ANALYSIS
The UV-Vis spectrum of reaction mixture (Graph 1- 4) was recorded as a function of a reaction
time (1 hr, 24 hr, 48 hr). The sample showed a similar behaviour with maximum absorption
peaks ranging between 410-430 nm due to characteristic surface plasmon absorption.
3.2 ANTIBACTERIAL STUDIES
The effect of silver nanoparticles synthesized using fruit extracts of T. chebula on bacteria was
found to be equivalent as compared with standards. The zone of inhibition (ZI) of silver
nanoparticles synthesized from different extracts of fruit of T. chebula and the standards is given
in Table 1.
4. DISCUSSION
Biosynthesis of nanoparticles by plant extracts is currently under exploitation and gaining
increasing importance. The development of biologically propogated experimental processes for
the synthesis of nanoparticles is evolving into an important branch of nanotechnology. The
present study deals with the synthesis of silver nanoparticles using various extracts of fruit of T.
chebula and aqueous Ag+ ions. The approach is cost effective alternative to conventional
methods of assembling silver nanoparticles. Formation and stability of silver nanoparticle in
aqueous colloidal solution are confirmed using UV-VIS spectral analysis. It is well known that
sliver nanoparticle exhibits yellowish brown color in aqeuous solution due to excitation of
surface plasmon vibrations in silver nanoparticles. As the extracts were mixed with aqueous
solution of sliver nitrate, it started to change the color from watery to reddish brown due to
reduction of silver ion, which indicated the formation of silver phytonanoparticles. It is generally
recognized that UV-VIS spectroscopy could be used to examine size and shape-controlled
phytonanoparticles in aqueous suspensions. Absorption spectra of silver phytonanoparticles
fromed in the reaction media has absorbance peak 420 nm and broading of peak indicated that
the particles are poly dispersed. Silver has been widely utilized for thousands of years in human
history. Its application includes utensils, dental alloy, currency, jewels, photography etc. Amongs
the various applications of silver, its disinfectant property has been exploited for hygienic and
medicinal purposes, such as treatment of dental illness, nicotine addiction and infectious diseases
like syphilis and gonorrhea. Sliver phytonanoparticle have been demonstrated to exhibit and
antimicrobial properties against bacteria with close attachment of the phytonanoarticles
themselves with the microbial cells and activities were found to be dose dependent in accordance
with the obtained results.
In conclusion, the antimicrobial activity of silver nanoparticles showed that silver nanoparticle
synthesized with different extracts of fruit of T. chebula have great potential to be used as
antimicrobial agent against different gram+ve and gram –ve bacteria. The highest effect was
found with silver nanoparticles synthesized from Aqueous Extract of fruit of T. chebula followed
by chloroform, methanol and pet. ether extracts mediated synthesized silver nanoparticles. All
the synthesized silver nanoparticles showed almost same ZI values as that are obtained with
standard references i.e Penicillin for garm+ve bacteria and Streptomycin for garm-ve bacteria.
The highest ZI value was observed to be 23.3 mm against Salmonella typhi with silver
nanoparticles synthesized from Aqu. extract and lowest as 11.8 mm against E.coli with silver
nanoparticles synthesized from Pet. Ether extract. The method employed for the preparation of
silver nanoparticles was easy and cost effective.
Acknowledgements
The authors would like to thank management of B. S. Anangpuria Educational Institutes for
providing the necessary support for successful completion of study.
REFERENCES
1. Nair LS, Laurencin CT. Silver nanoparticles: synthesis and therpeutic applications. J
Biomed Nanotechnol 2007; 3: 301-316.
2. Lee KS, El-Sayed MA. Gold and silver nanoparticles in sensing and imaging: sensitivity
of plasmon response to size, shape and metal composition. j phys chem B 2006; 110:
19220-19225.
3. Jain PK, Huang X, El-Sayed MA. Novel metals on the nanoscale: optical and
photothermal properties and some applications in imaging, sensing, biological and
medicine. Acc Chem Res 2008; 41: 1578-1586.
4. Vigneshwaran N, Ashtaputre NM, Varadarajan PV, Nachane RP, Paralikar KM,
Balasubramanya RH. Biological synthesis of silver nanoparticles using the fungus
Aspergillus flavus. Material letters 2007; 61: 1413-1418.
5. Frattini A, Pellegri N, Nicastro D, De Sanctis O. Effect of amino groups in the synthesis
of Ag nanoparticles using aminosilanes. Mater Chem & Phy 2005; 94: 148.
6. Textor T, Fouda MMG, Mahltig B. Deposition of durable thin silver layers onto
polyamides employing a heterogeneous Tollen’s reaction. App Surf Sci 2010; 256: 23372342.
7. Navaladian S, Viswanathan B, Viswanath RP, Varadarajan TK. Thermal decomposition
as route for silver nanoparticles. Nanoscale Res Lett 2007; 2: 44-48.
8. Sreeram KJ, Nidin M, Nair BU. Microwave assisted template synthesis of silver
nanoparticles. Bull Mater Sci 2008; 31(7): 937-942.
9. Starowicz M, Stypula B, Banas J. Electrochemical synthesis of silver nanoparticles.
Electrochem Commun 2006; 8(2): 227-230.
10. Liz-Marzan LM, Lado-Tourino I. Reduction and stabilization of silver nanoparticles in
ethanol by nonionic surfactant. Langmuir 1996; 12: 3585-3589.
11. Klaus T, Joerger R, Olsson E, Granqvist CG. Silver based crystalline nanoparticles,
microbially fabricated. J Proc Natl Acad Sci USA 1999; 96: 13611-13614.
12. Bhattacharya D, Gupta RK. Nanotechnology and potential of miroorganisms. Crit Rev
Biotechnol 2005; 25: 199-204.
13. Mohanpuria P, Rana NK, Yadav SK. Biosynthesis of nanoparticles: technological
concepts and future applications. J Nanopart Res 2007; 10: 507-515.
14. Gupta AK. Quality Standards of Indian Medicinal Plants. New Delhi: Indian Council of
Medical Research; 2003;1:205-211.
15. Vaidyaratnam PS. Varier’s Arya Vaidya Sala - Indian Medicinal Plants: A Compendium
of 500 species. Chennai: Orient Longman; 1994 (Reprint 2002) ;5: 263-4.
16. Kirtikar KR, Basu BD. Indian Medicinal Plants. 2nd Edition. Dehradun: International
Book Distributors, Book Sellers and Publishers; 1999; 2: 1020-1023.
17. Nadkarni KM. Indian material medica. Mumbai: Popular Prakashan; 1976; 1:1205-1210.
18. Anonymous. Indian Herbal Pharmacopeia 2000; 439-448.
19. Lee HS, Won NH, Lee H. MAPA 2006; 28: 48.
20. Sudarshan SR. Encyclopedia of Indian medicine. Mumbai: Popular Prakashan; 2005; 4:
92-93.
21. Kappor LD. Hand book of Ayurvedic medicinal plants : CRC press 2000; 322.
22. Rastogi, Mehrotra. Compend. India Med. Plants,PID, New Delhi, 1990;1:406-408.
UV-Visible Spectra of silver phytonanoparticles synthesised using
Pet Ether extract of Terminalia chebula fruit
2.5
Absorbance
2
1.5
1 Hr
24 Hr
1
48 Hr
0.5
0
300
320
340
360
380
400
420
440
460
480
500
520
540
Wave length
Graph No. 1: UV-Visible Spectra of silver phytonanoparticles synthesised using Pet. Ether extract of
Terminalia chebula fruit
UV-Visible Spectra of silver phytonanoparticles synthesised using
Chloroform extract of Terminalia chebula fruit
2.5
Absorbance
2
1.5
1 Hr
24 Hr
1
36 Hr
0.5
0
300
320
340
360
380
400
420
440
460
480
500
520
540
Wave length
Graph No. 2: UV-Visible Spectra of silver phytonanoparticles synthesised using Chloroform extract of
Terminalia chebula fruit
UV-Visible Spectra of silver phytonanoparticle synthesised using
Methanol extract of Terminalia chebula fruit
3.5
3
Absorbance
2.5
2
1 Hr
1.5
24 Hr
1
48 Hr
0.5
0
300
320
340
360
380
400
420
440
460
480
500
520
540
Wave length
Graph No. 3: UV-Visible Spectra of silver phytonanoparticles synthesised using Methanol extract of
Terminalia chebula fruit
UV-Visible Spectra of silver phytonanoparticles synthesised using
Aqueous extract of Terminalia chebula fruit
3.5
3
Absorbance
2.5
2
1.5
1
0.5
0
300
320
340
360
380
400
420
440
460
480
500
520
540
Wave length
Graph No. 4: UV-Visible Spectra of silver phytonanoparticles synthesised using Aqueous extract of
Terminalia chebula fruit
Antibacterial Activity of silver phytonanoparticles of Terminalia chebula Fruit
Extract
Conc. (g)
Pet ether
Chloroform
Methanol
Aqueous
Standard
50
100
150
200
300
50
100
150
200
300
50
100
150
200
300
50
100
150
200
300
50
100
150
200
300
10.2
11.2
12
12.6
13.2
14.1
16.2
18.3
21
23
10.1
11
11.6
12
12.5
14.5
16
18.6
21
22.8
17
21
22.5
25
29
9.3
10.5
12.2
12.8
13.2
15.5
16.9
18.5
20.2
22.1
9.3
10.5
11.2
12
12.3
14.2
15.9
17.3
19.5
22.3
16.5
20.3
23.5
26
28
9.8
10.4
11.4
12.2
12.9
14
15.1
17.2
20
22
10
11.2
12.3
12.9
13.1
15.2
17.3
19.8
21.3
23.2
16.2
19.3
22.4
25.5
27.3
9.6
10.4
11.2
12.1
12.9
15.2
17.3
19.3
21.3
22.2
8.9
9.8
10.4
11.2
12.2
14.9
15.9
17.3
20.5
23.3
17.2
18.5
21.2
23.5
25.6
9.8
10.4
10.9
11.3
11.8
15.1
17.2
19.2
21.2
23.3
9
9.8
10.4
11.2
12.3
14.5
16.1
18.3
20.3
23.1
16.2
18.5
20.5
23.3
25.3
9.2
10.5
10.9
11.5
12.2
12.5
15.5
18.3
20.4
22.8
9.2
9.8
10.6
11.3
12.1
14.2
16.1
17.9
19.2
21.5
18
20.2
22.2
23.8
25.2
Bacillus
Cerius
Staph
aureus
Pseudo
aerogenosa
Salmonella
typhi
E. Coli
Klebsiella
Pneumnae
Zone of Inhibition was measured in mm.
Table No. 1: Antibacterial Activity Silver Phytonanoparticles of Terminalia chebula Fruit