Preparation and characterization of chitosan – silver bionanocomposite through green synthesis of silver nanoparticles using Trigonella foenum-graecum Subhasree, RS; Prasanna, S. Bakshi; Selvakumar, D*; Kumar, NS and Padaki, VC Defence Bioengineering and Electromedical Laboratory (DEBEL), DRDO, Bangalore, India ABSTRACT
INTRODUCTION
CHARACTERIZATION OF BIONANOCOMPOSITE FILM
Metal nanoparticles are explored as alternative approach to effectively kill pathogenic microorganisms that are multidrug resistant. Silver is the metal of choice, especially in the nanoscale. An ultra low cost, eco-friendly method for synthesis of silver nanoparticles through extract of Trigonella foenum-graecum (Fenugreek) is reported here. The reduction of Ag+ from silver nitrate (precursor) to nano silver could be mediated through polyphenols and quercetin present in Trigonella. The reduction was confirmed by UV-visible spectroscopy showing surface plasmon resonance of silver nanoparticles at 458 nm. The minimum inhibitory concentration for the synthesized nanoparticles was found out to be 70 μg/mL and 80 μg/mL against E. coli and S. aureus respectively. The as prepared nanoparticles were used to enhance the bactericidal effects of chitosan biopolymer. The nanosilver incorporated chitosan (1% w/v) was cross linked using glutaraldehyde (1% w/w) and prepared as free standing films. The films were also characterized by FT-IR and SEM. The membranes were found to be permeable to water vapor. The bionanocomposite has the capacity to swell 1000%. In antimicrobial studies, the chitosan – silver bionanocomposite showed 99.99% reduction against E. coli and S. aureus within an hour of treatment. It is proposed to use this bionanocomposite in applications of wound healing due to its excellent swelling and antimicrobial properties.
Chitosan is the second most plentiful natural biopolymer and is relatively cheap. It has attracted considerable interest due to its biological properties, such as antimicrobial activity, antitumor activity and immune enhancing effect. However, the antibacterial activity of chitosan is limited.[1]
UV-Visible spectrum of Silver NP’s
Shake flask studies showing 99% bacterial reduction
Silver nitrate (AgNO3) R E D U C T I O N
40.0 38 36 34 32
Quercetin
30
Date: 5/7/12
Minimum Inhibitory Concentration : E. coli – 70 μg/mL & S. aureus – 85 μg/mL
28 26 24 22
MATERIALS Source of Plant extract: Aqueous extract of Trigonella foenum-greacum leaves (Fenugreek)
Table 1 showing percentage bacterial reduction
Silver nanoparticles
%T 20
Sample
18
% Bacterial reduction in 1 hr
16
E. coli
S. aureus
No reduction
No reduction
Chitosan film
78.36
79.52
Chitosan-silver film
99.71
99.17
14
Optimized reduction time : 90 mins Broad absorption band at λmax – 458 nm: Nanoparticles of 70-80 nm range 12
Control
10 8
Date: 5/7/12
6 4 2
1800.0 -1.0
40.0
1700
1600
1500
1400
1300
1200
1100 cm-1
1000
900
800
700
600
450.0
38
Mechanism of antibacterial action of Silver
Chitosan Chitosan-1A-110407.sp
36 34
DSK Chit SiO ZnO.sp
Nano-silver incorporated
32
chitosan
30
1
28
FT-IR spectrum of chitosan and chitosan-silver bionanocomposite films
% Transmittance
In recent times, the use of silver nanoparticles has gained popularity for its intensified antimicrobial property. The use of plant extracts for the synthesis of silver nanoparticles offers benefits of eco-friendliness and compatibility for biomedical applications. Chemical synthesis methods use reducing agents used that may be toxic and have adverse effects in medical applications. Green synthesis is advantageous over physical and chemical methods as it is cost-effective and no need to use high pressure, energy and temperature. Fenugreek (Trigonella foenum-graecum) a medicinal herb found to contain polyphenols and quercetin (3, 5, 7, 3’, 4’pentahydroxy flavone) that involved in the reduction of silver ions.[2]
26
1258 cm-1
24 22
%T 20 18 16 14
1332 cm-1
12 10 8
1652 cm-1
6 4 2 1800.0 -1.0
1700
1600
1500
1400
1300
1200
1100 cm-1
1000
900
800
700
600
450.0
Chitosan-1A-110407.sp DSK Chit SiO ZnO.sp
CHITOSAN: C=O at 1652 cm-1 and C-N at 1375 cm-1; C–O–C stretching at 1258 cm-1 CHITOSAN-SILVER NANOCOMPOSITE: Absence of peak at 1651 cm-1 due to silver particles bound to the functional groups of chitosan[3]
1 - Interruption of cell membrane 2 – Inhibition of enzymes 3 – Interruption of DNA strands
Metal Precursor: Silver nitrate (1 mM)
CONCLUSION
SCHEME OF WORK 1. Preparation T. foenum-greacum extract
4. Chitosan (1% w/v) in 1% acetic acid
Figure 1. SEM of Chitosan 5. Preparation CS +SNP films
3. Synthesis Silver nanoparticles
pH 4
FT-IR
CONTACT Anti microbia l
Characterizatio n
SEM
% Swelling
Ag NPs
1200
pH 7
1000
Plant Extract
Figure 2. SEM of Chitosan-Silver
1400 1200
Poster Design & Printing by Genigraphics® - 800.790.4001
Zone of Inhibition
+ Polyphenols
2. Optimization Plant extract : Metal Precursor
D. SELVAKUMAR Defence Bioengineering and Electromedical Laboratory (DEBEL), DRDO, CV Raman Nagar, Bangalore, India, PIN: 560 093. Email: dhanaselva@gmail.com
ANTIBACTERIAL CHARACTERIZATION
1000
pH 10
800
800
600 400 200
WVPT Swellin g
pH 4 - Repulsion between polymer chains lead to a better water diffusion pH 7 & pH 10 – Diffusion of water molecules is hindered by the hydrogen bonding
Chitosan and silver nanoparticles have proven wound healing properties individually. The synergistic effect of both was exploited to enhance antimicrobial activity of chitosan. FT-IR and SEM confirmed the incorporation of silver into chitosan biopolymer. These films were evaluated for water absorption capacity and antibacterial activity. The films have shown about 1000 % in water absorption capacity. It is proposed that the Chitosan-Silver nanoparticle composite films can be used as wound dressings.
REFERENCES 1. Cheng, et al., Molecules. (2011), 16, pp. 8304-8314 2. Honary, et al., Trop J Pharm Res. (2011), 10, pp. 70-76
0 1
2
3
4
5
6
7
Time in h
Figure 3. Swelling studies at pH 4, pH 7 and pH 10
3. Durian, et al., Appl Microbiol Biotechnol. (2011), 3, pp. 5120-5125