International Journal of Engineering Trends and Technology (IJETT) – Volume3 Issue 4 Number2–Aug 2012
Biosynthesis Of Silver Nanoparticles Using Morinda Citrifolia L. As Capping And Reducing
Agents
D.Rachel Evangelene Tulip1, Aishwarya2,K.K. Surya2, K. Krishna Devi2,R. Kousalya2
1
Bharath University, Selaiyur,Chennai-73
2
Dr.M.G.R. Educational and Research Institute, Chennai-95
ABSTRACT
Green synthesis of silver nanoparticles was carried out using boiling extract of Morinda
citrifolia as capping and reducing agents. Silver nano particles were produced with different
ratios of silver nitrate and plant extracts at 1mM concentration of silver nitrate. Reduction of
silver nitrate was monitored using UV measurements at different time intervals for 5 hrs at room
temperature, colour change was observed after 24 hours of incubation. Characterization of silver
nanoparticles was carried out. Size and shape were studied through XRD, FTIR, SEM and TEM
analysis. Antibacterial activity was carried out using Disc diffusion method for human
pathogenic bacteria such as Staphylococcus aureus, pseudomonas aeruginosa, Escherichia coli
and Shigella sonnei. The zone of inhibition was observed. The silver nanoparticles were toxic
against human pathogens. On comparison with the antibiotics alone nanoparticles associated
antibiotics outerperformed the bacterial effect.
1. INTRODUCTION:
Nanotechnology is the creation of functional materials, devices and systems, through the
understanding and control of matter at dimensions in the nanometer scale length ( 1-100nm),
where new functionalities and properties of matter are observed and harnessed for a broad range
of applications. Nanoparticles, generally considered as particles with size upto 100 nm, exhibit
completely new or improved properties as compared to the larger particles of the bulk material
that they are composed of based on specific characteristics such as size, distribution and
morphology.
Nanoscience is the world of atoms, molecules, macromolecules and macromolecular
assemblies and is dominated by surface effects such as Vander Waals force attraction, hydrogen
bonding, electronic charge, ionic bonding, covalent bonding, hydrophilicity and quantum
mechanical tunneling, to the virtual exclusion of macro-scale effects such as turbulence and
inertia.
The fascination of nanotechnology stems from the unique quantum and surface
phenomenon that matter exhibit at the nanoscale, making possible novel applications and
interesting materials. nanoparticles of noble metals, such as gold, silver and platinum are widely
applied in products that directly come in contact with the human body, such as shampoos, soaps,
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International Journal of Engineering Trends and Technology (IJETT) – Volume3 Issue 4 Number2–Aug 2012
detergents, shoes, cosmetic products and toothpaste, besides medical and pharmaceutical
application.
Novel metal nanocrystallites such as silver and gold provide a more interesting research
field due to their conduction and valence bonds in which electrons move freely. The free
electrons give rise to a surface Plasmon absorption band, which depends on both the particles
size and chemical surroundings. In the last five years, much effort has been expended on their
organization on surfaces for the construction of functional interfaces.
2. MATERIALS AND METHODS:
Silver nitrate solution purchased from HiMedia Laboratories Pvt. Limited, Mumbai, India. The
Morinda citrifolia was obtained from coastal regions of kerala.
2.1. PREPARATION OF AQUEOUS SILVER NITRATE:
1mM silver nitrate solution was prepared in 100 ml deionized water.
2.2. PREPARATION OF EXTRACT BY CONVENTIONAL METHOD:
The Morinda citrifolia leaves were washed several times with deionised water. 50gm of finely
cut Morinda citrifolia leaves were taken and boiled in 150ml of double distilled water for 3min
and filtered. Centrifuged at 6000rpm for 20mins. Collect the supernatant and store at 4°C.
2.3. PREPARATION OF EXTRACT BY HOMOGENIZATION METHOD:
THE Morinda citrifolia leaves were washed several times with deionised water. 50g of the
leaves were homogenized in 150ml water with the help of mortar and pestle anf filtered. Then
the filterate was centrifuged for 30 min at 6000rpm and supernatant was collected. Collect the
supernatant and store at 4°C.
2.4. SYNTHESIS OF SILVER NANOPARTICLES:
15ml of Morinda citrifolia extract was added to 9ml of 1mM AgNO3 separately and kept at room
temperature. Bioreduction of silver ions in the solution were monitored by measuring using UV
spectra of the solution at periodic intervals. The nanoparticle synthesized was confirmed by UV
spectra plasma curve. The solution was centrifuged and the particles were characterized.
2.5. UV –Vis SPECTRA ANALYSIS:
The bioreduction of Ag+ in aqueous solution was monitored by periodic sampling of aliquots
(0.2ml) of the suspension, then diluting the samples with 2ml deionized water and subsequently
measuring UV-Vis spectra, at the wave length of (400-500). UV-Vis spectra were recorded for 4
hrs.
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International Journal of Engineering Trends and Technology (IJETT) – Volume3 Issue 4 Number2–Aug 2012
2.6. RECOVERY OF SILVER NANOPARTICLES BY CENTRIFUGATION:
After bioreduction, the solution consisting of hydrosols of silver nanoparticles was subjected to
centrifugation at 6000 rpm for 20 min, and the supernatant was discarded. The pellet formed was
dissolved in 0.1 ml of toluene water and air dried.
3. RESULT AND DISSCUSION:
3.1. BIOSYNTHESIS OF SILVER NANOPARTICLES:
Biosynthesis of silver nanoparticles by the filtrate of Morinda citrifolia was confirmed by change
in the colour of the filtrate to brown after addiction of silver nitrate. This arises due to excitation
of surface Plasmon vibrations in the metal nanoparticles.
(a)
(b)
Figure 1: Synthesis of silver nanoparticles (a) Before reaction (b) After Reaction
3.2. UV-VIS SPECTRAL ANALYSIS:
The bioreduction of Ag+ in aqueous solution was monitored by periodic sampling of the
reaction mixture at regular intervals by using UV-Vis spectroscopy. Fig shows a strong
characteristic absorbance peak at around 450nm throughout the reaction period. Analysis by
spectrophotometer was made up to 5hrs experiment.
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International Journal of Engineering Trends and Technology (IJETT) – Volume3 Issue 4 Number2–Aug 2012
Figure 2.: UV-Vis spectroscopy of silver nanoparticle
.3.3. CHARACTERIZATION OF SILVER NANOPARTICLES:
3.3.1. XRD:
XRD analysis showed three distinct diffraction peaks at 27.56°, 31.92°, 45.75° and can be
indexed the angle values of (60), (100), (35) crystalline planes of sphere Ag. This analysis
revealed that nanoparticles are in orthorhombic crystals. The high peaks in the analysis indicate
the active silver composition with the indexing.
Figure 3. The XRD pattern of the siver nanoparticles formedin our experiment.
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3.3.2. FTIR ANALYSIS
FTIR spectral analysis showed array of absorbance bands in 500 cm-1 – 4000 cm-1 . The spectral
band peaks are along the range of between 600 cm-1 – 4000 cm-1 with prominent peaks at 674.3
cm-1 , 1072.1 cm-1, 1261.5 cm-1, 1651.7 cm-1, 2080.2 cm-1, 2926 cm-1 and 3398.3 cm-1 which
were interpreted for the identification of the functional moieties in the air dried silver
nanoparticles.
Figure 4: The FTIR graph of silver nanoparticles.
3.3.3. SEM ANALYSIS:
Scanning electron microscope analysis of the silver nitrate solution (Control) and reduced form
of silver nitrate solution are clearly distinguishable owing to their size difference. It is clear from
the SEM pictures that control silver nitrate particles are more than 1000 Mn size, where as silver
particles in the bioreduced colloidal suspensions measured 20-40nm in size.
Figure 5: SEM analysis of silver nitrate
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International Journal of Engineering Trends and Technology (IJETT) – Volume3 Issue 4 Number2–Aug 2012
Figure 6: SEM analysis of silver nanoparticle 1 µm
Figure 7: SEM analysis of silver nanoparticle 5 µm
3.2.3 TEM ANALYSIS:
TEM analysis revealed that the synthesized nanoparticles are stable in solution at room
temperature. The size of nanoparticle range from 20 - 40nm. The decrease in anisotrophy and
particle size is evident from the images. The TEM images revealed equal spherical shape.
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Figure 8: TEM analysis of silver nanoparticle in 150nm
3.4. ANTIBACTERIAL ACTIVITY OF SILVER NANOPARTICLES AGAINST HUMAN
PATHOGENIC BACTERIA:
3.4.1. DISC DIFFUSION METHOD:
Zone of inhibition in the plate showed that silver nanoparticles synthesized using filtrate have the
antibacterial activity against test pathogens namely Staphylococcus aureus, Pseudomonas
aeruginosa, Escherichia coli and Shigella sonnei. On comparison with the antibiotics alone
nanoparticles associated antibiotics outerperformed the bactericidal effect.
Antibacterial activity of silver nanoparticle with ciprofloxin
1. Staphylococcus aureus
2. Pseudomonas aeruginosa
11
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International Journal of Engineering Trends and Technology (IJETT) – Volume3 Issue 4 Number2–Aug 2012
3. Shigella sonnei
Organism
S.auerus
P. aeruginosa
S. sonnei
E. coli
Np
11
14
15
10
4. Escherichia coli
Np+ A
32
30
26
32
A
30
26
24
29
4. CONCLUSIONS:
Addition of 1mM silver nitrate solution into filtrate led the appearance of yellowish
brown colour as a resultant of formation of silver nanoparticles in the solution. The UV-Vis
absorption spectrum recorded for the reaction solution shows the characteristic surface Plasmon
resonance band for silver nanoparticles in the range of 420-460nm. The reddish brown colour
appears after 24hrs of incubation on the addition of the silver nitrate into liquid filtrate.
The SEM studies confirmed the formation of silver particles in the size range of 1520nm, a clear indication of the formation of silver nanoparticles. The characterization of silver
nanoparticles by TEM studies revealed the average grain size to be 35nm. XRD of silver
nanoparticles was with crystalline planes (60), (100), (35) corresponding t spherical shape of
silver nanoparticles. FTIR analysis was used to characterize the nature of capping ligands that
stabilizes the silver nanoparticles formed by bioreduction process. The FTIR spectrum showed
bands at 674.3cm-1 and 2926 cm-1 corresponds to –C-H bending and stretching vibrations, while
the band at 1072.1 is characteristic of amine (-C-N) and carboxylic (-C-O) stretching groups. The
absorbance band at 1261.5 is characteristic of –C-N amine group and –NO2 nitro compound
stretching. The absorbance band at 1651.7 is characteristic of –NO2 notro compound and –C=C
alkenes stretching. The absorbance band at 3398.3 is characteristics of amine –N-H and hydroxyl
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–O-H groups stretching and the absorbance band formed at 2080 is characteristics of alkynes –
C≡C groups stretching.
We have found that the silver nanoparticles synthesized in our study effectively
inhibitedthe growth and multiplication of human pathogenic bacteria like Staphylococcus aureus,
Pseudomonas aeruginosa, Eschericia coli and Shigella sonnei. The silver nanoparticles treated
cultures exhibited reduced/almost no metabolic rates when compared with untreated bacterial
cultures. This can be attributed to the inhibition activity of silver nanoparticles on the respiratory
enzymes (cytochrome oxidases , malate dehydrogenase and succinate dehyrogenase) or as a
result of complete destruction of the bacteria. But yet more experimental data in this regard is
necessary to support the idea.
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