Research Journal of Biotechnology
13 (9) September (2018)
Biotic synthesis of silver nanoparticles from
Momordica charantia (Cucurbitaceae) and its
characterization studies
Gandhiraj V.1*, Sathish Kumar K.2 and Narendrakumar G.3
1. Department of Biotechnology, Shri Andal Alagar College of Engineering, Mamandur, 603111, INDIA
2. Department of Chemical Engineering, SSN College of Engineering, Chennai, 603110, INDIA
3. Department of Biotechnology, Sathyabama University, Chennai, 600119, INDIA
*ecogandhi@gmail.com
Abstract
A collection of novel strategies was applied for the
biosynthesis of silver nanoparticles by using plants. In
the present study, green synthesis was carried using
Momordica charantia and the optimization of silver
nanoparticle synthesis was carried out by varying
varieties, solvent, parts of fruit, plant extract
concentration, metal ion concentration of silver nitrate
solution, pH, temperature, reaction time and reaction
condition. The characterization study was done using
UV-Visible spectrophotometer, SEM, TEM, FTIR
spectroscopy, EDX and XRD.
The results revealed that the shape of synthesized silver
nanoparticle is spherical and size ranges from 75-150
nm in SEM and 50-200nm in TEM. FTIR identified
nitro compounds, phenols, alcohols, aliphatic amines
and alkenes. The chemical compound analysis is done
using gas chromatography-mass spectroscopy (GC-
MS).
Keywords: Silver nanoparticles, biosynthesis, Momordica
charantia.
Introduction
The progress of green synthesis of nanoparticles is sprouting
into an important branch of nanotechnology1-4. Momordica
charantia is a tropical and subtropical vine of the family
Cucurbitaceae, widely grown in Asia, Africa and the
Caribbean for its edible fruit that has lot of medicinal
activity5-8. There are many varieties of bitter gourd that
differentiated in its shape (oblong, short), colour (different
shades of green), grow up to 20 cm in length, some may be
oval 10cm in length and wild variety is 3-5cm long.
Bitter gourd contains flavonoids, alkaloids, terpenoids,
proteins, polyphenols, anthoquinone, charatin,
momordicosides, ascorbic acid, vitamin C, carotenoids,
glycosides etc.9-12 It is used in the treatment of diabetes,
cancer, HIV, immune diseases, spermatogonic diseases,
anthrogonic diseases, ulcer and urogenic disorders13,14.
The objective of the present investigation is to prepare the
plant extract and rapidly synthesize silver nanoparticles
using biological methods followed by characterization using
UV-Vis spectrometer, SEM, EDAX, FTIR, TEM and XRD.
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Material and Methods
Preparation of Fruit Extract
Collection of Sample: The fresh fruits of Momordica
charantia were collected from the market of Chennai. The
taxonomic identification of the plant was authenticated by
the Botany CAS, Chennai, Tamilnadu, India. Silver nitrate
is purchased from Qualigens, Mumbai. All solutions were
freshly prepared using deionized water and were kept in the
dark to keep away from any photochemical reaction.
Glassware were properly washed with potassium chromate
and rinsed with soap oil and water.
Preparation of extract: The fresh Momordica charantia
was washed several times with normal water and then in
deionized water to remove dust particles. The seeds are
removed from the fruit and chopped into fine pieces dried in
shades until all the moisture content was removed. 80g of
dried fruit pulp was blended in homogenizer. 5g of powdered
fruit pulp was added into 50ml of water in 100ml conical
flask. The mixture was boiled for 45mins at 90ºC. The
aqueous extract was filtered using Whatmann filter paper
(No. 40) and centrifuged to remove impurities.
Preparation of Silver nitrate solution: Silver nitrate of
analytical grade is used to prepare silver nitrate solution.
5mM stock solution of silver nitrate solution (AgNO3) was
prepared by dissolving 0.845g of silver nitrate in 1000 ml of
deionized water12.
Synthesis of silver nanoparticle: 1ml of Momordica
charantia aqueous fruit extract was added into 9ml of silver
nitrate solution and kept in a magnetic stirrer for
homogenization of the reaction mixture and filtrate was
taken after filtration using Whatmann no. 1 filter paper. The
formation of silver nanoparticles was confirmed when the
colour change from pale yellow to reddish brown is
observed.9 The optimum factors for silver nanoparticles
synthesis were carried out in different condition such as
differing solvent, differing variety of fruit, differing fruit
extract concentration, temperature of extraction, reaction
time, reaction condition and different pH.
Optimization Studies
Effect of solvent and different parts of fruit: To study the
effect of solvent, aqueous and solvent extraction is done. The
aqueous extraction is done by adding 5g of dried fruit pulp,
whole fruit and seed powder into 50ml of deionized water
respectively and then kept in water bath for 45min at 100˚C.
Research Journal of Biotechnology
It is filtered using Whatmann No.40 filter paper7-9. The
ethanol extraction is done by adding 5g of dried fruit pulp,
whole fruit and seed powder respectively into 50ml of
ethanol and then kept in Rotatory shaker for 24 hrs. It is then
centrifuged at 4000 rpm for 10 mins at 25˚C. Then
supernatant is used as ethanol extract.
Effect of different variety of fruit: The wild and cultivable
type of Momordica charantia was used for extraction.
Effect of metal ion concentration: To study the effect of
metal ion concentration of AgNO3 solution, different
molarity such as 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 10 mM,
20 mM, 40 mM were prepared for synthesis of silver
nanoparticles.
Effect of fruit extract concentration: To study the effect
of fruit extract concentration, different concentrations of
fruit extract such as 10%, 20%, 30%, 40%, 50% were
prepared for silver nanoparticle synthesis.
Effect of extraction temperature: To study the effect of
temperature for extraction, different temperatures such as
40˚C, 50˚C, 60˚C, 70˚C, 80˚C, 90˚C, 100˚C were varied for
extraction.
Effect of reaction time for silver nanoparticle synthesis:
To study the effect of reaction time for synthesis of silver
nanoparticle, different reaction times such as 0, 15, 30, 60,
120, 1440 min were observed for silver nanoparticle
synthesis.
Effect of reaction condition for silver nanoparticle
synthesis: To study the effect of reaction condition for
synthesis of silver nanoparticle, the mixture of fruit pulp
extract and silver nitrate solution was kept in different
reaction condition such as sunlight, water bath and in dark
for the synthesis of silver nanoparticles.
Effect of pH: To study the effect of pH for synthesis of
silver nanoparticle, different pH such as 3, 4, 5, 6, 7, 8, 9
were varied for synthesis of silver nanoparticles.
Characterization of Silver Nanoparticles
To confirm the synthesis of silver nanoparticles, the
following methods are essential. The following
characteristic methods were used to characterize the silver
nanoparticles that were synthesized biologically4-6.
UV-Visible Spectroscopy: After color change from pale
yellow to reddish brown, it is analyzed in UV-Visible
spectrophotometer to confirm the formation of silver
nanoparticles. The absorbance reading from 200-700nm is
taken using spectrophotometer (ELICO SL 159).
Scanning Electron Microscopy: The silver nanoparticle
pellet was subjected to SEM analysis. The thin film of
solution was prepared on a carbon coated grid by dropping a
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very small amount of the sample on the grid, extra solution
was removed using a blotting paper and then the film on the
SEM grid was allowed to dry for analysis.
Transmission Electron Microscopy: Transmission
electron microscope was used to identify the internal
diameter and its morphology. HR-TEM measurements were
performed by Hitachi H-7650 instrument. By drop coating,
silver nanoparticle was prepared for HR-Transmission
electron microscope analysis on to carbon coated copper
TEM grids. The film on the TEM grids was allowed to stand
for 2 min following which the extra solution was removed
using a blotting paper and grid was allowed to dry prior to
the measurement.
Fourier Transmission Infra-Red Spectroscopy (FTIR):
FTIR (Fourier Transform Infra-Red Spectroscopy) is a
sensitive technique useful for identifying organic chemicals
in whole range of applications although it can also
characterize some inorganics including paints, adhesives,
resins, polymers, coatings and drugs. The FTIR is powerful
tool for isolating and characterizing organic contamination.
In FTIR, the most molecules absorb light in the bonds
present in the molecule and functional groups present in the
sample. FTIR is particularly useful for identification of
organic molecular groups and compounds due to the range
of functional groups, side chains and cross-links involved,
all of which will have characteristic vibration frequencies in
the infra-red range.
X-Ray Diffraction (XRD) measurements: The phase
purity and the crystalline structure of bio-synthesized silver
nanoparticles were investigated through X-ray Diffraction
technique using a Rigaku Multiflex X-ray powder
diffractrometer employing CuK𝛼 radiation. This thin silver
film was subjected to X-ray diffraction operating between
the angle of 10º and 80º with a scanning rate of angle 2
degree per minute. The average crystalline size of the silver
nanoparticles was calculated using a line broadening profile
of peak and Scherrer’s formula as follows:
D = 0.9(λ)/β Cos ϴ
where λ is the wavelength (1.5418Å), β is the full width half
maximum (FWHM) of corresponding peak and ϴ is the
angle of the diffraction peak.
Energy Dispersion Spectroscopy: Energy dispersive
analysis X-ray (EXD) spectrometer works using photon
nature of light. In the X-ray range, the energy of a single
photon is just sufficient to produce a measurable Pulse X-
ray. A semiconductor material is used to detect the X-ray
along with processing electronics to analysis the spectrum.
GC-MS Analysis: The GC-MS analysis was done to
identify the qualitative and quantitative data of compounds
from the plant extract and silver nanoparticles. The samples
were injected into the Agilent 7890B GC related to 5977a
Research Journal of Biotechnology
MSD mass spectrometry. The HP-5MS 5% phenyl methyl
silox column at 30m×250µm×0.25µm size was used. The
oven temperature was programmed as 50˚C for 2mins. Then
it was ramped up to 300˚C at 5˚C/min and then it was held
at 300˚C for 5min. The sample injected temperature is
250˚C. 1µl of sample is injected through auto sampler
(G4513A) and the injection mode is split less. The solvent
was delayed for 2mins. The helium gas flow rate was
1ml/min. the samples were scanned at the range of 40-
700m/z. The total run time was 57mins.The obtained results
were analyzed using NIST library.
Results and Discussion
Synthesis of Silver Nanoparticle: The colour change from
pale yellow to reddish brown indicates the synthesis of silver
nanoparticles.
Optimization Studies
Effect of different parts of fruit and solvent for
extraction: To study the effect of solvent, aqueous and
solvent extraction was done. The colour change from pale
yellow to reddish brown was observed in pulp and whole
fruit of both aqueous and ethanol extract. The aqueous and
solvent extraction of pulp does not give reddish brown
colour. Aqueous extract of pulp gives dark reddish-brown
color than others which indicates more synthesis of silver
nanoparticle than other. The UV-Visible analysis of different
parts of fruit and solvent for extraction is done. The aqueous
extract of pulp shows more silver nanoparticle synthesis than
others.
Effect of different variety of fruit: The study on effect of
different samples was done using wild and normal variety.
From them, wild variety of Momordica charantia fruit gave
rapid colour change than normal variety Momordica
charantia fruit which indicates the synthesis of silver
nanoparticles. The UV-Visible spectral analysis was
performed in two different samples such as wild and normal
variety of Momordica charantia fruit pulp (figure 3).
The broad spectra in the wild variety of Momordica
charantia fruit pulp sample indicates more stable synthesis
of silver nanoparticles than normal variety of Momordica
charantia fruit pulp.
Figure 1: Before and after synthesis of silver
nanoparticle
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Effect of metal ion concentration: The study on effect of
metal ion concentration was done using different molarity of
silver nitrate solution such as 1mM, 2mM, 3mM, 4mM and
5mM. From them, 5mM gave dark reddish colour than other
thus indicating the synthesis of more silver nanoparticles.
The UV-Visible spectral analysis was done by varying the
morality of AgNO3 solution such as 1mM, 2mM, 3mM,
4mM and 5mM. The 5mM AgNO3 solution gives more
formation of silver nanoparticles than other (figure 4).
Effects of fruit extract concentration: The study on effect
of fruit extract concentration was done using different
concentration of fruit pulp extract such as 10%, 20%, 30%,
40% and 50% per 5ml AgNO3. 5ml plant extract gave dark
reddish brown colour than other concentration. The UV-
Visible spectral analysis was done for different
concentration of fruit extract. The 1ml plant extract shows
more formation of silver nanoparticles than other fruit
extract concentration (figure 5).
Effect of pH: The study on effect of pH is done by varying
different pH such as 2, 3, 4, 5, 6, 7, 8 and 9. The pH 5 showed
reddish brown colour which indicates that at pH 5, the
stability of formed silver nanoparticles is good. The UV-
Visible spectral analysis was done for varying different pH
for synthesis of silver nanoparticle (figure 6).
Effect of reaction time for silver nanoparticle synthesis:
The study on effect of reaction time for synthesis of silver
nanoparticle is done by varying different reaction such as 0
hr, 15 mins, 30 mins, 1 hr, 2 hrs, 24 hrs for synthesis of silver
nanoparticles. The UV-Visible spectral analysis was done
for varying different reaction time for synthesis of silver
nanoparticle (figure 7).
Characterization of Silver Nanoparticles
Scanning Electron Microscopy: The silver nanoparticles
synthesized using Momordica charantia fruit pulp extract
were scanned using Scanning electron microscopy. The
figure shows the SEM image of the silver nanoparticle
synthesized using Momordica charantia fruit pulp extract.
The size of silver nanoparticle is observed to be in the range
between 75nm-150nm (figure 8).
EDAX analysis: The silver nanoparticle was exhibited an
optical band peak at 3kev. This confirms the presence of
silver metal (figure 9).
Transmission Electron Microscopy: The size and
morphology of AgNP are studied using TEM. The shape of
AgNP is observed spherical and its size ranges between
50nm and 200nm (figure 10).
Fourier Transmission Infra-Red Spectroscopy (FTIR)
analysis of plant extract: The strong and broad stretch (O-
H bonded) at 3350-3000 cm-1 indicates the presence of
alcohol. The strong and broad stretch (H bonded) at 3350-
3000cm-1 reveals the presence of phenol. The symmetric
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strong stretch at 1380-1243.64cm-1 indicates the presence of
nitro compounds. The medium C-N stretch at 1155-
1053.82cm-1 reveals the presence of aliphatic amines. The
medium stretch (N-H) at 3350-3800cm-1 indicates the
presence of Secondary amines. The strong stretch (C=O) at
1755-1650.10 cm-1 indicates the presence of carboxylic
acids. The stretch (C=C) at 1650.10-1641 cm-1 reveals the
alkene presence. Thus, FTIR results indicate that the nitro
compounds, phenols, alcohols, aliphatic amines, alkenes
may be responsible for the formation of silver nanoparticles
(figure 11, table 1).
FTIR analysis of silver nanoparticle: The strong and broad
stretch at 3750-3424cm-1 indicates the presence of alcohol
(O-H bonded). The strong and broad stretch at 3750-
3424cm-1 reveals the presence of phenol (H bonded). The
medium N-H bend at 1635.86cm-1 indicates the presence of
Secondary amines. The strong C-N stretch at 1394.28-
1253cm-1 indicates the presence of aromatic amines. The
medium C-N stretch at 1201-1139cm-1 reveals the presence
of aliphatic amines (figure 12, table 2).
X-Ray Diffraction analysis: Using silver nanoparticle
synthesized using Momsordica charantia fruit pulp extract
XRD pattern was recorded. The crystalline sizes of AgNPs
are estimated using Scherrer’s equation. The diffraction
peaks are indexed as (111), (220), (311) planes of a pure face
centered crystalline (FCC) structure of silver (figure 13).
GC-MS Analysis: The major absorption peak was observed
with retention time of 3.725mins. The mass spectral analysis
showed the molecular ion intensity of 999 with a base peak
at m/z 45. The other peaks include 43, 57, 27, 29, 47, 28, 44,
15 and 46. From the computer library NIST, the suggested
phytochemical was 2, 3 Butanediol. The other peaks
observed at various retention time include 2.525 mins, 2.882
mins, 3.119 mins, 3.216 mins, 3.424 mins, 3.491 mins, 3.758
mins, 3.892 mins, 4.724 mins, 4.821 mins, 5.237 mins,
22.649 mins, 28.199 mins, 31.549 mins, 34.431mins.
The other phytochemicals suggested by NIST library were
tris(cyclopentadienyl-cobalt)-hexapropenylbenzene, 2,3
Butanediol, tetracetyl-d-xylonic nitrile,
dihydroxydimethylsilane, 1,2-cyclopentanedione, 2,4-di-
tert-butylphenol, 3-phenyl-2-butanol, methyl 2,5,5,8a-
tetramethyl-3-oxo-3,4,4a,5,6,7,8,8a-octahydro-1-
naphthalenecarboxylate, 7,9-Di-tert-butyl-1-oxaspiro(4,5)
deca-6,9-diene-2,8-dione, 1-Ethylhexanoic anhydride
(figure 14).

1.2 Aqueous Extract

1

0.8

0.6

0.4

0.2

0
350 370 390 410 430 450 470 490
Whole Fruit Pulp seed

2.5 Ethanol Extract

1.5

0.5

0
350 400 450 500

Whole Fruit Pulp seed

Figure 2: UV-Visible graphs of different parts of fruit and solvent for extraction.

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GC-MS analysis of silver nanoparticle: The major
absorption peak was observed with retention time of
3.127mins. The mass spectral analysis showed the molecular
ion intensity 999 with a base peak at m/z 59. The other peaks
include 31, 29, 27, 42, 59, 41, 60 and 39, 28, 26. From the
computer library NIST, the suggested chemical compound
was 1-propanol. The other peaks observed at various
retention times include 3.127mins, 3.327mins, 3.959mins,
4.0*03mins, 5.095mins, 8.364 mins, 8.795 mins, 22.226
mins, 32.797 mins and 33.250 mins. The other chemical
compounds suggested by NIST library were isobutyl
alcohol, fluorotriethylsilane, isopentyl alcohol, propanoic
acid,2-hydroxy-2-methyl-, ethyl ester, tetraethyl silicate,
Diethyl dimethyl orthosilicate, 2,4-Di-tert-butylphenol,
Indazol-4-one,3,6,6-trimethyl-1-phthalazin-1-yl-1,5,6,7-
tetrahydro-. The n-propanol serves as a starting material for
the manufacture of insecticides, herbicides and medicines. It
is also used as disinfectant, antiseptic and as mouth wash
solution, high blood pressure and blocking beta receptors
that play an important role in regulating a range of bodily
functions (figure 15).

1.7
1.5
1.3
1.1
0.9
0.7
0.5
350 400 450 500 550 600
Wild var Normal Var
Figure 3: UV-Visible graph of different variety of fruit

Figure 4: UV-Visible graph of metal ion concentration

1 Varying PE Conc. (5mM AgNo3).

0.8
0.6
0.4
0.2
0
300 350 400 450 500 550 600 650

1% PE 5% PE 15% PE 10% PE 20% PE

Figure 5: UV-Visible graph of fruit extract concentration

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Figure 6: UV-Visible graph of different pH

3.5
3
2.5
2
1.5
1
0.5
0
300 400 500 600 700

0hr 15mins 30mins

60mins 2hrs 24hrs

Figure 7: UV-Visible graph of reaction time for of silver nanoparticle synthesis

Figure 8: SEM micrograph of silver nanoparticle synthesized from Momordica charantia fruit pulp extract
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Figure 9: EDX image of silver nanoparticle synthesized from Momordica charantia fruit pulp extract

(a) (b)

(c)
Figure 10: TEM image of silver nanoparticle synthesized from Momordica charantia fruit pulp extract

Table 1
Fourier Transforms Infra-Red analysis of plant extract
Characteristic
Type of Vibration Intensity Functional Group
Absorption (cm-1)
3350-3000 Stretch (O-H bonded) Strong, broad Alcohol
3350-3000 Stretch (H bonded) Strong, broad phenol
1380-1243.64 Symmetric stretch (N-O) Medium Nitro compounds
1155-1053.82 Stretch (C-N) Medium Aliphatic amines
3350-3800 Stretch (N-H) Medium Secondary amines
1755-1650.10 Stretch (C=O) Strong Carboxylic acids
1650.10-1641 Stretch (C=C) - Alkenes

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Figure 11: Fourier Transforms Infra-Red analysis of plant extract

Figure 12: Fourier transform infra-red analysis of Silver Nanoparticle

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Figure 13: XRD spectra of silver nanoparticle synthesized from Momordica charantia fruit pulp extract.

Figure 14: GC-MS analysis of plant extract

Figure 15: GC-MS analysis of silver nanoparticle

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Table 2
Fourier Transform Infra-Red analysis of Silver Nanoparticle
Characteristic Absorption
Type of Vibration
(cm-1)
3750-3424 Stretch (O-H bonded)
3750-3424 Stretch (H bonded)
1635.86 N-H bend
1394.28-1253 C-N stretch
1201-1139 C-N stretch
Conclusion
The silver nanoparticle was successfully synthesized by
optimization of different varieties of fruit, solvent, parts of
fruit, plant extract concentration, metal ion concentration of
silver nitrate solution, pH, temperature, reaction time and
reaction condition. The characterization was carried out by
using UV-Visible spectrophotometer, scanning electron
microscopy (SEM), Transmission electron microscopy
(TEM), Fourier transform infrared (FTIR) spectroscopy,
Energy dispersive ray analysis (EDX) and X-ray Diffraction
(XRD) to study the structural, morphological and elemental
characteristics of synthesized silver nanoparticle.
The shape of synthesized silver nanoparticle is spherical and
size ranges between 75-150nm in SEM and between 50-
200nm in TEM. The GC-MS analysis of silver nanoparticle
results revealed the presence of phytochemicals over it.
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(Received 19th July 2017, accepted 10th November 2017)