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Pak. J. Anal. Environ. Chem. Vol. 19, No. 2 (2018) 122 – 127

http://doi.org/10.21743/pjaec/2018.12.13

Synthesis of Copper Nanoparticles via Trigonella Foenum-
Graecum Seed Extract for Antibacterial Response

Tahira Moeen Khan
*
, Amat ul Mateen and Bushra Khan

Department of Chemistry, Lahore College for Women University, Jail Road, Lahore, Pakistan.
*Corresponding Author Email: tahira.moeen@lcwu.edu.pk

Received 02 April 2018, Revised 16 October 2018, Accepted 20 October 2018

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Abstract
In view of the immense capability of plants this work is planned to employ seed extract as a source
for the reduction of Cu ions in to Cu nanoparticles (Cu NPs). For this purpose seed extract of
Trigonella foenum-graecum (fenugreek seeds) was utilized as a substitute of classical methods.
This green path for synthesizing Cu NPS is easy, natural, low cost, sustainable and eco-friendly as
compared to conventional methods. In this experiment harmful chemical/physical methods for the
production of Cu nanoparticles is replaced by using minimum concentration of seed extract. The
stepwise characterization was done by using Atomic Absorption spectroscopy, UV-Vis
Spectroscopy, FTIR spectrophotometer and X-ray diffraction (XRD) which have given much
valuable information about these materials. Antibacterial activity of these nanoparticles is observed
at different concentration so their ZOI (Zone of inhibition) and MIC (minimum inhibitory
Concentration) was also calculated against four human pathogenic strains.

Keywords: Nanoparticles, Synthesis, Seed extract, FTIR, XRD, Bacterial culture.
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Introduction

Nanotechnology has achieved a prominent place in
today’s research. With reducing size (1-100 nm)
nanoparticles possess a higher surface to volume
ratio which is significant for catalytic reactivity
and other similar properties such as antimicrobial
activity in nanoparticles [1]. Nanoparticles can be
prepared by either physical methods including
pulsed laser ablation [2], vacuum vapor deposition
[3], pulsed wire discharge [4], mechanical milling
[5] and chemical methods involving chemical
reduction [6], micro emulsion techniques [7],
sonochemical reduction [8], electrochemical [9],
microwave assisted [10] and hydrothermal methods
[11].

he usage of lethal chemicals and by
products with in these methods make the
nanoparticles synthesis more challenging and
problematic. Moreover, these methods also
demand high energy and uneconomical
purifications. Green synthesis provides

advancement over both chemical and physical
methods as it is economical, eco-friendly, capable
for large scale synthesis and does not require
consumption of harmful chemicals, high energy,
temperature and elevated pressure [12]. Presently
green chemistry involve the biosynthesis through
microorganism and plant extracts [13, 14]. By
using natural extract of Aspalathus Linearis
crystalline perovskite ZnSnO3 nanoclusters, NiO,
Pd & pdO nanoparticles are biosynthesized
[15,16]. Sageretia thea & Moringa Oleifera natural
extracts are used as chelating agent to prepare ZnO
nanoparticle [17,18 ].

Copper nanoparticles exist in three forms
i.e. Cu metal nanoparticle, Cu(I) oxide
nanoparticle [Cu2O] and Cu(II) oxide nanoparticle
[CuO].These have catalytic [19], optical [20] &
sensing activities [21]. They are also helpful as
resistance materials [22], inorganic-organic
nanocomposite [23] & in solar cells [24]. Metal



Pak. J. Anal. Environ. Chem. Vol. 19, No. 2 (2018) 123

nanoparticles can show a good antibacterial
response. This antibacterial activity of Cu may be
accredited to their micron range size less than the
pore size of the bacteria and thus, they are capable
of easily crossing the cell membrane without any
hindrance [25].

Fenugreek, Trigonella foenum-graecum
(Greek hay) is an annual herb of the pea family
(Fabaceae) cultivated in North Africa, the Middle
East and Asia. It is used mostly as ingredient in
traditional medicine in Egypt, India and Asia.
Fenugreek helps to heal inflammation and its
extracts can be added in manufacturing cosmetics
and also in soaps due to its long history as anti-
tumorigenic, antioxidant, antidiabetic, and
antimicrobial activities. Its seeds Extract help
control in digestion, arthritis, diabetes, cholesterol,
cancer, heart attack, improvement in breast milk,
kidney and liver function [26, 27].

his research work aims to throw light on
the bio reduction of Cu

+2
ions into Cu

0

nanoparticles by employing aqueous Fenugreek
seeds extract under varied percentage compositions
and there by implementing eco-friendly chemistry
for the future research. To the best of our
knowledge this green synthesis of Copper
nanoparticles by using aqueous extract of
Fenugreek seeds is a novel research and has not
been reported earlier.

Experimental

Addition of the aqueous plant extract into
a solution of the appropriate metal salt initiates a
plant extract-facilitated green reduction. The
preparation of NPs takes place at room temperature
and completes within a few minutes.

Seed extract preparation

They were thoroughly washed with
distilled water, dried and crushed. The powder was
further used for preparation of 1 g/100 mL aqueous
seeds extract. This extract was boiled for 30
minutes approx. at 80-90ºC, shaked (5 c/s for 10
minutes) & filtered to obtain homogenized solution
which was kept at 4°C in 250 mL Erlenmeyer
flask. In each and every step of the experiment,

sterility conditions were maintained for the
efficacy and accuracy in results without any
impurity.

Biosynthesis of nanoparticles

For biosynthesis of nanoparticles, 2.0 mL
of the seeds extract was mixed with 20 µL of
freshly prepared 1×10

-2
M aqueous copper sulphate

pentahydrate solution in 250 mL Erlenmeyer flask
under continuous magnetic stirring. The Cu
nanoparticles obtained were purified by repeated
centrifugation method at 6,000rpm for 40 min
followed by dispersion of the pellet in deionized
water. Later the Cu nanoparticles were dried in an
oven at 90-100 ºC for 3-4 hours [28].

Table 1 shows the preparation of different
concentrations by varying the amount of seed
extract and keeping the concentration of
CuSO4.5H2O constant i.e., 1×10

-2
M.

Table 1. Procedure for the preparation of different concentrations

Concentration Procedure

5% 5mL of the seed extract (Fenugreek) was mixed
with 95mL of the 0.01M CuSO4 Solution.

15% 15mL of the seed extract was mixed with 85mL
of the 0.01M CuSO4 Solution.

25% 25mL of the seed extract was mixed with 75mL
of the 0.01M CuSO4 Solution.

Procedure for Antibacterial Activity

For antibacterial activity agar well
diffusion method was employed [25]. In each well
50uL of the sample was dispensed . Four bacterial
strains were selected including Pseudomonas
aeruginosa (ATCC 27853), Proteus mirabilis
(ATCC 7002), Agrobacterium tumefaciens (ATCC
5577) and Bacillus subtilis (ATCC 6051). For each
strain different concentrations 5%, 15% & 25%
was prepared and also measured the zone of
inhibition (ZOI) of each in mm [29].

Biosynthesized nanoparticles characterization

1mL of the colloidal solution of prepared
Cu NPS was diluted with 10 mL of the distilled
water and then this diluted solution was aspirated



Pak. J. Anal. Environ. Chem. Vol. 19, No. 2 (2018)124

into the atomic absorption spectrophotometer
through capillary tube to confirm the NPs
synthesis. UV-Vis spectral analysis was also done
between 200-700 nm. FTIR spectral studies was
done on Shimadzu IRTracer-100, fourier transform
infrared spectrometer between 4000 and 375 cm

-1

with resolution of 4 cm
-1

. The powder X-ray
diffraction was conducted on a Philips Analytical
XPERT diffractometer using a Cu Kα radiation (λ 
= 1.540598 °A) with a MINIPROP detector and
operating at 40 kV generator voltage and 40 mA
generator current. X-ray diffraction patterns were
recorded between 2θ = 5◦ and 79◦ with a step of 
0.04◦ and a time of 0.2 s by step.

Results and Discussion
Atomic absorption spectrometry

The presence of copper nanoparticles was
confirmed by concentration-absorption linear
relationship. Fig. 1 shows the increase in
absorption with the growing concentration of Cu
nanoparticles with gradual increase of seed extract
concentration.

Figure 1. Effect of the Fenugreek seed extract percentage
composition on the synthesis of Cu NPs.

Visual observation and UV-Vis spectroscopy

Reduction of Cu
+2

into Cu
0

during addition
of fenugreek seeds extract is monitored as a
consequence of the change in colour and thus can
be studied through UV-Vis spectroscopy. The
change in colour is because of the Surface Plasmon
Resonance (SPR). The SPR band was observed at
a maximum absorbance of approximately 560 nm
which was confirmed from the literature value
[30].

Different parameters were optimized
including concentration of seeds extract and
reaction time. It was identified that the yields of
copper nanoparticles were affected by varying
these parameters.

Effect of contact time at room temperature

Increasing the reaction time results in
gradual increase of absorbance which was
quantitatively monitored (Fig. 2). Furthermore, the
color intensity also increases with the duration of
incubation from faint blue to yellow brown and
then to deep brown as shown in (Fig. 3). This
increase in absorbance and color intensity is
attributed to the higher concentration of Cu NPs.
[31].

1.41

1.58

3.44

4.43

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

2 6 24 48

Time (hours)

C
o

n
c
en

tr
a

ti
o
n

o
f

C
u

N
P

s
(p

p
m

)

Figure 2. Cu NPs concentration at different time intervals with
15% seed extract added to 0.01M CuSO4 Solution

a b c d

A: 0.01M CuSO4 Solution
B: Aqueous seed extract
C: Addition of seeds extract into 0.01M CuSO4 Solution
D: Nanoparticles emulsion after 24 hrs.

Figure 3. Formation of Nps with colour change.

Increase in absorption intensity

As the concentration of the fenugreek seeds extract
increases, the absorption peak gets more sharpness
with slight increase in wavelength. If we increase
the extract concentration from 5% to 25%, there is

C
o

n
ce

n
tr

a
ti

o
n

o
f

C
u

N
P

s



Pak. J. Anal. Environ. Chem. Vol. 19, No. 2 (2018) 125

increase in intensity of absorption as presented in
Fig. 4.

0

0.05

0.1

0.15

0.2

0.25

0.3

7
0

0
6

7
8

6
5

6

6
3

4
6

1
2

5
9

0
5

6
8

5
4

6
5

2
4

5
0

2
4

8
0

6
8

8

6
6

6
6

4
4

6
2

2
6

0
0

5
7

8
5

5
6

5
3

4
5

1
2

4
9

0

6
9

8
6

7
6

6
5

4
6

3
2

6
1

0
5

8
8

5
6

6
5

4
4

5
2

2

5
0

0
4

7
8

Wavelength (nm)

5% 15%
25%

A
b

so
rb

a
n

c
e

Figure 4. For 5% seed extract absorbance is observed at 536nm
and with 25% at 560 nm

Fourier transform infrared spectroscopy (FTIR)

Fig. 5 shows FTIR spectrum of Cu
nanoparticles from Fenugreek seeds extract. A
wide band at 3358 cm

-1
is due to the OH group

indicating the presence of intermolecular hydrogen
bonding. A peak at 2358 cm

-1
is observed which

can be attributed to C≡C stretching vibrations i.e.
alkyne group present in phyto constituents of
extract. A band at about 1645 cm

-1
is due to C=O

stretching vibrations associated with amide I
functional group. The amide I band is directly
linked to the backbone conformation indicating the
presence of proteins in seeds extract [32].

Figure 5. FTIR spectrum of Cu nanoparticles synthesized from
Fenugreek seeds extract

X- Ray diffraction studies

The results of XRD pattern analysis
revealed three intense peaks in the whole spectrum

of 2 theta values ranging from 20 to 80 for the Cu
nanoparticles. The sample demonstrated a high
crystallinity level with diffraction angles of
28.86, 32.14 and 46.55 which correspond to the
characteristic of face-centred cubic of copper lines
indexed at (210), (111), and (222) Fig. 6. Peaks
for Cu Nps are compared with JCPDS literature.
Cu nanoparticles with different sizes were
obtained. By applying Debye–Scherrer equation to
the obtained XRD pattern of the Cu NPs, the
average nanoparticles size was found to be 14-17
nm, which may indicate a high surface area-to-
volume ratio of nanocrystals.Cu nanoparticles are
easily prone to oxidation and results in the
formation of CuO and Cu2O nanoparticles as
shown below [33].

2 0 3 0 4 0 5 0 6 0
0

5 0

1 0 0

1 5 0

2 0 0

2 5 0

3 0 0

in
te

n
s
it
y

(c
o

u
n

ts
)

A n g le ( 2 th e ta )

i n t e n s it y

d e m o d e m o d e m o d e m o d e m o

d e m o d e m o d e m o d e m o d e m o

d e m o d e m o d e m o d e m o d e m o

d e m o d e m o d e m o d e m o d e m o

d e m o d e m o d e m o d e m o d e m o

d e m o d e m o d e m o d e m o d e m o

d e m o d e m o d e m o d e m o d e m o

Figure 6. XRD plot of Cu NPs synthesized from fenugreek seeds
extract

Cu
+2

+ 2e- → Cu
0

+ Air (O2) → CuO/ Cu2O

But by using fenugreek seed extract as
reductant Cu NPs were not oxidized to CuO NPs.
Thus, from these observations it may be concluded
that fenugreek seed extract is not only an excellent
reducing agent but also a strong capping agent that
it coats the surface of Cu NPs avoiding its contact
with the air and hence no oxidation took place over
the surface of synthesized NPs.

Determination of MIC (minimum inhibitory
concentration)

Four bacterial cultures including Bacillus
subtilis (one Gram positive), Pseudomonas
aeruginosa, Proteus mirabilis, Agrobacterium
tumefaciens(three Gram negative bacteria) were



Pak. J. Anal. Environ. Chem. Vol. 19, No. 2 (2018)126

used to check the antibacterial potential of Cu
nanoparticles (5% to 25% concentration)
synthesized from fenugreek seed extract.
Ciprofloxacin was used as control against Gram
positive bacterium while amoxicillin for Gram
negative bacteria. 25% (Max) concentration give
maximum antibacterial potential i.e., 10 mm zone
of inhibition for Bacillus subtilis, 5 mm for
Pseudomonas aeruginosa, 9 mm for Proteus
mirabilis and 10 mm for Agrobacterium
tumefaciens Table 2. With 5% concentration which
is Minimum Inhibitary Concentration (MIC) 5 mm
ZOI was observed for Bacillus subtilis, 2 mm for
Pseudomonas aeruginosa, 3 mm for Proteus
mirabilis and 5 mm for Agrobacterium tumefaciens
Fig. 7.

a

c

b

d

A = Bacillus subtilis B = Pseudomonas aeruginosa
C = Proteus mirabilis D = Agrobacterium tumefaciens.

Figure 7. Observed antibacterial activities against four human
pathogenic strains

Conclusion

In this research a simple, convenient, cost
effective, significant and eco-friendly method for
the synthesis of copper nanoparticles through the
reduction of copper salts has been demonstrated.
Aqueous seed extract of fenugreek is used as
reducing and capping agent. Characterization of
Cu nanoparticles was done by AAS, UV
spectroscopy, FTIR spectroscopy and XRD
analysis. These nanoparticles were evaluated for

their antibacterial activity against one gram
positive and three gram negative bacterial strains.
These biologically synthesized copper
nanoparticles using seed extract showed sufficient
antibacterial potential. The minimum inhibitory
concentration of nanoparticles synthesized from
aqueous seed extract of fenugreek was 5%. In the
concluding remarks, the copper nanoparticles
synthesized using fenugreek seed extract would be
a better antimicrobial effective against various
bacterial species.

Acknowledgement

All this research is done and supported by
Lahore College for Women University Jail Road
Lahore, Pakistan.

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