J. Nig. Soc. Phys. Sci. 4 (2022) 835

Journal of the
Nigerian Society

of Physical
Sciences

Synthesis, Characterization and Antimicrobial Activities of
Copper-Tea Leaves (Camellia Sinensis) Extract Nanoparticles.

B. T. Iorhunaa,∗, T. T. Awuheb, I. C. Azuagaa, E Isaaca, F. Shuaibua, B. Yohannaa

a Chemical Sciences Department, Taraba State University, Jalingo, Taraba State, Nigeria.
b Science Department, Federal Polytechnic of Oil and Gas, Bonny Island, Rivers State, Nigeria.

Abstract

Nanoparticles, NPs synthesis has gained attention recently due to their ease of preparation (especially green synthesis), availability of raw materials
and usefulness. The green synthesis of copper nanoparticles (CuNPs) was done using Tea leaves extract-harvested from the Mambilla in Taraba
State-Nigeria. The phytochemical analysis of the tea leaves extract was done and found to contain phenols, steroids and saponins which could
have caused colour change, reduction of copper ions, capping and stabilisation of the synthesized CuNPs. The presence of NPs in the mixture was
identified by the change in colour of the mixture, λmax of the ultraviolet-visible spectrophotometry and spectra of the Fourier Transform Infrared,
FTIR spectrophotometry on the mixture. Antimicrobial studies of the synthesized CuNPs on the bacterial (Escherichia Coli) showed effective
toxic effects. This study showed that, Tea leaves extract is good for synthesizing copper nanoparticles that can be used as antimicrobial agents.

DOI:10.46481/jnsps.2022.835

Keywords: Nanoparticles, Characterisation, Antimicrobial, Copper, Tea Leaves.

Article History :
Received: 28 May 2022
Received in revised form: 19 July 2022
Accepted for publication: 27 July 2022
Published: 10 November 2022

c©2022 Journal of the Nigerian Society of Physical Sciences. All rights reserved.
Communicated by: B. J. Falaye

1. Introduction

Diseases causing organisms like fungi, bacteria, and
viruses which are resistant to many drugs have caused a lot
of issues Worldwide. This could be due to the unprescribed
use of antimicrobial drugs that have led to increasing cases of
drug resistance, thus bringing down the effectiveness of these
drugs when used for treatment. Therefore, new procedures
such as the development of alternative antimicrobial drugs or
modifying the existing drugs are being sorted with high interest
[1].

∗Corresponding author tel. no: +2347030479355
Email address: boniface.tersoo@tsuniversity.edu.ng (B. T.

Iorhuna )

Nanoparticles could occur naturally or they can be engi-
neered. They are very small in size and their particles sizes
vary between 1 - 100 nm. They are unique in their properties
and are highly valuable. Nanoscaled particles, display better
properties (such as; catalytic, magnetic, electrical, mechan-
ical, optical, chemical, biological, medical, etc). Because
these particles have high surface/volume ratio, they show
higher reactivity, mobility, dissolution properties, and strength
compare to other materials [2]. The design, synthesis and
manipulation of nanoparticles structure is an upcoming and
relevant field of research and technology [3]. Nanoparticles
obtained from metals have found a very good number of
applications in nature, they can be applied in many industries
(including catalysis) and medicine including; drug delivery,
cancer treatment, wastewater treatment and DNA analysis, as

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B. T. Iorhuna et al. / J. Nig. Soc. Phys. Sci. 4 (2022) 835 2

antibacterial agents, as biosensors, in solar power generation,
and many others [4].

Many methods (including chemical, physical and biolog-
ical) can be used in the synthesis of nanoparticles. Although
chemical method of synthesis takes a shorter period of time
to synthesize a large quantity of nanoparticles, this method
uses capping agents to stabilize the nanoparticles, and most
of these chemicals are toxic and can result to by-products
that are non-ecofriendly. Because we are all concerned with
the use of environmentally friendly synthetic approaches for
nanoparticles synthesis, this leads to the developing interest in
biological approaches which reduce the use of toxic chemicals
as by-products [5]. We shall prepare nanoparticles by green
synthesis of metallic nanoparticles using locally available
materials as a cost-effective and environmentally benign alter-
native to chemical and physical methods. Copper nanoparticles
(CuNPs) research has been of great interest in recent years due
to its applications in various fields of endeavour.

Tea leaves and many other plants (contain amino acids,
alkaloids, proteins, polysaccharides, phenolics, terpenoids,
flavones, and other active biomolecules) play important role
in nanoparticles synthesis, these plants’ phytochemicals
act as both reducing and capping agents [6]. Amongst the
biomolecules, phenols and flavonoids have unique chemical
properties and can reduce and wrap NPs. This could be because
of the presence of hydroxyl and carboxyl functional groups
that can bind to the metal [7].

Tea is a drink that is an infusion of processed leaves and
flowers of one of the varieties of an evergreen shrub botan-
ically called Camellia sinensis. Tea is a common beverage
that is drunk by many. Tea Leaves (Camellia Sinensis) is rich
in polyphenolic compounds, and has been reported as a nat-
ural source in the synthesis of nanoparticles [3]. Green Tea
is mainly composed by epigallocatechin, epigallocatechin-3-
gallate (EGCG), epicatechin and epicatechin-3-gallate which
act as reducing and capping agents [8, 9]. The green tea has
many medical benefits and the phytochemicals obtained from
Tea leaves have been used previously in cosmetic and thera-
peutic applications. The phytochemicals are mainly catechins
and caffeine which are water-soluble and can act as reducing
agents of metal ions, leading to the formation of capped metal-
lic nanoparticles [9].

2. Materials and Methods

2.1. Materials
Cu(NO3)2. 2.5H2O, NaOH and iron(III) chloride (LOBAAR

CHEMIE PVT Ltd), H2SO4 (JHDAR), Ethanol and iodine
solution (FenxichunAR), nutrients agar and acetic anhydrate
(QualikemsAR), deionized water, Tea Leaves (Camellia Sinen-
sis), Sieve (Cole Parmer-typed sieve of 0.80 mm mesh), pH me-
ter (Hanna Instrument H19024) , UV-visible (UV Apel 3000
UV USA) and FTIR spectrophotometers (Agilant Technologies
MicroLab PC).

2.2. Methods

2.2.1. Harvest and Preparation of Tea Leaves Extract
Fresh Tea Leaves were harvested around the premises

of Kakara Beverage of Sardauna Local Government Area of
Taraba State of Nigeria. It was dried at room temperature and
grounded into a powder form through sieve. The Tea Leaves ex-
tract was prepared by taking 10 g of grounded Tea Leaves and
dissolving it in a 500 mL beaker with the addition of 200 mL
of deionized water and stirring for 25 minutes, Figure 1. The
mixture was left to stand in a cupboard for 2 hours at room tem-
perature. The extraction was done by filtration using Whatman
filter paper [10].

2.2.2. Phytochemical Analysis of Tea Leaves Extract
Test for Steroids: this was done using the general proce-

dure; 5 mL of chloroform was added to 1 mL of the Tea Leaves
extract, then 5 mL of concentrated H2SO4 was then slowly
added. The upper layer turned red and the acid layer turned
yellow-green which showed the presence of steroids [11].

Test for Carbohydrates: to the fresh Tea Leaves Extract
were added a few drops of iodine solution, and no reaction was
observed indicating the absence of carbohydrates [10, 11].

Test for Flavonoids: these are tested according to this
method; to the 4 mL of the Tea Leaves Extract were added 1
mL of a 2 N NaOH(aq), no reaction was observed (no yellow
colour formation) indicating the absence of flavonoids [10, 11].

Test for Saponins: a test-tube containing 4 mL of aqueous
extract of Tea Leaves was vigorously shaken, and a 1 cm foam
layer was formed which indicated the presence of saponins
[11].

Test for Phenols: to a 2.5 mL of ethanol was added equal
volume of Tea Leaves extract and a few drops of iron(III) chlo-
ride. A yellow-greenish precipitate was formed indicating the
presence of phenols [10].

2.2.3. Synthesis of Copper Nanoparticles, CuNPs
Nanoparticles derived from the Tea Leaves extract were

synthesized using the methodology according to a previous
study with some modifications [10].

2.2.4. Characterization of the Tea Leaves Extract and CuNPs
The Tea Leaves Extract and CuNPs functional (chro-

mophore) groups were characterized by UV-visible absorption
and Fourier-Transform Infrared (FTIR) spectrophotometry. In
addition, the formation of the CuNPs was done with continuous
observation of the change in colour of the mixture [12].

2.2.5. Antibacterial Activity of the CuNPs
Agar disc-diffusion method was used to determine the an-

tibacterial activity of our prepared CuNPs against Escherichia
coli, a gram-negative bacteria. The microbial strain was ob-
tained from Federal Medical Center Jalingo, Taraba State, and

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B. T. Iorhuna et al. / J. Nig. Soc. Phys. Sci. 4 (2022) 835 3

Figure 1: (a) Fresh Tea Leaves, (b) Dried Tea Leaves and (c) Grounded Tea Leaves

was cultured in a nutrient broth for 24 hours to obtain a growth
phase of the strain bacteria. This actively growing bacterial cul-
ture was inoculated/spread into the Muller Hinton Agar (MHA).
The CuNPs solutions were prepared at varying concentrations;
0.031, 0.063, 0.125, 0.250 and 0.500 mg/mL (dissolved in
Dimethyl Sulfoxide, DMSO). The Whatman filter paper disks
were punched at 6 mm diameter and spread through with the
dissolved CuNPs and then placed to the MHA surface. Strepto-
mycin disc was used as a positive control while DMSO was
taken as the negative control. The plates were incubated at
37◦C for 34 and 48 hours. The effect of synthesized CuNPs
on the bacterial was evaluated in terms of zones of inhibition
measured, and recorded in millimeters using a ruler [10, 12].

3. Results and Discussion

3.1. Phytochemical Analysis of the Tea Leaves Extract
The phytochemical analysis of the Tea Leaves Extract is as

presented in Table 1.

Table 1: Phytochemical analysis of Tea Leaves Extract.

Phytochemicals Present Absent
Phenols ++
Steroids ++
Carbohydrates -
Anthocyanins ++
Flavonoids ++
Saponins -

Aqueous Tea Leaves Extract was analysed and the results
are presented in Table 1. This analysis showed phytochemicals
such as phenols, steroids, flavonoids, anthocyanins are present
in the Tea Leaves Extract while, carbohydrates and saponins
are absent in the Tea Leaves Extract. This is in line with the
branded; ’HighLand Tea’ (a product of Kakara Group of Com-
panies, Taraba State-Nigeria) which uses the Tea Leaves for
its products, and the other findings [13]. The phytochemicals
present in the Tea Leaves Extract could be responsible for the
reduction of Cu2+ ions and capping of the CuNPs.

3.2. Characterization of Tea Leaves Extract and CuNPs by UV-
visible Spectrophotometry

The interaction of NPs with wavelengths of light is depen-
dent on the size, shape and cluster state of NPs. Thus, UV-
visible spectroscopy is used for confirming the formation of
NPs [13]. In our synthesis, the reduction of Cu ions to CuNPs
by Tea Leaves Extract was observed by the change in colour of
the reaction mixture (from red to brown then darkened), Fig-
ure 2. The mixture (synthesized CuNPs) showed an absorption
peak at 400 nm (Figure 3a), confirming the presence of CuNPs,
which is in line with other works [12]. The shift in the ab-
sorption band from 440 nm of the Tea Leaves Extract (Figure
3b) to 400 nm of CuNPs (Figure 3a) further supports this claim
[1, 14].

Figure 2: Synthesis of copper nanoparticles.

The functional groups in the Tea Leaves Extract and CuNPs
were also analyzed by FTIR spectroscopy. The FTIR spectrum
of the Tea Leaves Extract (Figure 4) shows various peaks at;
3200 cm−1 (corresponding to O-H), 1540 and 1544 cm−1 (cor-
responding to C=C), while those at 1160 and 1087 cm−1 corre-
spond to stretching vibrations of hydroxyl and carboxyl func-
tional groups of phenols and carboxylic acids respectively. The
band at 1396 cm−1 corresponds to C−O−H bending [15, 16].
The FTIR spectrum of the synthesized CuNPs (Figure 5) on the
other hand shows a shift in some of the peaks and the resulting
peaks are narrower and less intense, because some of the active
compounds in the Extract took part in the reduction of Cu2+

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B. T. Iorhuna et al. / J. Nig. Soc. Phys. Sci. 4 (2022) 835 4

(a)

(b)

Figure 3: (a) UV-visible spectrum of synthesized copper nanoparticles, (b) UV-
visible spectrum of Tea Leaves extract.

Figure 4: FTIR spectrum of Tea Leaves extract

Figure 5: FTIR spectrum of synthesized copper nanoparticles

ions and capping of the CuNPs. The reduction and capping of
the CuNPs prepared could be due to the presence of phenols,

flavonoids, steroids or anthocyanins groups, which were seen
in the IR spectrum (Figure 4). Previous reports on Green syn-
thesis of NPs have shown the presence of these phytochemicals
[10].

3.3. Antibacterial Activity of the CuNPs

The antibacterial activity was done in terms of zone of
inhibition measured, and was recorded in millimeters using a
ruler as contained in Table 2.

Table 2: Antibacterial activity of CuNPs on Escherichia Coli.

Microorganism Nanoparticles
(mg/mL)

Sensitivity
(34hrs)
(mm)

Sensitivity
(48hrs)
(mm)

Escherichia
Coli

0.500 5.00 7.00

0.250 2.00 4.00
0.125 0.00 1.50
0.063 0.00 0.00
0.031 0.00 0.00

Bacterial strain, Escherichia coli was used to study the an-
timicrobial activity of the CuNPs obtained from Tea Leaves Ex-
tract. Table 2 presents the antibacterial activity of the CuNPs
on the bacteria in a well diffusion assay. The results showed
that CuNPs have very good antibacterial activity against Es-
cherichia coli. The inhibition zone diameter is related to the
magnitude of activity of CuNPs on microbes. It was observed
that, the CuNPs exhibited antibacterial activity at a concentra-
tion similar to that of the reference drug (streptomycin). The
effects of the CuNPs against bacterial could be due to the dual
activity nature of the nano-sized copper and the bioactive agents
of the Tea Leaves Extract on the surface of the CuNPs [17, 18].

4. Conclusion

Due to the phytochemicals present in Tea Leaves (Camellia
Sinensis) Extract; phenols, steroids, flavonoids, anthocyanins
etc, which are responsible for the reduction and capping of
CuNPs, it use in the synthesis of CuNPs has been developed.
Tea Leaves were used in producing copper nanoparticles. The
CuNPs synthesized were characterized by UV-visible and FTIR
spectrophotometry. The formation of CuNPs was observed by
colour change from red to brown (and darkened). The synthe-
sized CuNPs displayed excellent antibacterial activity against
Escherichia coli. The bioreduction process of the CuNPs is
an economic and ecofriendly, simple one-step method, and this
green synthetic protocol would be a better alternative to the ex-
isting methods. The use of Camellia Sinensis in the synthe-
sis of NPs could also expand its market beyond mere Tea pro-
duction/consumption, providing a means of livelihood for the
Mambilla people of Taraba State, of Nigeria.

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B. T. Iorhuna et al. / J. Nig. Soc. Phys. Sci. 4 (2022) 835 5

Acknowledgements

We greatly appreciate the Editor in Chief of this Journal for
his patience and encouragement

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