Hindawi
International Journal of Biomaterials
Volume 2022, Article ID 4106558, 9 pages
https://doi.org/10.1155/2022/4106558
Research Article
Monodispersed AgNPs Synthesized from the Nanofactories of
Theobroma cacao (Cocoa) Leaves and Pod Husk and Their
Antimicrobial Activity
Johnson Kwame Efavi ,1 Emmanuel Nyankson ,1 Kwaku Kyeremeh,2
Gloria Pokuaa Manu ,1 Kingsford Asare,1 and Nathaniel Yeboah1
1College of Basic and Applied Sciences, Department of Materials Science & Engineering, University of Ghana, Accra, Ghana
2College of Basic and Applied Sciences, Department of Chemistry, University of Ghana, Accra, Ghana
Correspondence should be addressed to Emmanuel Nyankson; enyankson@ug.edu.gh
Received 7 September 2021; Revised 7 November 2021; Accepted 21 December 2021; Published 2 February 2022
Academic Editor: Fu-Gen Wu
Copyright © 2022 Johnson Kwame Efavi et al. -is is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.
Silver nanoparticles (AgNPs) have been synthesized from the more chemically rich and diverse cocoa pod; the synthesis of silver
nanoparticles from cocoa leaves, which are less rich and have low diversity in bioactive molecules, is yet to be achieved. In this
work, AgNPs produced using the extracts of the cocoa leaf (CL) and cocoa pods (CP) have been investigated and their anti-
microbial activity against E. coli was evaluated. UV-visible absorption spectroscopy was used to examine the reduction of silver
ions in solution and the surface plasmon resonance of AgNPs. Scanning electron microscopy (SEM), energy-dispersive X-ray
spectroscopy (EDX), dynamic light scattering (DLS), X-ray diffraction (XRD), and Fourier transform infrared spectroscopy
(FTIR) were used to further characterize the nanoparticles.-e crystalline nature of AgNPs was confirmed by XRD, and the purity
and presence of elemental silver were determined by EDX. CL-AgNPs were observed to have a surface plasmon resonance of
425 nm, while CP-AgNPs had a surface plasmon resonance of 440 nm. CL-AgNPs had a significantly higher purity than CP-
AgNPs. With a shorter nucleation time, the intensity of the UV-Vis spectrum was always higher in the case of CL-AgNPs,
indicating a larger population of bioactive molecules available for CL-AgNPs synthesis. FTIR confirmed the presence of phenolic
compounds in the leaf and pod extract, implying that water-soluble polyphenolic and flavonoid chemicals are responsible for
nanoparticle reduction, capping, and stability. AgNPs generated from CL and CP extracts are polydispersed, with particle sizes of
10–110 nm and 20–680 nm, respectively, according to DLS. -e corresponding zeta potentials measured are −2.7mV for CL-
AgNPs and −0.93mV for CP-AgNPs. -e zeta potential values suggest that the particles have long-term stability. Furthermore,
CL-AgNPs outperformed CP-AgNPs in terms of antibacterial activity against Escherichia coli. CL-AgNPs were found to have a
maximal inhibitory zone of 21mm.
1. Introduction over a century and are now widely used in biomedical
sciences and engineering. -e efficacy of these nanometer
-e exploitation of quantum confinements and increased materials is size-, shape-, and concentration-dependent,
surface area [1] at the nanometer materials regime are at the making them appealing for biomedical applications [3].
forefront of the current and next-generation technologies to -ese materials can be synthesized and modified with a
solve some of the pressing needs of mankind in the area of variety of chemical functional groups, allowing them to be
energy, environment, food security, medicine, shelter, water, covalently linked with ligands, antibodies, and drugs of
and cybercrime [2]. interest, opening up a wide range of potential applications in
Metallic and metal composite nanoparticles, among magnetic separation, biotechnology, and preconcentration
several kinds of nanomaterials, have intrigued scientists for of target analytes, targeted drug delivery, and vehicles for
2 International Journal of Biomaterials
gene and more importantly diagnostic applications [4, 5]. molecule profiles and will thus produce different charac-
For example, the antibacterial activity of various metal teristics of nanoparticles that can be exploited in diverse
nanoparticles, such as silver colloids, is inversely propor- biomedical applications. In this work, AgNPs have been
tional to their size; the smaller the silver nuclei, the greater synthesized from the extract of the cocoa leaf (CL) and cocoa
the antibacterial activity. Furthermore, these nanoparticles’ pod (CP) and their characteristic features evaluated using
catalytic activity is influenced by their size, structure, shape, UV-Vis, XRD, FTIR, EDX/SEM, and DLS. -e biological
size distribution, and chemical-physical environment. As a activities of the nanoparticles have also been observed using
result, maintaining control over the size and size distribution E. coli in a disc diffusion method for antimicrobial testing.
is critical.
Electrochemical techniques, chemical reduction and 2. Materials and Methods
photochemical reduction, plasma arcing, ball milling, spray
pyrolysis, ultrathin films, thermal evaporate, pulsed laser 2.1. Materials. Silver nitrate was procured from Sigma-
desorption, lithographic techniques, sputter deposition, Aldrich of UK. Fresh "eobroma cacao (Cocoa) fruits and
layer-by-layer growth, diffusion flame methods, molecular leaves (see Figure S1 in the Supplementary Material) were
beam epistaxis, and other physical and chemical methods harvested from a farm in Akropong, Eastern Region, Ghana,
can all be used to synthesize and stabilize metal nano- and transported to the laboratory for further processing.
particles [6, 7]. -e experimental conditions, the kinetics of
metal ions interaction with chemical reducing agents, and
the adsorption processes of stabilizing agents have all been 2.2. Processing of Cocoa Husk Pod and Leaves into Liquid
shown to have a significant impact on the size, texture, Extract. -e cocoa pods and leaves were first washed and
stability, and properties of chemically/physically synthesized rinsed to remove any undesired foreign substances. To
metal nanoparticles [8, 9]. Furthermore, because current extract the beans from the pod, the cocoa pod was sliced and
nanoparticle processing methods are costly, the develop- opened. -e pod was chopped into pieces and air-dried for
ment of a synthesis approach that allows for precise control two weeks at room temperature. -e leaves were also air-
of size, shape, stability, and characteristics has been a major dried for one week. -e dried pod and leaves were then
focus. ground in a Philips electric blender to obtain fine powder
Synthesis of metal nanoparticles from biological sources following a similar standard method in other works [15].
(plant and microbe sources) has become a research area of About 15 g of the powdered (cocoa pod and cocoa leave
interest [8, 10]. -ese plants and microorganisms have powder) samples was weighed and transferred into beakers
bioactive compounds that can be used as a reducing and containing ca.100mL of distilled water. -e mixture was
stabilizing agent in one-pot nanoparticle production. boiled for about 10 minutes and cooled to ca. 25°C. -e
However, due to the difficulty in maintaining microbial filtrate extract was collected after the mixture was filtered
cultures [10] and the advantages plants provide in terms of twice with Whatman No.1 filter sheets. -e fresh plant
resource availability, security, reaction rate and convenience, extracts were stored in a refrigerator in 250mL conical flasks
and feasibility of the large-scale production, the synthesis of and used for further experiments within 24 hours.
nanoparticles from plant extract is preferred.
Numerous studies have been published on the utilization 2.3. Green Synthesis of Cocoa Pod and Cocoa Leaf AgNPs.
of plant extracts in the manufacture of noble metal nano- A 1mM aqueous solution of silver nitrate (AgNO ) was
particles, particularly silver nanoparticles (AgNPs). -is 3prepared for each plant extract obtained by measuring a
interest in plant extract methods stems from the ease of calculated mass of silver nitrate salts into a beaker. Distilled
processing, low cost, and environmentally friendly proce- water from a wash bottle was then added and stirred con-
dures that produce nanoparticles with unique properties that tinuously until it dissolved. -e solution was then trans-
can be used in biomedicine, fiber technology, electronics, ferred to a 250mL volumetric flask and diluted with more
food preservation, cosmetics, and other fields [11–13]. distilled water. In an Erlenmeyer flask, 200mL of 1mM
-e significance of landforms and rock types in estab- silver nitrate was measured, and 20mL of plant extract was
lishing unique regional distributions of plant ecosystems and added to the contents of the flask. -e solution was then
promoting evolutionary diversification is influenced by the monitored for colour change characteristic of silver nano-
geology of a given location [14]. It is also known that particle formation (reddish-brown, dark brown) caused by
ecosystems and habitats have a complicated interaction excitation of surface plasmon vibrations [16].
between physical and biological components, implying that
microorganisms responsible for the reduction and stabili-
zation of AgNPs during synthesis may differ depending on 2.4. Characterization of AgNPs: UV-Vis, XRD, FTIR, SEM,
soil type and environment. EDX, and DLS. UV-Vis spectrophotometry was used to
-e geological settings in Ghana are unique and different monitor the completion of silver ion bioreduction. -at is,
from places where AgNPs have already been synthesized 1mL samples of silver nitrate and plant extract solution
from plant parts; therefore, the bioactive molecular make-up mixture were taken at regular intervals, diluted with 2mL
of plant parts in such regions is expected to differ. -erefore, deionized water, and absorbance was measured using UV-
we hypothesize that the plant parts of cocoa in Ghana have Vis spectrophotometry while looking for a characteristic
different concentrations or proportions of bioactive peak of silver nanoparticles. In a GENESYS 10S UV-Vis
International Journal of Biomaterials 3
(version v4.005 2L5S048209) scanner, the sample was absorbance peaks of silver nanoparticles occur [19].
scanned between 200 and 700 nm wavelengths. Powder Figure 1(a) shows the absorption spectra of silver nano-
X-ray Diffractometer (XRD) patterns recorded using an particles formed from cocoa leaves broth, and Figure 1(b)
Empyrean PANalytical series 2 XRD with CuKα (1.54 Ǻ) shows cocoa pod broth. It can be observed that in both cases,
radiation source and a tube running at 40mA and 40 kV there was a steady increase in intensity with reaction time.
were used to determine the phase purity and crystallinity of Because the SPR band intensity and wavelength are
AgNPs. X’Pert Highscore plus database software was used to affected by factors that influence the electron charge density
identify the phases present in the samples. Energy-dispersive on the particle surface, such as metal type and structure,
X-ray spectroscopy (EDX)-Zeiss EVO LS10 scanning elec- particle size and shape, composition, and the dielectric
tron microscopy (SEM) with Oxford INCA X-act detector constant of the surrounding medium, as theoretically de-
was used to evaluate structural morphology and empirical scribed by Mie theory, the difference in the observed ab-
elemental compositions, chemical purity, and stoichiometry. sorption peak is expected [20, 21]. -is suggests that the
Using a Malvern Instrument ZEN 3600 Zetasizer Nano-ZX, kinetics of the CL-AgNPs and CP-AgNPs formation and the
the AgNPs were further characterized using dynamic light number of nanoparticles produced are different. It is also
scattering (DLS) and zeta potential to quantify the volume- observed that the absorption peaks did not shift to different
weighted hydrodynamic size and zeta potential, respectively. wavelengths as the reaction time changed for both leaves and
-e measurement temperature was kept constant at 25°C. cocoa pods, demonstrating that the as-synthesized silver
nanoparticles are uniformly distributed and stable with the
same properties [21].
2.5. Antibacterial Activity Test. Silver nanoparticles made XRD analysis was used to examine the phases present in
from cocoa leaves and those made from cocoa pods were the synthesized CL-AgNPs and CP-AgNPs. Figure 2 shows
tested for antibacterial activity against Escherichia coli the patterns of AgNPs formed from the broth of cocoa leaves
(E. coli). -e bacteria for this test were obtained from the and pods superimposed. Peaks were observed at 2θ� 38.18°,
University of Ghana’s Noguchi Memorial Institute utilizing 42.32°, 64.43°, 77.44°, and 81.52° for both CL-AgNPs and CP-
the disc diffusion methods outlined in [17]. AgNPs. -ese peaks are attributed to crystallographic planes
Small disks cut out from filter paper were soaked in silver (111), (200), (220), (311), and (222), which correspond to
nanoparticle solutions of the cocoa extracts. Filter papers characteristic Bragg reflections in silver [22].-e observance
were also soaked in distilled water, aqueous silver nitrate, of characteristic Bragg diffraction peaks in the silver
and plant extracts to serve as controls. -e filter papers were nanoparticles from both sources of extract confirms the
then air-dried. Plates containing E. coli inoculummixed with crystalline morphology of the nanoparticles. -ere is no
nutrient agar were then prepared. -e plates were then difference in the crystallographic planes responsible for the
partitioned for placements of filter paper. On one type of observed diffractions except that the intensity of the spectra
plate, 4 filter papers from distilled water treatment, AgNPs of CL-AgNPs is higher, indicating increased levels of
from cocoa leaves, and pod and AgNPs from chemical crystallinity.
synthesis were placed. On another type of plate (control It is reported that during the production of nanoparticles
plate), filter paper treatments of distilled water, cocoa plant from nanofactories of plant extract, AgNO3 dissociate into
extracts, and aqueous silver nitrate treatments were placed. Ag⁺ and NO3‾ in the aqueous medium and that the plant
In a growth chamber at 37°C, the plates were incubated for extracts contain a high level of bioactive molecules that acts
48 hours. For each type of filter paper treatment, the zones of as both reducing and stabilizing agents. -e plant extract
inhibition established after 48 hours were evaluated. releases bioactive compounds, which react with the aqueous
AgNO3 solution, forming a bioactive substrate complex as
+
3. Results and Discussion Ag ions join with the bioactive molecules. -e functional
groups released by the bioactive molecules interact with the
Studies have shown that metal nanoparticles have a strong silver ions, resulting in the formation of AgNP. -e silver
absorption band and produce a distinctive colloidal sus- nanoparticle then reacts with proteins produced by the plant
pension colour due to surface plasmon resonance (SPR) [18]. extract, resulting in protein-capped silver nanoparticles [21].
-e reaction of aqueous extracts of cocoa leaves and pods Plant extracts and AgNPs generated were analyzed using
with an aqueous solution of silver nitrate resulted in the Fourier transform infrared spectroscopy (FTIR) to detect the
bioreduction of silver ions to silver. -e extracts changed functional groups present that could be responsible for the
colour from pale yellow to dark brown (see Figures S2 (a and nanoparticles’ reduction and efficient capping and stability.
b, respectively) in the Supplementary material), showing the Figures 3(a) and 3(b) show the superimposition of FTIR
production of silver nanoparticles in the colloidal suspen- spectra of CL extract and the CL-AgNP spectra and that of
sion. -is colour change in the suspension confirms the CP extract and the corresponding CP-AgNPs, respectively.
formation of AgNPs as a consequence of surface plasmon -e spectra reveal strong absorption bands at 3338, 2123,
vibrations of AgNPs being excited [18, 19]. 1634, and 571 cm−1. For CL extract, the 3338 cm−1 band is
-e formation of AgNPs from both cocoa leaf and cocoa associated with hydroxyl (OH) groups, which consist of
pod extracts was confirmed using UV-Vis spectroscopy at alcohol and phenolic compounds with hydroxyl bonds [23].
absorbance peak wavelengths of 425 nm and 440 nm, re- -e 2123 cm−1 band is attributed to triple bond mono-
spectively. -is is consistent with the range at which substituted alkyne [23]. Due to aromatic ring deformation,
4 International Journal of Biomaterials
UV-Vis Absorption Spectra of CL-AgNPs UV-Vis Spectra of CL-AgNPs
3.5 2.5
24 h 24 h
3.0
2.0
2.5
1.5
2.0 4 h
1.5 1.0
2 h 4 h
1.0
0.5
0.5 2 h
0.0 0.0
400 500 600 700 400 500 600 700
Wavelength (nm) Wavelength (nm)
2 h 2 h
4 h 4 h
24 h 24 h
(a) (b)
Figure 1: (a) UV-Vis absorption spectra of CL-AgNPs. (b) UV-vis absorption spectra of CP-AgNPs.
XRD Spectra of CL-AgNps and CP-AgNPs they were actively involved in the bioreduction and stability
1600 (111) of CL-AgNPs. -is corroborates a number of phytochemicalstudies using cocoa plant, which showed that in cocoa, the
1400 main bioactive molecules are phenols or phenolic com-
1200 pounds [27].
From Figure 3(b), bands are assigned at 3307, 2118, and
1000 1634 cm−1 for both the CP extract and CP-AgNPs produced.
800 However, the bands are not as strong as those observed from
(200) the CL extract. -is could be owing to the fact that they
600 contain fewer polyphenol chemicals, which have been linked
400 (220) to nanoparticle reduction and stabilization [28].-ese bands
(222) 3307, 2118, and 1634 cm
−1, as discussed earlier, are assigned
200
a to alcohols/phenolic compounds (-OH), triple bond mon-
0 b osubstituted alkyne, and stretching vibrations of C�C
groups, respectively. -e appearance of the 1017 cm−1 band
0 20 40 60 80 100 on the CP-AgNPs spectra is associated with triple bond
2θ (º) alkynes [23].
CL-AgNps -e near disappearance of these bands after the bio-
CP-AgNPs reduction is indicative of the fact that polyphenol com-
Figure 2: (a) XRD spectra of CL-AgNPs. (b) XRD spectra of CP- pounds are primarily responsible for the bioactivity AgNPs
AgNPs. production [29]. Also, as seen in Figure 3(a), new bands also
appear in the spectra of the AgNPs at 1303, 961, and
the 1634 cm−1 band is connected with stretching vibrations 774 cm−1 wavenumbers. -is observation suggests that their
of C�C groups [24]. Aliphatic iodo compounds (C-I) stretch presence in the crude extract was overshadowed, causing
is attributed to the band at 571 cm−1 [25]. -e CL-AgNPs them not to be detected [29].
spectra show similar bands (3338, 2123, 1634, and 571 cm−1) -e above assignment of spectra and description shows
but with a shift towards a lower wavelength, as seen in Figure that the major chemical elements of cocoa, regardless of the
3(a). -is shows that the biomolecules in the extract and the plant parts, are a cocktail mixture of polyphenolics that
AgNPs formed have a greater interaction [26]. -e binding include flavonoids.-e IR spectra of crude cocoa leaves (CL)
of C�C groups with AgNPs is responsible for the shift and pods (CP) do not disappoint, with smooth broad peaks
observed at 1634 cm−1. -e emergence of phenolic and at 3338 cm−1 and 3307 cm−1, respectively, due to the lack of
flavonoid functional groups in the spectra of CL-AgNPs and any further peaks in the 1725–1700 cm−1 range, and are thus
the fact that band shifts and intensity decrease suggest that assigned to an alcohol O-H stretch vibration. -e peaks at
Intensity (au)
Absorbance (%)
Absorbance (%)
International Journal of Biomaterials 5
FTIR Spectra of CL and CL-AgNps FTIR Spectra of CP and CP-AgNps
110
CL CP
100
2123.22 100
3280.51 2118.75
90 90 CL-AgNps
CL-AgNps 3195.61 571.04 80 1017.24
572.68
80 774.39961.25
1303.32 70 1634.26
1634.64
70 60
50
60 3307.39
40
50 3338.99 30 426.83
4000 3000 2000 1000 0 4000 3000 2000 1000 0
Wavenumber (cm–1) Wavenumber (cm–1)
CL CP
CL-AgNps CP-AgNps
(a) (b)
Figure 3: (a) FTIR spectra of Cl and CL-AgNPs. (b) FTIR spectra of CP and CP-AgNPs.
50
80
40
60
30
40 20
20 10
0 0
C O Ag C O Cl Ag
Elements Elements
(a) (b)
Figure 4: (a) EDX analysis of CL-AgNPs. (b) EDX analysis of CP-AgNPs.
1634 cm−1 in Figures 3(a) and 3(b) were too low in wave- -e IR spectra of both CL-AgNPs and CP-AgNPs have
number to be assigned to a ketone; even for the instance of a fine structure because the synthesized particles are
conjugation to an unsaturated group or if assigned to an normally purified and freed from the rest of the chemical
aldehyde, the aldehydic hydrogen must be at components in the extracts. -e O-H stretch vibration
2900–2700 cm−1. -e O-H stretches in the spectra must be frequencies 3195 cm−1 and 3280 cm−1 detected in both the
substantially wider to assign these peaks at 1634 cm−1 to the CL-AgNPs and CP-AgNPs, respectively, are much
C�O of carboxylic acid. As a result, the peak at 1634 cm−1 broader than those in the spectrum of the crude raw
was unambiguously ascribed to a C�C stretch of an alkene extracts. -is is understandable since the broadness is
or, more likely, an aromatic. Absorption peaks at 2123 cm−1 indicative of the many geometric configurations that exist
and 2118 cm−1 for both CL and CP are possibly aromatic around the O-H bonds especially in the structure of the
overtone and combination bands representing most prob- synthesized silver nanoparticles. -e absorption peaks at
ably trisubstituted polyphenolic aromatic rings. In the IR 1303, 961, and 774 cm−1 in the fingerprint region of the IR
spectrum of crude CL and CP extracts, peaks at 571 cm−1 and spectrum of CL-AgNPs are difficult to assign unambig-
426 cm−1 are indicative of residual brominated compounds uously but are most likely related to the aromatics; these
like methyl bromide that form the major constituents of peaks have become characteristic of all the silver nano-
cocoa pesticides. -e IR spectra of CL and CP lack fine particles we have synthesized from CL. -e same obser-
structure due to the highly concentrated nature of these vation is also true for CP-AgNPs, where fingerprint
extracts. absorption peaks are seen at 1000 and 1017 cm−1.
Transmittance (%)
Norm. C. [wt%]
Norm. C. [wt%] Transmittance (%)
6 International Journal of Biomaterials
However, small amounts of the residual brominated -e measured zeta potential of the particles in colloidal
pesticides are still left even after purification of the syn- solution is also shown in Figure S3b in the Supplementary
thesized nanoparticles. -e reduction of silver ions in CL- Materials.-emovement of nanoparticles under the effect of
AgNPs and CP-AgNPs appears to be caused by phenolic an applied electric field and surface charges and the colloidal
hydroxyl ligands. medium alter the zeta potential values [31]. -e particles
Hence, we conclude that the silver (I) ions reduction have a measured average negative zeta potential of
during the synthesis of the nanoparticles is most probably −0.54mV, indicating that they are highly stable due to
facilitated by the polyphenolic natural products that are very electrostatic repulsive force between them [30].
characteristic of all cocoa extracts and known to be present -e particles distribution profile of the CP-AgNPs is also
even in refined cocoa products like chocolates, cocoa presented in Figure S4a (Supplementary Material) with the
powders, and cocoa butter. corresponding zeta potential measurement in Figure S4b in
EDX analysis of the AgNPs synthesized from both ex- Supplementary Materials. -e particles are polydispersed
tracts of cocoa leaves and the cocoa pod is presented in with two overlapping size populations. -e particles size
Figure 4. Signals of silver were detected in the EDX along distribution is characterized by a much larger aggregate with
with other elements of O, C, and Cl, with percentage a size range below 680 nm compared to CL-AgNPs, having a
compositions showing the purity of the synthesized Ag very narrow scatter towards smaller sizes and a broad size
nanoparticles. It is observed that there is a higher percentage scatter to larger particles. -e average calculated PDI is 0.74,
of Ag content in the CL-AgNPs than in CP-AgNPs. -e indicating a broader spread of the size distribution. -e
other signals present in the EDX may be coming from traces distribution further shows that 90% of particles have sizes
of compounds bound to the AgNPs. -is goes to confirm below ∼700 nm. -e particles have an average measured
that the bioactive molecules in cocoa leaves extract are more negative zeta potential value of −0.72mV, also pointing to
effective in reducing and stabilizing nanoparticles, resulting the fact that the particles are highly stable.-e negative value
in higher purity of Ag for the same processing variables. -e validates the particles’ repulsion and demonstrates their
full EDX spectrum of CL-AgNPs and CP-AgNPs can be stability basically because they do not show any disposition
found in the Supplementary Materials (see Figures S5 and to come together, thereby preventing agglomeration.
S6, respectively, in the Supplementary Materials). -is difference in particle sizes and the spread of the
-e morphology of the CL-AgNPs and CP-AgNPs is distribution as observed in CL-AgNPs and CP-AgNPs is
shown in the SEM image in Figure 5. AgNP aggregates can attributed to a higher population of bioactive molecules in
be seen in both cases; however, they are not in direct contact the leaf extract participating in the reduction of Ag+, yielding
even inside the aggregates, indicating that bioactive capping more abrupt nucleation and faster growth of the nano-
agents have stabilized the nanoparticles. It is observed in particles, while producing small agglomerates [32].
Figure 5(a) that the size of aggregation in CL-AgNPs is Using the agar well diffusion assay, the antibacterial
smaller than that of the CP-AgNPs depicted in Figure 5(b). activity of biosynthesized silver nanoparticles, AgNO3, from
By observation, CL-AgNPs have a greater and denser cocoa leaf extract and the cocoa pod was investigated against
nanoparticle population than CP-AgNPs. Gram-negative (E. coli) bacteria, and the zone of inhibition
-e size population distribution of the nanoparticles was was observed as shown in Figure 6. In the disk diffusion
analyzed using DLS techniques. It is a statistical analytical method, results were obtained by measuring the zone of
method that uses laser light scattered by Brownian motion inhibition after the incubation period.
nanoparticles in a colloidal suspension. Figure S3 in Sup- -e zones of inhibition after the incubation period were
plementary Materials shows the DLS pattern of silver inspected andmeasured on filter paper discs with CL-AgNPs
nanoparticles in colloidal suspension synthesized using (A), CP-AgNPs (B), chemically synthesized silver nano-
cocoa leaf extract (CL-AgNPs). -e particle size distribution particles (C), and distilled water as control (D). -e
profile shows two peaks indicating polydispersion of the chemically synthesized silver nanoparticles had an average
population (see Figure S3(a) in the Supplementary Mate- particle size of 25–450 nm [33]. -e antibacterial activity of
rials). -e particle size distribution is characterized by ag- the synthesized AgNPs against E. coli was excellent. As
gregates with size populations within 110 nm, having a very shown in Figure 6(a) (disc A), silver nanoparticles made
narrow scatter. -e mean polydispersity index (PDI), a from cocoa leaf extracts had the greatest inhibitory zone with
parameter for describing nanoparticle size distributions, a diameter of around 21mm.-is value of 21mm (2.1 cm) is
gives information on many size populations observed at the comparable to that of the measured zones when Ampicillin
same time. -is is calculated based on zeta potential mea- is used as a positive control in various investigations in our
surements and is 0.45 for the produced AgNPs. -is value of laboratory against strains of E. coli.
polydispersity index (PDI) of the CL-AgNPs shows that the On the other hand, there was no zone of inhibition in
nanoparticles have a restricted size distribution and the the negative control (distilled water). -ese findings also
technique used for the measurements is good [30]. In ad- imply that particle binding to bacteria is influenced by the
dition, statistically, the distribution profile in Figure S3a in surface area available for interaction. -e highest zone of
Supplementary Materials suggests that about 95% of the inhibition demonstrated by CL-AgNPs against CP-AgNPs
distribution is within 2 standard deviations from the value of suggests that the CL-AgNPs are of smaller sizes. Generally,
100 nm and that 30% of them have sizes above ∼20 nm and small nanoparticles have a wider surface area for bacterium
about 22% have sizes of ∼55 nm. contact than larger particles, resulting in increased
International Journal of Biomaterials 7
(a) (b)
Figure 5: (a) SEM of CL-AgNPs. (b) SEM of CP-AgNPs.
Bacteria
(a) (b)
Figure 6: (a) Zone of exhibition of antimicrobial activity. (b) Optical microscope image of the zone of exhibition of CL-AgNPs.
Table 1: Zones of inhibition of green synthesized AgNPs against antibacterial activity. Table 1 compares the microbial ac-
E. coli. tivity of the silver nanoparticles synthesized in this work to
that of other published works. It can be seen that the zone
Sample Zone of inhibition(mm) Reference
of inhibition for E. coli for the standard antibiotics is 8mm
and that the green synthesized AgNPs are all below our
CL-AgNps 21 -is article synthesized CL-AgNPs, thereby confirming the effective-
Pen-Strep (standard ness of the CL-AgNPs.
antibiotic) 8
GPP-AgNPs 1.8
GPF-AgNPs 3.0 [34] 4. Conclusion
RPP-AgNPs 2.5
RPF-AgNPs 4.1 -e characteristics of the respective nanoparticles (CL-
T. triangulare AgNPs 2.55 [35] AgNPs and CP-AgNPs) were evaluated using UV-Vis, XRD,
50 μg/ml AgNPs 12 FTIR, SEM, EDX, and DLS after AgNPs were effectively
100 μg/ml AgNPs 15 [36] synthesized from the cocoa plant leaf and pod extracts. -e
25 μg/ml AgNPs 7 phytochemicals of phenols in the plant extracts functioning
50 μg/ml AgNPs 9 [37] as reducing, stabilizing, and capping agents are responsible
100 μg/ml AgNPs 10 for the effective synthesis of AgNPs from the extracts, as
0.25mM AgNPs 0 confirmed by FTIR analysis. CL-AgNPs were observed to
0.5mM AgNPs 8.33 [38] have smaller particle sizes and higher purity in the
1mM AgNPs 16.67 10–100 nm range. -is indicates that the cocoa leaf extract
8 International Journal of Biomaterials
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