International Journal of Nanomedicine Dovepress

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ORIGINAL RESEARCH

Green Synthesis, Characterization and Antimicrobial

Activity of Copper Oxide Nanomaterial Derived
from Momordica charantia
This article was published in the following Dove Press journal:
International Journal of Nanomedicine

Hina Qamar 1 Background: In the emerging field of nanotechnology, copper oxide (CuO) nanomaterials
Sumbul Rehman 2 are considered to be one of the most important transition metal oxides owing to its
1 fascinating properties. Its synthesis from green chemistry principles is gaining importance
Dushyant Kumar Chauhan
Ashok Kumar Tiwari3 as next-generation antibiotics due to its simplicity, eco-friendliness, and cost-effectiveness.
In the present study, CuO nanorods (CuO NRs) were synthesized from the aqueous fruit
Vikramaditya Upmanyu3
extract of Momordica charantia and characterized using different analytical techniques.
1
Department of Zoology, Chaudhary Further, the biomedical therapeutic potential was evaluated against multi-drug resistant
Charan Singh University, Meerut, Uttar
Pradesh, India; 2Department of Ilmul microbial strains.
Advia (Unani Pharmacology), A.K. Tibbiya Materials and Methods: To synthesize CuO NRs, 0.1M of CuSO4.5H2O solution was
College, Aligarh Muslim University,
added to aqueous extract of Momordica charantia in a 1:3 (v/v) ratio (pH=11) and heated
Aligarh, Uttar Pradesh, India; 3Biological
Standardization Division, Indian at 50°C followed by washing and drying. The synthesized CuO NRs were subjected to
Veterinary Research Institute, Bareilly, characterization using different analytical techniques such as UV visible spectroscopy,
Uttar Pradesh, India
zeta sizer equipped with zeta potential, Fourier transform infrared spectroscopy (FTIR),
X-ray diffraction (XRD), scanning electron microscopy (SEM) equipped with energy-
dispersive X-ray spectroscopy (EDS) and transmission electron microscopy (TEM).
Further, the application as a biomedical therapeutic potential was evaluated in vitro
using well diffusion method against eleven multidrug-resistant clinical bacterial strains,
a fungus- Trichophyton rubrum and in ovo against the R2B virus using haemagglutination
(HA) test.
Results: Characterization was preliminarily done by the spectral study that confirms the
absorbance band at 245nm. FTIR analysis at 628 cm−1 peak identified copper oxide
vibration. SEM analysis revealed agglomerated particle clusters. However, with TEM
clear nanorods of average diameter of 61.48 ± 2 nm were observed. EDAX confirmed
CuO formation while XRD showed a typical monoclinic structure with 6 nm crystallite
size. Biological screening of CuO NRs showed significant results against both in vitro
and in ovo methods. Significant inhibitory activity (p<0.0001) was noted against most of
the resistant human pathogenic strains including both Gram-positive and Gram-negative
bacteria. The highest efficacy was observed against Bacillus cereus with a 31.66 mm
zone of inhibition. Besides, the therapeutic potential of CuO NRs against
Corynebacterium xerosis, Streptococcus viridians and R2B strain of Newcastle disease
is reported for the first time.
Correspondence: Dushyant Kumar Conclusion: Based on the present results, it could be expected that green synthesized CuO
Chauhan NRs would find potential applications in the field of nanomedicine.
Department of Zoology, Chaudhary
Charan Singh University, Meerut 250001, Keywords: copper oxide nanorods, CuO NRs, biosynthesis, antibacterial, antifungal,
Uttar Pradesh, India antiviral, Trichophyton rubrum, multidrug-resistance, TEM, R2B Newcastle disease virus
Tel +91 941 270 8983
Email drdushyant.zoology@gmail.com

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Introduction that threaten modern medicine where common infections

Worldwide, the use of nanomaterial in the biomedical field could become more deadly. Next to bacterial infections,
has attracted the increasing interest of researchers owing to CuO NRs were screened for antifungal activity against
their unique property when interacting with cells and tis- Trichophyton rubrum, which causes a superficial dermato-
sues at the molecular level with a high degree of specifi- phyte infection that has increased to an alarming level in the
city and improved efficacy to combat infectious diseases.1 last few years due to the resistance developed to many con-
Their small size, design flexibility and large surface-to- ventional antidermatophytic agents having an azole group.18
volume ratio make these materials of intense use.2,3 It is a Dermatophytes are pathogenic fungi belonging to the kerati-
known fact that metal oxide at the nanoscale exhibits nophilic group that requires keratin for their growth and
improved properties and thus during the past decade exten- infects the keratinized tissues of humans and animals such
sive research on different metal oxide nanomaterials such as hairs, nails, and skin.19 Though several methods have been
as copper oxide nanoparticles and nanorods, have been availed to control and prevent the growth of fungal cells yet
carried out to explore their applications in different inter- there is a vital need to explore this field utilizing
disciplinary fields.4,5 nanomedicines.20 Preceding, the antiviral activity of CuO
Copper oxide nanomaterials have been gaining interest NRs was also evaluated against R2B strain of Newcastle
as nanomaterial due to their unique optical, thermal, elec- disease virus (NDV) which is a fast-growing, single-stranded
trical, chemical and biological properties.6,7 These proper- RNA avian paramyxovirus that causes a contagious bird
ties impart a wide range of their applications in different disease which incurs huge economic loss throughout the
fields from the formation of sensors, storage devices, super- world to poultry farmers by inducing mortality, low egg
capacitors and infrared filters to the health and environment production and reduced amount of dietary proteins. Studies
sector.6,8 Also, the antimicrobial activity of CuO nanoma- report that NDV causes conjunctivitis in humans especially
terial has made them strong candidates to be used as ther- to the people who get exposed to poultry farms.21,22
apeutic agents.9 At present, researchers are facing a major
challenge in the healthcare sector to combat drug resistance.
Materials and Methods
In this regard, day-by-day several improved synthesis stra-
tegies are developing that includes physical, chemical and
Materials
Copper (II) sulfate pentahydrate (CuSO4.5H2O) and
biological processes.10 Though synthesis of CuO nanoma-
Dimethyl sulfoxide (DMSO) were obtained from Sigma-
terial is cost-effective as compared to silver (Ag), gold (Au)
Aldrich. Nutrient agar, nutrient broth medium and antimicro-
and platinum (Pt) nanoparticles, but to attain its stability is
bial disks were purchased from Hi-media Labs, Mumbai,
still a challenging task.11,12 Many studies have been per-
India. The clinical isolates of bacterial and fungal cultures
formed on the synthesis of CuO nanomaterial which include
were kindly provided by Microbiology Lab, Department of
vapor deposition13 to electrochemical reduction,14 radioly-
Ilmul Advia and Jawaharlal Nehru Medical College and
sis reduction,15 thermal decomposition16 and chemical
Hospital, Aligarh Muslim University, Aligarh and R2B strain
reduction.17 But these strategies are associated with the
of NDV from Indian Veterinary Research Institute,
contamination issues and harm ecosystems as well as
Izzatnagar, Bareilly (U.P). All bacterial strains were resistant
human health.5 Thus, to overcome these shortfalls, the
principle of green chemistry utilizing natural origin sources to Cefixime, Amoxyclav, Cefotaxime, Methicillin and
such as plants and microbes for the synthesis process may Ampicillin antibiotics.
prove to be a potential solution.12
In the present study, an attempt has been made to develop Methods
an eco-friendly, cost-effective technique for the synthesis of Preparation of Extract
CuO nanorods (CuO NRs) by reducing Cu2+ ions in the Fresh fruits of Momordica charantia (MC) were procured
copper sulfate solution using Momordica charantia (Bitter from the local market. After thorough washing, the fruit
gourd) fruit extract. The characterization of CuO NRs was was ground to reduce the particle size and was subjected to
done by standard physiochemical techniques. Further, the extraction by the reflux method using soxhlet apparatus to
biomedical applications of green synthesized CuO NRs obtain crude aqueous extract.23 To prepare the aqueous
were evaluated against multidrug-resistant bacterial strains extract, 1:10 w/v of the mixture in distilled water was

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taken and heated at 50 °C for 40 min. The extract was Transmission Electron Microscopy
filtered and stored at 4 °C until further use. Transmission electron microscopy (TEM) analysis was
done using a 200kV JEOL transmission electron micro-
Green Synthesis of CuO NRs scope (JEOL Ltd. Tokyo, Japan) to determine the size of
To synthesize CuO NRs, Mary, et al, 2019 protocol with the synthesized particles. For TEM analysis, the sample
slight modifications was followed. Briefly, 0.1M of was prepared by adding 20 μL of CuO nanomaterial solu-
CuSO4.5H2O solution was added to aqueous extract of tion on a Cu grid, dried at room temperature and subse-
M. charantia (AE-MC) in a 1:3 (v/v) ratio followed by quently analyzed under the microscope with different
pH adjustment to 11 by sodium hydroxide pellet. Then, the magnifications.
solution was heated at 50 °C till the color of solution
changes to brown which might indicate the formation of
Scanning Electron Microscopy and Energy-Dispersive
X-Ray Spectroscopy
CuO NRs. The solution containing synthesized CuO NRs
The morphology to determine shape lattice and chemical
was washed three times, each repeat followed by centrifu-
composition of synthesized nanoparticles were examined
gation at 5000 rpm for 10 minutes. Finally, the pellet was
by scanning electron microscope (SEM; Model No. JSM
collected, dried and stored at 4 °C till further use.24
6510LV, Make-JEOL, Japan) equipped with energy dis-
persive X-ray spectrometer (EDSX) followed by micro-
Physiochemical Characterization of CuO NRs
scopic imaging performed from 1000X to 30,000X with
To confirm the formation of CuO NRs different physio-
0.5–1µm resolution at15kV.
chemical characterization techniques were used.
Biological Screening of CuO NRs
UV-Vis Spectroscopy In vitro Antimicrobial Assay of CuO NRs Against
Optical properties were analyzed by visualization of peaks Multidrug-Resistant Bacterial Strains
obtained from UV-Vis spectral scan from 200 nm to 800 The susceptibility of CuO NRs against multidrug-resistant
nm in a UV-Visible spectrophotometer (Motras Scientific; bacterial strains was determined using Kirby-Bauer’s disk
UV Plus). diffusion and agar well diffusion method according to
CLSI (Clinical Laboratory Standard Institute) Guide-
Zeta Sizer and Zeta Potential lines (2009).25,26 Seven Gram-positive (Staphylococcus
For determining the size of CuO NRs, in aqueous medium aureus, Streptococcus mutans, Streptococcus pyogenes,
zeta size and zeta potential were measured using a Streptococcus viridans, Staphylococcus epidermidis,
Zetasizer (Nano-ZS, Model ZEN3600). Corynebacterium xerosis, and Bacillus cereus) and four
Gram-negative (Escherichia coli, Klebsiella pneumonia,
Fourier Transform Infrared Spectroscopy Pseudomonas aeruginosa, and Proteus vulgaris) multi-
For the fourier transform infrared spectroscopy (FTIR) drug-resistant clinical bacterial strains were used. About
analysis sample was grounded with KBr pellet and ana- 50µL of the test sample (concentration of CuO NRs 1.25
lyzed on a Perkin Elmer (Model: Spectrum Two) with mg/50 µL DMSO) was used against each strain swabbed
spectrum recorded in the range of 400–4000 cm−1. on nutrient agar plates followed by incubation at 37°C for
24 hrs. DMSO was used as negative control while 50μL
X-Ray Diffraction aqueous extract of M. charantia was used as positive
X-ray diffractometer (XRD, Model: Mini Flex II; Make: control and 10µg Streptomycin disk for Gram-positive
Rigaku) was used to determine the crystallite size, struc- and 10µg Norfloxacin disk for Gram-negative strains
ture and crystallinity of nanoparticle with Cu-Kα radiations were used as standard: positive control. Antimicrobial
(λ= 0.154 nm) in 2θ range from 20º to 80º followed by activity was assessed using a zone of inhibition (ZoI)
data analysis in PowderX software. The mean size of measured after the incubation period against each tested
nanocrystals for the particle was determined from the micro-organisms.
diffraction peaks corresponding to the most intensive
reflections according to the JCPDS (Joint Committee on In vitro Antifungal Assay of CuO NRs
Powder Diffraction Standards) database and International Antifungal activity of CuO NRs against Trichophyton
Centre for Diffraction Data (ICCD). rubrum was determined using agar well diffusion method.27

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Briefly, Sabouraud dextrose agar culture media plates were Group C:Low dose- Nanoparticle treated [0.9mL of
prepared, inoculated with the T. rubrum and incubated at CuO NRs (50µg/mL) followed by 0.1mL NDV inoculums]
25°C for 15 days for fungus growth. After 15 days, wells Group D:High Dose-Nanoparticle treated [0.9mL of
were punched in the agar plate using a core-borer and CuO NRs (100µg/mL) followed by 0.1mL NDV
100µL CuO NRs dissolved in DMSO was instilled in a inoculums]
well. The plates were left for 1 h at 37 °C to allow the The treated eggs were incubated at 37°C for 96 hours
diffusion of the test sample and then incubated at 25 °C for a followed by regular monitoring for embryonic develop-
week. DMSO was used as negative control while 50μL ment and viability status at every 24-hour interval. After
aqueous extract of M. charantia was used as positive con- 96 hours, all eggs were chilled for an hour at 4°C to kill
trol and 10μg Fluconazole disk was used as standard. After the embryo and evaluated for antiviral activity by haemag-
the incubation period, the zone of inhibition was measured glutination assay.
against tested fungus.
Haemagglutination Test
In Ovo Antiviral Activity of CuO NRs To confirm the presence of NDV in allantoic fluid, the
Virus Cultivation and EID50 Calculation haemagglutination test using 96 well microtiter plate was
In ovo antiviral screening of the CuO NRs was carried out carried out.28 Allantoic fluid from each egg was collected
in 9–10 day old sterilized embryonated chicken eggs separately. 50µL PBS solution was added in each well of
through allantoic cavity route on R2B strain of Newcastle the microtiter plate followed by the addition of 50µL of
Disease Virus (NDV) and their efficacy was assessed by 0.45µ filtered allantoic fluid in two-fold dilutions in sub-
haemagglutination (HA) test.28 Firstly, NDV was propa- sequent wells. Next, 25µL 10%RBCs was added to each
gated in eggs. Then, to determine the virus titer, embryo well and left for 45 min at room temperature. After the
infectious dose (EID50) following embryo % mortality and incubation period, the plate was observed for any visible
% viability testing was evaluated (Table 1).29 EID50 was agglutination reaction visualized as a sharp button or dif-
calculated using a mathematical technique devised by fused film. Three separate wells were treated as control as
Reed and Muench.30 To study antiviral activity, two dif- follows:
ferent combinations of CuO NRs concentrations with Self-Control: 50µL PBS+25µL 10%RBCs (for auto-
determined EID50/mL of virus inoculum were used. agglutination)
Working NDV concentration was prepared by diluting Negative Control: 50µL allantoic fluid lacking NDV
the NDV stock to 1:2000 in phosphate buffer saline +25µL 10%RBCs
(PBS) and injecting 0.1mL of inoculum in the allantoic Positive Control: 50µL allantoic fluid having NDV+
cavity by perching the hole at egg air sac space. Two 25µL 10%RBCs
different doses: first d1=50µg/mL and second d2=100µg/
mL of CuO NRs were prepared dissolved in 1% DMSO. Statistical Analysis
Eggs were grouped into four, each carrying 5 eggs as The experiment was performed in triplicates, Mean ± SEM
follows: (S.D) was compared with the standard used and analyzed
Group A:Negative control [1mL of 1% DMSO] statistically using graph-paid instat Dataset1.ISD software
Group B:Positive control [1mL of NDV inoculum] by Tukey Kramer Comparison test, One way ANOVA.

Table 1 In Ovo Evaluation of CuO NRs Antiviral Activity

Groups Mean±SEM (SD) % Viability

Untreated A Negative control 1mL of 1% DMSO 4.66±0.3 (0.57) 100

B Positive control 1mL of NDV inoculums 0±0 (0) 0

Treated C CuO NRs treated 0.9mL CuO NRs (50µg CuO NRs concentration dissolved in 1mL 2.66±0.88 (1.5) 53.2
(Low dose) 1% DMSO + 0.1mL NDV inoculums)

D CuO NRs treated 0.9mL CuO NRs (100µg CuO NRs concentration dissolved in 1mL 4±0.5 (1.0) 80
(High dose) 1% DMSO + 0.1mL NDV inoculums

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Results and Discussion

Green Synthesis of CuO NRs and UV-Vis
Spectroscopic Analysis
The present study deals with the rapid green synthesis of
CuO NRs from fruit extract of Momordica charantia.
There are a plethora of studies that reported the presence
of various bioactive compounds in M. charantia such as
triterpenes, alkaloids, steroids, phenols, etc. to list a few.31
In the present study, the mechanism of CuO NRs synthesis
can be hypothesized and explained by using the phenolic
content of fruit extract of M. charantia as they are known
to have the high reducing ability.32,33 It is well documen-
ted that the gallic acid is present as one of the key phenolic
components in M. charantia fruit extract which might
facilitate the reduction of Cu(OH)2 to nano-sized CuO.34
During the reaction, when pH is increased in the range of
11, the color of reaction changes from blue to brown and Figure 1 Sequential color change during the formation of CuO nanorods.

then to black.35 The changes in the color may be under-

stood in terms of the reaction between copper (II) sulfate 5
pentahydrate and sodium hydroxide to form copper (II) Single absorbance peak at λ= 245nm
hydroxide (blue), which in turn reacts with the gallic acid 4
resulting in the formation of dehydrogallic acid and copper
3
Absorbance

(I) oxide(brown).36
CuSO4: 5H2 O þ 2NaOH ! Na2 SO4 þCuðOHÞ2 þ5H2 O 2

1
2CuðOHÞ2 þC7 H6 O5 ! Cu2 OþC7 H4 O5 þ3H2 O
0
However, as reaction proceeds with time, the copper (I) 225 250 275
oxide was formed and finally CuO nanoparticles formation -1
occur by a series of following reactions (Figure 1).35–37 Wavelength (λ=nm)
 
Cu2 O þ OH þH2 O ! CuðOHÞ2 þCuOH Figure 2 UV visible spectral analysis of CuO nanorods.

2CuOH ! Cu2 OþH2 O (DLS) and was found to be 90 ± 5 nm (Figure 3). DLS
is widely used for determining the diameter of suspended
2CuOHþO2 ! 4CuO þ 2H2 O particles hydro dynamically based on the Brownian move-
Color changed from blue to brown due to excitation of sur- ment. It is the particle diffusion behavior within any fluid
face Plasmon resonance and presence of polyphenolics as and is measured by the fluctuations in light intensity that
antioxidant source arising as a result of π → π* transitions passes through a colloidal solution as a function of time.37
indicated the synthesis of CuO NRs.4 Further, the absorption Further, zeta potential is considered as a measure of
spectrum recorded at 200–600 nm showed a single absor- charges on the surface of nanoparticles. It indicates the
bance peak at 246 nm corresponded to the characteristic stability of colloidal dispersions. Colloids that have high
absorbance band of CuO NRs which was in accordance zeta potential are electrically more stable than colloids
with previous studies reported (Figure 2).4,36 with low zeta potential that tends to coagulate. The stabi-
lity is maintained by potent repulsive forces among the
Zeta Size and Potential of CuO NRs ultrafine particles.38 To measure zeta potential using elec-
An average hydrodynamic diameter of CuO NRs was trophoretic light scattering, nanoparticles were dispersed
measured by Zetasizer using dynamic light scattering in water. The results showed zeta potential to be −7.23

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6 the O–H and N–H bond stretching vibrations. The vibrations

Size distribution intensity might be due to phenolic compounds present in the solution.39
5
The small peak at 2071.8 cm−1 corresponds to the stretching
4 vibrations of the compounds containing C≡N bonds. The
Intensity (Percent)

sharp peak at 1612.8–1668.2 cm−1 corresponds to the pre-

3
sence of C=N or C=O stretching. Likely, peak at 1377.9–
2 1434.6 cm−1 denote sp3 C-H bending or acyl C-O (or phenol
C-O) stretching and 2700–2954 cm−1 denotes C-H stretching.
1
Furthermore, the peak at 1087.2–1125 cm−1 denotes alkoxy
0 C-O. The unsaturated C-H bending appears under 1000 cm−1
50 75 100 125 150
-1 Size (nm) that might be due to the presence of bioactive phytochemicals
such as triterpenes, proteins, steroids, carbohydrates, alkaloids
Figure 3 Zeta sizer measurement of CuO nanorods. and other compounds in the solution. These compounds might
impart capping which helps in maintaining the stability of
mV. Lower zeta potential value indicates the presence of CuO NRs. Moreover, a sharp peak found in the infrared
thin coating of M. charantia aqueous extract over the spectrum at low frequencies at 590.4–628.2 cm−1 corresponds
nanorods that might result in colloidal instability and to CuO vibrations which were in accordance with earlier
agglomeration. Furthermore, the negative value indicates reports.40
the formation of hydroxyl groups on the surface of parti-
cles upon dispersion in water.38
XRD Analysis of CuO NRs
The microcrystalline structure of CuO NR was analyzed
FTIR Analysis of CuO NRs using the XRD technique. The graph was prepared using
FTIR spectral analysis of CuO NRs showed different peaks at PowderX software. As shown in Figure 5, the characteristic
459–487.3 cm−1, 590.4–628.2 cm−1, 1087.2–1125 cm−1, XRD peaks were observed at 32.64, 35.1, 38.9, 48.9, 52.0,
1377.9–1434.6 cm−1, 1612.8–1668.2 cm−1, 2071.8 cm−1, 58.46, 62.9, 65.94 and 67.96 corresponding to 110, 002, 111,
2700–2954 cm−1 and 3384–3534.3 cm−1 (Figure 4). The 202, 020, 202, 113, 311 and 113 reflections respectively
broad peak observed at 3384–3534.3 cm−1 corresponds to which indicate the formation of typical monoclinic CuO

250

200 20.71.8
487.8-459 1434.6-1377.9
2954-2700
Transmittance (%)

150

628.2-590.4 1668.2-1612.8
100
1125-1087.2

50

3534.3-3384
0
0 500 1000 1500 2000 2500 3000 3500 4000 4500
Wave number (cm-1)

Figure 4 FTIR analysis of CuO nanorods.

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700

600
CuONRs peaks
500

Intensity count
400

300

200

100

0
20 30 40 50 60 70 80
2θ value

Figure 5 XRD pattern determination of CuO nanorods.

NR structure and are in agreement with the standard values β is full-width at half maximum (FWHM) of the peak in
reported by the JCPDS card no. 801268 and ICDD card no. radians and θ is the diffraction angle (degree).
801916 which was in accordance with previous studies
reported.40,41 However, other peaks are also denoted in the SEM/EDS Analysis of CuO NRs
figure. The average crystallite size was calculated to be 6 nm Microscopic SEM analysis revealed that particles might be
using Debye Scherrer’s equation. spherical. A clear morphology could not be depicted as
D ¼ Kλ=ðβ cosθÞ exhibited agglomeration and clumped to form clustered
particles (Figure 6).42,43 The EDX spectroscopy was
Where D is an average particle size (nm), K is the constant applied to quantify the elemental composition of synthe-
and equals to 0.94, λ is the wavelength of X-ray radiation, sized nanoparticles. The spectrum of CuO NRs given in

Figure 6 SEM analysis of CuO nanorods.

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Figure 7 confirms the existence of Cu, O and C. The peak analyzed using agar well diffusion method which was in
around 0.5 keV belongs to the binding energy of oxygen accordance with previous studies reported.20,41
(OKα); while peaks located at binding energies of 1, 8 and The antibacterial activity of CuO NRs was evaluated
9keV correspond to CuLα, CuKα, and CuKβ, respectively. against both Gram-positive and Gram-negative multidrug-
Additionally, a peak at 0.25keV corresponding to carbon resistant bacterial strains by measuring the zones of inhibition
(CKα) was also reported. The percentage of Cu, O, and C (ZoI) (Table 2). The efficacy of CuO NRs for all strains was
present in CuO NRs was found to be 54.51%, 31.50%, and found to be significant as compared to the standard drug
13.99% respectively. The appearance of a carbon peak in (Figure 9) with p<0.0001 in most of the cases. The aqueous
the sample verified the presence of carbon-based stabili- extract of M charantia did not show any activity. The highest
zers or it may be due to carbon tape used during the efficacy was observed against Bacillus cereus with a
process.44 31.66 mm zone of inhibition. The results for antifungal activity
were also satisfactory for Trichophyton rubrum and ZoI was
TEM Analysis of CuO NRs found to be 12 mm which is considered moderately significant
(Figure 10). It’s observed that the zone of inhibition was less in
TEM analysis revealed well dispersed rod-shaped nanopar-
fungi when compared with bacteria. This is because the fungal
ticles within the range of 61.48 ± 2 nm in diameter and
cell wall is more firm as it is made up of chitin, comprising of
400–500 nm in length (Figure 8). The figure shows that
polysaccharides having N-acetylglucosamine and a nitrogen
nanoparticle distributions are compatible with the Gaussian
group. Thereby, not allow easy passage of CuO NRs from the
distribution, noting that the distribution is skewed toward a
outer layer of the cell wall to the inner layer. However, the cell
small size. The most common nanoparticle size was 60 nm
wall of bacteria is made up of peptidoglycan (a polymer
and the range of nanoparticle sizes was between 50–57 nm.
having sugars and amino acids), which is less firm and allow
As shown by the selected area electron diffraction
easy passage of CuO NRs when compared with fungi.
(SAED) pattern it is clear that the prepared nanoparticles
Besides, among the bacterial strains, the antibacterial activity
are in a well defined crystalline form identical to the single-
of CuO NRs was found more effective for Gram-positive
phase. It indicates a reflection of the monoclinic CuO struc-
ture (Figure 8B) which is in accordance with the XRD pattern bacteria in comparison to Gram-negative. Previous studies
as mentioned above. The TEM results mentioned were in reported that CuO NRs penetrate inside the bacterial cell due
consistent with the previous studies reported.45,46 to the changes in membrane morphology that significantly
increases cell permeability and affect transport via plasma
membrane which results in cell death.47,48 To date, different
Antimicrobial Analysis of CuO NRs mechanisms responsible for the antibacterial activity of CuO
In the present study, antibacterial preventive and antifun- NRs have been reported. These include the generation of
gal curative efficacy of green synthesized CuO NRs was reactive oxygen species, protein oxidation, lipid peroxidation,
destruction of the cell membrane and DNA degradation in
bacterial cells.49 Moreover, the antibacterial activity of nano-
particles depends on its shape, size and oxidation number.49 In
the present study, nanorods have large surface to volume ratio
to interact with the cell membranes of microorganisms and
ceases their growth. It was also observed that the antimicrobial
efficacy of CuO NRs when evaluated after 4 months showed
the same ZoI which suggests that CuO NRs efficacy to kill
microbes does not decrease with time. Noteworthy, the anti-
fungal activity of CuO NRs against the Trichophyton rubrum
showed significant results (p<0.0001) when compared to stan-
dard drug fluconazole which is almost resistant to T. rubrum.
They are safe and more acceptable topically, as they have a
greater affinity toward amines and carboxyl groups on fungal
cell surfaces and their extremely large surface area provides
Figure 7 EDS analysis of CuO nanorods. better contact with the fungus. Also, copper ions released

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A TEM analysis depicting rod-shaped B SAED pattern of CuO NRs

structures
21

19

17

15
Number of particles

13

11

5
50 55 60 65 70 75

Particle size (nm)

C Statistical analysis of synthesized CuO NRs
Figure 8 TEM analysis of CuO nanorods.
Notes: (A) TEM analysis depicting rod-shaped structure. (B) SAED pattern of CuO NRs. (C) Statistical analysis of synthesized CuO NRs.

during the process may bind with DNA molecules and disrupt sphingolipid synthesis, trehalose, metabolic glyoxylate speci-
the helical structure, resulting in the lysis of dermatophytic fically the enzyme isocitrate lyase, mitogen-activated protein
cells.50 Though several biochemical processes got disrupted (MAP) kinase, high-osmolarity glycerol (HOG), cell wall
when copper ions penetrate inside the cells. Further, studies target 3-phosphoinositide-dependent protein kinase 1 (Pdk1)
are required to get ensure the mechanism by which nano- and an inhibitor VCN-01 of calcium signaling.52 Based on the
particles exhibit antifungal activity.51 It can be assumed that results obtained, the green synthesized CuO NRs can be
the novel pathways and targets that shall be considered while applied in combination with a lotion or a cream-based product
screening antifungal drugs may include calcineurin, RAS and as they have a size greater than 30nm, which is considered safe

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Table 2 In vitro Antimicrobial Assay Readings Against Various Multidrug Resistant Microbial Strains
Name of Microbial Strains Zone of Inhibition (in mm) Results Expressed as Mean±SEM (SD)

CuO NRs Positive Control Negative Control P value

Gram positive bacteria Staphylococcus aureus 28.66±0.66(1.15) 17.66±0.33(0.57) 6.66±0.33(0.57) <0.0001

Streptococcus mutans 28.66±0.33(0.5) 28±0.33(0.57) 6.33±0.33(0.57) <0.0001
Streptococcus pyogenes 25.66±0.66(1.15) 23±0.83(0.57) 6.33±0.33(0.57) <0.0001
Streptococcus viridians 27.33±0.33(0.57) 16.33±0.33(0.57) 6.33±0.33(0.57) <0.0001
Staphylococcus epidermidis 23±2.0(3.4) 7.33±0.03(0.57) 6.33±0.33(0.57) <0.0001
Corynebacterium xerosis 28.66±5.66(9.8) 19±0.63(0.57) 6.33±0.33(0.57) 0.0085
Bacillus cereus 31.66±1.2(2.08) 15±0.66(1.15) 6.33±0.33(0.57) <0.0001

Gram negative bacteria Escherichia coli 25.33±5.3(9.2) 20±0.33(0.57) 6.66±0.33(0.57) 0.013

Klebsiella pneumonia 24.66±2.6(4.5) 32±0.33(0.57) 6.33±0.33(0.57) <0.0001
Pseudomonas aeruginosa 25.66±2.33(4.04) 14±0.63(0.57) 6.33±0.33(0.57) 0.002
Proteus vulgaris 26.33±2.18(3.78) 16.33±0.33(0.57) 6.33±0.33(0.57) <0.0001

Fungus Trichophyton rubrum 12.54±0.33(0.57) 6.33±0.33(0.57) 6.33±0.33(0.57) <0.0001

for human health and does not enter the bloodstream through eggs was calculated by applying the Reed and Muench for-
the skin.20 Due to a high incidence of disease reoccurrence and mula (1938) and was found to be 107.5 per mL. After examin-
other side effects, CuO NRs can be considered as a better ing EID50, the determined EID50/mL of virus inoculum has
solution.20,53 been used against each concentration of CuO NRs to check the
antiviral activity (Figure 11). Following 96 hrs of incubation,
Antiviral Analysis of CuO NRs it was observed that no embryo death occurred in the negative
In the present study, titration was done to measure infectious control. Further, 53.2% viability occurred in 50 µg/mL con-
NDV concentration in the suspension (Table 3). The embryo centrations of CuO NRs treated eggs and 80% in 100µg/mL
infectious dosage (EID50) that killed 50% of NDV treated concentrations of CuO NRs treated eggs (Table 1). Based on

Figure 9 In vitro antibacterial activity of CuO nanorods.

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Table 3 Calculation of EID50

Dilution of NDV No. of Eggs Mortality Accumulated Numbers % of Mortality= % of Viability=
A/A+B X 100 B/A+B X100
24 h 48 h 72 h 96 h Infected eggs Noninfected eggs Total
(A) (B) (A+B)

−1
10 5 3 2 0 0 5 0 5 100 0
10−2 5 2 1 1 1 5 0 5 100 0
10−3 5 2 1 2 1 5 0 5 100 0
10−4 5 0 1 2 2 5 0 5 100 0
10−5 5 0 0 2 2 4 1 5 80 20
10−6 5 0 1 2 0 3 2 5 60 40
10−7 5 0 0 2 0 2 3 5 40 60
10−8 5 0 0 0 1 1 4 5 20 80
10−9 5 0 0 0 0 0 5 5 0 100
10−10 5 0 0 0 0 0 5 5 0 100

the results obtained, it’s concluded that 100 µg/mL concentra- nanoparticles, where it was found that the diameter of the
tions of CuO NRs were effective against NDV viral growth. CuO NRs was 61.48± 2 nm and the length ranged from
400–500 nm. Single crystalline and uniformly structures
have been seen through XRD and SAED patterns. The nano-
Haemagglutination Test (HA Test)
particles were confirmed by UV-Vis spectrum FTIR and EDX
In haemagglutination assay, negative control and eggs treated
results. Moreover, the results of physicochemical characteriza-
with 100µg/mL concentrations of CuO NRs showed HA
tions of CuO NRs also supplemented in vitro studies observa-
negative results up to 210 dilutions. In contrast, results for
tions. The green synthesized CuO NRs find potential
positive control eggs showed HA positive results up to
applications in the field of nanomedicine and could be used
210dilutions. The results indicated a significant effect of nano-
to develop targeted therapies against bacteria, fungi and
particles in preventing the growth of NDV in the allantoic fluid
viruses. The diameters of the prepared CuO NRs were smaller
of treated eggs in a dose-dependent manner.
than the pores of the bacterial cell walls, and therefore the
effect of these minutes lies in their effect on the enzymatic
Conclusions activities inside the cells, thereby inhibiting the growth of the
CuO nanorods were extracted from Momordica charantia fruit bacteria. The therapeutic potential of CuO NRs against
extract which is an environmentally friendly and cost-effective Corynebacterium xerosis, Streptococcus viridians and R2B
method. TEM analysis was used to study the sizes of strain of Newcastle disease is reported for the first time.

Figure 11 In ovo antiviral activity of CuO nanorods against R2B strain of

Figure 10 Antifungal activity of CuO nanorods against Trichophyton rubrum. Newcastle disease virus.

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C. xerosis and NDV, both are reported to cause conjunctivitis 5. Grigore ME, Biscu ER, Holban AM, Gestal MC, Grumezescu AM.
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