Journal of Inorganic and Organometallic Polymers and Materials (2021) 31:1846–1857

https://doi.org/10.1007/s10904-020-01837-7

Biogenic Production and Characterization of CuO Nanoparticles

by Carica papaya Leaves and Its Biocompatibility Applications
Kanika Dulta1 · Gözde Koşarsoy Ağçeli2 · Parveen Chauhan1 · P. K. Chauhan1

Received: 19 September 2020 / Accepted: 12 November 2020 / Published online: 18 January 2021
© The Author(s), under exclusive licence to Springer Science+Business Media, LLC part of Springer Nature 2021

Abstract
Nowadays, environmentally friendly, economical and efficient production of nanoparticles is increasingly important. The most
appropriate nanoparticle synthesis that can be used in this process is green synthesis performed with biological materials
(bacteria, fungi or plant). Metallic nanoparticles can be used in many applications and several studies have been conducted
to use these particles as antimicrobial and anticancer agents. However, there are limited studies on the green synthesis and
biocompatibility of nanocrystalline copper oxide. In this study extracts of Carica papaya leaves were used for the produc-
tion of copper oxide (CuO) nanoparticles (NPs). UV-Vis spectral analysis showed copper nanoparticles exhibit an absorp-
tion peak at around 250 nm. The CuO NPs were analysed with Fourier transform infrared spectroscopy (FT-IR), Scanning
electron microscope (SEM), X-ray diffraction (XRD) spectrum and Dynamic light scattering (DLS). The SEM and XRD
analysis exposed CuO NPs are spherical in shape with an average particle size of 77 nm. Further, as-formed CuO NPs exhibit
significant antioxidant and anticancer activity.

Keywords Green synthesis · Carica papaya · Copper oxide nanoparticles · Antioxidant · Anticancer

1 Introduction The use of metal oxide NPs in various applications is

becoming increasingly important. Metallic nanoparticle-
The nanoparticles have a size below 100 nm (nm) and exhibit supported catalysts have been necessary materials in a wide
optical, electronic, mechanical, magnetic and chemical prop- range of industries such as chemical resources, energy, and
erties that are considered to be much different and supe- environmental protection in recent years. Although theo-
rior than volumetric materials [1–5]. Nanoparticles, which retical chemists have suggested the significant potential and
account for about half of nanotechnology, are vital materi- uniqueness of metal cluster catalysts, limitations in synthesis
als that play a role in recent technologies such as catalysts, have prevented discussion from experimental investigations
chemical/biological analysis and optoelectronic devices [11].
[6–8]. Nanoparticles are not simple molecules itself and Nano-sized inorganic particles of either simple or com-
therefore composed of three layers. These can be listed as plex nature, display unique, physical and chemical proper-
follows; (a) The surface layer, which may be functionalized ties and represent an increasingly important material in the
with a variety of small molecules, metal ions, surfactants development of novel nanodevices which can be used in
and polymers. (b) The shell layer, which is chemically vari- numerous physical, biological, biomedical and pharmaceuti-
ous material from the core in all aspects, and (c) The core, cal applications [12–14].
which is essentially the central portion of the nanoparticle Copper oxide (CuO) is an important p-type semiconduc-
and usually refers the nanoparticle itself [9, 10]. tor with a band gap of 1.7 eV and Copper oxide nanoparti-
cles (CuO NPs) have gained significant interest and atten-
* P. K. Chauhan tion due to their chemical and physical characteristics [15].
chauhanbiochem084@gmail.com CuO nanoparticles are used in many applications, such as
the production of electronic devices, high surface area cata-
1
Faculty of Applied Sciences and Biotechnology, Shoolini lysts, preparation of thin films, in medicine especially the
University, Solan, HP 173229, India
treatment of cancer cells and antimicrobial formulation [16].
2
Faculty of Science, Department of Biology, Hacettepe
University, 06800 Ankara, Turkey

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Journal of Inorganic and Organometallic Polymers and Materials (2021) 31:1846–1857 1847

Metallic NPs can be synthesized different physical or solution with constant stirring at 50–60 °C. The color
chemical procedures [17–22]. However, green and biogenic change after nanoparticle synthesis (5 mM Copper sulphate
bottom-up synthesis attracting many researchers due to the and C. papaya leaves extract) was compared with the control
feasibility and less toxic nature of processes. These pro- solution. Colour change of the solution was monitored from
cesses are cost-effective and environmental friendly, where green to blackish brown on vigorous stirring for 24 h. After
synthesis of NPs is accomplished via biological systems the solution was cooled to 25 °C, a brown-black product
such as using plant extracts [10]. Today, nanoparticles such was gained. The solid portion in the product was separated
as silver, gold and copper oxide can be synthesized from by centrifugation at 10.000 rpm, 15 min., 4 °C conditions,
various plants by green chemistry [23–26]. washed twice with deionized water and the pellet was dried
Plants can produce secondary metabolites with strong in a hot air oven at 50 °C. Nanoparticles in the form of black
reducing potential. Thera are also more resistant to metal precipitates, which are allowed to dry on the watch glass,
toxicity than bacteria and algae. This gives the plants an were prepared by grinding for characterization (Fig. 1).
advantage for nanoparticle green biosynthesis [27]. Medici-
nal plants can change the shape and size of NPs [28]. Carica 2.3 Effect of Various Parameters on CuO NPs
papaya belongs to the family Caricaceae, commonly used Synthesis
in the diseases cure and management worldwide, especially
in tropical and subtropical parts of the world. Various parts The effect of various contents in the synthesis of CuO NPs
of the papaya plant are used for medical treatment. Medi- was investigated using different concentrations of copper
cines from the papaya plant are used in the treatment of sulfate (1 mM - 5 mM). Surface plasmon resonance (SPR)
many other diseases such as diabetes, eczema, warts, cancer peaks were recorded over spectrophotometer. The effect
and malaria [29–31]. In addition, Carica papaya leaf and of different concentrations (5-25 mL) of C. papaya leaves
fruit extract, which has a strong antioxidant and antimicro- extract on nanoparticle synthesis was determined by SPR
bial properties, have rich vitamins, phenols and proteolytic and UV-vis analysis. The effect of different pH (from pH 4
enzymes [32–34]. to pH 12), different temperatures (30 °C to 100 °C) and dif-
In this research, the biological synthesis of CuO NPs was ferent times (from 0 h to 48 h) on nanoparticle synthesis was
achieved by Carica papaya leaf extract grown in district measured spectrophotometrically. Stability of nanoparticles
Solan, Himachal Pradesh. The biosynthesized CuO NPs ensures that nanoparticles can be used for a long time. To
were characterized by various physicochemical characteri- determine the stability of the synthesized CuO NPs, they
zation techniques. Thereafter, the CuO NPs antioxidant, were stored at room temperature and analyzed by SPR read-
antimicrobial and anticancer activities were determined. ings for 7 days of interval for one month.

2.4 Characterization
2 Material and Methods
It is supported by UV-vis, where the particles obtained are
2.1 Preparation of Leaf Extract CuO nanoparticles (UV-1601, Shimadzu, Japan). FTIR
spectrum was reached by spectrum RX-1 instrument in dif-
In this study, C. papaya was used as a reducing agent for fuse reflectance mode functioned at a resolution of 4 cm−1.
the biosynthesis of CuONPs. 10 g of C. papaya plants were DynaPro Plate Reader (Wyatt Technology) was used for
washed and dried in shady area about 2 weeks and then Dynamic light scattering (DLS) analysis of copper oxide
powdered by grinder. The powdered material was boiled in nanoparticles, and Malvern Zetazier (Nano ZS90, UK) was
a 250 mL flask with 100 mL deionized water for 30 min at used for the particles size and stability. The form and mor-
60 °C and then cooled down to 25 °C. The solid was filtered phology of copper oxide NPs were revealed using a scan-
to remove particles and then again filtered through a 0.2 μm ning electron microscopy (SEM-Hitachi/s-4200 N). In addi-
Whatman filter paper. The obtained filtrate was stored at tion, with this device, and energy dispersive X-ray analysis
4 °C for use for the synthesis of CuO NPs. (EDAX)and elemental mapping were performed. XRD pat-
tern of CuO NPs was obtained using a powder X-ray dif-
2.2 Green Synthesis of Copper Oxide Nanoparticles fractometer (Philps X’Pert Pro X-ray diffractometer) with
Cu(Kα) radiations (1.5406 Å) in 2θ range from 10 °C to
For CuO NPs synthesis 10 mL of C. papaya leaves extract 80 °C was done using the same instrument at Indian Institute
was mixed 90 mL of 5 mM cupric sulphate (­ CuSO4.5H2O) of Technology, Mandi.

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1848 Journal of Inorganic and Organometallic Polymers and Materials (2021) 31:1846–1857

Plant extract
Keep at magnec Incubaon for 48h
srrer at 60oC and
CuSO4 centrifugaon
soluon

The pellet dried Pellet collecon

Characterizaon
in oven

Fig. 1  Green synthesis of CuO nanoparticles by Carica papaya leaf extracts

2.5 Quantification of Phenolic and Flavonoid 2005). Approximately 5 mg of each of the sample is dis-
Content solved in 5 mL methanol. 1 mL was combined with 9 mL
d-water from each of these normal solutions and eventually
Phenolic and flavonoid measurements of Carica papaya with 1 mL sodium nitrite (5%). Each mixture was left for
leaf extract (CP) and copper oxide nanoparticles (CuO NPs) 6 min for a continuous reaction, and 2 mL of 10% ­AlCl3
were measured according to procedures. solution was added and left for another 5 min. Then 1 M
sodium hydroxide was added to the mixtures by adding 2 mL
2.5.1 Total Phenolic Content each, and the mixtures were measured at 510 nm. Drawn for
determining the normal quercetin curve of the total flavonoid
Using the Folin-Ciocalteu test, the total phenolic contents of material (0 to 100 mg/mL). The total content of flavonoids
Carica papaya leaf extract and CuO NPs were determined. was measured as mg of equivalent ­C15H10O7 (quercetin) (mg
5 mg of the samples were dissolved in 5 mL of methanol. QE/ g) per gram of dry sample.
1 mL Folin-Ciocalteu reagent was diluted to 10 mL with
d-water. The samples were prepared by mixing 1 mL stand- 2.6 Antioxidant Assay
ard solution with 9 mL d-water. 1 mL of diluted Folin-
Ciocalteu reagent was added to each test tube and stand for 2.6.1 ABTS Radical Scavenging Assay
6 min. Distilled water was added to each test tube and the
tubes were incubated for 90 min at room temperature. After To determine ABTS radical scavenging assay, the method
incubation, 10 mL of 7% N ­ a2CO3 solutions were added to was described by [35]. The studied sample was incubated for
the reaction mixes and diluted to 25 mL. The absorbance of 12 h in the dark at room temperature with equal mixing with
the sample was measured with a 760 nm UV spectropho- 7 mmol/L ABTS solution and 2.4 mmol/L ­K2S2O8 solution.
tometer. Calibration curve of ­C7H6O5 (0 to 100 mg/mL) was The resulting solution was diluted with 1 mL ABTS solution
used for the total phenolic content and measured as equiva- to achieve absorbance of the units at 734 nm (0.706 ± 0.001).
lent milligram ­C7H6O5 (mg GAE/g) per dry sample. ABTS solution was prepared fresh for each experiment sepa-
rately. 1 mL of Carica papaya leaf extracts was reacted with
2.5.2 Total Flavonoid Content 2.5 mL ABTS solution for 7 min and taken spectrophotomet-
ric measurement at 734 nm. The ABTS scavenging capacity
Total flavonoid content was determined by the A
­ lCl3 colori- of the extract was compared with that of BHT and % inhibi-
metric assay according to the method reported (Meda et al. tion was calculated as;

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Journal of Inorganic and Organometallic Polymers and Materials (2021) 31:1846–1857 1849

ABTS radical scavenging activity (%) incubation, resazurin dye was added to each well and incu-
( )
= Acontrol − Atest ∕Acontrol × 100 bated for an additional 2 h. Bacterial reproduction was
showed color changes blue to purple pink or colorless. As
MIC value, the lowest concentration at which color change
Acontrol: the absorbance of ABTS radical+methanol occurred was accepted.
Atest: the absorbance of ABTS radical+sample extract/
standard 2.8 Determination of Cytotoxicity Studies of CuO
NPs
2.6.2 DPPH Radical Scavenging Assay
2.8.1 Cell Culturing and Maintenance
2 g of the fine powder of each sample was soaked in 20 mL
methanol with stirring for 48 h, filtered through Whatman The synthesized CuO NPs were also evaluated for cytotoxic
filter paper No. (1) to attain a clear filtrate. The filtrates were activity on Human colon cancer (HT-29) and Human Cervi-
evaporated and dried at 40 °C in water bath. DPPH solution cal cancer (HeLa cells) cell line was procured from National
was prepared by dissolving 20 mg DPPH in 100 mL metha- Centre for Cell Sciences (NCCS), Pune, India. Following
nol. 3 mL was taken from this solution, and its absorbance standard procedures, stock cells were cultured in DMEM
at 515 nm (control solution) was set to 0.75. To prevent supplemented with 10% inactivated Fetal Bovine Serum
free radicals, the DPPH stock solution was coated with alu- (FBS), 1% Penicillin/Streptomycin and Amphotericin-B
minum foil and incubated in the no light condition about (5 μg/mL) in a humidified atmosphere of 5% ­CO2 at 37 °C
24 h. 5 mg of each extract dissolve in 5 mL methanol for until confluent. The stock cultures were grown in tissue cul-
stock solutions preparation. Different dilutions (25, 50, 75 ture flasks, and all experiments were carried out in 96 well
and 100 μg/mL) were prepare. Approximately 2 mL of each Microtiter plates.
dilution was mixed with a solution of 2 mL DPPH and kept
for 15 min in darkness. Ascorbic acid has been used as a 2.8.2 In Vitro Cytotoxicity Assay
typical antioxidant compound in all the assays for compara-
tive analysis. The percentage inhibition of DPPH free radical MTT cell viability test was utilized for cytotoxicity assay
by plant extract was calculated [36]; described by [39]. The aqueous extract of C. papaya and
CuO NPs was tested for in vitro cytotoxicity by using MTT
%Inhibition = (Ac − As∕Ac) × 100 reagent (5 mg/mL). Cells (2000 cells/well) were seeded in
media (DMEM) in a 96 well plate followed by overnight
Ac: OD of the control incubation. Different concentrations (25, 50, 75, 100 μg/
As: OD of the extract / standard. mL) were added followed by a 24 h incubation. The optical
density was noted at 595 nm using a microplate reader. I­ C50
2.7 Antimicrobial Effect of Copper Oxide NPs values were calculated by GraphPad Prism 5.0.2.

Biological synthesized copper oxide NPs were tested on

Escherichia coli, Klebsiella pneumonia, Yersinia enteroco- 3 Results and Discussion
litica, Bacillus subtilis and Staphylococcus aureus. The anti-
microbial activity was determined by well diffusion method The synthesis of metal NPs from plant extracts has brought
[37]. 100 μL of culture was spread on Nutrient Agar plates. a new perspective to the medical field for the past decade. C.
The wells of 6 mm size were formed on the medium. CuO papaya is widespread throughout the world and available at
NPs were prepared at concentrations of 5 mg/mL and 20 mg/ all seasons. Firstly, the precursor ­(CuSO4.5H2O) reacts with
mL. As a positive control, 0.1 g/mL Ampicillin was used. hydroxyl anion(-OH) which is generated by water, and forms
The prepared nanoparticle solutions and Ampicillin were copper hydroxide. The CuO NPs were efficiently synthesized
poured into wells of 100 μL and incubated at 37 °C, 24 h. from C. papaya leaf extract with 5 mM ­CuSO4.5H2O con-
The experiment was repeated three times and the data pre- taining solution at 25 °C. Color change in solution is marker
sented as mean ± SE (standard error of the mean). of the formation of CuO NPs. While the color of the control
The minimum inhibitory concentration (MIC) of the solution did not change, the C. papaya leaf extract solu-
extract was evaluated by broth dilution method described tion containing 5 mM cupric sulfate began to change color
under [38] guidelines using 2, 3, 5-triphenytetrazolium after 24 h incubation. After 48 h of incubation, the samples
chloride. Samples was dissolved in DMSO were poured containing the extract turned dark blackish brown (Fig. 2).
into 96-well plates and incubated at 37 °C for 24 h. After

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Fig. 2  Visual observation of

CuO NPs synthesis (a) copper
sulphate solution (b) plant
extract (c) final color change

2.0 this study, nanoparticle synthesis was carried out at 60 °C

250 nm
at pH 6 by adding 5 mM copper precursor to different
1.5
plant extract concentrations (5 mL, 10 mL, 15 mL, 20 mL,
Absorbance (a.u.)

25 mL). The peak density at 285 nm is directly proportional

to the increase in plant extract concentration. Linear trend
1.0
was determined between 5 mL and 10 mL (Fig. 4b). The
high rate of phytochemicals compared to copper precursors
0.5 may have decreased the peak density. The lack of copper
ions caused the density to decrease in nanoparticle forma-
tion [43].
0.0
200 300 400 500 600 700 The temperature plays a significant role for the formation
Wavelength (nm) of NPs especially for biochemical reaction. 30, 50, 60, 80
and 100 °C temperatures were studied for production optimi-
Fig. 3  UV-Vis spectra showing absorbance CuO NPs synthesized at zation of CuO NPs. 10 mL of C. papaya leaves extract with
250 nm 5 mM ­CuSO4.5H2O was reacted at different temperatures at
pH 6. The highest peak intensity at 270 nm was observed at
60 °C (Fig. 4c).
3.1 UV–Vis Studies Among other properties, pH is very important in terms of
biochemical reactions in the formation of nanoparticles. In
The maximum absorption peaks of the CuO NPs in the UV- this work, pH 4, 6, 8, 10 and 12 were studied for production
vis spectrum at 250 nm are shown in (Fig. 3). These peaks optimization of CuO nanoparticles. 10 mL of C. papaya
point out the surface plasmon absorption of the metal oxide. leaves extract with 5 mM C ­ uSO4.5H2O was reached at 60 °C
The surface plasmon absorption in the metal oxide NPs is at different pH. By measuring the absorbance of nanoparti-
because of the collective swinging of the free conduction cles produced in different pH, the highest density at 275 nm
band electrons which is excited by the incident electro- was obtained at pH 6 (Fig. 4d).
magnetic radiation. This resonance was observed because The requirement of Cu 2+ to the two ­OH− ions in the
the incident light wavelength exceeded the particle diam- solution provides the formation of copper oxide NPs [44].
eter. Surface plasmon absorption band with a maximum at Therefore, if the pH value in the solution is low (pH < 10),
250 nm indicates the formation of CuO NPs [40–42]. the ­OH− concentration present will be insufficient to form
The size, shape and optical properties of nanoparti- the nanoparticles and the peak density will decrease at low
cles; varies depending on copper precursor concentra- pH. Also, while the pH of the solution was high (pH > 10),
tion, plant extract volume, pH and temperature. 10 mL of the intensity of the peaks decreased. In previous studies, it is
papaya extract was mixed to a concentration of 1 to 5 mM stated that concentration of ­Cu2+ ions decreases by increas-
­CuSO4.5H2O, incubated at 60 °C, then the absorbances of ing the pH of the solution [45].
the obtained CuO NPs were measured (Fig. 4a) As the peak In this study, CuO NPs were produced at different time
density at 280 nm increased with increasing from 1 mM to intervals (0, 12, 24, 48 and 72 h) and time optimization was
5 mM, the study continued with a copper precursor concen- determined. 10 mL of C. papaya leaves extract with 5 mM
tration of 5 mM. ­CuSO4.5H2O was reacted at 60 °C, 6 pH and different times.
Plant extracts have powerful inhibitory agents such as By measuring the absorbance of nanoparticles produced in
polyphenols and antioxidants. Therefore, plant extract con- different times, the highest density at 265 nm was obtained
centration is very important in nanoparticle synthesis. In at 72 h (Fig. 4e).

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Journal of Inorganic and Organometallic Polymers and Materials (2021) 31:1846–1857 1851

Fig. 4  (a) UV-Vis spectra of different substrate concentration on CuO ture conditions on CuO NPs synthesis (e) UV-Vis spectra of different
NPs synthesis (b) UV-Vis spectra of different plant extract concentra- time conditions on CuO NPs synthesis (f) UV-Vis spectra of synthe-
tion on CuO NPs synthesis (c) UV-Vis spectra of different pH condi- sized CuO nanoparticles stability
tions on CuO NPs synthesis (d) UV-Vis spectra of different tempera-

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1852 Journal of Inorganic and Organometallic Polymers and Materials (2021) 31:1846–1857

The stability of copper NPs is lower than that of silver 700

(Ag) and gold (Au) nanoparticles, so its stability is impor-
tant [46]. Stability of the produced CuO NPs by C. papaya 600
leaves extract was studied for a month at RT via SPR moni-

Intensity(a.u)
500 (110)
toring (Fig. 4f). As a result of a month measurements, no
reduction in the high stability density of the nanoparticles (111)
400
produced was observed.
300 (022)
3.2 FTIR Analysis (311)

200
FTIR spectrum was measurement in solid phase using 10 20 30 40 50 60 70 80
potassium bromide pellet technique in the range of 2 Theta (degree)
4000–400 cm−1. The CuO NPs produced by the bioactive
molecules in C. papaya leaves extract by reducing the pre- Fig. 6  XRD pattern of CuO nanoparticles
cursors were confirmed by FTIR.
Figure 5 The reduction of metal ions and the formation
of metal NPs may be caused by these flavonoids and pheno- 111, 022 and 311 of face-centered-cubic structure of CuO
lics. FTIR spectrum of copper oxide NPs by Carica papaya NPs with a monoclinic phase, confirmed by the International
showed up a band spectrum at 2954 due to N-H stretch- Center of Diffraction Data Card (JCPDS NO: 087122). XRD
ing of amine salts, 2850 due to carboxylic acid OH stretch, results showed that the synthesized CuO NPs are crystalline
2050 cm−1 N=C=S stretching, C=O stretching at 1745 cm−1 in nature. The average CuO NPs size calculated for the (110)
due to isothiocyanate and esters, 618 cm−1 can be assigned plane using the Scherrer formula is 77 nm. The XRD results
to vibrations of copper oxide, confirming the formation of of these synthesized CuO NPs are parallel to the literature
highly pure CuO NPs [47] [42]. We took a decision that the CuO NPs were crystalline
in nature cubic in format and without remarkable impurities.
3.3 XRD and DLS Analysis of CuO NPs Dynamic light scattering (DLS) measurements give infor-
mation about the surface charge and size of nanoparticles.
XRD analysis of nanoparticles synthesized from Carica Average particle size distribution of CuO NPs synthesized
papaya is shown in (Fig. 6) and (Table 1). Accordingly, by C. papaya leaves extract was measured as 30- 80 nm
the copper oxide phase of the CuO NPs is confirmed. XRD (Fig. 7).
analysis bared small distinct diffraction peaks at 32.816,
38.842, 61.403 and 71.189, that indexed the planes 110, 3.4 SEM and EDX Analysis

In Fig. 8a structural morphology of CuO NPs synthesized by

102 C. papaya leaves extract was determined using SEM analy-
sis. These nanoparticles synthesized according to this analy-
100 sis appear to be spherical. As the XRD analysis result sup-
ports, aggregations with an average size of less than 50 nm
% Transmittance

98 were observed. In SEM analysis, clumping and formation

of nanoscale are seen in the synthesized nanoparticles. The
96 measured nanosize can be changed by increasing the molar
618 concentrations.
94
1250
92 2850 Table 1  XRD parameters for CuO NPs by C. papaya
1754
90 2954 Crystallite size = 77 nm
Peak 2 Theta (2θ) d-spacing (Å)
4000 3500 3000 2500 2000 1500 1000 500
-1 (110) 32.20 2.78
Wavenumber(cm ) (111) 38.89 2.31
(022) 61.24 1.51
Fig. 5  Fourier-transform infrared spectrum of C. papaya (CP) and (311) 71.16 1.32
synthesized CuO NPs

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Journal of Inorganic and Organometallic Polymers and Materials (2021) 31:1846–1857 1853

40 atomic Cu and 31.91% atomic O elemental peaks are shown

in accordance with the baseline of copper oxide nanopar-
ticles. In previous studies, the copper oxide nanoparticles
30
composition was confirmed by EDX [48].
Intensity (% )

20 3.5 Biological Activity

Phenolic and flavonoid contents were measured in both the

10
crude plant extract and attached in NPs. The phenol content
of C. papaya extract was measured as 39.2 ± 1.89 mg GAE
0 / mg. This amount is 49.9 ± 1.96 mg GAE / mg in copper
0.1 1 10 100 1000 10000 oxide nanoparticles that are synthesized from the extract.
Size (d.nm) CuO NPs were evaluated for bound flavonoid content
(23.5 ± 1.60 mg of QE/mg). In our study, 29.2 ± 1.70 mg of
Fig. 7  DLS pattern of CuO nanoparticles QE / mg flavonoid content were measured in the C. papaya
extract (Fig. 9a).
Figure 8b shows the energy dispersive X-ray analysis In vitro radical scavenging capacity of the synthesized
(EDX) of CuO NPs synthesized by C. papaya. Copper peaks CuO NPs was evaluated by ABTS and DPPH analysis. The
in the sample indicate the presence of oxygen, which con- effects of free radical scavenging activity at different concen-
firms the formation of CuO NPs. The element percentages trations of copper oxide NPs were determined and compared
of the pure nanoparticles that are synthesized by C. papaya with ­IC50 values with respect to the ascorbic acid ­(C6H8O6)
are shown in (Fig. 8b). According to this figure, 68.09% (Table 2). A significant antioxidant activity was measured in

Fig. 8  (a) Representative SEM

images of synthesized CuO
nanoparticles at 2 μm (b) EDX
spectra of CuO nanoparticles

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1854 Journal of Inorganic and Organometallic Polymers and Materials (2021) 31:1846–1857

Fig. 9  Biological activity of C. papaya (C.P) and CuO NPs (a) Total phenolic and total flavonoid content (b) antioxidant by ABTS assay (c)
antioxidant by DPPH assay (d) Minimum inhibitory concentration

Table 2  Antioxidant activity IC50 value (μg/mL) in Escherichia coli and Klebsiella pneumonia, respectively.
of Carica papaya leaf The amount increase in CuO NPs does not have a significant
extract (CP), Copper oxide Sample ABTS DPPH
value on the antimicrobial effect of K. pneumonia. How-
nanoparticles (CuO NPs) and
Ascorbic Acid (AA) CP 152.1 128.0 ever, increasing the amount from 5 mg/mL to 20 mg/mL
CuO NPs 146.0 87.46 increased E. coli inhibition in direct proportion. Among
AA 28.73 29.24 the bacteria, the inhibition zone is higher in Gram negative
bacteria than Gram positive ones. Due to differences in cell
wall structure, the passage of nanoparticles through the cell
CuO NPs synthesized by C. papaya. The increase in sample wall is easier in Gram negatives than Gram positives [49].
concentration increases the cleaning efficiency of CuO NPs. The CuO NPs, which inactivate the enzymes in the bacterial
Phenolic compounds, saponins, tannins, and amino acids in cell membrane, cause bacteria to be inhibited [50]. The cyto-
these synthesized nanoparticles and this allows to have a toxicity of the CuO NPs was measured against (HT-29 cells
higher antioxidant effect than other biologically synthesized and HeLa cells lines) at a various concentration (25–100 μg/
nanoparticles (Fig. 9b, c). mL). The results showed that CuO NPs were potent to kill
The antibacterial effects of the Carica papaya and CuO cancers cells (HT-29 cells and HeLa cells lines). The cells
NPs by C. papaya were evaluated against Escherichia coli, viability decreased with increasing CuO NPs concentration
Klebsiella pneumonia, Yersinia enterocolitica, Staphylococ- (Fig. 10). The cytotoxic effect of the HeLa cell line was dose
cus aureus and Bacillus subtilis using agar well diffusion dependent in cultures, and the I­ C50 values were 171.4 μg/
assay. It has been reported that the synthesized CuO NPs mL for CP and 139.6 μg/mL for CuO NPs effectively. In
have a significant antimicrobial effect in all Gram-negative case of HT-29 cell line the ­IC50 values were 211.1 μg/mL
bacteria used in the study (Table 3). The highest antimicro- for CP and 93.4 μg/mL for CuO NPs. Over 50% inhibition
bial zone diameter formed by nanoparticles synthesized by of cell growth was accomplished with 100 μg/mL CuO NPs.
Carica papaya was measured as 11 ± 0.81 and 11.33 ± 0.47 Small size and spherical shapes of nanaoparticles may have

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Journal of Inorganic and Organometallic Polymers and Materials (2021) 31:1846–1857 1855

Table 3  The antibacterial effects of the C. papaya and CuO NPs

Zone diameter (mm)
S. No. Bacterial strains Plant extract (CP) Positive control CuO NPs Positive control Negative
(Ampicillin- (Ampicil- control
(5 mg/mL) (20 mg/mL) 0.1 g/mL) (5 mg/mL) (20 mg/mL) lin-0.1 g/mL) (water)

01. Escherichia coli 6.66 ± 0.94 7.33 ± 0.47 24.0 ± 0.81 8.66 ± 0.47 11.00 ± 0.81 21.00 ± 0.81 ND
02. Klebsiella pneu- 6.66 ± 0.47 7.66 ± 0.47 23.0 ± 0.81 11.0 ± 0.81 11.33 ± 0.47 23.3 ± 0.81 ND
monia
03. Yersinia entero- 6.66 ± 0.47 7.33 ± 0.47 23.0 ± 0.94 8.66 ± 0.47 9.00 ± 0.81 20.00 ± 0.81 ND
colitica
04. Staphylococcus 6.33 ± 0.47 6.33 ± 0.47 23.0 ± 0.00 – – 25.30 ± 0.94 ND
aureus
05. Bacillus subtilis 6.33 ± 0.47 6.66 ± 0.47 25.0 ± 0.00 6.33 ± 0.47 6.66 ± 0.47 25.00 ± 0.00 ND
MIC (μg/mL) Plant extract (CP) Ab (Antibiotic) CuO NPs

01. Escherichia 500 0.30 125 ND

coli
02. Klebsiella 500 0.30 125 ND
pneumonia
03. Yersinia 500 0.30 125 ND
enterocol-
itica
04. Staphylococ- 500 0.30 250 ND
cus aureus
05. Bacillus 500 0.30 250 ND
subtilis

Fig. 10  Anticancer effect of CuO NPs from leaf extract of C. papaya (a) HT-29 and (b) Hela cell

an effect on cytotoxic activity. For example, in a study by suppress the proliferation of HeLa cells. CuO NPs shows
Sivaraj et al., CuO NPs synthesized from A. indica have a the 56.8% of control growth at the highest concentration of
particle size of 26 to 30 nm and show very strong cytotoxic 80 μg/mL against HT-29 cell line compared to other treat-
activity in MCF7 breast cancer cells [51, 52]. In previous ments [53, 54].
study showed that the CuONPs can induce apoptosis and

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1856 Journal of Inorganic and Organometallic Polymers and Materials (2021) 31:1846–1857

4 Conclusion 8. V. Castagnola, J. Cookman, J.M. De Araújo, E. Polo, Q. Cai,

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