CALLUS-MEDIATED BIOSYNTHESIS OF AG AND ZNO NANOPARTICLES USING AQUEOUS CALLUS EXTRACT OF CANNABIS SATIVA: THEIR CYTOTOXIC POTENTIAL AND CLINICAL ...
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Green Processing and Synthesis 2021; 10: 569–584
Research Article
Mehreen Zaka*, Syed Salman Hashmi, Moiz A. Siddiqui, Lubna Rahman, Sadaf Mushtaq,
Haider Ali, Christophe Hano, and Bilal Haider Abbasi*
Callus-mediated biosynthesis of Ag and ZnO
nanoparticles using aqueous callus extract of
Cannabis sativa: Their cytotoxic potential and
clinical potential against human pathogenic
bacteria and fungi
https://doi.org/10.1515/gps-2021-0057 namely Mucor, Aspergillus flavus, Aspergillus fumigatus,
received June 07, 2021; accepted August 16, 2021 Aspergillus niger, and Fusarium solani, were utilized for
Abstract: In this paper, we have presented the method antifungal assay. Cytotoxicity assay was also performed
of green synthesis of ZnO and Ag-NPs using the callus using the HepG2 cell line. The results showed considerable
extract (CE) of medicinally important Cannabis sativa. antibacterial and antifungal activities. It also showed better
The synthesis of nanoparticles (NPs) was confirmed by cytotoxicity values as compared to the control.
UV-Vis spectroscopy, while as far as the size and shape of Keywords: Cannabis sativa, callus extract, green synth-
the NPs were concerned, they were validated using the esis, Ag-NPs, ZnO-NPs
techniques of X-ray diffraction and scanning electron micro-
scopy, respectively. The energy dispersive X-ray analysis
graph confirmed the constitution of elements along with
the surface chemical state of NPs. Fourier transform-infrared 1 Introduction
spectroscopy was utilized for the confirmation of biomole-
cules capping the NPs. In order to test the application of Apart from the selection of edible plants, including wheat,
these biosynthesized NPs on biological entities, four bac- rice, or maize, by earlier civilizations in order to obtain
terial strains, including Bacillus subtilis, Klebsiella pneu- improved yields and quality, Cannabis sativa has also
monia, Staphylococcus aureus, and Pseudomonas aerugi- been selected and grown artificially in large quantities,
nosa, were used. On the other hand, five fungal strains, making it the most renowned plant species on the earth.
According to Richard E. Schultes, who was responsible
for the origin of ethnobotany, although humans have
extensively used the products of this plant for more
* Corresponding author: Mehreen Zaka, Department of
Biotechnology, Quaid-i-Azam University, Islamabad-45320,
than 10,000 years, it has not yet been recognized in the
Pakistan, e-mail: mzaka@bs.qau.edu.pk positive way it deserves [1]. C. sativa belongs to a group of
* Corresponding author: Bilal Haider Abbasi, Department of herbaceous shrubs normally having a height of 1–2 m. It
Biotechnology, Quaid-i-Azam University, Islamabad-45320, was estimated that its cultivation first started in Russia in
Pakistan, e-mail: bhabbasi@qau.edu.pk about 4000 B.C. and is now widely used as a drug around
Syed Salman Hashmi, Lubna Rahman, Haider Ali: Department of
the world by millions of people in the form of marijuana,
Biotechnology, Quaid-i-Azam University, Islamabad-45320, Pakistan
Moiz A. Siddiqui: Department of Chemistry, Faculty of Sciences, hashish, or bhang. It has also been exploited as a source
Synergy Education, Faisalabad, Pakistan of fiber and oil. As far as its chemistry is concerned, it
Sadaf Mushtaq: Department of Biotechnology, Quaid-i-Azam contains approx. 480 compounds, belonging to various
University, Islamabad-45320, Pakistan; Functional Genomics chemical classes, including steroids, fatty acids, amino
Group, Institute of Biomedical & Genetic Engineering (IBGE), Sector
acids, alkaloids, terpenoids, and flavonoids [2]. The con-
G-9/1, Islamabad, Pakistan
Christophe Hano: LBLGC (Laboratoire de Biologie des Ligneux et des
centrations of these compounds vary according to the
Grandes Cultures) - INRAE USC1328, University of Orleans, type, age, variety, growth, and storage conditions of the
Department of Plant Sciences 45067 Orleans CEDEX 2, France tissue. Phytochemicals derived from C. sativa have been
Open Access. © 2021 Mehreen Zaka et al., published by De Gruyter. This work is licensed under the Creative Commons Attribution 4.0
International License.
570 Mehreen Zaka et al.
used in medicines as anti-vomiting, anti-spasm, anti-epi- toward a greener and a much safer synthesis approach
lepsy, anti-asthma, and as an appetite stimulant [3]. [17,18]. The green synthesis approach for the Ag-NP synth-
Due to the remarkable properties of nanoparticles esis involves the use of living organisms like microbes,
(NPs) such as electrical conductivity, mechanical strength, fungi, and plants or their products [19]. Ag-NPs have
as well as magnetic and thermal properties, NPs have been effectively employed in research for multiple applica-
found their way into research in almost every field. But tions, including optical devices, electronics, biosensors,
the problem associated with their synthesis is that it and biolabeling. Ag-NPs have also been proved to be a
requires processes such as laser ablation, inert gas con- potent antimicrobial and anti-cancer, wound-healing, water
densation, or chemical reduction, which adds to the toxi- treatment, and drug delivery agents [20].
city of the process [4]. In order to synthesize NPs, more Previously, the wild plant of C. sativa has been
human and environmentally friendly methods are pre- exploited for silver (Ag) and gold (Au) nano-synthesis,
ferred. Green synthesis involving plant extracts, micro- but no reports are available on the exploitation of the
organisms, and enzymes is a very promising alternative to callus extract (CE) for the NP synthesis [8]. The induction
the chemical and physical methods of NP synthesis [5]. Not of callus in the controlled in vitro conditions offers an
only can it minimize the toxicity of the particles, but it can increased quantity of secondary metabolites. The plant
also increase the quality of the product. Phyto-nanotech- growth regulators (PGRs) used as stress inducers in the
nology is also emerging as a simple, less expensive, time- callus induction are responsible for the increased sec-
friendly, and produces stable NPs [6]. Several types of NPs, ondary metabolites production under specific lab condi-
especially metallic NPs like silver (Ag) [7], gold (Au) [8], tions [12]. In this study, we have utilized Cannabis sativa
zinc oxide (ZnO) [9], copper oxide (CuO) [10], iron (Fe), (CS) CE to synthesize Ag and ZnO NPs. As the extract from
and lead oxide (PbO) [11] have been successfully prepared callus is supposed to have an increased quantity of sec-
using different plants. Out of these, silver nanoparticles (Ag- ondary metabolites, it is a useful source for capping,
NPs) are among the most widely synthesized NPs owing to reduction, and NP stabilization. The characterization of
their tremendous antimicrobial potential [8]. Similarly, zinc the biosynthesized NPs was done using UV-Vis spectro-
oxide nanoparticles (ZnO-NPs) are also popular in biome- scopy, Fourier transform-infrared spectroscopy (FT-IR),
dical research due to their biocompatibility [12]. X-ray diffraction analysis (XRD), scanning electron micro-
ZnO-NPs are regarded as metal oxide NPs having scopy (SEM), and energy dispersive X-ray analysis (EDX).
a diverse range of significant characteristics like bio-
compatible nature, cheap synthesis methods, and easy
accessibility [13]. The worldwide annual production of
ZnO-NPs is approximately 31,000–34,000 metric tons, 2 Materials and methods
which made these metal oxide NPs a popular choice
[14]. The semiconductor properties of ZnO-NPs due to
2.1 Plant collection
the presence of zinc and oxygen are promising. The
exciton binding energy ranges of ZnO-NPs are up to
Wild plants of Cannabis sativa (L.) were collected from
60 meV, and the bandgap is 3.37 eV. The tolerance of
the surroundings of Quaid-i-Azam University, Islamabad,
electric fields is increased and falls in detectable ranges.
and their fresh leaves were taken. In the next step, the
These NPs are effective to work alone as well as in the
leaves were washed two times, with high care so that all
form of complexes and bimetallic forms with other metals
the debris gets removed before taking it to the laminar
[12]. The ZnO-NP synthesis protocols are eco-friendly and
flow hood. Then, 1% mercuric chloride solution was
biocompatible, which make them a popular candidate to
applied on the leaves for about 15 s, and these were
be chosen for a variety of applications, including drug
then washed five times with autoclaved distilled water.
delivery, diagnosis, biological research, agricultural research,
The leaves were then dried on autoclaved filter paper.
biosensors, cosmetics, solar cells, semiconductors, and bio-
logical imaging probes [15].
Ag-NPs are the most common and widely used metal
NPs due to their multifarious properties [16]. Ag-NPs 2.2 Establishment of callus cultures from
have been successfully synthesized using both physical the wild leaf explant
and chemical approaches; however, the time-consuming,
expensive and toxic nature of these approaches make them Inoculation of the leaf explants having a size of about
less attractive, and hence, increasing focus is projected 1–2 cm was done on the MS (Murishage and Skoog,
Callus-mediated biosynthesis of Ag and ZnO nanoparticles 571
Table 1: C. sativa callus induction using different concentrations of resulting mixture was boiled for 8 min. After boiling, the
NAA and TDZ temperature of Cannabis CE was cooled to room tempera-
ture, and it was filtered through Whatman no. 1 filter
Sr No NAA (mg·L−1) TDZ (mg·L−1) paper. The filtrate was then stored at a temperature of
1 0.50 1.00 4°C after adjusting its volume to 100 mL.
2 1.00 1.00
3 1.50 1.00
4 2.00 1.00
2.4 Biosynthesis of ZnO-NPs
The plant extract and metallic salt concentrations, along
1962) medium, consisting of 8 g of agar and 30 g of with pH, temperature, and response time, play a vital role
sucrose. Different concentrations of PGRs for cannabis in the biosynthesis of metal NPs [24]. The precursor salt
callus induction have been reported in the literature used for the synthesis of ZnO NPs was zinc acetate dihy-
and were exploited [21–23]. Different combinations of drate (C4H6O4Zn). A 0.02 M zinc acetate dihydrate solu-
PGRs like IBA (indole-3-butyric acid), BA (6-benzylami- tion was prepared for the synthesis of ZnO NPs. In the
nopurine), Kn (kinetin), 2,4-D(2,4-dichlorophenoxyacetic first step, 1 mL of the sample extract was mixed sepa-
acid), NAA (1-naphthalene acetic acid), and TDZ (thidia- rately with 0.02 M zinc acetate (50 mL) solution under
zuron) were added into the medium. The combination of continuous stirring. The pH of the resulting mixture
NAA and TDZ gave the best response in callus induction was adjusted at 12 using sodium hydroxide (NaOH). Alka-
at different concentrations. Optimization under different loids, terpenoids, and flavonoids available in the extract
PGR concentrations was done according to the protocols caused the reduction. The resulting precipitates were white
available in the previous literature [22]. Briefly, the con- and pale, which were subjected to washing with ethanol
centrations used for optimization are given in Table 1. before being washed with distilled water twice (Figure 1a).
The best response was recorded at concentrations of The sample was then centrifuged for 15 min at 6,000 rpm
0.50 mg·L−1 NAA and 1.00 mg·L−1 TDZ. Under laminar prior to incubation at 60°C overnight to dry. Drying of the
flow, pH was balanced between 5.6 and 5.7, utilizing 1 N samples yielded powdered ZnO-NPs, which were then uti-
HCl and 1 N NaOH. The experiment was conducted using lized for characterization and biological assays.
three replicates such that the inoculation of the three
explants was done in each conical flask as one replicate
for one PGR concentration. These flasks were then placed 2.5 Biosynthesis of Ag-NPs
at 25°C for a 16/8 h photoperiod in a growth room.
For the synthesis of Ag-NPs, the protocols of Abbasi et al.
[8] were used with slight modifications. In brief, the calli
2.3 Calli extract preparation for the extract was separately mixed with 1 mM AgNO3 (pre-
biosynthesis of NPs cursor salt) in different ratios (1:2, 1:5, and 1:10). The
incubation of the blend was done at room temperature
For the CE preparation, 10 g of callus was added to 100 mL for 24 h. A color change from yellow to brown was
of distilled water in an Erlenmeyer flask (500 mL). The observed (Figure 1b), then the absorbance of each test
Figure 1: (a) Dried pale white precipitates of ZnO-NPs; (b) color change after the Ag-NP synthesis at different concentrations.
572 Mehreen Zaka et al.
sample was recorded through a UV-Vis spectrophoto- In the initial step, a small quantity of the sample was placed
meter in order to confirm the biosynthesis of Ag-NPs on a grid made of copper before coating it with carbon. This
from the calli extract. Once the Ag-NP biosynthesis was coat was then subjected to drying under a mercury lamp for
complete, the NPs were separated through centrifugation about 5 min; the samples were examined, and their photo-
at 12,000 rpm for 10 min. The isolated Ag-NPs were washed graphs were captured. EDX analysis was carried out to ana-
thrice using distilled water. lyze the elemental composition. The samples were coated
with carbon, dried, and further analysis was performed
using SEM equipped with an EDX detector.
2.6 Characterization techniques
2.7 Antibacterial activity
2.6.1 UV vis spectroscopy
For antibacterial activity assay, a well diffusion method
A Shimadzu UV-1650 PC Spectrophotometer was used to was used according to Bereksi et al. [25]. The bacteria
confirm the biosynthesis of Ag-NPs and ZnO-NPs. The against which the activity of these NPs was evaluated
light absorption spectra were provided using distilled included both Gram-positive and Gram-negative bacteria.
water as a reference. The surface plasmon resonance Gram-positive bacteria included Bacillus subtilis (ATCC:
peak of Ag-NPs and ZnO-NPs was noted. The spectra 6633) and Staphylococcus epidermidis (ATCC: 14990), and
were recorded at different times to check the absorbance Gram-negative bacterial strains included Klebsiella pneu-
and wavelength of the NPs. monia (ATCC: 4617) and Pseudomonas aeruginosa (ATCC:
9721). In the first step, the inoculation of bacterial strains
was done in 10 mL tryptic soy broth (TSB), which was
2.6.2 XRD analysis then incubated at 120 rpm for 24 h at 37°C temperature in
a shaking incubator. The inoculum was prepared and
The crystalline nature of the structures of Ag-NPs and standardized at 1 × 108 CFU·mL−1 for each bacterial strain.
ZnO-NPs was determined using an XRD (Model-D8 Advance, Further, on a Trypticase soy agar media plate, each of the
Germany) instrument. In this instrument, the cathode ray bacterial broth cultures (100 µL) was poured and spread in
releases the X-rays in the direction of the sample. For order to get a uniform bacterial lawn using a sterilized
the examination and study, 1 mg of each Ag-NPs and glass rod. In the case of NPs, in each of the 5 wells,
ZnO-NPs were taken. For determining the size of the 30 µL of each NP with serial dilutions of 5, 4, and 3 mg·mL−1
NPs, the Debye–Scherrer equation was used as follows: for Ag-NPs and 10, 5, and 4 mg·mL−1 for ZnO-NPs was intro-
duced. The positive control used in this experiment was
D = kλ / β cos θ (1)
Cefixime. The broth dilution method of Tai et al. [26] was
where λ is the X-ray wavelength (1.5418 Å), k is the shape utilized to calculate the corresponding MICs.
factor (0.94), θ is the Bragg’s angle, and β is the full width
at half-maximum in radians.
2.8 Antifungal activity
2.6.3 Fourier transform infrared spectroscopy (FTIR) For antifungal activity assay, the agar well diffusion method
of Nath and Joshi [27] was used with some slight modifica-
FTIR spectroscopy (Spectrum One, Perkin Elmer, Waltham, tions. Five fungal strains (pathogenic), including Aspergillus
MA, USA) was used for determining the functional groups fumigatus (FCBP-1264), Aspergillus niger (FCBP-0198),
attached to ZnO-NPs and Ag-NPs. In the FTIR spectroscope, Fusarium solani (FCBP-1114), Aspergillus flavus (FCBP-
KBr pellets were utilized, and the data was recorded in the 0064), and Mucor (FCBP-0041), were taken and the activity
spectral range of 500 to 3,500 cm−1. of NPs was investigated. The media used for the preparation
of broth cultures of pathogenic fungi was the Sabouraud
dextrose liquid medium (Oxoid: CM0147). After inoculating
2.6.4 SEM and EDX analysis the fungal strains in the broth, it was incubated in a shaking
incubator at a temperature of 37°C for 24 h. The optical
The morphology and the sizes of NPs were determined density (OD) of the broth cultures was adjusted to 0.5 before
using an SEM (MIRA3 TESCAN model), operated at 10 kV. carrying out the assay. About 50 µL of the broth culture
Callus-mediated biosynthesis of Ag and ZnO nanoparticles 573 Figure 2: Cannabis sativa callus induction stages: (a) initiation of callus, (b) callus formation 1.5 weeks after culture, (c) growth of the callus, and (d) cell death. from standardized cultures was taken and was spread on a 2.9 Cytotoxicity screening Petri plate containing the Sabouraud dextrose agar medium using a sterilized glass rod in order to obtain a steady lawn 2.9.1 Cell culture of pathogenic fungal strains. The positive control used in this experiment was Amphotericin B. The plates were incu- Dulbecco’s modified Eagle’s medium consisting of 10% bated at 37°C for 24–48 h, and the inhibition zones were fetal calf serum along with 100 U·mL−1 penicillin, L-glu- recorded after a certain period of time. tamine (2 mM), and 100 μg·mL−1 streptomycin at a Figure 3: UV-Vis absorption spectra of (a) Ag-NPs and (b) ZnO NPs.
574 Mehreen Zaka et al. Figure 4: XRD analysis of Ag-NPs and ZnO-NPs. temperature of 37°C and a humid atmosphere that con- 2.9.2 Cell viability assay tained 5% CO2 and 1 mM Na-pyruvate as a supplement was used for culturing human hepatocellular carcinoma In a sulforhodamine B (SRB) assay, ZnO-NPs and Ag-NPs cells (ATCC HB-8065). Apart from this, 0.5 mM trypsin/EDTA were tested to check their cytotoxic effect on the HepG2 was used at room temperature for 1 min in order to harvest cell line. For cytotoxic screening assay, NP samples were the cells. prepared by suspending them in deionized water. Then, a Figure 5: FTIR spectra of Ag-NPs and ZnO-NPs.
Callus-mediated biosynthesis of Ag and ZnO nanoparticles 575
Figure 6: SEM graphs of Ag and ZnO-NPs.
Figure 7: EDX analysis of Ag and ZnO-NPs.
plate containing 96 wells and having a density of 12,000 cells as positive and negative controls of the experiment,
per well was utilized for seeding HepG2 cells having more respectively. The OD values of only the media and only
than 90% confluency, where they were allowed to adhere the sample were represented by blanks and were con-
for 2 h at 37°C. NPs (200 µg·mL−1) were applied to these
cells for 24 h. Pre-chilled trichloroacetic acid (50%) was
used to attach these cells, followed by their incubation Table 2: EDX analysis (atomic percentages of elements)
for 1 h at 4°C. After incubation, the cells were rinsed
thrice using deionized water. The plates thus formed NPs Element Weight (%) Atomic (%)
were dried using dry air before staining the cells with Ag-NPs OK 33.14 76.97
0.01% SRB dye and then again incubated at room tem- Ag L 66.86 23.03
perature for 30 min. Acetic acid (1%) was then applied ZnO-NPs OK 28.05 61.43
Zn K 71.95 38.57
on the plates in order to remove any unbounded dye.
Total 100.00
Doxorubicin (34 µM) and untreated cells were designated
576 Mehreen Zaka et al.
sidered as controls. An Olympus CK2 light microscope While the cell inhibition percentage was determined
equipped with a digital camera was utilized to take the by the following formula:
snapshots. Using triplicates of each of the samples, this
Cell inhibition percentage (%) = 100 − Cell viability
experiment was repeated two times. Then, 100 µL of (3)
10 mM Tris having a pH of 8 was introduced into each percentage (%)
of the wells at room temperature for 5 min in order
to make SRB dye more soluble. A microplate reader
(Platos R 496, AMP) was then utilized to analyze the
absorbance values at a wavelength of 565 nm. The viabi-
2.10 Statistical analysis
lity percentage was calculated relative to the untreated
All the statistical calculations were done in triplicates. The
sample by the following formula:
values present in the text as well as in the figures were
Cell viability percentage (%) = statistically examined as mean ± SE (standard error).
Absorbance of sample − Absorbance of control Origin Pro (version 8.5) software was used to analyze all
× 100
Absorbance of untreated cells − Absorbance of media the graphs. The probability value was viewed to be notably
(2) contrasting at the point of P < 0.05 (95%).
Figure 8: Antibacterial activity of Ag-NPs at different concentrations (5, 4, and 3 mg·mL−1); Ab – antibiotics, CE – callus extract.
Callus-mediated biosynthesis of Ag and ZnO nanoparticles 577
3 Results and discussion
Table 3: Antibacterial activity of CE and callus-mediated Ag-NPs and ZnO-NPs against two Gram-negative (Klebsiella pneumoniae, Pseudomonas aeruginosa), and two Gram-positive bacteria
Precursor
8 ± 0.40
8 ± 0.40
8 ± 0.40
7 ± 0.35
salt
3.1 Callus induction
10 ± 0.50
14 ± 0.70
13 ± 0.65
7 ± 0.35
Different studies have shown the optimization of Cannabis
callus induction at different concentrations of PGRs [21–23].
CE
The protocol of callus induction was optimized by using the
(10 μg·mL−1)
PGR concentrations reported in the previous literature. The
30 ± 1.50
30 ± 1.50
30 ± 1.50
27 ± 1.35
Cefixime
development of callus cultures was done using leaf explants
from the wild C. sativa plant. Leaf portions of about 3–4 mm
ZnO-NPs
were cut out and were then cultured on the Murashige and
4 mg·mL−1
Skoog medium. The optimized callus was off-white and fri-
20 ± 1.00
15 ± 0.75
6 ± 0.30
6 ± 0.30
able obtained at concentrations of 1.00 mg·L−1 TDZ and
0.50 mg·L−1 NAA of PGRs (Figure 2).
5 mg·mL−1
12 ± 0.60
12 ± 0.60
15 ± 0.75
6 ± 0.30
3.2 UV-Vis analysis
Diameter of the inhibition zone (mm)
A preliminary confirmation of biosynthesis of Ag-NPs and
10 mg·mL−1
optimization of suitable CE to precursor salt ratio for the
18 ± 0.90
14 ± 0.70
14 ± 0.70
13 ± 0.65
biosynthesis using CE was carried out. The mixture of
AgNO3 and the extracts mixed in different ratios were
characterized using a UV-Vis spectrophotometer. The mix-
Precursor salt
ture for CE and AgNO3 in 1:2 displayed maximum absorbance
Gram negative
Gram positive
(2.188) at a wavelength of 382 nm. At 1:5, the maximum
6 ± 0.30
6 ± 0.30
9 ± 0.45
5 ± 0.25
absorbance (1.811) was recorded at 422 nm, while at 1:10
the maximum absorbance (1.284) was recorded at 430 nm.
Due to the optimized absorbance and a sharp peak near
(10 μg·mL−1)
28 ± 1.40
422 nm, 1:5 was selected for further experiments and charac-
30 ± 1.50
35 ± 1.75
35 ± 1.75
Cefixime
terization. Figure 3a shows the spectrophotometric analysis
of CE-mediated Ag-NPs at the aforementioned ratios. The
biosynthesis of ZnO NPs was also confirmed using spectro-
4 ± 0.20
5 ± 0.25
5 ± 0.25
5 ± 0.25
photometric analysis. Soon after the formation of precipi-
Ag-NPs
CE
tates, the mixture was subjected to a spectrophotometer.
The maximum absorbance was recorded at 362 nm, which
3 mg·mL−1
12 ± 0.60
10 ± 0.50
is a characteristic of ZnO NPs. Figure 3b represents the spec-
7 ± 0.35
7 ± 0.35
trophotometric analysis of CE-mediated ZnO NPs.
Values are average of triplicates ± standard error.
(Staphylococcus epidermidis, Bacillus subtilis)
4 mg·mL−1
14 ± 0.70
10 ± 0.50
8 ± 0.40
8 ± 0.40
3.3 XRD results
For determining the properties like film thickness, purity
Abbreviation: CE – callus extract.
of phase (crystalline nature), and the ordering of atoms in
5 mg·mL−1
12 ± 0.60
13 ± 0.65
11 ± 0.55
11 ± 0.55
amorphous materials, the XRD technique is applied. In
this analysis, the overall structure of the material is
observed based on the X-ray penetration. The distinct
peaks for silver were obtained at 38.10°, 46.42°, 69.27°,
Bacterial strains
Bacillus subtilis
Staphylococcus
Pseudomonas
and 76.87°, which correspond to the lattice patterns (111),
pneumoniae
epidermidis
aeruginosa
(200), (220), and (311) as discussed by Amin [28]. The
Klebsiella
peak at the (111) plane is the predominant orientation
because it is more intense as compared to other peaks.
578 Mehreen Zaka et al.
Figure 9: Antibacterial activity of ZnO-NPs at different concentrations (10, 5, and 4 mg·mL−1); Ab – antibiotics, CE – callus extract.
The results show that the synthesized Ag-NPs had a 3.4 FT-IR analysis
face-centered cubic (fcc) structure. Figure 4 represents
the XRD pattern for CE-mediated Ag-NPs and ZnO-NPs. The confirmation of capping of phytochemicals upon NPs
The 2theta values of the peaks obtained were 33.81°, 34.06°, was done by subjecting the powdered NPs to FT-IR ana-
35.91°, 47.19°, 56.29°, and 62.65° which corresponds to lysis. FT-IR analysis of C. sativa CE-mediated Ag-NPs
(100), (002), (101), (110), (103), and (200) planes of lattices, showed peaks at 3288.44, 2918.46, 2360.84, 2119.04, 1652.46,
respectively, as discussed by Siddiquah et al. [12]. A crystal- 1558.07, 1540.47, 1521.11, 1506.06, 1456.70, 1036.62, and
line structure for callus-mediated ZnO-NPs was revealed 667.73 cm−1 (Figure 5a), while C. sativa CE-mediated ZnO-NPs
through XRD data. The average size of Ag-NP and ZnO-NP showed peaks at 3367.44, 2926.43, 2360.55, 1662.53, 1658.50,
nanoparticles was calculated by using Debye–Scherer’s 1540.70, 1506.74, 1456.51, 1032.80, 871.86, and 666.85 cm−1
equation. First, individual peaks sizes were calculated, (Figure 5b). The peaks at 3288.44 and 3367.44 cm−1 show
and then the average of all the individual peaks in the graph O–H stretching in alcohols, the peaks at 2918.46 and
was taken, and the average size was found to be 14.85 nm 2926.43 cm−1 show C–H stretching of alkanes [7], the peaks
for Ag-NPs and 16.43 nm for ZnO-NPs. at 2360.84 and 2360.55 cm−1 show asymmetric C]OCallus-mediated biosynthesis of Ag and ZnO nanoparticles 579
Figure 10: Antifungal activity of Ag-NPs against five different fungal strains at different concentrations (5, 4, and 3 mg·mL−1);
Ab – antibiotics, CE – callus extract.
stretching [12], the peaks at 1652.46 and 1658.50 cm−1 show confirms the presence of elemental silver and a hint of the
C]C stretching in alkenes, the multiple peaks between presence of oxygen in the sample. A strong signal of ele-
1,400 and 1,600 cm−1 show C]C stretching in aromatic mental silver can be seen around 3 keV. Noohpisheh et al.
compounds, the peaks at 1036.62 and 1032.80 cm−1 show [31] also reported the synthesis of Ag-ZnO composites
C–O stretching, the peak at 871.86 cm−1 shows C–N stretching using Trigonella foenum-graecum leaf extract. In the
in amines [8], while the peaks at 667.73 and 666.85 cm−1 show case of ZnO-NPs, the presence of both elemental Zn and
C–Cl stretching in alkyl halides. The relationship between O is evident. Strong Zn signals were observed around
the primary and secondary metabolites and the Ag-NPs and 1 keV while two other signals can be seen at 8.6 and
ZnO-NPs can be clearly observed from the findings. This 9.6 keV. The signal for elemental O was obtained around
attachment of functional groups is due to the presence of 0.5 keV. No signals were found for any other metal in the
electrostatic forces between the positively charged zinc ions sample, which proves that the NPs were highly pure.
and negatively charged groups present in organic molecules. It The results are in agreement with Siddiquah et al. [12].
is the result of this binding force that makes these NPs per- The relative concentration of each elemental component
fectly suitable for different applications including the study of is given in Table 2 for Ag-NPs and ZnO-NPs. The data in
biological interactions [29] and drug delivery [30]. the table also show that the synthesized NPs were free of
any impurities.
3.5 SEM and EDX analysis
3.6 Antibacterial activity
Figure 6 shows the SEM images of Ag-NPs and ZnO-NPs,
respectively, while Figure 7 shows the EDX analyses of In antibacterial activity, inhibition against all the bacterial
Ag-NPs and ZnO-NPs, respectively. In the case of Ag-NPs, strains was observed after treatment with CE-mediated
highly aggregated NPs having roughly spherical shape Ag-NPs and ZnO-NPs. The experiment was performed at
was observed. Similar results were reported by Hashmi various concentrations, i.e., 5, 4, and 3 mg·mL−1 for Ag-NPs
et al. [7]. The ZnO-NPs synthesized were shown to be and 10, 5, and 4 mg·mL−1 for ZnO-NPs. It was found that
highly aggregated and needle-shaped stacked together CE-mediated Ag-NPs showed the highest activity against
in flower-like morphology. The EDX spectrum for Ag-NPs S. epidermidis with 14 mm zone followed by P. aeruginosa580 Mehreen Zaka et al.
with 12 mm zone (Figure 8). ZnO-NPs showed promising
B (10 μg·mL−1)
Amphotericin
Table 4: Antifungal activities of Ag-NPs, ZnO-NPs, and CE of Cannabis sativa analyzed against pathogenic fungi Mucor, Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, and
activity against K. pneumoniae with an inhibition zone of
40 ± 2.00
40 ± 2.00
20 ± 1.00
45 ± 2.25
25 ± 1.25
20 mm, P. aeruginosa with a maximum zone of inhibition
of 18 mm, followed by B. subtilis with 15 mm zone within
MIC ranges (300–120 μg·mL−1) (Table 3 and Figure 9). Our
Precursor results are in agreement with Lara et al. [32]. According to
10 ± 0.50
10 ± 0.50
15 ± 1.00
6 ± 0.30
5 ± 0.25
them, the bactericidal effect of Ag-NPs was potent against
the P. aeruginosa strain. Ag-NPs have significant antibac-
Salt
terial potential against various strains of bacteria, such as
Gram-negative and Gram-positive bacterial strains. Sev-
4 mg·mL−1
20 ± 1.00
15 ± 0.75
7 ± 0.35
7 ± 0.35
5 ± 0.25
ZnO-NPs
eral antimicrobial properties of Ag-NPs have been investi-
gated due to their chemical stability, wound-healing cap-
ability, catalytic activity, or high conductivity [33]. Ag-NPs
−1
exhibit antimicrobial properties that mainly rely on super-
12 ± 0.60
10 ± 0.50
30 ± 1.50
15 ± 0.75
6 ± 0.30
5 mg·mL
ficial contact; thus, they prevent respiratory chains by
inhibiting the enzymatic system and change the synthesis
pattern of DNA. Since the small size of NPs gives a large
−1
surface area as compared to other salts (including silver
15 ± 0.75 20 ± 1.00
12 ± 0.60
10 ± 0.50
30 ± 1.50
10 mg·mL
11 ± 0.55
Diameter of the inhibition zone (mm)
particulate), it thus provides a platform to bind with micro-
organisms via the cell membrane and enter inside a cell
[34]. The mode of antibacterial action of ZnO-NPs involves
6 ± 0.30
6 ± 0.30
7 ± 0.35
7 ± 0.35
entering into a bacterium and getting attached to its cell
CE
membrane. These NPs alter the process of production
of energy by affecting the cell membrane, thus releasing
Amphotericin B
the cell contents [35]. This result is in agreement with the
(10 μg·mL−1)
previous literature. Siddiquah et al. [12] reported that
45 ± 2.25
30 ± 1.50
30 ± 1.50
17 ± 0.85
35 ± 1.75
ZnO-NPs have a broad-spectrum bactericidal effect. Accord-
ing to Gunalan et al. [45], the bactericidal activity of the
biosynthesized ZnO-NPs is increased as compared to che-
Precursor
12 ± 0.60
mically synthesized ZnO-NPs. Owing to the high surface
7 ± 0.35
5 ± 0.25
5 ± 0.25
5 ± 0.25
area and chemical stability of ZnO-NPs, it is available to
salt
the cytoplasm of microbes and, as a result, the destruction
of cells leads to apoptosis [46,47]. Likewise, Nazir et al. [9]
−1
10 ± 0.50
6 ± 0.30
6 ± 0.30
4 ± 0.20
3 mg·mL
5 ± 0.25
examined that the biosynthesized ZnO-NPs by using CE of
Ag-NPs
Silybum marianum has greater potential against B. subtilis
and K. pneumonia, as compared to chemically synthesized
ZnO-NPs.
−1
6 ± 0.30
4 ± 0.20
4 mg·mL
7 ± 0.35
5 ± 0.25
5 ± 0.25
Values are average of triplicates ± standard error.
−1
3.7 Antifungal assay
6 ± 0.30
6 ± 0.30
6 ± 0.30 6 ± 0.30
6 ± 0.30 7 ± 0.35
6 ± 0.30 7 ± 0.35
5 mg·mL
Abbreviation: CE – callus extract.
The antifungal potential of callus-mediated Ag-NP and
ZnO-NP samples was evaluated by a modified agar well-
7 ± 0.35
5 ± 0.25
diffusion assay described previously by Ginovyan et al.
CE
[36] with some modifications. All the NPs showed different
activities. Antifungal activity was investigated for the given
Fusarium solani
Fusarium solani
Fungal strains
stock concentrations of 5, 4, and 3 mg·mL−1 for CE-mediated
Aspergillus
Aspergillus
Aspergillus
Ag-NPs and 10, 5, and 4 mg·mL−1 for CE-mediated ZnO-NPs.
fumigatus
Mucor
flavus
The NPs were evaluated against pathogenic fungi includ-
niger
ing Mucor (FCBP-0041), Aspergillus flavus (FCBP-0064),Callus-mediated biosynthesis of Ag and ZnO nanoparticles 581 Figure 11: Antifungal activity of ZnO-NPs against five different fungal strains at different concentrations (10, 5, and 4 mg·mL−1); Ab – antibiotic, CE – callus extract. Figure 12: Cell viability and cell inhibition percentage relative to the untreated control (mean ± SE). Aspergillus fumigatus (FCBP-1264) Aspergillus niger (FCBP- Amphotericin B. Ag-NPs caused the ATP content present 0198), and Fusarium solani (FCBP-1114). The callus Ag-NPs inside the cells of the microorganisms to drop by getting showed promising activity against A. niger with the maximum attached to the sulfur group containing proteins present in zone of inhibitions, i.e., 10 mm, followed by A. fumigatus and the cell membrane and ultimately damaging it. Another effect A. flavus with a 7 mm zone (Figure 10). CE-mediated ZnO-NPs of the NPs in microbes is blocking cell production by directly showed maximum antifungal activity against Mucor, and affecting the DNA [37]. The Ag-NPs successfully presented A. fumigatus with maximum zones of inhibition, i.e., 30 and antimicrobial activity, which can be very effective, especially 20 mm respectively, with MIC ranges from 300 to 120 μg·mL−1 against microorganisms resistant to conventional antimicro- (Table 4, Figure 11). The positive control involved here was bials [38]. The striking antifungal activity was in agreement
582 Mehreen Zaka et al.
Figure 13: Cytotoxicity effect of Doxorubicin 20 µM, MCX (Ag-NPs), and MCZ (ZnO-NPs) on HepG2 cells after 24 h.
with the earlier findings of Buszewski et al. [39]. According to distinctive chemical and physical characteristics of the
He et al. [48], the inhibition of fungal growth is initiated by metallic NPs but also on quantum size and shape [44].
the ZnO-NP interaction and ROS generation. Likewise, signif-
icant fungicidal activity has been reported by previous studies
[49,50], which is in agreement with our study. Our results
expanded our knowledge for the preparation of new antimi- 4 Conclusion
crobial agents with synergistic amplification of the antibac-
terial process against clinical bacteria. Green synthesized NPs have been the center of contem-
plation among scientists in the current era. Plants have
emerged as the most widely used reducing and capping
agents for NPs synthesis; however, extract from wild
3.8 Cytotoxicity assay plants may result in non-uniform NP morphology. CEs
serve as a better reducing and capping agent and result
For the screening of in vitro cytotoxicity of NPs and in more uniform NPs morphology, and therefore, Cannabis
extracts, SRB assay involving HepG2 cell line was applied. sativa CE was used for the fabrication of Ag-NPs and
The formation of hydroxyl (OH−) ions impacts the ionic ZnO-NPs. Cannabis sativa CE contains crucial bioactive
strength and pH of NPs and is directly involved in the compounds, including flavonoids and terpenoids, which
repulsion and attractions of particles [40]. About 200 µg·mL−1 could enhance the therapeutic potential of NPs. Both Ag-
of CE-mediated ZnO-NPs and Ag-NPs was applied on the NPs and ZnO-NPs were characterized using standard tech-
cells for 24 h. In our study, ZnO-NPs showed enhanced niques like UV-Vis spectroscopy, XRD, FTIR, SEM, and
cytotoxicity (20.07%) as compared to Ag-NPs (29.13%) EDX. The synthesized Ag-NPs and ZnO-NPs showed potent
(Figure 12). Morphological changes were observed in cytotoxic potential against HepG2 cancer cell lines, which
ZnO-NP-treated cells in comparison with Ag-NPs. Cell elon- can be attributed to the surface chemistry and morphology
gation and other structural changes were observed in var- of NPs. The synthesized NPs also showed potency against
ious cell lines (SH-SY5Y, U937, Hs888Lu, and differentiated pathogenic bacterial and fungal strains. Overall, the
SH-SY5Y) caused by cytotoxic ZnO-NPs [41]. These changes current study revealed ZnO-NPs to be more efficient anti-
may also harm some processes involving the movement bacterial, antifungal, and cytotoxic agents in compar-
within the cells like migration, invasion, and substrate ison to Ag-NPs. Such huge potential of NPs against
attachment [42]. As shown in Figure 13, the positive control microbes could be a bright future prospect considering
(doxorubicin) presented elevated levels of cytotoxicity at the increasing issue of antibiotic resistance; however,
a concentration of 20 µM. Cytotoxicity related to ZnO-NPs there is still a lot of work to be done in order to analyze
has been reported by Akhtar et al. [43] as a result of the efficacy of NPs as effective antimicrobials and anti-
the formation of reactive oxygen species in HepG2 cells. cancer agents in vivo as well as their mode of delivery
Therapeutic properties of NPs not only depend upon the and possible side effects.Callus-mediated biosynthesis of Ag and ZnO nanoparticles 583
Acknowledgments: Moiz A. Siddiqui acknowledges Ahmad [8] Abbasi BH, Zaka M, Hashmi SS, Khan Z. Biogenic synthesis of
Wahab, Grade 12 student at Beaconhouse College Program Au, Ag and Au–Ag alloy nanoparticles using Cannabis sativa
and Research trainee at Synergy Education, Faisalabad, for leaf extract. IET Nanobiotechnol. 2017;12(3):277–84.
[9] Nazir S, Zaka M, Adil M, Abbasi BH, Hano C. Synthesis, char-
his assistance in collecting the plant data used in this
acterisation and bactericidal effect of ZnO nanoparticles via
research and his help in the data analysis. chemical and bio-assisted (Silybum marianum in vitro plant-
lets and callus extract) methods: a comparative study. IET
Funding information: The authors state that there was no Nanobiotechnol. 2018;12(5):604–8.
funding involved. [10] Chowdhury R, Khan A, Rashid MH. Green synthesis of CuO
nanoparticles using Lantana camara flower extract and their
potential catalytic activity towards the aza-Michael reaction.
Author contributions: Mehreen Zaka and Syed Salman RSC Adv. 2020;10(24):14374–85.
Hashmi devised the idea of the project and performed [11] Muhammad W, Khan MA, Nazir M, Siddiquah A, Mushtaq S,
most of the lab experiments and drafted the manuscript; Hashmi SS, et al. Papaver somniferum L. mediated novel
Moiz A. Siddiqui contributed to data analysis and under- bioinspired lead oxide (PbO) and iron oxide (Fe2O3) nano-
particles: In-vitro biological applications, biocompatibility and
standing of chemistry, and also took part in manuscript
their potential towards HepG2 cell line. Mater Sci Eng C.
writing; Lubna Rahman carried out the antimicrobial assays;
2019;103:109740.
Sadaf Mushtaq performed anticancer assay; Haider Ali [12] Siddiquah A, Hashmi SS, Mushtaq S, Renouard S, Blondeau JP,
assisted Mehreen Zaka in manuscript editing and review; Abbasi R, et al. Exploiting in vitro potential and characteriza-
Christophe Hano and Bilal Haider Abbasi supervised the tion of surface modified Zinc oxide nanoparticles of Isodon
project and gave valuable suggestions throughout the rugosus extract: Their clinical potential towards HepG2 cell
line and human pathogenic bacteria. EXCLI J. 2018;17:671.
drafting of the paper and review. All authors gave final
[13] Riaz HR, Hashmi SS, Khan T, Hano C, Giglioli-Guivarc’h N,
approval for publication. Abbasi BH. Melatonin-stimulated biosynthesis of anti-micro-
bial ZnONPs by enhancing bio-reductive prospective in callus
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