biomolecules
Article
The Antimicrobial, Antioxidant, and Anticancer Activity of
Greenly Synthesized Selenium and Zinc Composite
Nanoparticles Using Ephedra aphylla Extract
Mustafa Mohsen El-Zayat 1 , Mostafa M. Eraqi 2,3 , Hani Alrefai 4,5, * , Ayman Y. El-Khateeb 6 ,
Marwan A. Ibrahim 3,7, * , Hashim M. Aljohani 8,9 , Maher M. Aljohani 10,11
and Moustafa Mohammed Elshaer 12
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Citation: El-Zayat, M.M.; Eraqi,
M.M.; Alrefai, H.; El-Khateeb, A.Y.;
9
Ibrahim, M.A.; Aljohani, H.M.;
Aljohani, M.M.; Elshaer, M.M. The
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Antimicrobial, Antioxidant, and
Anticancer Activity of Greenly
Synthesized Selenium and Zinc
Composite Nanoparticles Using
Ephedra aphylla Extract. Biomolecules
2021, 11, 470. https://doi.org/
10.3390/biom11030470
Academic Editor: Joseph Erlichman
Received: 25 January 2021
Accepted: 16 March 2021
Published: 22 March 2021
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Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
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*
Unit of Genetic Engineering and Biotechnology, Faculty of Science, Mansoura University,
Mansoura City 35516, Egypt; mzayat2001@yahoo.com
National Research Center, Department of Microbiology and Immunology, Veterinary Research Division,
Dokki Giza 12622, Egypt; m.eraqi@mu.edu.sa
Department of Biology, College of Science, Majmaah University, Majmaah 11952, Saudi Arabia
Medical Biochemistry Department, Faculty of Medicine, Mansoura University,
Mansoura City 35516, Egypt
Department of Internal Medicine, Infectious Diseases Division, College of Medicine,
University of Cincinnati, Cincinnati, OH 45267, USA
Department of Agricultural Chemistry, Faculty of Agriculture, Mansoura University,
Mansoura City 35516, Egypt; aymanco@mans.edu.eg
Department of Zoology, Women’s College, Ain Shams University, Cairo City 11566, Egypt
Department of Molecular Genetics and Biochemistry, College of Medicine, University of Cincinnati,
Cincinnati, OH 45221, USA; aljohahm@mail.uc.edu
Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, Taibah University,
Medina City 42353, Saudi Arabia
Department of Pathology, College of Medicine, Taibah University, Medina City 42353, Saudi Arabia;
Maher.aljohani@yahoo.com
Department of Pathology and Laboratory Medicine, Ministry of The National Guard-Heath Affairs,
Medina City 42353, Saudi Arabia
Department of Microbiology at Specialized Medical Hospital, Mansoura University,
Mansoura City 35516, Egypt; moustafaelshaer@mans.edu.eg
Correspondence: alrefahd@ucmail.uc.edu (H.A.); m.ab.ibrahim@mu.edu.sa (M.A.I.);
Tel.: +1-513-9759-195 (H.A.); +966-541-267-818 (M.A.I.)
Abstract: The current work aimed to synthesize selenium and zinc nanoparticles using the aqueous
extract of Ephedra aphylla as a valuable medicinal plant. The prepared nanoparticles were characterized by TEM, zeta potential, and changes in the phytochemical constituents. Hence, the phenolic,
flavonoid, and tannin contents were reduced in the case of the prepared samples of nanoparticles
than the original values in the aqueous extract. The prepared extract of Ephedra aphylla and its
selenium and zinc nanoparticles showed high potency as antioxidant agents as a result of the DPPH•
assay. The samples were assessed as anticancer agents against six tumor cells and a normal lung
fibroblast (WI-38) cell line. The selenium nanoparticles of Ephedra aphylla extract revealed very strong
cytotoxicity against HePG-2 cells (inhibitory concentration (IC50 ) = 7.56 ± 0.6 µg/mL), HCT-116
cells (IC50 = 10.02 ± 0.9 µg/mL), and HeLa cells (IC50 = 9.23 ± 0.8 µg/mL). The samples were
evaluated as antimicrobial agents against bacterial and fungal strains. Thus, selenium nanoparticles showed potent activities against Gram-negative strains (Salmonella typhimurium, Pseudomonas
aeruginosa, Klebsiella pneumoniae, and Escherichia coli), Gram-positive strains (Bacillus cereus, Listeria
monocytogenes, Staphylococcus aureus, and Staphylococcus epidermidis), and the fungal strain Candida
albicans. In conclusion, the preparation of nanoparticles of either selenium or zinc is crucial for
improved biological characteristics.
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
Keywords: Ephedra aphylla; aqueous extract; selenium; zinc; nanoparticles; antioxidant; oxidation;
cytotoxic; antimicrobial; cancer
4.0/).
Biomolecules 2021, 11, 470. https://doi.org/10.3390/biom11030470
https://www.mdpi.com/journal/biomolecules
Biomolecules 2021, 11, 470
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1. Introduction
Plants are rich in different types of natural compounds. About 25% of the prescription
products in the world originated from wild or cultivated plants [1]. Recent research in
synthetic chemistry reported a good potential of natural compounds to provide better
aspects of treatment and prevention of many diseases [2–5]. Plant-derived anticancer
drugs, such as vincristine, vinblastine, camptothecin, and taxol, are a part of the battle
against tumor cells [1]. The continuing search for new antitumor natural compounds is a
promising avenue for its prevention or treatment [6]. Plant compounds such as alkaloids,
phenolics, flavonoids, phenyl-propanoids, and terpenoids have also been reported to have
anticancer activity [7,8].
Green synthesis of nanomaterials using plant extracts as a furious source of natural
compounds like carbohydrates, phenolics, flavonoids, tannins, and alkaloids [9,10] that
could act as safe reducing and stabilizing agents in addition to their ability to maintaining aseptic environments throughout the process [11,12]. Therefore, medicinal plants of
therapeutic potential could control the dimensions and forms of the biosynthesized nanomaterial [13,14]. Selenium and zinc are vital trace elements in living organisms that play
an important role in antioxidant defense, immune regulation, and antitumor for human
health. Biosynthesized zinc and selenium nanoparticles play a great advantage due to their
higher degradability, lower toxicity, and ability to clear from the body [15–18].
Ephedra is the only genus in the family Ephedraceae. It comprises 40 species distributed
worldwide. In Egypt, the genus Ephedra is represented by five species [19]. They are
characterized by their medicinal importance. Many of these were applied in traditional
medicine for treating bronchial disorders and asthma [20,21].
E. aphylla is distributed along the eastern Mediterranean region up to the Arabian
Peninsula. It is a large shrub that grows in the cracks of limestone cliffs or nearby valleys
in the sand and usually grows in juniper forests with Pistacia, Opuntia, Daphne linearifolia,
Artemesia, and Thymelaea hirsuta. E. aphylla is present in the Egyptian protected area of Wadi
El-Gemal-Hamata in the National Reserve of Egypt [22].
Ephedra aphylla has been reported as being rich with valuable phytochemicals such as
alkaloids like ephedrine, pseudoephedrine, N-methylephedrine, and 6-methoxykynurenic
acid, and hordenine [22,23], phenolics and flavonoids, i.e., di-C-glucosyl flavone, 2,2′ di-O-β-glucopyranosyl-vicenin, di-O-glycoside, herbacetin 3-O-α -rhamnopyranoside-8O-β-glucopyranoside, vicenin, 7-methoxy-4-oxo-1,4-dihydroquinoline-2-carboxylic acid,
ephedralone, 4-hydroxybenzoic acid, (E)-3-(4-hydroxyphenyl)acrylic acid, 3,4-dihydroxybenzoic acid, and 7-methoxy-herbacetin [24,25].
Some of the phytochemical constituents in Ephedra aphylla possess larvicidal activity
and could be used for controlling disease-caused by mosquitoes [26]. E. aphylla showed
strong antiproliferative potential against the breast cancer cell lines MFC7 and T47D that
may be assigned to the active phytochemicals in the plant, such as ephedrine alkaloids
and herbacetin [27]. Ephedrine has been described to suppress hepatocyte growth factor
(HGF)-induced cancer cell motility by preventing both HGF-induced phosphorylation of
c-Met and its tyrosine kinase activity [28]. It was also reported that Ephedra extracts exhibit
antimetastatic and antitumor effects by suppressing the hepatocyte growth factor-c-Met
signaling pathway through the inhibition of c-Met tyrosine kinase activity [28].
The intent of the current work was to biosynthesize selenium and zinc nanoparticles
of the aqueous extract of Ephedra aphylla as a source of reducing components, and to assess
their biological characteristics as antioxidant, anticancer, and antimicrobial agents.
2. Materials and Methods
2.1. Materials
2.1.1. Chemicals and Reagents
Folin-Ciocalteau reagent (Fluka, Biochemical Inc., Bucharest, Romania), Gallic acid
(Biomedical Inc., Orange City, FL, USA), 1,1-Diphenyl-2-picrylhydrazyl (DPPH• ), aluminum chloride, sodium hydroxide, sodium nitrite, catechin, vanillin, hydrochloric acid,
Biomolecules 2021, 11, 470
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ascorbic acid, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT), RPMI1640 medium, and DMSO were purchased from Sigma Aldrich (St. Louis, MO, USA).
Sodium Carbonate (El-Nasr Pharmaceutical Chemicals, Cairo, Egypt). Fetal Bovine serum
(FBS) (GIBCO, UK). ZnSO4 (Andenex-Chemie, Hamburg, Germany), SeSO4 (Alpha
Chemika, Panvel, Maharashta, India). Doxorubicin, Ceftazidime and Ampicillin-Sulbactam
were purchased from Merck (Darmstadt, Germany).
2.1.2. Plant Materials
The stems of Ephedra aphylla were collected from their authentic habitats at Saint
Catherine Protectorate, South Sinai, Egypt. The plant was taxonomically identified by
Mustafa El-Zayat and authenticated by Boulos [29].
2.2. Phytochemical Analysis
The active constituents were extracted using the same trend of usage in folklore
medicine as hot infusions. Twenty grams of the dried plant stems were mixed with 200 mL
deionized water with shaking for 30 min in a water bath system at 70 ◦ C. The produced
extract was filtered, and the filtrate was kept at 4 ◦ C for further use.
The total phenolic, flavonoid, and tannin components of the aqueous extract of Ephedra
aphylla stems were quantitatively estimated.
2.2.1. Total Phenolic Contents
The total phenolic constitutes were assessed by the Folin-Ciocalteu procedure progressed by Wolfe et al. [30,31], in which the procedure involved the use of gallic acid as
a standard. The investigated values of total phenolic constitute in the aqueous extract of
Ephedra aphylla, and its nanoparticles with zinc and selenium were quantified as equivalents
in milligram of gallic acid/dried plant extract in gram using a standard curve (y = 0.0062x,
r2 = 0.987).
2.2.2. Total Flavonoid Contents
The total flavonoids constitutes were appreciated by a colorimetric assessment using
aluminum chloride as conveyed by Zhishen et al. [32] with the use of catechin as a standard.
The values of total flavonoid constitutes were quantified as equivalents of catechin in
milligram per dried plant extract in gram using a standard curve (y = 0.0028x, r2 = 0.988).
2.2.3. Total Tannin Contents
The total tannins constitutes were assessed using vanillin-hydrochloride assay [33,34],
and the values of the anticipated samples were quantified as equivalents of tannic acid in
gram/dried plant in 100 g.
2.3. Preparation of Metal Nanoparticles
The procedure from Devasenan et al. [35] was applied with an insignificant amendment in the synthesis of the metal nanoparticles. Each selenium sulfate (1 mmol) or zinc
sulfate (1 mmol) was dissolved in deionized water (20 mL), and the solution was added
gradually in portions to a well-stirred plant extract (20 mL). After the complete addition of
the metal aqueous solution, the mixture was stirred for an extra 2 h at room temperature.
The formed nanoparticles were acquired in the equimolar ratio in both cases.
2.4. Structure Characterization of the Metal Nanoparticles
2.4.1. Transmission Electron Microscope (TEM)
The physical properties and chemical structure, i.e., particle’s size, shape, surface
nature, crystal structure, and morphological data of the prepared nanoparticles, were
identified as conveyed by Otunola et al. [36] using TEM (JEOL TEM-2100, Tokyo, Japan) at
the Electron Microscope Unit, Mansoura University, Egypt. The analysis was run with a
200 nm magnification value.
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2.4.2. Nanoparticles Characteristic via Zeta Potential
The surface charge of the prepared selenium and zinc nanoparticles in the suspension
was characterized by applying Zeta potential technique using Malvern Instruments Ltd.
Zeta Potential Ver. 2.3 (Kassel, Germany) according to Bhattacharjee, [37] at the Electron
Microscope Unit, Mansoura University, Egypt. The process is significant for studying the
surface nature of nanoparticles, and the stability of these particles can be expected to last
for long-term periods [38].
2.5. Potential Biological Applications
2.5.1. Antioxidant Activity—DPPH Assay
The antioxidant capacity of the aqueous extract of Ephedra aphylla and its selenium
and zinc nanoparticles was investigated following the DPPH• colorimetric method using
ascorbic acid as a standard by way of the assay reported by Kitts et al. [39]. The serial
dilution of each sample was prepared by mixing the sample with methanol in an equivalent
amount. The DPPH• solution was prepared in a concentration of 0.135 mM and mixed
with each sample in the serial dilution with a volume of 1 mL. after the addition of DPPH•
solution; the samples were kept in the dark for 30 min at room temperature. The absorbance
of each sample was measured at 517 nm in the next step. The % DPPH• remaining was
calculated using Equation (1):
% DPPH• remaining = [DPPH• ]T /[DPPH• ]T = 0 × 100
(1)
The values of % DPPH• remaining were plotted versus mg extract/mL using an exponential curve to identify the inhibitory concentration “IC50 ”. IC50 indicates the constitutes
amount of antioxidants needed to decrease the initial concentration of DPPH• solution by
50%. The values of IC50 point out the inverse relationship with the antioxidant capacity of
the tested sample [40].
2.5.2. Antimicrobial Activity Procedure
The antimicrobial potential of the studied Ephedra aphylla extract and the prepared zinc
and selenium nanocomposites were estimated using the agar well diffusion assay [41,42].
Ceftazidime (CAZ) and Ampicillin-Sulbactam (SAM) were used as standard antibiotics.
The tested microbial and fungal species were Salmonella typhimurium (ATCC® 14028™),
Pseudomonas aeruginosa (ATCC® 9027™), Staphylococcus epidermidis (ATCC® 12228™), Klebsiella pneumonia (ATCC® 10031™), Bacillus cereus (ATCC® 11778™), Staphylococcus aureus
(ATCC® 6538™), Escherichia coli (ATCC® 10536™), Listeria monocytogenes (ATCC® 19115™),
and Candida albicans EMCC number-105. The obtained strains were of animal origin and
obtained from the Microbiological Resources Centre (MIRCEN), Faculty of Agriculture,
Ain Shams University.
2.5.3. Anticancer Activity Procedure
Six human tumor cell lines specifically; Hepatocellular carcinoma (HePG-2), Epithelioid cervix carcinoma (Hela), Epidermoid larynx carcinoma (HEP2), Human prostate
cancer (PC3), Mammary gland carcinoma (MCF-7), and Colorectal carcinoma (HCT-116),
were acquired from the ATCC holding company for biological products and vaccines
(VACSERA), Cairo, Egypt. Doxorubicin was used as a stock chemotherapeutic anticancer
drug. The chemical reagents RPMI-1640 medium, MTT, DMSO (Sigma co., St. Louis, MO,
USA), Fetal Bovine Serum (FBS; Gibco Life Technologies, Paisley, UK).
Standard colorimetric MTT assay was applied to assess the cytotoxicity of the investigated samples by measuring the cell growth following the procedure conveyed by Bondock
et al. [43]. Concisely, the reduction in the yellow color of MTT (2-(4,5-dimethylthiazol2-yl)-3,5-diphenyl-2H-tetrazol-3-ium bromide) to purple formazan was achieved by mitochondrial succinate dehydrogenases of living cells. The cell strains were grown in
RPMI-1640 medium with 10% fetal bovine serum. Antibiotics, penicillin (100 units/mL)
Biomolecules 2021, 11, 470
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and streptomycin (100 µg/mL) were added at 37 ◦ C in a 5% CO2 incubator. Cell lines
were seeded in a 96-well plate at a density of 1.0 × 104 cells/well at 37 ◦ C for 48 h under
5% CO2 . After incubation, cells were treated with different concentrations of the tested
samples and incubated for an additional 24 h. After 24 h of drug treatment, MTT solution
(5 mg/mL, 20 µL) was then added and incubated over again for 4 h. DMSO (100 µL)
was then added to each well to dissolve the produced violet formazan. The colorimetric
evaluation was reverent, and the absorbance values were measured at 570 nm utilizing
a plate reader (EXL 800, New York, NY, USA). The IC50 values were calculated using
nonlinear regression (sigmoid type), analyzed using the Origin 8.0® software (OriginLab
Corporation, https://www.originlab.com/ (accessed on 19 March 2021)). Relative cell
viability in percent was calculated from Equation (2):
% Relative cell viability = [Sample absorbance/Control absorbance] × 100
(2)
3. Results and Discussion
The chemical constitutes of aqueous stem extract of Ephedra aphylla, and its sources
comprise a wide range of diverse privileged secondary metabolites, in which they are
potentially reducing materials for the biogenic production of nanoparticles [44]. The
present study demonstrated that Ephedra aphylla stem extract is filled with phenolics
(131.55 mg gallic acid equivalent/g dry extract), flavonoids (27.51 mg catechin equivalent/g
dry extract), and tannins (64.91 mg gallic acid equivalent/g dry extract) that could be
utilized for the reduction and stabilization of selenium and zinc metal ions and the green
synthesis of their nanoparticles. Polyphenol compounds have an electron resonance
hybrid effect, as they play the role of biological reduction in salts ions and convert them
into nanoparticles, as well as having a role in stabilizing those particles in a stable, nonprecipitating form [45,46]. Flavonoids as a subclass of phenolics are difficult to break,
and therefore they are used in the bio-reduction in zinc and selenium ions, and their
conversion into nanoparticles where the flavonoids aggregate and bind on the surface of
the nanoparticles and neutralize their charges to zero-valent molecules in the nanometer
range, and thus new compounds are formed that have very small sizes, thus increasing
their surface area and are active, effective, and unique, chemically and biologically [45–47].
Regarding the prepared nano selenium and nano zinc, there were marked decreases in the
phenolics (26.85 and 40.63 mg gallic acid equivalent/g dry extract, respectively), flavonoids
(7.09 and 2.98 mg catechin equivalent/g dry extract, respectively), and tannins (15.82 and
15.05 mg gallic acid equivalent/g dry extract, respectively) (Table 1).
Table 1. The phytochemical analysis of Ephedra aphylla extract and its selenium and zinc nanoparticles (NPs).
Phytochemical Analysis
Samples
Ephedra aphylla extract
Ephedra aphylla + SeNPs
Ephedra aphylla + ZnNPs
Phenolic Contents “mg Gallic
Acid Equivalent/g Dry Extract”
Flavonoid Contents “mg Catechin
Equivalent/g Dry Extract”
Tannin Contents “mg Gallic Acid
Equivalent/g Dry Extract”
131.55
26.85
40.63
27.51
7.09
2.98
64.91
15.82
15.05
3.1. Characterization of the Prepared Nanoparticles
3.1.1. Transmission Electron Microscope (TEM)
TEM technique was applied for characterizing digital images of the prepared selenium
and zinc nanoparticles of Ephedra aphylla, which provided a good view of morphological
particles. The samples were analyzed at a higher spatial resolution (200 nm). Figure 1
shows the TEM micrographs and size distributions of the prepared selenium and zinc
nanoparticles. Figure 1a shows the formation of the spherical and tetragonal shapes of
the selenium nanoparticles, in addition to zinc nanoparticles (Figure 1b), which seemed
to be spherical particles only, which indicated the crystallinity of the particles, supported
by the emission diffraction of the selected area. The size of the selenium particles is
Biomolecules 2021, 11, 470
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ranged from 13.95 to 26.26 nm, while the size of the zinc particles is ranged from 8.34
to 15.20 nm. TEM allowed the assessment of agglomeration and/or aggregation of the
constituent nanoparticles. The zinc nanoparticles were more aggregated than the selenium
nanoparticle providing a large surface area of selenium nanoparticles that would increase
their efficiency when applied as a cytotoxic agent.
(a)
(b)
Figure 1. TEM micrographs of the prepared selenium and zinc nanoparticles of the extract of Ephedra aphylla. (a) TEM micrographs and size distributions for selenium nanoparticles synthesized by Ephedra aphylla extract at a 200 nm magnification
value. (b) TEM micrographs and size distributions for zinc nanoparticles synthesized by Ephedra aphylla extract at a 200 nm
magnification value.
3.1.2. Zeta Potential Analysis
The significance of this analysis lies in the possibility of studying the nature of the
particles present on the surface of the nanoparticles, and thus, these particles could be
expected to be stable long-term. It was found that the nanoparticles had a charge on their
surface, being able to attract a thin layer of ions opposite them in the charge, and from
here, this analysis was applied with the zeta potential technique to define the nature of
the charge on the surface of the nanoparticles. The nanoparticles contained a double layer
of ions which were transferred as they diffused into solution. The electrical potentials
at the borders of the double layer were defined as the zeta potential of the particles and
− to −100 mV. The selenium and zinc particles synthehad values ranging from +100 mV
−
sized with the Ephedra aphylla extract had zeta potential values
of −−5.61 and −8.78 mV
(Figure 2), which were highly stable because nanoparticles with zeta potential values less
than +25 mV or greater than −25 mV generally have a high degree of stabiliity as described
by Honary and Zahir [38].
−
Biomolecules 2021, 11, 470
(a)
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(b)
Figure 2. Zeta potential charts of the prepared selenium and zinc nanoparticles of the extract of Ephedra aphylla. (a) Zeta
potential of the prepared nano selenium synthesized by Ephedra aphylla extract. (b) Zeta potential of the prepared nano zinc
synthesized by Ephedra aphylla extract.
3.2. Biological Potentials
3.2.1. Antioxidant Activity
The water extract of the Ephedra aphylla stems showed an IC50 of 0.053 mg/mL, while
/
the zinc and selenium nano-solution of Ephedra aphylla extract showed lower antioxidant
activity with IC50 values of 0.213 and 0.296 mg extract/mL that was compared to ascorbic
acid as a strong antioxidant with an IC50 of 0.022 mg extract/mL.
The obtained results indicated that Ephedra aphylla extract was rich in phenolics,
flavonoids, and tannins that possess reasonable antioxidant activity and are well known
for their antioxidant potential based mainly on their structure, especially the number and
attitude of the hydroxyl groups that are of importance for green synthesis as reducing
agents due to their ability to reduce ions into nanoparticles [48,49]. Utilizing the groups
responsible for antioxidant activity in the extract during biosynthesis led to a decrease in
antioxidant activity.
3.2.2. Anticancer Activity
The anticancer activities of polar and nonpolar extracts of naturally occurring E. aphylla
against MFC7 and T47D cell lines have been recently reported [28]. It was reported
that nano-selenium and nano-zinc in their zero-oxidation state exhibit lower toxicity
and excellent bioavailability and could be stabilized by encapsulation into suitable nanovehicles [50,51]. They have a wide range of medical applications like antioxidant properties,
the capability of reducing oxidative stress [52], chemopreventive activity as a potential
anticancer drug, and antimicrobial effects [53,54]. The hollow spherical selenium nanoparticles (SeNPs) reduce the risk of selenium toxicity [55]. The results of many studies indicate
that Nano-Se can be more helpful in cancer chemoprevention as a potential anticancer
drug [56,57] as well as an anticancer drug delivery carrier [58]. Moreover, the immunostimulatory effect of nanoscale selenium has been confirmed [59]. SeNPs have also shown
remarkable anticancer activity [60–62] and exhibit high potential in cancer chemotherapy
and as drug carriers [63]. The anticancer effects of SeNPs are mediated through their
ability to inhibit the growth of cancer cells through the induction of cell cycle arrest at the S
phase [56]. Cell membrane plays an important role in SeNPs-induced toxicity in cancer
cells. SeNPs treatment changes the biomechanical properties of cancer cells; in particular,
they remarkably decrease the adhesion force and Young’s modulus [64]. Besides unique
anticancer efficacy, SeNPs have been proved to present a better selectivity between normal
and cancer cells [65].
Besides their direct anticancer effects, SeNPs have been pointed to as potential anticancer drug delivery carriers [56]. A key factor that usually contributes to nanomaterialbased drug cytotoxicity is cellular uptake [56]. The nano-size of these materials allows an
efficient uptake by various cell types and selective drug accumulation at target sites [66].
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Zinc nanoparticles have great potential in cancer therapy. It has been reported that ZnNPs
induce selective killing of cancer cells where ZnNPs selectively induce apoptosis in cancer cells, which is likely to be mediated by reactive oxygen species via the p53 pathway,
through which most anticancer drugs trigger apoptosis [67].
In this work, the cytotoxic activities of the prepared Ephedra aphylla extract and its
selenium and zinc nanoparticles were evaluated using an MTT assay. The samples were
tested in vitro against six tumor cells, i.e., HepG-2, MCF-7, HCT-116, PC3, HeP2, and HeLa
cell lines. Doxorubicin was selected as a reference drug, comparing the results of the tested
samples against the different cancer cells. IC50 values express the concentrations that induced 50% of the death of tumor cells in µg/mL. The IC50 values are inversely proportional
to the efficiency of the sample to inhibit the growth of the cancer cells. Therefore, a potent
cytotoxic agent would require the lowest concentration and IC50 values.
The results of in vitro cytotoxicity are listed in Table 2, in which the scale of the cell’s
viability or potency of the samples was mentioned in Table 2. The results demonstrated, in
general, that the prepared selenium and zinc nanoparticles had potent cytotoxicity against
the diverse tumor cell lines than the original extract. Accordingly, the nanoparticles had a
large surface size that increased the efficiency of the sample to inhibit the growth of the
tumor cells. Moreover, the selenium nanoparticles of Ephedra aphylla revealed the most
potent cytotoxicity against HePG-2 cell lines with an IC50 of 7.56 ± 0.6 µg/mL. Besides,
strong cytotoxicity was recorded for selenium nanoparticles of Ephedra aphylla with an IC50
of 15.65 ± 1.4 µg/mL against MCF-7 tumor cell lines, indicating high effectiveness of the
selenium nanoparticles in the first order, accompanied by those of zinc nanoparticles that
had moderate cytotoxicity against MCF-7 cell lines with an IC50 of 29.32 ± 2.2 µg/mL.
Selenium nanoparticles of Ephedra aphylla revealed strong cytotoxicity against HCT-116
cancer cell lines with an IC50 of 10.02 ± 0.9 µg/mL. On the other hand, the tested samples
were less potent anticancer agents against PC3 cancer cell lines, but selenium nanoparticles
of Ephedra aphylla remained strong cytotoxic agents with an IC50 of 18.63 ± 1.5 µg/mL.
Selenium nanoparticles of Ephedra aphylla also showed strong cytotoxicity against HeP2
and HeLa tumor cell lines with an IC50 of 12.10 ± 1.2, and 9.23 ± 0.8 µg/mL, respectively.
In addition, all the tested samples revealed weak cytotoxic activities against the normal
lung fibroblast (WI-38) cell line. The results indicated the potential for the application of
these samples as anticancer drugs. Briefly, the Ephedra aphylla seemed to be an effective
cytotoxic agent against all the tested tumor cell lines as used as a simple extract or its
isolated zinc or selenium nanoparticles. The results showed that using selenium metal ions
to prepare nanoparticles of the extract of Ephedra aphylla had higher efficiency in inhibiting
the growth of cancer cells more than the use of zinc metal ions.
Table 2. Cytotoxic activity of the prepared samples against the diverse human tumor cells.
Samples
Doxorubicin
Ephedra aphylla
Ephedra aphylla+ SeNPs
Ephedra aphylla+ ZnNPs
Selenium sulfate
Zinc sulfate
(a)
In Vitro Cytotoxicity, IC50 ± SD (µg/mL) (a)
HePG-2
MCF-7
HCT-116
PC3
HeP2
HeLa
WI-38
4.50 ± 0.2
27.72 ± 2.1
7.56 ± 0.6
17.46 ± 1.1
32.98 ± 2.3
36.75 ± 1.9
4.17 ± 0.2
36.77 ± 2.8
15.65 ± 1.4
29.32 ± 2.2
48.24 ± 2.7
53.89 ± 2.4
5.23 ± 0.3
47.77 ± 3.3
10.02 ± 0.9
33.74 ± 2.7
59.50 ± 3.1
59.26 ± 3.3
8.87 ± 0.6
42.76 ± 3.1
18.63 ± 1.5
32.36 ± 1.9
54.83 ± 2.8
62.86 ± 3.2
8.54 ± 0.6
30.53 ± 2.4
12.10 ± 1.2
22.95 ± 1.1
39.04 ± 1.9
44.1 ± 2.1
5.57 ± 0.4
23.92 ± 1.7
9.23 ± 0.8
21.65 ± 1.8
33.26 ± 1.6
34.62 ± 1.8
94.94
>100
>100
96.76
>100
>100
IC50 : inhibitory concentration (µg): 1–10 (very strong), 11–20 (strong), 21–50 (moderate), 51–100 (weak), and above 100 (non-cytotoxic).
The results of selenium and zinc sulfate solutions (Table 2) demonstrated that both
samples displayed moderate activities against HePG-2 cell lines (IC50 = 32.98 ± 2.3 and
36.75 ± 1.9), HeP2 cell lines (IC50 = 39.04 ± 1.9 and 44.1 ± 2.1), and HeLa cell lines
(IC50 = 33.26 ± 1.6 and 34.62 ± 1.8) relative to the results of the reference standard, extracted sample, and the prepared nanoparticles. Besides, the solutions of selenium and
zinc sulfate revealed relatively weak cytotoxicity against MCF-7, HCT-116, and PC3 tumor
Biomolecules 2021, 11, 470
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cell lines. The results of the investigated salt solutions reflect the efficiency of the extracted
Ephedra aphylla, and its selenium and zinc nanoparticles on the cytotoxicity in comparison
with the results of the salt solution samples. Concisely, the salts, in general, did not impact
the cytotoxicity against the tested tumor cell lines.
A comparative relationship of the results of the extract of Ephedra aphylla and its
selenium and zinc nanoparticles against HepG-2, MCF-7, HCT-116, PC3, HeP2, and HeLa
cell lines with the results of Doxorubicin is specified in Figure 3. It is worth mentioning
that the formation of metal nanoparticles enhanced the cytotoxic characters of the extract
of Ephedra aphylla by different ranges. The difference in the results between selenium and
zinc nanoparticles or the extract of Ephedra aphylla itself depended on the type of tumor
cell lines or the nature of the nanoparticles of the used metal ions, as it was characterized as having small particles and higher aggregation. Generally, the extract of Ephedra
aphylla was an efficient cytotoxic agent against HeLa cell lines with effective cytotoxicity
(IC50 = 23.92 ± 1.7 µg/mL) when compared to the other tested cell lines. The selenium
nanoparticle of the extract of Ephedra aphylla was the most potent cytotoxic agent against the
other tested samples and its results against the diverse cell lines (IC50 = 9.23 ± 0.8 µg/mL).
The potent cytotoxicity of zinc nanoparticles was also noted against HePG-2 cell lines
(IC50 = 17.46 ± 1.1 µg/mL). Subsequently, the extract of Ephedra aphylla and its metal
nanoparticles were applicable for the inhibition of cancer cell growth against HePG-2 and
HeLa cell lines rather than the other tested tumor cells.
70
60
IC50 Values
50
40
30
20
10
0
HePG-2
MCF-7
HCT-116
PC3
HeP2
HeLa
Human Tumor Cells
Doxorubicin
Ephedra aphylla
Ephedra aphylla+ SeNPs
Ephedra aphylla+ ZnNPs
Selenium sulfate
Zinc sulfate
Figure 3. Comparison of the IC50 values of the tested samples against human cancer cells.
Figure 4 shows the inhibition % of the Ephedra aphylla extract and its metals nanoparticles against all the tested human tumor cells at different concentrations. The samples were
prepared as a serial dilution of seven concentrations starting from 100 µg/mL. The results
demonstrated that the use of a higher concentration of a sample increased the efficiency of
the sample to inhibit the growth of the cancer cells. In consequence, the extract of Ephedra
aphylla displayed potent cytotoxicity with inhibition percentages ranging from 63.6% to
76.4% at a concentration of 100 µg/mL, while the use of 1.56 µg/mL of the same Ephedra
aphylla extract resulted in non-cytotoxic characteristics against all the tested cancer cell
lines. The prepared selenium nanoparticles of Ephedra aphylla verified the strongest potency
against all the tested cancer cell lines with inhibition percentages ranging from 76.3% to
91.7% at the higher concentration, but it remains has weak activities at the lower concentration (1.56 µg/mL) against HePG-2, HCT-116, HeP2, and Hela tumor cell lines. The results
of zinc nanoparticles of the extracted Ephedra aphylla demonstrated the capability of the
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nanoparticles to improve the anticancer behavior of the extract against all the tested cancer
cell lines. The percent of inhibition was also calculated for the selenium and zinc sulfate
solutions at different concentrations (1.56–100 µg/mL). All the solutions of the metal salts
displayed moderate activities at the higher level of concentrations (100 µg/mL) with the
percentage of inhibition ranging from 40.4% to 48.5% for the selenium sulfate solution, and
33.7% to 40.4% for the zinc sulfate solution against diverse tumor cell lines. The results
of the percentage of inhibition agreed with that of the plant extract and its selenium and
zinc nanoparticles.
% Inhibition
Percent of average relative viability of cells
a
b
c
d
e
f
g
h
Figure 4. Cont.
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i
j
k
l
Figure 4. Comparison of the inhibition percentage with the percent of average relative viability of tumor and normal cells at
different concentrations. Where: (a,b) for Doxorubicin, (c,d) for Ephedra aphylla extract, (e,f) for Ephedra aphylla + SeNPs,
(g,h) for Ephedra aphylla + ZnNPs, (i,j) for Selenium sulfate solution and (k,l) for Zinc sulfate solution.
Figure 4 shows the percent of average relative viability of all tumor cell lines at
different concentrations. The percent viability of tumor cells is inversely proportional to
the inhibition percentage at the same concentration. The results were matched with that
recorded for the Ephedra aphylla extract and its selenium and zinc nanoparticles against
diverse human tumor cell lines. The results of average relative viability of tumor cell lines
showed non-cytotoxic effects at lower concentrations 1.56–12.5 µg/mL for both solutions
of metal salts. The average relative viability of both solutions of metal salts at the higher
concentration (100 µg/mL) demonstrated weak cytotoxic effects of both solutions of metal
salts against all human tumor cell lines with a cell viability average range from 51.5–59.6%
for the solution of selenium sulfate, and 59.6–66.3% for the solution of zinc sulfate. The
principal aspect affecting the results of cytotoxicity was the efficiency of the extracted
Ephedra aphylla, and its selenium and zinc nanoparticles. It seemed obvious that the salt
solutions had an inoperative impact on the results obtained.
3.2.3. Antimicrobial Activity
The antimicrobial potential of Ephedra aphylla aqueous extract in addition to the
prepared selenium and zinc nanoparticles solutions were tested against several pathogenic
microbial isolates using an agar well diffusion assay, as presented in Table 3 and Figure 5.
The Ephedra aphylla aqueous extract expressed no antimicrobial activity against any of
the tested microbes. It was noted that selenium nanoparticles of the aqueous extract of
Ephedra aphylla are potent antimicrobial agents, and their derived components, such as
selenium sulfide, are mostly applied in medicine for the treatment of infectious diseases,
e.g., Malassezia and Tinea versicolor. Nevertheless, the excess amount of selenium led to toxic
effects and selenosis. Therefore, the current research was focused on reducing cell toxicity
and develop the bio-functional characteristics of selenium. Consecutively, nanotechnology
has provided a safe approach to decrease the toxicity and enhance the functionality of
selenium over biosynthesis [68].
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Table 3. Antimicrobial activity of the greenly synthesized selenium nanoparticles using Ephedra aphylla stem extract on
various pathogenic microbial strains.
Pathogenic
Bacterial Strains
Inhibition Zones Measured in Millimeters (a)
Plant Extract
Nano-Zinc Composite
Nano-Selenium Composite
Standard Antibiotic
SAM
CAZ
Gram-negative bacteria
Salmonella
typhimurium
Pseudomonas
aeruginosa
Klebsiella pneumoniae
Escherichia coli
-
16
39.3
15
20
-
17
20
R
19
-
21
20
38.3
47
15
15
28
24
Gram-positive bacteria
Staphylococcus
epidermidis
Bacillus cereus
Staphylococcus aureus
Listeria
monocytogenes
-
-
31
15
8
-
14
19
21
36.3
13
12
7
20
-
20
26.7
34
13
19.33
17
18
Fungi
Candida albicans
(a)
-
-
The diameter of the well (8.0 mm) is included in the measured zone of inhibition, Ceftazidime (CAZ) and Ampicillin-Sulbactam (SAM).
The prepared selenium nanoparticles displayed a broad and proficient antimicrobial impact against Gram-positive bacterial strains, i.e., S. aureus, S. epidermis, B. cereus,
L. monocytogenes, Gram-negative bacterial strains, i.e., P. aeruginosa, E. coli, S. typhimuriu,
and K. pneumonia, and fungal species, i.e., C. albicans, in which the results were compared
with the standard antibiotics (Gentamycin, and Clotrimazole).
The higher concentration of nanoparticles of zinc displayed accomplished growth inhibition as a result of their impact on the growth of pathogenic bacterial species. Accordingly,
it was proposed that at low concentrations of zinc, it behaved as bacteriostatic; however, at
the higher concentrations, the material had bactericidal influence. The proposed discussion
is in agreement with the results of numerous earlier researches [69,70].
As conveyed in the earlier reports, it was found that the nanoparticles of zinc demonstrated a wide range of antibacterial characteristics. Therefore, the antibacterial behavior
was improved due to the reduced size of the particles and hence increased the surface
area of the particles and increased its efficiency in inhibiting bacterial growth [71]. The
mechanism of action, in this case, is still not clarified, and the antibacterial activity of zinc
nanoparticles is a result of these points: (i) The cell membrane has electrostatic interaction
with the nanoparticles of zinc; (ii) cellular internalization of zinc nanoparticles; (iii) The
role and impact of reactive oxygen species such as phenolic components. Concisely, the
interaction could occur between the zinc nanoparticles and the cell membrane with the
change in the cell membrane permeability against the tested nanoparticles. Therefore,
induced oxidative stress took place owing to the entrance of the nanoparticles into the cell,
causing growth inhibition and cell death [72].
The prepared zinc nanoparticles exhibited a broad and efficient antimicrobial spectrum
against Gram-positive bacteria, i.e., S. aureus, B. cereus, L. monocytogenes; Gram-negative
P. aeruginosa, E. coli, S. typhimurium, K. pneumonia, and expressed no activity against the
fungal species C. albicans and the Gram-positive bacteria S. epidermidis.
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(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
(i)
Figure 5. Photos of the antimicrobial activity of the wild Ephedra aphylla water extract and the synthesized nanoparticles using
well diffusion assay against different pathogenic microbial starins as presented in subfigures (a): Salmonella typhimurium,
(b): Bacillus cereus, (c): Klebsiella pneumoniae, (d): Escherichia coli, (e): Staphylococcus epidermidis, (f): Pseudomonas aeruginosa,
(g): Staphylococcus aureus, (h): Listeria monocytogenes and (i): Candida albicans where, Code 1 = Ephedra aphylla aqueous extract;
Code 1 Se = greenly synthesized selenium nanoparticles; Code 1 Zn = greenly synthesized zinc nanoparticles.
Nowadays, bacterial infections are a serious threat because of the antibiotic resistance
crisis that has been correlated to the overuse and misuse of antibiotics in addition to the
lack of new medications development. The development of antibiotic-resistant bacteria
such as S. aureus has minimized the effectiveness of antibiotics for treatment and has
increased the need for the discovery of new antimicrobial drugs of natural origin [73–75].
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This study provided evidence for the synergetic relation between Ephedra aphylla extracts
and the produced selenium and zinc nanoparticles, and the obtained results revealed that
the resulted products from our study could be used as an alternative for antibiotics as it
was clear that these products were more efficient than the compared antibiotics, in addition
to their broad spectrum of antimicrobial activity against antibiotic-resistant bacteria such
as S. aureus.
4. Conclusions
The present work provides a green technique for the synthesis of selenium and zinc
nanoparticles from an extract of Ephedra aphylla stems. The selenium and zinc nanoparticles had higher stability and various desired morphologies due to the presence of certain
chemical constituents such as phenolic, flavonoid, tannin, and alkaloids content that were
responsible for nanoparticle biosynthesis and stability. The results of this study indicated
that the prepared nano-solutions expressed potent antimicrobial and anticancer activities
along with reduced antioxidant characters. The most potent cytotoxic results were recorded
for the nano-selenium solution of the plant extract with high potency against HePG-2,
MCF-7, HCT-116, PC3, HeP2, and Hela cell lines (IC50 = (7.56 ± 0.6)–(18.63 ± 1.5) µg/mL),
relative to the reference standard along with high inhibition percentages and low relative
viability at different concentrations (1.56–100 µg/mL). The selenium nanoparticles displayed potent antimicrobial activities against diverse bacterial and fungal species with
improved inhibition zone diameter within 19.33–39.33 mm. The antioxidant characters
and phytochemical constitutes were found to be more reduced than in the original extract. Hence, the nano-selenium solution of the extracted Ephedra aphylla stems could be
widely applied in the future in various biological implementations in medicine, cosmetic
manufacturing, food processing, and many other applications.
Supplementary Materials: The following are available online at https://www.mdpi.com/2218-2
73X/11/3/470/s1, Table S1: Cytotoxic activity of some compounds against human tumor cells
(% inhibition). Table S2: Percent of average relative viability of cells.
Author Contributions: M.M.E.-Z., M.M.E. (Moustafa Mohammed Elshaer), M.M.E. (Mostafa M.
Eraqi) and A.Y.E.-K. proposed the research concept, carried out experimental model development,
plant material collection, accomplished the phytochemical analysis, prepared and characterized the
metal nanoparticles, investigated the antimicrobial and cytotoxic activities, participated in writing
and critical reviewing of the final manuscript. H.A., M.A.I., H.M.A. and M.M.A. provided the
biochemical, biological and molecular analyses for the study, data curation and statistical analysis,
contributed to the interpretation of the results, reviewing and revising of the manuscript. All authors
contributed, reviewed and revised the article and critically appraised it. All authors have read and
agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data is contained within the article and Supplementary Material.
Acknowledgments: The authors would like to thank the Deanship of Scientific Research at Majmaah
University, Kingdom of Saudi Arabia, for supporting this work under Project Number (R-2021-51).
Conflicts of Interest: The authors declare no conflict of interest.
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