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Article

Cytotoxic Activity of Zinc Oxide Nanoparticles Mediated by Euphorbia Retusa

by
Abdulsalam A. Alqahtani
1,
Gouda H. Attia
2,*,
Abdelbaset Elgamal
3,
Mohamed Aleraky
4,
Mahmoud Youns
5,
Ammar M. Ibrahim
6,
Randa Abdou
7,8,
Ibrahim Ahmed Shaikh
9 and
Mohamed A. El Raey
10,*
1
Department of Pharmaceutics, College of Pharmacy, Najran University, Najran 55461, Saudi Arabia
2
Department of Pharmacognosy, College of Pharmacy, Najran University, Najran 55461, Saudi Arabia
3
Department of Chemistry of Natural and Microbial Products, National Research Centre, Dokki, Cairo 12311, Egypt
4
Department of Clinical Pathology, College of Medicine, Najran University, Najran 55461, Saudi Arabia
5
Department of Biochemistry, Faculty of Pharmacy, Helwan University, Cairo 11795, Egypt
6
Applied Medical Sciences College, Najran University, Najran 55461, Saudi Arabia
7
Department of Pharmacognosy, Faculty of Pharmacy, Helwan University, Cairo 11795, Egypt
8
Department of Pharmacognosy, Faculty of Pharmacy, Umm Al-Qura University, Makkah 24382, Saudi Arabia
9
Department of Pharmacology, College of Pharmacy, Najran University, Najran 55461, Saudi Arabia
10
Department of Phytochemistry and Plant Systematics, National Research Centre, Dokki, Cairo 12311, Egypt
*
Authors to whom correspondence should be addressed.
Crystals 2022, 12(7), 903; https://doi.org/10.3390/cryst12070903
Submission received: 16 May 2022 / Revised: 20 June 2022 / Accepted: 20 June 2022 / Published: 24 June 2022
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Abstract

:
Background: Cancer is a dangerous threat that creates extremely high rates of death and morbidity in various regions of the world. Finding suitable therapeutics to improve cancer therapy while avoiding side effects is critical. The most appropriate innovative therapeutics, which combine natural ingredients and nanomaterials, can improve the biological activity of cancer chemotherapeutics. Methods: Phenolic profiling using high-resolution mass spectrometry and the synthesis of zinc oxide nanoparticles was achieved through the reaction of zinc acetate with Euphorbia retusa extract. The characterization of ZnONPs was performed by UV, IR, Zeta potential, XRD, SEM, and TEM. The cytotoxic activity of the ZnONPs was evaluated using a SRB assay against lung, liver, and breast cancer cell lines. Moreover, the mechanism of cytotoxic activity was evaluated in the form of caspase-8 promoters and anti-inflammatory mechanisms using the Western blot method. Results: The high-resolution LC/MS/MS of the E. retusa led to the identification of 22 compounds in the plant for the first time. The Er-ZnONPs had hexagonal shapes, were approximately 100 nm in size, and consisted of aggregated particles of about 10 nm. The E. retusa ZnONPs exhibited cytotoxic activity against HA-549 (IC50 = 22.3 µg/mL), HepG2 (IC50 = 25.6), Huh-7 (IC50 = 25.7), MCF-7 (IC50 = 37.7), and MDA-MB-231 (IC50 = 37). Conclusions: E. retusa are rich in phenolics that are capable of synthesizing ZnONPs, which possess cytotoxic activity, via caspase-8 promotion and anti-inflammatory mechanisms.

1. Introduction

Cancer is a broad term for a collection of diseases defined by unregulated cell differentiation that results in malignant tumors that not only invade surrounding bodily areas, but also spread to distant sections via the lymphatic system or bloodstream. With an annual incidence rate of 14 million new cases and 8 million deaths, it is the second largest cause of mortality in adults globally. Around 200 distinct forms of cancer are recorded [1].
In recent years, scientists have developed a new cancer treatment pathway based on the concept of nanotechnology. Nanotechnology is a science that combines techniques and instruments from various fields, such as chemistry, biology, medicine, and engineering. This sector has the potential to significantly improve drug bioavailability, reducing the toxicity associated with the high doses that are generally necessary for optimal response and the ability to transport chemicals to specific organs [2].
Nanoparticles (NPs) are attractive materials for biomedical applications because of their small sizes and high surface areas. Unfortunately, some benign materials, when reduced to the nano-scale, may develop toxicity. If these NPs display only selective toxicity to given biological systems they can be used in biomedical applications. Fortunately, the use of green synthesis methods to prepare NPs reduces their toxicity, which makes them useful for biomedical applications, such as cancer therapy [3]. Chemical methods for the synthesis of nanoparticles frequently produce hazardous by-products that are detrimental to the environment. In comparison to these chemical approaches, the green synthesis method has many advantages, and in the last decade, it has attracted the attention of researchers investigating economical and environmentally friendly forms of nanoparticle production [4]. Zinc oxide nanoparticles are safe materials that were proven to have cytotoxic activity through in vivo and in vitro experiments, and were approved by the FDA to be used as food additives or as food packaging materials [5]. Most chemotherapy cancer drugs are accompanied by adverse side effects for patients, including headache, fatigue, weakness, hair loss, nausea, vomiting, diarrhea, abdominal cramps, memory impairment, and numbness; therefore, there is an urgent need for new therapeutics [3]. The use of plants for the synthesis of metal nanoparticles is a novel and promising approach. Green synthesis is the synthesis of nanoparticles using plants or their constituents. It is a straightforward procedure that takes less time to synthesize nanoparticles and does not necessitate the use of additional reduction and capping agents. The phytochemicals and biomolecules found in plant extracts, such as phenolics flavonoids, tannins, terpenoids, and polysaccharide, are responsible for the reduction of metal ions into metal nanoparticles, as well as acting as capping and stabilization agents for synthesized metal nanoparticles [6]. Green synthesis using plant extracts to form metal nanoparticles is an expanding field of study for a wide range of biomedical applications of nanoparticles in various fields. Metal nanoparticles made from green synthesis have countless biological applications. As a result, it is assumed that synthesizing metal nanoparticles from plant extracts and evaluating their biological activity would be of interest [7]. Euphorbia retusa Forssk. belongs to the family Euphorbiaceae. It is also known as Sula retusa, Euphorbia cornuta, or Euphorbia kahirensis.
E. retusa was used in folk medicine for the treatment of infantile eczema, warts, and trichiasis. It was reported also to have antioxidant [8], antimicrobial [9], analgesic, anti-inflammatory [10], and hepatoprotective activities [11]. The goals of this research are to pursue of cancer treatment pathway based on the modern technique of green synthesis, as a safe and cost-effective technique for the synthesis of zinc oxide nanoparticles using hydralcoholic extracts of Euphorbia retusa, to test their activity against breast, lung, and liver cancer cell lines, and to evaluate the mechanism of action by using inflammation and caspase-8-promoter markers.

2. Results and Discussion

To identify the nature of the reducing agent used for the preperation of the zinc oxide nanoparticles, the phytochemical profiles of the aqueous methanolic extracts from the E. retusa aerial parts was performed by high-resolution LC-MS/MS. Twenty-two phytochemical constituents were tentatively identified for the first time from E. retusa. They mainly comprised hydroxy aliphatic acids, phenolic acids, and flavonoids (Table 1 and Figure 1). The plant components were identified based on their molecular formulas, molecular ions, mass/ mass fragmentation, and comparisons with previously published data.
Compound 6 has the molecular formula C13H14O11 and a daughter ion at m/z 169 [M-H-176]−, suggesting 6 to be gallic acid-1-O-glucouronide. Compounds 10 and 13 exhibited molecular ions at m/z 355 and were tentatively identified to be ferulic acid-1-O-hexoside and ferulic acid-4-O-hexoside, respectively. The LC/MS/MS fragmentation chromatogram of 13 (ferulic acid-4-O-hexoside) showed daughter ions at m/z 311 [M-H-44]−, which suggest the presence of free carboxylic groups. Compound 22 has a molecular ion at 501, molecular formula C25H26O11, and daughter ions at m/z 337 [M-H-164]−, which is characteristic of coumaroyl quinic acid and 163 [M-H-164-191]−, suggesting 22 to be dihydrocaffeoyl coumaroyl quinic acid (Figure 2).

2.1. Characterization of Synthesized ZnONPs

2.1.1. UV Analysis

The green synthesis of the ZnONPs using the aqueous ethanolic extract from the E. retusa aerial parts was examined by UV–visible spectroscopy. When the hydroalcoholic extract from the E. retusa was added to the filtered zinc acetate solution, the color turned from brownish red to faint yellow, demonstrating the complete transformation of the Zn(CH3COO)2 to Er-ZnONPs. The prepared Er-ZnONPs were investigated by UV–Vis spectroscopy, which showed a broad absorption band at 280 and 340 nm (Figure S1, supplementary data) suggesting the formation of Er-ZnONPs. The corresponding band gap was calculated as 3.65 eV using the formula Eg = 1240/λ [1].

2.1.2. Characterization of Functional Groups (FT-IR)

The FT-IR spectra of Er-ZnONPs are illustrated in Figure 3. The stretching bands at 3384 cm−1 were due to the OH stretching vibration band. Thus, the presence of phytochemicals, such as polyphenols, on the surface of colloids makes the synthesized metallic nanoparticles stable, as well as acting as a capping agent [23,24]. The peak at 2980 cm−1 agrees with the C-H stretching of the asymmetry vibration. The peaks at 1585 and 445 cm−1 belong to Zn–O stretching and deformation vibration, respectively [25]. The functional groups present in the extract were determined by LC/MS (Table 1).

2.1.3. Zeta Potential of Synthesized Er-ZnONPs

The Er-ZnONP colloids showed stability in the neutral aqueous medium (di-distilled water) because they possessed a Zeta potential value of −30.7 mV, as shown in Figure 4.

2.1.4. X-ray Diffraction (XRD) Pattern Analysis

The crystalline nature of the Er-ZnONPs was confirmed by an X-ray diffraction (XRD) pattern. Distinguishing peaks at (2θ) angles of 31.77, 34.43, 36.26, 47.55, 56.60, 62.88, 66.39, 67.96, and 69.10°, accompanied by indices of (100), (002), (101), (102), (210), and (103), respectively, were observed, as shown in Figure 5. The XRD spectra of the Er-ZnONPs showed that the synthesized nano-ZnO had a hexagonal shape and matched with the card no. COD 2300450 pattern reported by M. Schreyer et al. in 2014 [26]. The particle size average was calculated, according to the Scherrer equation, to be 3.23 nm.

2.1.5. Transmission and Scanning Electron Microscopy (SEM and TEM) Studies

The SEM image obtained for the Er-ZnONPs was used to explore the surface morphology, and it showed that the Er-ZnONPs particles were elongated, spherically agglomerated shapes with diameters ranging from 48 to 170 nm, with an average size of 87 nm (Figure 6a). The larger sizes observed by SEM were due to the agglomeration of the small nanoparticles together with the high resolution.
TEM studies were performed to investigate the nature of the Er-ZnONP particle sizes and their crystallinity. As shown in Figure 6a, the particles consisted of large hexagonal crystals of about 100 nm (Figure 6b). These hexagonal crystals contained small-particle aggregates with an average size of 3.47 nm. It should be noted that these data were similar to the X-ray results.

2.1.6. Cytotoxic Activity

Because of their safety for normal cells and harmful selectivity towards cancer cells, zinc oxide nanoparticles offer unique features as anticancer medicines [27].
In our study, the cytotoxic activity of the Er-ZnONPs was evaluated in vitro against lung, liver, and breast cancer cell lines. The SRB colorimetric assay was used to evaluate the cellular proliferation, with steadily increasing Er-ZnONP dosing for 72 h. It showed that the lung cancer A549 was the most affected cell line (IC50 22.3 ± 1.11 µg/mL), followed by the two hepatic cancer cell lines, HepG2 and Huh-7 (IC50 values of 25.6 ± 1.23 and 25.7 ± 1.02 µg/mL, respectively), followed by the breast cancer cell lines, MDA-MB231 and MCF-7 (IC50 values of 37 ± 3.45 and 37.7 ± 2.01 µg/mL, respectively). The lung cancer cell line, H460, was the least affected after treatment (IC50 51.4 ± 3.27 µg/mL), as shown in Table 2.
The effects of increasing Er-ZnONP concentrations on the protein levels of the caspase-8 and COX-2 are shown in Figure 7a,b. Our results showed clearly that the Er-ZnONPs significantly increased the expression of the apoptotic protein, caspase-8 (Figure 7a), and significantly decreased the expression of the anti-inflammatory protein, COX-2 (Figure 7b), in a dose-dependent manner. Our results demonstrate that apoptosis induction was among the mechanisms triggering Er-ZnONPs’ inhibitory effects on the breast and hepatic cancer cell lines. Furthermore, the Er-ZnONPs increased the caspase-8 expression in all the tested cell lines compared to the ß-actin. Furthermore, the results showed that COX-2 protein expression is deregulated after the cellular treatment of breast and hepatic cancer cell lines with Er-ZnONPs, demonstrating that Er-ZnONPs are selective COX-2 expressions and that their anti-inflammatory effects play a role in their chemotherapeutic action.
For the cytotoxic activity, it was reported that the zinc oxide nanoparticles showed safety towards normal cells and harmful selectivity towards cancer cells. Thus, zinc oxide nanoparticles offer unique features as anticancer medicines [27].
In our study, the cytotoxic activity of the Er-ZnO NPs was evaluated in vitro against lung, liver, and breast cancer cell lines. The SRB colorimetric assay was used to evaluate the cellular proliferation with steadily increasing Er-ZnONP dosing for 72 h. It showed that the lung cancer A549 was the most affected cell line (IC50 22.3 ± 1.11 µg/mL), followed by the two hepatic cancer cell lines, HepG2 and Huh-7 (IC50 values of 25.6 ± 1.23 and 25.7 ± 1.02, respectively), followed by the breast cancer cell lines, MDA-MB231 and MCF-7 (IC50 values of 37 ± 3.45 and 37.7 ± 2.01, respectively). The lung cancer cell line H460 was the least affected after treatment (IC50 51.4 ± 3.27), as shown in Table 1. It is obvious that our results are promising compared to those in previous reports of, such as the green-synthesized ZnONPs using Luffa acutangula peel extract 10–20 nm in size, examined by Ananthalakshmi et al. (2019) for their activity against the human cancer cell lines Huh-7 and MCF-7, which showed IC50s of 40 µg/mL and 121 µg/mL, respectively [28]. In another example, the ZnONPs synthesized by Umamaheswari et al., in 2021, using Raphanus sativus, showed IC50 of 40 μg/mL against the A549 cell line [29].
These results were due to the extract’s action on the surface of nanoparticles. E. retusa has been shown to contain anticancer metabolites, such as coumarines, flavonoids, ellagitannins, and phenolic acids [30,31,32,33]. An immunoblotting experiment using caspase-8 and COX-2 was used to investigate the mechanism of action. Caspase-8 is a protease from the caspase family that is involved in apoptosis during normal development and adulthood. The inactivation of caspase-8 has been seen in a range of human malignancies, which may enhance tumor development and resistance to existing treatments. As a result, caspase-8 appears to be a promising target for restoring tumors’ faulty apoptotic systems and overcoming resistance [34]. On the other hand, COX-2 is widely expressed in a variety of malignancies, where it plays a pleiotropic and multidimensional function in carcinogenesis and cancer cells’ resistance to chemotherapy and radiation. COX-2 is secreted into the tumor microenvironment by cancer-associated fibroblasts (CAFs), macrophage type 2 (M2) cells, and cancer cells (TME). COX-2 enhances the apoptotic resistance, proliferation, angiogenesis, inflammation, invasion, and metastasis of cancer cells by inducing CSC-like activity. COX-2-mediated hypoxia in the TME, as well as its favorable associations with YAP1 and antiapoptotic mediators, all help cancer cells withstand chemotherapy treatments [35]. Based on these findings, raising caspase-8 expression while decreasing COX-2 expression is critical in cancer treatment, battling resistance, and chemoprevention. The effects of increasing concentrations of Er-ZnONPs on levels of the caspase-8 and COX-2 proteins are shown in Figure 7a,b. Our results show clearly that Er-ZnONPs significantly increased the expression of the apoptotic protein, caspase-8 (Figure 7a), and significantly decreased the expression of the anti-inflammatory protein COX-2 (Figure 7b) in a dose-dependent manner. Our results demonstrate that apoptosis induction was among the mechanisms triggering the Er-ZnONPs’ inhibitory effects on the breast and hepatic cancer cell lines, since the Er-ZnONPs increased the caspase-8 expression in all the tested cell lines compared to the ß-actin. Furthermore, the results showed that COX-2 protein expression is deregulated after the cellular treatment of breast and hepatic cancer cell lines with Er-ZnONPs, demonstrating that Er-ZnONP are selective COX-2 expressions, and that their anti-inflammatory effects play a role in their chemotherapeutic action.

3. Materials and Methods

3.1. Plant Materials

The plant materials were collected and authenticated as reported by [36]. In total, 100 g air-dried powdered Euphorbia retusa aerial parts underwent extraction with 500 mL aqueous MeOH (3:1) via ultrasonic assisted extraction method, and the extract was filtered and evaporated under vacuum by rotavapor to yield 18.5 g of extract.

3.2. Zinc Oxide Nanoparticle Biosynthesis (ZnO NPs)

Nanoparticles of ZnO were biosynthesized using Gouda et al. (2020)’s method [25], according to which 1 g of the E. retusa hydroalcoholic extract was mixed with 5 g zinc acetate solution (soluble in 500 mL of bi-distilled H2O) and subsequently heated for 20 min at 100 °C under stirring, before a few drops of NH4OH solution were added until a yellowish white precipitate was formed. The reaction mixture was left for half an hour to complete the reaction. The precipitate was centrifuged at 4000 rpm and washed twice by bi-distilled H2O, and subsequently washed by ethanol to remove faint yellowish powder of Er-ZnONPs.

3.2.1. UV-Visible Spectral Analysis

UV spectrophotometer, Shimadzu, UV-1601(Shimadzu Corporation, Japan) used to examine preparations of Er-ZnONPs. The UV spectra recorded between 200 and 400 nm.

3.2.2. Fourier Transform-Infrared (FT-IR Analysis)

The Er-ZnONP functional groups were characterized using a FT-IR 6100 spectrometer (Jasco, Japan) in the range of 4000–400 cm−1.

3.2.3. Zeta Potential Measurements

Synthesized nanoparticles’ stability and charges were achieved using Zetasizer Nano-ZS (Malvern, UK) laser diffractometer.

3.2.4. X-ray Diffraction

X-ray diffraction (XRD) examination was performed on the formed solid materials using a Bruker D8 Advance Diffractometer (Bruker AXS, Karlsruhe, Germany) with Cu Ka radiation (k = 1.54). Over a 2-theta range of 10–90, the XRD pattern of ZnONPs was acquired using energy-dispersive x-ray spectroscopy.

3.2.5. Transmission Electron Microscopy (TEM)

Transmission electron microscopy was used to examine the particle sizes and general shapes of zinc oxide nanoparticles (JEOL-JEM-1011, Japan). Drops of nanoparticle suspension were placed on a carbon-coated copper grid, and the solvent was allowed to drain slowly before the transmission electron microscopy image was taken.

3.3. Cytotoxicity Assay

3.3.1. Cell Culture

The hepatocellular (HepG2, Huh-7), lung (A549, H460), and breast cancer (MCF-7, MDA-MB231) cell lines were developed from Nawah-Scientific Inc. (Mokatam, Cairo, Egypt). Cells were kept in either RPMI media (H460) or DMEM (HepG2, Huh-7, A549, MCF-7 and MDA-MB231) and mixed with 100 mg/mL of streptomycin, 100 units/mL of penicillin, and 10% of heat-inactivated fetal bovine serum in humidified, 5% (v/v) CO2 atmosphere at 37 °C.

3.3.2. SRB Cytotoxicity Assay

Cell viability was assessed by SRB assay. Aliquots of 100-microliter cell suspension (5 × 103 cells) were placed in 96-well plates and incubated in complete media for 24 h [37,38,39]. Cells were treated with another aliquot of 100-microliter media containing Er-ZnONPs at various concentrations (0.01, 0.1, 1, 10, 100 ug/mL). The sulforhodamine B (SRB) assay was performed to evaluate the cellular protein content [40,41,42,43,44,45]. Briefly, cell lines were incubated and gradually treated for 72 h with DMSO or increasing doses of Er-ZnONPs. Next, cells were fixed with trichloroacetic acid (TCA) (10%) and stained with SRB fluorescent dye for 30 min. The excess dye was repeatedly washed with 1% acetic acid and the SRB bound to cellular proteins was then dissolved in 10 mM Tris base. The absorbance was measured at 510 nm in a reader (Molecular Devices, Sunnyvale, CA, USA).

3.3.3. Immunoblotting Method

Cells were washed in PBS and lysed in boiling sample buffer (62.5 mM Tris-HCl pH 6.8, 1% SDS, 10% glycerol, and 5% β-mercaptoethanol) for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The lysates were boiled for 5 min with lamellae buffer, and the proteins were separated by SDS-PAGE and transferred to an Immobilon membrane (Millipore). The antibodies used were antigen-affinity-purified polyclonal sheep IgG anti-human COX-2 and Caspase-8 (Santa Cruze). After incubation in 5% non-fat dry milk, Tris-HCL, 0.1% Tween 20 for 1 h, COX-2, and caspase-8 polyclonal antibodies were added to one of the membranes containing specimen samples and incubated at 4 °C overnight. Appropriate secondary antibodies were incubated for 2 h at room temperature. After washing twice n 1 × TBS-T, densitometric analysis of the immunoblots was performed to quantify the amounts of COX-2 and caspase-8 against control sample by total protein normalization using image-analysis software on the ChemiDoc MP imaging system (version 3) produced by Bio-Rad (Hercules, CA, USA) [46].

4. Conclusions

The aerial parts of Euphorbia retusa are rich in phenolic metabolites, including phenolic acids and its conjugates, flavonoids, flavonoid glycosides, and sulfated metabolites. These metabolites acts as reducing agents for synthesizing ZnONPs. These nanoparticles possess hexagonal shapes with particle sizes of around 100 nm and consist of small particles of around 3.23 nm with good stability and high crystallinity, as shown by sharp XRD peaks. In this study, Er-ZnONPs were synthesized using hydroalcoholic extracts from the aerial parts of Euphorbia retusa, which revealed a distinctive anticancer activity on hepatocellular (HepG2, Huh-7), lung (A549, H460), and breast cancer (MCF-7, MDA-MB231) cell lines. The Er-ZnONPs were characterized by UV, FT-IR, Zeta potential, XRD, SEM, and TEM. The results also revealed that the Er-ZnONPs significantly increased the expression of the protein, caspase-8, and significantly decreased the expression of the anti-inflammatory protein, COX-2, in a dose-dependent manner. Hence, further studies of Er-ZnONPs are required to prove its role in the treatment of cancer, in fighting drug resistance, and as a chemopreventive agent.

Supplementary Materials

The following are available online at https://www.mdpi.com/article/10.3390/cryst12070903/s1, Figure S1: UV of Er-ZNONPs, Figure S2: Cell viability graphs.

Author Contributions

Conceptualization, G.H.A., M.A.E.R. and A.E.; methodology, M.A.E.R., A.E., M.Y. and M.A.; software, A.A.A., R.A., A.M.I. and I.A.S.; validation, A.E., G.H.A. and R.A.; formal analysis, M.A.E.R. and A.A.A.; investigation, I.A.S. and A.A.A.; resources, G.H.A. and A.A.A.; data curation, M.Y.; writing—original draft preparation, A.E., G.H.A. and M.A.E.R.; writing—review and editing, M.A. and R.A.; visualization, M.Y. and A.E.; supervision, G.H.A. and M.A.E.R.; project administration, M.A. and A.M.I.; funding acquisition, G.H.A. and A.A.A. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the Deanship of Scientific Research at Najran University, under the General Research Funding program, grant code NU/-/MRC/10/362.

Data Availability Statement

Not applicable.

Acknowledgments

The authors are thankful to the Deanship of Scientific Research at Najran University for funding this work under the General Research Funding program, grant code NU/-/MRC/10/362.

Conflicts of Interest

The authors declare no conflict of interest.

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Crystals 12 00903 g001 550
Figure 1. LC-MS profile of aqueous methanolic extract from E. retusa aerial parts.
Figure 1. LC-MS profile of aqueous methanolic extract from E. retusa aerial parts.
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Crystals 12 00903 g002 550
Figure 2. LC/MS/MS chromatogram of dihydrocaffeoyl coumaroyl quinic acid.
Figure 2. LC/MS/MS chromatogram of dihydrocaffeoyl coumaroyl quinic acid.
Crystals 12 00903 g002
Crystals 12 00903 g003 550
Figure 3. FT-IR spectra of Er-ZnONPs.
Figure 3. FT-IR spectra of Er-ZnONPs.
Crystals 12 00903 g003
Crystals 12 00903 g004 550
Figure 4. Zeta potential analysis of synthesized Er-ZnONPs.
Figure 4. Zeta potential analysis of synthesized Er-ZnONPs.
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Crystals 12 00903 g005 550
Figure 5. XRD spectrum of synthesized Er-ZnONPs.
Figure 5. XRD spectrum of synthesized Er-ZnONPs.
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Crystals 12 00903 g006a 550Crystals 12 00903 g006b 550
Figure 6. (a) SEM analysis of synthesized Er-ZnONPs; (b) TEM analysis of synthesized Er-ZnONPs.
Figure 6. (a) SEM analysis of synthesized Er-ZnONPs; (b) TEM analysis of synthesized Er-ZnONPs.
Crystals 12 00903 g006aCrystals 12 00903 g006b
Crystals 12 00903 g007 550
Figure 7. (a) Effect of Er-ZnONPs on caspase-8. (b) Effect of Er-ZnONPs on COX-2 proteins.
Figure 7. (a) Effect of Er-ZnONPs on caspase-8. (b) Effect of Er-ZnONPs on COX-2 proteins.
Crystals 12 00903 g007
Table
Table 1. Tentative identification of secondary metabolites from E. retusa aerial parts utilizing high-resolution LC-MS/MS.
Table 1. Tentative identification of secondary metabolites from E. retusa aerial parts utilizing high-resolution LC-MS/MS.
NoRt[M-H]−FragmentationsMolecular FormulaErrorProposed StructuresRef.
12.01191.0551173, 147, 111C7H12O60.115Quinic acid[12]
22.17295.0668179, 133, 115C10H16O100.827Malic acid-2-O-hexoside[13]
32.30341.1089179, 161, 143C12H22O111.062Dihexoside[14]
43.47315.0718153, 123, 109C13H16O90.782Protocatechuic acid-1-O-hexoside[15]
53.52329.0883167, 152, 123C14H18O91.591Vanillic acid-1-O-hexoside[14]
63.53345.0466169, 125C13H14O111.362Gallic acid–1-O-glucouronide
73.62153.018145, 109C7H6O40.235Protocatechuic acid[15]
84.10137.023793C7H6O30.379Hydroxybenzoic acid[15]
94.11183.0288168C8H8O50.010Methyl gallate[16]
104.12355.1038193, 178, 149C16H20O91.441Ferulic acid-1-O-hexoside[15]
114.31163.0026119C9H8O30.015p-Coumaric acid[17]
124.33385.1139223, 208, 193C17H22O100.977Sinapic acid-1-O-hexoside[14]
134.34355.1035311, 193C16H20O91.411Ferulic acid-4-O-hexoside[15]
144.39179.02318135C9H8O44.551Caffeic acid[14]
154.41449.1093287, 269, 179C21H22O11 1.462Dihydrokaempferol–O-hexoside[18]
164.42353.0880289, 191, 179, 173C16H18O91.291Chlorogenic acid[12]
175.35447.0935301C21H20O111.312Quercetin-O-rhamnoside[19]
185.36461.1669315, 299C22H22O111.557Isorhamnetin-O-rhamnoside[20]
195.95491.08334301, 179, 151C22H20O131.323Quercetin-3-O-glucouronide methyl ester
205.97273.007193, 178, 149C10H10O7S-Ferulic acid 4-O-sulphate[21]
216.01417.0669285C20H18O102.08Kaempferol-O-pentoside[22]
226.32501.1408337, 175, 163, 119C25H26O111.662Dihydrocaffeoyl coumaroyl quinic acid
Table
Table 2. IC50 values (ug/mL) for the effect of Er-ZnONPs on different cancer cells representing different tumor types. Results are represented as mean value (M) ± S.E.M of at least three independent experiments (three replicates each).
Table 2. IC50 values (ug/mL) for the effect of Er-ZnONPs on different cancer cells representing different tumor types. Results are represented as mean value (M) ± S.E.M of at least three independent experiments (three replicates each).
Cell LineCancer TypeIC50 (µg/mL)
HepG2Hepatocellular carcinoma25.6 ± 1.23
Huh-7Hepatocellular carcinoma25.7 ± 1.02
A-549Lung cancer22.3 ± 1.11
H460Lung cancer51.4 ± 3.27
MCF-7Breast cancer37.7 ± 2.01
MDA-MB-231Breast cancer37 ± 3.45
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Alqahtani, A.A.; Attia, G.H.; Elgamal, A.; Aleraky, M.; Youns, M.; Ibrahim, A.M.; Abdou, R.; Shaikh, I.A.; El Raey, M.A. Cytotoxic Activity of Zinc Oxide Nanoparticles Mediated by Euphorbia Retusa. Crystals 2022, 12, 903. https://doi.org/10.3390/cryst12070903

AMA Style

Alqahtani AA, Attia GH, Elgamal A, Aleraky M, Youns M, Ibrahim AM, Abdou R, Shaikh IA, El Raey MA. Cytotoxic Activity of Zinc Oxide Nanoparticles Mediated by Euphorbia Retusa. Crystals. 2022; 12(7):903. https://doi.org/10.3390/cryst12070903

Chicago/Turabian Style

Alqahtani, Abdulsalam A., Gouda H. Attia, Abdelbaset Elgamal, Mohamed Aleraky, Mahmoud Youns, Ammar M. Ibrahim, Randa Abdou, Ibrahim Ahmed Shaikh, and Mohamed A. El Raey. 2022. "Cytotoxic Activity of Zinc Oxide Nanoparticles Mediated by Euphorbia Retusa" Crystals 12, no. 7: 903. https://doi.org/10.3390/cryst12070903

APA Style

Alqahtani, A. A., Attia, G. H., Elgamal, A., Aleraky, M., Youns, M., Ibrahim, A. M., Abdou, R., Shaikh, I. A., & El Raey, M. A. (2022). Cytotoxic Activity of Zinc Oxide Nanoparticles Mediated by Euphorbia Retusa. Crystals, 12(7), 903. https://doi.org/10.3390/cryst12070903

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