Characterization and biological activities of synthesized zinc oxide nanoparticles using the extract of Acantholimon serotinum

2.1 Plant extraction procedure

Aerial parts of Acantholimon serotinum Rech.f.& Schiman-Czeika were collected from Baft to Khabr, Kerman Province, Southeast of Iran, identified according to the standard keys, and deposited in MIR herbarium under voucher number 1968. Dry powdered plant material was extracted using a maceration technique with methanol 80% and then dried in the oven (Vacucell, Einrichtungen GmbH) at 40°C [17–19].

2.2 Biosynthesis of ZnO-NPs

The amount of 10 mL of plant extracts sample (100 µg/mL in distilled water) was added to 100 mL of 0.1 M zinc sulfate (ZnSO₄) aqueous solution. An aqueous solution of NaOH was gradually added into the solution under the steady stirring until the pH of the solution reached to 8 to attain smaller size particles [20]. The solution was kept on a magnetic stirrer at 60°C for 6 h. The yellowish-brown color of the solution indicated the formation of the particles. The solution was centrifuged at 10,000 rpm for 10 min, the supernatant was discarded, and the pellet was washed using deionized water 4–5 times; afterward, it was washed by ethanol around 3 times to remove organic impurities. After washing, the samples were dried in an oven at 50°C.

2.3 Characterizations of ZnO-NPs

The formation of ZnO-NPs was monitored using a UV-Vis spectrophotometer (PerkinElmer, Germany) at 300–600 nm. Field emission scanning electron microscopy (FESEM) (Quanta 200, USA) was used for examination of the size, morphology, and distribution of synthesized samples. Crystallographic properties of ZnO NPs were explored using the X-ray diffraction (XRD) technique within 2θ = 10–90 using the XRD instrument (Rigaku, Ultima IV, Tokyo, Japan) with a Cu LFF λ = 1.540598 A as a radiation source. The obtained pattern from the XRD was then analyzed using X’Pert High Score Plus software, and the chemical composition, crystalline structure, and size of the NP were identified. The size of the particles was calculated using the Debye–Scherrer equation:

where D is the crystalline size, λ is the wavelength of X-ray used, β is the full line width at the half maximum (FWHM) elevation of the main intensity peak, and θ is the Bragg’s angle.

The NPs and plant extract were subjected to Fourier transform infrared spectra (FT-IR) spectrometric analysis to specify the functional groups in the extract that may be responsible for reducing ions to NPs.

2.4 Cell culture and cytotoxic activity

To examine the cytotoxic activity of ZnO-NPs, human colon cancer (Caco-2), neuroblastoma (SH-SY5Y), breast cancer (MDA-MB-231), and embryonic kidney (HEK-293) cell lines were cultured with Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 µg/mL penicillin and 100 µg/mL streptomycin and incubated at 37°C (5% CO₂). The attached cells were trypsinized for 3–5 min to get the individual cells. The cells were counted and distributed in a 96-well plate with 5,000 cells in each well. The plate was incubated for 24 h to allow the cells to form ∼70–80% confluence as a monolayer. Cytotoxicity of ZnO-NPs was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. For this, cells were exposed for 48 h to different concentrations (10, 20, 40, 80, 160, 320, and 640 µg/mL) of ZnO-NPs. To detect the cell viability, 20 µL MTT solution was added to each well and incubated for 3 h. Then, the MTT solution was removed and 100 µL DMSO was added to each well followed by 15 min incubation. The optical density of the formazan product was taken at 499 nm in a microplate reader (BioTek-ELx800, USA), and the percentage of cell viability was calculated as follows: A_treatment/A_control × 100% (where, A = absorbance). The mean of three absorbance values were calculated for each concentration. The date was used to determine IC₅₀ value, a concentration that a cytotoxic agent induces a 50% growth inhibition [21].

2.5 Intracellular ROS detection

The level of intracellular ROS was assessed by measuring the oxidation of 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA). DCFH-DA diffuses through the cell membrane and is deacetylated by cellular esterase to the non-fluorescent DCFH. Intracellular ROS can oxidize DCFH to the fluorescent 2,7-dichlorofluorescein (DCF); therefore, the intensity of fluorescence is directly proportional to the levels of intracellular ROS [22,23]. Briefly, 25 × 10³ cells were cultured in 96-well microplates. After 24 h, the medium was removed and replaced by a medium containing 10 µM of DCFH-DA (Sigma-Aldrich, Germany), and cells were kept in a humidified atmosphere (5% CO₂, 37°C) for 45 min. To measure intracellular ROS, cells were exposed to various concentrations of ZnO-NPs (25–200 µg/mL) or 600 µM H₂O₂ as a positive control. After 3 and 24 h, fluorescence was measured at an excitation wavelength of 485 nm and an emission wavelength of 538 nm (FLX 800; BioTek). Results were expressed as the percentage of fluorescence intensity relative to untreated control cells [23,24].

2.6 In vitro apoptosis/necrosis assay

PE-conjugated-Annexin V/7-AAD assay (BD Biosciences kit) was used to quantitatively determine the percentage of cells within a population that are actively undergoing apoptosis/necrosis. At >90% confluence, the HEK-293 cells (4 × 10⁵ cells/6 cm dish) were incubated with prepared ZnO-NPs at 60 µg/mL concentration. Untreated HEK-293 cells were used as a negative control. Following treatment for 48 h, both adherent and floating cells were collected and washed with PBS. The cell pellets were suspended in 100 µL Annexin V binding buffer, 5 µL PE-Annexin V, and 5 µL 7-AAD. The tube was gently vortexed and incubated for 15 min in dark condition. Following the addition of 400 µL binding buffer, the samples were used for flow cytometric analysis by BD FACS Calibur™ (BD Biosciences, San Jose, CA, USA) at an excitation wavelength of 488 nm. PE was detected in fluorescence channel 2 (FL2): 575/30 band pass filter (BP) and 7-AAD were detected in channel 3 (FL3): 620/30 BP [25,26].

2.7 Analysis of apoptosis-related gene expression

An SYBR Green real-time quantitative PCR was carried out to compare the expression levels of Bax and Bcl-2 mRNAs in untreated and ZnO-NPs-treated HEK-293 cells. HEK-293 cells were seeded into 6 cm dishes (5 × 10⁴ cells/well) and incubated for 24 h, then the cells were treated with 60 µg/mL ZnO-NPs for 48 h. Total cellular RNA was extracted from cells using the total RNA isolation kit (DENAzist Asia, Mashhad, Iran) according to the manufacturer’s instructions. The quantity and quality of RNA were assessed using a Nanodrop and agarose gel electrophoresis. The cDNA was synthesized using M-MuLV reverse transcriptase (Cat. No. EP0441; Thermo Scientific, Wilmington, USA), according to protocol. The primers used for real-time PCR were as follows: forward 5′-CCCGAGAGGTCTTTTTCCGAG-3′ and reverse 5′- CCAGCCCATGATGGTTCTGAT-3′ for Bax, and forward 5′-CATGTGTGTGGAGAGCGTCAA-3′ and reverse 5′-GCCGGTTCAGGTACTCAGTCA-3′ for Bcl-2. Also, the sequence of the forward primer for the internal control gene beta-2-microglobulin (β2M) was 5′-CTCCGTGGCCTTAGCTGTG-3′ and that of the reverse primer was 5′-TTTGGAGTACGCTGGATAGCCT-3′.

The expression of the target genes was studied using an Analytik Jena, a real-time PCR system (Germany). Each PCR amplification reaction was performed in 20 µL reaction mixture containing 10 µL of 2× SYBR Green master mix (Cat. No. 5000850-1250; Amplicon, UK), 0.5 µL of each primer (0.25 µM), 1 µL cDNA (50 ng), and 8 µL double-distilled water. After denaturation at 95°C for 15 min, 40 cycles were followed by 95°C for 15 s, 60°C for 20 s, and 72°C for 10 s in PCR cycling condition. The amplification stage was followed by a melting stage that temperature was increased in steps of 1°C for 10 s from 61°C to 95°C.

A comparative threshold cycle method was used to determine the relative expression level of the target gene. For this, the mean threshold cycle value of β2M as a reference gene was subtracted from the mean threshold cycle value of the target genes (Bax and Bcl-2) to obtain ΔCT and fold change in the target gene of ZnO-NPs-treated cell relative to the untreated control sample was calculated according to the following equation: Fold change = 2^(−ΔΔCT) where ΔΔCT is calculated by the following equation: ∆CT_{test sample} − ∆CT_{control sample} [27,28].

2.8 Determination of antibacterial property

The antibacterial potential of synthesized ZnO-NPs was tested against Enterococcus faecalis (ATCC 29212), Staphylococcus aureus (ATCC 25838), Pseudomonas aeruginosa (ATCC 27853), and Escherichia coli (ATCC 11333) bacteria using the agar well diffusion method. Briefly, the Muller Hinton agar plates were inoculated with 1 mL (10⁸ colony-forming units) of bacterial cultures using spread-plating. After drying the plates, wells of size 6 mm have been made on Muller–Hinton agar plates using gel puncture. 100 µL of various concentrations (500, 1,000, 3,000 µg/mL) of ZnO-NPs was poured in each well. The culture plates were incubated at 37°C for 24 h. After the growth period, the plates were removed and the antibacterial activity was measured based on the inhibition zone (millimeters) around wells poured with the nanoparticle. Ciprofloxacin (25 mg/mL) and deionized water were used as a positive and negative control, respectively. The experiment was repeated three times [29,30].

2.9 Statistical analysis

The one-way analysis of variance (ANOVA) was used to determine whether there are any statistically significant differences between the means of control and treatments. The data were shown as mean ± SD and p < 0.05 accepted as the minimum level of significance.

3.1 Characterizations of ZnO-NPs

UV-Vis spectra showed a strong peak at 380 nm confirming the ZnO-NPs synthesis (Figure 1a).

Figure 1

Characterization of green synthesized ZnO-NPs prepared using methanol extract of the A. serotinum plant. The UV-Vis spectrum of ZnO-NPs synthesized by A. serotinum and color change of zinc sulfate to ZnO-NPs (a), FESEM image (b), X-ray powder diffraction pattern of the green synthesized ZnO-NPs (c) and FT-IR spectrum for plant extract and synthesized ZnO-NPs (d).

The size of sphere-like shaped ZnO-NPs was measured in the range of 20–80 nm using the FESEM image (Figure 1b). The crystalline structure of the ZnO NPs revealed the presence of distinct line broadening of XRD peaks with no remarkable shift in the diffraction peaks indicating that the crystalline product was without any impurities. The XRD peaks were observed at 31.76°, 34.42°, 36.25°, 47.51°, 56.59°, 62.85°, 66.37°, 67.94°, 69.08°, 72.5°, 76.9°, 81.3°, and 89.63°, which are indexed in crystal system of hexagonal phase with a space group of P63mc and reference code of 01-079-0206. The findings are in accordance with the results of Mahendra et al. that they reported the green synthesis of ZnO-NPs of Cochlospermum religiosum was without any impurities in the crystalline product [8]. The crystalline size of the bio-synthesized ZnO NPs was found to be about 16.3 nm from the FWHM of the most intense peak corresponding to 101 plane located at 36.25° using Debye–Scherrer equation (Figure 1c). The FT-IR was analyzed in the range of 0–4,000 cm⁻¹ to recognize the various functional groups present in the synthesized NPs and plant extract. In the case of AgNPs, the intense absorption band at 3406.23 cm⁻¹ was occurred due to O–H which was related to the adsorption of water on the surface of ZnO-NPs. The intense peaks at 2853.86 and 2923.61 cm⁻¹ indicate the presence of C–H stretch. Bands at 1362.71 cm⁻¹ assigned to –C–H bending vibrations absorption bands 1048.81 and 1126.02 cm⁻¹ indicate the significant presence of C–O stretch which is a functional group of alcohols, carboxylic acids esters, and ethers. The absorption at 600 cm⁻¹ indicates the presence of ZnO-NPs. FT-IR results for Plant extract showed a broad range at 3411.42 corresponding to O–H stretch, H-bonded in alcohols, and phenols. Weak peaks obtained at 2931.61 depicted the presence of C–H stretch alkane group. The band at 1617.59 cm⁻¹ in plant extract was due to the presence of and C–C stretch (in–ring), C–N stretch of amide I in proteins or C═O stretch in polyphenols which was shifted to 1699.59 cm⁻¹ in ZnNPs due to the proteins or polyphenols that may have been linked to NPs by the amine or phenolic groups. Medium peaks obtained at 1448.60 and 1040 cm⁻¹ illustrated C–H bend alkanes group. Weak bonds obtained at 650–1,000 cm⁻¹ represented ═C–H bend alkenes group (Figure 1d) [31].

3.2 Determination of cytotoxic effect of synthesized ZnO-NPs

MTT is a colorimetric assay based on the mitochondrial succinate dehydrogenase activity of viable cells [32]. MTT assay was performed to investigate the in vitro cytotoxic property of ZnO-NPs. Caco-2, SH-SY5Y, MDA-MB-231, and HEK-293 cell lines were treated with different concentrations of synthesized ZnO-NPs for 48 h to determine the inhibitory percentage. Based on the results, cell viability was proportional to the concentration of the ZnO-NPs. ZnO-NPs showed very potent cytotoxic activity in a way that the viability of Caco-2, SH-SY5Y, MDA-MB-231, and HEK-293 cell lines decreased by 50% following 48 h of incubation with 61, 42, 24, and 60 µg/mL concentration of ZnO-NPs, respectively. The obtained concentrations values corresponding to a survival rate of 50% is defined as the IC₅₀ values for each cell lines (Figure 2).

Figure 2

Anti-proliferative activity of ZnO-NPs. SH-SY5Y, HEK 293, Caco-2, and MDA-MD-231 cell lines were treated with different concentrations of ZnO-NPs (5, 10, 20, 40, 80, 160, 320, 640 µg/mL) for 48 h and subjected to MTT assay and cell viability was calculated.

3.3 Determination of intracellular ROS

Results obtained from ROS generation in HEK-293 cells exposed to H₂O₂ (600 µM) and different concentrations of ZnO-NPs (25, 50, 200 µg/mL) for 3 and 24 h are shown in Figure 3. A statistically significant induction in ROS generation was measured in HEK-293 cells exposed to H₂O₂ as a positive control for oxidative stress. Cells that were treated with ZnO-NPs for 3 h did not increase ROS as compared to untreated control (Figure 3a). However, the ROS level increased 2-fold after exposure to 200 µg/mL ZnO-NPs for 24 h when compared to control cells (Figure 3b).

Figure 3

Evaluation of ROS generation in HEK-293 cells using the fluorescent probe DCF. Cells were treated with different concentrations of ZnO-NPs (25, 50, 200 µM) and positive control (600 µM H₂O₂) for 3 h (a) and 24 h (b). Data are expressed as a percent of values of untreated control cells and are means ± SD of three independent experiments. * indicates a statistically significant difference from corresponding untreated control cells (p < 0.05).

3.4 Apoptosis/necrosis assessment using annexin V-PE and 7-AAD

Annexin-V/7-AAD detection kit takes advantage of the fact that phosphatidyl serine (PS) translocate from the inner (cytoplasmic) leaflet of the plasma membrane to the outer (cell surface) leaflet soon after the induction of apoptosis and that the Annexin V protein has a strong, specific affinity for PS [33]. Moreover, late apoptotic cells and necrotic cells lose their cell membrane integrity and are permeable to vital dyes such as 7-AAD [34]. Hence, the annexin V^+/7-AAD⁻ cells detect early stage of apoptosis, annexin V^+/PI⁺ cells exhibit late stage of apoptosis, annexin V-/7-AAD+ cells represent necrosis, and annexin V⁻/7-AAD⁻ cells show live cells. Flow cytometric analysis of Annexin V-PE staining showed 96% of HEK-293 control cells were alive. There are about 25% and 14% increases in late apoptotic and necrotic cell population in HEK-293 cells treated with ZnO-NPs, respectively, as compared with untreated ones (Figure 4).

Figure 4

Flow cytometric analysis of ZnO-NPs -induced HEK-293 cells apoptosis. Flow cytometry dot plots of Annexin V-FITC/7-AAD double staining of control HEK-293 cells without treatment (a) and after treatment with ZnO-NPs (b).

3.5 Analysis of apoptosis-related gene expression

In this study, the expression of pro-apoptotic and anti-apoptotic genes at the mRNA level in ZnO-NPs-exposed HEK-293 cell line was studied using quantitative real-time PCR (Figure 5). Our findings show that the mRNA level of Bax was significantly upregulated (6.8-fold), while the expression of the Bcl-2 was significantly diminished (178-fold) in cells treated with ZnO-NPs when compared with normal cells. These results confirm that the exposure of ZnO-NPs induced substantial apoptosis in HEK-293 cells.

Figure 5

Gene expression ratio of Bcl-2 and Bax in HEK-293 cells exposed to ZnO-NPs compared to untreated control cells. HEK-293 cells were treated with ZnO-NPs (60 µg/mL) for 48 h. Expression of Bax and Bcl-2 were considered as 1 in untreated control HEK-293 cells and the level of genes after treatment with ZnO-NPs are represented as the fold change over the untreated controls.

3.6 Antibacterial activity

Owing to bacterial resistant to antibiotics and metal ions, scientists have focused on using NPs for killing the pathogenic bacteria [35]. In this report, the anti-microbial activity of synthesized ZnO-NPs against four bacteria strains was studied by the agar well diffusion method and results are given in Table 1. According to the zone of inhibition, ciprofloxacin showed good inhibitory activity for all the tested bacterial strains and synthesized ZnO-NPs showed weak antibacterial activities. About 3,000 µg/mL was recorded as the lowest concentration at which antibacterial activities on two Gram-positive strains: E. faecalis and S. aureus and two Gram-negative strains: P. aeruginosa and E. coli were demonstrated. We found no antibacterial activity at a lower concentration of ZnO-NPs.