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

Abstract The present study reports the synthesis of ZnO-NPs using Acantholimon serotinum extracts followed by characterization and evaluation of biological activities. Field emission scanning electron microscope revealed irregular spherical morphology with a size in the range of 20–80 nm. The X-ray diffraction analysis confirmed the synthesis of highly pure ZnO NPs with a hexagonal shape and a crystalline size of 16.3 nm. The UV-Vis spectroscopy indicates the synthesis of ZnO-NPs. FT-IR confirmed the presence of phytocomponents in the plant extract, which was responsible for nanoparticle synthesis. According to MTT results, the biosynthesized ZnO-NPs showed cytotoxic effects on human colon cancer Caco-2 (IC50: 61 µg/mL), neuroblastoma SH-SY5Y (IC50: 42 µg/mL), breast cancer MDA-MB-231 (IC50: 24 µg/mL), and embryonic kidney HEK-293 (IC50: 60 µg/mL) cell lines. Significant reactive oxygen species (ROS) generation was measured by the DCFH-DA assay after 24 h incubation with ZnO-NPs (200 µg/mL). ZnO-NPs caused apoptotic and necrotic effects on cells, which was confirmed by Annexin V-PE/7-AAD staining and 6.8-fold increase in pro-apoptosis gene Bax and 178-fold decrease in anti-apoptosis gene Bcl-2. The well diffusion method did not show effective growth inhibition activities of the ZnO-NPs against bacteria. In conclusion, the ZnO-NPs induce cytotoxicity in cell lines through ROS generation and oxidative stress.


Introduction
Nanoparticles (NPs) have been introduced in many fields of biology, medicine, and material science and have been harnessed for application in diverse fields such as tissue engineering, drug design, and gene therapy to develop new therapeutic approaches over the last decade [1][2][3]. Metal NPs research has recently received special attention due to their unusual properties when compared with bulk metal [4]. For example, zinc oxide nanoparticles (ZnO-NPs) have become one of the most popular metal oxide NPs in industrial, pharmaceutical, and biological applications [5]. Physical and chemical methods that are used for the synthesis of metal NPs such as ZnO-NPs are expensive and toxic. Green biosynthesis of ZnO-NPs is the use of reducing and capping agents obtained from plant material that eliminates the use of noxious chemicals with toxic effects [6,7]. Furthermore, many scientists have been attracted to use such resources for the biosynthesis of metal NPs due to the ease of production, diversity in size and shape, and enhanced biocompatibility of NPs relative to other methods. Until now, green synthesis of ZnO-NPs using different plant extracts and their potential applications in biology were reported [8,9] Acantholimon Boiss is a genus in the family Plumbaginaceae composed of approximately 200 species that most of them are distributed in Irano-Turanian phytogeographic region [10][11][12][13]. It was shown that plants from the Plumbaginaceous family contain secondary metabolites including plumbagin, lignin, saponins, anthocyanin, quinines, alkaloids, simple phenolic, tannins, and flavonoids that are responsible for their biological effects [14,15]. Until now, little attention has been given to the identification of phytochemicals and biological activities of the genus Acantholimon [16]. Recently, cytotoxic, antioxidant, and antibacterial activities of three species of Acantholimon including A. austro-iranicum, A. serotinum and A. chlorostegium were investigated by Soltanian et al. [17].
In this study, ZnO-NPs were green synthesized using A. serotinum methanol extract for the first time. Synthesized ZnO-NPs were characterized by various techniques, and cytotoxic activity against several types of cancer and normal cell lines and antibacterial activity against two gram-positive (Enterococcus faecalis and Staphylococcus aureus) and two gram-negative (Pseudomonas aeruginosa and Escherichia coli) bacteria were evaluated. In addition, this study showed that the exposure of cells to ZnO-NPs leads to reactive oxygen species (ROS) generation, upregulation of Bax and downregulation of Bcl-2, and finally apoptosis/necrosis induction.

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 (ZnSO 4 ) 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.

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.

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% CO 2 ). 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 IC 50 value, a concentration that a cytotoxic agent induces a 50% growth inhibition [21].

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 × 10 3 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% CO 2 , 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 H 2 O 2 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 × 10 5 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.

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 × 10 4 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′-CCCG AGAGGTCTTTTTCCGAG-3′ and reverse 5′-CCAGCCCATGA TGGTTCTGAT-3′ for Bax, and forward 5′-CATGTGTGTGG AGAGCGTCAA-3′ and reverse 5′-GCCGGTTCAGGTACTCAG TCA-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-NPstreated 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: ΔCT test sample − ΔCT control sample [27,28].

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 (10 8 colonyforming 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].

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.

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

Determination of cytotoxic effect of synthesized ZnO-NPs
MTT is a colorimetric assay based on the mitochondrial succinate dehydrogenase activity of viable cells [32].

Determination of intracellular ROS
Results obtained from ROS generation in HEK-293 cells exposed to H 2 O 2 (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 H 2 O 2 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).

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).

Analysis of apoptosis-related gene expression
In this study, the expression of pro-apoptotic and antiapoptotic genes at the mRNA level in ZnO-NPs-exposed HEK-293 cell line was studied using quantitative realtime 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.

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.
The bioreduction of ZnO-NPs using methanol extracts of A. serotinum was investigated in this study. Synthesized ZnO-NPs were characterized using UV-Vis spectroscopy, FT-IR, and FESEM. UV-Vis spectra suggested the presence of a strong peak at 380 nm confirming the NPs synthesis. FESEM micrograph demonstrated the presence of spherical NPs with a size range of 20-80 nm. FT-IR confirmed the presence of some functional groups such as free hydroxyl, aromatic, carbonyl, primary amine, and carboxylic acid in the plant extract which were responsible for nanoparticle synthesis. FT-IR revealed that proteins or other soluble organic compounds in the extract may bind with zinc ions and reduce the zinc ions to NPs. After characterization, cytotoxicity, ROS production, induction of apoptosis and necrosis and antibacterial activities of green bio-synthesized ZnO-NPs were investigated in this study. The evaluation of the antiproliferative/cytotoxic activity against human colon carcinoma (Caco-2), neuroblastoma (SH-SY5Y), breast (MDA-MB-231), and embryonic kidney (HEK-293) cells showed the great cytotoxic potential of green ZnO-NPs with IC 50 value of 61, 42, 24, and 60 µg/mL. In our previous research, the cytotoxicity of methanol extract of A. serotinum was tested on various cancer cell lines [17]. When the IC 50 of the ZnO-NPs and extract were compared, the cytotoxic potential of synthesized ZnO-NPs was higher than the A. serotinum extract (IC 50 values of extract for neuroblastoma, breast, and colon cancer cell lines were calculated as 328, 403, and 600 µg/mL) [17]. It might be due to the synergetic effects of biomolecular groups derived from A. serotinum that adhered to the process of ZnO-NPs synthesis [45]. Moreover, nanosized particles may improve their stability and cell penetration leading to enhanced bioavailability and cytotoxicity [46]. As a result, cytotoxic effects induced by ZnO-NPs can be attributed to both nanosized zinc and bioactive phytocompounds attached on the surface of ZnO-NPs [46]. This is the first investigation to introduce the cytotoxicity of photosynthesized ZnO-NPs using methanol extract of A. serotinum against cancer and normal cell lines. Cytotoxicity and anticancer effects of biosynthesized ZnO-NPs using another plant extracts such as Punica granatum, Silybum marianum, Tectona grandis, Tamarindus indica, Nepeta deflersiana, and Albizia lebbeck also supported results obtained from this study [9,24,30,39,40,47]. Unfortunately, these findings suggest that the A. serotinum ZnO-NPs did not show specificity and selectivity toward the cancerous cells when compared with the normal cells; therefore, it is not selective enough to be useful as an anticancer compound. Moreover, we demonstrated that synthesized ZnO-NPs induce the generation of ROS in cells. Our results are in good agreement with the recent reports that have shown NPs such as ZnO-NPs can stimulate ROS formation [24,[48][49][50]. Therefore, it is approved that the toxicity of ZnO-NPs is induced by the generation of ROS. Free radicals generated by ZnO-NPs would oxide and modify macromolecules including proteins, enzymes, membrane lipids, and DNA which subsequently result in oxidative damage of organelles and cell apoptosis. Staining the cells with Annexin V and 7-AAD solution showed that the cytotoxicity of ZnO-NPs toward HEK-293 cells was mediated by apoptosis and necrosis induction. In other reports, apoptotic and necrosis induction was also observed after exposure of cells to plantsynthesized ZnO-NPs [48,[50][51][52].
Further studies to illuminate the ZnO-NP-induced apoptosis was conducted by expression analysis of Bax and Bcl-2. The BCL-2 protein family consisting of antiapoptotic and pro-apoptotic members acts as a critical life-death decision point within the common pathway of apoptosis. BCL-2 as an anti-apoptotic member of this family prevents apoptosis. In contrast, pro-apoptotic members of this family, such as BAX lead to caspase activation and trigger apoptosis [53]. Real-time PCR results showed that the expression of pro-apoptotic Bax gene was significantly upregulated, while the expression of the anti-apoptotic Bcl-2 gene was significantly reduced in cells treated with ZnO-NPs when compared with normal cells. In accordance with our results, previous researches also showed the upregulation of Bax and downregulation of Bcl-2 during apoptosis induction by NPs such as ZnO-NPs [27,28]. Figure  6 shows the mechanism of the toxicity of ZnO-NPs.
Owing to bacteria resistance to antibiotic and metal ions, scientists have focused on the development of other methods such as NPs for killing the pathogenic bacteria [35]. Photosynthesized ZnO-NPs in this research showed weak antibacterial activity.
It has been reported that NPs interact with the bacterial cell wall or membrane and release metal ions that result in the disruption of the cell permeability and production of ROS inside the cell. This can damage DNA and denature proteins that finally trigger apoptosis or cell death [42]. Antibacterial activities of ZnO-NPs depend on morphology, particle size, powder concentration, specific surface area, etc. [30]. For example, smaller NPs having a large surface area available for interaction have more antibacterial effect than the larger nanoparticle. On the other hand, it is demonstrated that when the concentration of plant extract increase, antimicrobial activities of green synthesized NPs increase because the presence of the plant extract on the surface of the NPs enhance nanoparticle solubility. An increase in solubility leads to permeation of NPs through the bacterial cell wall, disturbance in cell metabolism, and finally cell death [29]. Since properties of ZnO-NPs and synthesis condition can affect on antibacterial properties, biosynthesized ZnO-NPs with various plant extracts such as Cassia alata, Tectona grandis, Cochlospermum religiosum, Albizia lebbeck, Punica granatum, and Silybum marianum showed different antibacterial activities [8,9,30,[39][40][41]54]. Weak antimicrobial activity of ZnO-NPs that was seen in our results may be due to agglomeration of ZnO-NPs in the solution using van der Waals forces and superficial effects [55,56].
In conclusion, plants have some biomolecules and bioreducing agents such as enzymes, proteins, flavonoids, terpenoids, and cofactors that provide a versatile, economical, and eco-friendly method to fabricate metal NPs [57,58]. ZnO-NPs synthesized with natural plant extracts have broad applications in the biomedical and industrial fields. In this study, ZnO-NPs were synthesized using a methanol extract of A. serotinum for the first time. The synthesized NPs exhibited potential cytotoxic activities against cancer and normal cell lines. The production of ROS analyzed using DCFH-DA assay is an essential mechanism through which ZnO-NPs induce oxidative stress and apoptosis. 7-AAD and Annexin-V dyes demonstrated that ZnO-NPs have the potential to induce apoptosis and necrosis. Apoptosis induction was further evaluated by expression analysis of two important members of BCL-2 family including Bax and Bcl-2. The antibacterial activity of the ZnO-NPs was examined on four bacterial stains, and it was discovered that ZnO-NPs synthesized by A. serotinum have weak antibacterial activity. Hence, it is concluded that the synthesized ZnO-NPs show cytotoxicity by the generation of ROS, leading to oxidative stress and eventually cell death.