Biosynthesis of Zinc Oxide Nanoparticles Using Capparis spinosa L. Fruit Extract: Characterization, Biocompatibility, and Antioxidant Activity

Abstract

Recent years have seen an increase in the acceptance of green chemistry as a viable method for producing nanoparticles. The aim of this work was to investigate the cytotoxicity, antioxidant effect, and hemocompatibility of zinc oxide nanoparticles (ZnO NPs) produced utilizing Capparis spinosa L. (C. spinosa L.) fruit extract as a powerful reducing agent. The reaction rate and ZnO NPs’ shape concerning extract concentration, contact time, pH, and temperature were investigated. The production and characterization of ZnO-NPs were confirmed by ultraviolet spectroscopy (U-V), Dynamic Light Scattering (DLS), zeta potential, Field Emission Scanning Electron Microscope (FESEM), X-ray Diffraction (XRD), and Fourier Transform Infrared Spectroscopy (FTIR). The antioxidant activity of ZnO NPs was performed by DPPH assay; hemocompatibility of the erythrocytes and cell-cytotoxic properties were investigated using MTT assay. XRD confirmed the production of highly pure, crystalline ZnO-NPs, while FESEM analyses revealed that these particles were agglomerated and spherical, with an average size of about 37.49 nm. Moreover, UV-Vis absorption was revealed at 366 nm, and tests of ZnO NPs’ ζ potential and DLS revealed values of −44.76 mV and 116.80 nm, respectively. ZnO NPs exhibited good biocompatibility with RBCs in hemolytic activity experiments and did not exhibit any hemolytic reaction at doses ranging from 7.5 to 120 μg/mL. The study concluded that greenly generated ZnO NPs had good antioxidant activity and little cytotoxicity and can thus possibly be developed as a useful therapeutic option.

1. Introduction

Nanotechnology is a rapidly expanding branch of technology that holds enormous potential for the chemical [1], medicinal [2], engineering [3], and food-processing industries [4]. This rapid development of nanotechnologies suggests that nanoscale production will soon be used in almost every area of science and technology. Metal oxide NPs have attracted a great deal of interest in a variety of disciplines, including biological [5], industrial [6], optical [7], catalytic [8], pharmaceutical [9], and healthcare applications [10] due to their exceptional surface area and nanoscale size properties. It is speculated that the widespread use of these metal oxide nanoparticles may expose people to their possibly dangerous environment. Because of the potential for cell membrane leakage caused by their toxicity, more environmentally friendly synthesis routes must be developed. Eco-friendly, sustainable materials, including plant extracts, bacteria, fungi, and enzymes, have several advantages in terms of eco-friendliness and compatibility for biomedical applications, as risky chemicals are not required for the production of ZnO NPs. ZnO nanoparticles are an important class of metal oxide nanoparticles exhibiting exciting biological and photocatalytic properties due to their small size and enhanced surface chemical reactivity [11]. However, the majority of conventional techniques for producing ZnO NPs are based on wet chemical route options, which demand the use of numerous hazardous chemicals during laborious, prolonged multistep processes that result in the production of vast quantities of hazardous byproducts and dangerous chemical waste [12]. In recent years, the use of more sustainable green techniques for preparation has been a catalyst for replacing harmful traditional chemicals with more environmentally friendly extracts from various natural materials, such as plants, fungi, bacteria, and algae, to prepare ZnO NPs; These NPs have demonstrated comparable and even higher activities compared to conventional ones. For instance, Eucalyptus [13], Phlomis [14], pomegranate [15], Syzygium cumini [16], and Ziziphus [17] have been used as plant part extracts for the green biosynthesis of ZnO NPs, with plant extracts utilized as capping agents and stabilizers to both maintain the generated nanoparticles’ stability and prevent their aggregation [18]. ZnO-NPs produced using green methods are safe for the environment and have also been used for biomedical purposes due to their antioxidant, antibacterial, and anticancer properties [19,20]. Selecting the appropriate plant source to prepare the extracts is a crucial step to obtaining ZnO NPs with the correct physicochemical properties, since phenolic chemicals in plant extracts play a crucial role in nanoparticle formation and development. It is important to note that C. spinosa L. is one of the Middle East region’s most widely used plants and is distinguished by a variety of medicinal properties. The plant boasts numerous long chains of natural compounds that could serve as capping and stabilizing agents and preclude the nanoparticles from aggregating [21,22]. Thus, this study aimed to investigate a simple and effective method for producing potent, eco-friendly ZnO nanoparticles utilizing high-purity fruit extract from C. spinosa L. as a reducing and stabilizing agent. Moreover, the morphological and structural features of the green biosynthesized ZnO NPs were characterized using Field Emission Scanning Electron Microscopy (FESEM), X-ray Diffraction (XRD), and Fourier Transform Infrared Spectroscopy (FTIR). In addition, the antioxidant activity, cytotoxicity, and hemocompatibility were evaluated.

2. Materials and Methods

2.1. Materials and Reagents

Zinc acetate dihydrate (Zn(CH₃COO)₂·2H₂O) with a purity of 99%, DPPH (Cat. No. D9132), Sodium dodecyl sulfate (SDS), phosphate-buffered saline (PBS), and MTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) were obtained from Sigma-Aldrich Co., St. Louis, MO, USA. All chemical reagents were utilized as received without any further purification.

2.2. Fruit Extraction

C. spinosa L. was collected from Ali Algharbi, Maysan Governorate, Iraq, in April and June 2022, and botanists at the University of Misan confirmed the authenticity. The extract of C. spinosa L. fruits was derived in accordance with [23], with a few modifications. The C. spinosa plant’s fresh fruits were collected, washed with tap water, and then washed again with deionized water. Then, the fruit samples were left to dry. In a pristine electric blender, the dried fruits were ground into a fine powder. Then, 100 mL of deionized water was added to 40 g of powdered dried fruit, which was then boiled for two hours at 90 °C while being magnetically agitated. The solution of extract was filtered using sterile Whatman No. 1 filter paper after cooling to room temperature and stored at 4 °C for further analysis.

2.3. Synthesis of ZnO NPs

ZnO NPs were synthesized following in accordance with [24], with some modifications. A hot plate with a magnetic stirrer was used to heat 30 mL of C. Spinosa L. fruit aqueous extract to 85 °C. When the mixture reached 60 °C, three grams of zinc acetate dehydrate was added, and the mixture was then allowed to boil until the extract was converted into a paste. Then, it was placed in a ceramic crucible cup and heated for three hours at 350 °C in a furnace. The gathered synthesized ZnO NP powder was centrifuged after being re-suspended in deionized distilled water. The powdered substance (ZnO NPs) was used in subsequent experiments.

2.4. Characterization of ZnO NPs

2.4.1. UV–Vis Spectroscopy

The obtained NPs were analyzed using a UV-Vis Spectrophotometer (UV-1800 UV-Vis Spectrophotometer, Shimadzu, Tokyo, Japan) in the 200–800 nm range to demonstrate that synthesized green ZnO NPs were produced.

2.4.2. XRD

The crystalline structure of the powdered ZnO NPs was examined using a Bruker D8 Advance diffractometer (Billerica, MA, USA) with CuKα radiation (λ = 1.5418 Å).

2.4.3. FTIR

An FTIR instrument (a Shimadzu Instrument, Kyoto Japan) was utilized to examine the samples over the wavelength range of 400–4000 cm1 in order to identify the functional groups, responsible for the reduction of Zinc ions and the stabilization of the synthesized nanoparticles.

2.4.4. FESEM

The morphology and size of the obtained ZnO NPs were determined using FESEM (TESCAN) at 30 kV acceleration voltages. The thin film of the obtained biosynthesized zinc oxide nanoparticles was prepared over the grid of a cover slide by applying a small amount of material, allowing it dry at room temperature, and then imaging using FESEM.

2.4.5. DLS and Zeta Potential

ZnO NPs were examined for size and surface charge using a particle size analyzer (Malvern, UK) through DLS and zeta potential.

2.5. MTT Assay

To evaluate the effect of ZnO NPs on cell proliferation in vitro, a Healthy L929 fibroblast cell line (ATCC, USA) was used. The cells were essentially grown in DMEM medium containing 10% fetal bovine serum [2]. Cells were plated into 96-well dishes with 100 mL of DMEM and kept at 37 °C in a humidified 5% CO₂ incubator. To investigate the effect of ZnONP concentration and exposure period on cell viability, different concentrations of green-produced ZnO NPs (7.5, 15, 30, 60, and 120 μg/mL) were added to each well and mixed with distilled water before being incubated for 24, 48, and 72 h. The diluent was used in the same quantity in the control cells (distilled water). Following the exposure period, the media in each well was changed to a new medium (100 mL) containing 5 mg/mL of MTT solution at 24, 48, and 72 h. After being incubated for four hours in the dark, the formazan crystal of the MTT reduction was dissolved in DMSO, and the absorbance was then determined using a microtiter plate ELISA reader. ZnONP effect was assessed using the proportion of reduced MTT dye control absorbance at 570 nm. Each experiment was performed three times. The viability of the cells was determined according to their capacity to transform the yellow dye MTT into a blue formazan crystal. The following formula was used to determine the percentage of cell viability.

$$\%\mathrm{cellular} \mathrm{viability}=\left(\mathrm{OD} \mathrm{specimen}/\mathrm{OD} \mathrm{control}\right)\times 100$$

2.6. Hemolytic Activity Assay

The hemolysis test was utilized to assess how green ZnO NPs and the fruit extract from C. Spinosa affected RBCs. Fresh human red blood cells were obtained from healthy 20–30-year-old donors. Centrifugation was used to prepare the blood sample, which required 8 min at 1500 rpm. A clean pellet was then obtained by washing three times with PBS. The collected RBCs were suspended in PBS (10% v/v). All samples were combined with various concentrations of C. Spinosa L. and ZnO NPs (7.5, 15, 30, 60, and 120 μg mL⁻¹) and incubated at 37 °C for 60 min. Following this, all samples were centrifuged at 5000 rpm for 4 min. The sample supernatant was then transferred to a 96-well plate. Using a plate reader, the absorbance values of the supernatants were calculated at 570 nm. The positive control was SDS in PBS (0.1%), and the negative control was PBS solution. The following formula was used to calculate the percentage of hemolysis in RBCs [25].

$$H\%=\frac{S.a-N.C.a}{P.C.a-N.C.a}\times 100\%$$

where H indicates hemolysis, S indicates sample, a indicates absorbance, C indicates control, N indicates negative, and P indicates positive.

2.7. Antioxidant Evaluation

Using the DPPH free radical scavenging method, the antioxidant potency of the ZnO NPs and C. spinosa L. extract was assessed, with ascorbic acid acting as the positive control. The standard solution of ascorbic acid was employed as a positive reference, and 1 mL of the biosynthesized ZnO NPs solution was diluted with methanol to obtain various concentrations (7.5–120 μg/mL). The mixture was then stirred and left in the dark at room temperature. After an hour of incubation, the absorbance (A) of each sample was measured using a UV-visible spectro-photometer at 520 nm to determine the measures of radical scavenging activity. The following equation was used to calculate the percentage inhibition (%) [26]:

$$\mathrm{Inhibition} \% =\frac{\left(A of C-A of S\right)}{\left(A of C\right)}\times 100\%$$

where A is the absorbance, C is the control, and S is the sample.

2.8. Statistical Analysis

One-way ANOVA was employed in the statistical method to assess group differences, and the Student t-test and Tukey’s test were applied to compare the groups. The data were displayed as the mean ± SD of triplicates.

3. Results

3.1. Characterization of the Synthesized ZnO NPs

3.1.1. Visual Inspection

The formation of ZnO NPs was confirmed by a visual color change and UV-Vis spectral analysis. As shown in Figure 1, the colors of the zinc acetate solutions changed to dark reddish-brown after microwave irradiation. This indicates that the metabolites in the aqueous extract of C. spinosa L. could be responsible for the reduction of zinc ions for synthesizing ZnO NPs. Comparing the experimental studies in the literature, similar color changes were observed in the green synthesis of ZnO NPs [27]. The biological foundation of green synthesis techniques depends on several factors, including the solvent, temperature, pressure, and pH levels (acidic, basic, or neutral). Functional groups in plant metabolites, such as amine, hydroxyl, and carbonyl, can interact with metal ions and reduce molecules to nanoscale size [28]. This is due to the availability of potent phytochemicals in various plant extracts, particularly in fruits, such as flavonoids, polyphenols, ketones, aldehydes, amides, alkaloids, carboxylic acids, saponins, and vitamins.

3.1.2. UV-Vis Spectral Analysis

To further verify the results, the formation and stability of the synthesized ZnO NPs were determined by measuring the absorption spectrum in the 200–800 nm wavelength range against the aqueous extract of C. spinosa L. as the reference. As seen in Figure 2, the maximum absorption peaks at 374 nm confirmed the green synthesized ZnO NPs. Furthermore, a peak with 290 nm was observed in the absorption spectra of the aqueous extract of C. spinosa L. The obtained absorption peaks in the visible range are formed from the excitation of free electrons due to the color change [28]. The position and shape of the absorption peaks of metallic nanoparticles are strongly related to particle size and morphology. A sharp, single absorption peak corresponds to spherical nanoparticles [29]. Metal ions are transformed into metal nanoparticles by the phytochemicals that are found in the plant extract. As a result, the plant extract has stabilizing and reducing capabilities. UV-Vis spectroscopy was used to observe the progression of this reaction [30]. When electromagnetic waves interacted with electron conduction band oscillation in the spectrum of UV-visible spectroscopy, peak absorption was coupled by the surface plasmon resonance (SPR), accurately simulating metal ion reduction and nanoparticle creation. Because there are numerous OH groups available, it is possible to make nanoparticles. The fruits of C. spinosa L. contain large amounts of alkaloids such as capparisine A, capparisine B, and capparisine C, as well as other flavonoid compounds such as rutin, quercetin, and tetrahydroquinoline acid, which are known for their ability to reduce metal ions and act as stabilizing agents in the nanoscale dimension [22]. These compounds are also known to be antioxidants and free of harmful substances.

3.1.3. XRD Analysis of the Resulting ZnO Nanoparticles

The XRD pattern of green-produced ZnO NPs using C. spinosa L. is illustrated in Figure 3. The XRD diffraction peaks at 2θ = 31.81°, 34.42°, 36.24°, 47.55°, 56.66°, 62.96°, 66.42°, 67.93°, 69.12°, 72.62°, and 77.02° correspond to (100), (002), (101), (102), (110), (103), (200), (112), (201), (004) and (202) planes, respectively. These peaks match those of (JCPDS card No. 36-1451), confirming the hexagonal wurtzite structure of the production of ZnO nanoparticles [31].

3.1.4. FTIR Analysis

The FTIR analysis was performed to confirm the presence of the functional groups of the metabolites of C. spinosa L. over the surface of the synthesized nanoparticles. The FTIR spectrums of the green synthesized ZnO NPs are shown in Figure 4. The stretching vibration of the hydroxyl group(O-H) is related to the peak in the FTIR spectrum that appears between 3291 and 3464 cm⁻¹, which is attributed to the phenolic compound in the plant extract that is involved in the synthesis of NPs [32]. The peak at 1635 cm⁻¹ in the FTIR spectrum of C. spinosa L. extract represents the stretching bands of (C=C) and (C=O) functional groups; an identical peak can also be seen at this location in the FTIR spectra of ZnO NPs produced at various temperatures. The organic functional groups at 1635 cm⁻¹ may not have entirely broken down because ZnO NPs’ annealing duration was barely one hour. This results in the vibrational peak we observed in our sample. This finding is consistent with the information provided by Faisal, Shah et al. [33]. The peak at 2977 cm⁻¹ is caused by the aliphatic (C-H) groups. The peaks at 1380 and 1382 cm⁻¹ indicate the (C-O-H). Between 492 and 422 cm⁻¹, metal-oxygen (Zn-O) stretching can be seen [34]. As a result, it can be said that the C. spinosa L. fruit extract has functional groups with (O-H) and (C=O) that operate as a capping, reducing, and stabilizing agents for ZnO NPs made biosynthetically. As efficient capping agents, the biomolecules in the plant extract aid in the production of nanoparticles. Several processes, including electrostatic stability, steric stabilization, hydration force stabilization, and van der Waals forces, appear to be used by the capping agents to stabilize NPs. The function and applications of nanoparticles depend on their stability.

3.1.5. The FESEM Results

FESM analysis demonstrated that the synthesized ZnO NPs were almost spherical and monodispersed. The synthesized ZnO NPs had an average size of 37.49 nm (Figure 5). The FESEM picture of the obtained ZnO NPs shows their homogeneous distribution and nearly spherical shape. The large majority of the ZnO NPs that were produced appeared extremely spherical and in the nanometer range. The green synthesis nanoparticles, which exhibit considerable aggregation development, were infrequently present in the generated ZnO NPs. This is due to the increased surface area and strong affinity of biosynthetic NPs, which accumulated or aggregated together.

3.1.6. DLS and Zeta Potential Analysis

The hydrodynamic diameters, zeta potentials, and polydispersity indexes (PDIs) of the synthesized ZnO NPs were determined by DLS analysis. The size distribution histogram of DLS showed that the average hydrodynamic diameters of the ZnO NPs were measured to be 116 ± 8.0 nm (Figure 6b). DLS measures the diameter of the particles in solution based on the Brownian motion. DLS analysis measures particle size including the hydration layer of water molecules; therefore, the average diameters of the synthesized ZnO NPs were greater than the diameters measured with FESEM and XRD analysis [35]. The ZnO NPs showed zeta potentials of −44 ± 76 mV (Figure 6a). It is known that a high negative charge prevents aggregation of nanoparticles via electrostatic repulsion and increases the stability of particles. PDI values of the synthesized ZnO NPs were calculated as 0.452 ± 0.092. A PDI value less than 0.7 indicates monodisperse particle size distribution [36]. It is well known that a nanomaterial’s physicochemical properties, such as its size, shape, surface area, zeta potential, and composition, have a substantial impact on the results of its cytotoxicity. Considering that ZnO NPs exhibit size-dependent substantial cytotoxicity, more soluble and smaller NPs offer more useful toxicological information on nanomaterials in this context. The biological activity of ZnO-NP is significantly influenced by their shape, size, and concentration. DLS average sizes are larger than those determined from SEM images; this is likely because ZnO NPs observed in SEM are only viewed on the surface of the agglomerates. In contrast, DLS measures the particles’ as three-dimensional, which might include a biomolecular coating. Such discrepancy between SEM and DLS average size has been reported previously and is related to the specifics of the method used. In order to examine the surface charge and stability of the synthesized NPs, a zeta-potential investigation was conducted. The results revealed that the synthesized NPs have good stability, as evidenced by the emergence of a distinct peak at roughly −44 ± 76 mV. The effective stability of ZnO NPs and reduction of metal ions may be due to the extract’s high protein and flavonoid content.

3.2. Cytotoxicity Results

To assess the cytotoxic effect of greenly obtained ZnO NPs utilizing C. spinosan. The MTT assay was used for various concentrations of ZnO NPs, including 7.5, 15, 30, 60, and 120 gmL⁻¹ on L929 normal fibroblast cells and cultured for different periods: 24, 48, and 72 h (Figure 7).

3.3. Hemolysis Test

To test the biocompatibility of ZnO nanoparticles in blood, hemolytic activity testing was performed (Figure 8). Hemolysis results from direct or indirect harmful effects against the RBC membrane and is a reliable indicator of a substance’s biological incompatibility. One of the tests for determining a biomaterial’s hemolytic potential is the determination of whether it is safe when in contact with blood [37] The red blood cells undergo hemolysis when, as a result of the broken cells, they free hemoglobin. We primarily used the ZnO nanoparticles made from the fruit extract of C. spinosa L. for the hemolysis experiment. In the other cytotoxicity tests, this ZnO NPs sample displayed the least harmful effects of the other samples. ZnO nanopowders as well as extracts of C. spinosa L. showed low hemolytic activity compared to the control, at doses ranging from 7.5 to 120 μg/mL, as shown in Figure 6. These results support the possibility that future in vivo investigations using green synthesized ZnO NPs generated using Capricorn spinosa L. fruit extract could yield promising results for medication administration.

3.4. Antioxidant Activity Analysis of ZnO-NPs

The capacity of ZnO-NPs to scavenge free radicals was assessed using the DPPH test. Free radicals such as DPPH have been widely employed to examine the antioxidant strength of inorganic substances and nanomaterials [38]. The DPPH solution was made with a deep violet hue that, after the addition of ZnO-NPs, turned pale yellow, which often denotes the antioxidant capacity of ZnO-NPs [39]. ZnO NPs and C. spinosa L. extract were evaluated for their antioxidant activity using the DPPH technique, with ascorbic acid acting as the standard reference. According to

Table 1. Ascorbic acid, ZnO NPs, and plant extract IC50 values.

Sample	IC50 (μg/mL)
C. spinosa fruit extract	81 ± 0.054
ZnO-NPs	43.68 ± 0.04
Ascorbic acid	26.4 ± 0.05

and Figure 9, the biosynthesis of ZnO NPs using C. spinosa L. extract displayed good antioxidant activity at dosages of 7.5–300 μg/mL, which varied from 23% to 96%. IC50 was about 43.68 ± 0.04 μg/mL compared with 81 ± 0.054 μg/mL for plant extract. However, there was a difference in the antioxidant potential between C. spinosa L. extract and the ZnO NPs. While both samples reacted directly and reduced the wide range of free radicals of DPPH, there was a difference in antioxidant capability between the extract and ZnO NPs, which may be caused by the chemical composition of each sample analyzed [40]. Our results are consistent with those of a recent work by Thavamurugan, S et al. [41] that investigated the antioxidant activity of Ag NPs using C. spinosa fruit extract. Previous studies have demonstrated the significant antioxidant activity of C. spinosa L. extract [42].

The antioxidant potential of biosynthesized ZnO NPs may be connected to the complex of various antioxidant metabolites that are found within cells in plants and protect biological substances such as flavonoids and phenolic chemicals from oxidation and damage. After the physicochemical interaction of the metal ions with the functional groups of the plant extract, these compounds created a coating covering for the ZnO NPs, giving them a spherical morphology and a greater surface area [43].

4. Conclusions

The advancement of nanotechnology as a credible, environmentally sustainable technique for producing a broad range of biocompatible materials and nanomaterials, including metal/metal oxide nanomaterials and nanocomposites, depends on the development of eco-friendly green methods for producing nanoparticles. Green synthesis is therefore seen as an essential strategy to reduce the negative impacts associated with the traditional methods of synthesis for nanoparticles that are routinely utilized in laboratories and industry. The particular phytochemicals generated from plant parts such the fruits, leaves, roots, and seeds have been used to synthesize ZnO-NPs. Natural plant extracts provide an inexpensive and environmentally friendly substitute for the use of intermediate base groups. The present study demonstrated the cost-effective and eco-friendly synthesis of ZnO NPs using C. spinosa L. extract as an effective reducing and capping agent. Through UV–Vis spectroscopy, the synthesis of the NPs was confirmed by peaks at 374 nm, whereas DLS and FESEM indicated the size, shape, and stability of the NPs as well as presence of Zn²⁺ ions. The capping of the synthesized ZnO NPs with bioactive compounds of C. spinosa L. was confirmed with FTIR analysis. In addition, the crystal structure of the NPs was confirmed by XRD analysis. The ZnO NPs showed no cytotoxic activity against normal cells in a dose-dependent manner. Our data indicated that the synthesized ZnO NPs have an antioxidant effect using DPPH assay. The ZnO NPs have hemolysis activity. Zinc oxide nanoparticles (ZnO NPs) biosynthesized by using C. spinosa L. extract exhibited significant antioxidant properties, low cytotoxicity on L929 normal fibroblast cells, and good biocompatibility with RBCs. ZnO NPs did not exhibit any hemolytic reaction at doses ranging from 7.5 to 120 μg/mL; thus, they can be used as a stable and safe substitute for synthetic substances in the pharmaceutical and biomedical research sectors.