1 Introduction

Nanoparticulate Zinc oxide is the most common nanoparticles among the various metal oxide [1]. It is a n-type semiconductor, known for its wide band gap properties. Among its interesting properties are its non-toxic nature [2], good durability, enhanced thermal and chemical stability [1]. In addition, its low cost, natural abundance, single oxidation state, and superior biocompatibility [3] make it a strong candidate for biological applications. Studies have shown that ZnO NPs exhibit high antioxidant activity and can be used in studies involving different diseases such as cancers, inflammations, and cell malignancy [4, 5]. The widespread use of NPs in modern medicine has been attributed to their unique interaction with biological cells vis-a-viz their penetration of cell walls and disturbance of biochemical pathways [6].

Naturally, the metabolic processes of normal cells result in the release of reactive oxygen species (ROS) that plays a very important role in different metabolic signaling pathways of the cells [7]. However, overproduction of ROS causes oxidative stress, which, of course, leads to the damage of deoxyribonucleic acid (DNA) molecules [8, 9]. The electrons in the mitochondria react with oxygen (O2) and form hydroxyl radical (HO−), superoxide anion (O2−), and hydrogen peroxide (H2O2), among others [10]. This subsequently alters the gene signaling pathways and promotes the development of various cancer cells [11, 12]. Globally, approximately 19.3 million people were diagnosed with cancer in 2020, with over 10 million deaths attributed to the disease [13]. Even though the advanced technology currently used to detect and treat cancer has improved the survival rate of cancer patients [14], the high cost associated with the healthcare system remains a concern, especially for individuals from undeveloped countries and rural areas [15]. Thus, there is an urgent need to investigate other alternative potential drugs with high antioxidant and anticancer properties that are less expensive, eco-friendly, and non-toxic such as green synthesized nanoparticles.

Currently, several cancer treatment practices are being developed and are under evaluation in clinical trials, while some have already been introduced in clinical practices [16]. Unfortunately, most of these treatments have constraints associated with negative side effects [17]. Hence, studies that focus on materials with anticancer potency continue to garner research interest. The study of green NPs synthesized with the aid of plant extracts that act as mediating/stabilizing agents has gained great attention in different fields including cosmetics, biotechnology, engineering, medicine, and agriculture [18,19,20]. ZnO is one of the most extensively studied NPs and green synthesized ZnO NPs have been widely applied in various applications where they showed remarkable performance, especially in different biological applications [19, 21]. This is due to their unique corrosion resistance, optical and structural state, and large surface area-to-volume ratio [22, 23]. In this study, ZnO NPs were used to test their efficacy to inhibit the proliferation of HEK 293 and HeLa cell lines. The selection of HEK 293 and HeLa cells in the anticancer studies is based on their distinct biological characteristics and relevance to cancer research. For example, HEK 293 Cells is often used as a non-cancerous control to compare the selective cytotoxicity of anticancer agents. It provides insights into the potential toxicity and biocompatibility of the tested nanoparticles in normal human cells. On the other hand, HeLa Cells are selected because they represent an aggressive cancer cell model, widely known for their high proliferation rate and resistance mechanisms. These cell lines are immortal human cells that can survive through series of cell generations [24, 25], making them good model for evaluating anticancer efficacy.

The current study aims to utilize the bioactive compounds present in the aqueous extract of V. erioloba seed as a mediating agent. The V. erioloba previously known as Accacia erioloba, is a tree plant belonging to the Fabaceae family. It originates from Southern Africa [26], some parts of Australia, and the Arabian Peninsula [27, 28]. Different parts of Vachellia trees have been reported to contain interesting bioactive compounds such as tannins, phenolic acids, flavonoids, prussic acid, and non-starch polysaccharides among others, which have been demonstrated to contain medicinal properties [29]. In the current study, these phytochemicals are successfully utilized as mediating agents for the synthesis of ZnO NPs. It is the first attempt to synthesize any form of NPs using V. erioloba seed extract. The potency of V. erioloba-mediated ZnO NPs to inhibit the proliferation of cancer cells as well as deter the production of free radicals was explored.

2 Experimental

2.1 Plant collection and chemicals

The dried pods of V. erioloba were collected by hand-picking from the Molelwane farm of the North-West University and dispersed to extract the seeds. Zinc acetate dihydrate [Zn(CH3COO)2•2H2O], sodium hydroxide (NaOH), and all other reagents were purchased from Merck (PTY) LTD, South Africa.

2.2 Preparation of V. erioloba aqueous seed extract

The V. erioloba pods were pounded inside a sac and the seeds were removed from the pods. Then the seeds were ground into fine powder using a milling machine. Afterwards, 250 g of the seed powder was weighed and mixed with 1000 mL of distilled water and blended into a homogeneous suspension. Finally, the mixture was filtered to produce a clean extract (Scheme 1) and then stored at 4 °C in a refrigerator for further use.

Scheme 1
scheme 1

Preparation of aqueous extract of V. erioloba seeds

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2.3 Synthesis of ZnO nanoparticles

The ZnO NPs were synthesized following methods described by Mthana et al. [21]. However, different volumes (20 and 30 mL) of the extract were used to synthesize the ZnO NPs which are denoted as ZnO (20) and ZnO (30) respectively. Briefly, 20.05 mM of zinc acetate solution was mixed with 20 mL of seed extracts in a beaker and the solution’s pH was adjusted to neutral (pH 7) using NaOH solution. The mixture was then stirred for 2 h at 85 °C using a magnetic stirrer. After the reaction, the solution was centrifuged at 5000 rpm for 15 min and the supernatant was discarded. The remaining product was washed with a mixture of distilled water and ethanol (1:1) and centrifuged three times to remove any impurities. Afterward, the product was dried overnight in a fume hood and then calcined at 400 °C for 2 h (Scheme 2) to afford a white product which was labeled as ZnO (20). The same procedure was repeated using 30 ml plant extract and the product was labeled ZnO (30).

Scheme 2
scheme 2

Green synthesis of ZnO nanoparticles using extracts of V. erioloba seeds

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2.4 Characterization of nanoparticles

Fourier transform infrared (FTIR) spectroscopy (Cary 670 FTIR spectrometer, Agilent Technologies) was employed (in the wavelength range of 400–4000 cm−1) to analyse the functional groups present in V. erioloba seed powder. A Bruker D8 Advanced X-ray diffractometer (Karlsruhe, Germany) with a proportional counter that uses Cu Kα radiation (k = 1.5405 Å; nickel filter), was used to identify the phase and crystalline nature of the ZnO NPs. A Quanta FEG 250 scanning electron microscope (SEM) with a 30 kV acceleration voltage was used for the analysis of the external morphology. A TECNAI G2 (ACI) transmission electron microscope (TEM) with a 200 kV accelerating voltage was used to examine the internal morphology of the ZnO NPs. The absorption property of the synthesized ZnO NPs was measured using a Cary 30 UV–vis spectrophotometer (Agilent Technologies) from 200 to 800 nm.

2.5 Anticancer analysis of the ZnO nanoparticles

The anticancer potential of ZnO NPs was evaluated  against HEK293 and HeLa cell lines (ATCC, USA). Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) with 10% Fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin in 25 cm2 flasks. The MTT assay was conducted in 96-well plates seeded with 2.5 × 103 cells/well in 100 μL DMEM. After overnight incubation at 37 °C, cells were treated with ZnO NPs (10–100 μg/mL) and further incubated for 48 h, with 5-fluorouracil (5-FU) as a standard. Following medium replacement with 10% MTT reagent for 4 h, formazan crystals were dissolved in dimethyl sulfoxide (DMSO), and absorbance at 570 nm was measured using a Mindray MR-96A microplate reader. DMSO served as a blank. Triplicate experiments were averaged to calculate cell viability following Mthana et al. [21].

2.6 Evaluation of antioxidant activity

The antioxidant activity was analyzed following a modified version of previously reported protocols [21, 30, 31]. The study employed the standard in-vitro 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay to assess the free radical scavenging activity of ZnO NPs, with ascorbic acid as the positive control. A 12.8 mg DPPH solution in 50 mL DMSO was prepared, shaken, and stored in the dark. ZnO NPs and ascorbic acid (10 mg each) were dissolved in 10 mL DMSO and sonicated. Scavenging activity was tested at concentrations of 3.13–50 µg/mL, with control samples lacking ZnO NPs [32]. Measurements were performed in triplicate, recording absorbance at 540 nm with an AMR microplate reader. IC50 values for ZnO NPs and the standard were determined using sigmoidal curves from percentage inhibition versus concentration via linear regression [33]. The inhibition percentage was calculated using Eq. 1.

$$\% {\text{ Inhibition}} = \frac{{\left( {AC - AS} \right)}}{AC} \times 100$$
(1)

where Ac is the control absorbance and As is the absorbance of the test sample (ZnO NPs and standard) [34, 35].

3 Results and discussion

3.1 Functional group analysis

The functional groups present in V. erioloba seed extract were studied by using FTIR analysis to determine the possible phytochemicals involved in the mediation of the NPs and the spectrum is presented in Fig. 1a. The spectrum showed different peaks at 3277, 2921, 1632, 1533, 1338, 1243, and 1035 cm−1. The peak at 3277 cm−1 is attributed to the stretching vibrational frequency of the hydroxyl (O–H) group from either phenols, polysaccharides, or tannins [36]. The symmetrical and asymmetrical stretching vibrations of the (C–H) group are responsible for the peaks at 2921 and 2861 cm−1 respectively, which may be due to the presence of saturated hydrocarbons. Vibration due to C = C in the organic molecules is responsible for the peak around 1533 cm−1 [6]. The peak at 1632 is associated with the carbonyl group (C = O) due to the presence of ketones, aldehydes, or carboxylic acids. The C–C and C-N groups may be attributed to the stretching vibrations at 1383 and 1243 cm−1 respectively [37], while the stretching vibrations of the C-O group is responsible for the band at 1035 cm−1.

Fig. 1
figure 1

The FTIR analysis of a V. erioloba seed extract and b ZnO Nps

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In the spectrum of the ZnO NPs (Fig. 1b), a broad peak is observed at 3358 cm−1 which is attributed to the O–H stretching vibration and indicates the existence of hydroxyl (OH) group in the ZnO NPs. The source of this could be from residual water molecules that were adsorbed on the surface of the nanoparticles [38]. The peak at 1546 cm−1 is typically attributed to the C = O stretching (asymmetric), and this may be due to carbon dioxide (CO2) molecules that were assimilated from the surrounding atmosphere during the synthesis processes of the nanoparticles [38]. This is corroborated by the peak around 1012 cm−1, attributed to C–O stretching vibration [39]. Most metal oxides exhibit an infrared fingerprint region below 1000 cm−1, which includes characteristic M–O vibration bands [40]. In this case, the Zn–O vibration is indicated by the peaks in the range 674–445 cm−1. The FTIR result agrees with reports from previous studies on the green synthesis of ZnO NPs using plant extracts [41].

3.2 X-ray diffraction studies of V. erioloba ZnO nanoparticles

Figure 2a and b shows the diffraction pattern of ZnO (20) and ZnO (30) NPs respectively. The diffraction patterns of both ZnO NPs indicated a high degree of crystallinity similar to previous reports [42]. The diffraction peaks for ZnO (20) appeared at 2θ values of 32.01°, 34.67°, 36.46°, 47.75°, 56.79°, 63.04°, 67.52°, 68.14°, 69.25°, 72,73°, 77.14° and 81.57°, which corresponded to the lattice planes of (100), (002), (101), (102), (110), (103), (112), (200), (201), (004), (202) and (104) respectively. Similarly, these lattice planes were respectively identified in the ZnO (30) at 2θ values of 31.93°, 34.57°, 36.38°, 47.63°, 56.67°, 62.89°, 67.51°, 68.0°, 69.12°, 72.56°, 76.98° and 82.12°. The peaks match that of hexagonal wurtzite structure with JCPDS card no. 36–1451, having a lattice constant of a = b = 3.242 Å and c = 5.205 Å [43], confirming the succesfull synthesis of ZnO NPs. Debye-Scherer equation presented in Eq. 2 was used to calculate the average crystallite size of both ZnO (20) and ZnO (30) NPs [44] and the average size was found to be about 19.45 and 24.60 nm, respectively.

$$D = \frac{{{\text{K}}\lambda }}{{\beta {\text{COS}}\theta }}$$
(2)

where, D = crystilite size (nm), K = 0.9 (Scherrer constant), λ = 0,15,406 Å, β = Full width at half maximum (FWHM), and θ = Bragg’s angle of reflection.

Fig. 2
figure 2

ZnO nanoparticles synthesized using a 20 and b 30 mL of V. erioloba seed extracts

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3.3 SEM, TEM, EDX and elemental mapping results of V. erioloba mediated ZnO nanoparticles

The morphological characterization of ZnO NPs synthesized using 20 and 30 mL of V. erioloba seed extract are presented in Fig. 3. The SEM micrographs of ZnO (20) (Fig. 3a) and ZnO (30) (Fig. 3b) show that both NPs were spherical with a high degree of agglomeration. This mass collection of NPs might be due to the interaction between the particles as a result of a strong kinetic energy emanating from calcination at high temperatures [45]. Also, the high agglomerations are due to the formation of metallic bonds that are difficult to disrupt, which increases the surface reactivity [46]. The TEM micrographs of ZnO (20) (Fig. 3c) and ZnO (30) (Fig. 3d) show the internal morphology of ZnO NPs which further confirm that both NPs were indeed spherical with obvious agglomeration. The average particle size of ZnO (20) (Fig. 3e) and ZnO (30) NPs (Fig. 3f) were found to be 17.92 and 21.14 nm, respectively.

Fig. 3
figure 3

a, b SEM of ZnO (20) and ZnO (30), c, d TEM of ZnO (20) and ZnO (30), e, f particle size distribution histogram of ZnO (20) and ZnO (30)

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Figure 4a presents the elemental composition of ZnO (20) obtained from the EDX analysis of the sample. The spectrum shows that the main elements are Zn and O. The results of the elemental mapping of ZnO (20), (Fig. 4b) confirm that Zn (Fig. 4c), and O (Fig. 4d) elements are uniformly distributed across the nanoparticles surface. Similarly, the EDX spectrum of the ZnO NPs obtained using 30 mL of plant extract is presented in Fig. 5a, while elemental mapping results of the NPs (Fig. 5b), showing Zn, and O elements are presented in Fig. 5c and d respectively. The EDX spectrum of ZnO (30) NPs prepared using 30 mL of V. erioloba seed extracts were also mainly composed of Zn and O. The presence of P may be attributed to the high concentration of the extract, and the mapping results confirmed they Zn and O are evenly distributed.

Fig. 4
figure 4

a EDX spectrum, b Elemental mapping of ZnO, c Zn, and d O elements of ZnO (20) NPs prepared using 20 mL of V. erioloba seed extracts

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Fig. 5
figure 5

a EDX spectrum, b Elemental mapping of ZnO, c Zn, and d O elements of ZnO (30) NPs prepared using 30 mL of V. erioloba seed extracts

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3.4 UV–visible studies of ZnO nanoparticles synthesized using V. erioloba seed extract

The UV–visible absorption spectra of ZnO (20) and ZnO (30) (Fig. 6a) show a maximum absorption peak around 374 and 380 nm respectively. These peaks further confirm the successful formation of ZnO NPs and could be due to intrinsic transmissions of electrons from the valence band of the ZnO to the conduction band [47]. Similar maximum absorption peaks were reported for ZnO NPs synthesized using seed extracts of Caesalpinia crista [48] and Artocarpus hirsutus [49]. The band-gap energies of both ZnO NPs were determined using Tauc’s relation shown in equation (Eq. 3).

$$\alpha {\text{hv}} = \left[ {{\text{A}}\left( {{\text{hv}} - {\text{Eg}}} \right)} \right]^{{\text{n}}}$$
(3)

where α is the absorption coefficient, A is a constant, h is Planck’s constant, ν is the photon frequency, Eg is the optical band gap, and n = 1/2 for a direct band gap semiconductor. The calculated band gap energy values of ZnO (20) and ZnO (30) NPs were 3.25 and 3.31 eV, respectively (Fig. 6b). These values are slightly lower than the band gap of bulk ZnO (3.4 eV) and higher than that reported by Dobrucka, Długaszewska [50].

Fig. 6
figure 6

a Absorption spectra and b Tauc plot of ZnO (20) and ZnO (30) nanoparticles

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3.5 Anticancer activity of the nanoparticles

Different concentrations of V. erioloba seed extract, as well as the V. erioloba mediated ZnO NPs were tested for their efficacy in inhibiting the proliferation of cancer cells (HEK 293 and HeLa), and the results were compared to the widely used standard drug (5-Fluorouracil) (Table 1). The results are presented in Fig. 7a and b for the HEK 293 and HeLa cells, respectively. The results revealed a concentration-dependent proliferative activity of test samples against the progression of all cancer cells, with the lowest (10 µg/mL) test sample concentration having higher cell viability. This could probably be due to the low concentration of test samples which were not sufficient to kill a majority of the cells. The trends observed in the current study were in line with those reported by other studies on ZnO NPs [21, 51,52,53]. Figure 7c shows the IC₅₀ (Half-Maximal Inhibitory Concentration), which indicates the concentration of each of the ZnO NPs and the standard reference drug required to inhibit the proliferation of HEK 293 and HeLa cells by 50% under the defined experimental conditions. The results show that the ZnO (20) exhibited higher anticancer activity on HEK 293 cells, which are in a similar range with the activity of the standard drug (IC50 values: 5.27 and 6.05 µg/mL, respectively). Their anticancer activity was better than that of ZnO (30) (IC50 values: 14.19 µg/mL) and seed extract (IC50 values: 22 µg/mL). This could be due to their smaller particle size compared to that of ZnO (30). Smaller NPs possess a larger surface area to volume ratio, which improves the material’s performance by the domination of atoms on the material surface [54]. With regards to HeLa cells, the ZnO (30) and ZnO (20) showed better activity to inhibit the growth of cells (IC50 values: 11.14 and 14.40 µg/mL, respectively) compared to the standard drug (IC50 values: 17.48 µg/mL), and seed extract (IC50 values: 29.72 µg/mL). The green ZnO NPs have been widely reported to influence many cancer cells in vitro [52, 53, 55]. This may be due to the semiconductor nature of ZnO NPs, which disrupts the cell membrane and allows the passage of Zn2+ ions into the cell [56]. The ability of Zn2+ to activate the production of ROS in the mitochondria might have led to oxidative stress and eventually kills the cells [57, 58]. A comparison of the cytotoxicity of the reported ZnO NPs with different reported green synthesized ZnO NPs is presented in Table 2.

Table 1 Anticancer activity of the green ZnO NPs and V. erioloba seed powder
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Fig. 7
figure 7

Anticancer activity on a HEK 293, b HeLa cells, and c IC50 for ZnO (20), ZnO (30), 5-FU and seed powder

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Table 2 Comparison of cell viability on cytotoxicity
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3.6 Antioxidant activity of the nanoparticles

The antioxidant capacity of ZnO NPs was assessed via the DPPH assay at different concentrations and the results are shown in Table 3 and Fig. 8a. ZnO-NPs and ascorbic acid reduced the deep violet DPPH solution to pale yellow, indicating radical scavenging activity. Both ZnO (20) and ZnO (30) NPs exhibited a dose-dependent increase in activity. Similar observations were reported on ZnO NPs synthesized using extracts of Eucalyptus globulus [62], Berberis aristate [33], and Capsicum chinense [21]. Table 4 presents a comparison of the antioxidant potency of the reported ZnO NPs with other green synthesized ZnO NPs earlier reported. With reference to the obtained IC50 values, presented in Fig. 8b, the radical scavenging activity of ascorbic acid (IC50 values: 20.89 µg/mL) was shown to be higher than that of ZnO (30) (22.91 µg/mL) and ZnO (20) (64.57 µg/mL). The results show that the standard drug exhibited better activity than both ZnO NPs in scavenging DPPH free radicals, consistent with studies that reported lower IC50 values for ascorbic acid than green-synthesized ZnO NPs [36]. However, ZnO (30) revealed a higher inhibition potency compared to ZnO (20) with a large difference in the IC50 values of 41.66 µg/mL. This suggests a higher stability of ZnO (30). Due to their stability, ZnO (30) could have a large surface area to volume ratio which increased their surface chemistry [63], thus resulting in high antioxidant activity.

Table 3 Antioxidant activity (%) of green ZnO nanoparticles
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Fig. 8
figure 8

a Antioxidant activity and b IC50 for ascorbic acid, ZnO (20) and ZnO (30)

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Table 4 Comparison of the DPPH free radical scavenging activity
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4 Conclusion

In conclusion, ZnO NPs were successfully synthesized using different volumes (20 and 30 mL) of V. erioloba seed extract. Their structural and optical characterization was established using XRD, SEM, TEM, and UV–visible absorption techniques. The compositional purity was ascertained by EDX while the uniform distribution of the elemental components was confirmed by Mapping. Both ZnO NPs were highly crystalline with a hexagonal wurtzite structure. The internal and external morphology showed both NPs to be spherical with average particle sizes of 17.92 and 21.14 nm for ZnO (20) and ZnO (30). Comparatively, the ZnO (20) displayed higher anticancer activity against HEK 293 cells than ZnO (30), while ZnO (30) has higher anticancer activity against HeLa cells. The antioxidant assay showed that ZnO (30) has a higher radical scavenging activity compared to ZnO (20), however, they were both lower than the ascorbic acid (used as standard in the assay). Nevertheless, ZnO (30) stands a chance to be a safe and stable alternative to synthetic chemicals in pharmaceutical and biomedical research. Thus, further research is a necessity to validate its compatibility on normal cells.