The XRD data can establish the crystallinity and purity of the nanostructures. XRD patterns are the results of the diffraction process of the X-rays on various crystallographic planes of the system. Figure 2 depicts the XRD pattern of ZnO NPs. The synthesized ZnO NPs have good crystallinity and purity, which are evident from the sharp peaks.

The diffraction peaks were found to arise from the miller planes of (100), (002), (101), (102), (110), (103), (112), which corresponds to the angle 2θ = 31.7°, 34.7°, 36.2°, 47.4°, 56.7°, 63.1°, 68.1° respectively. This confirms that the ZnO NPs possess a hexagonal phase consistent with the Wurtzite structure [JCPDS No: 89-7102] (El-Belely et al. 2021). The XRD data indicate that plant extracts significantly impacted the development of ZnO NPs with good crystalline nature, as evident from the peak intensities (Pillai et al. 2020). Further investigations to the structure using the Scherrer equation divulge that ZnO NPs have an average particle size ~ 20.2 nm. The surface crystallization of organic constituents from the plant extract on ZnO NPs is responsible for the presence of minute peaks (El-Belely et al. 2021).

Phytochemicals present in the bioresource employed for the synthesis are reported to be responsible for the formation of nanoparticles from their metal precursor. Different classes of phytochemicals such as flavonoids, chalcones, anthocyanins can aid in the formation of ZnO NPs from precursors (Jayachandran et al. 2021). The evidence of these moieties can be easily obtained from the FTIR spectra of the sample, along with information about the vibrational modes of the ZnO NPs as represented in Fig. 3. The presence of ZnO from inter-atomic vibrations of the lattice is confirmed by the peak at 835 cm−1. Phytochemicals present in the plant extract give the rest of the peaks. All these peaks are in accordance with green synthesized ZnO NPs reported in the literature. The vibrational band above 3000 cm−1 arises from the O–H bond stretching and can be due to the phenolic contents present in the extract of S. dulcis. The vibration at 2340 cm−1 occurs from the nitrile group (CN) expected from the nitrogen compounds of the plant. The carbonyl (C = O) bond of unsaturated carbonyl compounds and stretching of C = C bonds from aromatic moieties are linked to the peak at 1680 cm−1. Stretching of C-H mode for alkanes are responsible for 1350 cm−1. These results implies the role of phenolic, flavonoids, and other phytochemical compounds present in the S.aureus extract can serve as the bio-reductant through stabilization of the zinc salts which finally gives control of the ZnO size on green synthesis (Elumalai and Velmurugan 2015). Thus, it is clear that ZnO NPs are produced from the metal precursor through the phytochemical constituents present in plant extract as a reducing agent.

It has been reported that several phytochemicals present in the plant extract serve as stabilizing and reducing agents for forming ZnO NP’s from the precursor. The hydroxy and oxo substituents on the plant metabolites can coordinate with the Zn2+ ion, making the process feasible. Here we demonstrate a plausible mechanism synthesis of ZnO NPs using precursors through the phytochemicals from S. dulcis as depicted in Fig. 4. 2-Hydroxy-2H-1,4-benzoxazol-3-one was selected as the phytochemical for proposing the mechanism since this is an active constituent present in all parts of the S. dulcis (Jiang et al. 2021). The mechanism involves deprotonation of the hydroxyl functional group, which creates a negatively charged oxygen. The zinc ion is getting chelated to this charge center through the formation of the complex, which finally produces ZnO NPs inconsistent with previously reported mechanisms in literature(Thi et al. 2020; Ansari et al. 2020; Selim et al. 2020).

S. dulcis extract-induced production of ZnO NPs: a possible mechanism

The structural characteristics and surface morphology of ZnO were studied using field-emission scanning electron microscopy. Figure 5 shows the FESEM pictures of the green-produced ZnO NPs.

ZnO NPs with pebble-like shape as shown by FESEM

The low-resolution image (Fig. 5a) indicates significant agglomeration for the ZnO NPs which is a characteristic feature of the plant extract-mediated synthesis. On the other hand, the high resolution in the image (Fig. 5b) clearly represents that ZnO NPs possess pebble-like morphology at the nanoscale. These nanopebbles are not homogenous and thus possess a diverse size range. The reason for agglomeration can be due to the existence of phytochemical moieties on the surface of the particles or due to the experimental conditions in the synthesis like pH of the medium, temperature etc., (Bandeira et al. 2020). The change in temperature affects crystal growth by variation in the nucleation process. Also, the increase in pH of the reaction medium from acidic to neutral and to the basic range will cause a decrease in agglomeration (Alias et al. 2010). This can be correlated to the slightly acidic nature of the S. dulcis leaf extract, which may cause slight agglomeration as observed in the ZnO NPs morphology.

Agar well diffusion method was employed for antibacterial and antifungal activity. In this method, diffusion of ZnO NPs into the medium was allowed to make interaction with the test organisms seeded in the petri-plates (Bauer et al. 1959a, b). As a result of the confluent lawn of development, the zones of inhibition (ZOI) will be uniformly round, as shown in Fig. 6.

Inhibition activity of ZnO NPs on a S. aureus, b E. coli, c C. albicans and d A. niger

ZOI is measured in millimeters which gives the extent of antimicrobial action of the sample against respective pathogens. The antibacterial activity was analyzed using Staphylococcus aureus and Escherichia coli for gram-positive and gram-negative stains, respectively. Also, the antifungal activity was analyzed using two common fungal pathogens such as Aspergillus niger and Candida albicans. Table 1 shows the ZOI values in different concentrations (500 μg/mL, 250 μg/mL, 100 μg/mL and 50 μg/mL). PC is positive control used which is a Gentamicin antibiotic.

It is clear that at 500 μg/mL, the ZnO NPs showed slightly higher antibacterial activity than Gentamicin on S. aureus. The activity decreases with a decrease in concentration, and a similar pattern is observed for E. coli also, which implies the dose-dependent action of ZnO NPs. However, in the case of E. coli, the activity was more than S.aureus for all the concentrations except 500 μg/mL. Similarly, for the antifungal activity, ZnO NPs showed ZOI values higher than that of Amphotericin B, which was used as a positive control at 500 µg/mL for C. albicans. No activity was observed for all the other concentrations, as summarized in Table 2. The results are significantly good on comparing with previous reports (Abdelhakim et al. 2020; Raj et al. 2021).

The activity of ZnO NPs against tested bacteria and fungi were represented in Fig. 7. However, ZnO NPs have shown high activity for A. niger for all the concentrations and comparable activity at 50 μg/mL than that of Amphotericin B. This discrepancy in the activities may be because of differences in cell wall compositions and the membrane structure.

ZOI values of ZnO NPs on a S. aureus, b E. coli, c C.albicans and d A. niger

Various mechanisms have been accounted for in the literature to explain the action of ZnO NPs against pathogens. The production of significant amounts of reactive oxygen species is one main inhibitory route (ROS). High oxidizing property and reactivity makes ROS to intervene in biological activities. Another study indicates the existence of multiple metabolic pathways for the action of ZnO NPs against microorganisms. This involves various media-dependent biomolecular pathways such as (i) destruction of cell integrity, (ii) ROS species generation, (iii) Zn(II) ion release to the biological species (Espitia et al. 2012). Further, other factors like concentration, morphology, specific surface area and particle size, could also influence the antibacterial action of ZnO NPs (Sirelkhatim et al. 2015). The ROS species include OH−, H2O2, and O22− produced on the ZnO surface, which are responsible for the cell death of microbial species through the destruction of cellular components. Here, H2O2 molecules are mainly responsible for bacterial cell death due to their ability to penetrate to the cell wall and triggering cellular damage. OH− and O22− cannot penetrate the cell wall due to the electrostatic barrier due to the negatively charged bacterial cell wall, which is not a challenge for H2O2 permeation. The higher bactericidal activity also results from the continuous release of peroxides by the ZnO NPs in the growth media (Sirelkhatim et al. 2015).

Reports suggest that the action of ZnO NPs against S. aureus and E. coli is inversely related to the size of ZnO NPs. Therefore, a decrease in the size will lead to better antibacterial activity against both S. aureus and E. coli which implies the activity is size-dependent. Also, the increased activity with the concentration of ZnO is attributed to the lactate dehydrogenase leakage resulting from mitochondrial dysfunction (Jeng and Swanson 2006).

In our knowledge, detailed mechanistic insights into the action of ZnO NPs against fungus is still in the infant stage, which needs to be elucidated in detail (Sun et al. 2018). Factors like crystallographic parameters and surface charge density also need to be thoroughly investigated to gain insights into biocidal activity. It has been reported that the presence of phytochemical components on the surface of ZnO NPs plays a vital role in enhancing antimicrobial efficiency (Ayoughi et al. 2011). The antifungal action of ZnO NPs could also be due to the disruptive action and permeation of the cell membrane which affects the cell membrane integrity and thereby leading to damage and cell death (Jamdagni et al. 2018).

Our results indicate that the combinations of antibiotics with ZnO NPs would tremendously enhance the activity through synergistic action. The ZnO NPs possess antibacterial and antifungal capacity for both gram-positive and negative stains and against common fungi. However, the cytocompatibility tests of the synthesized nanoparticles have to be studied with normal cell lines to apply for biomedical applications. The results are very useful for applying the ZnO NPs for agricultural sector since some of these pathogens exist as a potential challenge (Julian et al. 2018; Park and Ronholm 2021). These aspects will be covered in our future works.

The enhanced antioxidant capacity of ZnO NPs can be attributed to the presence of various classes of phytochemicals on the surface derived from the plant extract used for synthesis (Ravichandran et al. 2016). DPPH assay provides a convenient and direct method to determine antioxidant efficacy. Here we have analyzed the in-vitro antioxidant capacity of the prepared ZnO NPs using DPPH free radical scavenging method. DPPH assay is a very reliable and facile method to monitor the antioxidant potential of nanoparticles (Khorrami et al. 2019). The principle of DPPH assay is that antioxidant activity is directly correlated to radical scavenging action which is reflected as the disappearance of DPPH in the sample under consideration. The disappearance of DPPH is monitored through a UV–visible spectrometer which directly gives the extent of antioxidant activity. A strong absorption maximum at 517 nm with purple color is attributed to DPPH (Table 3). The synthesis of DPPH is confirmed by yellow color formation when hydrogen is absorbed from an antioxidant that is stoichiometric in terms of the quantity of hydrogen atoms consumed in the process. Higher radical scavenging ability is reflected through lower absorbance values of the reaction mixture. As a result, the antioxidant activity can be determined using the following formula while monitoring the decrease in UV absorption at 517 nm.

$$\mathrm\left(\mathrm\right)= \frac-\mathrm}} \times 100.$$

Figure 8 represents the antioxidant potential of ZnO NPs at various concentrations against the inhibition percentage using Ascorbic acid as the reference standard. It is evident that even at a low concentration of 10 μg/mL, ZnO NPs have around 41% antioxidant efficacy compared to ascorbic acid. The IC50 value was found to be 1.78 μg/mL. IC50 value is considered as a critical parameter for analyzing the antioxidant efficacy of a sample. It is defined as the amount of sample needed to reduce the concentration of DPPH by half of its initial value (Sánchez-Moreno et al. 1998). Thus, a very less value of 1.78 μg/mL clearly indicates that ZnO NPs are having excellent antioxidant properties.

DPPH assay for antioxidant activity of ZnO NPs

The antioxidant activity of ZnO NPs has been explained by literature in several ways. It can be attributed to the development of stable DPPH molecule in the medium through electron density charge transfer phenomenon from the oxygen to nitrogen center of DPPH (Madan et al. 2016). Another possibility is the surface generation of electron–hole pairs in high quantity on ZnO NPs. This process leads to the creation of hydroxyl and hydrogen radicals through water splitting through the generation of high redox potential, which finally leads to the formation of a stable DPPH molecule (Sun et al. 2011; Kiran Kumar et al. 2014). All these results show the synthesized ZnO NPs possess a good extent of antioxidant activity.