**4. Discussion**

The UV-Vis absorption peaks exhibited all the ZnNPs are way below the characteristic excitonic absorption peak value of 370 nm attributed to intrinsic band-gap absorption phenomena exhibited by the bulk ZnO under valence to conduction band electron transitions [36,38]. Similar reports of blue-shifted UV vis absorption peaks have been reported for ZnNPs synthesized through sol-gel [25,39], microemulsion route [37], and green synthesis approach [40]. Moreover, a similar UV-Vis peak at 347 nm has been observed for the starch (CA)-ZnNPs on laser ablation synthesis in an aqueous starch solution for 15 to 20 min which could be ascribed to the formation of a layered nanocomposite comprised of starch-β-Zn(OH)<sup>2</sup> sheets [41]. The occurrence of a single sharp peak indicates the formation of monodispersed ZnNPs and therefore, the narrow size distribution pattern of the synthesized ZnNPs [42]. The dual or triple peaks indicate the polydisperse nature of the aqueous nano-sol probably due to the formation of larger agglomerates by coalescence during or post-nucleation process [43]. The albumin (CA) ZnNMs showed a sharp peak at 220 nm, and multiple stout peaks at 400, 620, 700, and 760 nm wavelength which showcased the highly polydisperse nature of the albumin (CA) ZnNMs aqueous sol. However, the small area under these multiple peaks is also indicative of the presence of variable larger-sized ZnNPs in low amounts.

On the estimation of the bandgap energy of different ZnNMs according to Einstein equation; Energy = hC/λ, where h = Plank's constant (6.626 × 10 −34 Joules sec), C = Velocity of light (3 × 10 <sup>8</sup> m s −1 ) and λ = wavelength (nm), the calculated values were 5.90, 5.64, 3.76 and 3.50 eV for the sharp and distinct absorption peak wavelengths of 210, 220, 330 and 350 nm respectively. These values are quite high compared to the moderate band-gap energy (3.35 eV) of ZnO which corresponds to UV and deep blue region indicating O2p to Zn3d electron transitions [44]. These blue shifts in the band-gap energy may be ascribed to the inadvertent occurrence of other metal atom impurities that may have altered the electronic movement across the valence and conduction bands [45]. Similar blueshifts have been observed on doping of zinc with IIIa group metal atoms [46] and 3d transitions metals [45].

The particle size of less than 10 nm have been reported for NaOH-ethanol reaction mix [43] while ZnNPs generated from NaOH-isopropanol reaction mixture formed particles with the mean size dimensions of 189.0 ± 6.0 and 447.0 ± 22.0 nm by sonochemical and hydrothermal techniques respectively [47]. However, unlike Chen et al. [30] the ZnNPs size was larger for the albumin (CA) ZnNPs while similar size distribution and average size have been observed for thiourea (RA) [26], cellulose (CA) [29], and starch (CA) ZnNPs [27].

The use of a high concentration of ZnCl<sup>2</sup> can lead to the formation of hexagonal layered plate-like crystals of simonkolleite [18]. However, the XRD spectra for NaOH and thiourea reducing agents exhibited the presence of only hexagonal zincite phase crystals. Similar diffraction peaks have been reported for ZnNPs obtained by green synthesis from fruit parts of *Citrullus colosynthis* [14] and cotton linter pulp [11]. The XRD spectra of the albumin (CA) ZnNMs showed a very broad peak spanning over 30 to 50 20◦ diffraction region with several substantially indistinct peaks within this range indicating the occurrence of mixed crystal phase. As Bovine serum albumin (BSA) exhibits affinity to adsorb or form protein corona structure on the surface of the ZnNPs [19,20], it may have restrained the coalescence of the smaller-sized particles to form larger aggregates during the nucleation process. However, the transmission electron micrograph indicated the formation of platelike hexagonal ZnNPs (Figure 2).

The DPPH FRSA of the ZnNMs may be ascribed to the transfer of electron density from oxygen atom in ZnNMs to N-atom odd electron in DPPH compound [48]. The antioxidant properties further depict the efficiency of the redox-catalysis reactions, electronic configuration, surface-interface effect, and biocompatibility of the ZnNMs. From the results obtained for the FRSA potential, it appears that the NPs of a particular size is critical beyond which any further reduction in the size of the NPs does not contribute significantly to the antioxidant behavior. These observations are in line with the anticipated size-dependent phenomena and larger surface area to volume ratio to be involved in improved neutralization/deactivation of the hydroxy (•OH) radicals [49]. Similar to observations of this study, the report on electron spin resonance (ESR) spectroscopy study of 4 to 15 nm AuNPs, showcased maximum antioxidant activity by 9 nm NPs and not by NPs with a size smaller than 9 nm [49]. Therefore, it can be argued that the dose [50,51] or concentration [52], specific surface, and crystallinity status [49] of the ZnNMs are relatively more critical features for altering the efficiency with which the ZnNMs interact with the DPPH radicals.

The fungal radial growth inhibition in the agar-well diffusion assay for the three test fungi indicated superior antimycotic activity of NaOH (RA) derived ZnNPs. Similar radial growth inhibition has also been reported for ZnO NPs by He et al. [11] for *Penicillium expansum* and *Botrytis cinerea*. The hyphal thinning effects can be observed for most of the ZnNMs evaluated in the study which appears to be a characteristic feature of any fungal tissue in response to nanoparticle challenge [53,54]. However, the morphological manifestations such as breakage of the cell wall and leakage of the cytoplasmic contents, and appearance of swollen hyphal cells indicate the alteration in the osmotic conditions and formation of physical pores in the cell wall/membrane of the treated fungal cells [19,23]. Coherent to the results of the mycelial inhibition in agar well assay as described in the previous Section 3.3.1, maximum impact on the hyphal morphology was recorded for the *Fusarium oxysporum*. However, the hyphal fragmentation can be observed in *Macrophomina phaseolina*, unlike the agar well assay which indicates the subtle cytological changes that occur in the fungal hyphae on treatment with ZnNMs. A substantially low concentrations of the ZnNMs have been evaluated in this study unlike the studies performed on *Pythyium* [23], *F. graminearum, A. flavus*, and *P. citrinum* [19], and *Aspergillus flavus* [14].

The genomic DNA fragmentation may be attributed to the physical and chemical properties of the NPs [55] including the size, concentration [56,57], chemistry [56–58], and surface functionalization [59]. Though within a fungal cell the predominant mechanism of degradation of the cellular DNA by ZnNPs is through formation of reactive oxygen species (ROS) which causes extensive DNA scissoring and fragmentation [7]. However, direct interactions of ZnNPs with DNA molecules involve the binding of DNA with ZnO nanoparticles to nucleobases [60]. The specific conjugation of the DNA on ZnO NPs surface has been further identified by Das et al. [61] evidenced through varied conductivity and mobility under electric field on an agarose gel. The results of this study effectively demonstrate that the ZnNPs can intercalate with the DNA and exhibit non-photocatalytic DNA

degradation unlike the sunlight-induced fragmentation of *Leishmania* DNA on incubation with Ag-doped ZnO NPs [62].

#### **5. Conclusions**

This study provides evidence on variation in the anti-mycotic, anti-oxidant, and DNA degrading effect of the ZnO nanoparticles generated through wet chemical synthesis methods by using different reducing or complexing/capping agents. Both the anti-oxidant and anti-mycotic potentials were not observed to follow strict nanoparticle size-dependence. Thus, the NaOH (RA) derived ZnNPs had zincite crystal phase and the particle size distribution ranging from 10 to 90 nm and possessed the highest antioxidant and antimycotic activities. This study also illustrates the subtle hyphal morphological changes occurring on the use of lower concentration of the ZnNPs which may span over a variety of manifestations ranging from thinning, fragmentation, swelling, and lysis of the fungal hyphae. These alterations in the hyphal morphology could be attributed to variability in the functionalizations or chemical functional groups present on the surface of the ZnNPs. Further, the fungal DNA-NaOH (RA) ZnNPs incubation study enunciated the DNA degradation and exacerbation of the total genomic DNA of the test pathogenic fungi in a concentration and time-dependent manner with complete degradation of the *Macrophomina phaseolina* genomic DNA after 24 h of incubation. It could also be inferred from the study that the DNA damaging effect of the ZnNPs is also fungal genera/species-specific and may vary according to the fungal culture being studied. Therefore, this work establishes the impact of the appropriate nano-crystallite size dimensions and surface functionalizations as the key factors that vary the anti-mycotic, anti-oxidant, and DNA degrading potentials of ZnNPs.

**Author Contributions:** Conceptualization, A.K.; methodology, A.K.; validation, A.K., P.M., J.K.; formal analysis, J.K., M.T., P.B., P.M.; investigation, A.K., P.M.; resources, A.K., M.A.A.; data curation, A.K.; writing—original draft preparation, A.S., A.K.; writing—review and editing, K.A.A.-E., M.A.A.; visualization, A.K.; supervision, A.K.; project administration, A.K.; funding acquisition, K.A.A.-E., A.S., M.A.A. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Rashtriya Krishi Vikas Yojana (RKVY), Government of India, Grant ID (RKVY-12, PC-1320-32) to Anu Kalia. Moreover, the current research was partly supported by the Science and Technology Development Fund (STDF), Joint Egypt (STDF)-South Africa (NRF) Scientific Cooperation, Grant ID. 27837 to Kamel Abd-Elsalam.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data is contained within the article.

**Acknowledgments:** The authors thank the Director of Research for the allocation of funds in the RKVY scheme.

**Conflicts of Interest:** The authors declare no conflict of interest.
