Zinc oxide nano falcates of sickle shape have been synthesized from Prunus cerasifera pomological extract as a reducing cum stabilizing agent via novel, biomimetic and non-toxic route. Zinc oxide nano falcates were analyzed via ultraviolet spectroscopy, Fourier transform infrared analysis, X-ray powder diffraction, scanning electron microscopy and atomic force microscopy. Highly stable zinc oxide nano falcates synthesized at 200°C and 400°C calcination temperatures expressed intense UV-vis peak at 398 nm. Phenolic and amino groups were revealed by FTIR in pomological extract. Wurtzite crystalline structure of zinc oxide nano falcates was confirmed by XRD with average crystal size of 4.93 nm. SEM sizes ranged between 72.11-120 nm and 56.57-107.70 nm, respectively and shown higher polydispersity levels for two calcination temperatures. Augmented photocatalytic degradation of methyl red and bromophenol blue under direct solar irradiance shown pseudo first order kinetics (R2= 0.99 and 0.96). Furthermore, biomedical and agriculturally important pathogenic strains i.e., Xanthomanas axonopodis pv. citri and Pseudomonas syringae, Aspergillus niger, Aspergillus flavus, Aspergillus fumigatus, Aspergillus terreus, Penicillium chrysogenum, Fusarium solani and Lasiodiplodia theobromae were remarkably inhibited. Enhanced photocatalytic and antimicrobial activity reveals zinc oxide nano falcates promising prospects in nano bioremediation of polluted water and conversion into green nano pesticides.
Modern age is essentially being dominated by nanotechnology, which has emerged as a novel technology by provisioning the advanced materials of nano range of medicinal and commercial importance. ZnO is a semi-conductor (II-IV) of wide band gap, has an energy band gap of 3.37 eV. Among metallic nanoparticles (NPs), ZnO NPs have been used in myriad of catalytic applications for larger surface area . ZnO as a catalyst has been synthesized on commercial scale for agrarian, skin protection and beautifying purposes owing to its antimicrobial and anti-oxidant properties [2,3,4]. ZnO NPs have been synthesized via physical and chemical routes posing challenges in terms of environmental toxicity and complicated operating conditions. Thus, ZnO NPs via plant biomimetic route has been employed as a green and novel method for last few decades. Phytosynthetic route for ZnO NPs is ecologically safe and cost effective demonstrating its potential for substitution of chemical and physical routes [5,6].
Prunus cerasifera Ehrh. (P. cerasifera) also named as cherry plum is an angiospermic plant belonging to family Rosaceae. P. cerasifera is an important medicinal plant. All prune fruits are reservoirs of phytochemicals i.e., polyphenols, anthocyanins, carotenoids, flavonoids, acids of organic make up, fibrous and enzymatic content, benzene containing compounds, tannins, minerals (K, P, Ca, Mg) and Vit. A, B, C and K . P. cerasifera fruit also has considerable phenolic content, many types of anthocyanins and antioxidant components . Various types of anthocyanins found in P. cerasifera fruit are cyanidin-3-galactoside, cyanidin-3-xyloside, cyanidin-3- glucoside, cyanidin-3-rutinoside, peonidin-3-glucoside, peonidin-3-rutinoside, pelargonidin-3-glucoside and yanidin-3-(6”-acetoyl) glucoside . Phenolic content like quercetin, quercetin glucoside, quercetin rutinoside, quercetin arabinopyranosyl, epicatechin, procyanidin C, chlorogenic acid, xyloside, catechin, procyanidin B1, quercetin galactoside and procyanidin B2 has been found in P. cerasifera fruit .
ZnO nanoparticles have been synthesized with reducing phytochemicals of different plants. However, studies regarding ZnO nanoparticles synthesized from P. cerasifera is nonexistent. Herein, ZnO nano falcates phytosynthesized from reducing agents of P. cerasifera fruit has been reported. Pomosynthetic (fruit mediated) ZnO nano falcates (PZO NFs) have been characterized via UV-vis, FTIR, SEM, XRD and AFM. Furthermore, PZO NFs were analyzed for their photocatalytic potential in degrading methyl red (MR) and bromophenol blue (BB) dyes direct solar irradiance exploiting its nanobioremediation potential. Dose dependent in vitro biological activity of PZO NFs was evaluated for bacterial and fungal strains i.e., X. citri, P. syringae, A. niger, A. flavus, A. fumigatus, A. terreus, P. chrysogenum, F. solani and L. theobromae. In vitro biological activity of PZO NFs has been done to evaluate its favorability in terms of green nano bactericide and fungicide.
Zinc nitrate, potassium bromide and nutrient agar (NA) culture media were purchased from Merck, Germany. Methyl red and bromophenol blue were purchased from BDH, England. Potato dextrose agar (PDA) culture media was purchased Liofilchem, Italy. All chemicals used in investigation were of analytical grade and have been used without further purification.
2.2 Fruit extract preparation
P. cerasifera ripened fruits were collected from trees located on farm sides in June, 2016 (summers) from Ali Zai area of Parachinar valley (latitude: 33°53’1.29”N, longitude: 70°6’35.49”E), head quarter of Kurram Agency, Federally Administered Tribal Areas, Pakistan (Figure 1a). Parachinar is famous for P. cerasifera production, utilized for a myriad of pharmacological and edible purposes. Fruit samples were transported to lab in tightly closed polyethylene bags. The ripened fruits exhibited yellow to reddish peel color, yellow pulp with characteristic sweet and sour taste and unique aroma similar to that of plum. For sterilization, fruit samples were washed three times with tap water for removal of deposited dust and environmental pollutants. Fruits were then spread on clean sheets in the shade for moisture removal and drying. Fruits were shade-dried for avoiding any possible phytoconstituents reaction with light. Upon complete drying, fruits were collected and washed with distilled water followed by oven drying at 100°C in Oven (UN110, Memmert, Germany). Fruits were then ground into fine powder with the help of pestle and mortar, sieved and stored in sealed polyethene bags.
For aqueous filtrate preparation, 10 g of fruit powder was weighed on weighing balance (UX6200H, Shimadzu, Japan) and extracted with 1000 mL of double distilled water in a 1000 mL conical flask. This mixture was then heated at 30°C with constant magnetic stirring on hotplate (MSH 20D, Wisestir, Germany) for 10 min. It was followed by double filtration with Whatman No. 1 filter paper (pore size: 11 μm), refrigerated at 4°C for utilization as a reducing agent in PZO NFs synthesis. The P. cerasifera fruit extract (PCFE) obtained was dark brown in color with characteristic cherry plum odor. The yield of doubly filtered PCFE was calculated as follows:
where W1 represents the PCFE weight after double filtration while W2 is the P. cerasifera powder taken initially. The percentage yield for 10 g of P. cerasifera fruit extracted with 1000 mL was estimated to be 89.88% expressing the solvent i.e., double distilled water’s higher efficiency in extraction of reducing agents from P. cerasifera.
2.3 Pomosynthesis of ZnO nano falcates
PZO NFS were synthesized from aqueous extract of P. cerasifera fruit. Prior to addition of Zn(NO3)2, 30 mL of P. cerasifera fruit extract was boiled at 60-80°C on magnetic stirring and heating. After 20 min of heating, 3 g of Zn(NO3)2 was added in solution at 60°C. Heating and stirring of this mixture was continued till conversion of the solution to deep yellowish suspension. The paste like suspension was centrifuged at 6000 rpm (C0060-230V, Labnet International, Inc. USA) and washed with ethanol followed by placing in two ceramic crucibles for hot air drying at 120°C in oven for 2 h . Obtained products were calcinated at two temperatures i.e., 200 and 400°C for 2 h for obtaining PZO NFs furnace (D550, Ney Vulcan, USA) . Calcined white powder was further ground with pestle and mortar for characterization.
PZO NFs were analytically examined via UV-Vis spectrophotometer (1602, Biomedical services, Spain) with spectra recorded in range of 200-800 nm. The photocatalytic dye degradation potential of PZO NFs was also monitored via UV-Vis. The interplay of functional groups was checked by Fourier Transform Infrared, FTIR spectrophotometer (8400, Shimadzu, Japan). Crystal structure of PZO NFs was characterized by Bruker AXS D-8 powder X-ray diffractometer (Shimadzu, Japan), operated at 40 kV, 20 mA, with CuKa radiation (λ = 1.5406 Å). Morphological features of PZO NFs were characterized by scanning electron microscopy (SEM JOEL JSM-6490, Germany) and atomic force microscopy (AFM (NT-MDT Solver Pro, Russia).
2.5 PZO NFs photocatalytic activity
Photocatalytic dye degradation potential of PZO NFs was examined for methyl red and bromophenol blue under direct solar irradiance for 30 min from 12:00-12:30 PM on a sunny day with an average intensity 68-73 Klux (LT300, Extech, UK). Methyl red and bromophenol blue solutions (10 mL) were separately mixed with 15 mg of PZO NFs. Degradation was monitored by UV-Vis spectrophotometer and alleviating absorbance was recorded for calculating the rate of reaction and degradation percentages by:
Where Ai represents the dyes’ initial absorbance while Af is the dyes’ final absorbance after addition of PZO NFs.
2.6 Antibacterial and antifungal assay
Culturing media i.e., PDA and NA, petri plates, and filter paper discs were autoclaved and then dried in oven prior to use for all experiments. All the experiments were carried out in triplicate and the final values obtained for zone of inhibition were presented as mean values along with standard deviations. Values of P<0.05 were considered to indicate a statistically significant difference. Antibacterial and antifungal assays were done with the PZO NFs calcined at 400°C due to their smaller size by preparation of PZO NFs stock solution in methanol for obtaining the final concentration of 100 mg/mL and the volume used in different dose was 5, 10, 15, 20 μL. Stock solution was sonicated for 30 min and assays were strictly done within 1-2 h of sonication.
Antibacterial potential of PZO NFs was tested against X. citri and P. syringae by standard Kirby–Bauer disc diffusion assay. Prior to PZO NFs inoculation, the bacterial test strains were grown in NA broth for 24 h in incubator at 37 °C. The NA plates were prepared followed by sterilization and solidification in laminar flow cabinet (Streamline, Singapore) at 45°C. The bacterial cultures grown overnight were spread on the solidified plates with help of sterile loop for obtaining bacterial lawns. The autoclaved and dried filter paper discs were picked up with help of a sterile forcipes and inserted on NA plates. Discs on control set were loaded with 10 μL of Zn(NO3)2 salt solution, 10 µL of standard antibacterial drug Ampicillin and 10 μL P. cerasifera fruit extract as a control while the discs on another set of NA plates was loaded with 2, 4, 6 and 10 μL of PZO NFs with 24 h incubation time at 37°C in incubator (Sanyo MR-153, GeminiBV, Netherlands). On the next day, the zone of inhibition were measured with help of meter ruler and recorded by taking mean values in mm and compared with standard Ampicillin. PZO NFs were also tested for their fungicidal activity against pathogens i.e., A. niger, A. flavus, A. fumigatus, A. terreus, P. chrysogenum, F. solani and L. theobromae by standard Kirby–Bauer disc diffusion assay. The fungal organisms were grown on PDA for 72 h. Fungal culture (200 μL) was poured onto the PDA with help of sterile spreader for obtaining fungal lawns. Discs on control set were loaded with 10 μL of Zn(NO3)2 salt solution, 10 μL of standard Amphotericin B and 10 μL P. cerasifera fruit extract as a control while the discs on another set of PDA plates was loaded with 5, 10, 15 and 20 µL of PZO NFs and were incubated for 72 h. The zones of inhibition were noted and recorded and compared with standard Amphotericin B.
The conducted research is not related to either human or animals use.
3 Results and discussion
Present research synthesized ZnO nano falcates through a complete green pathway. The synthetic route utilized the biochemicals found in P. cerasifera as an alternative to chemical stabilizers e.g., sodium borohydride, trisodium citrate, glucose. Additionally, these biochemicals capped the ZnO nano falcates thus terminating the need for chemical capping agents e.g., polyvinyl pyrrolidone, ethylenediaminetetraacetic acid, polyvinyl alcohol. However, zinc nitrate has been used as ZnO source in one pot synthesis. Since all Prunus fruits are highly rich in reducing agents thus have been utilized for biogenic synthesis of different metallic nanoparticles e.g. P. amygdalus , P. domestica , P. persica , P. serotina , P. armeniaca , P. yedoensis . Only one research has been reported for the synthesis of ZnO with juice extract of P. cerasus , however no data has been reported for ZnO fabrication with P. cerasifera. Various studies identified P. cerasifera growing in regions like Serbia , Pakistan , China  and France . P. cerasifera in these regions has been used for biological investigations however, current investigation utilized P. cerasifera for facile and biomimetic synthesis of PZO NFs synthesis. P. cerasifera has a widespread distribution and fruits found in different regions predominantly vary in color and size. The wild as well as the cultivated P. cerasifera fruit distributed all over the world are highly polymorphic for various characteristics. P. cerasifera is exceedingly adaptable to transforming ecological conditions.
3.1 Pomoreductant role of P. cerasifera
Although, ZnO nanoparticles have been synthesized with variety of plant species but present investigation specifically employed P. cerasifera fruit for pomoreduction of PZO NFs primarily due to its highly prevalent spatial distribution, unique phytochemical composition and enhanced antimicrobial activity. P. cerasifera has a plenty of significant chemical resource but it is underutilized  making it a suitable candidate for current bioprospecting in PZO NFs one pot synthesis.
P. cerasifera (Figure 1b) occurrence in European and Asian countries in wild as well as domesticated varieties contributes to its widespread distribution. However, the sampled fruits utilized in present research represents wild variety possessing higher innate pest resistance. Thus, enhancing its availability on large scale for extraction of reducing agents for prunosynthesis of PZO NFs. P. cerasifera fruit is also available on commercial scale both in fresh and dried forms in all seasons.
Furthermore, P. cerasifera fruit was also preferred due to its exceptional chemical composition which surpasses many plant species in not only reducing but also stabilizing of PZO NFs. Dried fruit powder utilized for PZO NFs synthesis comprised of higher quantities of antioxidant phytochemicals actively involved in capping of PZO NFs. Drying augments the quantity of reducing sugars due to dehydration. P. cerasifera phytochemicals involved in prunosynthesis of PZO NFs are sugars (glucose, fructose, sucrose, sorbitol), amino acids (total amino acids and aspartic acid), vitamins (ascorbic acid (C), thiamin (B1), riboflavin (B2), niacin (B3), α-tocopherol (e), carotenoids (lutein, α-carotene, β-carotene), organic acids (malic and quinic acid), phenolic compounds (neochlorogenic acid, chlorogenic acid, caffeic acid, coumaric acid, anthocyanins, catechins, rutin) . Total antioxidant capacity (TAC) method evaluation reveals P. cerasifera to possess antioxidant potential up to 0.267 mg Gallic acid equivalent (GAE) kg-1  in addition to the polyphenol chlorogenic acid’ s main contribution towards antioxidant potential.
P. cerasifera, due to its inherent antimicrobial potential against bacterial and fungal strains was utilized for augmenting the inhibition power of PZO NFs. Higher quantities of tannins in P. cerasifera can combine with proteins inducing the inhibition of plant microbial enzymes and viruses thus acting as natural pesticide [26,27].
3.2 Synthesis and characterization of PZO NFs
PZO NFs formation was confirmed by the yellowish colored suspension formed and later precipitation occurred during reaction. PZO NFs have shown a broad peak in range of 350-375 nm depicting the electronic vibrations in this region which were further intensified signifying the uninterrupted PZO NFs synthesis. Final surface plasmon resonance induced by the mutual vibration of electrons, PZO NFs exhibited lambda maximum at 398 nm (Figure 2a). Zn(NO3)2 salt was reduced and stabilized in presence of P. cerasifera fruit extract without any addition of chemical reducing agents. Such reduction cum stabilization express the anti-oxidant power of phytochemicals found in P. cerasifera fruit extract. PZO NFs have diffraction peaks at 2θ values of 34.7o, 35.6o, 48o, 55.21o, 61o and 68o, which can be credited to (002), (012), (110), (013) and (201), respectively. Comparison with JCPDS card no. 36-1451 and peaks confirmed the single phase hexagonal (Wurtzite structure) for PZO NFs (Figure 2b). Crystalline nature of PZO NFs confirms the validity of pomosynthetic route as green and efficient method for getting nano ZnO. Furthermore, the average crystallite size pf PZO NF was determined to be 4.93 nm from Scherrer equation:
where, D is representing the average of crystal size in Å, K (0.9) is shape factor, λ is wavelength of X-ray Cu Ka (1.5406 Å) radiation, θ is the Bragg angle and β is the corrected line expressing NPs broadening.
FTIR analysis was done to comprehend the involvement of functional groups involved in stabilization and reduction of PZO NFs by P. cerasifera fruit extract (Figure 3a, b). FTIR spectra was recorded between the wavenumber spanning over a range of 4000–400 cm-1. FTIR spectra of P. cerasifera fruit extract confirmed the presence of O–H stretching with absorption bands at 3343 and 2930 cm-1 signifying polyols in it. Broad peaks at 1732 and 1628 cm-1 represents C=O and N-H of ketones and amines respectively. Relatively smaller and broader peaks at 1400, 1261 and 1078 cm-1 of C-C, C-N and C-N stretch represents the presence of aromatics, aromatic amines and aliphatic amines. Detected bands signify different types of polyols i.e. phenols and flavonoids, proteinaceous and terpenoids profusion in P. cerasifera fruit extract. FTIR spectra for PZO NFs synthesized at different calcination temperatures expressed the loss of few peaks and emergence of new peaks when reduced and stabilized by P. cerasifera fruit extract. PZO NFs (200°C) have shown absorption bands around 3484, 2851, 2101, 1244 and 835 cm-1 of alcohol/ phenols, alkanes, alkynes, aliphatic amines and alkyl halides. PZO NFs (400°C) expressed variance in absorption bands due to better conversion into PZO NFs at elevated temperatures with 3507, 3404, 2851, 2764 and 1325 cm-1 of O–H, C–H, H–C=O, C–H and C–N stretching mode. Phenolic compounds in P. cerasifera fruit extract interact with PZO NPs during reduction and stabilization and induce transmittance shifts.
|FTIR peaks (cm-1)||Bond||Functional groups|
|P. cerasifera fruit powder|
|3343||O–H stretch, H–bonded||Alcohol, phenol|
|2930||O-H Stretch||Carboxylic acid|
|1261||C–N stretch||Aromatic amines|
|1078||C–N stretch||Aliphatic amines|
|800||C–Cl stretch||Alkyl halides|
|744||C–Cl stretch||Alkyl halides|
|611||–C=C–H: C–H bend||Alkynes|
|PZO NFs (calcined at: 200°C)|
|3484||O–H stretch, H-bonded||Alcohol, phenol|
|1244||C–N stretch||Aliphatic amines|
|835||C–Cl stretch||Alkyl halides|
|802||C–Cl stretch||Alkyl halides|
|PZO NFs (calcined at: 400°C)|
|3507||O–H stretch, H–bonded||Alcohol, phenol|
|3404||O–H stretch, H–bonded||Alcohol, phenol|
|2764||H–C=O: C–H stretch||Aldehydes|
|1325||C–N stretch||Aromatic amines|
|1117||C–N stretch||Aliphatic amines|
|835||C–Cl stretch||Alkyl halides|
|629||–C=C–H: C-H bend||Alkynes|
PZO NFs were analyzed for surface morphology and size ranges by scanning electron microscopy (SEM JOEL JSM-6490, Germany). PZO NFs were highly influenced by variation in calcination temperatures. Nearly smooth surface morphology with larger sized nano falcates in range of 72.11 -120.00 nm were detected for PZO NPs calcined at 200°C. Figure 4 (a-e) shows nano falcate of sickle like hooked shape nano ZnO at different resolutions. PZO NFs calcined at 400°C (Figure 4 f-j) expressed rough surface morphology  with roughly nano falcates in protrusion like network with irregular orientations. Nano-falcates obtained at 400°C were not evenly distributed like that of 200°C but has an alleviated size ranges of 56.57-107.70 nm thus revealing the sensitivity of ZnO nano falcates towards calcination temperatures. Topographical and surface roughness of nano falcates was analyzed via atomic force microscopy. AFM was conducted on variable magnification ranges (Figure 4k-o). 2D and 3D micrographs show the smooth nano falcates stabilized and caped by phytoconstituents of P. cerasifera fruit extract. AFM revealed the polydispersity and slightly elongated shapes of nano falcates.
3.3 Photocatalytic activity of PZO NFs
Dyes have been known as the environmental pollutants since they have very good solubility in water thus rendering it unsuitable for consumption by human and other organisms. In many cases dyes become persistent organic pollutants. Consequently, efforts have been done for the pretreatment of dyes particularly in textile industry. Removal of these dyes through physicochemical or radiation reduction is associated with release of even more toxic degradation products thus challenging the credibility of these processes. Thus, the environmental integrity is further deteriorated instead of betterment . Photocatalytic phenomenon is governed by photons that possess harmonious behavior or surpass the band gap energy of semiconductor of interest . The conversion of persistent organic pollutants to harmless products via photocatalytic pathway in case of metal oxides is mainly dependent upon the incidence of light radiations. Such radiations are taken up by the metal oxides for excitation of electrons from valence to conduction band. The consequent hole developed in valence band is then associated with generation of radicals after absorption of electron. Dyes are oxidized to their leuco forms (achromatic form) by the previously generated radicals signifying the conversion of environmentally perilous dyes to benign components . ZnO has been utilized for photocatalytic degradation of chemicals due to its elevated activity in UV region . Ecologically toxic pollutants have been efficiently degraded with ZnO owing to its innocuous nature, economic favorability and augmented photo degradative potential .
Methyl red and bromophenol blue are heterocyclic aromatic dyes (Figure 5a, b). Degradation of these dyes was studied in absence and presence of PZO NFs with direct exposure to solar irradiance. (Figure 6a, b). Dye reference solutions without PZO NFs remained unaffected by solar irradiance. Photocatalytic dye degradation for both dyes was monitored via alleviating UV-Vis absorption peaks. Upon addition of PZO NFs, the degradation was completed in less than an hour with complete color disappearance signifying the remarkable photocatalytic potential of PZO NFs. Methyl red, significant textile dye possessing the most reactive nature photo catalytically degraded by ZnO nanoparticles within 35min has also been reported . Results revealed 96.77% of bromophenol blue in 50 minutes exceeded methyl red with 82.65% in degradation in same exposure time (Figure 6c). However, photo degradation with commercial ZnO has also exhibited discoloration extents up to 99% of MR producing no absorption peak. Such discoloration signifies the MR transformation to simpler organic form due to elimination of main chromophore azo group in aqueous solution . Comparative analysis of ZnO with commercial as well as prepared ZnO NPs, the PZO NFs are good in terms of efficiency and handiness.
Variety of physicochemical methods i.e. absorptive , photocatalytic  and biological degradation  have been devised for conversion of highly toxic and persistent organic pollutant bromophenol blue. However, these technologies have not been successful so far due to their alleviated efficiency, higher cost and operational complications . Thus, such challenges have been addressed and BB in the present research has been photodegraded with PZO NFs as photo catalysts in conformity with all principles of Green chemistry. Degradation percentages up to 84.2% in 30min , 95% in 90 min  and 64% in 160 min  have been achieved with ZnO photo catalysts suggestive of PZO NFs with 96.77% as exceedingly efficient over conventional ZnO NPs. Such photo degradation is governed by the coverage of the active sites of PZO NFs leaving only fewer photons reaching PZO NFs’ surface. This can be attributed to the maximum light absorption by BB molecules instead of PZO NFs . However, the competition between BB molecules and water molecules for photo commenced H+ inducing a diminution sin OH• and other reactive species generation. PZO NFs excelled in photo degradation of BB due to stronger electrostatic forces in form of adsorptive interactions between cationic PZO NFs’ surface and anionic dye solution . Figure 6d, e illustrated the reaction kinetics (ln (At/Ao) vs time for determining the order of photocatalytic reaction. The reduction caused by PZO NFs was found to be pseudo first order kinetics. There was a linear relationship between ln (At/Ao) vs time with R2 = 0.99 and 0.96 for methyl red and bromophenol blue respectively. Thus PZO NFs synthesized via green route were revealed to be efficient candidates for nano bioremediation at low cost and easy mode of synthesis.
3.4 Antimicrobial efficacy
Nanoscale ZnO particles have been significantly employed for bacterial inhibition at a global scale due to advances in nanotechnology . Though, ZnO nanoparticles based bacterial inhibition has been rigorously studied, despite this fact the accurate toxicological mechanism is yet contentious and needs thorough investigations for polemical mechanisms given so far. PZO NFs in this regard have not been reported for the tested strains. Thus, present study is the first investigation of P. cerasifera fruit mediated ZnO NFs’ potency against pathogenic bacterial and fungal strains. PZO NFs were evaluated for their bacterial and fungicidal efficacy via by standard Kirby–Bauer disc diffusion assay and the microbial growth prevention was quantified from the zone of inhibition (ZOI) in mm. PZO NFs were loaded on discs in dose dependent manner with 2, 4, 6 and 10 μL (Table 2). Dose dependent in vitro antibacterial assay of PZO NFs exhibited elevated activities against plant pathogens i.e., X. citri and P. syringae in comparison to the standard drug. P. cerasifera fruit extract produced commendable zone of 12.03 mm in comparison to 14.03 mm produced by standard drug. Thus showing the bioprospects of P. cerasifera fruit extract against X. citri, however the clearance zone for the later strain was smaller comparatively. Overall PZO NFs inhibited the growth of all nine microbes with higher clearance zones against bacterial strains, such obliterative mechanism can be due to PZO NFs direct interaction with bacterial cell walls and ultimate destruction of cellular entirety [45,46,47,48] in addition to the generation of inhibitory Zn2+ ions ions [49,50] and ROS generation . Augmented growth clearance zones against two bacterial strains expresses the PZO NFs potential in inhibition as well as the bacterial strains’ susceptibility towards PZO NFs [52,53,54]. Due to thinner cell walls found in all Gram negative bacteria which is easily susceptible to the facile rupture and quicker assimilation. The concentration of PZO NFs expressed a positive correlation with inhibition and the highest concentration exceeded the standard drug in inhibiting both pathogenic strains [55,56]. Such an enhanced antibacterial potency of PZO NFs significantly portrays the higher susceptibility of test microbes and decreased resistance thus favoring the development of synthesized PZO NFs into bactericidal agents on commercial scale. PZO NFs utilization for agricultural biocidal and biomedical purpose is advantageous over commercially available ZnO antibacterial agents because they exhibit no toxicity at lower concentrations to human cells but possess higher toxicities against bacterial strains . In case of human disease, ZnO has been shown to provide protection against E. coli induced intestinal ailments by inhibition of E. coli adhesion and internal residence . ZnO NPs have succeeded in reducing up to 99% of bacterial growth .
|Bacterial Strain||Salt sol. (10 μL)||Standard[*](10 μL)||PCFE (10 μL)||PZO NFs (2 μL)||PZO NFs (4 μL)||PZO NFs (6 μL)||PZO NFs (10 μL)|
|Fungal cultures||Salt sol. (10 μL)||Standard [**](10 μL)||PCFE (10 μL)||PZO NFs (5 μL)||PZO NFs (10 μL)||PZO NFs (15 μL)||PZO NFs (20 μL)|
PZO NFs loaded discs in dose dependent manner revealed variable and higher zones of inhibitions for all fungal pathogens i.e. A. niger, A. flavus, A. fumigatus, A. terreus, P. chrysogenum, F. solani and L. theobromae. The results obtained for PZO NFs’ highest dose are comparable with those of the standard drug. For all the pathogenic fungal cultures, P. cerasifera fruit extract shown enhanced antifungal activity (Table 2). The dose dependent in vitro assay expressed highest inhibition against L. theobromae (24.02 mm) and lowest against A. terreus (15.01 mm) (Figure 7a). Linear relationship between PZO NFs concentration and antifungal activity enhancement can be attributed to the consequent incremental ROS production as reported earlier [60,61,62]. Toxicological pattern of PZO NFs against targeted strains may vary depending upon various factors in addition to the morphological and size ranges of PZO NFs. PZO NFs with hexagonal (Wurtzite structure) crystals contained edges and corners as reactive surfaces [63, 64]. Defect sites are known for their elevated reactivity and catalysis of ROS production that directly increases the antimicrobial activity . PZO NFs antifungal activity is in agreement with reported studies on different fungal strains; however, seven fungal strains tested with PZO NFs have never been reported before. ZnO NPs synthesized via myriad of synthetic routes has been employed for inhibition of A. flavus, A. fumigatus and A. niger but no reports have been found on Prunus cerasifera mediated PZO NFs. Thus, PZO NFs can effectively control the harmful fungal strains and can be used for agricultural and biomedical purposes .
Metallic nanoparticles have been investigated against a wide range of microbes but data on ZnO nanomaterials as antifungal agents against agricultural pests is scanty. Higher food spoilage induced by fungal species on global scale is not only an economical but environmental concern, for which green antifungal treatments needs to be developed . Targeted fungal strains are not only phytopathogenic in nature but also source of human ailments e.g., A. fumigatus can cause hypersensitivity pneumonitis and A. terreus causes invasive aspergillosis. Thus, PZO NFs’ antifungal potential against these pathogens offers facile, biomimetic and inexpensive treatment. Though deeper insights are required for comprehension of accurate antifungal mechanism of PZO NFs, but based upon similar reports on ZnO NPs, it is assumed that PZO NFs interact with fungal cell inducing structural deformation and interfering with the cellular functionalities . Thus, such distortions ultimately results into fungal cell death signifying the antifungal potential of PZO NFs . Synthetic route and conditions needs to be kept uniform for ensuring the antifungal activity of PZO NFs . Comparatively higher fungicidal activity in terms of ZOI was obtained due to the bioreduction of PZO NFs with P. cerasifera fruit phytoconstituents which not only bio-capped the PZO NFs but also stabilized them for longer durations. Such remarkable inhibition by PZO NFs makes them suitable choice for biomedical and agricultural treatments . P. cerasifera fruit mediated metallic nanoparticles in addition to PZO NFs have been found effective against variety of microorganisms  thus minimizing the need for bactericides and fungicides with reported toxicities and harmful impacts on environment [73,74].
Plants being the natural reservoirs of chemicals, can be used for development of advanced nanoscale materials through biomimetic routes. Facile, unprecedented and nontoxic method was adopted for fabrication of zinc oxide nano falcates from reducing agents of P. cerasifera pomological extract. Fabrication and stabilization of zinc oxide nano falcates is attributed to the phenols and amines abundantly present in P. cerasifera. Zinc oxide nano falcates of wurtzite structure has lambda maximum at 398 nm. Zinc oxide nano falcates exhibited 4.93 nm average crystallite size. Methyl red and bromophenol blue were efficiently degraded with zinc oxide nano falcates i.e., 82.65 and 96.77% in 50 min. Zinc oxide nano falcates are effective against the tested bacterial and fungal strains with zone of inhibitions as high as 24 mm. P. cerasifera fabricated zinc oxide nano falcates are expected to be of extensive applications in remediation of chemicals and plant protection against pathogens.
Authors are grateful to Fatima Jinnah Women University, Rawalpindi, Pakistan for provisioning of all required chemicals and analytical facilities.
Conflict of interest: Authors state no conflict of interest.
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