Investigation of chemogenic and biogenic derived nano ZnO activity on excision wound in rat model: a comparative study

Zinc nitrate hexahydrate (Zn(NO₃)₂.6H₂O; 99.9%), Zinc acetate dihydrate (Zn(CH₃.COO)₂.2H₂O; 99.9%), Sodium carbonate anhydrous (Na₂CO₃, 99.9%) were obtained from Sisco Research Laboratories Pvt. Ltd (SRL), Mumbai. All the solutions and aqueous extract were prepared using double distilled water. Healthy fruits of Pomegranate (Punica granatum L.) were collected from local market of Bangalore, India.

2.2.1. Processing of plant material

2.2.2. Synthesis of nano ZnO

2.2.2.1. Chemogenic synthesis of ZnO NPs

Chemogenic ZnO nanoparticles (ZnO-C) was prepared by precipitation method reported earlier [29]. In brief, 0.1 M Zinc nitrate hexahydrate was added dropwise to 0.12 M sodium carbonate solution under vigorous stirring. The precipitate formed was collected by centrifuging at 10 000 rpm for 10 min The resultant precipitate was washed with distilled water for two times and ethanol for one time. The milky white colour solids were then dried at 100 °C for 6 h and calcined at 600 °C for 2 h.

The reactions that occur during the chemogenic synthesis of ZnO NPs can be explained as follows; Synthesis of zinc oxide nanoparticles was carried out by using zinc nitrate hexahydrate and sodium carbonate as precursors. In this process, zinc carbonate is formed as intermediate product (white precipitate), which when calcinated ∼350 °C forms zinc oxide by decomposition of zinc carbonate producing CO₂ [29–31]. The reaction of zinc oxide formation are shown as follows [32]:

$$\begin{eqnarray*}&&{\rm{Zn}}{({{\rm{NO}}}_{3})}_{2}\cdot 6{{\rm{H}}}_{2}{\rm{O}}+{{\rm{Na}}}_{2}{{\rm{CO}}}_{3}\to {{\rm{ZnCO}}}_{3}+2{{\rm{NaNO}}}_{3}+6{{\rm{H}}}_{2}{\rm{O}}\end{eqnarray*}$$

$$\begin{eqnarray*}&&{{\rm{ZnCO}}}_{3}\to {\rm{ZnO}}+{{\rm{CO}}}_{2}\uparrow \end{eqnarray*}$$

2.2.2.2. Biogenic synthesis of ZnO NPs

For the biogenic synthesis of ZnO nanoparticles (ZnO-B), 0.2 M of zinc acetate dihydrate was dissolved in 70 mL of distilled water and stirred for few minutes. 30 ml of Pomegranate peel extract (PPE) was added to this solution and mixed homogenously at 60 °C for 40 min. Further, it was cooled to room temperature and centrifuged at 10 000 rpm for 10 min. The resultant precipitate was washed with distilled water for two times and ethanol for one time. The obtained yellowish white precipitate was dried overnight at 60 °C and calcined at 500 °C for 2 h [25].

Pomegranate peel extract is known to majorly contain punicalagin and gallic acid along with other phenolic compounds such as ellagic acid, chlorogenic acid, caffeic acid, punicalin, apigenin, quercetin, pelargonidin, cyanidin, granatin A, granatin B etc, that are proven to be involved in biosynthesis of various metal and metal oxide nanoparticles [33–35]. Negatively charged atoms present in gallic acid and punicalagin can donate electrons resulting in stabilization of the positively charged Zn²⁺ complex ions. Further, calcination at 500 °C leads to decomposition of Zn²⁺ complex ions to stable nano zinc oxide. The reaction mechanism is as follows [36]:

$$\begin{eqnarray*}&&\begin{array}{l}{\rm{Punica}}\,{\rm{granatum}}\,{\rm{peel}}\,{\rm{extract}}\\ \,+{{\rm{Zn}}}^{{2}^{+}}\mathop{\longrightarrow }\limits^{{\rm{Stirring}}}\,\left[{\rm{Punica}}\,{\rm{granatum}}/{{\rm{Zn}}}^{{2}^{+}}{\rm{complex}}\right]\end{array}\end{eqnarray*}$$

$$\begin{eqnarray*}&&\begin{array}{l}\left[{\rm{Punica}}\,{\rm{granatum}}/{{\rm{Zn}}}^{{2}^{+}}{\rm{complex}}\right]\\ \,+{{\rm{O}}}_{2}\left({\rm{g}}\right)\mathop{\longrightarrow }\limits^{500\,{}^{^\circ }{\rm{C}}}{\rm{ZnO}}\,{{\rm{NP}}}_{{\rm{s}}}+{{\rm{CO}}}_{2}\left({\rm{g}}\right)\end{array}\end{eqnarray*}$$

Characterization studies of synthesized ZnO nanoparticles were carried out using various analytical techniques. Crystalline phases were analysed by Powder x-ray diffraction (XRD) using Rigaku Ultima IV, Japan diffractometer with Cu K_α radiation (λ = 1.541 Å).The morphology of the samples were analysed by scanning electron micrographs (SEM) using Carl Zeiss ULTRA55, Germany Field Emission Scanning Electron Microscopy (FESEM). Since ZnO is insulating, gold sputtering was done prior to electron micrography to get clear images. Transmission electron microscopy (TEM) was performed on a Jeol/JEM 2100 (accelerated voltage upto 200 kV, LaB₆ filament) Transmission electron microscope.

2.4.1. Preparation of formulation

2.4.2. Evaluation of wound healing activity

Excision wound model was used to evaluate the wound healing activity of synthesized Chemogenic and biogenic ZnO nanoparticles (i.e., ZnO-C and ZnO-B). Entire animal study and experimental protocol was conducted as per the Institutional Animal Ethics Committee for the purpose of control and supervision of experiments on animal guidelines, India (Approval number IAEC: 1165/bc/08/CPCSEA/17/2015). Excision of wounds were made as described by Morton and Malone [37]. Animals were anaesthetized with anesthetic ether and placed in operation table in its natural position. A square wound of about 500 mm² was made on depilated ethanol-sterilized dorsal thoracic region of rats. After wound creation, experimental animals (i.e., Male albino Wistar rats) weighing 110 to 150 g were divided into 6 groups of 8 rats each. Out of six groups of animals, group—I received ointment base (control); Group-II were treated with a 5%w/w povidone–iodine ointment; Group-III were treated with ZnO-B ointment (1%w/w); Group-IV were treated with ZnO-B ointment (2%w/w); Group-V were treated with ZnO-C ointment (1%w/w) and Group-VI were treated with ZnO-C ointment (2%w/w).

The wound contraction was studied by tracing the raw wound area subsequently on day 1, 3, 7, 11, 14, 17 and 21. During course of excision wound model analysis, there was no death of any rats in the study groups. Scar residue, area and time of complete epithelisation were also measured.

The percentage of wound contraction was calculated as:

$$\begin{eqnarray*}&&{\rm{Percentage}}\,{\rm{of}}\,{\rm{wound}}\,{\rm{contraction}}=\displaystyle \frac{{\rm{wound}}\,{\rm{area}}\,{\rm{on}}\,{\rm{day}}\,0-{\rm{wound}}\,{\rm{area}}\,{\rm{on}}\,{\rm{day}}\,{\rm{n}}}{{\rm{wound}}\,{\rm{area}}\,{\rm{on}}\,{\rm{day}}\,0}\times 100\end{eqnarray*}$$

Where n stands for number of days of treatment (3rd, 7th, 11th, 14th, 17th and 21st day)

Results are expressed as the mean ± S.D (n = 8). Statistical significance (p) was calculated by one-way ANOVA followed by Dennett's test employing statistical software, GraphPad, InStat 3 and by comparing treated group with control. p values <0.05 mean between paired observations were considered statistically significant.

2.4.3. Histopathological analysis

A 3200 μg ml⁻¹ stock solution of Ciprofloxacin was prepared by dissolving 0.32 mg of (Ciprofloxacin) in 0.1 ml of sterile acidified water (0.1 N HCl solution). Similarly, stock solutions of test samples (ZnO-C and ZnO-B) were prepared by dissolving 1.0 mg of test sample in 0.313 ml of DMSO separately. The stock solutions were diluted was serially diluted by two-fold dilution to obtain a series of working stock (WS) solutions.

One day before the initiation of experiment, the bacterial strains (Escherichia coli, Staphylococcus aureus, Bacillus subtilis and Pseudomonas aeruginosa) were picked from isolated colonies, inoculated into sterile cation adjusted Muller Hinton Broth (CAMHB) tubes and incubated overnight at 37 °C to exponential-phase. Post incubation, the cultures were identified by Gram staining and optical density is adjusted to that of a 0.5 McFarland Standard (Approximately equivalent to a bacterial suspension 1.0 × 10⁸ CFU ml⁻¹), and diluted to ∼1.0 × 10⁷ CFU ml⁻¹.

Micro dilution method was used for determination of MICs as per Clinical and Laboratory Standard Institute (CLSI) [R F Cockerill. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; Approved Standard—Ninth Edition. Clinical and Laboratory Standards Institute (CLSI). 32 (2012) 1–68]. The MIC assay was performed in 96 well microtitre plates. 150 μl of CAMHB broth containing working stock solutions of test samples (ZnO-C and ZnO-B) of varying concentrations were added into each well. Ciprofloxacin was used as reference compound and DMSO served as vehicle control. Each well was inoculated with 50 μl of bacterial culture (∼1.0 × 10⁷ CFU ml⁻¹). The plates were incubated at 37 °C for 24 h. Post incubation, plates were visually examined for turbidity and the optical density was read at 630 nm in a Spectrophotometer. The assay was done in triplicates and the obtained results were statistically analysed using ANOVA software.

For in silico wound healing and antimicrobial activity studies, Glycogen synthase kinase-3β (GSK-3β) and glucosamine-6-phosphate synthase (GlcN-6-p synthase) are considered as target enzymes respectively. The structure of the target enzymes was obtained from Protein Data Bank (PDB ID; 1Q5K and PDB ID; 2VF5). Wurtzite structure of Zinc oxide was obtained as PDB file using Vesta software. Active pockets for proteins were acquired from CASTp server [38]. Intermediator steps, such as energy minimization, protein and ligands preparation and grid box creation were completed using Graphical User Interface program AutoDock Tools (ADT). AutoDock/Vina was employed for docking using protein and ligand information along with grid box properties in the configuration file. AutoDock/Vina employs iterated local search global optimizer [39, 40]. During the docking procedure, both the protein and ligands are considered as rigid. After the successful completion of the docking runs, different conformations of the ligands known as binding modes were obtained with their respective binding affinity and the stable one which happens to be the one with the lowest binding energy was picked and aligned with receptor structure for further analysis [41].

XRD patterns of the products obtained by chemogenic and biogenic methods of synthesis are shown in figures 1(a) and (b) respectively. The two products are labelled as ZnO-C and ZnO-B respectively for chemogenic and biogenic derived products henceforth. All the planes of the diffraction pattern matches with wurtzite structure and can be readily indexed to standard data (JCPDS70-0134). Keen observation of the diffraction pattern reveals that ZnO-B shows relatively broad peaks compared to ZnO-C. Otherwise, diffraction peak positions in both the samples are found to be similar with no significant variation. In order to evaluate the influence of synthesis method on crystallite size of the products, the average crystallite size (D) of ZnO-C and ZnO-B are calculated by x-ray peak broadening method using Full width half maximum of the diffraction peaks by Scherrer's formula [42]. From Scherrer's method the crystallite size of the products ZnO-C and ZnO-B are found to be 19 and 13 nm respectively. Further, it is also interesting to note that the peak intensity of the ZnO-C is found to be significantly higher compared to ZnO-B. This suggests that by varying the method of synthesis, crystallinity of the samples are also varied. The lower crystallinity and crystallite size of ZnO-B can be attributed to the lower synthesis temperature for biogenic method compared to chemogenic process. Lower crystallite size can also be explained based on the phytoconstituents acting as capping agents which may restrict the crystallite growth [43].

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Various parameters such as peak shift, peak broadening, peak asymmetry etc of the XRD pattern can be used to evaluate the crystal structure of the material under investigation using Rietveld refinement method [44, 45]. In the present study Rietveld refinement for the experimentally obtained XRD data are carried out in order to evaluate the influence of method of synthesis on structural parameters. The output of the refinement carried out for ZnO-C and ZnO-B using FULLPROF program is shown in figure 2. Using the pseudo-Voigt function various parameters are fitted with experimental data set [46]. Integrated intensity of the diffraction peaks as a function of structural parameters was considered and following Marquardst least squares procedure refinement was carried out. Refinement was performed to minimise the difference between calculated and experiment powder diffraction patterns. The final refinement analysis shows that the experimental and calculated PXRD patterns obtained by the Rietveld refinement are in good agreement with each other. The model crystal structure generated using Rietveld refinement parameters finally obtained is shown in figure 3. The refined parameters, reliable factors calculated are summarized in table 1.

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The scanning electron micrographs of ZnO-B and ZnO-C are shown in figures 4(a)–(d) respectively. SEM micrographs of ZnO-B exhibits porous and fluffy morphology. Very fine and near spherical particles forming inter connected foamy structures are found to be highly agglomerated. Higher magnification SEM images (figure 4(b)) of ZnO-B shows that agglomerated particles are found to form clusters of various sizes. On the other hand, micrographs of ZnO-C are found to have larger particle agglomerates with more number of voids in their structure. Careful observation of micrographs also reveals the surface structure of ZnO-C also found to be rich with numerous pores and voids. However, unlike ZnO-B, individual particles could not be differentiated in ZnO-C due to high agglomeration nature of particles. The likely reason for higher agglomeration in ZnO-C may be due to higher calcination temperature required in this process compared to ZnO-B. The difference in surface morphology between ZnO-B and ZnO-C may be attributed to the difference in the mechanism of particle formation and the variation in synthesis temperature of the two methods. It is observed that biogenic synthesis using plant extracts is a controlled equilibrium process resulting in energetically favourable spherical shape particles [43]. Sudhasree et al [2014] have reported that biogenic nickel nanoparticles synthesised using extract of Desmodium gangeticum were of reduced size and monodispersed when compared to chemogenic nickel nanoparticles [47].

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The difference in the morphological features and extent of agglomerations in ZnO-C and ZnO-B can be explained as follows; Phyto-extracts have been extensively used for synthesis of various nanoparticles and these extracts contain phytoconstituents such as polyphenols, polysaccharides, proteins, terpenoids, flavones, amino acids, vitamins and alkaloids etc which act as both reducing and capping agents leading to the formation of nanoparticles. Phenols and flavonoids in particular have unique property to cap nanoparticles, thus preventing agglomeration of nanoparticles [48, 49]. Further, hydroxyl and carboxyl groups of phenolic compounds have strong affinity to bind to metals and metal ions leading to reduced agglomeration [50].

Pomegranate peel extract is known to majorly contain punicalagin and gallic acid along with other phenolic compounds such as ellagic acid, chlorogenic acid, caffeic acid, punicalin, apigenin, quercetin, pelargonidin, cyanidin, granatin A, granatin B etc, that act as act as reducing and stabilizing agents in bio-mediated synthesis of various metal and metal oxide nanoparticles [33–35]. Recently, Sukri et al [36] reported the role of gallic acid and punicalagin as stabilizing agents for the synthesis of ZnO nanoparticles. On the other hand Farrokhnia et al [51] showed that the hydroxyl groups of gallic acid can act as capping agents during the synthesis of AgNPs. Further, Wang et al [52] also reported that use of gallic acid helps in stabilization and prevention of agglomeration of gold nanoparticles. Therefore, from these literature reports we infer that, the reason for lesser agglomeration in ZnO-B compared to ZnO-C is due to the presence of phytoconstituents in general, gallic acid and punicalagin in particular that act as capping agents.

Figures 5(a), (b) and (d), (e) shows Transmission electron micrographs of ZnO-B and ZnO-C respectively. TEM micrographs of ZnO-B shows particles are nearly spherical and are uniform in size. However, particles are found to be agglomerated with uniform distribution of particles. The particles in ZnO-B are found to be in the range of 10–20 nm. However, from ZnO-C TEM images, it can be seen that the particles are uneven in size and the shape of the particles are also not uniform. The average sizes of the particles are found to be in the range of 60–80 nm and a few particles are found to be >100 nm. The bigger particle size of ZnO-C compared to ZnO-B might be due to the difference in the calcination temperature and the mechanism of particle formation. HRTEM images of ZnO-B and ZnO-C (figures 5(c) and (f) respectively) shows clear well resolved lattice fringes showing highly crystalline nature of the products. The interplanar distance from HRTEM images is found to be 0.27 nm corresponding to (002) plane in ZnO [53].

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Wound healing effects of ZnO-C and ZnO-B formulations of 1% and 2% along with control (only ointment base) and standard group (povidone–iodine ointment (5%)) were evaluated by excision wound model (figure 6). The apparent changes in wound contraction area are tabulated in table 2. On the 17th day significant wound closure rate was observed for the test groups compared to control group. 2% ZnO-B (89.05 ± 0.47%) exhibited highest wound closure rate compared to standard (88.08 ± 0.72%) and control group (75.76 ± 2.149%). On the 21st day, 2% ZnO-B exhibited 95.57 ± 1.031% wound contraction compared to standard ointment (93.29 ± 0.391%) and control (81.54 ± 0.67%). Whereas 1% ZnO-C (92.84 ± 1.091%), 2% ZnO-C– (93.95 ± 1.143%), 1% ZnO-B—(93.29 ± 0.391%) exhibited relatively less wound closure rate compared to 2% ZnO-B—(95.57 ± 1.031%). The results obtained suggest that 2% ZnO-B had the highest wound healing property.

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Sudheesh Kumar et al [54] have reported the wound healing potential of β-Chitin hydrogel/ chemogenic nano ZnO composite bandage. ZnO was prepared by chemogenic precipitation method. Composite bandage treated wounds showed 70% and 85% wound closure after 14 days and 21 days respectively. Arshad et al [55] have reported the wound healing activity of chemogenic nano ZnO embedded thiolated chitosan-alginate bandage. Wound closure of ∼80% was observed after 12 days of treatment and complete healing was reported after 28 days. Yadav et al [20] have reported the wound healing activity of Trianthema portulacastrum Linn. plant extract mediated biogenic zinc oxide nanoparticles using excision wound model. The percentage wound closure rate of ZnO on 6th day was found to be ∼27%. Significant wound closure rate was observed only form 9th day onwards. Nano ZnO treated wounds showed ∼67%, ∼80% and ∼85% wound closure after 12, 15 and 17 days respectively.

In the present study, significant wound closure rate was observed from 7th day itself for both biogenic and chemogenic ZnO treated wounds. Wound healing activity of ZnO-B was comparable to that of ZnO-C. Percentage wound closure was ∼80%, ∼85% and ∼93% after 14, 17 and 21days for both ZnO-B and ZnO-C treated wounds. The obtained results indicate that the activity of synthesized biogenic and chemogenic ZnO nanoparticles are higher than that reported for chemogenic ZnO and comparable to that of biogenic ZnO reported in literature. Absence of impurities and smaller size of synthesized zinc oxide nanoparticles attributes to significant wound healing activities observed in the present study.

Histopathological examination of excision wound tissue was performed on 21st day and the micrographs are shown in figure 7. The micrograph revealed that animals treated with only ointment base (i.e., control group) showed normal skin epidermis. Dermis showed proliferation of fibroblasts and mononuclear inflammatory infiltrates with thin walled congested vessels indicating incomplete healing of wounds (figure 7(a)). Sections of animals treated with standard povidine iodine exhibited complete re-epithelialization and focal mild mononuclear inflammatory infiltrates with thin walled congested vessels ((figure 7(b)). Histopathological section of animals treated with 2% ZnO-C also revealed mild mononuclear inflammatory infiltrates, complete re-epithelialization with proliferation of fibroblasts and thin-walled vessels (figure 7(c)). On the other hand, sections of animals treated with 2% ZnO-B showed epidermal re-epithelialization with proliferation of fibroblast and mononuclear inflammatory infiltrates, new blood vessels and significant increase in collagen formation in the dermis (figure 7(d)) [56].

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Similar histopathological observations have been reported for nano ZnO treated wounds. Yadav et al [20] observed enhanced collagen formation with less inflammatory cells, re-epithelialization and keratinization for biogenic ZnO treated wounds compared with control and standard. Arshad et al [55] have reported complete re-epithelialization and intact epidermis of nano ZnO treated wounds. Topical application of biogenic Ag nanoparticles showed creation of fibroblast rich granulation tissue under the wounds with complete re- epithelialization [57].

Several mechanisms have been proposed for the wound healing activity of nano ZnO like migration of keratinocytes towards wound area by the released Zn²⁺ ions inducing epithelialization of wounds [54, 58]; reduction in oxidative stress and inflammatory reactions resulting in quick formation and deposition of collagen fibers, tissue granulation and reconstruction of epithelial lining [20]; anti-inflammatory effect due to inhibition of protein [20]. In the present case, wound healing activity of ZnO nanoparticles might be due to the antibacterial activity coupled with interaction with specific enzymes responsible for wound healing activity. In order to prove this antibacterial activity and molecular docking studies are performed and the results are discussed in the following sections.

Antibacterial activity of ZnO-B and ZnO-C nanoparticles were evaluated against 3 bacterial strains (i.e., Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli). Minimum Inhibition Concentration (MIC) for standard drug (i.e., Ciprofloxacin) against Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli was found to be 8 μg ml⁻¹, 8 μg ml⁻¹ and 4 μg ml⁻¹ respectively. From MIC values and comparing with CSLI standards, it can be inferred that bacterial strains are moderately resistant (figure 8) [59]. The MIC of ZnO-B on Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli was 50 μg ml⁻¹, 60 μg ml⁻¹, and 30 μg ml⁻¹ respectively. On the other hand ZnO-C showed MIC values of 60 μg ml⁻¹, 80 μg ml⁻¹ and 50 μg ml⁻¹ respectively for Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli. Compared to the standard drug, ZnO-B and ZnO-C showed higher MIC against all the tested strains. However, it is worth noticing that ZnO-B nanoparticles showed higher antibacterial activity compared to ZnO-C nanoparticles. Higher antibacterial activity of ZnO-B nanoparticles compared to ZnO-C can be attributed to smaller size of ZnO-B compared to ZnO-C. Smaller size nanoparticles can penetrate the bacterial cell membrane easily and cause membrane leakage and cell death. Similar observations are also reported for nickel nano particles by Sudhasree et al [47]. Where authors reported higher bacterial activity of biogenic derived Ni nanoparticles compared to chemically synthesized nickel nanoparticles. Further, it should also be noted that relatively higher antibacterial activity was observed against Gram negative strains than Gram positive strains. This is due to the fact that Gram negative bacteria exhibits less resistance to nanoparticle mediated toxicity on cell membrane interaction, because of thin peptidoglycan layer compared to Gram positive bacteria [60]. Antibacterial activity of ZnO-B and ZnO-C nanoparticles may be for the following reasons (i) interaction of nano ZnO with bacterial cells leading to ROS generation and ROS causes membrane leakage of proteins and nucleic acids by enhancing lipid peroxidation on membrane [61]. (ii) Zn²⁺ ions released from the nanoparticles might also damage the cell membrane and interact with intracellular components destructing bacterial cell integrity [62, 63]. Similar results and various other pathways of antibacterial activity/ mechanism and wound healing are also reported by various research groups [64–66].

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Molecular docking can predict the affinity and activity of small molecule drug to their protein targets. GSK-3β is a widely expressed serine/threonine kinase encoded by 2 genes, GSK3A and GSK3B. GSK-3β controls the progression of wound healing and fibrosis by modulating ET-1 levels suggesting that targeting the GSK-3β pathway or ET-1 may be of benefit in controlling tissue repair and fibrogenic responses in vivo [67].

Molecular docking comparision of Zinc oxide with standard drug Povidone iodine against the GSK-3β protein, a wound healing biomarker, is presented in figure 9 and table 3. Povidone iodine exhibited a binding affinity of −5.1 Kcal mol⁻¹. Formation of H–bond was absent and seven residues were involved in hydrophobic interactions. On the other hand, the binding site of ZnO on GSK-3β includes five residues involved in the hydrogen bond and five residues involved in hydrophobic interactions. ZnO exhibited binding affinity of −5.6 Kcal mol⁻¹ with 7 H–bonds and hydrophobic interactions with Leu227, Gly230, Ile228, Tyr216 and Ser215. The strong binding is due to the existence of seven hydrogen bonds with three of them having bond length <3.00 Å. As evident from zero binding site similarity observed, it can be concluded that ZnO docked at completely different sites as compared to Povidone iodine. Overall analysis of the comparative insilico studies indicates that, ZnO with higher binding energy, protein-ligand complex stabilized by multiple H–bonds and good hydrophobic interactions with amino acids can be considered as effective compound for GSK inhibition.

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In addition to the docking studies of GSK-3β protein, in silico molecular docking was also performed for glucosamine-6-phosphate synthase (GlcN-6-P synthase). The enzyme GlcN-6-P synthase is involved in the formation of N-acetyl Glucosamine, which is an essential building block of bacterial and fungal cell walls [68]. Hence, GlcN-6-P can be a promising target for antibacterial and antifungal drug discovery. Ciprofloxacin has been reported to be a good inhibitor of GlcN-6-P synthase [69]. Considering GlcN-6-P synthase as the target receptor, comparative molecular docking studies of Zinc oxide with standard drug Ciprofloxacin was performed and the results are presented in figure 10 and table 4. Standard antibiotic Ciprofloxacin exhibited binding affinity of −7.0 Kcal mol⁻¹ with 2 H–bonds (Thr302, Ser401) and hydrophobic interactions with Ala400, Gln348, Ser303, Cys300, Asb354, Val605, Thr352 and Val399. On the other hand, ZnO exhibited binding affinity of −4.9 Kcal mol⁻¹ with 8 H–bonds and hydrophobic interactions with Ser303, Thr302, Gly301 and Ser604. The binding site of ZnO on GlcN-6-P synthase includes five residues involved in the hydrogen bond as against only two residues in case of Ciprofloxacin. Overall analysis of the in silico studies indicates that ZnO with higher protein-ligand complex stabilized by multiple H–bonds and good hydrophobic interactions can be considered more effective than the standard drug Ciprofloxacin for inhibition of GlcN-6-P synthase. The obtained results correlate to the observed antibacterial activity. It can be concluded that ZnO exhibits remarkable antibacterial activity through the inhibition of GlcN-6- P synthase.

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