Phytochemical Profiling, Molecular Docking, ADMET Analysis of Zingiber Officinale Peel Extract for the Biogenic Synthesis of ZnO and Fe-Doped ZnO Nanoparticles with Antibacterial and Photocatalytic Potential

Abstract

Industrial dye effluents are the predominant source of water pollution, while the increasing prevalence of bacterial infections poses a serious public health threat. Nanoparticles offer promising avenues for addressing these challenges. Therefore, this study sought to synthesize ZnO (ZNP) and Fe-doped ZnO (F-ZNP) nanoparticles using Zingiber officinale peel extract and investigate their antimicrobial and photocatalytic potential, along with molecular insights into the antibacterial activity of phytochemicals through in silico studies. Phytochemical profiling of the Z. officinale peel extract was performed using gas chromatography–mass spectrometry (GC–MS). The physicochemical characteristics of the biogenic ZNP and F-ZNP were analyzed using X-ray diffraction (XRD), scanning electron microscopy (SEM), UV–visible (UV–vis) spectroscopy, and thermogravimetric and differential thermal (TG/DT) analyses. In silico analysis of phytochemicals was performed to assess their interactions with bacterial proteins (GyrB and FabH) and to predict their pharmacokinetic properties. XRD analysis confirmed the hexagonal wurtzite structure with a mean crystallite size of 31.92 nm for ZNP and 23.49 nm for F-ZNP. SEM micrographs revealed a flake-like morphology with an average particle size of 47 and 35 nm for ZNP and F-ZNP, respectively. UV–vis spectroscopy revealed absorption edges at 363 and 369 nm, with corresponding band gaps of 3.15 and 3.10 eV for ZNP and F-ZNP, respectively. TG/DT thermographs demonstrated excellent thermal stability, with minimal weight loss of only 2.70% for ZNP and 5.21% for F-ZNP up to 800 °C. The dynamic light scattering technique revealed hydrodynamic diameters of ∼433 nm (ZNP) and ∼422 nm (F-ZNP) with minor aggregation. Zeta potentials of +7.27 mV (ZNP) and +17.44 mV (F-ZNP) indicated low and improved colloidal stability, respectively, demonstrating enhanced dispersibility upon Fe doping. The biogenic ZNP exhibited a moderate methylene blue degradation rate of 56.24%, while F-ZNP resulted in an outstanding photocatalytic degradation rate of 91.39% within 120 min. Moreover, F-ZNP demonstrated excellent photocatalytic reusability, maintaining a high performance (∼78%) even after three cycles. The biogenic ZNP and F-ZNP exhibited substantial antibacterial activity against both Gram-positive and Gram-negative bacterial strains, showing a zone of inhibition of 10 to 16 mm. These results were corroborated by favorable binding affinity with bacterial proteins −7.2 to −5.0 kcal/mol), and the bioactive molecules were safe based on ADMET analysis. Moreover, both ZNP and F-ZNP exhibited excellent biocompatibility with hemolysis percentages remaining below 2% across all tested concentrations (25–400 μg/mL). Notably, F-ZNP demonstrated slightly enhanced hemocompatibility compared to ZNP. Therefore, our findings suggest a facile and sustainable route to synthesize ZNP and F-ZNP using the ginger peel extract, focusing on their safe application in antibacterial therapy and wastewater treatment.

1. Introduction

Certainly, water pollution is a pressing and serious global issue with organic dye contamination being even more severe. Annually, over seven million tons of dyes are produced worldwide, of which nearly 10% are released into the environment through industrial wastewater. Indeed, as reported by the World Bank, textile dyes are responsible for about 20% of industrial water pollution. , A significant concern is that the discharge of industrial dyes profoundly affects public health due to their potential carcinogenic, mutagenic, or teratogenic effects. Furthermore, they pose huge threat to the ecosystem as the discharge of industrial dyes can disrupt aquatic life, harm biodiversity, and disturb the natural balance. Like industrial water pollution, another public health concern that requires urgent attention is the rise of multidrug-resistant (MDR) bacterial strains, mostly arising from the excessive and improper use of antibiotics. This alarming situation severely undermines the effectiveness of existing antibiotics, making bacterial infections intractable. Reported in 2020, a study highlighted that this situation is responsible for more than 6 million deaths directly or indirectly. The World Health Organization has already underscored this era as ‘post antibiotic era’, emphasizing the urgency of alternative antimicrobial strategies. , In these contexts, nanotechnology offers a promising solution by enabling the development of multifunctional nanomaterials capable of addressing both issues through a single, synergistic strategy.

Metal oxide nanoparticles (MONPs) have garnered remarkable attention as they effectively address these challenges. Conventional methods such as chlorination, membrane filtration, ion exchange, adsorption, reverse osmosis, and ultrafiltration often fall short, typically converting dyes from one phase to another rather than achieving complete degradation. In contrast, MONPs involve photodegradation, a sustainable and eco-friendly technique, and operate under ambient conditions, enabling the complete mineralization of dye pollutants into less harmful end products. − Additionally, due to nanoscale dimensions, high surface-to-volume ratio, biocompatibility, and prolonged functional stability, MONPs are increasingly explored in therapeutic applications. , Among the wide range of MONPs, ZnO nanoparticles exhibit a wide band gap of 3.37 eV, excellent intrinsic electron mobility of 300 cm²·V^–1·s^–1, and a strong excitonic binding energy of 60 meV. Additionally, it possesses a remarkably high melting point of 2248 K and a cohesive energy of 1.89 eV, distinguishing it as a versatile material for various applications in the fields of catalysis, electronics, energy, agriculture, and environment, among others. − Moreover, the Food and Drug Administration 21CFR182.8991 classifies ZnO as a generally recognized as safe compound for human and animal, supporting their potential in biomedical fields such as drug delivery, cancer therapy, antimicrobial therapeutic, and biosensors. −

However, ZnO nanoparticles have relatively high excitation energy and rapid recombination rate of photogenerated electron–hole pairs, which collectively affect their antimicrobial and photocatalytic performance. , Consequently, the strategy such as doping addresses these limitations by introducing a small quantity of impurities deliberately incorporated into the ZnO matrix. Previously various dopants, including Al, Mn, Mg, Ag, Ni, Co, Cu, and others, have been incorporated into ZnO to enhance charge separation and modify electronic, structural, and morphological properties. Iron (Fe), being a transition metal, is considered a prominent dopant for ZnO due to its d-electrons, which can easily overlap with the valence band of ZnO. This interaction modifies the band gap of nanoparticles, ultimately enhancing the photocatalytic and antimicrobial properties. −

Nanoparticles can be synthesized via various methods such as hydrothermal synthesis, ultraviolet radiation, thermal decomposition, plasma-assisted physical vapor deposition, electrochemical processes, microwave-based synthesis, and so on. However, these methods are often costly and energy-intensive and involve frequent usage of toxic and hazardous chemicals. Additionally, the obtained nanoparticles often lack homogeneity and have a tendency to agglomerate. − In green synthesis, biological elements including microbes and plant derivatives such as flowers, seeds, leaves, and fruits are used. These biological elements function as natural reducing and capping agents, facilitating the formation of highly homogeneous, nanoscale particles. − Prior studies have demonstrated that biogenic nanoparticles exhibit superior biological and photocatalytic activities compared with conventionally synthesized counterparts. For instance, Haque et al. reported superior antibacterial and photocatalytic activity of ZnO nanoparticles synthesized using Azadirachta indica (neem) compared to the sol gel method. Similarly, Vasiljevic et al. highlighted that the concentration of green extract influences the biological and photocatalytic properties of ZnO nanoparticles. Basically, the bioactive compounds form an outer coating around the nanoparticles, effectively creating a core–shell structure. This modification significantly enhances the biological properties of the nanoparticles. Apart from this, the biogenic approach diminishes the reliance on toxic and hazardous chemicals and also offers a significant reduction in energy consumption and operational costs. Consequently, it represents a sustainable, eco-friendly alternative to conventional nanoparticle synthesis methods. ,

Ginger (Zingiber officinale) is one of the most commonly consumed dietary spices in many regions and has long been valued in traditional medicine for treating colds, nausea, inflammation, and cardiovascular diseases. It is rich in bioactive compounds, including gingerols, zingerones, shogaols, and flavonoids with strong antioxidant and antimicrobial properties. To date, several studies have explored the synthesis of ZnO nanoparticles by using different parts of ginger. However, ginger peel, often regarded as a domestic agrowaste, has not been explored, largely despite its potential as an effective reducing and capping agent. Prior research studies lacked comprehensive phytochemical profiling of the ginger peel extract, leaving it unclear which specific phytochemicals play a crucial role in nanoparticle formation and stabilization. − In addition, several studies have explored the biogenic synthesis of ZnO and Fe-doped ZnO nanoparticles; many lack comprehensive characterizations and thorough evaluation of their application-specific properties. Therefore, the present study provides an in-depth analysis of both ZNP and F-ZNP, encompassing their structural, morphological, optical, and thermal properties, as well as their photocatalytic reusability and hemocompatibility. The pharmacokinetics, toxicity, and drug-likeness of the bioactive compounds were further assessed using molecular docking simulations and ADMET profiling, forecasting their potential as lead candidates for safe biomedical applications. Overall, this study aims to develop biogenic ZnO-based nanoparticles with improved antibacterial and photocatalytic performance, offering the potential for environmentally sustainable and biologically safe applications.

2. Materials and Methods

2.1. Materials

Zinc acetate dihydrate [Zn­(CH₃COO)₂·2H₂O] and iron­(II) chloride [FeCl₂], serving as the source materials for Zn and Fe, were obtained from Sigma-Aldrich (Merck, Germany) with a purity of ≥ 98%. Additionally, sodium hydroxide (NaOH), methylene blue (MB) (C₁₆H₁₈ClN₃S), and ethanol (C₂H₅OH) were sourced from the same supplier. All chemicals utilized in this study were of analytical grade. Z. officinalewas collected from Mirpur kachabazar, Dhaka, Bangladesh and was systematically identified by MD. Mahfuzur Rahman, Botanist (MSc, Department of Botany, Jagannath University, Bangladesh).

2.2. Methods

2.2.1. Preparation of the Z. officinale Peel Extract

Domestic agro-waste Z. officinale peel was thoroughly washed with water to remove dirt and contaminants and subsequently dried and ground into a fine powder. A total of 20 g of the powdered peel was extracted in 100 mL of water under the reflux condition of 80 °C for 3 h. The extract was then filtered using Whatman filter paper no. 1 and stored at 4 °C for further usage.

2.2.2. Biosynthesis of ZNPs and F-ZNPs

ZNP was synthesized using the Z. officinale peel extract, following a prior method reported by MuthuKathija et al. with slight modifications. First, a 0.1 M solution of zinc acetate dihydrate was prepared in 50 mL of deionized water (DI) and stirred vigorously for 20 min. Then, 20 mL of the aqueous Z. officinale peel extract was added dropwise under continuous stirring. To adjust the pH to 12, NaOH was added gradually. The mixture was stirred for an additional 3 h at 70 °C, during which whitish lotion-like precipitation became evident. After the reaction was completed, the solution was left to age for 24 h. The precipitate was collected via centrifugation at 6000 rpm for 15 min, washed multiple times with DI water to remove residual impurities, and dried at 60 °C for 2 h. The dried powder was then calcined in a muffle furnace at 400 °C for 2 h, ground into a fine powder, and stored for further characterization. A similar approach was used to synthesize F-ZNP. In this case, FeCl₂ was introduced alongside zinc acetate dihydrate, maintaining a molar ratio according to the formula Fe _x Zn_1–x O, where x = 0.03.

The yield of synthesized nanoparticles can be estimated using eq .

$$\mathrm{y}\mathrm{i}\mathrm{e}\mathrm{l}\mathrm{d}(\%)=\frac{\mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t}\mathrm{o}\mathrm{f}\mathrm{n}\mathrm{a}\mathrm{n}\mathrm{o}\mathrm{p}\mathrm{a}\mathrm{r}\mathrm{t}\mathrm{i}\mathrm{c}\mathrm{l}\mathrm{e}\mathrm{s}}{\mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t}\mathrm{o}\mathrm{f}\mathrm{p}\mathrm{r}\mathrm{e}\mathrm{c}\mathrm{u}\mathrm{r}\mathrm{s}\mathrm{o}\mathrm{r}\mathrm{s}\mathrm{a}\mathrm{l}\mathrm{t}}\times 100\%$$ 1

3. Characterization

The crystallographic analysis of ZNP and F-ZNP was investigated by X-ray diffraction (XRD) analysis. An X-ray diffractometer [Rigaku Smart Lab, Japan] with CuKα radiation (λ = 1.54 Å) was employed for this purpose. The investigation was performed using the 1D scan mode at a speed of 10.0° /min and increments of the step interval of 0.01 with scanning ranges (2θ) of 0°–80°. The surface morphology and particle size of the biogenic nanoparticles were examined by using scanning electron microscopy (SEM) (Carl Zeiss AG, EVO18, UK). Energy-dispersive X-ray spectroscopy (EDS) was conducted to analyze the elemental composition by using a JCM-6000PLUS (JEOL, Japan) instrument. Thermogravimetric (TG) and differential thermal (DT) analyses of the nanoparticles were carried out using a thermoanalyzer (STA 8000, PerkinElmer, Netherlands). The measurements were performed in an air atmosphere, with the temperature maintained from 30 °C to 800 °C at a heating rate of 10 °C/min. The optical property of nanoparticles was investigated within the wavelength range of 300–600 nm using a spectrophotometer (SP-UV-500DB, Spectrum instrument, Germany). Hydrodynamic size and ξ potential were measured by dynamic light scattering (DLS) analysis (ZSU5700, Malvern Panalytical, UK).

3.1. GC–MS Analysis

Gas chromatography–mass spectrometry (GC–MS) analysis of the Z. officinale peel extract was performed employing a mass spectrometer (TQ 8040, Shimadzu, Kyoto, Japan) and a gas chromatograph (Shimadzu) which was equipped with a Rxi-5 MS capillary column (30 m length, 0.32 mm diameter, and 0.25 μm film thickness) interfaced with DB-1 (J & W). In the column, helium gas was used as a carrier gas with a flow rate of 0.6 mL/min at a constant pressure (90 kPa). The oven temperature was set at 70 °C (0 min); 10 °C, 150 °C (5 min); 12 °C, 200 °C (15 min); and 12 °C, 220 °C (5 min), with a clamp time of 10 min. From the clear extract, 1 μL of the sample was injected in a splitless mode with a mass spectral range of 50–550 m/z. The MS start time was set at 5.00 min and an end time of 50.00 min. The spectrum obtained from the volatile compound was compared and matched with the National Institute of Standards and Technology (NIST) library.

3.2. Photocatalytic Activity

The photocatalytic activity of ZNP and F-ZNP was evaluated according to a method previously demonstrated by Faisal et al., with slight modifications. A cationic dye, MB, was used as a model organic dye. Initially, a 20 ppm solution of MB dye was prepared in 50 mL of distilled water, and subsequently, 5 mL of the solution was taken to record its absorbance spectrum in between 400 and 800 nm. Then, 25 mg of the catalyst (ZNP/F-ZNP) was added to the remaining 45 mL of MB dye solution and subjected to vigorous stirring. The mixture was kept in the dark to reach adsorption–desorption equilibrium before being exposed to sunlight. The solution was centrifuged at 9500 rpm for 15 min to separate the catalyst, and the absorbance was recorded between 400 and 800 nm. This process was repeated every 20 min for a total duration of 120 min. It should be noted that the photocatalytic experiment was conducted under direct sunlight on the rooftop of the Biomedical and Toxicological Research Institute, Bangladesh Council of Scientific and Industrial Research (BCSIR), Dhaka 1205, Bangladesh. The weather was clear and sunny, ensuring consistent exposure during the test, which was conducted between 10 AM and 1 PM based on weather forecasts.

The photocatalytic efficiency was determined using eq .

$$\mathrm{p}\mathrm{h}\mathrm{o}\mathrm{t}\mathrm{o}\mathrm{d}\mathrm{e}\mathrm{g}\mathrm{r}\mathrm{a}\mathrm{d}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}(\%)=\frac{A_0-\mathrm{A}}{A_0}\times 100\%$$ 2

In this equation, A ₀ and A are the initial and final absorbance at different time intervals of MB dye, respectively.

3.3. Antibacterial Assay

The in vitro antibacterial activity of ZNP and F-ZNP was examined by the disk diffusion method. The Gram-negative strains, Pseudomonas aeruginosa (ATCC27853), Shigella flexneri (clinical isolate), and Serratia marcescens (MT912977), along with the Gram-positive strain, Staphylococcus aureus (ATCC25923), were tested. First, all the bacterial strains were subcultured in nutrient agar media at 37 °C. To prepare bacterial inocula, the isolated colonies were dispersed in 0.9% sterile saline until the turbidity matched with the 0.5 McFarland standard. Subsequently, with the help of a sterile cotton swab, the bacteria were uniformly spread onto Mueller–Hinton agar. Standard sterile discs (5 mm in diameter) were loaded with ZNP and F-ZNP suspensions at concentrations of 100, 200, and 300 μg/mL. As a positive control, ciprofloxacin (5 μg/disc) was employed. However, methanol was used as the negative control. The prepared discs were carefully positioned on the inoculated agar plates and incubated at 37 °C for 24 h. The antibacterial efficacy was assessed by measuring the zone of inhibition (ZOI) in mm scale.

3.4. Hemolysis Assay

The biocompatibility of ZNP and F-ZNP was evaluated via hemolysis assay using fresh human red blood cells (hRBCs) following a prior study demonstrated by Faisal et al. Blood samples were collected from a healthy volunteer with consent using tubes containing ethylenediaminetetraacetic acid (EDTA) as an anticoagulant. The collected blood sample was then centrifuged to separate the red blood cells. It was further washed three times with phosphate-buffered saline (PBS, pH 7.2) to remove residual plasma and impurities. Discarding the supernatant, a suspension was prepared by mixing 200 μL of the washed erythrocytes with 9.8 mL of PBS. This erythrocyte suspension was then combined with varying concentrations of the biogenic ZNP and F-ZNP in Eppendorf tubes. The mixtures were incubated at 35 °C for 1 h to allow interaction between the nanoparticles and the red blood cells. Following incubation, the samples were centrifuged at 1000 rpm for 10 min to separate the cells from the supernatant. A portion (200 μL) of the resulting supernatant was transferred to a 96-well plate, and the absorbance was recorded at 540 nm. In this evaluation, Triton X-100 (0.5%) was used as a positive control, while PBS served as a negative control. The percentage (%) of hemolysis was calculated employing Formula .

$$\%\mathrm{h}\mathrm{e}\mathrm{m}\mathrm{o}\mathrm{l}\mathrm{y}\mathrm{s}\mathrm{i}\mathrm{s}=\left(\frac{\mathrm{a}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{r}\mathrm{b}\mathrm{a}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{o}\mathrm{f}\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}-\mathrm{a}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{r}\mathrm{b}\mathrm{a}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{o}\mathrm{f}\mathrm{n}\mathrm{e}\mathrm{g}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{e}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}}{\mathrm{a}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{r}\mathrm{b}\mathrm{a}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{o}\mathrm{f}\mathrm{p}\mathrm{o}\mathrm{s}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{e}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}-\mathrm{a}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{r}\mathrm{b}\mathrm{a}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{o}\mathrm{f}\mathrm{n}\mathrm{e}\mathrm{g}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{e}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}}\right)\times 100\%$$ 3

3.5. Molecular Docking

3.5.1. In Silico Docking

For docking purposes, the current study retrieved 3D crystal structures of GyrB and FabH (PDB: 4URO for S. aureus and 2X3E for P. aeruginosa), respectively, using the RCSB Protein Data Bank (PDB) (https://www.rcsb.org/). The 3D structures of ligands (targeted phytochemicals) were taken from PubChem (http://pubchem.ncbi.nlm.nih.gov/) and saved in the SDF format. To prepare these proteins for further analysis, all crystal structures of the proteins were prepared by removing heteroatoms, water molecules, and cocrystallized ligands using BIOVIA Discovery Studio software (https://discover.3ds.com/discovery-studio-visualizer-download). The modified receptors were subjected to energy minimization utilizing the Swiss-PDB Viewer (4.1.0). Then, polar hydrogen atoms and Kollman charges were added by using MGL Tools 1.5.6. Finally, the receptors were saved in a PDB format for further docking. For docking, first we identified the probable binding pocket of the targeted protein using the CASTp web server (http://sts.bioe.uic.edu/castp/index.html?3igg), which measures geometric and topological properties of protein structures. After identifying the pocket amino acid residue, we uploaded the protein into PyRx software as a macromolecule and phytochemicals as a ligand. We then selected the amino acid residues associated with binding pockets to delineate the docking grid. Finally, docking was performed to assess the docking scores between the targeted proteins and phytochemicals, describing insights into their potential interactions.

3.5.2. In Silico Drug-Likeness/ADME and Toxicity Analysis of Selected Phytochemicals

To predict the substantial pharmacokinetic and toxicological properties, we assessed ADMET (absorption, distribution, metabolism, excretion, and toxicity) properties for all 10 selected phytochemicals and ciprofloxacin as a standard drug using SwissADME (http://www.swissadme.ch/), AdmetSAR (https://lmmd.ecust.edu.cn/admetsar2/), and PROTOX-II (https://tox-new.charite.de/protox_II/) online servers. The SwissADME server was accustomed to estimating drug-likeness (Lipinski’s rule of five) features, including molecular weight (MW), number of rotatable bonds, number of hydrogen bond donors and acceptors (HBA), calculated octanol–water partition coefficient (ClogP), pharmacokinetics (ADME), and oral bioavailability. The AdmetSAR server was used to calculate the Caco-2 cell permeability, hERG inhibition, AMES mutagenesis, and biodegradation. In addition, the PROTOX-II Web server was used to evaluate the toxicity (e.g., hepato-, nephro-, cardio-, immunotoxicity, lethal dose 50 (LD50), etc.) of these selected phytochemicals.

4. Results and Discussion

4.1. GC–MS Analysis

Phytochemical profiling of the Z. officinale peel extract identified the presence of eighty (80) compounds, as shown in the GC–MS chromatogram (Figure ). Of these, 35 major compounds, along with their name, PubChem compound identifier (CID), molecular formula, MW, retention time, peak area (%), and chemical nature are depicted in Table . The nature of the identified molecules belongs to diverse chemical classes, including phenols, alkaloids, terpenes, amines, esters, fatty acids, amino acids, vitamins, hormones, and so on. Our results were in line with previous phytochemical profiling of the Z. officinale rhizome extract. − The bioactive phytochemicals of zinger can be used as reducing and stabilizing agents for nanoparticle synthesis and have multifaceted biomedical and environmental applications that were reflected in our study.

1.

GC–MS chromatogram of the aqueous extract derived from Z. officinale peel. The X axis represents retention time (min), and TIC stands for total ion chromatogram.

1. Phytochemical Profiling of the Z. officinale Aqueous Peel Extract Using GC–MS Analysis.

CID	name	molecular formula	molecular weight (g/mol)	r. time	area	compound nature
97704	3,6-dimethylpiperazine-2,5-dione	C6H10N2O2	142.16	9.078	36,762	heterocyclic diketone
11368	(−)-norephedrine	C18H24ClNO3	337.8	9.078	38,427	alkylbenzene
541442	2-heptanamine, 5-methyl-	C8H19N	129.24	9.078	42,354	aliphatic amine
31211	2-butanone, 4-(4-hydroxy-3-methoxyphenyl)-	C11H14O3	194.23	10.699	3,693,173	phenolic compounds
62465	phenol, 4-ethyl-2-methoxy-	C9H12O2	152.19	10.699	3,693,173	phenolic compounds
137860	6-methylnicotinic acid	C7H7NO2	137.14	10.699	3,693,173	pyridine alkaloid
445070	2,6,10-dodecatrien-1-ol, 3,7,11-trimethyl-	C15H26O	222.37	12.988	15,460	sesquiterpenoid
1549778	5,9-undecadien-2-one, 6,10-dimethyl-, (Z)-	C13H22O	194.31	12.988	15,460	monoterpene ketone
5950	alanine	C3H7NO2	89.09	13.873	21,891	amino acid
8181	hexadecanoic acid, methyl ester	C17H34O2	270.5	13.87	1,114,454	fatty acid
6997344	2-pentanamine, (2S)-	C5H13N	87.1	15.256	29,225	aliphatic amine
6567	1,2-propanediamine	C3H10N2	74.13	15.256	29334	diaminoalkane
5363265	cis-7, cis-11-hexadecadien-1-yl acetate	C18H32O2	280.4	15.847	29294	ester
5358322	5,7-dodecadiene, (Z, Z)-	C12H20O	180.29	15.847	29294	unsaturated hydrocarbon
137645	bicyclo [3.3.1] nonan-2-one	C9H14O	138.21	15.847	29294	bicyclic ketone
5363282	Z, E-7,11-hexadecadien-1-yl acetate	C18H32O2	280.4	15.847	29294	carboxylic ester
135398658	folic acid	C19H19N7O6	441.4	15.847	29294	vitamin
426589	2,6,6-trimethyl-bicyclo [3.1.1] hept-3-ylamine	C10H19N	153.2	15.847	29294	bicyclic amine
5816	epinephrine	C9H13NO3	183.2	15.847	29294	hormone
8201	methyl stearate	C19H38O2	298	15.961	768329	ester
9240	3-azabicyclo [3.2.2] nonane	C8H15N	125.21	16.514	38871	bicyclic amine
67678	cystine	C6H12N2O4S2	240.3	16.514	38871	amino acid
108965	ethyl homovanillate	C11H14O4	210.23	17.065	1,270,415	phenolic ester
2998	nonivamide	C17H27NO3	293.4	17.065	1,270,415	synthetic capsaicinoid
16928	homovanillyl alcohol	C9H12O3	168.19	17.065	1,270,415	phenolic compound
95415	ethanone, 2-hydroxy-1,2-bis(4-methoxyphenyl)-	C16H16O4	272.29	17.065	1,270,415	phenolic compound
126537	3,6-dimethyl-2,3,3a,4,5,7a-hexahydrobenzofuran	C10H16O	152.23	17.919	770663	polycyclic ether
70942	3,3-dimethylpiperidine	C7H15N	113.2	18.76	262970	alkaloid
5365371	13-docosenamide, (Z)-	C22H43NO	337.6	18.758	1,036,196	fatty acid
5280435	phytol	C20H40O	296.5	18.76	262970	terpenoid fatty alcohol
17369	2H-azepin-2-one, hexahydro-1-methyl-	C7H13NO	127.18	18.76	262970	heterocyclic amide
85779	2-hexadecanol	C16H34O	242.4	22.715	55292	fatty alcohol derivative
3893	dodecanal	C12H24O2	200.3	24.225	22876	fatty acid

4.2. Biosynthesis of Nanoparticles and the Hypothesized Mechanism

ZNP and F-ZNP were biosynthesized using the Z. officinale peel extract, as illustrated in Figure a. Regarding the preparation of F-ZNP, the molar ratio of Fe/Zn was maintained at 0.03/0.97, corresponding to Fe_0.03 Zn_0.97O. It is evident from the phytochemical profiling of the Z. officinale peel extract that it is rich with abundant bioactive compounds containing various functional groups such as hydroxyl (–OH), carbonyl (CO), carboxyl (–COOH), and amine (–NH₂). These phytochemicals act as the reducing and the capping agents during biosynthesis. The plausible mechanism of ZNP and F-ZNP syntheses is depicted in Figure b. The reaction between the initial precursors and the phytochemicals leads to the formation of reduced metallic compounds. Later, these phytochemicals facilitate nucleation and further growth by acting as both capping and stabilizing agents. During this process, generally there occurs a gradual color change of the solution that indicates the precipitate formation. The obtained precipitate was then subjected to drying and calcination, ultimately yielding ZNP and F-ZNP.

2.

(a) Schematic representation of the biogenic synthesis process and (b) the plausible reaction mechanism of the formation of ZNP and F-ZNP.

4.3. X-ray Diffraction Analysis

The structural and phase analyses of ZNP and F-ZNP were conducted using the XRD technique. As depicted in Figure a, the diffraction patterns exhibited well-defined peaks at Bragg angle, 2θ values of 31.7°, 34.3°, 36.2°, 47.4°, 56.5°, 62.8°, 66.2°, 67.8°, 69.04°, 72.58°, and 76.9°. These peaks resemble the (100), (002), (101), (102), (110), (103), (200), (112), (201), (004), and (202) crystallographic planes, respectively. Compared to the standard reference pattern (JCPDS-96-900-4180), both ZNP and F-ZNP possess a hexagonal wurtzite structure, as shown in Figure b, with a P63mc space group, affirming their phase purity and structural consistency. The absence of any secondary phase formation in the diffraction patterns of F-ZNP confirms the successful incorporation of the Fe dopant into the ZnO lattice through an effective ionic substitution. The inset in Figure a reveals that the incorporation of Fe dopant into the ZnO lattice leads to a shift in the crystallographic plane (101) along with a reduction in crystallinity. Prior studies suggest that, generally, the larger dopant causes a peak shift toward lower angles, whereas smaller dopants result in a shift toward higher angles. Since the Fe dopant can be found in two charge states, Fe²⁺ (0.77 Å) and Fe³⁺ (0.645 Å), their substitution behavior significantly influences the peak position. In this study, the observed shift of the (101) peak toward a lower angle suggests that Zn²⁺ ions (0.74 Å) are predominantly replaced by Fe²⁺ ions. ,

3.

(a) XRD patterns of ZNP and F-ZNP, confirming crystalline nature (b) representative model of the hexagonal crystal structure.

The crystallite size (D) of ZNP and F-ZNP was calculated using the Debye–Scherrer eq .

$$D=\frac{0.9\lambda }{\beta \cos \theta }$$ 4

Here, θ is the Bragg diffraction angle, β denotes the full width at half-maxima, and the wavelength of X-ray, λ = 1.5406 Å.

The average crystallite size of ZNP and F-ZNP was calculated to be 31.92 and 23.49 nm, respectively. It should be noted that the Debye–Scherrer method estimates the crystallite size without counting lattice strain, making it less precise. Therefore, this study incorporated the Williamson–Hall (W–H) plot to determine crystallite size while taking lattice strain into account for improved accuracy. The mathematical expression of the W–H method is expressed in eq .

$$\beta \cos \theta =\epsilon (4\sin \theta )+\frac{\mathrm{k}\lambda }{D}$$ 5

Here, β is the FWHM, ε is the strain, D is the crystallite size, and λ is the wavelength. Comparing the equation with standard equation of straight line, y = mx + c, it is found that the slope, m = lattice strain (ε) while intercept c = $\frac{\mathrm{k}\lambda }{D}$ .

The estimation of crystallite size and lattice strain of ZNP and F-ZNP is illustrated in Figure a,b. The crystallite size of ZNP is determined to be 37.59 nm, with a corresponding lattice strain of 0.000656 N·m^2–. Upon Fe doping, the lattice strain increases to 0.00123 N·m^2–, while a reduction in crystallite size has been observed, which decreases to 33.18 nm.

4.

Estimation of crystallite size of (a) ZNP and (b) F-ZNP using the Williamson–Hall (W–H) plot based on XRD analysis.

Apart from these variations, the incorporation of an Fe dopant in ZnO induces several interrelated changes. These are summarized in Table , which provides comparative insights into the effect of Fe on the structural properties. The values were calculated using eqs –.

$$\mathrm{d}\mathrm{i}\mathrm{s}\mathrm{l}\mathrm{o}\mathrm{c}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{d}\mathrm{e}\mathrm{n}\mathrm{s}\mathrm{i}\mathrm{t}\mathrm{y},\delta =\frac{1}{D^2}$$ 6

$$\mathrm{d}‐\mathrm{s}\mathrm{p}\mathrm{a}\mathrm{c}\mathrm{i}\mathrm{n}\mathrm{g},\mathrm{d}=\frac{\mathrm{n}\lambda }{2\sin \theta }$$ 7

2. Crystallographic Insights of ZNP and F-ZNP.

structural parameters	ZNP	F-ZNP
structure	hexagonal	hexagonal
space group	P63mc	P63mc
FWHM at (101)	0.300	0.408
lattice parameter	a = b = 3.247 c = 5.211	a = b = 3.254 c = 5.2138
dislocation density (nm–2)	9.81 × 10–4	1.81 × 10–3
c/a ratio	1.605	1.602
cell volume (Å3)	47.65	47.91
crystallite size (nm) (Scherrer method)	31.92727	23.49091
crystallite size (nm) (W–H method)	37.59	33.18
lattice strain (Nm2–)	0.000656	0.00123

Lattice parameter for the wurtzite hexagonal crystal structure

$$\frac{1}{d^2}=\frac{4}{3}\times \frac{h^2+\mathrm{h}\mathrm{k}+k^2}{a^2}+\frac{{\mathrm{l}}^2}{c^2}$$ 8

$$\mathrm{u}\mathrm{n}\mathrm{i}\mathrm{t}\mathrm{c}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{v}\mathrm{o}\mathrm{l}\mathrm{u}\mathrm{m}\mathrm{e},v=\frac{\sqrt{3}}{2}{\mathrm{a}}^2\mathrm{c}$$ 9

Here, a = b and c represent the lattice parameters; d denotes the interplanar distance corresponding to the Miller indices (hkl); and v refers to the unit cell volume.

4.4. Morphological and Elemental Analysis

The particle size and morphological characteristics of ZNP and F-ZNP were investigated through SEM analysis and are shown in Figure a,b. The SEM micrographs suggest that both samples exhibit a similar flake-like structure with a tendency to agglomerate. Previously, Aliannezhadi et al. synthesized ZnO nanoparticles using the Z. officinale extract in varying quantities. Notably, the sample prepared with 5 mL of the extract exhibited a morphology closely resembling that observed in this study. However, variations in the extract quantity resulted in morphological changes, suggesting that the morphology is dependent on the amount of the plant extract used. The particle size of biosynthesized ZNP and F-ZNP was measured using ImageJ software. It is shown in Figure c,d that the average particle size of ZNP and F-ZNP is determined to be 47 and 35 nm, respectively. Previous studies have suggested that the presence of terpenoids, flavonoids, and other phytochemicals may play a significant role in determining the small particle size of nanoparticles. It is evident that the incorporation of Fe dopants into ZnO led into a reduction in particle size, which, in turn, contributed to an increase in the surface area of the nanoparticles, a finding that was similarly reported by Algarni et al. As evident in XRD analysis, for F-ZNP, a noticeable shift of the (101) diffraction peak toward lower 2θ angles was observed, indicating lattice expansion. This shift suggests that Zn²⁺ ions in the crystal lattice were predominantly replaced by Fe²⁺ ions, which possess a comparatively larger ionic radius. Such substitution leads to lattice distortion due to ionic size mismatch, thereby inducing strain within the crystal structure. This strain contributes to the generation of structural defects, particularly oxygen vacancies, which play a crucial role in limiting crystal growth. As a result, the formation of smaller crystallites is facilitated, consistent with the observed reduction in the particle size. Our observation correlates with prior studies where incorporation of Fe dopants into the ZnO matrix resulted in smaller particle size. ,

5.

(a,b) SEM micrographs and (c,d) corresponding particles size distribution of biogenic ZNP and F- ZNP.

The elemental composition of ZNP and F-ZNP was determined by employing EDS analysis. The EDS spectrographs presented in Figure a,b show the presence of Zn and O elements in the nanoparticles, without any significant impurities detected. Additionally, the presence of Fe dopants in F-ZNP was confirmed, as illustrated through elemental color mapping in Figure c.

6.

(a,b) EDS spectrographs of biogenic ZNP and F-ZNP and (c) elemental color mapping of biogenic F-ZNP, ensuring the presence of Fe dopants.

4.5. Thermogravimetric Analysis

TG/DT analysis was conducted to examine the thermal stability of ZNP and F-ZNP. Hence, the samples underwent gradual heating within temperatures 30 °C to 800 °C, and the corresponding weight loss was recorded. The outcomes are presented in Figure a,b. For ZNP, a rapid weight loss was noticed at temperatures ranging from 40 °C to 100 °C, beyond which no notable loss occurred. However, F-ZNP exhibited rapid weight loss between 45 °C and 400 °C, after which the trend stabilized. According to previous studies, weight loss up to 300 °C is primarily attributed to the evaporation of water content. In addition, between 300 to 600 °C, the weight loss occurred due to the decomposition of organic components (Z. officinale) which was absorbed by nanoparticles. −

7.

TG/DT thermographs of biogenic (a) ZNP and (b) F-ZNP.

The DT curves exhibit corresponding endothermic and exothermic peaks, which indicate phase transitions and thermal decomposition processes, including the evaporation of water, degradation of precursors, and breakdown of organic elements. Our findings suggest that the total weight loss of ZNP and F-ZNP was 2.70% and 5.21%, respectively, indicating the remarkable thermal stability of both samples. The higher residual mass in both ZNP and F-ZNP can be attributed to their metallic content. Although both ZNP and F-ZNP were synthesized, maintaining similar parameters, their thermal behaviors exhibited noticeable differences. Apart from the residual moisture and organic constituents, the incorporation of Fe dopant plays a significant role in altering thermal characteristics. As evidenced by our particle characterization analyses, the presence of Fe induced several structural and morphological changes that are likely responsible for the observed variations in thermal stability and decomposition patterns. Moreover, an increase in temperature can facilitate the formation of new phases, potentially altering the thermal behavior of the material. Previous studies have indicated that introducing dopant ions into the ZnO lattice significantly modifies its thermal characteristics compared to pure ZnO. ,

4.6. Optical Properties

The optical absorption spectra of ZNP and F-ZNP were measured in the range 300–600 nm, and the results are presented in Figure a. Notably, two prominent peaks, one at 363 nm for ZNP and another at 369 nm for F-ZNP, are observed that suggest the characteristic absorbance peak of wurtzite hexagonal ZnO. A slight red-shift in the absorption peak of F-ZNP was observed, supporting the successful incorporation of the Fe dopant into the ZnO crystal lattice and resulting in lattice distortion.

8.

(a) Absorption edges of ZNP and F-ZNP with inset highlighting the absorption peak shift and (b) Tauc plots used for the estimation of band gap of both samples.

This result aligns with previous findings by Roguai et al., who similarly reported a red-shift upon the incorporation of Fe dopant into the ZnO structure.

The energy band gap values were calculated using the Tauc plot (eq ) based on the optical absorption spectrum shown in Figure b.

$${(\alpha \mathrm{h}\nu )}^2=\mathrm{A}(\mathrm{h}\nu -E_g)$$ 10

where α is the absorption coefficient, h is Planck’s constant, ν is the frequency of incident light, A is a material-dependent constant, and E _g represents the energy band gap. The calculated band gap of ZNP is 3.15 eV, while F-ZNP exhibits a slightly reduced band gap of 3.10 eV. Several factors may contribute to this variation, including the band structure, sp-d exchange interactions, differences in synthesis and processing methods, as well as the presence of lattice strain and defects. Similar trends have been reported in previous studies, where the incorporation of Fe dopant led to a reduction in the band gap. ,

4.7. Hydrodynamic Size and Zeta (ξ)-Potential Analysis

The hydrodynamic size distribution and colloidal stability of ZNP and F-ZNP were characterized by using the DLS technique. As shown in Figure a,b, the average hydrodynamic diameters of ZNP and F-ZNP were determined to be 433.3 and 422.2 nm, respectively, with corresponding polydispersity indices (PDI) of 0.3337 and 0.3227. These values indicate a moderately polydisperse distribution. In both cases, minor secondary peaks of lower intensity were observed, suggesting the presence of a small population of larger aggregates or agglomerates.

9.

(a,b) Hydrodynamic diameter and (c,d) corresponding ξ- potential of biogenic ZNP and F-ZNP, respectively.

It is important to note that the intensity (%) in DLS measurements does not directly correspond to the particle number or mass fraction. This is due to the Rayleigh scattering principle, where the scattering intensity scales with the third power of the particle radius (∝R ³). As a result, even a small number of larger particles can disproportionately influence the DLS signal. Therefore, the secondary peaks are likely attributable to a limited number of agglomerates and do not significantly skew the overall distribution. Overall, the data suggest that both ZNP and F-ZNP predominantly exhibit a monodisperse population with minor aggregation. A notable variation exists between the particle sizes obtained from DLS and those measured via SEM analysis. This discrepancy arises because DLS measures the hydrodynamic diameter, which encompasses not only the nanoparticle core but also the surrounding solvation shell, surface-bound phytochemicals, and capping agents. Besides, due to the nonspherical shape of ZNP and F-ZNP, the average particle size can be overestimated in DLS measurements. ξ -potential measurements, presented in Figure c,d, provide additional insight into colloidal stability. The ZNP sample exhibited a ξ- potential of +7.27 mV, while F-ZNP showed a significantly higher value of +17.44 mV. Although both values fall below the ± 30 mV threshold commonly associated with highly stable colloids, ξ- potentials exceeding ± 15 mV is indicative of moderate stability, supporting the improved dispersibility of F-ZNP. These findings are in agreement with existing literature, which has reported comparable trends.

4.8. Photocatalytic Activity

The photocatalytic efficiency of ZNP and F-ZNP was investigated by measuring the photodegradation of MB dye under sunlight irradiation at different time intervals (20, 40, 60, 80, 100, and 120 min). As shown in Figure a,b, over time, a gradual decrease in the absorbance spectra of both samples was observed, indicating the decomposition of MB in the presence of nanoparticles under sunlight irradiation. The photodegradation efficiency of ZNP and F-ZNP was meticulously calculated and is illustrated in Figure c. Initially, ZNP demonstrated a relatively slow degradation of 2.25% after 20 min. However, a significant increase was observed, reaching 31.82% at 40 min. With longer exposure to sunlight, the photodegradation efficiency continued to improve, attaining 56.24% after 120 min and establishing ZNP as a promising photocatalyst.

10.

(a,b) MB degradation of biogenic ZNP and F-ZNP under sunlight, (c) degradation vs time graph, (d) pseudo-first-order kinetic model for both samples.

Compared with ZNP, F-ZNP exhibited significantly enhanced photocatalytic performance. The initial degradation of MB dye was 17.34%, underscoring a higher efficiency than ZNP. This degradation rate continued to increase progressively over time, ultimately reaching a remarkable 91.39% after 120 min of sunlight exposure. The photodegradation rate of ZNP and F-ZNP can be measured by plotting the ratio of the MB dye concentration during the initial concentration to irradiation concentration [ ln C ₀/C] vs time (t), as depicted in Figure d. The calculated rate constant (k) was 0.0074 for ZNP and 0.02031 for F-ZNP. The higher value of k for F-ZNP indicates a significantly improved photocatalytic efficiency compared to pure ZnO. The half-time (t ^1/2) value which is needed to degrade the MB concentration to half of its initial value is estimated using the following Formula :

$$t^{1/2}=\frac{\ln 2}{K}$$ 11

The calculated half-time values were 93.67 and 34.128 min for ZNP and F-ZNP, respectively.

Our findings suggest that the Fe dopant significantly enhances the photodegradation efficiency of ZnO. Of several underlying factors, the tuning of the band gap is considered a key contributing factor, which plays a crucial role in this context. Generally, the incorporation of Fe into the ZnO structure results in a narrower band gap, enabling better absorption of visible light. Besides, the interaction of Fe with ZnO leads to the formation of a Schottky barrier, which lowers the Fermi level, facilitating more efficient charge separation. This mechanism effectively suppresses electron–hole recombination, thereby further enhancing the photocatalytic activity of F-ZNP. − To fully understand this phenomenon, the photocatalytic mechanism of ZnO nanoparticles must be clearly explained.

When light with an energy equal to or greater than the band gap of ZnO is illuminated, electrons (e^–) in the valence band excite and jump into the conduction band leaving an equal number of holes (h⁺) in the conduction band. This phenomenon is termed as charge separation, which leads to the formation of electron–hole pairs (e^–/h⁺). Now, electron–hole pairs (e^–/h⁺) can be further recombined or interact with the surfaces of the pollutants. Basically, the positive holes (h⁺) react with water and, through an oxidation reaction generate •OH^– radicals; concurrently, a reduction reaction occurs between free electrons and oxygen which eventually produce superoxide anion (O_2• ^–) radicals. Finally, these reactive oxygen species (ROS) decompose the pollutants through an oxidation reaction. ,, A schematic representation of the photocatalytic mechanism is illustrated in Figure .

11.

Schematic illustration of the photocatalytic degradation of MB by ZNP and F-ZNP under sunlight. The process involves ROS generation via photogenerated electrons and holes. Visual images confirm faster MB decolorization with F-ZNP, indicating improved photocatalytic activity.

A major limitation of ZnO is its wide band gap, which allows absorption in the UV region (below 380 nm), covering approximately 5% of the solar spectrum. Besides, ZnO exhibits a rapid recombination rate of photogenerated electron–hole pairs, which eventually hinders its photocatalysis activity. The addition of an Fe dopant effectively addresses these challenges by narrowing the band gap and forming the Schottky barrier, as previously discussed. Simply, the Schottky barrier serves as an electron sink, slowing down recombination and enhancing charge separation. ,, This reduction in band gap is further supported by UV analysis that shows a reduction of 0.05 eV after the addition of the Fe dopant into ZnO in our context.

Previously, Algarni et al. reported that Fe doping in the ZnO matrix influences charge separation and recombination rate by introducing defects such as oxygen vacancies, as validated through photoluminescence (PL) analysis. In our study, Fe doping led to an increase in the lattice strain, which is likely attributable to the formation of lattice defects. These defects are likely to influence charge carrier dynamics, potentially enhancing charge separation and suppressing the recombination rate in F-ZNP. Another critical factor influencing photocatalytic efficiency is the particle size and shape. Our findings show that while the surface morphology of ZNP and F-ZNP appears similar, F-ZNP possesses better shape homogeneity and relatively smaller particle size (Figure ). These lead to a larger surface area, enhancing the interaction between the nanoparticles and pollutants. On the other hand, DLS analysis suggests that F-ZNP exhibited a relatively smaller hydrodynamic particle size and a more stable zeta potential, indicating better colloidal stability and dispersion behavior in aqueous media. These phenomena perhaps contributed to the outstanding photodegradation efficiency of F-ZNP. , Vasiljevic et al. reported a study in which iron titanate (Fe₂TiO₅), a hybrid material derived from TiO₂ and Fe₂O₃, exhibited notable MB dye degradation under natural sunlight. The study basically highlighted the influence of several factorssuch as band gap, particle size, and electron–hole recombination rate as well as external parameters such as initial dye concentration and solution pH on photocatalytic activity. Several studies have reported the enhancement of photocatalytic performance through Fe doping into the ZnO system. Chai et al. synthesized ZnO and Fe-doped ZnO nanoparticles using the Hibiscus rosa-sinensis leaf extract and reported improved photocatalytic degradation of palm oil mill effluent with the Fe-doped sample. Algarni et al. investigated the effect of Fe doping concentration in ZnO nanoparticles, synthesized from the olive leaf extract, on the degradation of methyl orange, with 5% Fe-doped ZnO showing the highest activity. In another study, Radha et al. reported enhanced photodegradation of MB dye upon Fe incorporation into the ZnO lattice. Consistent with previous reports, our study demonstrates that F-ZNP exhibits enhanced photocatalytic activity compared with ZNP. Therefore, F-ZNP can be considered a promising candidate for sustainable photocatalytic applications, particularly in environmental remediation.

4.8.1. Photocatalytic Reusability

It is evident that F-ZNP exhibited outstanding photodegradation efficacy compared to ZNP. It certainly underscores the effectiveness of the Fe doping strategy in developing an outstanding photocatalyst. Now, in this part of our study, we further investigated the reusability and photostability of F-ZNP to assess its practical applicability. The photodegradation performance was examined over three consecutive cycles, using the same catalyst (F-ZNP). As shown in Figure a, during each cycle, the photodegradation rate was on the rise with respect to time of irradiation. Figure b shows that F-ZNP achieved an impressive degradation efficiency of 88.12% within 120 min during the first cycle. Although a gradual decrease was observed in subsequent cycles83.17% in the second and 77.92% in the thirdthe catalyst retained a significant proportion of its initial activity. This observation suggests that F-ZNP can be effectively recovered and reused without substantial change in efficacy. It should be noted that during each cycle, there occurs an accumulation of dye on the catalyst that perhaps reduces the active sites, eventually resulting in a decrease in photocatalytic activity.

12.

(a) Reusability performance of F-ZNP at time interval 40, 80, and 120 min during three consecutive cycles, (b) maximum efficiency after end (120 min) of each cycle.

The significance of the reusability of F-ZNP is far-reaching, in terms of economic and environmental aspects. One of the primary concerns in nanoparticles is their potential toxicity with uncontrolled release into the aquatic system. However, the reusability assessment of F-ZNP demonstrates the efficient recovery and sustained activity of F-ZNP over multiple cycles. This certainly reduces its risk of toxicity in the aquatic environment by minimizing secondary contamination and mitigating the risks of bioaccumulation and long-term ecological impact. On the other hand, from a circular economic view, the reusability of nanoparticles certainly offers substantial benefits by extending catalyst lifespan, lowering synthesis frequency and production costs, and reducing associated waste generation. Therefore, F-ZNP not only demonstrates exceptional photocatalytic performance but also holds significant promise for sustainable environmental and economic feasibility.

4.9. Antibacterial Activity

The antibacterial activity of ZNP and F-ZNP was evaluated against both Gram-positive and Gram-negative bacterial strains. The ZOI was measured at varying concentrations (100, 200, and 300 μg/mL) and compared with ciprofloxacin (5 μg/mL) as the positive control and methanol as the negative control. The results are summarized in Table .

3. Measured ZOI against Different Bacterial Strains.

sample name	bacteria name	zone of inhibition (mm)
		ciprofloxacin 5 μg/mL (positive control)	methanol (negative control)	100 μg/mL	200 μg/mL	300 μg/mL
ZNP	P. aeruginosa	31	0	11	13	16
ZNP	S. flexneri	27	0	10	12.5	15
ZNP	S. aureus	24	0	7	9	10.5
ZNP	S. marcescens	35	0	9.5	12	15
F-ZNP	P. aeruginosa	31	0	8	14.5	16
F-ZNP	S. flexneri	27	0	8.5	11	12.5
F-ZNP	S. aureus	24	0	0	8	10
F-ZNP	S. marcescens	35	0	7.5	10	13.5

Figure shows that both ZNP and F-ZNP exhibited notable antibacterial efficacy against all pathogens; however, Fe doping did not lead to a significant enhancement as their antibacterial performance remained comparable. For ZNP, the highest ZOI was observed against P. aeruginosa (16 mm) at 300 μg/mL, followed by S. flexneri (15 mm), S. marcescens (15 mm), and S. aureus (10.5 mm). In contrast, F-ZNP demonstrated variable efficacy, showing an equal ZOI against P. aeruginosa (16 mm at 300 μg/mL) but reduced inhibition against S. flexneri (12.5 mm) and S. marcescens (13.5 mm) compared to ZNP. However, the ZOI against S. aureus was relatively close to ZNP, measuring 10 mm at the highest concentration of 300 μg/mL. Additionally, the antibacterial activity was found to be concentration-dependent. Notably, both ZNP and F-ZNP exhibited greater efficiency against Gram-negative bacterial strains. Generally, the outer lipopolysaccharide membrane of Gram-negative bacteria is known to enhance resistance to antimicrobial agents. However, exceptions have been reported in the literature. For instance, Rabbi et al. previously observed that Gram-positive bacterial strains exhibited greater resistance to Ag nanoparticles.

13.

Antibacterial activity of ZNP and F-ZNP showing ZOI against different pathogens.

Although the exact mechanism of antibacterial activity is not yet fully understood, it generally follows several pathways, as illustrated in Figure . First, in direct interaction, nanoparticles disrupt the bacterial cell wall, causing leakage of intracellular components. Second, in aqueous solutions, nanoparticles release ions that interact with the bacterial membrane, facilitating nanoparticle entry and further cellular damage. For instance, Zn²⁺ ions inhibit glycolytic enzymes by oxidizing thiol groups due to their strong affinity for sulfur. Lastly, the generation of ROS via electron–hole pair formation induces oxidative stress, damaging DNA and proteins, ultimately leading to bacterial cell death. ,

14.

Plausible antibacterial mechanism of biogenic nanoparticles. Three pathways are depicted such as direct interaction, ROS generation, and metal ion release.

4.10. Molecular Docking with Bacterial Proteins

The present study sought to reveal the antimicrobial potential of employing molecular docking analysis. The proteins gyrase B, GyrB (4URO), and 3-oxoacyl-(acyl carrier protein) synthase III, FabH (2X3E), crucial for bacterial replication and fatty acid synthesis, respectively, have been considered as ideal targets for novel antibiotic development. , The 3D structures of target proteins (4URO and 2X3E), along with their active sites, and chemical structures of selected phytochemicals (ligands) are indicated in Figure .

15.

Chemical structures of the ten selective phytochemicals and 3D structure of target proteins and their active sites.

Overall, 80 compounds were identified in GC–MS outputs, of which one-third of the compounds interacted with Gram-positive (S. aureus) and Gram-negative (P. aeruginosa) bacterial proteins (GyrB and FabH, respectively). However, from these interacted molecules, ten (10) compounds (Figure ) have drug-likeness properties and pass in silico toxicity assessment, also demonstrating substantial binding affinity with the target proteins that are depicted in Figure . The ten compounds were: 1. 2-butanone, 4-(4-hydroxy-3-methoxyphenyl), 2. phenol, 4-ethyl-2-methoxy-, 3. 2,6,10-dodecatrien-1-ol, 3,7,11-trimethyl-, 4. 5,9-undecadien-2-one, 6,10-dimethyl-, (z)-, z, 5. e-7,11-hexadecadien-1-yl acetate, 6. 2,6,6-trimethyl-bicyclo[3.1.1]­hept-3-ylamine, 7. ethyl homovanillate, 8. homovanillyl alcohol, 9. ethanone, 2-hydroxy-1,2-bis­(4-methoxyphenyl)-, and 10. phytol, which had a similar or lower binding affinity (kcal/mol) than the standard drug ciprofloxacin. The binding energy range of selected phytochemicals was −7.2 to −5.0 kcal/mol, whereas ciprofloxacin was −7.9 to −7.1 kcal/mol (Table ).

4. Average Binding Affinity and Non-bonding Interaction of Selected Bioactive Compounds of the Z. officinale Aqueous Peel Extract and Ciprofloxacin Ligands with Target Proteins (FabH and GyrB).

name	binding affinity (kcal/mol)	residues in contact	interaction type	distance (Å)
2X3E_2-butanone, 4-(4-hydroxy-3-methoxyphenyl)- (compound 1)	–6.0	GLY82	conventional hydrogen	2.06117
		ASP106	carbon hydrogen	3.50789
		CYS85	pi-sulfur
4URO_2-butanone, 4-(4-hydroxy-3-methoxyphenyl) -(compound 1)	–6.1	VAL88	conventional hydrogen	2.19043
		ASP89	conventional hydrogen	2.45169
		ARG144	conventional hydrogen	2.48809
		ASP89	pi-anion	4.64068
		TYR15	conventional hydrogen	2.60735
		PRO16	pi-alkyl	5.4533
2X3E_phenol, 4-ethyl-2-methoxy- (compound 2)	–5.4	ASP106	conventional hydrogen	2.26834
4URO_phenol, 4-ethyl-2-methoxy- (compound 2)	–5.1	GLU201	conventional hydrogen	3.54593
		NOV2000	alkyl	3.92708
2X3E_2,6,10-dodecatrien-1-ol,3,7,11-trimethyl-(compound 3)	–5.6	PRO87	conventional hydrogen	2.54177
		GLY82	carbon hydrogen	3.54751
4URO_2,6,10-dodecatrien-1-ol, 3,7,11-trimethyl-(compound 3)	–5.6	GLU92	conventional hydrogen	2.96213
		NOV2000	alkyl	4.21244
		PHE204	pi-alkyl	4.28441
2X3E_5,9-undecadien-2-one, 6,10-dimethyl-, (Z)- (compound 4)	–5.2	GLY82	conventional hydrogen	2.08908
4URO_5,9-undecadien-2-one, 6,10-dimethyl-, (Z)-(compound 4)	–5.8	TYR229	conventional hydrogen	2.22545
		ARG200	carbon hydrogen	3.54273
		NOV2000	alkyl	4.32022
2X3E_ Z, E-7,11-hexadecadien-1-yl acetate (compound 5)	–5.2	ARG201	conventional hydrogen	2.51185
		CYS85	alkyl	4.63999
		ALA195	alkyl	5.00413
4URO_ Z, E-7,11-hexadecadien-1-yl acetate (compound 5)	–5.0	NOV2000	conventional hydrogen	2.39156
		TYR229	conventional hydrogen	2.74459
		ARG200	alkyl	4.48923
		PHE204	pi-alkyl	4.86595
2X3E_2,6,6-trimethyl-bicyclo [3.1.1] hept-3-ylamine (compound 6)	–5.7	PHE84	conventional hydrogen	2.9718
		CYS85	conventional hydrogen	2.56579
4URO_2,6,6-trimethyl-bicyclo [3.1.1] hept-3-ylamine (compound 6)	–5.4	GLU201	conventional hydrogen	1.91551
		GLN197	conventional hydrogen	2.07167
		NOV2000	alkyl	4.25337
2X3E_ ethyl homovanillate (compound 7)	–5.7	ARG200	conventional hydrogen	2.41764
		PHE204	pi–pi stacked	4.20355
		TYR229	pi–pi t-shaped	5.89761
		NOV2000	pi-alkyl	5.44365
4URO_ ethyl homovanillate (compound 7)	–5.4	LYS93	conventional hydrogen	2.94348
		ARG200	conventional hydrogen	2.21359
		GLN197	conventional hydrogen	2.44664
		PHE204	conventional hydrogen	4.88481
2X3E_homovanillyl alcohol (compound 8)	–5.7	PHE84	conventional hydrogen	2.47367
		ASP106	conventional hydrogen	2.1879
4URO_ homovanillyl alcohol (compound 8)	–5.1	ARG200	conventional hydrogen	2.31043
		GLN91	carbon hydrogen	3.51553
		NOV2000	pi-alkyl	4.24725
2X3E_ ethanone, 2-hydroxy-1,2-bis(4-methoxyphenyl) -(compound 9)	–7.2	CYS85	conventional hydrogen	3.09289
		ARG201	conventional hydrogen	2.05696
		VAL196	carbon hydrogen	3.59814
		ALA195	pi-alkyl	4.21319
4URO_ ethanone, 2-hydroxy-1,2-bis(4-methoxyphenyl) -(compound 9)	–6.9	ARG200	conventional hydrogen	2.42975
		PHE204	pi–pi stacked	3.75623
		VAL101	alkyl	4.22022
		NOV2000	pi-alkyl	5.39982
2X3E_phytol (compound 10)	–5.9	CYS85	alkyl	5.45589
4URO_phytol (compound 10)	–5.8	GLU201	carbon hydrogen	3.42487
		NOV2000	alkyl	4.4858
		ARG200	alkyl	5.17088
		PHE204	pi-alkyl	4.11348
2X3E_1-ciprofloxacin	–7.9	GLY82	conventional hydrogen	2.09787
		CYS85	conventional hydrogen	2.20319
		ASP106	conventional hydrogen	2.1134
		SER108	carbon hydrogen	3.49095
		SER80	carbon hydrogen	3.24732
4URO_ ciprofloxacin	–7.1	ARG200	conventional hydrogen	2.91325
		GLN91	carbon hydrogen	3.31379
		NOV2000	pi-sigma	3.64986
		VAL101	alkyl	4.95315
		ILE102	alkyl	5.3058

Table recapitulates the average binding energy, the amino acid residues in contact, interaction types, and bond distances for the ten most potential molecules interacting with GyrB and FabH. Notably, most of the compounds interacted with active site residues of target proteins with favorable binding energy. For instances, 2-butanone, 4-(4-hydroxy-3-methoxyphenyl)- was interacted with some active site residues of FabH, including GLY82, ASP106, CYS85, and of GyrB, including VAL88, ASP89, ARG144, TYR15, and PRO16 with the binding energy of −6.0 to −6.1 kcal/mol, respectively. In addition, among the ten molecules, ethanone, 2-hydroxy-1,2-bis­(4-methoxyphenyl)- strongly interacted with FabH and GyrB with the binding energy of −7.2 to −6.9 kcal/mol, respectively. The binding affinity of the other phytochemicals with GyrB and FabH is shown in Table . Figure exhibits both the three-dimensional and two-dimensional binding interaction between phytochemicals and the bacterial (4URO and 2X3E) proteins. The binding pattern embracing various types of bonds include conventional hydrogen, carbon hydrogen, hydrophobic (alkyl, π-alkyl, π-sigma, and π-sulfur), and/or electrostatic (π-anion, π-π-stacked, and π-π-T-shaped) that facilitate the stability and specificity of the interactions between biomarkers and phytochemicals. Our results were consistent with previous findings of Kiranmayee et al. and Alarfaj et al. According to their study, active molecules of the plant extract exhibited potent antibacterial activity and possessed a drug-likeness property that closely aligned with our study, suggesting a promising avenue for novel antibiotic development.

16.

Three-dimensional and two-dimensional interaction profiles depicting the binding of selected bioactive compounds to the bacterial proteins 4URO and 2X3E. The phytochemicals include: compound 1. 2-butanone, 4-(4-hydroxy-3-methoxyphenyl), compound 2. phenol, 4-ethyl-2-methoxy-, compound 3. 2,6,10-dodecatrien-1-ol, 3,7,11-trimethyl-, compound 4. 5,9-undecadien-2-one, 6,10-dimethyl-, (z)-, z, compound 5. e-7,11-hexadecadien-1-yl acetate, compound 6. 2,6,6-trimethyl-bicyclo [3.1.1] hept-3-ylamine, compound 7. ethyl homovanillate, compound 8. homovanillyl alcohol, compound 9. ethenone, 2-hydroxy-1,2-bis­(4-methoxyphenyl)-, and compound 10. phytol.

4.11. In Silico Physicochemical and Pharmacokinetics Prediction

In silico physicochemical and pharmacokinetics (ADME) analysis of selected phytochemicals is a prediction to check whether bioactive molecules can be suitable for oral medication and subjected to further clinical trials. Indeed, this prediction is based on Lipinski’s rule of five (RO5) and favorable ADME (absorption, distribution, metabolism, and excretion) properties. − The current study assessed in silico drug-likeness and ADMET properties of the GC–MS profiling chemicals; the compounds (ten phytochemicals) that meet the Lipinski’s rule of five and have favorable pharmacokinetics and toxicity features were further chosen for molecular docking. Table demonstrates the Lipinski rule of five, solubility, topological polar surface area (TPSA), and bioavailability of the selected molecules, that unveil considerable binding affinity with the bacterial proteins. From the tabulated data, it has been shown that almost all of the phytochemicals follow Lipinski’s RO5 and have excellent bioavailability scores, while two compounds showed one RO5 violation that is considerable. Regarding pharmacokinetics, all of the selected compounds have high gastrointestinal (GI) absorption and blood–brain barrier (BBB) penetration as their TPSA was <60 Ų, whereas the standard drug ciprofloxacin was BBB negative (Table ). The compounds with high GI absorption and BBB penetration capacity could be promising candidates for treating neurological disorders. , In case of solubility, most of the compounds had good solubility except phytol as their log S range was between −1 and −5 (Table ). Surprisingly, all of the compounds demonstrated high skin permeability (log Kp, −8.0 to −1.0), Caco2 positive, and a P-glycoprotein (Pgp) inhibitory effect, whereas the ciprofloxacin violated these parameters. Hence, the promising drug candidates may unveil good absorption, oral bioavailability, and effectiveness. , In metabolism, 50% (5/10) of the molecules unveiled a CYP1A2 inhibitory effect, which can significantly influence the metabolism of caffeine and antipsychotic medications, resulting in elevated plasma levels and potential drug interactions. ,

5. Prediction of Physicochemical and Drug-Likeliness Properties of the Selected Ligands from GC–MS-Identified Bioactive Compounds of the Z. officinale Aqueous Peel Extract.

compound	M.W. (g/mol)	no. of RB	no. of HBAs	no. of HBDs	lipophilicity (Clog P)	solubility (Log S)	TPSA	bioavailability	Lipinski rules of violations	Lipinski
2-butanone, 4-(4-hydroxy-3-methoxyphenyl)	194.23	4	3	1	1.79	–3.10	46.53	0.55	0	yes
phenol, 4-ethyl-2-methoxy-	152.19	2	2	1	2.02	–2.73	29.46	0.55	0	yes
2,6,10-dodecatrien-1-ol, 3,7,11-trimethyl-/trans,trans-farnesol	222.37	7	1	1	4.32	–3.15	20.23	0.55	0	yes
5,9-undecadien-2-one, 6,10-dimethyl-, (Z)-	194.31	6	1	0	3.60	–3.18	17.07	0.55	0	yes
Z, E-7,11-hexadecadien-1-yl acetate	266.42	14	2	0	5.09	–4.59	26.30	0.55	1	yes
2,6,6-trimethyl-bicyclo[3.1.1]hept-3-ylamine	153.26	0	1	1	2.15	–1.67	26.02	0.55	0	yes
ethyl homovanillate	210.23	5	4	1	1.60	–2.84	55.76	0.55	0	yes
homovanillyl alcohol	168.19	3	3	2	1.14	–2.18	49.69	0.55	0	yes
ethanone, 2-hydroxy-1,2-bis(4-methoxyphenyl)-	272.30	5	4	1	2.29	–4.59	55.76	0.55	0	yes
Phytol	296.53	13	1	1	6.25	–5.51	20.23	0.55	1	yes
Ciprofloxacin	331.34	3	5	2	1.10	–3.50	74.57	0.55	0	yes

6. Predicted Pharmacokinetics Properties of Bioactive Compounds from the Z. officinale Peel Aqueous Extract Using SwissADME and admetSAR Online Server.

compound	GI absorption	BBB	Caco-2	Pgp substrate	Log Kp (cm/s)	CYP1A2 inhibitor	CYP2C19 inhibitor	CYP2C9 inhibitor	CYP2D6 inhibitor	CYP3A4 inhibitor
2-butanone, 4-(4-hydroxy-3-methoxyphenyl)-	high	yes	yes	no	–6.70	yes	no	no	no	no
phenol, 4-ethyl-2-methoxy-	high	yes	yes	no	–5.81	yes	no	no	no	no
2,6,10-dodecatrien-1-ol, 3,7,11-trimethyl-	high	yes	yes	no	–3.81	yes	no	yes	no	no
5,9-undecadien-2-one, 6,10-dimethyl-, (Z)-	high	yes	yes	no	–4.86	no	no	no	no	no
Z, E-7,11-hexadecadien-1-yl acetate	high	yes	yes	no	–3.66	yes	no	yes	no	no
2,6,6-trimethyl-bicyclo[3.1.1]hept-3-ylamine	high	yes	yes	no	–5.60	no	no	no	no	no
ethyl homovanillate	high	yes	yes	no	–6.86	no	no	no	no	no
homovanillyl alcohol	high	yes	yes	no	–6.99	no	no	no	no	no
ethanone, 2-hydroxy-1,2-bis(4-methoxyphenyl)-	high	yes	yes	no	–6.49	yes	yes	no	no	yes
Phytol	low	yes	yes	no	–2.29	no	no	yes	no	no
Ciprofloxacin	high	no	no	yes	–9.09	no	no	no	no	no

The notable cytochrome P450 polymorphic enzymes that are involved in various drug metabolisms include CYP2C19, CYP2C9, and CYP2D6. According to the tabulated data, only one compound (Ethanone, 2-hydroxy-1,2-bis­(4-methoxyphenyl)−) was a CYP2C19 inhibitor, and three compounds (2,6,10-dodecatrien-1-ol, 3,7,11-trimethyl, Z, E-7,11-hexadecadien-1-yl acetate, and phytol) were CYP2C9 inhibitors. Indeed, if these enzymes are inhibited, they may affect drug metabolism and cause undesirable side effects. Additionally, CYP3A4, the key CYP enzyme, is probably responsible for the metabolism of more than half of all pharmaceutical products. Of the molecules that were identified, only one (ethanone, 2-hydroxy-1,2-bis­(4-methoxyphenyl)–) exhibited inhibitory activity, indicating that the remaining phytochemicals may have improved metabolism, making them strong drug candidates.

4.12. Toxicity Prediction

One of the most imperative criteria in modern drug design is the ability to forecast the toxicity of lead compounds because a strong drug candidate should have sufficient efficacy and experience therapeutic success following oral delivery. In Table , hERG inhibition, organ toxicity, fish and protozoan toxicity, biodegradation, Ames mutagenicity, LD₅₀, and toxicity class of the selected phytochemicals and one standard drug (ciprofloxacin) were anticipated.

7. Toxicity Analysis of the Phytochemicals of the Z. officinale Aqueous Peel Extract Using PROTOX-II and admetSAR Online Server .

compound	hERG inhibition	hepatotoxicity	nephrotoxicity	respiratory toxicity	cardiotoxicity	immunotoxicity	fish toxicity	Tetrahymena Pyriformis toxicity	biodegradation	ames mutagenesis	LD50 (mg/kg)	toxicity class
2-butanone, 4-(4-hydroxy-3-methoxyphenyl)-	–0.89	–0.51	–0.56	–0.92	–0.83	0.90	↓0.84	↓0.82	+0.52	–0.92	2580	5
phenol, 4-ethyl-2-methoxy-	–0.91	–0.71	–0.59	–0.93	–0.80	–0.82	↓0.55	↓0.85	+0.52	–0.95	1930	4
2,6,10-dodecatrien-1-ol, 3,7,11-trimethyl-	–0.83	–0.79	–0.74	–0.81	–0.84	–0.99	↓0.92	↓0.98	+0.89	–0.91	5000	5
5,9-undecadien-2-one, 6,10-dimethyl-, (Z)-	–0.83	–0.72	–0.83	–0.98	–0.90	–0.99	↓0.92	↓0.99	+0.91	–0.95	5000	5
Z, E-7,11-hexadecadien-1-yl acetate	–0.93	–0.79	–0.58	–0.99	–0.82	–0.93	↓0.95	↓0.99	+0.90	–0.90	8400	6
2,6,6-trimethyl-bicyclo[3.1.1]hept-3-ylamine	–0.83	–0.90	–0.92	+0.70	–0.80	–0.97	↓0.95	↓0.85	–0.79	–0.86	530	4
ethyl homovanillate	–0.80	–0.74	+0.62	–0.90	–0.79	–0.88	↓0.69	↓0.93	+0.80	–0.93	1400	4
homovanillyl alcohol	–0.75	–0.86	–0.52	–0.66	–0.71	–0.64	↑0.85	↓0.89	+0.83	–0.90	1930	4
ethanone, 2-hydroxy-1,2-bis(4-methoxyphenyl)-	–0.90	–0.54	+0.57	–0.64	–0 0.73	–0.99	↓0.88	↓0.99	–0.61	–0.90	3000	5
phytol	–0.84	–0.79	–0.74	–0.81	–0.84	–0.99	↓0.92	↓0.97	+0.89	–0.91	5000	5
ciprofloxacin	–0.69	–0.65	+0.95	+0.91	–0.82	–0.91	↓0.99	↓0.82	–1.00	+0.88	2000	4

^ahERG = human ether a-go-go-related gene inhibition; the value indicates probability score and the sign (± and ↑/↓) indicates positive/negative and high/low, respectively; LD 50 = LD50 (mg·kg–1).

From the tabulated data, none of the compounds were used in hERG inhibition, hepatotoxicity, cardiotoxicity, and immunotoxicity-positive, making them excellent candidates for further drug development due to their reduced cardiac arrhythmias, liver, heart, and immune safety. Additionally, most of the phytochemicals exhibited high fish half-maximal toxicity (FHMT) and Tetrahymena pyriformis toxicity (TPT) values, demonstrating that relatively high concentrations are required to elicit toxic effects in aquatic and protozoan models. This indicates a lower environmental toxicity burden, augmenting their aptness for therapeutic or environmental applications. An exception is homovanillyl alcohol, which showed comparatively lower FHMT however, higher TPT, suggesting further evaluation of its environmental safety.

Besides, the Ames test corroborated that no identified molecules were projected to have mutagenic potential on human organisms except standard ciprofloxacin. According to ProTox II toxicity estimation, the selected ten molecules LD50 values were in the range of 530–8400 mg/kg, and approximately 50% of molecules (5/10) may have mild toxicity (class: 5), four molecules including ciprofloxacin may have moderate toxicity but not be acutely lethal at low doses (class: 4), and one molecule is nontoxic (class: 6). Therefore, from the above drug-likeness properties, pharmacokinetic profiles, and toxicity assessments, the selected ten molecules exhibit strong potential as promising drug candidates for further in vitro and in vivo trials as well as for nanoparticle-based drug delivery and environmental applications.

4.13. Hemolysis Assay

The hemocompatibility of biogenic ZNP and F-ZNP was evaluated at various concentrations (25–400 μg/mL) using hRBCs, and the findings are presented in Figure . As shown in the figure, both ZNP and F-ZNP demonstrated concentration-dependent hemolytic responses. However, both samples exhibited lower hemolytic activity even at higher concentrations (400 μg/mL). The maximum hemolysis was observed approximately at 1.54 ± 0.05% and 1.39 ± 0.02% for ZNP and F-ZNP, respectively.

17.

Percentage (%) of hemolysis of ZNP and F-ZNP at various concentrations (25–400 μg/mL). Data are presented in mean ± standard error, where for ZNP, n = 3 and for F-ZNP, n = 2.

According to the American Society for Testing and Materials (ASTM) guidelines, material exhibiting <2% hemolysis is classified as nonhemolytic, 2–5% as slightly hemolytic, and >5% as hemolytic. The findings indicate that both ZNP and F-ZNP are nonhemolytic, with F-ZNP showing slightly improved hemocompatibility. This finding aligns with earlier reports on the biocompatibility of ZnO nanoparticles. For example, Faisal et al. reported a hemolysis level of 3.58 ± 0.11% at 400 μg/mL for biogenic ZnO, which falls within the slightly hemolytic range according to ASTM guidelines.

In comparison, the present study recorded lower hemolysis values for both ZNP (1.54 ± 0.05%) and F-ZNP (1.39 ± 0.02%), indicating better compatibility with hRBCs. As discussed by Shoudho et al., the presence of bioactive compounds from green elements which is Z. officinale in our context can influence the biocompatibility, potentially forming an organic layer around the biogenic nanoparticles that reduces direct interaction with erythrocyte membranes. These findings support the safe biomedical applicability of biogenic ZNP and F-ZNP in nanotherapeutic applications.

5. Conclusion

Herein, we performed the successful biogenic synthesis of ZNP and F-ZNP using Z. officinale peel extract. The phytochemical profiling of Z. officinale peel extract confirmed the presence of different bioactive compounds that served as reducing and capping agents during the synthesis. XRD analysis ensured the hexagonal wurtzite structure of ZNP and F-ZNP without any presence of a secondary phase. The peak shift and the alteration of lattice strain further suggest the successful incorporation of Fe dopants into the ZnO matrix. The crystallite size was calculated to be 31.92 nm for ZNP and 23.49 nm for Fe-ZNP. SEM micrographs revealed a flake-like morphology for both ZNP and F-ZNP. The incorporation of Fe dopants led to a noticeable reduction in the particle size, further influencing the structural attributes of the nanoparticles. The UV analysis showed a blue-shift in the absorption edge upon Fe incorporation, with a corresponding decrease in the band gap from 3.15 eV (ZNP) to 3.10 eV (F-ZNP). Thermal analysis demonstrated excellent stability, with minimal weight loss up to 800 °C. DLS analyses confirmed improved colloidal stability and dispersibility of F-ZNP compared to ZNP, highlighting the effect of Fe doping. F-ZNP exhibited elevated photodegradation, occurring 91.39% decomposition of MB dye within 120 min, significantly outperforming pure ZnO (56.24%). The kinetic model further validated the improved degradation efficiency, with F-ZNP displaying a half-life (34.13 min) much lower than that of ZNP (93.67 min). Moreover, F-ZNP exhibited outstanding photocatalytic reusability performance with minimal loss in efficacy after three cycles. Additionally, both ZNP and F-ZNP exhibited substantial antibacterial activity, with the highest ZOI (16 mm) against P. aeruginosa and considerable ZOI (10 mm) against S. aureus. This antibacterial result was further elucidated through molecular docking of bacterial proteins (GyrB, FabH) with GC–MS identified phytochemicals as the bioactive compounds were capped with synthesized nanoparticles and elicited antimicrobial activity. The 10 selected molecules revealed noteworthy binding affinity (−7.2 to −5.0 kcal/mol) with target proteins and also demonstrated favorable drug-likeness, pharmacokinetics, and toxicity features, suggesting potent biomedical and environmental applications. Moreover, both ZNP and F-ZNP demonstrated excellent biocompatibility in hemolysis assays. However, further studies are necessary to comprehensively assess their toxicity profiles in both human and aquatic systems. Above all, this study highlights the immense prospects of F-ZNP as a highly efficient photocatalytic and antibacterial agent. Furthermore, it underscores the significance of the Z. officinale peel extract as a sustainable and eco-friendly green element for nanoparticle synthesis, offering a promising approach for advanced functional materials in environmental and biomedical applications.

The data supporting the findings of this study are presented within the article, and all characterization data are provided in the Supporting Information file.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c09902.

• Raw data associated with material characterizations (XLSX)

A.M.: conceptualization, methodology, investigation, formal analysis, software, writingoriginal draft, data curation, and writingreview and editing. H.A.: conceptualization, methodology, investigation, data curation, software, writingoriginal draft, supervision, project administration, and visualization. E.I.R.: formal analysis, investigation, data curation, and writingreview and editing. A.I.: investigation, data curation, and writingreview and editing. E.P.L.: investigation and writingreview and editing. M.A.S.M.: resources and writingreview and editing. S.I.: visualization, validation, and writingreview and editing.

This work was supported by funding from the Bangladesh Council of Scientific and Industrial Research (BCSIR) through an R&D project (ref no. 39.02.0000.011.14.169.2023/877; 17.09.2023) and from the Ministry of Science and Technology, Government of the People’s Republic of Bangladesh (Project ID: R&D-2420198; ref. No. 39.00.0000.012.02.010.24–42; Date: March 04, 2025).

The authors declare no competing financial interest.