Protective mechanisms of Azadirachta indica against titanium dioxide nanoparticle-induced reproductive toxicity through antioxidant and endocrine axis regulation

Received: 07-Aug-2026, Manuscript No. mp-26-193447; Accepted: 13-Oct-2026, Pre QC No. mp-26-193447 (PQ); Editor assigned: 10-Aug-2026, Pre QC No. mp-26-193447 (PQ); Reviewed: 21-Sep-2026, QC No. mp-26-193447; Revised: 09-Oct-2026, Manuscript No. mp-26-193447 (R); Published: 20-Oct-2026 , DOI: 10.5281/zenodo.23729718

Introduction

Nanotechnology plays a crucial role in contemporary science and significantly contributes to the synthesis and enhancement of nanoparticles (Yang, et al. 2021). Nanoparticles are present in our environment in both natural and synthetic forms. Naturally occurring nanoparticles emerge from sources like volcanic eruptions, Windblown particles, and sea spray. These fine particles are often dispersed through natural processes like wind and ocean currents. On the other hand, many nanoparticles are engineered for specific purposes. These artificial particles are developed in laboratories and industries for use in technology, medicine, and manufacturing (Chintagunta, et al. 2021). The use of nanoparticles in consumer products from textiles to cosmetics to paints offers convenience and efficiency, but it also introduces significant environmental and health concerns (Hong, et al. 2018).

Among the many nanoparticles utilized today, titanium dioxide stands out. It is a transition metal with a color ranging from white to silver and holds great importance due to its broad applications in products like tiles, filtration systems for air and water and self-cleaning windows (Ziental, et al. 2020, Bakare, et al. 2016). Materials integrated into common accessories can enter the human body with regular use. Once inside, they are quickly distributed across many organs and tissues, traveling through the circulatory system. This fast dispersion enables the substances to reach cellular targets efficiently. The effects on cells can be profound, as these materials may disrupt or modify normal cell activity. Li, et al. 2016 examined this process, noting the swift distribution and cellular impact. Their findings highlight potential health risks associated with these materials.

Reproduction ensures continuity of life by transmitting genetic traits from parents to offspring. Male reproductive organs have specific functions: sperm production, maintenance, and transport. Titanium dioxide, prevalent in many modern products, may enter the body and accumulate in these organs, affecting critical cells like Sertoli cells and those responsible for sperm development. This leads to a decline in sperm production (Suthar, et al. 2023).

The modern resurgence of herbal medicine highlights its enduring value. As global populations seek integrative healthcare options, plant-based treatments offer promising avenues for developing new, effective, and safer pharmacological agents (Pathan, et al. 2025).

Neem, a revered medicinal plant in traditional systems like Ayurveda, holds potent antibacterial and antifungal properties, making it a natural remedy for treating skin diseases such as eczema, psoriasis, acne, and infections, with its leaves, oil, and extracts cleansing the skin and preventing microbial growth without harsh chemical side effects (Jamshidi, et al. 2025). Neem’s antiviral and antiparasitic attributes contribute significantly in treating ailments like malaria, chickenpox, and intestinal worms, as compounds like azadirachtin and nimbin found in neem interfere with pathogen development and strengthen the body’s resistance to infections. (Khan, et al. 2024) Its strong anti-inflammatory and immune-boosting abilities support the treatment of chronic illnesses like arthritis and joint pain, where neem helps reduce swelling and pain, enhances blood circulation, and offers a natural alternative to synthetic medications for managing autoimmune and inflammatory conditions (Aini and Andri, 2025). Secondary metabolites in neem plants, including alkaloids, flavonoids, saponins, tannins and total phenolic compounds, play a significant role against diseases and environmental stress. Alkaloids exhibit antimicrobial activity that helps suppress the growth of harmful pathogens. Flavonoids and total phenols function as powerful antioxidants, reducing oxidative damage and enhancing defense responses. Saponins and tannins contribute to disease resistance by disrupting pathogen development and strengthening natural protective barriers. Collectively, these bioactive compounds make neem a valuable natural source of remedial agents for disease management while improving plant resilience under adverse conditions (Mshelmbula, et al. 2025). Lastly, neem supports blood purification and liver detoxification, which indirectly benefits conditions like diabetes and cardiovascular disease by regulating blood sugar levels, improving metabolic activity, and aiding in the removal of toxins, thus enhancing overall systemic health naturally (Pathak and Avijit, 2024).

Materials and Methods

Ethics and patient admission information

The all procedure of animal study was carried out in compliance with the Ethics Review Ethics approval number, Ref. No. GCUF/ ERC/3122, dated 14-05-2024.

Patient approval has not been obtained as it is performed on animals.

Plant sample collection and extraction methodology

Leaves of Azadirachta indica (Neem) were sourced and thoroughly washed to eliminate contaminants. The leaves were sun-dried and ground into a fine powder using a Sigma grinder. For extraction, 500 g of powdered leaves were soaked in 1.5 L methanol at ambient temperature for seven days with daily stirring. The methanolic extract was filtered by muslin cloth and concentrated via rotary evaporation at 40°C. The residue was reconstituted in 0.9% NaCl, ultra-sonicated for 20 minutes at 40°C, and vortexed for 1 minute before oral administration (Alzohairy, et al. 2016).

Determination of LC₅₀ of plant extract

Male Sprague Dawley rats were exposed to A. indica at doses of ‘1000-5000 mg/kg’ BW in six replicates, with mortality recorded every 12 hours over ‘96 hours’. The LC₅₀ was determined following standard (Nagano, et al. 2021) protocols, without feeding the rats during the test.

Quantitative analysis of plant extracts

Plants secondary metabolites contents were quantified by following the method of (Saleem, et al. 2021).

Alkaloids: 1 g of each plant sample along with 200 ml CH₃COOH and C₂H₅OH (20%) were mixed in a breaker and filtered after 4 hours. It was then heated in water bath (Memmert) at 90°C until solution remained 1/4th of original solution. Precipitates were filtered with NH₄OH, oven dried and weighted by using weight balance (Shimedzu).

Following formula was used to calculate alkaloids: Alkaloids%=Weight of precipitate/Weight of original sample × 100

Flavonoids: 5 g plant sample (A. indica) was extracted with 80% methanol and then filtered with Whatman filter paper. Filtrate was heated into water bath (Memmert) for 30 min, evaporated in incubator (JSR-100T) at 30°C and weighed in mg/100 g.

Saponins: Plant sample (10 g) and 200 ml ethanol (20%) were taken in a Flask (Pyrex), mixed and placed on shaker (KS-130B, Basic) for 30min. After that time it was then heated into water bath (Memmert) at 50°C for 3 hours. Extraction was completed with 200 ml ethanol (20%) after filtration. The mixture was again evaporated into water bath (90°C) until 30 ml volume was attained. 20 ml diethyl ether and 30 ml filtrate were shaken together vigorously and only bottom ether layer was used for n-butanol extraction. 50 ml n-butanol was added in funnel along with bottom layer of ether and again shaked very well on shaker. Further, upper layer of n-butanol extract in funnel was washed two times with 5% Sodium Chloride (NaCl) buffer (10 ml). The remaining mixture was evaporated to dry into water bath and oven dried it at 40°C. Percentage of saponins was evaluated using the formula;

Saponins%=Weight of residues/Weight of plant material used × 100

Tannins: Plant sample (1 g) along with double distilled water (10 ml) were kept in 250 ml flask and placed on shaker (KS-130B, basic) at 1000 rpm for one hour. Afterwards, filtration was completed in 50 ml flask to make 50 ml volume. 5 ml of filtrate, 2 ml of diluted (10 fold) iron chloride (0.1 M) in hydrochloric acid (0.1M) and K4[Fe(CN)6]•3H2O (0.008M) were mixed and then placed at 37°C. Absorbance were noted at 395 nm within 10 minutes in mg/100 ml.

Total phenols: Plant sample (2 g) and 50 ml ether was taken in flask and boiled for 15 minutes. 5 ml of boiling solution and 10 ml double distill water was added in a flask and stirred gently. 5 ml NH4OH and 2 ml CH3(CH2)CH2OH was also added with constant stirring, It was kept at room temperature for 30 minutes for color observation and absorbance was noted at 505 nm and expressed as mg/100 g of sample.

Dosage and administration of drugs

Forty-eight healthy male Sprague Dawley rats weighing ‘200-220 g’ were obtained and kept in stainless steel cages under controlled environmental conditions (’20-23°C’, ‘12h’ light/dark cycle, ~’5 %’ humidity) for a two-week acclimation period. The rats were divided into six groups, each comprising eight animals (n=8). Group I served as the negative Control (C) and was given only distilled water. Group II, the placebo group (S), received saline solution (‘0.9% ‘saline). Group III, TiO2NPs treated group@ 120 mg/kg’ BW (represented by G*) was exposed to TiO2 nanoparticles (120 mg/kg’ BW) via intraperitoneal injection. Groups IV-VI were treated with the same TiO2NPs dose plus low (G1=‘100 mg/kg’), medium (G2=‘150 mg/kg’), or high (G3=‘300 mg/kg) doses of A. indica, administered orally for 90 consecutive days.

Sample collection

Following the 91-day experimental period, the rats were subjected to an overnight fast, anesthetized with ketamine hydrochloride (30 mg/kg body weight), and subsequently euthanized for sample collection. Blood samples were collected in gel-based coagulation tubes for testosterone analysis (Jannyketchuly, et al. 2016) while testes were excised, weighed using a Shimadzu Sartorius balance, and processed for further evaluation. One testis portion was frozen at ‘-80°C’ for sperm and cell viability studies, and another was preserved in a fixative mixture for histopathological examination, following Institutional Animal Ethics Committee (IAEC) approved animal care guidelines.

Body and somatic index

Body weight was measured weekly using a weighing balance (Shemazu 230), while testis weight was recorded post-sacrifice of the rats.

Hormonal parameter

Testosterone level: Testosterone levels were measured using a commercial kit, each for 25 μL of samples (blood), standards and controls were added to coated 96-well plates. Then ‘100 μL’ of conjugate reagent was added, shaken for ‘30 min’ and incubated at room temperature for ‘1 h’. After washing, ‘100 μL’ of 3,3',5,5'-Tetramethylbenzidine (TMB reagent) was added and shaken for ‘1 min’ before adding ‘100 μL’ stop solution. Absorbance was measured at ‘450 nm’ and testosterone concentrations were calculated using a standard curve (Akdogan, et al. 2004).

Luteinizing hormone: Blood samples were obtained from the experimental animals immediately after sacrifice. Following clot formation, serum was isolated by centrifugation and preserved at -80°C until biochemical analysis. Serum levels of Luteinizing Hormone (LH) were quantified using a commercial Enzyme-Linked Immunosorbent Assay (ELISA) kit (Cat. No. CK-E90904) in accordance with the manufacturer's instructions. Absorbance values for the standards and serum samples were recorded at a wavelength of 450 nm using a Bio-Rad ELISA microplate reader.

Oxidative stress markers

Oxidative stress was analyzed through Malondialdehyde (MDA), lipid peroxidation, Superoxide Dismutase (SOD), Catalase (CAT), Glutathione Peroxidase (GPx), and Reduced Glutathione (GSH) levels were measured using standard established protocols (Demirci and Sahin, 2019).

Homogenate preparation: A 0.25 g sample of liver tissue was homogenized in 2.5 mL of 0.1 M Tris-HCl buffer (pH 7.4) using a bullet blender under chilled conditions (4°C) to preserve enzymatic activity. The homogenate was subsequently centrifuged at 10,000 rpm for 20 minutes at 4°C to separate the cellular debris. The resulting supernatant was carefully collected, stored at -20°C, and later utilized for the assessment of oxidative stress biomarkers.

Estimation of Malondialdehyde (MDA): Malondialdehyde (MDA), a secondary by-product of Lipid Peroxidation (LPO), was quantified following the method of Ohkawa, et al. 1979. Briefly, the reaction mixture consisted of 0.2 ml tissue homogenate, 0.2 ml of 8.1% Sodium Dodecyl Sulfate (SDS), 1.5 ml of 20% acetic acid (pH 3.5), and 1.5 ml of 0.8% Thiobarbituric Acid (TBA), with distilled water added to obtain a final volume of 4 ml. The mixture was incubated at 95°C for 60 minutes in a water bath to allow color development. After cooling, 1 ml distilled water and 5 ml of n-butanol: Pyridine (15:1, v/v) were added, followed by vigorous mixing and centrifugation at 4000 rpm for 10 minutes. The absorbance of the upper organic layer was recorded at 532 nm using a spectrophotometer, and MDA levels were expressed as nmol per gram of tissue.

Estimation of lipid peroxidation: A 0.5 mL aliquot of freshly prepared tissue homogenate was combined with 0.9 mL of Ferrous Oxidation-Xylenol Orange (FOX) reagent. The reagent consisted of butylated hydroxytoluene (88 mg), xylenol orange (7.6 mg), and ammonium iron sulfate (9.8 mg), dissolved in a methanol and sulfuric acid mixture at a ratio of 90:10 (v/v). Following thorough mixing, the samples were incubated at 37°C for 30 minutes to allow color formation. The absorbance of the developed color was then measured at 560 nm using a spectrophotometer. Lipid peroxidation (LPO) levels were calculated and expressed as mM per gram of tissue.

Superoxide Dismutase (SOD): A 10% (w/v) liver homogenate was prepared in ice-cold 50 mM phosphate buffer (pH 7.4) and centrifuged at 10,000 × g for 15 minutes at 4°C, after which the supernatant was collected. The reaction mixture consisted of phosphate buffer (pH 7.8), 0.1 mM Ethylenediaminetetraacetic Acid (EDTA), 13 mM methionine, 75 μM NBT, and 2 μM riboflavin. An appropriate volume of liver supernatant was added to this mixture and exposed to fluorescent light for 10-15 minutes to initiate the reaction. Absorbance was measured at 560 nm, and one unit of SOD activity was defined as the amount of enzyme required to produce 50% inhibition of Nitroblue Tetrazolium (NBT) reduction.

Estimation of Catalase (CAT): Catalase (CAT) activity in liver tissue homogenate is commonly determined spectrophotometrically by monitoring the decomposition of hydrogen peroxide (H₂O₂). Fresh liver tissue is homogenized in ice-cold phosphate buffer (50 mM, pH 7.0) and centrifuged to obtain a clear supernatant. The reaction mixture typically contains phosphate buffer, a defined concentration of H₂O₂ (e.g., 10-30 mM), and an appropriate volume of tissue homogenate, and the decrease in absorbance is recorded at 240 nm for 1-3 minutes using a Ultraviolet (UV)-visible spectrophotometer. Catalase activity is calculated from the rate of H₂O₂ degradation using its molar extinction coefficient and expressed as units per milligram of protein.

Estimation of Glutathione Peroxidase (GPx): Glutathione Peroxidase (GPx) activity in liver homogenate was determined using a coupled assay method. Briefly, liver tissue was homogenized in ice-cold phosphate buffer (0.1 M, pH 7.4) and centrifuged at 10,000 × g for 15 min at 4°C to obtain the supernatant. The reaction mixture contained phosphate buffer, reduced Glutathione (GSH), reduced Nicotinamide Adenine Dinucleotide Phosphate (NADPH), glutathione reductase, and hydrogen peroxide as substrate, and the decrease in absorbance was monitored at 340 nm. GPx activity was calculated from the rate of NADPH oxidation and expressed as units per mg protein.

Estimation of Reduced Glutathione (GSH) levels: Reduced Glutathione (GSH) levels in liver tissue homogenates were quantified using the classical Ellman’s method. Briefly, liver samples were homogenized in ice-cold phosphate buffer (0.1 M, pH 7.4) and centrifuged at 10,000 × g for 15 minutes at 4°C to obtain the supernatant. An aliquot of the supernatant was reacted with 5,5′-Dithiobis (2-Nitrobenzoic Acid) (DTNB), and the formation of the yellow chromophore (TNB) was measured spectrophotometrically at 412 nm. GSH concentration was calculated from a standard curve prepared using reduced glutathione and expressed as μmol GSH per gram of tissue.

Histology

A small portion of gonadal tissue from both control and treated groups was collected and fixed using Sera’s solution (formaldehyde, ethanol, and glacial acetic acid). The samples were then dehydrated through a graded alcohol series, followed by clearing with cedar wood oil at room temperature. Embedding was carried out in multiple steps involving benzol and paraplast at ‘50°C’. Once embedded, the tissue was sectioned into 5 μm slices using a microtome (Histo-line MR 2258). To assess tissue morphology and histopathological changes, the prepared sections were stained with Hematoxylin and Eosin (H and E) and subsequently examined micro-scopically (Irais, et al. 2021).

Statistical analysis

The Data in this study was expressed as mean ± Standard Error (SE). Statistical significance was determined using Minitab software (minitab15). Body weights, organ weights, Hormonal parameter and oxidative stress Biomarker were analyzed through one way ANOVA by using general linear model to found out the effect of various treatment of A. indica on different parameter at (p<0.05) compared using the Duncan test. Histological changes in testis were assessed through visual inspection of photographic images from control and treated groups.

Results

LC₅₀ of plant extract

As presented in Fig. 1, the LC₅₀ value of A. indica for Sprague Dawley rats over ‘96 h’ was found to be ‘2155.21 mg/kg’ body weight.

Figure 1: Mortality response of Sprague-Dawley rats to acute 96-hour exposure to Azadirachta indica.

Quantitative analysis of plant extracts

The phytochemical analysis of A. indica revealed the presence of several important secondary metabolites with varying concentrations (See in Tab. 1). Among the analyzed compounds, alkaloids were detected at the highest concentration (9.42%), indicating that they are the predominant secondary metabolite in the plant. Flavonoids were recorded at 3.24 mg/100 g, followed by tannins at 2.88 mg/100 mL and total phenols at 2.36 mg/100 g, demonstrating moderate levels of these bioactive constituents. In contrast, saponins were present at the lowest concentration (1.55%), suggesting that they are comparatively less abundant than the other metabolites. Overall, the findings indicate that A. indica is particularly rich in alkaloids while also containing appreciable amounts of flavonoids, tannins, total phenols, and saponins, all of which may contribute to its biological and therapeutic properties.

Note: Mean values having different alphabets (A and B) in rows are significantly different (p<0.05).

Table 1. Quantitative analysis of secondary metabolites in Azadirachta indica.

Body and somatic index

The G* group (Positive control group) showed a marked drop in body weight compared to untreated and plant-treated rats. High-dose A. indica (‘300 mg/kg’) effectively countered TiO₂-NPs toxicity, more than the low (‘100 mg/kg’) and medium (‘150 mg/kg’) doses. In low-dose groups, weight rose in month one then declined, while medium-dose rats showed early loss followed by later gain (see Tab. 2).

Note: AW: Animal Weight, OW: Organ Weight, SD: Standard Deviation, a: Similar to each other, different from b and c,  b: Different from a, similar within itself,  c: Different from both a and b,  ab: Similar to both a and b.d; Different from a,b and c

Table 2. Mean body weight and testicular somatic index (g; Mean ± SD) of Sprague-Dawley rats following 90-day Azadirachta indica exposure

The testicular somatic index was significantly lower (p<0.05) in the positive control group, whereas high-dose A. indica significantly restored it compared with the positive control and low-dose groups (see Tab. 2).

Hormonal parameter

Tab. 3 illustrates a marked reduction (p<0.05) in testosterone and luteinizing hormone levels from the positive control group (G*) when compared to the non-treated and other treated groups. However, administration of a high dose of A. indica (G3) led to a significant rise (p<0.05) in both testosterone and luteinizing hormone levels in the gonads of these rat relative to the positive control group. Low and medium doses caused limited changes, whereas G3 exhibited the most significant hormonal restoration.

Note: SD: Standard Deviation, a: Similar to each other, different from b and c, b: Different from a, similar within itself, c: Different from both a and b, ab: Similar to both a and b.d; Different from a,b and c

Table 3. Hormonal profile of Sprague-Dawley rats: Serum testosterone and luteinizing hormone responses to 90-day Azadirachta indica exposure.

Oxidative stress markers

The results showed that TiO₂-NP exposure significantly increased MDA and LPO levels, indicating enhanced oxidative stress, while antioxidant markers (SOD, CAT, GPx, and GSH) were also significantly elevated compared with the control group (p<0.05). Plant extract treatment at 100, 150, and 300 mg/kg body weight significantly reduced these TiO₂-NP-induced alterations in a dose-dependent manner. The magnitude of reduction was greater at higher extract doses, with the 300 mg/kg dose producing the most pronounced protective effect. At this dose, most oxidative stress parameters were restored to values close to those of the control group. Overall, the plant extract effectively attenuated TiO₂-NP-induced oxidative stress and improved the antioxidant status of the treated groups (Tab. 4).

Note: MDA: Malondialdehyde, LPO: Lipid Peroxiation, SOD: Superoxide Dismutase, CA: Catalase, GPx: Glutathione Peroxidase, GSH: Glutathione, SD: Standard Deviation
Mean values having different alphabets in rows are significantly different (p<0.05).

Table 4. Oxidative stress markers of Sprague-Dawley rats in responses to 90-day Azadirachta indica exposure.

Histology

The histopathological assessment of testicular tissue is summarized in Tab. 5. The control and saline-treated groups exhibited normal testicular architecture, characterized by intact seminiferous tubules, well-organized seminiferous epithelium, normal Sertoli cells, and active spermatogenesis. No signs of seminiferous tubule destruction, degeneration, necrosis, vacuolation, torsion, or basement membrane damage were observed in these groups. In contrast, the positive control group treated with TiO₂ nanoparticles (TiO₂-NPs) showed severe histopathological alterations (+++). These changes included marked destruction and degeneration of seminiferous tubules, extensive Sertoli cell damage, vacuolar degeneration of the seminiferous epithelium, sloughing of germinal epithelium, basement membrane disruption, testicular atrophy, arrested or impaired spermatogenesis, and pronounced necrosis. These findings confirm the toxic effect of TiO₂-NPs on testicular tissue. Administration of A. indica extract at a low dose resulted in considerable histological damage, with moderate to severe alterations (++ to +++) comparable to those observed in the positive control group. However, treatment with the medium dose of A. indica extract significantly reduced the severity of these lesions, showing only mild histopathological changes (+). Notably, animals treated with the high dose of A. indica extract (300 mg/kg) demonstrated near-normal testicular architecture, with preservation of seminiferous tubules, intact basement membranes, normal Sertoli cells, and restoration of spermatogenesis. No significant pathological alterations were observed in this group, similar to the control and saline groups. Overall, these findings indicate that A. indica extract exerts a dose-dependent protective and remedial effect against TiO₂-NP-induced testicular toxicity, with the 300 mg/kg dose providing maximum protection and restoring normal testicular histology.

Table 5. Histological scoring of tissue alterations in control, saline, positive control (TiO₂ NPs), and A. indica-treated groups (100, 150, and 300 mg/kg).

Fig. 2 shows histology of control (A-D) shows normal Seminiferous Tubules (ST) and Sertoli Cells (SC). TiO₂-NPs @120 mg/kg (E-J) caused severe changes: Torsion (T), Vacuolar Degeneration of Seminiferous epithelium (VDS), Seminiferous Tubule Degeneration (STD), Testicular Atrophy (TA), Arrested/Impaired Spermatogenesis (AS) and Necrosis (N) shown in Fig. 3. A. indica @100 mg/kg (K&L) showed Seminiferous Epithelium Sloughing (SES) and Destruction of Basement Membrane (DBM); @150 mg/kg (M&N), animals treated with the high dose of A. indica extract (300 mg/kg) demonstrated near-normal testicular architecture, with preservation of seminiferous tubules, intact basement membranes, normal Sertoli cells, and restoration of spermatogenesis (O-P) shown in Fig. 4.

Figure 2: Photomicrograph (H and E; X400) of the testis tissue in control (non-treated) group (A and B) and saline group (0.9% saline solution) showing normal Sertoli cell and seminiferous tubules in Sprague Dawley rats.

Figure 3: Photomicrograph (H and E; X400) of the testis tissues in Sprague Dawley rats with intraperitoneal injection of TiO₂-NPs @ 120 mg/kg BW (E-J).

Figure 4: Photomicrograph (H& E; X400) of the testicular tissues of Sprague Dawley rats orally treated with different doses of A. indica along with intraperitoneal injection of TiO₂-NPs @ 120mg/kg BW.

Conclusion

Titanium dioxide nanoparticles (TiO2-NPs) significantly impair male reproductive health, reducing body weight, testicular somatic index, hormone levels, and cell viability in rats. High-dose A. indica (300 mg/kg) effectively mitigated these toxic effects, showing the greatest improvement across all parameters. Histopathological analysis confirmed the protective role of neem extract against TiO2-induced testicular damage. Therefore, A. indica offers a promising natural remedy against nanoparticle-induced reproductive toxicity.

Acknowledgements

I am sincerely grateful to my department and institute for their valuable support, guidance, and assistance throughout the completion of this research work. Their cooperation and encouragement played an important role in making this research possible. We also thank the anonymous reviewers for their valuable comments and suggestions, which helped improve the quality of this paper.

Data Availability Statement

Data will be made available on request from the Corresponding author.

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