Zinc oxide nanoemulsions prevent kidney stone formation and protect renal function in rats

Kidney stones pose a significant issue and may result in severe problems such as blockage of the urinary system, hydronephrosis, infections of the urinary tract, and bleeding¹. The incidence of kidney stones ranges from 1 to 15% worldwide². Kidney stones have a high recurrence rate, with around 50% of patients encountering one within 10 years of treatment. The annual expense of treating kidney stones in the United States was $2.1 billion in 2000, and it is expected to approach $4 billion by 2030^3,4. Increased risk of kidney stones relates to high salt intake, inadequate water intake, insufficient consumption of dairy products, low intake of tea, consumption of oxalate-rich meals, and consumption of processed foods⁵.

Numerous factors, including eating choices, a sedentary lifestyle, diabetes mellitus, obesity, hypertension, and metabolic syndrome, increase the possibility of developing kidney stones^6,7. Traditional medicine has extensively used medicinal herbs for thousands of years to address many health-related conditions⁸. The World Health Organization (WHO) states that most of the global population relies on traditional medicine as their main source of basic healthcare. The utilization of medicinal plants is gradually rising owing to their effectiveness and few adverse effects in contrast to current synthetic pharmaceuticals⁹.

Cymbopogon Proximus (C. Proximus, CP), often called Halfabar, Maharaib, and Alazkher, is notable for its strong aroma and extensive presence in Egypt and northern Sudan. The local community has always depended on this plant for its capacity to promote smooth muscle relaxation, leading to its widespread use as a diuretic and antispasmodic agent. Moreover, it has shown hypoglycemic, antipyretic, bronchodilatory, antibacterial, anticonvulsant, and antiemetic properties, further emphasizing its medicinal potential¹⁰. Furthermore, both in vivo and in vitro investigations have shown the advantageous pharmacological impacts of Cymbopogon spp., including its ability to combat cancer, protect the heart, reduce inflammation, serve as an antioxidant, regulate blood sugar levels, inhibit cholinesterase activity, and exhibit antibacterial and antifungal properties¹¹. Currently, nanotechnology is anticipated to serve as the foundation for several biotechnological advancements in the 21 st century and is considered the next industrial revolution. Nanomaterials have been referred to as a remarkable advancement in contemporary medicine and have generated significant attention in recent years¹².

Nanotechnology encompasses diverse scientific applications, including sunscreens, diagnostic procedures, antimicrobial bandages, medicine delivery, and antibiotics¹³. The green production of nanoparticles (NPs) involves using plants or plant components to biologically synthesize metallic NPs, instead of employing hazardous substances that cause environmental harm¹⁴. Many bioactive compounds found in plants, including alkaloids, terpenoids, flavonoids, amino acids, enzymes, vitamins, proteins, and glycosides, may also play a role in reducing, forming, and stabilizing metal nanoparticles¹⁵. Zinc oxide nanoparticles (ZnONPs) are intriguing among metal nanoparticles because of their remarkable features, including a broadband gap, substantial binding energy, and strong piezoelectric capability¹⁶. The synthesis of Zinc oxide nanoparticles is commonly used due to their ecologically benign nature, cost-effectiveness, and lower toxicity than other synthetic processes. Researchers have shown that various plant extracts may be used to create ZnONPs with antimicrobial properties¹⁴ effectively. ZnONPs have gained considerable interest among various nanomaterials owing to their adaptability in several fields such as agriculture, cosmetics, biology and medicine, and environmental pollution management¹⁷. This work aims to develop a novel, eco-friendly, and non-toxic approach of ZnO nanoemulsions to assess the protective effects against ethylene glycol-induced renal stones and rat kidney tissue damage in male albino rats. ZnO@CP nanoemulsions were created using high-speed homogenization and the wet chemical precipitation technique. The plant source used for this process is the crud C. Proximus (CP) oil extract, free from harmful chemicals.

Collection of plant materials and Preparation of the crude oil extract

C. Proximus Grass (CP) was purchased from the local market in Cairo, Egypt. 50 g of CP underwent two rounds of washing with tap water followed by distillation. After being exposed to air for 24 h under a hood, the CP plant was crushed into a fine powder.CP crude extract was produced by dissolving 200 mg of CP powder in 100 ml of hot water in a shaker at 150 rpm overnight at 35 °C. Then, the extract was filtered using Whatman No. 1 filter paper. Finally, the extract was stored in a refrigerator at 4 °C for further investigation.

Determination of total phenolic content (TPC) and total flavonoid content (TFC)

TPC of the aqueous CP extract was assessed using the Folin-Ciocalteu reagent as described by¹⁸. In brief, a reaction mixture was prepared by combining 0.5 mL of extract, 5.0 mL of distilled water, and 0.5 mL of the Folin-Ciocalteu reagent [100 g of sodium tungstate (Na₂WO₄. 2H₂O,) and 25 g of sodium molybdate, (Na₂MoO₄. 2H₂O, in 700 mL of distilled water)]. After 3 min, 2.5 mL of a 20% Na₂CO₃ solution was added. The mixture was thoroughly agitated and allowed to develop colour for 1 h. in darkness. Absorbance was measured at 725 nm using a Hitachi UV-Vis U 3000 spectrophotometer (Tokyo, Japan), with each measurement conducted in triplicate. Results were expressed as mg GAE (gallic acid equivalents)/g dry extract following the establishment of a calibration curve for gallic acid. TFC were assessed using the previously reported method¹⁹. Briefly, a mixture was made in a 10 ml test tube containing 0.3 ml of the extract, 4 ml of 30% methanol, 0.15 ml of NaNO₂ (0.5 M), and 0.15 ml of AlCl₃ (0.3 M). After 5 min, 1 ml of 1 m NaOH was added, and the absorbance of the reagent sample at 510 nm was measured. TFC was calculated in milligrams of rutin equivalents per 100 g of dry plant powder (mg. RE/100 g).2.3 Synthesis of ZnONPs.

The synthesis of ZnONPs was performed using a modified version of the established method²⁰. The synthetic mixture was prepared from two different solutions: solution A and B; solution A contained 2.95 g of zinc nitrate [Zn (NO₃)2·6H₂O] dissolved in 40 mL of ddH₂O.The pH was adjusted to 7, 9, or 11 using solution B contained 7.22 mmol of NaOH dissolved in 320 µL of ddH₂O. Solution B was added dropwise to solution A under vigorous and constant stirring at 90 °C for 6 h. The solution was separated from the sediment using centrifugation at 15,000 rpm for 5 min. The precipitated particles were subjected to several washes by distillation with water to remove any residual impurities. After 5 h of drying at 70 °C, a beige powder composed of ZnONPs was formed and obtained in Scheme 1A.

Fabrication of ZnO@CP NEs

First, 20 mg ZnONPs solution was added under stirring to the former solution to obtain a homogenous mixture (high-speed homogenizer IKA, Staufen, Germany) at 12,000 rpm for 5 min. Then, tween 80 (0.2% v/v) was subtly added to create a tween 80-coated ZnONPs film. 10 mL of the oil crude extract of the plant (hydrophobic phase) was added drop by drop to 30 mL of tween80/ZnONPs mixture solution. After a 10-minute incubation period, 30 mL of distilled water (the volume of the hydrophobic phase) was added drop by drop to the hydrophilic phase. The product was left for 5 min without agitation to separate the layers. Finally, after removing the oil phase, the aqueous phase and the nanocarriers were separated by centrifugation (Hermle-Labnet) at 6,000 rpm for 10 min. Each sample at each step was frozen in the freezer at − 20° and powdered by a freeze dryer²¹. (Scheme 1B).

Diagrammatic representation of the multifunctional of (A) ZnONPs and (B) ZnO@CP nanoemulations preparation process.

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Physico-chemical characterization

Ultraviolet-visible spectrometry (UV-Vis)

A tiny quantity of the samples was dispersed again in about 10 mL of DIH₂O and introduced into a 1 cm path quartz cell to evaluate the optical characteristics of both chemically and greenly produced CP, ZnONPs, and ZnO@CP NEs. The sample was subjected to scanning from 200 to 800 nm using a UV-Spectrophotometer (Shimadzu UV-1700, Tokyo, Japan), and the absorbance at the peak wavelength was measured.

Fourier transform infrared spectroscopy (FTIR)

FTIR spectra were acquired with a Shimadzu IR Tracer-100 FTIR spectrometer. Each sample was amalgamated with crystalline KBr at a ratio of 1:100 (sample: KBr). A spectrum was obtained for all samples within the wavenumber range of 4000–400 cm⁻¹.

Transmission electron microscope (TEM)

TEM was performed after diluting various ZnONPs and ZnO@CPNEs samples with dH₂O at a 1:1 µL/mL⁻¹ ratio. A mixture drop was added with a micropipette onto a carbon-coated grid, and filter paper was used to dry the sample on the grids for 3–5 min. The grids were imaged using TEM (JEOL JEM-2010, Tokyo, Japan). The ImageJ program was calibrated using TEM image for measuring the size distribution of NPs²².

Dynamic light scattering (DLS)

The average values of particle size (PS) polydispersity index (PDI), and Zeta potential (ζ) for the formulations were ascertained using DLS using a Malvern Instruments apparatus. The PS and PDI, which quantify the dispersion of the nanoparticle population, were assessed using the Malvern Mastersizer 2000MU (Malvern, UK, detection limit 0.01–1,000 μm). The ζ value of ZnONPs and ZnO@CP NEs was assessed using the Zetasizer 2000 (Malvern, UK). The ZnONPs and ZnO@CP NEs suspensions were diluted with double-distilled water (1:100) to achieve a homogeneous dispersion before analysis.

Determination of antioxidant activity

The antioxidant activity of the CP, ZnO NPs, and Zno@CP NEs were evaluated using a DPPH test. The aqueous extract, ascorbic acid, and DPPH solution were diluted in methanol to achieve a 2 mg/mL concentration. The aqueous extract was diluted in 0.125 to 3 mg/mL concentrations. Ascorbic acid, unloaded ZnO NPs, and ZnO@CP NEs were diluted in concentrations at 1.95 to 1000 µg/mL. Subsequently, 10 µL of DPPH solution was introduced to each well and incubated at room temperature in the dark for 30 min. Following the incubation period, the level of light absorption was quantified at a wavelength of 517 nm. Ascorbic acid served as the reference standard. The following formula was used to determine the scavenging capacity: The multiple absorbance readings were taken three times. Then calculate the mean (average) absorbance for each concentration. Then, calculate the standard deviation to show the variability within replicates. Then plot the mean absorbance values to create your dose-response curve. The IC50 value was determined by performing experiments using a range of inhibitor concentrations to see what concentration reduces a biological process by 50%, through manual linear regression analysis of the data points.

Where: As is the sample’s absorbance, and A0 is the absorbance of the negative control (methanol and DPPH solution).

Biocompatibility assays in vitro

The cytotoxic effect of ZnO NPs and ZnO@CP NEs formulation was assessed normal mouse tail fibroblast cell line (L929 purchased from the American Type Culture Collection (Rockville, MO, USA)) using the MTT⁹. 100 µL of the cell line was grown in 96-well plates at a density of 1.1 × 10⁴ cells per well for 24 h at 37 °C. Subsequently, 100 µL of RPMI media containing varying concentrations of ZnO NPs and ZnO@CP NEs (10–200 µg/mL) were administered to each well for 48 h, incubated at 37 °C with 5% CO₂. Subsequently, 30 µL of MTT (5 µg/mL) was added to each well, and after a 4-hour incubation at 37 °C, 50 µL of DMSO was added to each well to dissolve the purple formazan crystals. Ultimately, the absorbance of each well was evaluated with an ELISA reader at a wavelength of 630 nm. Subsequently, viability was determined utilizing Eq. (2).

As = Absorbance of the nanoparticles, Ac = Absorbance of the control²³.

In vivo study

Experimental design

Thirty-six healthy adult male Wistar rats, weighing (250–270 g), were housed in standard laboratory conditions with controlled humidity and temperature (22 ± 2 °C) and a 12-hour light/dark cycle (lights on from 7:00 am to 7:00 pm), with unrestricted access to food and tap water. After one week of acclimatization, experimental volunteers were randomly assigned to six groups (n = 6 per group).

In group I: Negative control group (NC), rats were orally saline (5 ml/k g body weight). In Group II : Positive control group (PC), rats were orally with 1% ethylene glycol. Group III, Effect Stander Drug Group (EG), the rats were orally with cystone (100 mg/kg body weight). Group IV rats with kidney stones were treated with CP orally twice daily at 0.05 mg/kg body weight²⁴. Group V, rats with kidney stones treated with unloaded ZnO NPs, orallyat 200 µg/ml divided into two periods each day²⁵. Group VI, rats with kidney stones treated with ZnO@CP NEs, orally 250 µg/ml divided twice a day²⁶. After four weeks of receiving each treatment, rats were sacrificed by cervical decapitation under ketamine anesthesia. Then, the kidneys were meticulously dissected, except for the right kidney, which was divided into two longitudinal halves. The homogenized half was used for the biochemical test, while the other was examined under a light microscope.

Biochemical examination of urine and renal tissue

The urine sample underwent centrifugation and dialysis for processing. Urine samples underwent centrifugation at 2000 rpm for 10 min utilizing a centrifuge. A cellulose dialysis membrane (Sigma, USA) was employed for overnight dialysis against distilled water. The resulting solution was subsequently utilized to assess protein and enzyme activities. The components and characteristics of stone formation were analyzed using unwashed samples²⁷. Serum creatinine and urea levels in the blood were measured using a colorimetric technique according to²⁸ outlined in a commercial kit provided by Diamond Diagnostics. Uric acid levels were quantitatively according to²⁹. Assessed using a commercial kit from Spinreact, Spain. To determine the liver function of these animals, we estimated the AST and ALT activities by the colorimetric kits (CAT No. 260-001 and CAT No. 264-001).

Histopathological examination

To investigate structural alterations in kidney tissue, we followed the protocol recently published by Neumann et al. (2020)³⁰. Kidney biopsy cores were immediately immersed in 10% neutral-buffered formalin for 18–24 h at room temperature. After fixation, tissues were transferred to an automated tissue processor and subjected to ascending alcohol concentrations (70%, 95% and two changes of absolute ethanol, 1 h each), followed by two 1 h changes of xylene and two 1 h infiltrations in molten paraffin melting point 58 °C (Wako Pure Chemical Industries, Osaka, Japan). The processed cores were then embedded in paraffin with the long axis oriented vertically to ensure cross-sectional representation of glomeruli and tubules. Sections were cut at 3 μm on a rotary microtome (Yamato Kohki Industrial, Saitama, Japan), and floated on a 45 °C water bath containing a 1% gelatin additive to eliminate wrinkles. The ribbons were then mounted on positively charged slides and dried at 60 °C for 30 min to promote adhesion. For routine histopathology, periodic acid–Schiff (PAS) staining was performed exactly as described by Neumann et al.: 0.5% periodic acid for 5 min, Schiff’s reagent for 30 min, a warm water rinse at 40 °C, Mayer’s haematoxylin for 1 min, a brief dip in 0.5% ammonium hydroxide for bluing, sequential dehydration in graded alcohols, two 1 min changes in xylene and coverslipping with Cytoseal. Quality control included the routine inclusion of a normal rat kidney section as a positive control; the absence of residual cloudiness in the final absolute ethanol rinse verified complete dehydration. to be examined under a light microscope. The microscope slides (size 76 × 26 mm, thickness 1.0 mm: Muto Pure Chemical, Tokyo, Japan) were digitised with a 20× objective, yielding a pixel resolution of 0.24–0.49 μm px⁻¹. Morphometric analysis was carried out using the open-source QuPath platform. Glomeruli were first identified at low magnification (10×) and then examined at 40× oil immersion. For each glomerulus, endocapillary cellularity and mesangial matrix expansion were scored on a 0–3 semi-quantitative scale³¹. Tubulointerstitial changes were assessed in ten consecutive cortical fields at 20× magnification: tubular atrophy was defined as a reduction in tubular diameter with thickened basement membranes, and interstitial fibrosis was quantified as the percentage of trichrome-positive cortical area using colour deconvolution.

Statistical analysis

The experiments were conducted in triplicate, and the data were reported as the mean ± standard diffusion (SD). The program GraphPad PRISM (version 8) was used to calculate EC₅₀ values. The results underwent a one-way analysis of variance (ANOVA), followed by Duncan’s multiple range test. Mean comparisons were conducted using Duncan’s multiple range test in SPSS (version 21.0, Chicago, USA).

Determination of TPC and TFC

Numerous medications are used to reduce the incidence of hypercalciuria and hyperoxaluria, which lead to the production of calculi. These medications include thiazide as a diuretic and alkali citrate, but their limited efficacy and poor tolerability make them unpromising³². Investigating novel pharmacological treatments for kidney stone management is valuable due to the drawbacks of surgical techniques and the lack of options in pharmacotherapy. Urolithiasis can be effectively treated using various medicinal herbs with diuretic, antispasmodic, and antioxidant properties. These medicinal herbs also limit crystal nucleation, aggregation, and crystallization³³. Moreover, phenolic compounds exhibit various biological properties, such as antibacterial, anticancer, and anti-inflammatory effects, which may be related to their antioxidant activity^34,35. Furthermore, we examined the amounts of TPC and TFC in the CP oil crud extract (Table 1). Quantitative investigation revealed that the TFC of the CP was substantially greater (22.04 ± 0.58 mg RT/g) than that of the TPC (21.73 ± 1.27 mg GAE/g). The results shown are consistent with those previously reported by³⁶. These results maybe due to the proportions and quantities of the extraction ingredients varying depending on the type and the polarity of the solvents used in the extraction process, which in turn affect the qualitative structure and physicochemical activity of the extracts as confirmed in previous reports by Gaweł-Bęben et al. 2015³⁷. Numerous studies have shown the various health benefits of flavonoids, including their anti-inflammatory, anti-allergic, analgesic, antiviral, anticancer, and cardioprotective and hepatoprotective properties. Flavonoids provide several medicinal benefits due to their distinctive molecular composition³⁸. Our results indicate that phenolic acids and flavonoids are likely the primary contributors to antioxidant activity, as evidenced by the significant correlation between the IC50 values of free radical scavenging activity and the phenolic or flavonoid contents in various soluble fractions of CP extracts.

Characterization of ZnO NPs

UV–Vis spectroscopy

At room temperature, we examined the UV-visible absorbance spectra of CP, ZnO NPs, and ZnO@CP NEs in order to examine their optical absorption properties. The wavelength range in which the observations were made was 300–800 nm. The results are displayed in Fig. 1A. The ZnONPs wavelength of 370 nm had the maximum absorbance, which is comparable with the previously published results by Sumanth et al.(2020)³⁹, and Balogun et al. (2020)⁴⁰, and fits into the distinctive band of ZnONPs nanoscale. Moreover, the ZnO@CP NEs absorption spectra revealed two absorption peaks at 345 nm and 370 nm. At this peak, separate bands of zinc colloids were seen, indicating that the CP effectively decreased the zinc ion. Moreover, CP absorbance was detected at 340 nm in wavelength.

FTIR

The formation of an emulsion layer at the surface of unloaded ZnONPs was verified, and the interactions between lipid molecules and ZnONPs surfaces for ZnO@CP NE were investigated using FTIR spectroscopy. FTIR measurements were performed at room temperature utilizing the KBr technique, with a wave number range of 4000 to 400 cm^–1. The findings of this study are presented in Fig. 1B. The crude CP displayed characteristic bands at 3440 cm⁻¹ (O-H stretching), 2360 cm⁻¹ (C = O stretching), and 1500 cm⁻¹ (C-O stretching)⁴¹. Similar patterns were observed in the FTIR spectra of ZnO@CP NE, which included most peaks identified for the CP, while the ZnO bond vibration peak was diminished at 619 cm⁻¹. Additionally, several extra bands were identified, including a broad peak at 3360 cm⁻¹ (O-H stretching) and peaks at 1640 cm⁻¹ (H-O-H bending) and 1110 cm⁻¹ (C-O stretching), which can be ascribed to water. In the FT-IR spectrum of the ZnO@CP NE sample, intense peaks at 2940 and 2350 cm⁻¹ were observed, corresponding to the stretching vibrations of –CH and C = O groups in the hydrophobic tail of the phospholipid. This observation confirms the formation of the phospholipid layer on the surface of pristine unloaded ZnONPs⁴². Moreover, the FTIR analysis indicated that the homogenization emulsification procedure did not affect the structure of the CP and ZnONPs components. The results demonstrated the successful creation of lipid coats on the surface of unloaded ZnONPs.

(A) the absorption spectra within the UV-visible, and (B) FTIR spectrum of unloaded ZnONPs and ZnO@CP NEs.

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TEM

The TEM micrographs reveal that the wet-chemical aqueous precipitation synthesis method produced of unloaded ZnONPs and ZnO@CP NEs and the histogram’s average particle size are shown in Fig. 2A-D. As demonstrated in Figures. 4 A and C, the synthesized unloaded ZnONPs agglomerated in bundles or irregularly shaped NPs. The unloaded ZnONPs occurred in lengths varying from 26 nm ​– 95 ​nm. The average length of the nanorods was observed to be 67.92 ​nm. However, the TEM results of ZnO@CP NE indicate the existence of non-agglomerated irregularly shaped NPs with a size range of 106–193 nm. The average length of the nanorods was observed to be 142.35 ​nm. (Fig. 2B and D). These results were confirmed by Alhudhaibi et al. (2024)²⁰. We may infer from the aforementioned TEM findings that the lipid bilayer can self-assemble on the surface of inorganic nanocrystals to provide a comprehensive and thick covering layer, hence preventing aggregation. and the interaction of the ZnO@CP NEs with the watery solution.

DLS

The DLS technique was used to determine the particle size and charge of unloaded ZnONPs, and ZnO@CP NEs were generated using the wet-chemical aqueous precipitation synthesis method. Figure 5A shows the PS distribution of unloaded ZnONPs and ZnO@CP NEs ranging from around 71.55 nm and 145.8 nm, PDI value was 0.336 and 0.281 (Fig. 2E), and ζ value was​– 20.9 mV and ​– 23.4 mV respectively, (Fig. 2F). The PS of the nanoparticles is increased because the DLS measures the core-shell covering the metal ions in the particles. The fact that the core-shell coated metal ion may obstruct the nanoparticles’ ability to engage with their target makes this research intriguing⁴³. The DLS analyzer revealed a wide range of frequencies that validate the reduction in size of the nanoparticles when correlated with the distinct peak (370 nm) observed in the ultraviolet-visible spectrum.

TEM image and size distributions of synthesized (A and C) unloaded ZnONPs and (B and D) ZnO@CP NEs, (E) PS and (F) ζ value of synthesis unloaded ZnONPs and ZnO@CP NEs measurement by DLS.

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Determination of antioxidant activity

CP, unloaded ZnONPs, and ZnO@CP NEs preparation solutions were measured by UV absorption at 517 nm using DPPH radical scavenging to determine the antioxidant capability of the samples compared to stander antioxidant drug ascorbic acid (IC50 = 2.62 µg/mL⁻¹). According to Fig. 3C, the IC50 of CP and unloaded ZnONPs are almost identical, with IC50 = 1.24 mg/mL⁻¹ (Table 1), and 45.63 µg/mL⁻¹ (Fig. 3), respectively. Compared to CP and unloaded ZnONPs, ZnO@CP NEs showed a higher antioxidant activity rate after 24 h (IC50 = 28.75 µg/mL⁻¹). This is consistent with the expected results, which were that the antioxidant activity of the ZnO@CP NEs was mainly ascribed to the antioxidant activity of CP. This study is in agreement with the study of⁴⁴. Adetuyi et al., (2024) reported a similar tendency in leaves with good free radical scavenging capacity due to higher DPPH radical inhibition and lower IC50 value⁴⁵. Several reports indicated that the antioxidant potential of CP may be related to the concentration of their phenolic compounds, including phenolic acids, flavonoids, anthocyanin, and tannins⁴⁶. In addition, Imath et al. (2025) presented an environmentally friendly synthesis of ZnO NPs using Fioria vitifolia leaf extracts that exhibited good antioxidant activity for IC50 values of 42 µg/mL⁴⁷.

Cell viability

The cytotoxicity of unloaded ZnONPs and ZnO@CP NEs were assessed using the L-929 mouse epithelial cell line using the MTT test, with findings shown in Fig. 3. The cytotoxicity rate escalated with higher unloaded ZnONPs and ZnO@CP NEs (Fig. 3). A notable cytotoxic impact began at 200 µg/mL and 250 µg/mL of ZnONPs and ZnO@CP NEs, respectively. In addition, the relative cell viability of unloaded ZnO NPs and ZnO@CP NEs presented 76.17% and 76.92%; the minimal acceptable toxicity level of 70% was observed. Literature indicates that the material is deemed non-cytotoxic to the cells if the relative cell viability exceeds 70%⁴⁸. Consequently, the ZnO nanoparticle might be used as a feed supplement, using a dosage rate of up to 200 µg/mL⁻¹ and 250 µg/mL⁻¹of unloaded ZnO NPs and ZnO@CP NEs, respectively. Hence, unloaded ZnO NPs and ZnO@CP NEs exhibited no cytotoxicity towards L929 cells and may serve as effective drug-delivery vehicles⁴⁹. Our results were similar to Geetha et al.(2020), who reported that the cytotoxicity study revealed a significant decrease in cell viability in L929 cells upon exposure to ZnO nanoparticles in a dose-dependent manner, with a maximum concentration of 180 µg/mL²⁵. Lipid-coated ZnO NPs are more likely to reduce cytotoxicity because the hydrophobic tail of the CP crude oil extract prevents ZnO NPs from dissolving and maintains their physical and chemical properties while also promoting good dispersion of the ZnO NPs as colloidal suspension in aqueous solution. The lipid layer also plays a significant role in the sustained release and can decrease Zn²⁺ cation⁴².

(A) Antioxidant activity measured by DPPH. (B and C) Cell viability image and percentage of unloaded ZnO NPs and ZnO@CP NEs on normal L929 cell lines after 24 h.

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Evaluation of calcium oxalate crystallization in urine by light microscopy

In susceptible rats, calcium oxalate nucleation started in supersaturated urine as the first step towards urolithiasis, since the developed nuclei either grow and/or aggregate to a large size, causing a pathological condition (Fig. 4A).

Microscopic examination of urine samples revealed dense calcium oxalate crystals in the positive-control rats. In contrast, animals treated with either plant extracts, cystone showed a high density of calcium oxalate crystals in the positive control rats, which two nano-formulations unloaded ZnO NPs (200 µg/ml) and ZnO@CP NEs (250 µg/ml) showed markedly fewer crystals, approaching the crystal-free appearance of healthy controls that remained stone-free throughout the experiment (Fig. 4B). The representative microscopic images of rats treated with CP, unloaded ZnO NPs, and ZnO@CP NEs are shown in Fig. 4D and E, and 4F, respectively, further confirming the reduced calcium oxalate crystallization in these treatment groups. The pictures were randomly selected due to the similarity of results⁵⁰.

(A) Anti-renal stone-prone effect of CP, unloaded ZnONPs, and ZnO@CP NEs in vivo. (B) Morphologically different microscopically identified crystal shapes of calcium oxalate. Envelope (pyramid)-shaped crystals of calcium oxalate dihydrate (COD) (red arrow). Weddellite crystals of calcium oxalate dihydrate (COD-W) (green arrow). Thin hexagonal lozenge (COM-TL) crystals of calcium oxalate monohydrate (blue arrow); (a) Group I NC group, rats were orally saline (5 ml/kg body weight). (b) In group II PC group, rats were administered orrlay1% ethylene glycol. (c) Group III, EG group, rats were orally with cystone (100 mg/kg body weight). (d) Group IV, rats with kidney stones treated with CP orally (5 mg/k g body weight) divided into two times a day. (e) Group V, rats with kidney stones treated with unloaded ZnO NPs, orally and 200 µg/ml divided into two times a day (f) Group VI, rats with kidney stones treated with 250 µg/ml ZnO@CP NEs.

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Effect of treatments on kidney function in rats

Renal function assessed via serum urea, uric acid, and creatinine was significantly improved (p < 0.05) in rats treated with CP, unloaded ZnO NPs (200 µg/ml.), or ZnO@CP NEs (250 µg/ml) compared with the untreated control group with 0.88 ± 0.28, 0.75 ± 0.0 and 0.95 ± 0.012 for creatinine, 37 ± 1.63, 35.6 ± 1.24 and 46.5 ± 1.63 for serum urea and 3.2 ± 0.20, 3.5 ± 0.12 and 3.8 ± 0.08 for uric acid, respectively, as illustrated in Fig. 5.

Compared with untreated controls (creatinine 1.20 ± 0.02 mg/dL, urea 67 ± 1.6 mg/dL, uric acid 5.2 ± 0.21 mg/dL), rats given CP, unloaded ZnO NPs (200 µg/mL) or ZnO@CP NEs (250 µg/mL) exhibited markedly improved renal function, with mean serum levels falling to 0.88 ± 0.28, 0.75 ± 0.01 and 0.95 ± 0.01 mg/dL for creatinine; 37 ± 1.63, 35.6 ± 1.24 and 46.5 ± 1.63 mg/dL for urea; and 3.2 ± 0.20, 3.5 ± 0.12 and 3.8 ± 0.08 mg/dL for uric acid, respectively (p < 0.05; Fig. 5).

The influence of treatments on (A) blood urea, (B) uric acid, and (C) serum creatinine levels data are reported as mean ± SD (n = 3). Values in each column followed by letters are significantly different ^{a, b,} (p < 0.05); statistical differences between samples in the Duncan test.

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Effect of treatments on liver biomarkers

ALT and AST enzymes usually are located in the cytosol of hepatocytes. When liver cells are damaged, these enzymes are released into the plasma, increasing their activities. These enzymes are a valuable marker of the extent and type of hepatocellular damage⁵¹. Green et al. (2005) observed that ethylene glycol is rapidly absorbed and metabolized in the liver via alcohol dehydrogenase and aldehyde dehydrogenase, converting it to glycollic acid⁵². Glycollic acid is then oxidized to glycolate and oxidized to oxalic acid by glycolate oxidase. Elevated dosages of ethylene glycol, particularly when given as an oral bolus, result in the plasma’s saturation-dependent buildup of glycollic acid, suggesting that glycolate oxidase is a rate-limiting enzyme in ethylene glycol metabolism. Our results showed that the effects of CP, unloaded ZnONPs, and ZnO@CP NEs on the liver function markers in the serum of the tested rats are shown in Figs. 6A and B, respectively. Rats received either CP, unloaded ZnONPs, and ZnO@CP NEs showed lower serum levels of ALT (30.66 ± 1.24, 34.33 ± 1.69, 37.66 ± 1.24 IU/L, respectively) compared to control (52.8 ± 1.63 IU/L) (p < 0.05) (Fig. 6A). The serum concentrations of AST in response to CP, and ZnO@CP NEs were also significantly decreased (32.33 ± 2.05, 32.66 ± 1.24, IU/L, respectively) compared NC (group ⅠⅠ) (36.16 ± 0.24 IU/L) (p < 0.05). In comparison, unloaded ZnO NPs showed a little increase (38.66 ± 1.24 IU/L)(p < 0.05) compared to NC (group ⅠⅠ) (Fig. 5B)⁵³. Abdel-Wareth et al. (2020) noted that ZnONPs improved the digestibility of nutrients, liver/kidney functions, and semen characteristics concentration in male rabbits⁵⁴. Furthermore, Mutawakel et al. (2021) showed CP has hepatoprotective on a model of acute carbon tetrachloride hepatitis in rats⁵⁵. Al-Oqail et al. (2020) demonstrated that pretreatment of cytotoxicity and oxidative stress in human liver cells with P. sativum offered protective properties by elevating the cell viability⁵⁶.

shows the influence of treatments on (A) ALT and (B) AST levels. Data are reported as mean ± SD (n = 3). Values in each column followed by letters are significantly different ^{a, b}, (p < 0.05), statistical differences between samples in the Duncan test.

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Histological examination

The findings based on the histological examination of the kidney are shown in Fig. 7. Microscopic observation showed apparent intact, well-organized histological structures of renal parenchyma, with many records of apparent intact renal tubular segments with intact tubular lining epithelium (arrow), intact renal corpuscles (star), and intact vasculatures in NC group (Fig. 7A). The PC group showed evident wide areas of degenerative tubular changes (red arrow) with moderate tubular dilation (black star), significant high interstitial inflammatory cells infiltrate (arrowhead), and many congested vasculatures (red star) (Fig. 7B). EG group showed moderate alternated records of tubular degenerative changes (red arrow) with lost luminal border integrity, moderate tubular dilatation (black star) accompanied with occasional figures of intraluminal casts (yellow star) and congested vasculatures (red star) (Fig. 7C). Furthermore, CP group showed marked tubular cystic dilatations as well as dilatation of Bowman’s spaces (black star) with mild degenerative changes of the tubular epithelium (red arrow), focal moderate figures of intraluminal casts (yellow star), and moderate records of interstitial inflammatory cell infiltrates (arrowhead) which is consistent with the findings of other investigations Essa et al. (2024), who reported that, concerning the results of histopathological studies, it indicated good effects of CP on the kidney and liver (Fig. 7D)⁵⁷. Unloaded ZnONPs samples showed mild focal tubular degenerative changes of lining epithelium (red arrow) with mild dilatation as well as mild dilatation of Bowman’s spaces (black star) accompanied by mild records of interstitial inflammatory cells infiltrates (arrowhead) and occasional sporadic records of luminal casts in some tubular segments (yellow star) (Fig. 7E). The alterations in the nucleus of the lining cells of renal tubules were less pronounced in the ZnO@CP NEs group. Almost the same records as the normal group without abnormal histological changes (Fig. 7F). In accordance with previous studies by El-Shenawy et al. 2019⁵⁸. There was a significant improvement in renal histological abnormalities in the rats treated with ZnO@CPNEs; nevertheless, there was a minor deterioration of the renal epithelial tubes and glomeruli, as well as localized regions of less widespread glomerular necrosis. Zinc oxide nanoparticles have been shown in previous studies to be capable of facilitating the repair of epithelial cells⁵⁹. According to Bashandy et al. (2018) ZnO NPs exhibiting free radical scavenging activity resulted in a reduction of lipid peroxidation and an enhancement of antioxidant enzymes (CAT and SOD), which contributed to the improvement of renal injury and a decrease in pathological changes observed in the ZnO@CP NEs group of rats⁶⁰. The results align with⁶¹ which also reported that ZnONPs have no harmful effect on kidney tissue.

Furthermore, ZnO@CP NEs possibility mechanism inhibits calcium oxalate (CaOx) crystallization maybe due to ZnO@CP NEs functions as a multi-modal crystallisation antagonist that operates simultaneously in the bulk solution and at the renal-cell interface as, (1) Aggregation disruption: ZnONPs obtained via chemical-mediated synthesis retains surface hydroxyl groups (–OH) that, at urinary pH (~ 6), are protonated to –OH₂⁺, giving the particles a positive charged⁶².When the oil nano-emulsion was linked onto the surface of ZnO@CP NEs, polyphenols from C. proximus (chlorogenic acid, isoorientin, caffeic acid)⁶³. displace these labile protons and create a dense, negatively charged corona (ζ ≈ − 25 mV). which can create an anionic shell that repels negatively charged oxalate (C₂O₄²⁻) via electrostatic interactions. This phenomenon contributes to the prevention of calcium oxalate kidney stones by reducing the aggregation and facilitating renal clearance, though other mechanisms like antioxidant and anti-inflammatory activities also play a role⁵⁷. (2) The administration of ZnO nanoemulsion had noticeable effects, including increased urine volume and pH, a reduction in urinary calcium oxalate crystals, decreased excretion of calcium, oxalate, and phosphate, and enhanced elimination of urinary magnesium. These effects might arise from the dual functionality of C. proximus, known for its diuretic and renal antispasmodic properties⁶⁴. (3) Anti-oxidative epithelial protection: The potent antioxidative activity found in C. proximus, attributed to bioactive compounds like flavonoids and carotenoids⁶⁵. The potentially enhances antioxidant defenses, raises kidney glutathione levels, and aids in kidney tissue restoration following nephrotoxicity⁶⁶. Additionally, the anti-inflammatory properties of C. proximus and its ability to protect nephrons might also contribute to these observed effects⁶⁷. Furthermore, calcium oxalate monohydrate (COM) crystals trigger NADPH-oxidase-dependent ROS production. The ZnO core scavenges superoxide via conduction-band electrons (e_CB⁻ + O₂•⁻ → •O₂⁻), while the polyphenol mantle quenches hydroxyl radicals by hydrogen-atom donation. The combined antioxidant effect suppresses redox-sensitive transcription factors (NF-κB, AP-1), down-regulating osteopontin and MCP-1 expression and thereby reducing crystal adhesion to renal tubular cells⁶⁸. On the other hand, following endocytosis, the mildly acidic lysosomal environment (pH 5.5) dissolves < 5% of the ZnO mass, raising intracellular Zn²⁺. This concentration up-regulates metallothionein-1 and Nrf2, reinforcing cellular antioxidant defenses and inhibiting oxalate-induced apoptosis without cytotoxicity⁶⁹. Collectively, these complementary actions convert the bulk urinary environment into a metastable zone unfavourable to CaOx crystallisation while simultaneously shielding renal epithelia from crystal-mediated injury.

Histological changes of kidney structure in different experimental groups, stained with H&E (A) NC group, rats were orally saline (5 ml/k g body weight). (B) In group II PC group, rats were orally 1% ethylene glycol. (C) Group III, EG group, rats were orally with cystone (100 mg/kg body weight). (D) Group IV, rats with kidney stones treated with CP, orally (5 mg/k g body weight) divided into two times a day. (E) Group V, rats with kidney stones treated with ZnO NPs, orally and 200 µg/ml divided into two times a day (F) Group VI, rats with kidney stones treated with ZnO@CP NEs (250 µg/ml).

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Author notes

1. These authors contributed equally to this work: Sherif M. Ragab and Mohamed Sharaf.

Authors and Affiliations

1. Department of Biochemistry, Faculty of Agriculture, Al-Azhar University, Nasr, Cairo, 11751, Egypt

   Mohamed Sharaf, Sherif M. Ragab, Zakaria H. Saad & Waled Abd-Elhamed

2. Department of Medical Laboratories Techniques, College of Health and Medical Techniques, Al-mustaqbal University, Hilla, 6163, Iraq

   Ameer Mezher Hadi

3. Dairy Department, Faculty of Agriculture, Al-Azhar University, Cairo, 11751, Egypt

   Abd El-Mageed M. M & El-Sayed A. G. M

4. Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Suez Canal University, Ismailia, 41522, Egypt

   Tamer H. Hassan

5. Biology Department, College of Science, Imam Mohammad ibn Saud Islamic University (IMSIU), P.O. Box 90950, Riyadh, 11623, Saudi Arabia

   Amr Elkelish, Mohammed AL-Zharani & Hassan Rudayni

6. Departement Biomédical et Santé publique, Institut de Recherche en Sciences de la Santé́(Santé́Santé́(IRSS), Centre National de la Recherche Scientifique et Technologique (CNRST), Ouagadougou, Burkina Faso

   Abdou Azaque Zoure

Contributions

M.S, S.R, S.Z, W.A, A.M, A.S, T.H, A.E, H.A, and A.Me wrote the main manuscript text and S.Z, W.A, A.M, A.S, T.H, A.E, H.A, prepared figures. All authors reviewed the manuscript.

Corresponding authors

Correspondence to Amr Elkelish or Abdou Azaque Zoure.

Competing interests

The authors declare no competing interests.

Ethics approval

All animal experiments were performed following the guidelines and approved by the Animal Ethics Committee of the Faculty of Pharmacy, Suez Canal University, Egypt [approval number: 202208MRA1], and all procedures were carried out in accordance with the applicable rules and regulations. The study was carried out in accordance with ARRIVE guidelines.

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