MaterialsChemicals and plant materials

Ethylene glycol and ammonium chloride were purchased from Sigma Co. (Poole, Dorset, UK); medium-viscosity sodium alginate (3500 cps); calcium chloride CaCl2 was supplied from Merck Chemicals Co. and was used as the crosslinker; polysorbate 80 Fenton Chemicals. ENDORE, MP.

Cymbopogon proximus, Petroselinum crispum seeds, and GA were purchased from the local market in Giza, Egypt.

Animals, diet and ethical statement

Twenty-four male Sprague Dawley (SD) rats weighing between 182 and 200 g (3–4 months) were sourced from the Animal House at the Veterinary Medicine faculty in Egypt. Stringently adhering to the National Institutes of Health Guide for the Care and Use of Laboratory Animals (Publication No. 85 − 23, revised 1985), all procedures and protocols concerning the care and handling of the animals were followed. Furthermore, ethical clearance for the present study was granted by the ethics committee of Veterinary Medicine at Cairo University, Egypt (ethical approval number: [Vet CU 09092023754]. Upon arrival, the rats underwent a one-week acclimatization period before being sorted into four groups (n = 6 per group). Each rat was housed in an individual polypropylene cage, ensuring optimal living conditions. Environmental parameters were meticulously regulated, maintaining a controlled temperature range of 24 ± 2 °C and relative humidity levels between 40% and 60%. The rats were subjected to a consistent 12-hour light-dark cycle and were allowed unrestricted access to both food and water throughout the entire duration of the study.

The diet employed in this study adhered to the AIN-93 diet guidelines [23], ensuring a well-balanced nutritional composition. The diet consisted of specific components: 12% of the total composition derived from casein as a protein source, 10% from corn oil, 10% from sucrose, 58.5% from maize starch, 5% cellulose, 3.5% from a salt mixture, and 1% from a vitamin mixture. Both the salt and vitamin mixtures were meticulously formulated in accordance with the AIN-93 guidelines [23].

MethodsPreparations and characterization of emulsion and nanogel emulsionPreparation of crude ethanol extracts of C. Proximus and P. Crispum seeds

The air-dried aerial parts of C. proximus and P. crispum seeds were initially cleaned and subsequently ground into a course powder using an electric grinder. Fifty grams (50 g) of each plant powder was soaked and stirred with an electric blender separately in 200 ml of ethyl alcohol 80% at a ratio of 1:4 (powder/solvent). Then, it was transferred into an airtight plastic container and kept for at least 48 h. Then, the mixture was filtrated and the solvent was removed using a vacuum rotary evaporator and the extract will be obtained [24]. The produced extract will be saved in a refrigerator at 4 oC to avoid any contaminations until the time of use.

Preparation of an emulsion using ethanolic extracts of C. Proximus and P. Crispum seeds with GA

To prepare an emulsion from P. crispum seeds and C. proximus ethanolic extracts with GA, 4 g of GA powder were dissolved in distilled water at a concentration of wt/v %. Then the GA solution was blended with P. crispum seeds and C. proximus extracts with blend ratio of 1:1:1. The obtained emulsion was stirred for 2 h using a magnetic stirrer until completely mixed.

Phytochemical analysis using gas chromatography-mass spectrometry (GC/MS)

The phytochemical analysis of the ethanolic extracts from C. proximus with P. crispum seeds was conducted via GC/MS. The analysis was performed using a fused silica capillary column (30 m, 0.251 mm, 0.1 mm film thickness) with a Thermo Scientific Trace GC Ultra/ISQ Single Quadrupole MS, TG-5MS. Helium gas was utilized as the carrier gas at a steady flow rate of 1 mL/min in an electron ionization device with an ionization energy of 70 eV for GC/MS detection. The temperature of the injector and MS transfer line was adjusted to 280 oC. The oven was set to start at 45 °C and hold it for two minutes. It was then programmed to increase to 150 °C at a pace of 7 °C per minute. After that, it was set to increase to 270 °C at a rate of 5 °C per minute (hold it for two minutes) and finally to 310 °C as the final temperature at a rate of 3.5 °C per minute (hold for 10 min). A percent relative peak area was used to evaluate the quantification of all the components that were discovered. Based on a comparison of the compounds’ relative retention times and mass spectra with the NIST, and WILLY library data of the GC/MS instrument, a preliminary identification of the compounds was carried out.

Preparation of a nanogel emulsion using ethanolic extracts of C. Proximus and P. Crispum seeds with GA

The methodology of Sundararajan et al. [25] was modified to prepare nanogel emulsions using concentrated extracts of C. proximus and parsley seeds. Nanogel emulsion quantification was developed using a low-energy method. GA (4% wt./v) was prepared by dissolving the required amount in deionized water and stirred by a magnetic stirrer (800 rpm/1 h). Also, 2% (wt./v) sodium alginate was prepared, added to the GA solution, and stirred for 1 h until completely dissolving. In addition, the two extracts were added in the same concentration ratio. Four grams of each extract were dissolved in fifty milliliters of 50% v/v ethanol to create the extract solution. 2% polysorbate 80 was then added, and the mixture was stirred. To the previously prepared coarse extract polysorbate the resultant aqueous GA alginate solution was added dropwise at a flow rate of 1 mL/min. 7.5 ml of 18 Mm of aqueous CaCl2 solution was added dropwise (140 ml/30 sec) to the gum Arabic alginate solution under gentle stirring before addition to the extract polysorbate solution. The mixture was agitated for ten hours at 1200 rpm. After that, the nano gel emulsion was maintained at room temperature (20 °C), and its stability was assessed [26]. Figure 1(a and b) shows a macroscopic study of the prepared gum Arabic solution and the two-extract mixture respectively. Furthermore, Fig. 1 (c and d) shows the difference between the prepared micro and nano emulsions of GA, C. proximus and P. crispum seed extracts.

Fig. 1

Macroscopic studies of (a) GA, (b) C. proximus and P. crispum seeds extracts, (c) C. proximus and P. crispum seeds extracts/GA emulsion, (d) C. proximus and P. crispum seeds/GA nanogel emulsion

Particle size analysis and zeta potential measurement

The average size distribution and zeta potential of the GA loaded with C. proximus and P. crispum seed extracts nanogel emulsion were determined by dynamic light scattering (DLS) with a Zeta Sizer (Malvern Instruments, UK) and a Zeta Potential analyzer (Nicomp 380 ZLS, USA), respectively. Measurements were made on the aqueous suspension of the NPs. The emulsion sample (2 drops) was diluted in 2 mL of water and put into a cuvette to measure the particle size. A capillary cell (25 µL) held the sample for the zeta potential measurement, which was subsequently diluted in two milliliters of water. The Stokes-Einstein relation and its corresponding polydispersity index (PDI) were used to calculate the particle size as a Z-average. Zeta potential and particle size were measured three times for every sample.

Design of the experimental studyInduction of urolithiasis

The evaluation of antiurolithiatic activity was conducted using a urolithiasis animal model induced by the simultaneous administration of ethylene glycol and ammonium chloride in male SD rats. This experimental approach is as per earlier reported methods, with some modifications.

In a 14-day protocol involving the provision of EG at a concentration of 0.75% v/v and AC at a concentration of 1% w/v supplied through drinking water ad libitum access, this induction protocol aimed to induce urolithiasis, thereby stimulating the formation of calcium oxalate crystals [27].

Preventive study model: dosing and grouping

The rats were divided into four groups (n = 6) and orally administered either the emulsion or nanogel emulsion using an oral tube. Throughout the experimental study, all rats had ad libitum access to both drinking water and diet.

Group 1: In the normal control group, rats had ad libitum access to drinking water for 21 days.

Group 2: Urolithiasis group: rats had unrestricted access to drinking water for 21 days. Starting from day 8 and continuing until day 21, they were given drinking water containing 0.75% EG V/V along with 1% AC W/V to induce urolithiasis.

Group 3: Emulsion group, rats were orally administered C. proximus and P. crispum seeds ethanolic extracts/GA emulsion (600 mg/kg b.w./day) for 21 days. From day 8 to day 21, they received a dose of 0.75% EG V/V + 1% AC in their drinking water.

Group 4: Nanogel emulsion group: rats were orally administered C. proximus and parsley seeds ethanolic extracts/GA nanogel emulsion (600 mg/kg b.w./day) for 21 days. From day 8 to day 21, they received a dosage of 0.75% EG V/V + 1% AC in their drinking water.

Fig. 2

Illustrates a schematic diagram depicting the experimental design for inducing urolithiasis in rats through EG and AC in drinking water for 14 days. Daily oral administration of either the emulsion (600 mg/kg) and nanogel emulsion (600 mg/kg) was conducted for 21 days. Following the last treatment, urine samples were collected from each rat. Twenty-four hours later (day 22), blood samples were obtained. Finally, the rats were sacrificed for kidney harvesting

Body weight and food intake measurement

During the study period, all groups of rats received a balanced diet. Weekly records were kept for each rat, documenting their body weight and food intake. At the end of the study, we calculated the total food intake, body weight gain, and feed efficiency ratio.

Collection and assessment of urine samples

Individual metabolic cages were assigned to each rat for the collection of 24-hour urine samples on the 21st day of evaluation, the rats had unrestricted access to drinking water. Following the urine collection, immediate assessments were made for urine pH using pH test strips (Macherey-Nagel GmbH & Co. KG, Düren, Germany) and urine volume. A drop of urine was dispensed onto a clean glass slide and examined under a light microscope (Olympus microscope, model CX41, Japan). Photomicrographs were captured for each urine sample. The micrographs were subsequently employed to estimate the quantity of crystals present. The grading system for the severity of crystalluria was as follows: “-” indicating no crystals observed; “+” denoting 1 to 5 crystals per high power field (x400); “++” indicating 6 to 20 crystals per high power field; and “+++” indicating more than 20 crystals per high power field.

The 24-hour urine samples were divided into two halves and stored at -20 °C. Prior to storage, one half was acidified with 1–2 drops of 5 M HCl, while the other remained non-acidified. Urine parameters including urea (Fawcett and Scott,1960) [28], creatinine (Larsen, 1972) [29], uric acid (Barham and Trinder, 2009) [30], total protein (Rheinhold, 1953) [31], albumin (Doumas et al., 1971) [32], were analyzed from the non-acidified urine samples. The acidified sample was employed for the assessment of urinary biochemical parameters such as calcium (Kessler and Wolfman) [33], phosphorus (El-merzabani et al.) [34], potassium (Sunderman) [35], sodium (Henry et al.) [36], and magnesium [37], all of which were colorimetrically assayed using standard commercial kits acquired from Salucea Co., Netherlands. These procedures were conducted in accordance with the manufacturer’s instructions. Urinary oxalate levels were determined using a quantitative rat oxalate colorimetric assay kit (Elabscience Biotechnology Co., Ltd., China, catalog No. E-BC-K892-M) at a wavelength of 550 nm for urinary oxalate analysis, following the manufacturer’s instructions.

Blood sampling and kidney collection

Twenty-four hours after the final treatment, each rat underwent anesthesia, administered as a combination of ketamine (100 mg/kg) and xylazine (10 mg/kg) following an overnight fasting period. Approximately 2 ml of blood was then drawn from the retro-orbital venous plexus of the rats’ eyes and collected in clear test tubes. Subsequently, the blood samples underwent centrifugation at 3000 rpm/min at 4 °C for 10 min using a laboratory centrifuge (2k15, Sigma, Germany). The resulting sera were stored at -20 °C until further analysis of the biochemical parameters.

Following euthanasia by cervical dislocation, their kidney specimens were promptly excised, immersed in a cold saline solution (0.9% NaCl), and gently blotted dry using filter paper. The specimens were then weighed.

The left kidney specimens, fixed in 10% neutral buffered formalin, were prepared for histopathological and immunohistochemistry examinations. Simultaneously, the right kidney specimens were immersed in cold phosphate buffer (0.1 M; pH 7.4) to create a 20% homogenate using a tissue homogenizer (MPW-120, BitLab Medical Instruments, Poland). This homogenization process was followed by centrifugation at 4000 rpm/min for 10 min at 4 °C. The resulting supernatant was carefully collected and stored at -80 °C for subsequent measurement of CAT, GSH, and MDA levels. All experimental procedures strictly adhered to approved ethical guidelines.

The kidney index was calculated using the following formula: kidney index = (kidney weight/body weight) ×100.

Assessment of serum and renal biomarkers

Serum kidney function indices, including urea (Fawcett and Scott,1960) [28], creatinine (Larsen, 1972) [29], uric acid (Barham and Trinder, 2009) [30], total protein (Rheinhold, 1953) [31], albumin (Doumas et al., 1971) [32], calcium (Kessler and Wolfman, 1964) [33], phosphorus (El-merzabani et al., 1977) [34], potassium (Sunderman, 1958) [35], and sodium (Henry et al., 74) [36], were assessed using commercial kits from Salucea Co., Netherlands. MDA levels were determined according to the method of Satoh, 2022 [38], while CAT and GSH were analyzed following the method outlined by Luck, 1978 and Sedlak & Lindsay, 1968 [39, 40], respectively. These analyses utilized a commercially available colorimetric kit (Biodiagnostics, Egypt; catalog no. MD 25 29, CA 25 17, and GR 25 11) according to the manufacturer’s instructions. Optical density measurements for all parameters were conducted using a spectrophotometer (Shimadzu UV-2401 PC, Australia).

A. Histopathological and immunohistochemical evaluations

The kidney specimens, preserved in formalin, underwent a standard processing procedure involving dehydration in varying alcohol concentrations, clearance in xylol, and embedding in paraffin. Subsequently, thin sections measuring 4–5 μm were obtained from the paraffin blocks, followed by staining using Hematoxylin and Eosin (H&E) [41].

For immunohistochemical assessment, the paraffin sections underwent antigen retrieval through microwave heating for 25 min at 720 W. Post-retrieval, they were incubated overnight at 4 °C with primary antibodies: rabbit monoclonal anti-rat caspase-3 (1:1000 dilution, Abcam, ab184787, Cambridge, MA, USA), and mouse monoclonal anti-rat TNF-α (1:500 dilution, Abcam, ab220210, Cambridge, MA, USA). Following PBS washing, sections were then incubated at room temperature for 30 min with respective biotinylated secondary antibodies at a 1:200 dilution (Dako Corp.), and streptavidin/ALP alkaline phosphatase complex, also at a 1:200 dilution (Dako Corp.). Visualization of antibody binding sites was accomplished using DAB (Sigma), followed by PBS washing and counterstaining with Hematoxylin for 2–3 min. The final steps involved sample dehydration in escalating ethanol solutions, double xylene soaking for 5 min each at room temperature, mounting, and examination under a high-power light microscope [42].

Quantification of marker expression’s positive brown area was conducted by measuring the percentage area across seven high-power microscopic fields using Image analysis software (Image J, 1.46a, NIH, USA).

B. Renal crystal deposition

The evaluation of renal crystal deposits followed a graded scale: zero indicated the absence of crystal deposits, 1 denoted the presence of crystal deposits at the papillary tip, 2 indicated crystal deposits at the cortico-medullary junction, and 3 signaled crystal deposits in the cortex. In instances where crystals were found in multiple locations, the scores were aggregated to derive a comprehensive final score [43].

Statistical analysis

Statistical analyses were conducted using SPSS version 25. The results obtained from the animal experiment were expressed as mean ± standard error (SE) and subjected to statistical scrutiny using one-way analysis of variance (ANOVA), followed by the Duncan test for post hoc analysis. A significance level of P ≤ 0.05 was applied to ascertain statistical significance. In instances where frequency data were involved, the Kruskal-Wallis H test, a nonparametric analysis, was employed, followed by the Mann-Whitney U test for further assessment. Median values were reported for the nonparametric data.