Pentylenetetrazol (PTZ) was administered orally (35 mg/kg, sub-convulsive) up to 11 doses on alternate days for 22 days. Following a injected parenterally, the animals were monitored for 30 min. PTZ-induced seizures were assessed and graded using the Fischer and Kittner’s methodology (Fischer, 1998).

No convulsive activity was observed

After each PTZ sub-convulsive dose, the mean of seizure score was calculated (Saleem et al., 2022).

Irrespective of gender, Swiss albino mice were split into six groups, each having six animals (n = 6). PTZ, 35 mg/kg, was administered orally every alternate day for 22 days to group II, the disease control group, whereas group I, the normal control group, received 0.5 mL (0.9%) normal saline. Group III (standard control) was administered diazepam, (2 mg/kg, orally) every alternate day for 22 days. Animals of treatment groups IV, V, and VI were administered Rosa moschata leaf extract (RMLE) orally at the doses of 150 mg/kg, 300 mg/kg, and 600 mg/kg. On the starting, in the middle, and at end of the study, changes in weight and behavior were noted. On the 22nd day of administration of the last dose, blood samples of all animals were collected to perform hematological and biochemical analyses, and animals were sacrificed by cervical dislocation under light anesthesia (3% isoflurane). In order to test oxidative stress biomarkers (levels of catalases, superoxide dismutase [SOD], malondialdehyde [MDA], glutathione [GSH], and glutathione peroxidase (GPx)) and neurotransmitters (dopamine, noradrenaline, and serotonin), the brain from each group was isolated. Normal saline was used for rinsing brain samples and preserved at pH 7.4 in cold phosphate buffer. Tissues of segregated vital organs were sliced and conserved in 10% formalin to ascertain histopathology (Saleem et al., 2022).

Animals in each group were examined and behavioral experiments were conducted to observe treatment impact on cognition, memory, learning, neuromuscular coordination, and locomotor functions after 22 days of the study. Following the behavioral tests, brain tissues of the animals were extracted, cleaned with cold normal saline, and kept at -80°C for additional biochemical analysis prior to animal scarification.

It was performed to examine how RMLE and PTZ could affect memory recall. The device was a circular pool, 45 cm in height, 150 cm in diameter, and 30 cm in depth. The water pool had a central platform and four equally sized quadrants and its temperature was 28°C. The animals were placed on a platform that was visible and accessible and trained to get out of water. The platform was then turned invisible to determine final readings. Animals were gently placed in a water pool from various angles and provided 120 s to escape. The escape latency was recorded (Othman et al., 2022).

Elevated pulse maze (EPM) test was conducted to examine the learning and cognitive capacities of mice. The apparatus included an open ceiling and two opposed open and shut arms of 50×10×40 cm. By raising the arms, mice were put near the border of the open arm to gauge the amount of anxiety. Time spent in opening and closing the arms was recorded on days 8 and 2 (Hooijmans et al., 2023).

The foundation of Y-Maze activities is the natural habit of mice to alternate arms when investigating a new location. The purpose of this activity was to examine short-term memory. To assess spatial working memory, a spontaneous alternation task was employed. Through the use of their natural curiosity, rodents explored unexplored arm, which was thought to be a modification to other arms. Y-Maze with spontaneous modification version was used. This labyrinth was open to animals for 6 to 8 min. Measurements included total arm inputs, total arms, finished triads, and alteration rate (Ju et al., 2022).

The main purpose of this test was to evaluate the mice’s ability to move, their automatic functions, and their exploratory behavior. The apparatus comprised square wooden boxes painted with white resin on a glass on one side wall to watch animal’s movement and activities. The floor of the open field apparatus was separated and equally divided into 16 squares to distinguishing central squares. Mice were gently placed at the center and their movements were observed. Different parameters, for example, total number of lines crossed, rearing, grooming, defecation, urination, cleaning, freezing behavior, and inclination for a specific quadrant were observed (Saleem et al., 2019).

Investigating neuromuscular power of rodents is an easy undertaking. To track neuromuscular activity in the forearm, a hanging test was conducted. Stainless steel rods, raised 150 cm from the ground on wooden supports, were used to make the device. Each treatment group’s execution of mice with stainless steel rods was timed individually (Saleem et al., 2019).

Brain tissues were isolated after completion of experimental period. These were washed and preserved in a biomedical freezer (-80°C) using ice cold normal saline. A 1:10 fraction of phosphate buffered saline (PBS) was used to create a homogenized solution of brain tissues. To obtain clear supernatant, the solution was continuously centrifuged for 10 min at 4,000 rpm and 4°C (Saleem et al., 2019).

Trichloroacetic acid (TCA, 10%) was added into 1-mL homogenate. This combination was further processed by adding 5,5 dithiobis-2-nitrobenzoic acid (DTNB) to make it soluble. This was achieved by first adding sodium phosphate buffer solution (0.1 M), 4 mL, pH 7.4, to the mixture. The amount of GSH was measured using an absorbance measurement at 412 nm (Saleem et al., 2022).

The solution mixture contained brain homogenate (0.1 mL), sodium azide (0.2 mL), ethylenediaminetetraacetic acid (EDTA), and hydrogen peroxide (H₂O₂). After adding TCA to halt the reaction, the supernatant was separated by centrifuging at 2,000 rpm for 10 min. After adding 0.5 mL of DTNB and 4 mL of sodium hydrogen phosphate (disodium phosphate), the top layer was separated and coalesced. The absorbance was measured at 412 nm (Saleem et al., 2022),

Malondialdehyde concentration was used to gauge the degree of lipid peroxidation. Thiobarbituric acid combination served as its foundation. Centrifugation of homogenate was done for 5 min at 1,500 rpm for supernatant. Supernatant, 1 mL, and 3 mL of TBA were combined. In order to initiate the reaction, 0.38% weight/weight (w/w) TBA, 2.5 mL of 0.25-M HCL, and 15% TCA were added. The assay mixture was vigorously shaken for 15 min, and kept in an ice bath of chilled water. Then, the test fluid was centrifuged at 3,500 rpm for 5 min. In order to assess the upper coating of the solution, a spectrophotometer set at 532 nm was used (Saleem et al., 2022). MDA was determined through subsequent formulary:

where

Vt = assay mixture total volume (4 mL),

Vu = aliquots volume,

Y = absorbance,

1.56 × 10⁵ = extinction molar coefficient,

Wt = brain weight (in g)

The tissues homogenate supernatant (0.1 mL) was added to create the mixture product. Phosphate buffer, 2.8 mL, and 10 mL of pyrogallol solution were used. The saturation of the solution was evaluated at 312 nm using UV spectrophotometer. Regression line equation of standard SOD activity was used to determine SOD levels (Saleem et al., 2022).

In this procedure, 250-µL phosphate buffer with pH 7.0, 250 µL of brain homogenate, and 0.03 mol/L H₂O₂ were added in reaction mixture. After an incubation of 5 min, titinyl sulfate was added and absorbance was measured at 420 nm (Gharaghani et al., 2022).

Catalase (CAT) activity was determined by the resulting formulary:

where

δ0.D: change in absorbance per minute

E: H₂O₂ extinction coefficient (0.071 mmol cm^-1)

Elman’s reagent, 100 µL, acetylthiocholine iodide (AChl), 20 µL, and 0.4 mL of brain homogenate were combined with 2.6 mL of phosphate buffer (pH 8.0). The absorbance was noted at 412 nm

where

A = absorbance shift/60 s,

Co = Brain homogenate concentration,

R = Substrate hydrolyzed/min/g tissue.

Tissue homogenate (0.2 mL) was mixed with 4.5 mL of Reagent 1 for estimation, and the resulting product combination was incubated for 10 min. Reagent 2 (1 part of 2N Folin-phenol and 1 part of H₂O) 0.5 mL was added after the mixture was incubated for 30 min. Spectroscopic investigation of this solution was carried out at 660 nm. Different BSA concentrations were utilized to create regression line (Kejriwal et al., 2014).

For nitrite level estimation, 2 mL each of brain homogenate and Griess reagent were mixed in a test tube. The Griess reagent was prepared by dissolution of equal amount of 2.5% phosphoric acid, 0.1% N-1-naphthyl ethylene amine dihydrochloride, 1% sulfanilamide, and homogenate. Absorbance was calculated at 546 nm after 10 min of incubation.

The homogenates of brain tissues were precisely produced using 5 mL of HCl butanol. Then, 10-min centrifugation was carried out at 2,000 rpm. After centrifugation, the supernatant was mixed with 0.31-mL HCl and 2.5-mL heptane, rapidly shaken, and centrifuged again for 10 min at 2,000 rpm. The mixture was separated into two layers following centrifugation, and the aqueous layer at the bottom (0.2 mL) was used for further analysis of neurotransmitter. Both, the aqueous and organic layers were separated during centrifugation. Aqueous layer was used to determine the amount of neurotransmitters (Saleem et al., 2021).

Serotonin was estimated by adding equal volume of aqueous phase, that is, O-phthaldialdehyde (0.25 mL). It was heated at 100ºC for 10 min. HCl was used as a blank. Absorbance was noted at 440 nm (Rahman and Eswaraiah, 2008).

Aqueous segment, 0.2 mL, was combined with EDTA solution (0.1 mL) and 50 μL of 0.4-M HCl. Addition of 0.1 mL Na₂SO₃ (100 μL) caused oxidation, which was then incubated for 15 min. Addition of 100 μL of iodine solution began the oxidation process. After that, 0.1 mL of acetic acid was added and the mixture was kept at 100°C. After cooling of the reaction mixture, absorbance was measured for both neurotransmitters at 350 nm and 450 nm, respectively. Before adding iodine solution, the blank sample was generated by adding Na₂SO₃ (Miziak et al., 2022).

Phosphate-buffered salt solution, 2.6 mL, with pH 8, was mixed in 100 µL of 2,4 dithiobisnitrobenzoic acid (DTNB) and 20 µL of AChl with a little amount of brain homogenate (0.4 mL). 2,4dithiobisnitrobenzoic acid (DTNB) created yellow coloration with thiocholine, and absorbance was determined at 412 nm (Saleem et al., 2021),

Where

Cο = tissue concentration,

A= absorbance change.

Cervical dislocation caused death of animals. The brain samples of all the animals were removed. Other vital organs of each animal, such as the heart, liver, lungs, spleen, and kidney, were also removed and weighed separately on weighing scales. All organs, including brain samples, were preserved in 10% formalin solution. Slides were prepared for the microscopic examination of cell damage of the brain and other vital organs, and digital pictures were taken with an optical microscope under polarized light (Saleem et al., 2022).

The wire hanging test results showed that compared to the standard group and animals in the RMLE 150, 300, and 600 mg/kg treatment groups and the control group, animals in the PTZ treatment group significantly (p < 0.05) fell from the wire hanging apparatus, demonstrating how RMLE therapy protected neuromuscular strength in a dose-dependent manner (Figure 2).

It was possible to study exploratory behavior, anxiety, and movement of the kindled epileptic animal model by providing open field environment. Compared to the control group, the kindled seizure model’s locomotion was reduced considerably (p < 0.05). RMLE therapy, however, significantly reduced the animals’ sad and aberrant neuropsychiatric behavior, which was not significantly different from the diazepam group. RMLE treatment groups underwent exploration while recovering in a dose-dependent manner (Figure 3).

Following the training session, measurements of animals’ escape latency were conducted on day 16, 18, 20, and 22. It was shown that kindled model mice took considerably (p < 0.05) more time to locate submerged platform and exit water. Compared to the standard group, treatment groups, and control groups, the escape latency of the intoxicated group on day 22 was noticeably longer with more time spent near the edge of the pool (Figure 4).

The kindled mice’s cognitive function was evaluated on day 22 of the experiment, and it was discovered that the transfer latency of PTZ-insulated mice was considerably (p < 0.05) stretched, compared to the groups that received the vehicle, and standard drug R. moschata 150, 300, and 600 mg/kg treatments. The performance of animals in terms of locomotion, exploration, and cognition was significantly improved by the RMLE therapy (Figure 5).

To assess mice cognitive capabilities and propensity for exploration, the Y-maze experiment was used. We observed and kept track of the total the number of triads, number of arm entries, and the proportion of spontaneous modifications. Mice that received PTZ displayed a severe loss in cognition and mobility. PTZ-treated mice had considerably (p < 0.05) less arm entries, triads, and spontaneous alteration proportions, compared to the control, RMLE 150, 300, and 600 mg/kg treatment, and diazepam-treated mice groups. The current research showed that RMLE medication significantly reversed cognitive deterioration in a dose-dependent manner (Figure 6).

Oxidative stress was brought on by PTZ therapy in the kindling mice model. The concentrations of the first-line antioxidant enzymes decreased considerably (p < 0.05 ). RMLE therapy (600, 300, and 150 mg/kg), however, (p < 0.05) restored considerably in a dose-dependent manner the levels of SOD, GSH, CAT, and proteins of treatment groups. In the kindling model, PTZ administration substantially (p < 0.05) elevated the levels of lipid peroxidation and nitrite. Treatment with RMLE reduced MDA and lipid peroxidation levels in a dose-dependent manner, although not significantly, compared to the control group (Table 1).

In estimation of catalase level, considerable (p < 0.001) decrease was found in the disease control group, compared to the control group. The diazepam group meaningfully (p < 0.01) overturned alteration in catalase level. When RMLE extract was administered for consecutive 22 days, momentous (p < 0.001) dose-dependent increase was observed in catalase level at RMLE 150, 300 and 600 mg/kg (Table 1).

Pentylenetetrazol caused momentous (p < 0.001) lessening of the GSH level in brain homogenate of disease animals, compared to the control group. GSH level meaningfully (p < 0.001) reached near to control value in standard group. The GSH level was significantly recovered in treatment groups (p < 0.001) at all dose levels shown in Table 1.

In disease animals, a noteworthy (p < 0.001) lessening of GPx level in brain tissue homogenate was observed, compared to the control group. GPx level meaningfully (p < 0.001) reached near to the control value in standard group. Substantial (p < 0.001) improvement was detected at RMLE dose of 150 mg/kg. Correspondingly, extract dose of 300 mg/kg (p < 0.01) and 600 mg/kg (p < 0.001) also presented substantial improvement (Table 1).

Noteworthy (p < 0.001) decline was observed in the MDA level of brain tissue homogenate of the disease-induced group, compared to the control group. MDA concentration expressively (p < 0.001) reached near the control value in the standard group. Significant (p < 0.001) improvement was detected at RMLE dose of 150 mg/kg. Extract dose at 300 mg/kg (p < 0.01) and 600 mg/kg (p < 0.001) also displayed substantial improvement when RMLE extract was administered for consecutive 22 days (Table 1).

Protein level decreased considerably (p < 0.001) in disease group animals. Standard control group significantly (p < 0.001) increased protein level. Significant improvement in total protein level was detected in the RMLE treatment groups of 150 mg/kg (p < 0.05), 300 mg/kg (p < 0.01), and 600 mg/kg (p < 0.001) (Table 1).

Owing to a considerable increase in the amount of acetyl cholinesterase (p < 0.05), the level of acetylcholine (ACh) was noticeably lowered in PTZ-affected mice. RMLE therapy, however, dose-dependently restored the level of ACh and cognitive function via modulating the level of acetyl cholinesterase in the treatment group that was nonsignificant in contrast to the disease control and control groups (Figure 7A).

Dopamine level measured in brain tissue homogenate decreased substantially (p < 0.001) in disease control animals. Dopamine level was restored significantly (p < 0.001) in standard and 600 mg/kg dose group. RMLE groups presented a substantial dose-dependent recovery of dopamine level (p < 0.05) at 150 mg/kg and (p < 0.001) at 300 mg/kg, compared to the disease control group (Figure 7B).

Decrease in serotonin level in epilepsy is correlated to mood disturbances and dyskinesia. Serotonin level pointedly (p < 0.001) reduced in PTZ-treated group, rather being normal in the control group. RMLE extract-treated groups at 150 mg/kg, 300 mg/kg, and 600 mg/kg showed significant improvement in serotonin levels. Standard group also showed remarkable (p<0.001) increase in serotonin level (Figure 7C).

The noradrenaline level estimated in brain tissue homogenate was considerably (p < 0.001) reduced in PTZ-treated groups, compared to the control group animals. Noradrenalin level was restored pointedly (p < 0.001) in the standard group. Animals treated with RMLE extract 150 mg/kg, 300 mg/kg, and 600 mg/kg exhibited a dose-dependent recovery of noradrenaline (p < 0.05), compared to the disease control group (Figure 7D).

The control group values of hemoglobin (Hb, 13.5±0.51), hematocrit (HCT, 7.82±0.02), and red blood cells (RBC, 47.4±0.57) remained within the normal range, compared to RMLE treatment animals (Hb, M: 12–17: F: 12–16, and HCT 35–55%). However, Hb (11.2±0.57), RBC (4.6±0.12), and hematocrit (42.3±0.57) dropped markedly (p < 0.001) in the PTZ treatment group. White blood cells (WBC, 13±0.57), neutrophils (77±0.52), lymphocytes (23.5±0.53), monocytes (05±0.32), and platelets (1,147±8.80) increased significantly (p < 0.001) in PTZ treatment group, compared to the control group values of WBC (10.6±0.32), neutrophils (69.5 ± 0.57), lymphocytes (18.3 ± 0.57), monocytes (03 ± 0.39), and platelets (344 ± 8.27), but were in normal range (Table 2). At various doses, RMLE extract had no negative impact on hematological markers.

Compared to the values of the control group (22±0.32), blood urea level (40±0.57) in the PTZ treatment group was increased significantly (p < 0.001). RMLE extract-treated groups at the doses of 150 mg/kg, 300 mg/kg, and 600 mg/kg showed notable (p < 0.001) respective decrease in blood urea level of 37±0.33, 29 ±0.57, and 24±0.53 (Table 3).

The serum creatinine level (1.2±0.02) in PTZ treatment groups was noticeably (p < 0.001) higher than the control group value (0.8±0.03). Treatment with RMLE at the doses of 150 mg/kg, 300 mg/kg, and 600 mg/kg resulted in significant dose-dependent (p < 0.001) lowering of the respective serum creatinine level of 1.1±0.02, 0.9±0.02, and 0.6±0.03 (Table 3).

The PTZ treatment group had a substantially higher serum bilirubin level (0.23±0.01) compared to the control group (0.9±0.06) (p < 0.001).

RMLE extract treatment at the doses of 150 mg/kg, 300 mg/kg, and 600 mg/kg resulted in a dose-dependent significant (p < 0.001) decrease in respective blood bilirubin levels of 0.20±0.04, 0.13±0.02, and 0.10±0.01 (Table 3).

In PTZ treatment group, alanine transferase (ALT) level (32 U/L) increased considerably (p < 0.001), compared to the control group (21 U/L). Alanine transferase level decreased considerably (p < 0.001) at RMLE extract doses of 150 mg/kg, 300 mg/kg, and 600 mg/kg (Table 3).

The analyzed control groups showed no granules according to histopathological analysis. However, hippocampus slices of the PTZ-treated group showed aberrant neuronal organization, neuronal loss and degeneration, presence of apoptotic bodies, proliferation of inflammatory mediators, and mild edema. However, RMLE treatment at doses of 150 mg/kg, 300 mg/kg, and 600 mg/kg significantly increased the structural integrity of the hippocampus (Figure 8).

Treatment with RMLE had no discernible negative effect on the histopathology of vital organs. The cardiac fiber architecture of the extract treated groups was similar to that of the control group. However, in the PTZ treatment group, vascular congestion in myocardial fibers was moderately severe. The histopathology of cardiac muscles demonstrated that RMLE had no adverse effect (Figure 9).

RMLE extract had no toxic effects on the histology of nephrons. The therapy groups exhibited normal renal parenchyma, much like the control group. However, renal parenchyma and kidney cell nuclei in the PTZ treatment group presented moderate levels of necrotic alterations and congestion (Figure 10).