Toxicity (acute and subacute) assessment and in-vivo antiurolithiatic activity of ethanolic extract of Caesalpinia bonducella seed in albino Wistar rat

INTRODUCTION Urolithiasis also termed kidney stone or nephrolithiasis, is a common worldwide disease with a high prevalence and recurrence rate. Chronic diseases, including diabetes mellitus, obesity, kidney disease, metabolic disorder, and hypertension, have a high tendency to form stones compared with healthy people (Besiroglu and Ozbek, 2019). It causes pyelonephritis or acute renal failure and severe acute back pain (Namburu et al., 2017; Yasui et al., 2017). Urolithiasis typically occurs between the age of 20 and 60 years and is more occurrences in hot climates. It affects nearly 10% of men and 6% of women over their lifetime. The recurrence rate is 50% within 5–10 years and 75% within 20 years of initial treatment (Dawson and Tomson, 2012; Yasui et al., 2017). The overall recurrence rate is high in male (70%–80%) as compared with female (47%–60%) (Ahmed et al., 2018; Wang et al., 2019). Approximately, 80% of stones are calcium-containing stones which are found as pure calcium oxalate (CaOx) (50%), pure calcium phosphate (1%), or a mixture of both (45%), and other stones are struvite (10%), uric acid (9%), and cystine (1%) (Bashir and Gilani, 2009; Namburu et al., 2017). CaOx (CaC2O4) stones are found as CaOx monohydrate or CaOx dehydrate form. Struvite stones are also called triple phosphate or infection stones or magnesium ammonium phosphate and develop Toxicity (acute and subacute) assessment and in-vivo antiurolithiatic activity of ethanolic extract of Caesalpinia bonducella seed in albino Wistar rat


INTRODUCTION
Urolithiasis also termed kidney stone or nephrolithiasis, is a common worldwide disease with a high prevalence and recurrence rate. Chronic diseases, including diabetes mellitus, obesity, kidney disease, metabolic disorder, and hypertension, have a high tendency to form stones compared with healthy people (Besiroglu and Ozbek, 2019). It causes pyelonephritis or acute renal failure and severe acute back pain (Namburu et al., 2017;Yasui et al., 2017). Urolithiasis typically occurs between the age of 20 and 60 years and is more occurrences in hot climates. It affects nearly 10% of men and 6% of women over their lifetime. The recurrence rate is 50% within 5-10 years and 75% within 20 years of initial treatment (Dawson and Tomson, 2012;Yasui et al., 2017). The overall recurrence rate is high in male (70%-80%) as compared with female (47%-60%) (Ahmed et al., 2018;Wang et al., 2019). Approximately, 80% of stones are calcium-containing stones which are found as pure calcium oxalate (CaOx) (50%), pure calcium phosphate (1%), or a mixture of both (45%), and other stones are struvite (10%), uric acid (9%), and cystine (1%) (Bashir and Gilani, 2009;Namburu et al., 2017). CaOx (CaC 2 O 4 ) stones are found as CaOx monohydrate or CaOx dehydrate form. Struvite stones are also called triple phosphate or infection stones or magnesium ammonium phosphate and develop Toxicity (acute and subacute) assessment and in-vivo antiurolithiatic activity of ethanolic extract of Caesalpinia bonducella seed in albino Wistar rat when the urease-producing bacteria such as proteus cause the urine pH higher than 7.2 by ammonia (urea convert in ammonia in presence of urease enzyme) (Ahmed et al., 2018).

Plant material and extraction
The plant seeds samples were collected from the local market in Varanasi, India. A voucher specimen was deposited (number 201901) and authenticated by Dr. Ashwani K. Kushwaha, Department of Dravyguna, Faculty of Ayurveda, Institute of Medical Sciences (IMS), Banaras Hindu University (BHU), Varanasi. The coarsely powdered seeds of C. bonducella (100 g) were soaked in 1 l of 95% ethanol for a week with occasional shaking. The crude extract was obtained by filtration through filter paper. The filtrate was evaporated by rotatory evaporator to give a viscous brownish dark mass and the extract was preserved in a refrigerator at 2°C-8°C for use in the experiments (Bashir and Gilani, 2009). The yield value of the extract was 6% (w/w). The extract was suspended in 0.5% carboxy methyl cellulose (CMC) for pharmacological screening.

Animals
Adult albino Wistar rats, 12 weeks old, 130-150 g bodyweight, were used for the study. They were placed under standard conditions (22°C ± 3°C) and allowed free access to standard food pellets and water. After 1 week of acclimatization, rats were used for the experiment. Rats were handled according to the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) guidelines and procedures were approved by the ethical committee of the Institute of Medical Sciences, BHU, Varanasi, India (2211, 07.11.2020 for the care and use of Laboratory Animals.

Acute oral toxicity
The acute toxicity was performed according to the Organisation for Economic Co-operation and Development (OECD) (423, December 17, 2001) test guidelines. Rats were divided into two groups (n = 3). Group I (control) received p.o. vehicle (0.5% CMC), while Group II was treated with ECB at a single dose of 2,000 mg/kg p.o.

Behavioral changes and mortality
Animals were observed for food intake, water intake, bodyweight, behavioral changes, seizure, hair loss, allergic reaction, hazardous symptoms, and number of deaths during the entire experiment.

Hematological and biochemical analysis
Animals were anesthetized with chloroform once the toxicity investigation was completed, and blood was taken via retro-orbital puncture with Ethylenediamine tetraacetic acid (EDTA) coated capillary tube. Hematological parameters were determined by Auto-Haematology analyser (Arkray Autocell Plus). The serum parameters were determined by using standard diagnostic kits.

Histopathological studies
After completion of toxicity study duration, all animals were anesthetized with chloroform and sacrificed. The organs, like liver, heart, lungs, and kidney, were collected and preserved in 10% formalin solution. The preserved organs were fixed in melted paraffin wax, then allowed to solidify. The tissue sections were prepared using a microtome (thickness of 4 µm). After staining with hematoxylin and eosin, the pathological changes of the tissue sections were examined and identified under microscope [Olympus Magnus Inverted (INVI) inverted microscope] in comparison with the control group (Ekanayake et al., 2016).

Sodium oxalate (SO) induced urolithiasis
The antiurolithiasis activity of ECB was evaluated by SO induced urolithiasis model (Araujo et al., 2020;Elhan et al., 2014;Tzou et al., 2016) with slight modification. Rats were divided into five groups (n = 5). Group I (normal control) received vehicle 0.5% CMC p.o. once daily for 21 days. Group II, III, IV, and V received SO (70 mg/kg per day, i.p.) for 7 days. Group II (induced control group) received vehicle 0.5% CMC p.o. once daily for next 14 days. Group III, IV, and V received p.o. 200, 400 mg/kg dose of ECB and 750 mg/kg dose of cystone, respectively, once daily for next 14 days.

Glycolic acid (GA) induced urolithiasis
The antiurolithiasis activity of ECB was evaluated by GA induced urolithiasis model (Mitra et al., 1998;Sathya and Kokilavani, 2012;Shehzad et al., 2021;Tzou et al., 2016) with slight modification. The rats were divided into five groups (n = 5). Group I (normal control) received vehicle 0.5% CMC p.o. once daily for 21 days. Group II, III, IV, and V received 3% v/v GA per day, p.o. for 7 days. Group II (induced control) received vehicle 0.5% CMC p.o. once daily from for next 14 days. Group III, IV, and V received p.o. 200, 400 mg/kg dose of ECB and 750 mg/kg dose of cystone, respectively, once daily for next 14 days.

Body weight
In EG induced urolithiatic model, changes in bodyweight of individual rats were measured on the 1st, 14th, and 28th days while, in the GA and SO induced urolithiatic model, the body weight was measured on 1st, 7th and 21st days.

Urine analysis
At the end of experiment, all animals were kept in metabolic case for 24 hours urine collection. A drop of conc. Hydrochloric acid (HCl) was mixed in urine to prevent microbial growth (Patel and Acharya, 2020). Urine samples were used to measurement of urine volume and then quantitative estimation of calcium, phosphorus, and magnesium. Urine pH was analyzed by pH meter (Eutech Instruments CyberScan pH 510)

Biochemical analysis
Animals were anesthetized with chloroform once the toxicity investigation was completed, and blood was taken via retro-orbital puncture with capillary. After centrifugation at 5,000 g for 15 minutes, serum was collected and stored in a deep freezer (Patel and Acharya, 2020). The serum parameters were determined by using diagnostic kits.

Histopathological studies
At the end of experiment, all rats were anesthetized with chloroform and sacrificed and the kidneys were collected and preserved in 10% formalin. Kidney sections were prepared as the above-described procedure. The pathological changes in kidney sections were identified by comparison with control group and photographs were taken by microscope (Olympus Magnus INVI inverted microscope).

Statistical analysis
The data were expressed as mean ± SEM and analyzed by using software GraphPad Prism 8.0.2. ordinary one-way and two-way analysis of variance with Dunnett's multiple comparisons test was applied.

RESULTS AND DISCUSSION
In order to investigate the effect of medicinal plants on urolithiasis, we chosen C. bonducella that have been claimed to have diverse therapeutic characteristics. Extraction of C. bunducella seed were performed in ethanol (95%) based on the literature studies stating that ethanol (95%) can offer superior extraction results for phenolic like chemicals because it dissolves the most polar and non-polar compounds. Extraction was done using the cold percolation process, and the operation was repeated three times with new solvents for a greater yield. Semisolid extracts were obtained and kept in the freezer for scientific investigations. In this investigation, ethanolic solvent yielded higher extract yields for C. bonducella (6%).
The signs and symptoms of toxicity, including morbidity or mortality, were not observed in both acute and subacute oral toxicity. LD 50 of ECB could be greater than 2,000 mg/kg because  there was no death recorded in rats during treatment. In acute and subacute toxicity study, the bodyweight of both the treated and normal control group were gradually increased and no significant change in mean body weight gain (Table 2 and 4). The results of hematological and biochemical parameters of treated groups were showed non-significant (p > 0.05), similar and within range as compared with normal control group in both acute (Table 1 and 2) and subacute oral toxicity study (Table 3 and 4). The results of hematological and biochemical parameters of all groups were found to be normal and nearly similar to previously reported studies (Han et al., 2015;Porwal et al., 2017;Silva et al., 2011). The histopathology of the liver showed no inflammation, normal hepatocytes cords, no acute or chronic damage in both acute (Fig.  1D), and subacute oral toxicity (Fig. 4) study. The histopathology of the kidney showed normal glomeruli, tubules, vessels with no features of damage or inflammation in both acute (Fig. 1A), and subacute oral toxicity (Fig. 2). The section of lungs showed normal patent alveoli lined by pneumocytes and there is no collapse, exudate, inflammatory infiltrate, or necrosis in the lung parenchyma in both acute (Fig. 1B) and subacute oral toxicity (Fig. 3). The section of the heart showed cardiomyocytes arranged in pseudosyncytium with normal thickness and there is no myonecrosis or inflammation in both acute (Fig. 1C) and subacute oral toxicity (Fig. 5).
with the induced control group (Table 5). Administration of EG, GA, and SO caused induction of renal stone formation and led to a significant decrease in body weight in the induced control group when compared with the normal control group. EG, GA, and SO caused body weight loss. After treatment with the 200 mg/kg of C. bonducella, slight body weight gain was observed; and treatment with 400 mg/kg of C. bonducella, maximum body weight gain was observed which was similar to cystone-treated group. Ahmed et al. (2013); Bawari et al. (2020); Bayir et al. (2011), Shehzad et al. (2021; Kaushik et al., (2019) reported that lithogenic treatment causes a decrease in body weight. EG, GA, and SO administration cause significant (p < 0.05 to p < 0.0001) increase in the urinary calcium, phosphorus level and significant (p <0.05 to p < 0.0001) decrease in the urine pH, urine volume, urinary magnesium level in the induced control group as compared with the normal control group. Treatment with 200 and 400 mg/kg of C. bonducella extract causes a significant (p < 0.05 to p < 0.0001) decrease in the levels of urinary calcium and phosphorus along with an increase in magnesium urinary level as compared with the induced control group (Table 6). The lithogenic substance decreases the urinary pH by its metabolism in the acidic compound which causes urinary acidosis. The decrease in urinary pH favors the CaOx precipitation by decreasing CaOx solubility and saturation level in urine which promotes stone formation. Low urinary output also plays a major role by decreasing the volume of urine which got easily supersaturated with oxalates and calcium ions (Patel and Acharya, 2020). The urinary pH and volume of the induced control group were decreased significantly and urine gets supersaturated with CaOxs, initiating stone formation. Volume and pH of urine in C. bonducella extract (200 and 400 mg/kg) and cystone-treated group was found significantly increased, which indicates that CaOx solubility and saturation level were increased in urine. Maximum recovery in urinary parameters were seen with highest dose (i.e., 400 mg/kg) which is similar with cystonetreated group.
Urinary magnesium works as an inhibitor in CaOx stone formation. Magnesium preferentially binds with oxalate and forms a stable complex that is highly water-soluble (Bano et al., 2018;Kaushik et al., 2019). A low level of magnesium is seen in the induced control group and after treatment with the C. bonducella extract, a nearly normal Mg level was resumed. Increased levels of calcium and phosphorous in the urine favor the growth of CaOx crystals. Phosphorous as phosphates was deposited on CaOx stone (Kaushik et al., 2019). The level of   calcium and phosphorous was decreased in the C. bonducella extract-treated group.
The serum biochemical parameters like uric acid, urea, creatinine, BUN, ALP level were significantly (p < 0.05 to <0.0001) increased in the induced control group as compared to the normal group. However, these biochemical parameters were found to be significantly (p < 0.05 to <0.0001) decreased in both C. bonducella extract and cystone-treated groups as compared to the induced control group (Table 7). Maximum recovery in biochemical parameters were seen with highest dose (i.e., 400 mg/ kg) which is similar with cystone-treated group. Stone deposition in tubules and glomerulus obstruct the outflow of urine and decreases   Urine pH 6.73 ± 0.10 5.42 ± 0.12 6.40 ± 0.08 **** 6.60 ± 0.14 **** 6.71 ± 0.11 **** Data are expressed as mean ± SEM (n = 5). * p < 0.05; ** p < 0.01; ***p < 0.001; ****p < 0.0001 compared with induced control group II.   the glomerular filtration rate which leads to the accumulation of nitrogenous waste products (urea, BUN, creatinine, and uric acid) in the blood (Bano et al., 2018;Bayir et al., 2011;Kaushik et al., 2019). However, the accumulation of nitrogenous waste in blood was reduced on treatment with C. bonducella extract. ALP level is a marker for kidney damage. In urolithiasis, apical membranes of renal tubular epithelial are damaged because of CaOx crystals deposition. This membrane damage leads to a rise in ALP (Kaushik et al., 2019).
The histopathological section of the kidney showed a mild increase in glomerular hypercellularity with severe tubular hydropic degeneration and casts in EG induced control group (Fig. 6B); moderate increased glomerular hypercellularity with moderate tubular hydropic degeneration and casts in GA induced control group (Fig. 7B); moderate increased glomerular hypercellularity, mild tubular hydropic degeneration and casts in SO-induced control group (Fig. 8B) as compared with the respective normal control group. In C. bonducella extract (200 and 400 mg/kg) treated rat's kidney section showed that glomerular hypercellularity, tubular hydropic degeneration, and casts were recovered in a concentration-dependent manner (Figs. 6C and D,7C and D,8C and D); and rats in the cystone-treated group were also recovered (Figs. 6E, 7E and 8E) as compared with the respective induced control group.

CONCLUSION
From the above results, it can be concluded that the ethanolic extract of C. bonducella has no toxic effect on albino Wistar rats in an acute and subacute oral study. 400 mg/kg of C. bonducella seed extract showed preventive effect in experimentally EG, GA, and SO-induced urolithiasis by normalizing imbalanced electrolytes in urine, nitrogenous substances in serum, and decreasing supersaturation of CaOx. Therefore, it is concluded that 400 mg/kg of C. bonducella seed extract could be used as a therapeutic agent to prevent urolithiasis and attenuate the problems associated with it.