Protective Effect of Acacia Ferruginea Bark Extract against Paracetamol-Induced Nephrotoxicity in Rats

Introduction

Kidneys are two organs shaped like beans behind the peritoneum, positioned between the twelfth thoracic vertebra and the third lumbar vertebra.¹ Kidneys regulate the body's internal balance by controlling osmotic balance, electrolyte levels, and acid-base balance. They manage blood pressure through sodium excretion and hormone production, like renin, which helps maintain fluid volume and pressure.²

Nephrotoxicity refers to the rapid deterioration or dysfunction of kidney function caused by the harmful effects of medications and chemicals.³ According to a WHO report from 2018, there was a 32% rise in kidney failure deaths, with 1.2 million fatalities in 2015.⁴

The compounds used to bring nephrotoxicity in lab animals, i.e., paracetamol (PCT), are considered as positive control. PCT overdose can cause nephrotoxicity due to its reactive byproduct, N-acetyl-p-benzoquinoneimine (NAPQI), which is formed via cytochrome P450 activity. Excess of this compound depletes intracellular glutathione (GSH) reserves, causing oxidative stress and cellular damage. This damage extends to the kidneys, causing acute tubular necrosis (ATN), characterized by renal tubular cell destruction and impaired kidney function. The oxidative stress from elevated NAPQI levels contributes to renal impairment.⁵

Vitamin C and Silymarin act as antioxidants that help prevent or reduce organ toxicity from chemically induced drugs.^6,7 There is significant interest in antioxidant strategies to mitigate renal toxicity.

Acacia ferruginea bark decoction is used in gargle preparations, and the bitter bark has applications as a hot anthelmintic and in treating conditions like itching, leukoderma, ulcers, stomatitis, and blood-related disorders.^8,9 Additionally, the ethanolic extract of its leaves shows hepatoprotective, larvicidal, antiulcer, and antitumor activities.^9,10,11,12 LC-MS/MS analysis of extract derived from the acetone bark extract identified 12 components, including rosmanol, catechin, ellagic acid, and procyanidin B1.¹³

The research investigates the nephroprotective properties of A. ferruginea bark ethanolic extract against paracetamol-induced nephrotoxicity in rats; it also evaluates its in vivo antioxidant activity through lipid peroxidation and reduced glutathione assays. Silymarin and vitamin C will serve as standard drugs. The semi-auto analyzer determines the physical parameters and biochemical markers in blood samples. Based on this, the study aimed to evaluate the protective role of ethanolic extract of A. ferruginea bark (EEAFB) against kidney damage caused by Paracetamol in rats.

Materials and Methods

Drugs and chemicals

Fresh bark of A. ferruginea was obtained from Kana-kapura, Karnataka, and was verified by the Central Ayurvedic Research Institute, Bengaluru, with Code No: Authentication/SMPU/CARI/BNG/2023-24/2260. Paracetamol was procured from KAPL, Bengaluru, and all remaining chemicals and reagents utilized are obtained from certified sources.

Extract preparation

The ethanolic extract of A. ferruginea bark was prepared using the Soxhlet apparatus. Fresh bark was collected, cleaned, shade-dried, roughly ground, and kept in a sealed container. The powdered bark was placed into the thimble of the Soxhlet apparatus, and ethanol was used as the solvent for extraction until the solvent turned colorless. The resulting stock extract was semisolid, dark brown, and kept in a sealed glass jar at 4ºC in the refrigerator.^14,15

Experimental subjects

Adult Albino Wistar rats of either sex weighing between 150-200 g were utilized for nephrotoxicity and acute toxicity research. The subjects were sourced from DTL, Bengaluru, and acclimatized in the Government College of Pharmacy, Bengaluru animal house facility. Rats are accommodated in polypropylene cages (32 x 16cm) with stainless steel grill tops and were bedded with paddy husk and provided commercial pelleted rat food (Shri Venkateshwara Enterprises, Bangalore) and had access to water. The studies were conducted following CCSEA guidelines and received approval from the IAEC with reference number GCP/IAEC/DOP/2023- 2024/92 dated 07.10.2023.

Acute toxicity study¹⁶

This study was conducted on female rats weighing 150 to 200 g maintained in standard laboratory environments, following OECD 423 guidelines. Rats were orally administered a maximum of 2000mg/kg body weight (b.w.) of ethanolic extract of A. ferruginea bark (EEAFB). Over 48 hours, the animals were assessed for general behavior, indications of discomfort, and nervous manifestations.

Experimental protocol for paracetamol-induced nephrotoxicity model:¹⁷

The study included seven groups of rats, each group containing six animals. Group 1 was the untreated normal negative control, while Group 2 was administered paracetamol (750 mg/kg) alone. Groups 3 and 4 were administered vitamin C (200 mg/kg) and silymarin (100 mg/kg), accordingly, an hour before paracetamol (750 mg/kg). Groups 5, 6, and 7 received EEAFB at the dose of 100, 200, and 400 mg/kg, accordingly, an hour before paracetamol (750 mg/kg). The treatments are provided orally for seven consecutive days.

On day 8, blood samples were drawn from all rats via the retro-orbital sinus to measure nephrotoxic biomarkers, including total serum protein, blood urea nitrogen (BUN), creatinine, urea, and uric acid, using a fully automated autoanalyzer. The rats were then euthanized under anesthesia, and their kidneys were collected to estimate reduced glutathione (GSH) levels and lipid peroxidation and for histopathological studies to assess nephroprotective activity in the kidney.

Measurement of physiological and biochemical indicators of blood serum

Kidney weight and body weight measurements were recorded. Serum creatinine, urea, uric acid, and total serum protein assay were conducted according to the procedure provided in the assay kit, after calibrating the instrument with reagent as a blank and using standards for urea, uric acid, creatinine, and total protein. Blood samples from rats were obtained from retro-orbital sinus and centrifuged at 10,000 rpm at 4ºC for 10 minutes. Serums were utilized for biochemical assessments with commercially available kits for urea, uric acid, creatinine, and total protein (Swemed Diagnostics, Bengaluru).^18,19,20,21 BUN was calculated by multiplying the serum urea concentration by 0.47. All parameters were carried out according to the specifications given by the manufacturer.

Measurement of Glutathione²²

GSH is estimated by taking 10% of 0.75 ml homogenate of tissue, deproteinized with 4% of 0.75 ml sulfosalicylic acid. After 1 hour at 4ºC, the sample is centrifuge at 1200 rpm for 20 minutes. Subsequently, 0.2 ml of supernatant is combined with 5.4 ml of 10 mM DTNB (5,5'-Dithiobis-(2-nitrobenzoic acid)) and 0.1M phosphate buffer (pH 7.4). The end point yellow color is measured at 412 nm, and GSH is determined using a 13.6 x 10³ M⁻¹ cm⁻¹ extinction coefficient.

Measurement of lipid peroxidation (LPO)²³

LPO was estimated by measuring the thiobarbituric acid reactive substances (TBARS) as malondialdehyde. A mixture of 0.2 ml Kidney homogenate, 1.5 ml of 20% acetic acid, 1.5 ml of 0.5% thiobarbituric acid, and 0.2 ml of 40% sodium dodecyl sulfate was boiled at 95ºC for 60 minutes to develop a pink color, then centrifuge at 3500 rpm for 10 minutes. The supernatant was recorded at 532nm, and the TBARS was assessed with a 1.56 x 10⁵ M⁻¹ cm⁻¹ molar extinction coefficient and quantified as nanomoles per mg of tissue.

Histopathological studies²⁴

Upon conclusion of the study, all rats were humanely euthanized with the use of approved anaesthesia, and the kidneys were dissected out and placed in a 10% buffered neutral formalin solution taken in a specimen storage container. After 24 hours of contact time, it was given for slide preparation and interpretation.

Statistical analysis

The data were evaluated using one-way ANOVA with Tukey’s post-hoc test for multiple comparisons, employing GraphPad Prism software. Results are exhibited as Mean ± SEM. Statistical significance is denoted by *P < 0.05, **P < 0.01, ***P < 0.001, ###P < 0.001, and ns for non-significant differences.

Results

EEAFB had a percentage yield of 7.65%. The acute oral toxicity evaluation confirmed no fatalities among the animals, even at the 2000mg/kg dose, leading to the conclusion that the extract is safe. According to the CCSEA guideline 423, 2000 mg/kg is considered the LD50. Consequently, 200mg/kg, 100mg/kg, and 400mg/kg were selected as therapeutic doses, half the therapeutic dose, and double the therapeutic dose, respectively, for further studies.

Nephroprotective activity screening

Physiological and biochemical parameters estimation in blood serum

The PCT group of rats has shown a reduction in the body's weight and an elevation in the weight of the kidney in comparison to the normal negative control group. Treatment for standard groups with vitamin C, and silymarin, and EEAFB with a dose of 100, 200, and 400mg/kg resulted in a significant rise in body weight and reduced in kidney weight compared to the disease-positive control group (Table 1).

Biochemical parameters

The biochemical indicators like uric acid, urea, creatinine and BUN levels are significantly elevated and total serum protein levels were reduced in the disease control (paracetamol) compared to the normal negative control group. Pretreatment of vitamin C and Silymarin and EEAFB 100mg/kg, 200mg/kg, and 400mg/kg has decreased levels of uric acid, urea, BUN, and creatinine significantly and raised levels of total serum protein compared to the paracetamol group (Table 2).

Assay of endogenous antioxidants

The paracetamol group of rats exhibited a significant reduction in GSH and an elevated LPO level compared to the negative control group. Pretreatment with vitamin C and silymarin and EEAFB with 100, 200, and 400mg/kg,b.w., for 7 days increased GSH and a significant decrease in LPO levels compared to paracetamol disease control of rats (Table 3).

Discussion

This study examined the effects of EEAFB in preventing nephrotoxicity caused by paracetamol in rats. Several parameters, including general parameters, serum parameters, kidney tissue parameters, and histopathological findings, were analyzed to assess the impact of EEAFB on nephrotoxicity.

The disease group showed a 19.05% reduction in body weight and a 50.01% elevation in kidney weight compared to the normal negative control. The vitamin C and silymarin groups improved body weight by 17.11% and 13.30% and reduced kidney weight by 23.69% and 29.17%, respectively, compared to the disease group. EEAFB treatment at 100, 200, and 400mg/kg showed dose-dependent improvements in the weight of the body (14.92%, 19.10%, and 20.82%) and reductions in kidney weight (8.56%, 23.01%, and 29.14%) in comparison with the disease group. The 400 mg/kg dose showed the most significant effect, leading to improvements in both body weight and kidney weight compared to the other groups.

Paracetamol significantly increased biomarker enzyme levels like uric acid, BUN, urea, and creatinine while reducing total serum proteins. Pretreatment with EEAFB at 100, 200, and 400mg/kg b.w. decreased urea (6.76%, 28.40%, 32.30%), uric acid (10.79%, 14.71%, 45.59%), creatinine (9.25%, 26.54%, 63.00%), and blood urea nitrogen (7.71%, 10.08%, 42.37%) in comparison to the paracetamol group. Vitamin C and Silymarin pretreatment also decreased urea (29.83%, 44.79%), uric acid (35.79%, 42.18%), creatinine (53.40%, 58.82%), and blood urea nitrogen (39.99%, 53.57%) while increasing total serum proteins (59.96%, 48.21%). EEAFB increased total serum protein levels by 11.68%, 29.66%, and 79.34%, with the 400 mg/kg dose showing the most significant improvement.

For antioxidant screening, EEAFB, GSH, and LPO levels were assessed. The disease group showed reduced GSH and increased LPO levels compared to the normal group. Vitamin C and Silymarin treatment restored GSH levels by 60.80% and 42.99%, and reduced LPO by 48.41% and 53.95%, respectively, compared to the disease group. EEAFB treatment at 100, 200, and 400mg/kg b.w. significantly increased GSH levels (16.90%, 20.56%, 37.89%) and decreased LPO levels (40.28%, 30.91%, 19.69%) in relation to disease and standard group, with the 400mg/kg showing the most significant improvement.

Conclusion

This study revealed that the ethanol extract of Acacia ferruginea bark (EEAFB) offers nephroprotection in paracetamol-induced nephrotoxicity in rats. EEAFB, particularly at 400 mg/kg, significantly enhanced body weight, kidney function, and histopathology. These findings indicate that EEAFB may act as a natural therapy for kidney protection. Further investigation is required to understand its mechanisms and clinical applications. This study contributes valuable knowledge toward developing natural remedies for kidney disorders.

Conflicts of interest

The authors have no conflicts of interest to report.

Acknowledgement

The authors would like to use the KAPL in Bengaluru to give drug samples. They also appreciate the support from the Government College of Pharmacy's Principal for essential research facilities.