Chebulic acid attenuates ischemia reperfusion induced biochemical alteration in diabetic rats

Abstract

Context: Diabetic nephropathy is one of the important microvascular complications of diabetes; however, the main problem remains is the control of progression of nephropathy in diabetes. Chebulic acid was selected, as tannins from Terminalia chebula are used as antidiabetic, renoprotective, antioxidant, hypotensive and an α-glucosidase inhibitor.

Objective: In this study, we evaluated the effect of chebulic acid on ischemia reperfusion induced biochemical alteration in diabetic rats.

Materials and methods: Chebulic acid (CA) was isolated from T. chebula; LD₅₀ and acute toxicity studies of CA were done. Renal ischemia and reperfusion technique was used to induce nephropathy in diabetic rats. Glibenclamide (10 mg/kg) was used as diabetic standard; CA at doses of 25 and 50 mg/kg were administered for 28 days and various biochemical parameters were monitored.

Results: The LD₅₀ was found to be 251 mg/kg; 25 and 50 mg/kg doses were selected as no toxic symptoms were observed at both doses, except slight diarrhea. CA significantly (p < 0.001) reduced the glucose, creatinine, urea nitrogen, glycosylated hemoglobulin, proteinuria, urine albumin excretion, glomerular filtration rate (GFR), and increased serum insulin and glycogen level. CA also restored glucose 6-phosphate dehydrogenase, glutathione, superoxide dismutase, catalase and malondialdehyde levels. Improvement in kidney was also noted in histopathological studies.

Conclusions: The statistical data indicated that chebulic acid at both doses (25 and 50 mg/kg) improves biochemical alterations caused by renal ischemia in diabetic rats.

Keywords::

• Diabetic nephropathy
• streptozotocin

Introduction

Diabetic nephropathy is one of the important microvascular complications of diabetes mellitus and the most common cause of chronic kidney disease (CKD) and end-stage renal disease (ESRD) in many countries (Ha et al., 2008). Nephropathy usually becomes clinically evident after 15–25 years of diabetes (Rabkin, 2003). Nephropathy develops in about 20–40% of all diabetic patients, with approximately 40% of patients with type 1 diabetes and 5–15% of patients with type 2 diabetes (DeFronzo, 1995). The exact cause of diabetic nephropathy is unknown, but a variety of factors contribute to the renal damage; hyperglycemia is a common etiologic factor in diabetic patients with nephropathy, but a genetic predisposition and smoking contribute as well, hypertension and microalbuminuria are also familial marker of risk (Evans & Capell, 2000; De Boer et al., 2011).

Terminalia chebula Retz. (Combretaceae), native to India and Southeast Asia, has been used traditionally to treat various diseases (Lee et al., 2007). In the modern era it has been used as a cytoprotective (Saleem et al., 2002), antidiabetic (Murali et al., 2004; Kumar et al., 2006), renoprotective (Rao & Nammi, 2006), antioxidant (Sabu & Kuttan, 2002; Naik et al., 2004), and cardioprotective (Suchalatha & Devi, 2005).

Previous studies showed that tannins isolated from T. chebula are cytoprotective (Lee et al., 1995), antioxidant (Klika et al., 2004), hypotensive (Lin et al., 1993), and α-glucosidase inhibitors (Gao et al., 2007). Chebulic acid (CA) has been used as hepatoprotective (Lee et al., 2007) and it causes reduction of advanced glycation end products in epithelial cells (Lee et al., 2010). Although antidiabetic and renoprotective effect of extract have been verified, the effect of CA on ischemia reperfusion induced biochemical alteration in diabetic rats has not been published. Therefore, the present study investigates the protective effect of CA (25 and 50 mg/kg) on animal model of diabetic nephropathy.

Materials and methods

Chemicals

All the chemicals, reagents, and solvents used in the activity were of analytical grade. Diagnostic kits used were procured from Lab Care Diagnostic (India) Pvt. Ltd. Serum insulin was analyzed by commercial pathology laboratory (Choksi Laboratories Limited, Indore, India).

Extraction and isolation

Dried fruits of T. chebula were purchased from a local market, Mandsaur, India, identified by Prof. Gyanendra Tiwari, Scientist, Government College of Horticulture, Mandsaur (Madhya Pradesh, India) and a voucher specimen (BRNCP/TC/009/2009) was deposited in the herbarium of Department of Pharmacognosy, B. R. Nahata College of Pharmacy, Mandsaur (Madhya Pradesh, India). T. chebula fruits (10 kg) were coarsely powdered; defatted with petroleum ether (60–80°C); dried and filled in a Soxhlet apparatus for extraction with absolute ethanol as solvent. The extraction was carried out for a period of 72 h. The extract obtained was dried in vacuum to remove excess solvent. The ethanol extract obtained was resuspended in H₂O, and then extracted successively with n-hexane, chloroform, ethyl acetate, and n-butanol. Extract obtained after extraction with ethyl acetate was dried. Dried compound was subjected to column chromatography (silica gel) and eluted by methanol in ethyl acetate (30:70, v/v). The obtained fraction was further purified by Sephadex LH-20 column chromatography using methanol as the eluent. RP-HPLC (Waters, USA) with C-18 column (5 µm, 250 × 4 mm, Lichro Cart, Merck, Germany) was used for further separation and estimation; formic acid and acetonitrite were used as eluent and quantification of compound was done using calibration curve of standard compound. Data of FTIR, ¹H NMR and MS spectroscopy of Lee et al. (2007), were used to identify the compound.

Animals

All the animals were procured from animal house of B.R.N.C.P. Mandsaur. Healthy adult Wistar albino rats of either sex weighing between 200–250 g were used for ischemia-reperfusion (ESRD) model. The animals were stabilized for 1 week; they were maintained in standard condition at room temp; 60 ± 5% relative humidity and 12 h light–dark cycle. They had been given standard pellet diet and water ad libitum throughout the course of the study. All the animal studies were carried out in Department of Pharmacology and Toxicology, B.R.N.C.P. Mandsaur; with permission from Institutional animal ethical committee (IAEC reg. no. 918/AC/05/CPCSEA). The animals were handled gently to avoid stress, which could result in an increased adrenal output.

Acute oral toxicity study and LD₅₀

The Miller and Tainter (graphical) method was used to calculate LD₅₀. The Probit values were plotted against log dose and then dose corresponding to Probit 5 was calculated. 1/5th and 1/10th of LD₅₀ was selected as dose for further study (Ghosh, 2008; Randhawa, 2009).

The rats were randomly divided into three equal groups (n = 6/group); Group I served as control and received distilled water; Group II received single oral dose of CA (25 mg/kg) and Group III received single oral dose of 50 mg/kg of CA. During the treatment, animal were observed daily for overt signs of toxicity (salivation, lachrymation, squinted eyes, writhing, convulsions, tremors, yellowing of fur, loss of hair), stress (erection of fur and exophthalmia), behavioral abnormalities (impairment of spontaneous movement, climbing, cleaning of face and ataxia and other postural changes) and aversive behavior (biting and scratching behavior, licking of tail, paw and penis, intense grooming behavior and vocalization) and diarrhea for 24 h and then daily for 14 days. Food consumption was monitored daily and body weights were recorded weekly. On the 14th day, animals were sacrificed and all the organs were removed for gross pathological examination.

Induction of diabetes

Animals were fasted overnight; a single i.p. injection of freshly prepared streptozotocin (50 mg/kg in 0.1 M citrate buffer pH 4.5) was injected. Following the STZ injection, rats were given drinking water supplemented with sucrose (15 g/L) for 48 h, to limit early mortality as stores of insulin are released from damaged pancreatic islets. The diabetes was confirmed by estimation of blood glucose level (BGL) at the third day. Rats having BGL more than 250 mg/dL were used for future studies. Diabetic rats should be given daily subcutaneous injections of long-acting insulin (2–4 U/rat, Human Mixtard, Abbott India Ltd., Mumbai, India) to maintain blood glucose levels in a desirable range. A relapse period of 2 weeks was give to rats before surgery (Nangle et al., 2006; Kumar & Subramanian, 2008; Tesch & Allen, 2007).

Induction of ischemia

The animals were anesthetized by an i.p. injection of ketamine (80 mg/kg; Neon Lab Ltd., Mumbai). During the operation the animals were placed on a thermo pad, which kept the body temperature at 37.5°C. An incision slightly deviated from midline towards left side was made and the left renal artery was located and dissected free from its surrounding structures. After a recovery period of 10 min, renal ischemia was evoked by clamping the left renal artery for 30 min. Subsequently the abdomen was sutured and the animals were returned to their cages. Animals were watched for 1 h after surgery for the recovery. Animals were given 1–2 mL normal saline and Pethidine HCl (10 mg/kg, Neon Lab. Ltd., Mumbai, India) after recovery. Aseptic conditions were maintained for 1 week with special care. Treatment was started next day of surgery. Animals were treated for 28 days (Melin et al., 1997; Wongmekiat et al., 2007).

The animals were randomly divided into the seven groups (n = 6); the treatment to each group was as described below.

• Group I: Normal control rats (NC).

• Group II: Diabetic control (DC).

• Group III: Nephropathy control (NeC); unilateral ischemia in normal rats.

• Group IV: Diabetic nephropathy control (DNC); unilateral ischemia in diabetic rats.

• Group V: Diabetes standard (DStd); Diabetes + ischemia rats given glibenclamide (10 mg/kg body weight/day/rat) in aqueous solution orally for 28 days.

• Group VI: Treatment group I (CA 25); Diabetes + ischemia rats given chebulic acid (25 mg/kg body weight/day/rat) in aqueous solution orally for 28 days.

• Group VII: Treatment group II (CA 50); Diabetes + ischemia rats given chebulic acid (50 mg/kg body weight/day/rat) in aqueous solution orally for 28 days.

Biochemical analysis

Blood urea nitrogen (BUN), urinary urea nitrogen (UUN), plasma (PC) and urine creatinine (UC), urine protein (UP), urinary albumin excretion (UAE), urinary glucose (UG), glycosylated hemoglobulin (GHb) and glucose 6-phosphate dehydrogenase (G6PDH) were assessed using commercially available diagnostic kits. Blood glucose level (BG) was assessed using glucose strips (Dr. Morphen’s glucostrip). Serum insulin was analyzed by commercial pathology laboratory (Choksi Laboratories Limited; Indore, India).

The method of Maiti et al. (2004) was used to estimate hepatic glycogen level. Kidneys were isolated and washed with chilled normal saline; Aslan et al. (2007) and Wang et al. (2008) methods were used to estimate reduced glutathione (GSH) and lipid peroxidation (MDA), respectively. The methods of Dhir and Kulkarni (2008) and Sharma et al. (2007) were adopted to estimate superoxide dismutase and catalase.

Histopathological studies

Kidney were collected and immediately fixed in 10% formalin, dehydrated in a graded ethanol (50–100%) series, cleared in xylene, and embedded in paraffin. Sections (4–5 µm) were prepared and stained with hematoxylin and eosin (HE) dyes for photomicroscopic observations. Sections were examined in a blinded fashion in all treatment groups.

Statistical analysis

All values are presented as mean ± SEM. CA (treated groups) were compared with all set of diseased control groups (DC, NeC, DNC). Statistical analysis of data was performed by one way analysis of variance (ANOVA) followed by Tukey’s t-test; p value <0.05 was considered as statistically significant.

Results

Isolation and characterization

The compound was isolated from T. chebula as yellow powder with a yield of 0.07% w/w (735 mg), purity of 97.04% w/w, and melting point of 195–208°C. The IR spectrum showed the main peaks at 3560-3500, 3000–3100, 1600, 725-680 (data not shown). ¹H NMR data showed (400 MHz, deuterium oxide) δ 6.75 (s, 1H), 5.50-5.41 (m, 2H), 3.98 (t, J = 5.1 Hz, 1H), 3.38 (q, J = 5.0 Hz, 1H), 3.06 (dd, J = 12.5, 5.1 Hz, 1H), 2.73 (dd, J = 12.5, 5.1 Hz, 1H). From these data, the compound was identified as chebulic acid.

Acute toxicity and LD₅₀

LD₅₀ of chebulic acid was found to be 251 mg/kg (Log LD₅₀ = 2.40); 25 and 50 mg/kg doses were used for entire study. In an acute oral toxicity study, CA treated animals did not show any change in their behavioral, neurological and autonomic pattern at both doses. Mild diarrhea was observed at both doses. There was no significant difference in the body weights and food consumption when compared to the vehicle treated group. Also, no gross pathological changes were seen. Thus, it was concluded that CA was safe at both doses (25 and 50 mg/kg).

Effect of CA on biochemical parameters

Table 1 shows that the blood glucose level of DC and DNC group was increased by nearly 5-fold above the normal range; no change was observed in the NeC group. Treatment with CA at both dose levels showed a significant reduction in blood glucose level. CA (50 mg/kg) exhibited a maximum glucose lowering effect (78.88%) at the end of study when compared to DNC.

Table 1.  Effect of Chebulic acid on the biochemical profile of blood.

Parameters	NC	DC	NeC	DNC	DStd	CA 25	CA 50
BG (mg/dL)	111.8 ± 2.78	500.8 ± 19.70	104.7 ± 4.08	502.5 ± 8.85	108.67 ± 1.14^a,c,***	128.6 ± 2.83^a,c,***	106.2 ± 3.91^a,***
BUN (mg/dL)	17.67 ± 0.67	32.67 ± 1.71	34.50 ± 1.09	34.17 ± 0.79	19.45 ± 0.50^a,c,***	24.33 ± 0.24^a,d,***	18.33 ± 0.55^a,d,***
SC (mg/dL)	0.348 ± 0.012	1.082 ± 0.018	1.087 ± 0.023	1.255 ± 0.030	0.392 ± 0.045^a,c,***	0.458 ± 0.020^a,d,***	0.375 ± 0.013^a,d,***
GHb (% HbA1C)	5.17 ± 0.60	12.83 ± 0.70	4.50 ± 0.43	13.00 ± 0.89	5.87 ± 0.58^a,c,***	6.58 ± 0.67^a,c,***	5.67 ± 0.33^a,b,***
G6PDH (m/g Hb)	5.562 ± 0.39	14.48 ± 0.23	5.01 ± 0.38	15.88 ± 0.43	5.98 ± 0.38^a,c,***	8.82 ± 0.33^a,d,***	6.26 ± 0.29^a,c,***
SI (U/mL)	14.50 ± 0.76	5.83 ± 0.79	14.33 ± 0.67	5.50 ± 0.43	10.86 ± 1.95^a,c,***	9.74 ± 0.56^a,c,***	11.67 ± 0.17^a,c,***
SGly (mg/gm wet tissues)	58.67 ± 2.20	25.50 ± 1.19	56.83 ± 0.75	23.83 ± 1.30	49.19 ± 0.72^a,c,***	36.87 ± 0.63^a,d,***	47.17 ± 0.60^a,d,***

N = 6; where ^ap < 0.001 vs diabetic control (DC); ^bp < 0.05, ^cp < 0.01, ^dp < 0.001 vs nepropathy control (NeC); cp < 0.01; ^***p < 0.001 vs diabetic nephropathy control (DNC); Value expressed in mean ± SEM.

There was a significant increment in serum blood urea nitrogen of the diseased group (DC, NeC and DNC) as compared to the normal animals; after 28 days treatment, CA 50 mg/kg (18.33 ± 0.55) brought BUN almost to the of normal level (17.67 ± 0.67).

CA showed a dose-related significant (p < 0.001) reduction in serum creatinine, GHb and G6PDH compared to diseased groups (Table 1). Insulin level was decreased in streptozotocin treated groups; it was decreased up to 1/3rd in DC (5.83 ± 0.79) and DNC groups (5.50 ± 0.43) whereas no effect was seen in the NeC group (14.33 ± 0.67). Administration of CA (25 and 50 mg/kg) caused a significant increase in serum insulin level at the end of study. Almost a 50% depletion of glycogen was observed in diseased animals (DC and DNC); Administration of CA at doses of 25 and 50 mg/kg for 28 days resulted in a significant (p < 0.001) increase in the glycogen level. However, the values were not restored to normal at any dose level (Table 1).

Table 2 depicts the effect on the levels of creatinine, protein, albumin, urea nitrogen and glucose in urine of treated animals. After the 28 days treatment, a significant (p < 0.001) reduction was observed in excretion of biochemicals in the urine of treated groups. A fall of 69% in UC, 63% in UP, 40% in UAE, 36% in UUN, 73% in UG and 88% in GFR was observed after 28 days of treatment with CA (50 mg/kg) as compared to the DNC group.

Table 2.  Effect of chebulic acid on the biochemical profile of urine.

Group	UC (mg/dL)	UP (mg/24 h)	UAE (mg/24 h)	UUN (mg/dL)	UG (mg/24 h)	GFR (mL/min)
NC	10.00 ± 0.57	20.00 ± 0.93	2.92 ± 0.07	66.33 ± 2.23	0.0 ± 0.0	0.67 ± 0.06
DC	38.67 ± 1.54	129.71 ± 5.37	10.06 ± 0.28	108.54 ± 5.85	3459 ± 76.42	6.25 ± 0.45
NeC	36.50 ± 1.60	89.50 ± 1.97	9.81 ± 0.37	105.53 ± 2.88	0.0 ± 0.0	5.70 ± 0.32
DNC	40.83 ± 0.60	131.50 ± 5.39	10.85 ± 0.40	114.33 ± 3.75	3509 ± 71.54	7.86 ± 0.41
DStd	12.40 ± 1.68^a,c,***	80.64 ± 3.84^a,***	6.40 ± 0.37^a,b,***	75.67 ± 2.05^a,b,***	823 ± 30.98^a,b,***	0.89 ± 0.92^a,c,***
CA 25	16.17 ± 1.22^a,b,***	67.83 ± 1.31^a,b,***	7.51 ± 0.18^a,b,***	79.67 ± 0.95^a,b,***	1088 ± 55.15^a,b,***	1.26 ± 0.84^a,b,***
CA 50	12.33 ± 2.67^a,b,***	48.26 ± 2.46^a,b,***	6.45 ± 0.20^a,b,***	73.24 ± 1.89^a,b,***	956 ± 45.38^a,b,***	0.96 ± 0.54^a,b,***

N = 6; where ^ap < 0.001 vs diabetic control (DC); ^bp < 0.001 vs nepropathy control (NeC); ^***p < 0.001 vs diabetic nephropathy control (DNC); Value expressed in mean ± SEM.

Effect of CA on in vivo antioxidant

After 28 days, it was observed that the diseased animals (DC, NeC, DNC) showed a significant reduction in catalase, GSH and SOD levels; whereas lipid peroxidation was increased as compared to NC groups (Figure 1a–1d). When compared to diseased rats, CA at both doses showed significant (p < 0.001) but dose-dependent effect. CA 50 mg/kg caused an increase of 47% in catalyze level, 65% in GSH level, and 59% in SOD level whereas MDA level was decreased by 53% as compared to DNC rats.

Figure 1.  Effect of chebulic acid on (a) catalase level, (b) GSH, (c) MDA and (d) SOD level of experimental groups. Values are expressed as mean ± SEM; where N = 6; ^£p < 0.001 vs diabetic control (DC); ^$p < 0.001 vs nepropathy control (NeC); ^***p < 0.001 vs diabetic nephropathy control (DNC).

Effect of CA on histology of kidney

Histological examination of kidney sections showed necrosis of glomerulous with damaged proximal and convoluted tubules; diffused mesangium and thicker basal membrane of DC, NeC and DNC (Figure 2b–2d) as compared to normal rats. Treatment with CA (25 and 50 mg/kg) showed reduction in glomerular damage improvement in tubules and a lesser degree of mesangial matrix increment and basal membrane thickening was observed (Figure 2 e and 2f).

Figure 2.  Effect of chebulic acid on histopathology of kidney (H and E × 100); (a) NC, normal glomeruli; (b) DC, diabetic control; (c) NeC, nephropathy control; (d) DNC, diabetic nephropathy control; (e) DStd, glibenclamide treated group (10 mg/kg); (f) CA 25, chebulic acid treated group (25 mg/kg); and (g) CA 50, chebulic acid treated group (50 mg/kg).

Discussion

The present manuscript describes the protective effect of chebulic acid on ischemia reperfusion induced biochemical alteration in diabetic rats. The LD₅₀ of CA was found to be 251 mg/kg. No serious toxic and adverse effects were noted at the 25 and 50 mg/kg doses of CA.

In the animal model STZ caused features resembling human diabetes, whereas ischemia caused symptoms resembling nephropathy and also increased oxidative burden on the body (Steffes et al., 1978).

Results of this study have shown that CA attenuated the cachectic condition along with polydipsia, polyuria and polyphagia which were commonly observed in diabetic nephropathy (Kawanishi et al., 1994; Casey et al., 2005).

Hyperglycemia is directly linked to the development of diabetic nephropathy as it causes series of biochemical disturbances in kidney. Hyperglycemia causes activation of the polyol pathway, nonenzymatic glycation, glucose autoxidation and de novo synthesis of diaglycerol leading to protein kinase C and phospholipase A₂ activation, resulting in hyperfusion and hyperfilteration and affecting glomerular permeability. Strict control of blood glucose level was found to be beneficial in controlling and preventing functional as well as structural abnormalities (Reddi & Camerini-Davalos, 1990; Larkins & Dunlop, 1992). The results of this study showed a significant (p < 0.001) reduction in blood glucose levels attribute to the antihyperglycemic effect of CA.

Hyperglycemia also leads to nonenzymatic glycation, resulting in structural and functional changes in soluble and insoluble protein molecules such as hemoglobin in blood. Uncontrolled GHb augments development and progression of microvasular and macrovascular complications. Glycation itself generates oxygen derived free radicals, further increasing the risk of proteinuria and renal failure. Strict control of glucose causes a subsequent reduction in GHb level leading to reduction in inflammatory mediator accumulation in kidney matrix as well as less accumulation of mesangial matrix and less thickening of glomerular basement membrane (Jovanovic & Peterson, 1981; Krishnamurti & Steffes, 2001). CA showed a prominent effect on reduction of glycosylate hemoglobulin, which further confirms an antidiabetogenic effect.

A reduction in the concentration of catalyses, GSH, SOD, and G6PDH, and an elevation in lipid peroxidation (MDA) was observed in animals with diabetes and its complications. Ameliorating oxidative stress by normalized activity of G6PDH, GSH, MDA, SOD, catalyze and resorting NADPH can be beneficial in diabetic nephropathy and prevents its progression (Sharma et al., 2006; Liu et al., 2008). In our study, administration of CA causes a significant increase in catalyze, GSH and SOD whereas G6PDH and MDA were significantly decreased. Thus, it is reasonable to conclude that CA restored antioxidant potential of treated animals.

A low level of insulin and hyperglycemia causes derangement of glycoprotein metabolism and thickening of basement membrane (Spiro & Spiro, 1971). A previous study showed that the antihyperglycemic effect of T. chebula was due to an increase in insulin level suggesting its insulinogenic activity due to stimulation of insulin secretion from remnant β cells or from regeneration of β cells (Chattopadhyay, 1999; Pari & Latha, 2002; Kumar et al., 2006). From these results, it can not be ignored that CA increases secretory β cells in islets and reduces blood glucose level in diabetic control and DNC rats.

Conversion of glucose to glycogen in liver is dependent on extracellular glucose concentration and on availability of insulin. CA significantly restored the level of hepatic glycogen, which further confirms the insulinogenic effect of CA and regulation of glycogenesis by CA (Kumar & Subramanian, 2008).

Alteration of urinary albumin, blood and urinary nitrogen urea, creatinine, urinary protein and glucose excretion was usually observed in diabetic nephropathy indicating progressive damage to glomerular and tubular cells resulting in decline in GFR (Rossing et al., 1994; Liu et al., 2008). In the present study, the treatment group showed a significant decrease in urea, creatinine and urinary excretion. The results contribute to the renoprotective effect of CA.

Significant reduction was observed in glucose and albumin excretion of CA treated animals. CA at both doses was unable to abolish the damage fully suggesting that the treatment regimen is only preventive and altered glomerular activity cannot be revert totally.

Glomerular filtration rate (GFR) and creatinine clearance are directly dependent on hemodynamic factors. Renal damage is due to hyperglycemia and oxidative stress is caused an elevation in GFR (Hostetter et al., 1981; Kelly et al., 2000). Animals treated with CA indicated a significant decrease in GFR which could be due to improvement in renal function and decreased kidney burden.

In present study, histopathology showed damaged proximal convoluted tubules, mesangial cells, dilated tubules and glomerular necrosis in kidney of diseased (diabetic, nephropatic and diabetic nephropathy) rats. CA was able to diminish the necrosis of glomerular, proximal convoluted tubule, mesangial cell, endothelial cells etc., but neither of the treatment regimens was able to normalize the renal damage. The observed effect may be due to promising control of hyperglycemia and urinary burden by chebulic acid.

Conclusion

The present study shows that chebulic acid was effective in controlling elevated metabolic parameters, oxidative stress and renal damage, supporting its beneficial effect in diabetic nephropathy. Further studies are in progress to determine exact mechanism of action(s) responsible for the activity.

Acknowledgments

The authors would like to express their gratitude to Ret. Prof. Dr. D. N. Srivasatva, Ex-Head, Department of Pharmacology and Veterinary Sciences, J. N. K. V., Jabalpur, Madhya Pradesh, India for their expert opinion on the histopathology.

Declaration of interest

The authors declare no conflicts of interest.