Protective effect of the combination of Hydroalcoholic extracts of Asparagus Racemosus, Centella Asiatica, Plumeria rubra with Glibenclamide in Experimentally Induced Diabetic Nephropathy in rats

 

Amruta Vidyadhar Yadav*, Chandrashekhar Devidas Upasani

Department of Pharmacology, SNJB’s Shriman Sureshdada Jain College of Pharmacy,

Affiliated to Savitribai Phule Pune University, Neminagar, Chandwad, Maharashtra 423101

*Corresponding Author E-mail: yadavamruta4@gmail.com, cdupasani@gmail.com

 

ABSTRACT:

Objective: The current investigation analysed the effect of combination of standardized hydroalcoholic extracts of Asparagus racemosus (AR), Centella asiatica (CA) and Plumeria rubra (PR) on glibenclamide (GB) administered in experimentally induced diabetic nephropathy (DN) in Wistar rats. Material and methods: DN was induced in laboratory rats by alloxan monohydrate (160 mg/kg i.p). Rats were given the combination of AR, CA and PR called as ACP (100, 200 and 400mg/kg) along with GB (5mg/kg). Group 1-4 were control, diabetic control, standard GB (10mg/kg), GB (5 mg/kg), while groups 5-7 were given ACP (100, 200 and 400mg/kg) along with GB (5mg/kg) once daily for 30 days. Blood glucose, serum insulin, uric acid, Blood Urea Nitrogen (BUN), creatinine, superoxide dismutase (SOD), albumin, malondialdehyde (MDA), and kidney histopathological examination was done in all experimental animals. Results: ACP along with GB improved the derailed glycaemic status, renal biochemical, oxidative stress parameters in animals. Co-administration of ACP (400mg/kg) with GB (5mg/kg) significantly (p<0.01) decreased BUN, creatinine, uric acid, significantly increased (p<0.05) SOD, significantly (p<0.01) decreased MDA, significantly increased (p<0.05) serum insulin when compared with diabetic control. Histopathological observations corroborated with biochemical parameters.

Conclusion: Enhancement in the effectiveness of GB was observed with ACP (400mg/kg). Thus, ACP confers protection by improving renal, oxidative parameters and the restoration of morphological abnormalities in the kidneys.

 

KEYWORDS: Alloxan, Insulin, Glucose, Diabetic nephropathy, Antioxidant.

 

 


1. INTRODUCTION:

Diabetic nephropathy (DN) is the fundamental reason of renal failure. The pathogenesis of DN lies in the interplay of metabolic and hemodynamic abnormalities and disturbances in the renal microcirculation. Hemodynamic factors include renin angiotensin aldosterone, endothelin systems and factors that increase glomerular pressure. Metabolic factors cause changes in polyol metabolism, nonenzymatic glycosylation and protein kinase C (PKC) activity causing extracellular matrix accumulation.1 Cases of end stage renal disease (ESRD) in India are approximately 150 to 200 per million population yearly, where diabetes is a major contributor in 30 to 40% of the patients.2 Effective treatment for DN appears to be angiotensin converting enzyme inhibitors, angiotensin receptor antagonists or their combination.3 Although these treatments retard the progression of nephropathy to ESRD in diabetics, but usually does not prevent it. Dialysis and renal transplantation are not affordable many a times by majority of Indian population. Therefore, alternative and safe therapy is needed that will be helpful in reducing the requirement of dialysis and in postponing the renal transplantation. Numerous plant extracts mediate protective effects on kidney functions in diabetes mellitus. Several therapeutic uses of Asparagus Racemosus (AR) root are mentioned in Ayurveda. It has been used as galactagogue, carminative, aphrodisiac, diuretic, rejuvenator, antiseptic. It is also reported to be effective in nervous disorders, dysentery, tumours, hyperdipsia, diarrhoea, neuropathy, cough, infectious diseases. Ethanolic extract of AR exerted significant antihyperlipidemic, antidiabetic and antioxidant activity in streptozotocin induced diabetes.4 Plumeria rubra (PR) is abundantly found in India. In Mexico, the decoction of the flowers was traditionally used for treating diabetes.5 It also possesses antimicrobial, antipyretic, anticancer, antioxidant, antianxiety effect.6 Centella asiatica (CA) is native to tropical areas of Asia used as a medicinal herb in India, China, Srilanka. It is called brain food in India as it revitalizes brain cells. The ethanolic and methanolic extracts of CA was effective in alloxan induced diabetes.7 These plants and its extracts have been used traditionally, but search of literature does not manifest any proof of its usage in DN. Therefore, the present study is aimed at to investigate the protective effect of the combination of Hydroalcoholic Extracts of AR, CA, PR with glibenclamide in Experimentally Induced DN in Rats.

 

2. MATERIALS AND METHODS:

2.1 Chemicals:

Standardized hydro-alcoholic extracts of PR, AR and CA were procured from Kuber Impex ltd. Indore. Alloxan monohydrate was procured from Sigma Aldrich. The kidney function, glucose estimation and other kits were purchased from Biolab.

 

2.2 Animals:

Male Wistar rats (190-255g) were procured from the National Institute of Biosciences. They were subjected to standard temperature (25±2°C), humidity (60%), feed, water. Permission of Institutional animal ethics committee was obtained as per IAEC letter no. ACP/IAEC/2018/01.

 

2.3 Preparation of AR+ CA+ PR (ACP) combination:

The powdered extracts of AR, CA and PR was mixed in the ratios of their effective doses that was obtained by the studies done previously. They were mixed together in 200:100:200mg proportion respectively and resulting mixture was named as ACP. Such formulated ACP was used further at the dose of 100, 200 and 400mg/kg and coadministered with GB 5mg/kg once a day for 30 days to animals.

 

3. EXPERIMENTAL PROTOCOL:

Alloxan monohydrate 160mg/kg i.p. was used for induction of DN. Blood glucose was estimated after 48 h and rats estimating blood glucose greater than 280mg/dl were chosen as diabetic animals for studies and six groups were made consisting 6 animals in each group.

Group I: Control: Animal received normal saline p.o.

Group II: Diabetic control: Animals received alloxan 160mg/kg and normal saline p.o.

Group III: GB-10: Diabetic animals received GB 10mg/kg p.o.

Group IV: GB-5: Diabetic animals received GB 5mg/kg p.o.

Group V: ACP GB-100: Diabetic animals received GB 5mg/kg and ACP 100mg/kg p.o.

Group VI: ACP GB-200: Diabetic animals received GB 5mg/kg and ACP 200mg/kg p.o.

Group VII: ACP GB-400: Diabetic animals received GB 5mg/kg and ACP 400mg/kg p.o.

 

All the animals were treated as per the experimental design given above for 30 days. Urine samples of all the experimental animals were collected and measured on last day using metabolic cages and body weight is also measured. Blood samples were collected by retro orbital vein puncture, and was centrifuged at 7000rpm at 4şC for 15 min to get serum using REMI cooling centrifuge. The serum was subjected to measurement of Blood glucose, insulin, creatinine, uric acid, albumin, BUN, MDA, SOD immediately after collection. Animals were sacrificed, one kidney was kept in a 10% neutral formalin buffer for histopathology while second one was used for estimation of MDA, SOD.8,21

 

3.1 Estimation of insulin:

Insulin was measured by ELISA method.9 Enzyme conjugate solution (100μl) and sample (25μl) were mixed. Incubation was done for 2 h at 37°C. Each well was washed with buffer solution that removed unbound enzyme followed by adding 3, 3, 5, 5-tetramethylbenzidine (200μl). It was then incubated for 15 min at 37°C. Stop solution (50μl) was mixed. Absorbance was noted at 450nm using microplate reader.

 

3.2 Estimation of creatinine:

Creatinine was analysed by Jaffe’s method.10 Serum sample (1000μl) and picric acid reagent (3000μl) were mixed followed by boiling for 1 min. It was cooled and centrifuged to obtain clear supernatant. Supernatant (2000 μl) was mixed with 0.75 N NaOH (500μl). The combination was kept at 37°C for 15 min. Colour change was noted at 520 nm UV spectrophotometer.

 

3.3 Estimation of uric acid:

Uric acid was estimated by uricase end point method,11 working reagent (1000μl) was mixed with serum (20μl) and incubation was done at 37 °C for 10 min. The colour changed was recorded at 546 nm.

 

3.4 Estimation of albumin:

Albumin estimation was done as per Bromocresol green (BCG) method.12 BCG reagent (1000μl) was mixed with serum sample (20μl) and incubation was done at 37°C for 10 min. Colour change was recorded at 546nm.

 

3.5 Estimation of BUN:

Urea was measured by Diacetylmonoxime (DAM) method.13 Acid reagent (1500μl) was mixed with test sample (20μl) and DAM (1500μl) reagent. The mixture was heated for 10 min and then cooled for 5 min. Colour change was recorded at 540nm.

 

3.6 Tissue homogenate:

After the removal of kidney, it was cleaned with cold saline and homogenized in cold tris HCl buffer at 10% w/v concentration. The solution was subjected to centrifugation at 7000rpm at 25 min. The supernatant obtained was analysed for MDA and SOD.

 

3.6.1 Estimation of SOD:

Kidney homogenate (100μl) was mixed with tris-HCl buffer till 3 ml volume. Pyrogallol (25μl) in 10mM HCl was added to it. Change in absorbance at 420nm was noted at 1 min interval for 3 min. Result was written as SOD units per mg of protein.14

 

3.6.2 Estimation of MDA:

Kidney homogenate (2ml) was added to 10% w/v trichloroacetic acid (2ml). The solution was subjected to cooling and precipitate formed was separated by centrifugation at 2500rpm. Supernatant (2ml) was added to 0.67% thiobarbituric acid (2ml). The mixture was heated and cooled for 5 min. The colour change was noted at 532nm. Result was written as nmol of MDA/mg protein.15

 

3.7 Histopathology studies:

Fixation of kidney was done in 10% neutral formalin for 48 h. This was followed by processing in ascending grades of alcohol. Xylene was used for clearing and paraffin wax for embedding the tissue. The tissue blocks were sectioned with the Rotary Microtome. Slides were stained with Haematoxylin and Eosin and examined under the microscope by Pathologist to study histopathology.

 

3.8 Statistical analysis:

One-way analysis of variance followed by Dunnett post test (ANOVA; Graph Pad PRISM®, Version 5.0, San Diego, CA, USA) was applied for determination of significant differences between the groups. A value of P<0.05 was considered significant.

 

4. RESULTS:

Table 1 illustrates the effect of ACP GB on blood glucose, insulin, uric acid, BUN, albumin. The blood glucose levels were significantly decreased in all groups treated with ACP GB. Significant increase in serum insulin levels were observed in group III (GB-10) and in group VII (ACP GB-400). BUN was decreased significantly in all the groups. Serum uric acid was decreased significantly in all groups. Serum albumin levels was increased significantly in all the groups except group IV (GB-5) and group V (ACP GB-100). Table 2 illustrates the effect of ACP GB on body weight, MDA, SOD. Body weight was significantly increased in all groups. The MDA levels were reduced significantly in group III (GB-10) and in group VII (ACP GB-400). SOD was significantly elevated in group III (GB-10) and in group VII (ACP GB-400). Table 3 illustrates the effect of ACP GB on urine volume, serum creatinine and creatinine clearance. There was significant reduction in urine volume in all the groups. There was significant reduction in serum creatinine levels in all groups except group V (ACP GB-100). Creatinine clearance was significantly increased in group III (GB-10) and in group VII (ACP GB-400).


 

 

Table 1. Effect of ACP GB on blood glucose, insulin, BUN, uric acid, creatinine, albumin

Groups

Blood glucose (mg/dl)

Serum insulin (μU/ml)

BUN(mg/dl)

Serum uric acid (mg/dl)

Serum albumin (g/dl)

I Control

95.5±4.01

13.23±0.35

12.07±1.01

5.76±0.62

5.08±0.37

II Diabetic control

321.5±23.7***

8±0.31***

22.91±0.66***

16.38±0.85***

2.34±0.22***

III GB-10

103.8±4.32***

10.36±0.32***

14.64±1.49***

7.23±0.73***

4.39±0.25***

IV GB-5

124.3±5.47***

9.07±0.26ns

17.6±0.46**

8.92±0.12*

2.78±0.20ns

V ACP GB-100

128±11.8***

8.53±0.40ns

17.14±0.87**

9.53±1.93*

2.64±0.24ns

VI ACP GB-200

115±7.93***

9.36±0.30ns

10.82±0.30***

4.57±0.29***

5.18±0.41***

VII ACP GB-400

107±5.19***

9.72±0.55*

10.46±0.30***

4.35±0.73***

4.57±0.29***

Values are expressed as mean ± S.E.M (n=6) Group II is compared with group I. Group III, IV, V, VI, VII are compared with group II. * P< 0.05, **P< 0.01, ***P< 0.001, ns=non-significant

 

 

 

Table 2. Effect of ACP GB on body weight, MDA, SOD

Groups

Body weight (g)

MDA (nmol/mg)

SOD(U/mg)

I Control

236±1.77

0.91±0.20

0.74±0.03

II Diabetic control

183.8±3.47***

2.45±0.24***

0.22±0.02***

III GB-10

232.5±1.45***

1.23±0.19***

0.62±0.02***

IV GB-5

222.8±2.12***

2.72±0.20ns

0.27±0.03ns

V ACP GB-100

221.3±2.51***

2.93±0.20ns

0.22±0.02ns

VI ACP GB-200

222.3±1.68***

2.60±0.19ns

0.28±0.02ns

VII ACP GB-400

224.2±3.71***

1.47±0.16**

0.34±0.01*

Values are expressed as mean ± S.E.M (n=6) Group II is compared with group I. Group III, IV, V, VI, VII are compared with group II. * P< 0.05, **P< 0.01, ***P< 0.001, ns=non-significant

 


Figure 1 shows the effect of ACP GB on kidney histopathology. Diabetic control rats (Fig. 1.1) produced nephropathic lesions with degeneration of tubules (DT), lymphocytic infiltration (LI), mesangial matrix (MM) expansion. GB-10 (Fig.1.2) showed mild to moderate DT as compared to GB-5. GB-5 (Fig. 1.3) developed DT with MM expansion. ACP GB-100 (Fig. 1.4) and ACP GB-200 (Fig. 1.5) developed nephropathic lesions in kidneys characterized by DT and LI. ACP GB-400 (Fig. 1.6) revealed less severe and degenerative changes characterized by DT as compared to above groups.


 

Table 3. Effect of ACP GB on urine volume, serum creatinine, creatinine clearance

Groups

Urine volume (ml)

Serum creatinine (mg/dl)

Creatinine clearance (ml/min)

I Control

9.06±0.46

0.74±0.13

0.39±0.09

II Diabetic control

21.50±1.42***

3.84±0.28***

0.05±0.01***

III GB-10

12.68±0.65***

1.25±0.22***

0.29±0.05**

IV GB-5

18.30±0.61**

3.12±0.17**

0.10±0.01ns

V ACP GB-100

18.08±0.65**

4.69±0.27ns

0.09±0.01ns

VI ACP GB-200

16.40±0.47***

2±0.26***

0.19±0.03ns

VII ACP GB-400

14.58±0.29***

1.87±0.25***

0.24±0.03*

Values are expressed as mean ± S.E.M (n=6) Group II is compared with group I. Group III, IV, V, VI, VII are compared with group II. * P< 0.05, **P< 0.01, ***P< 0.001, ns=non-significant

 

Fig.1 Effect of ACP GB on histopathology of kidney

 


5. DISCUSSION:

Herbal drugs are widely used for the management of diabetes over centuries. In addition to the mainstream treatment, diabetic patients also consume herbal medicines with antidiabetic properties. These may mediate beneficial as well as detrimental effects in effective management of the disease. Thus, the potential interaction of herb and drug can be a double-edged sword with risks as well as benefits.16 Administration of herbal formulation can significantly influence the sequence of glucose tolerance in normal and diabetic animals.17 Alloxan administration induces several pathophysiological conditions like beta cell destruction, islets necrosis, hyperglycaemia, oxidative stress that have similar characteristics to that of human diabetes. It induces the generation of ROS that mediate cellular damage. The cascade of events lead to deterioration of the glomeruli, tubular cells and assembling of extracellular matrix indicative of DN.18 It causes diabetes by destruction of the beta cell resulting in the decline in endogenous insulin release, inturn causing decline in glucose utilization.19 Alloxan treated groups exhibited significant augmentation in blood glucose. The extermination of the insulin secreting beta cells by alloxan resulted in hyperglycaemia. Maintenance of normal glycemic status is indeed challenging in the characteristic environment of diabetes and chronic kidney disease. 20 In GB treated diabetic rats, decline in glucose while increase in insulin was recorded. This might be due to the insulin secretagogue action along with improved uptake of peripheral glucose and decrease in endogenous production of glucose.21 Our study was in agreement with these reports that also stated the hypoglycemic effect of GB attributing to its insulin stimulatory effect from beta cells of pancreas. Co-administration of ACP with GB depleted the blood glucose in diabetic rats. Previous studies reported the antidiabetic activity of AR ascribed to the suppression of α-amylase and α-glucosidase enzyme.4 The antioxidant and hypolipedemic effect of flavone glycoside present in PR was responsible for its antidiabetic effect.22 Depletion in serum insulin was seen in alloxan administered rats. ACP GB 400 raised serum insulin. The antidiabetic effect may be ascribed to insulin release from the existing pancreatic cells. Previous studies reported significant stimulatory effects of AR on insulin secretion.23 It might facilitate the insulin secretion along with GB and other extracts in combination.

 

Alloxan induced diabetes caused kidney dysfunction indicated by the increase in uric acid, BUN, creatinine.24 Purine catabolism leads to the production of uric acid in liver, muscle, adipose tissue. An imbalance in the level of production, absorption and excretion increased serum uric acid.25 Creatinine is a creatine metabolite found in skeletal muscle. The creatine per unit of skeletal muscle and its breakdown rate is consistent and is indicative of skeletal mass. Urea is produced by liver and is major nitrogenous end product of protein breakdown.26 Disturbance in the nitrogen balance and reduced protein production elevates blood urea. Depletion in the filtering potential of kidneys might raise BUN, creatinine, uric acid levels in diabetic rats. Reduction in BUN, creatinine, uric acid levels by the combination treatment of ACP and GB possibly by increasing its clearance from the blood is an indication of the restoring ability of the kidneys. The alteration in the biochemical values suggested its role in renoprotection in diabetes. Low serum albumin is linked with insulin resistance. Some investigations revealed the correlation of insulin and albumin production. Insulin mediates effect in the synthesis of albumin. Insulin augmented albumin gene transcription and mRNA production in a concentration dependent mode. While, insulin deficit declined this process and reduced albumin.27 Significant reduction of serum albumin in diabetes substantiated with the previous studies.28 ACP GB administration increased serum albumin that may be the result of insulin secretion and insulin mediated protein synthesis.

 

Polyuria is the principle symptom of diabetes evidenced due to osmotic diuresis and impairment in the reabsorption of glucose by proximal tubules.29 Significant decrease in urine volume was found by ACP GB treatment that might be due to the reduction in glucose level or osmotic diuresis. Body weight in diabetic animals was notably less than normal animals. Diabetes is accompanied with weight loss owing to muscle wasting and protein deprivation. Reduction in insulin decreased glucose as a source of energy, that resulted in gluconeogenesis attributing to weight loss. Diabetic rats treated with ACP GB showed improvement in body weight that may be due to decrease in gluconeogenesis, muscle wasting and improvement in glucose level. The data obtained was in accordance with previous investigations.28

 

Diabetes mellitus is characterized by impairment in glucose metabolism resulting in elevation in blood glucose and formation of free radicals.30 Various types of free radicals are frequently formed for specific metabolic needs and quenched by an efficient antioxidant system of body. Oxidative injury of cells may result if the amount of free radicals exceeds the antioxidant pathways.31Treatment with an antioxidant is needed if the body fails to balance increased oxidative stress.32 Antioxidant-based formulations are utilized for the prevention and treatment of diseases like atherosclerosis, diabetes, stroke and cancer.33

 

Creatinine clearance was significantly reduced in diabetic control group. The decrease in creatinine clearance rate revealed successive development of kidney failure. Treatment with ACP GB significantly increased the creatinine clearance indicating its ability to clear the creatinine from the circulation into the urine. Oxidative stress and lipid peroxidation is linked with alloxan administration. Lipid peroxidation is the outcome of the reaction of free radicals with polyunsaturated fatty acids.34 Increase in TBARS damages DNA induced by H2O2.35 MDA is the product of the oxidation of polyunsaturated fatty acids that acts as an indicator for oxidative stress.36 Many products are formed by lipid peroxidation that get transported from the organ into the circulation and get excreted in urine. MDA is one of the product and biomarker of ROS damage. It determines the extent of cellular toxicity. It is formed as a result of the reaction between molecular oxygen and polyunsaturated fatty acids. As biologic membranes are abundant in polyunsaturated fatty acid they are susceptible to ROS attack.37 Increase in glucose gives rise to oxidative stress and increases tissue MDA levels. Decreased SOD is also aligned with the rise in oxidation. Glucose elevates the superoxide radical by mitochondrial electron-transport. Superoxide radicals lead to vascular cell damage by PKC activation, hexosamine and advanced glycation end products. SOD eliminates superoxide by converting it into H2O2 and molecular oxygen. Depletion of SOD activity in diabetes mediated superoxide overproduction and renal cell injury.38 In ACP GB treated rats, decrease in MDA and rise in SOD was seen. It was reported that the polysaccharides present in AR possessed the scavenging abilities that inhibited lipid peroxidation, prevented SOD and protein thiols from inactivation attributing to the antioxidant activity.39 The antioxidant effect of CA was due to polyphenols, triterpenoids and flavonoid content.40 High phenolic content and flavone glycoside in PR was responsible for the antioxidant activity and decrease in MDA.6,22 Synergistic effect of these components might be responsible for the abolition of lipid peroxidation and elevation of SOD activity. The presence of mesangial matrix expansion within the glomeruli indicated glomerulopathy in diabetic kidneys. Glomerulopathy is the prominent structural change in DN, identified by glomerulosclerosis and mesangial matrix accumulation. Histopathological observation also revealed the mitigatory effect of ACP in combination with GB. Further, these histological observations corroborated with the biochemical parameters. Our study demonstrated, that the lower dose i.e ACP GB 100 was not sufficient to cause significant changes in all biochemical parameters. The effect of ACP GB 400 was comparable to that of GB 10 and was more pronounced in comparison with GB 5. An additive effect was mediated by ACP GB 400 as compared to GB 5.

 

6. CONCLUSION:

In this study, it is observed that ACP has shown significant renoprotection in DN. This might be assigned to its antihyperglycemic, antioxidant effect along with the restoration of morphological injuries to the kidney. The study also suggested that ACP potentiated the effect of GB, implicating its dose reduction that may also reduce the chances of its side effects.

 

7. ACKNOWLEDGEMENT:

The study did not receive any financial support. The authors are thankful to MCE Society’s Allana College of Pharmacy, Pune for the provisions related to the work.

 

8. ETHICAL STATEMENT:

The study protocol was approved by Institutional Animal Ethical Committee (ACP/IAEC/2018/01).

 

9. CONFLICT OF INTEREST:

Authors do not have conflict of interest.

 

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Received on 15.12.2020                Modified on 16.04.2021

Accepted on 08.06.2021               © RJPT All right reserved

Research J. Pharm.and Tech 2022; 15(4): 1614-1620.

DOI: 10.52711/0974-360X.2022.00270