THE PROTECTIVE ROLE OF RAPHANUS SATIVUS ROOTS IN REVERSING THE RENAL ALTERATIONS AND OXIDATIVE DAMAGE AGAINST STREPTOZOTOCIN INDUCED DIABETES IN RATS

THE PROTECTIVE ROLE OF RAPHANUS SATIVUS ROOTS IN REVERSING THE RENAL ALTERATIONS AND OXIDATIVE DAMAGE AGAINST STREPTOZOTOCIN INDUCED DIABETES IN RATS

P. Shanmuga Priya ^* and N. Jayshree

Institute of Pharmacology, Madras Medical College, Chennai - 600003, Tamil Nadu, India.

ABSTRACT: Background: Oxidative stress and hyperglycemia are two major factors implicated in the development of renal damage. Roots of Raphanus sativus have been reported to possess both ant diabetic and antioxidant activities. So, the present study was aimed to investigate whether aqueous root extract of Raphanus sativus (ARRS) is effective in reversing the renal alterations and oxidative damage against streptozotocin (STZ) induced diabetes in rats. Methods: Rats were divided into five groups of six animals each. Group I is normal control which receives distilled water (p.o). Remaining animals were injected with STZ (60 mg/kg; i.p) to induce diabetes. 3 days after injection, animals with blood glucose levels greater than 250 mg/dl were divided into 4 groups: Group II received distilled water, Group III received glibenclamide (GLB; 5 mg/kg) and group IV and V received 100 and 200 mg/kg of ARRS at oral route for 8 weeks. At the end of 8 weeks, blood glucose, glycated hemoglobin (HbA1c), albumin, creatinine, blood urea nitrogen (BUN), malondialdehyde (MDA) and glutathione (GSH) levels were analyzed in all the groups. Results: ARRS (100 and 200 mg/kg) treatment in diabetic rats showed a significant reduction in blood glucose, HbA1c and also reversed the albumin, creatinine, BUN, MDA and GSH levels as compared to untreated diabetic rats. Conclusion: From the present study, it is concluded that the aqueous root extract of Raphanus sativus possesses promising renoprotective activity against diabetes-induced renal alterations.

Hyperglycemia, Oxidative stress, Aqueous root extract of Raphanus sativus, Streptozotocin, Advanced glycation end products, Kidney markers

INTRODUCTION: Diabetes mellitus is characterized by hyperglycemia resulting from defects in insulin secretion, insulin action or both. Sustained hyperglycemia is further, associated with the development of many complications in diabetic patients.

Renal damage is one of the major microvascular complications and is reported to develop in 30-40% of patients with diabetes and has become a leading cause of end-stage renal failure worldwide ¹. It is characterized by the presence of pathological quantities of albumin in the urine, diabetic glomerular lesions and decline in glomerular filtration rate ².

Poor glycemic control, oxidative stress, and accumulation of advanced glycation end products (AGE) play a significant role in the development of renal dysfunction in diabetic nephropathy (DN) ¹. Antihypertensive such as angiotensin-converting enzyme inhibitors, angiotensin-1 antagonists and AGE inhibitor, aminoguanidine are the currently available treatments for progressive diabetic nephropathy.

The currently available drugs, though useful, are associated with side effects ². Hence, the demand for herbal medicines is increasing day by day. Raphanus sativus commonly known as radish is a root vegetable. The phytoconstituents present in leaves and roots of radish are alkaloids, nitrogen compounds, coumarins, gibberellins, glucose-inolates, oilseed compounds, organic acids, phenolic compounds, quercetin, pigments etc. ³ Leaves and roots of Raphanus sativus have phenolic compounds that have been associated with antidiabetic effects ^{3, 4}. Catechin, a phenolic compound present in radish, significantly enhances insulin secretion. Aqueous extract of Raphanus sativus roots has insulin-like components ⁵. Antidiabetic activity of Raphanus sativus roots has been reported earlier ⁶.

Thus, the aim of the present study was to evaluate the protective effects of aqueous extract of Raphanus sativus roots in reversing the renal alterations and oxidative damage against streptozotocin-induced diabetes in rats.

MATERIALS AND METHODS:

Procurement and Authentication of Plant: The plant was collected from the market of Chennai, India in the month of August 2018 and authenticated by Dr. K.N. Sunil Kumar, Research Officer and HOD of Pharmacognosy, Siddha Central Reseach Institute (Central Council for Research in Siddha), Chennai.

Preparation of Plant Extract: The freshly collected roots were chopped, shade dried and powdered. The powdered roots were macerated with water for 7 days using 6% chloroform as a preservative. The obtained aqueous extract was evaporated to dryness for in-vitro and in-vivo studies.

Drugs and Chemicals: The standard drug, glibenclamide was procured from Pharmafabrikon Ltd, Madurai. STZ was purchased from Lab Chemicals, Chennai. Blood glucose was analyzed using a one-touch Accu-chek glucometer. HbA1c, albumin, creatinine and BUN estimation were done using biochemical kits obtained from Coral Clinical Systems, Goa, India. All the chemicals and solvents used were of analytical grade.

In-vitro Evaluation of Antioxidant Activity:

DPPH Radical Scavenging Assay: The free radical-scavenging activity of ARRS was measured by DPPH radical scavenging assay. For this, 1 ml of DPPH solution (0.1 mM) in methanol was added to different concentrations (200-1000 µg/ml) of extract. The reaction mixture was incubated in dark conditions at room temperature for 20 min. After 20 min, the absorbance was measured at 517 nm using a UV-Visible spectrophotometer. A control was prepared without adding extract. Rutin was used as a standard. The % radical scavenging activity of the extract was calculated using the formula ⁷.

% RSA = [Abs _Control-Abs _sample/Abs _Control] × 100

Where
RSA = Radical scavenging activity

Abs_control = Absorbance of the DPPH radical

Abs_sample = Absorbance of DPPH + ARRS.

The antioxidant activity of ARRS was expressed as IC₅₀ and compared with the standard.

Superoxide Radical Scavenging Activity: It was assayed by spectrophotometric method. 1ml of nitro blue tetrazolium solution, 1 ml of NADH solution, 0.1 ml of ARRS (10 mg in 0.1 ml dimethyl sulfoxide and 0.9 ml of phosphate buffer) and 0.1 ml of phenazine meth sulfate solution were mixed and incubated at 25 °C for 5 min. After 5 min, the absorbance was read at 560 nm. One unit of enzyme activity is defined as the amount of enzyme that gave 50% inhibition of nitroblue tetrazolium reduction in one min ⁷.

% Inhibition = A560 nm of blank - A560 nm of test / A560 nm of blank × 100

In-vivo Studies:

Animals: Albino Wistar rats of either sex weighing 130-150 g were used for the study. They were maintained under standard laboratory conditions (22 ± 3 °C, 12-h light/dark cycle) supplied with standard pellet food and water given ad libitum in Animal Experimental Laboratory, Madras Medical College, Chennai.

The animals cared in accordance with the guidelines provided by the CPCSEA. Approval for the study was obtained from the Institutional Animal Ethics Committee (Approval no. 34/18).

Experimental Design: Experimental design 6 Albino Wistar rats were included in group I.

Group I: Distilled water (p.o; 8 weeks) the remaining rats were injected with streptozotocin (60 mg/kg; i.p) dissolved in ice-cold citrate buffer. Three days after streptozotocin injection, blood was withdrawn from the tail vein and animals with blood glucose levels greater than 250 mg/dl were used for further studies and were grouped as.

Group II: Distilled water (p.o; 8 weeks)

Group III: GLB (5 mg/kg; p.o; 8 weeks)

Group IV: ARRS (100 mg/kg; p.o; 8 weeks)

Group V: ARRS (200 mg/kg; p.o; 8 weeks)

At the end of 8 weeks, rats were placed in the metabolic cages for 24 h urine collection and urine collected was estimated for albumin, creatinine, and urea nitrogen levels. Rats were sacrificed with high doses of isoflurane and blood samples were collected through cardiac puncture and were estimated for blood glucose and HbA1c levels. The remaining blood samples are centrifuged at 3000 rpm for 10 min to obtain clear sera. Albumin, creatinine and BUN levels were estimated in serum. Kidneys were excised without any damage and stored in 10% neutral buffered formalin. Half of the kidneys were used for histopathological studies. The remaining kidney was homogenized and was used for the estimation of MDA and GSH levels.

Blood Analysis:

Estimation of Bood Glucose: Blood glucose was measured using a one-touch glucometer.

Estimation of Glycated Hemoglobin: Glycated hemoglobin level was measured by the turbidity method as described in the product insert of Coral clinical systems. The HbA1c is measured as a percentage of total hemoglobin in human whole blood (% HbA1c). In the first stage of the reaction: the HbA1c in the sample reacts with the anti-HbA1c specific antibody to form soluble antigen-antibody complexes. Then the polyhapten is added. The polyhapten reacts with the specific antibody excess from the first reaction, producing insoluble immune complexes which can be measured turbid metrically at 340 nm ⁸.

% HbA1c = 91.5 × HbA1c (g/dl) / Hb (mg/dl) + 2.15.

Serum Analysis:

Estimation of Albumin: Albumin content was estimated by bromocresol green method using the kit procured from the Coral clinical systems. Briefly, 1 ml of reagent was added to 0.01 ml of the sample and the solutions were mixed well and incubated for 5 min. Absorbance was read at 630 nm after incubation. The anionic dye-bromocresol green binds to albumin present in the sample, forming a green-colored complex. The measured absorbance was compared with the standard and the albumin content was expressed in g/dl ⁹.

Estimation of Creatinine: Creatinine content was estimated by modified. Jaffe method using the kit purchased from Coral clinical systems. Briefly, 0.5 ml of the sample was added to 5 ml of reagent and the solutions were mixed well. The mixture was kept in boiling water for 40 sec and 0.2 ml of 2.5 M sodium hydroxide was added. Initial absorbance (520 nm) A₁, for the standard and test exactly after 30 sec was read. Another absorbance A2 of the standard and test exactly 60 sec later was also read. The change in absorbance for both the standard and the test was calculated and the creatinine content was expressed in mg/dl ¹⁰.

Estimation of BUN: 0.01 ml of sample was added to the acid, urea and diacetyl monoxime reagent 1 ml and was kept in boiling water 100 °C for 10 min. Urea present in the sample reacts with diacetyl monoxime in the presence of thiosemicarbazide to form a purple-colored complex, which was measured at 520 nm. The absorbance was compared with that of the standard and the urea nitrogen content was expressed as mg/dl ¹¹.

Urine Analysis: Urine albumin, creatinine, and urea nitrogen was estimated using the same method as in serum analysis

Kidney Homogenate Analysis:

Preparation of Kidney Homogenate: Kidneys were washed with saline, and homogenate (10% w/v) was prepared with 0.1 M phosphate buffer and centrifuged at 3000 g for 10 min at 4 °C. The supernatant fraction was used for the determination of MDA and GSH levels ¹².

Estimation of MDA: To 2.5 ml of tissue homogenate, 0.5 ml of saline (0.9% sodium chloride) and 1.0 ml (20% w/v) of trichloroacetic acid were added and centrifuged for 20 min at 4000 rpm. The mixture was incubated at 95 °C for 1 h. Finally, an equal volume of n-butanol was added to the mixture and the contents were centrifuged for 15 min at 4000 rpm ¹². The absorbance of the organic layer was measured at 532 nm. The level of MDA is expressed as nanomoles of MDA/g of tissue

Nanomole of MDA/ g of tissue = V×A₅₃₂/0.156 × g of tissue

V = Final volume of the test solution A =    Absorbance at 532 nm 0.156 = Extinction coefficient of MDA/M/cm.

Estimation of GSH: 0.5 ml of tissue homogenate was mixed with 1.5 ml of 0.2 M tris buffer (pH-8.2) and 0.1ml of 0.01M dithiobis trinitrobenzoic acid and this mixture was made up to 10.0 ml with 7.9 ml of absolute methanol. The above reaction mixture was centrifuged at approximately 300 rpm at room temperature for 15 min. The absorbance of the supernatant was read in a spectrophotometer against a reagent blank (without sample) at 412 nm ¹³.

Mg of GSH/ g of tissue = A₄₁₂ × dilution factor/1.36 X 10⁴ × 100

1.36 × 10⁴ = Extinction coefficient of GSH

Histopathological Studies: The kidneys from the animals were rinsed in ice-cold 0.9% saline and were fixed in 10% formalin embedded in paraffin and cut into 5 µm thick sections using a microtome. Sections were mounted on glass slides using standard techniques. The sections were stained with Hematoxylin-Eosin (H and E) and were examined under a microscope using 40 × magnifications.

Statistical Analysis: All the data are expressed as mean ± SEM (n = 6). Statistical analysis was performed by one-way ANOVA followed by Dunnet's test using Graph pad prism software version 8.0.1. The results were considered statistically significant if p < 0.05.

RESULTS:

In-vitro Evaluation of Antioxidant Activity:

DPPH Radical Scavenging Activity: The effect of ARRS on DPPH scavenging assay are shown in Table 1 and Fig. 1 (A) ARRS and standard rutin showed concentration-dependent scavenging activity on DPPH radical at different concentrations (200-1000 µg/ml).

IC₅₀ of ARRS (586.98 µg/ml) was marginally higher than that of the reference standard (532.93 µg/ml). These results indicate that the ARRS exhibited the ability to quench the DPPH radicals proving that the extract has antioxidant activity.

Superoxide Radical Scavenging Activity: The effect of ARRS on superoxide radical scavenging assay is shown in Table 1 and Fig. 1B. ARRS and standard ascorbic acid showed concentration-dependent scavenging activity on superoxide radical at different concentrations (200-1000 µg/ml).

IC₅₀ of ARRS (87.20 µg/ml) was marginally higher than that of the reference standard (33.61µg/ml). These results indicate that the ARRS exhibited effective antioxidant activity.

In-vivo Studies:

Blood Analysis:

Estimation of Blood Glucose: The effect of ARRS on blood glucose levels is shown in Tab1e 2 and Fig. 2A.

TABLE 1: EFFECT OF ARRS ON DPPH AND SUPEROXIDE RADICAL SCAVENGING ASSAY

Values are expressed in mean ± SEM of triplicate determinations

The blood glucose levels were significantly increased (P < 0.001) in the untreated diabetic rats when compared to normal rats. The diabetic rats which were treated with glibenclamide and ARRS (100 and 200 mg/kg) showed a significant decrease (P < 0.001) in blood glucose as compared to untreated diabetic rats.

FIG 1: EFFECT OF ARRS ON (A) DPPH AND (B) SUPEROXIDE RADICAL SCAVENGING ASSAYS

Though there was a decrease in the raised glucose levels in the glibenclamide and ARRS (100 and 200 mg/kg) treated groups, the levels were still significantly (P < 0.001) higher than the normal levels.

Estimation of HbA1c: The effect of ARRS on HbA1c levels is shown in Tab1e 2 and Fig. 2B. The HbA1c was significantly increased (P < 0.001) in the untreated diabetic rats when compared to normal rats. The diabetic rats which were treated with glibenclamide and ARRS (100 and 200 mg/kg) showed a significant decrease (P < 0.001, P < 0.01 and P < 0.001) in HbA1c levels as compared to untreated diabetic rats. Even though the HbA1c levels showed an improvement in the glibenclamide and ARRS (100 mg/kg) treated rats, the levels were still significantly (P < 0.001) higher than the normal levels. But in the ARRS (200 mg/kg) treated group of animals, the HbA1c levels were restored to normal levels and there was no significant difference compared with normal values.

Serum Analysis:

Estimation of Serum Albumin: The effect of ARRS on serum albumin levels are shown in Table 2 and Fig. 2C. The serum albumin levels were significantly decreased (P < 0.001) in the untreated diabetic rats when compared to normal rats. The diabetic rats which were treated with glibenclamide and ARRS (100 and 200 mg/kg) showed a significant increase (P < 0.001) in serum albumin levels as compared to untreated diabetic rats. Though, there was a rise in the decreased serum albumin levels in ARRS (100 mg/kg) treated group, the levels were still significantly lower (P < 0.001) than the normal levels. But, in the glibenclamide and ARRS (200 mg/kg) treated groups, the serum albumin levels were restored to normal levels and there was no significant difference compared to normal values.

Estimation of Serum Creatinine: The effect of ARRS on serum creatinine levels are shown in Table 2 and Fig. 2D. The serum creatinine levels were significantly increased (P < 0.001) in the untreated diabetic rats when compared to normal rats. The diabetic rats which were treated with glibenclamide and ARRS (100 and 200 mg/kg) showed a significant decrease (P < 0.001) in serum creatinine levels as compared to untreated diabetic rats. Though there was a decrease in the raised serum creatinine levels in the glibenclamide and ARRS (100 and 200 mg/kg) treated groups, the levels were still significantly higher (P < 0.01, P < 0.001 and P < 0.01) than the normal levels.

Estimation of serum BUN: The effect of ARRS on serum BUN is shown in Table 2 and Fig. 2 E. The serum BUN levels were significantly increased (P < 0.001) in the untreated diabetic rats when compared to normal rats. The diabetic rats which were treated with glibenclamide and ARRS (100 and 200 mg/kg) showed a significant decrease (P < 0.001) in serum BUN levels as compared to untreated diabetic rats. Even though the serum BUN levels showed an improvement in the glibenclamide and ARRS (100 mg/kg) treated rats, the levels were still significantly higher (P < 0.01 and P < 0.001) than the normal levels. But in the ARRS (200 mg/kg) treated group of animals, the serum BUN levels were restored to normal levels and there was no significant difference compared to normal values.

Estimation of Serum BUN: The effect of ARRS on serum BUN is shown in Table 2 and Fig. 2 (E). The serum BUN levels were significantly increased (P < 0.001) in the untreated diabetic rats when compared to normal rats. The diabetic rats which were treated with glibenclamide and ARRS (100 and 200 mg/kg) showed a significant decrease (P < 0.001) in serum BUN levels as compared to untreated diabetic rats. Even though the serum BUN levels showed an improvement in the glibenclamide and ARRS (100 mg/kg) treated rats, the levels were still significantly higher (P < 0.01 and P < 0.001) than the normal levels. But in the ARRS (200 mg/kg) treated group of animals, the serum BUN levels were restored to normal levels and there was no significant difference compared to normal values.

TABLE 2: EFFECT OF ARRS ON METABOLIC PARAMETERS AND SERUM BIOCHEMISTRY

Table represents the metabolic parameters and serum biochemistry of the animals from five groups; NC-normal control, DC- diabetic control, GLB - glibenclamide. Values represent the mean ± SEM of the samples (n = 6).  NS- non significant, ##P < 0.01 and ###P < 0.001 compared to normal control group. ** P < 0.01 and *** P < 0.001 compared to diabetic control group

FIG. 2: EFFECT OF ARRS ON (A) BLOOD GLUCOSE, (B) HBA1C, (C) SERUM ALBUMIN, (D) SERUM CREATININE (E) SERUM BUN. NC NORMAL CONTROL, DC DIABETIC CONTROL, GLB GLIBENCLAMIDE Values are expressed as mean ± SEM (n = 6). NS, ^##P < 0.01 and #^##P < 0.001 compared to normal control group. ^** P < 0.01 and ^*** P < 0.001 compared to diabetic control group

Urine Analysis:

Estimation of Urine Albumin: The effect of ARRS on urine albumin levels is shown in Table 3 and Fig. 3A. The urine albumin levels were significantly increased (P < 0.001) in the untreated diabetic rats when compared to normal rats. The diabetic rats which were treated with glibenclamide and ARRS (100 and 200 mg/kg) showed a significant decrease (P < 0.001) in urine albumin levels as compared to untreated diabetic rats.

Even though the urine albumin levels showed an improvement in the glibenclamide and ARRS (100 and 200 mg/kg) treated rats, the levels were still significantly higher (P < 0.01, P < 0.001 and P < 0.05) than the normal levels.

Estimation of Urine Creatinine: The effect of ARRS on urine creatinine levels are shown in Table 3 and Fig. 3B. The urine creatinine levels were significantly decreased (P < 0.001) in the untreated diabetic rats when compared to normal rats. The diabetic rats which were treated with glibenclamide and ARRS (100 and 200 mg/kg) showed a significant increase (P < 0.001) in urine creatinine levels as compared to untreated diabetic rats.

Even though the urine creatinine levels showed an improvement in the glibenclamide and ARRS (100 mg/kg) treated rats, the levels were still significantly lower (P < 0.05 and P < 0.001) than the normal levels. But in the ARRS (200 mg/kg) treated group of animals, the urine creatinine levels were restored to normal levels and there was no significant difference compared to normal values.

Effect of ARRS on Urea Nitrogen: The effect of ARRS on urea nitrogen is shown in Table 3 and Fig. 3C. The urea nitrogen levels were significantly decreased (P < 0.001) in the untreated diabetic rats when compared to normal rats. The diabetic rats which were treated with glibenclamide and ARRS (100 and 200 mg/kg) showed a significant increase (P < 0.001) in urea nitrogen levels as compared to untreated diabetic rats.

Though there was a rise in the decreased urea nitrogen levels in the glibenclamide and ARRS (100 and 200 mg/kg) treated groups, the levels were still significantly lower (P < 0.001) than the normal levels.

Kidney Homogenate Analysis:

Estimation of MDA: The effect of ARRS on renal MDA levels is shown in Table 4 and Fig. 4A. The renal MDA levels were significantly increased (P < 0.001) in the untreated diabetic rats when compared to normal rats. The diabetic rats which were treated with glibenclamide and ARRS (100 and 200 mg/kg) showed a significant decrease (P < 0.001) in renal MDA levels as compared to untreated diabetic rats.

Though there was a decrease in the raised MDA levels in the glibenclamide and ARRS (100 and 200 mg/kg) treated groups, the levels were still significantly higher (P < 0.001) than the normal levels.

Estimation of GSH: The effect of ARRS on renal GSH levels is shown in Table 4 and Fig. 4B. Renal GSH levels were significantly decreased (P < 0.001) in the untreated diabetic rats when compared to normal rats. The diabetic rats which were treated with glibenclamide and ARRS (100 and 200 mg/kg) showed a significant increase in (P < 0.001) in renal GSH levels as compared to untreated diabetic rats. Though there was a rise in the decreased GSH levels in the glibenclamide and ARRS (100 and 200 mg/kg) treated groups, the levels were still significantly (P < 0.001) lower than the normal levels.

TABLE 3: EFFECT OF ARRS ON URINE BIOCHEMISTRY

Table represents the urine biochemistry of the animals from five groups; NC-normal control, DC- Diabetic control, GLB - Glibenclamide. Values represent the mean ± SEM of the samples (n = 6).  NS- non significant, ^#P < 0.05, ^##P < 0.01and #^##P < 0.001 compared to normal control group. ^*** P < 0.001 compared to diabetic control group

FIG. 3: EFFECT OF ARRS ON (A) URINE ALBUMIN, (B) CREATININE (C) UREA NITROGEN. NC – NORMAL CONTROL, DC-DIABETIC CONTROL GLB - GLIBENCLAMIDE values are expressed as mean ± SEM (n = 6). Ns, ^#p < 0.05, #^#p < 0.01 and ^###p < 0.001 compared to normal control group.  ^***p < 0.001 compared to diabetic control group

TABLE 4: EFFECT OF ARRS ON MDA AND GSH LEVELS

Table represents the oxidative stress parameters of the animals from five groups; NC-normal control, DC- diabetic control, GLB - Glibenclamide values are expressed as mean ± SEM (n = 4). ^### p < 0.001 compared to normal control group. ^***p < 0.001 compared to diabetic control group

FIG. 4: EFFECT OF ARRS ON (A) RENAL MDA, (B) GSH LEVELS. NC - NORMAL CONTROL, DC - DIABETIC CONTROL AND GLB - GLIBENCLAMIDE values are expressed as mean ± SEM (n = 4). ^### p < 0.001 compared to normal control group. ^***p < 0.001 compared to diabetic control group

Histopathological Studies: The histopathology of the kidneys was carried out and the photograph of the slides was given in Fig. 5A-5E. A. Photomicrograph of sections from normal rats showed normal glomerular architecture represented by thin glomerular basement membrane (G), mesangium (M) and tubules (T) with healthy epithelial cells. B. Diabetic control rats showed massive thickening of the glomerular basement membrane (G), mesangium (M) and degenerative tubules (T). C. GLB treated group showed a mild improvement in reversing the thickening of the glomerular basement membrane (G) and mesangial expansion (M). The kidney tissue showed improved tubular epithelial cells with less congestion of blood vessels (T). D. ARRS (100 mg/kg) treated group revealed an improvement in glomerular structure represented by a thin glomerular basement membrane (G) and mesangial expansion (M). Tubules showed regenerative changes as compared to the diabetic control group. E. ARRS (200 mg/kg) treated group revealed a significant improvement in glomerular structure. The kidney tissues showed a thin glomerular basement membrane (G) with mesangium (M). Tubules also showed regenerative changes with healthy epithelial cells (H and E 40).

FIG. 5 A-E: HISTOPATHOLOGY OF KIDNEYS

DISCUSSION: Diabetes mellitus is a group of metabolic disorders which is characterized by high blood glucose level over a prolonged period. Diabetes-related complications can be divided into vascular and nonvascular complications. The vascular complications of diabetes mellitus are subdivided into microvascular (retinopathy, neuropathy and nephropathy) and macrovascular (coronary heart disease, peripheral arterial disease, cerebrovascular disease) complications. Non-vascular complications include gastroparesis, infections, skin changes and hearing loss ¹⁴.

Among the diabetic complications, diabetic nephropathy is considered as one of the most critical and life-threatening complications and has become the leading cause of chronic kidney disease in the developed world. Chronic continuous hyperglycemia has been mainly implicated in the pathogenesis of diabetic nephropathy.

However, other factors such as increased oxidative stress, chronic inflammation, impaired insulin signaling, dyslipidemia, renal polyol formation and accumulation of advanced glycation end-products can also significantly contribute to the onset and progression of diabetic nephropathy. The high rates of incidence, high mortality rate and relatively poor prognosis associated with diabetic nephropathy have attracted much interest and research on this subject ¹⁵.

Currently used drugs for this condition are the antidiabetic agents such as peroxisome proliferator-activated receptor inhibitors, DPP-4 inhibitors and sodium-glucose cotransport-2 inhibitors. Anti-hypertensive agents particularly those targeting the renin-angiotensin system such as ACE inhibitors and angiotensin receptor-1 antagonists are reported to be the most effective treatments for progressive renal damage. Novel agents such as renin inhibitors, endothelin inhibitors, phosphodiesterase inhibitors, aldose reductase inhibitors, AGE inhibitors and glycosaminoglycans are widely used.

Among the novel agents, AGE inhibitor, aminoguanidine, is currently used for attenuating albuminuria, mesangial expansion and collagen deposition in diabetic rats ². However, the current therapeutic approach towards alleviating renal damage has not been fully effective which may be due to the complexity involved in the pathogenesis of chronic kidney dysfunction in renal damage, in addition to the unpleasant side effects that accompany their use. The reliance on diabetic patients on plant-based therapy for treatment is gradually increasing in recent years. This is due to their relative availability, perceived effectiveness, low cost and fewer side effects ¹⁵. So, a compound with anti-diabetic and antioxidant activities will be the better option for the treatment of DN. Raphanus sativus is a root vegetable commonly known as radish. Anti-diabetic ⁶, anti-hyperlipidemic ¹⁶, anti-urolithiasis ¹⁷ and antioxidant ¹⁸ activities have been reported previously in leaves and roots of Raphanus sativus. Raphanus sativus has various phytoconstituents such as alkaloids, nitrogen compounds, coumarins, gibberellins, glucosino-lates, organic acids, pigments, flavonoids, phenolics, polysaccharides and quercetin ³. Among them, phenolic compounds are the ones that might be responsible for its anti-diabetic activity ⁴.

Catechin, a phenolic compound present in radish roots significantly enhances insulin secretion⁵. Hence it was felt that the Raphanus sativus would be the ideal plant that has both anti-hyperglycemic and antioxidant activity for the treatment of diabetes-induced renal damage.

The literature review indicated that this activity has not so far been evaluated. In order to confirm the antioxidant activity of aqueous root extract of Raphanus sativus, in-vitro DPPH and superoxide radical scavenging assays were carried out. Our data reveal that, in DPPH and SOD scavenging assays, ARRS shows good antioxidant activity which might be due to the presence of phenolic compounds. Poor glycemic control plays a significant role in the development of DN¹.

It has been already reported that phenolics in plants are the main compounds responsible for anti-diabetic activity ⁴. Roots of Raphanus sativus have been already evaluated for antidiabetic activity ⁶. Streptozotocin has been widely used to induce diabetes in rodent models. It is a broad-spectrum, aminoglycoside antibiotic and acts as a nitric oxide donor. STZ is mostly used as an investigational drug for diabetes research due to its specific toxicity associated with pancreatic β cells.

Because of its structural features, STZ gets selective entry into the β-cells of the islets of Langerhans via the low-affinity glucose transporter glucose transporter-2 in its plasma membrane and causes methylation and fragmentation of DNA, inhibits DNA synthesis and finally causes irreversible necrosis of β cells ¹⁹.

For our study also diabetes was induced using the STZ model. The results from our study confirm the anti-diabetic activity of ARRS. It was found that untreated diabetic rats show elevated blood glucose levels, while treatment with ARRS reduced the elevated blood glucose levels to almost normal levels. ARRS has phenolics along with insulin-like components in the aqueous extract which might be responsible for providing effective anti-diabetic activity. In longstanding hyperglycemia, excess glucose combines with free amino groups on lipids, proteins and nucleic acids to form AGE. AGE results in the expression and activation of a number of transcription factors implicated in the development of diabetic nephropathy, including nuclear factor-kappa-β and PKC. AGE also contributes to the release of pro-inflammatory cytokines and expression of growth factors such as TGF-β and connective tissue growth factor by interacting with specific receptors ²⁰.

Our results revealed that the diabetic rats treated with ARRS showed significantly decreased HbA1c levels as compared to diabetic control rats. HbA1c content in the ARRS (200 mg/kg) treated group was almost restored to the normal levels. It has been reported that the compounds rich in phenolics and polysaccharides are known to possess antiglycation activity ²¹. Since ARRS is rich in phenolics and polysaccharides these could be responsible for its antiglycation activity.

The selective marker for glomerular injury is urinary albumin levels. Treatment with ARRS has restored the elevated urine albumin levels and brought them to normal. Biochemical analyses revealed that the untreated diabetic rats have increased levels of urea and creatinine in serum with a subsequent decrease in urine indicating the presence of renal damage has occurred. ARRS treatment restored urea and creatinine levels in both serum and urine. Hyperglycemia is known to induce oxidative stress, which plays a critical role in kidney damage. Increased oxidative stress leads to the production of reactive oxygen species. The reactive oxygen species so formed damage the lipids in the cell membrane causing lipid peroxidation. MDA and GSH levels were assessed to evaluate the oxidative stress. MDA is a byproduct of lipid peroxidation. GSH is a tripeptide, non-enzymatic biological antioxidant present in the liver. It protects cells against free radicals, peroxides and other toxic compounds. Glutathione also plays an important role in the kidney and takes part in a transport system involved in the reabsorption of amino acids ⁷. Lipid peroxidation product malondialdehyde was observed to be increased in renal tissue homogenates while GSH levels were in a declined state in diabetic animals. ARRS treatment decreased the MDA levels and increased GSH levels due to potential antioxidant activity. It has already been reported that quercetin is a strong antioxidant and it has the ability to attenuate renal damage in rats ²².

Roots of Raphanus sativus have already been reported to be rich in quercetin and this might have a renoprotective role in STZ induced diabetic mellitus in rats. Increased glucose levels result in the activation of metabolic pathways leading to increased oxidative stress ²³. This, in turn, results in the overproduction and accumulation of AGE responsible for enhancing the risk of developing glomerular diseases.

Mesangial cells grown on advanced glycosylation end product-modified matrix proteins demonstrate increased production of fibronectin and a decrease in proliferation ²⁴. These factors eventually lead to the thickening of the glomerular basement membrane and mesangial matrix expansion ²⁵. In the histopathological reports of our study, there was thickening of the glomerular basement membrane and mesangial expansion in renal tissues of untreated diabetic rats indicative of renal damage. Treatment with ARRS showed an improvement in glomerular structure. However, the best renoprotection was obtained with ARRS (200 mg/kg) treatment, which is characterized by a thin glomerular basement membrane with normal mesangium.

CONCLUSION: From this study, it is concluded that the aqueous root extract of Raphanus sativus possesses promising renoprotective activity in diabetes-induced renal alterations. The activity was more pronounced at the oral dose of 200 mg/kg as compared to 100 mg/kg. However, this activity was not found to be superior to the activity exhibited by glibenclamide. Further studies are required to confirm whether still higher doses of ARRS will be seen to be more effective in the treatment of diabetic nephropathy either alone or in combination.

ACKNOWLEDGEMENT: I thank Dr. N. Jayshree, professor of Pharmacology, Madras Medical College, for her great support and encouragement.

CONFLICTS OF INTEREST: The authors declare that there are no conflicts of interest.

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    How to cite this article:

    Priya PS and Jayshree DN: The protective role of Raphanus sativus roots in reversing the renal alterations and oxidative damage against streptozotocin induced diabetes in rats. Int J Pharm Sci & Res 2020; 11(1): 420-31. doi: 10.13040/IJPSR.0975-8232.11(1).420-31.

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