Therapeutic effect of Acacia nilotica pods extract on streptozotocin induced diabetic nephropathy in rat

Phytomedicine 19 (2012) 1059–1067

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Therapeutic effect of Acacia nilotica pods extract on streptozotocin induced diabetic nephropathy in rat Enayat A. Omara a,∗ , Somaia A. Nada b , Abdel Razik H. Farrag a , Walid M. Sharaf a , Sayed A. El-Toumy c a b c

Pathology Department, National Research Center, 12622 Dokki, Cairo, Egypt Pharmacology Department, National Research Center, 12622 Dokki, Cairo, Egypt Chemistry of Tannins Department, National Research Center, 12622 Dokki, Cairo, Egypt

a r t i c l e Keywords: Acacia nilotica Streptozotocin Biochemical Histopathological Antioxidant activity

i n f o

a b s t r a c t The aim of the present study was to examine the effect of aqueous methanol extract (150 and 300 mg/kg body weight) of Acacia nilotica pods in streptozotocin-induced diabetic rats for 60 days, and its biochemical, histopathological and histochemical study in the kidney tissues. Diabetic rats exhibited hyperglycemia, elevated of serum urea and creatinine. Significant increase in lipid peroxidation (LPO), superoxide dismutase (SOD) and reduced glutathione (GSH) was observed in diabetic kidney. Histopathological examination revealed infiltration of the lymphocytes in the interstitial spaces, glomerular hypertrophy, basement membrane thickening and tubular necrosis with loss of their brush border in some of the proximal convoluted tubules in diabetic rats. Acacia nilotica extract lowered blood glucose levels, restored serum urea and creatinine. In addition, Acacia nilotica extract attenuated the adverse effect of diabetes on LPO, SOD and GSH activity. Treatment with Acacia nilotica was found to almost restore the normal histopathological architecture of kidney of streptozotocin-induced diabetic rats. However, glomerular size and damaged area showed ameliorative effect after treatment with the extract. In conclusion, the antioxidant and antihyperglycemic properties of Acacia nilotica extract may offer a potential therapeutic source for the treatment of diabetes. © 2012 Elsevier GmbH. All rights reserved.

Introduction Diabetic nephropathy, a major long-term complication of diabetes mellitus, is the most common cause of end-stage renal disease requiring dialysis worldwide (King et al. 1998) and is becoming a staggering challenge to public healthcare systems due to the prohibitive costs of renal replacement therapy that could become unaffordable even for developed countries. Early diabetic nephropathy is characterized by hypertrophy of the glomeruli and tubular epithelial cells, thickening of basement membranes and enhanced renal blood flow and glomerular hyperfiltration (Hostetter 2001). This is accompanied by increased protein excretion and subsequent development of progressive glomerulosclerosis, with accumulation of extracellular matrix proteins in the glomerular mesangium thickening of glomerular and tubular membranes, and tubulointerstitial fibrosis, all of them contributing to the inexorable progressive deterioration of renal function (Parving et al. 2000). Free radicals have been shown to be harmful as they react with important cellular components such as proteins, DNA and

∗ Corresponding author. Tel.: +20 2 33371362; fax: +20 2 33370931. E-mail address: [email protected] (E.A. Omara). 0944-7113/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.phymed.2012.07.006

cell membrane (Mantena et al. 2008). The body on the other hand, requires free radicals for immune system responses. However, an overload of these molecules has been linked to certain chronic diseases of heart, liver and some form of cancers (Prakash et al. 2007). All organisms contain anti-free radical defense system, which includes antioxidant enzymes such as catalase, peroxidase and superoxide dismutase and antioxidants like ascorbic acid and tocopherol. All over the world, people depended on herbs for the treatment of various ailments before the advent of modern medicine. In Egypt, many plants are used today in folk medicine and are sold at herbal vendors and shops (Abdel-Azim et al. 2011). The ancient Egyptians were familiar with many medicinal herbs and aware of their usefulness in the treatment of various diseases. They used the plant organs such as roots, rhizomes, flowers, leaves, fruits, seeds, and oils. They applied their medicaments in the form of powders, pills, suppositories, creams, pastes, and ointments (Shahat et al. 2001; Dagmar 2006). However, scientific evidence for the medicinal properties of such plants is not always demonstrated. The genus Acacia comprises about 1350 species (Seigler 2003), distributed through the tropics and to some extent in the temperature regions. A. nilotica is a multipurpose tree of Fabaceae family that is used extensively for the treatment of various diseases, e.g. cold, bronchitis, diarrhea, dysentery, biliousness, bleeding piles and

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leucoderma (Ambasta 1994). It is used by traditionally in treatment of various cancer types of mouth, bone and skin. In West Africa, the bark and gum are used against cancers and/or tumors (of ear, eye, or testicles) and indurations of liver and spleen, the root for tuberculosis, the wood for smallpox and the leaves for ulcers (Kalaivani and Mathew 2010). A. nilotica has been reported to have many biological activities including antihypertensive, antispasmodic (Gilani et al. 1999), antidiabetic (Ahmad et al. 2008), hypochlesterolemic, and it had a beneficial effect on the hyperlipidemia associated with hyperglycemia (Laakso 1995; Maciejewski et al. 2001) and decreased the risk of liver failure associated with diabetes mellitus (Ahmad et al. 2008). Nubians in South Egypt believe that diabetic patients may eat as much food rich in carbohydrates without risk, if they regularly take powdered pods of A. nilotica (Boulos 1983). A. nilotica is reported to be rich in tannins and polyphenols (Caster and Cowan 1988; Kumar 1983). Plants have played a major role in the introduction of new therapeutic agents. A medicinal plant, Galega officinalis, led to the discovery and synthesis of metformin (Cusi and Defronzo 1998). It is our opinion that instead of random search of plants, a selective search based on traditional knowledge would be focused and productive and certainly more economic. In the present study was designed to investigate the effect of aqueous methanol extract of A. nilotica pods on streptozotocin-induced diabetic in rat including kidney functions, antioxidant parameters, and histopathological examinations for the renal tissue. Materials and methods General NMR experiments were performed on a Bruker AMX 400 and 500 instruments with standard pulse sequences operating at 400, 500 MHz in 1 H NMR and 100, 125 MHz in 13 C NMR. Chemical shifts are given in ı values (ppm) using tetramethylsilane as the internal standard and DMSO-d6 as solvent at room temperature. HRESI-MS was taken on a Micromass Autospec (70 eV) spectrometer. UV spectral data was measured on a Shimadzu 240 spectrometer in MeOH. Paper chromatography Whatman 1, using solvent systems A (15% AcOH) and B (n-BuOH:AcOH:H2 O, 4:1:5, upper layer). Compounds were visualized by exposure to UV light (365 nm), before and after spraying with AlCl3 and Naturestoff-polyethylene glycol reagents. Plant material Pods of Acacia nilotica were collected in April 2009 from the Upper Egypt. Identification of the plants was confirmed by Prof. Dr. Ibrahium El-Garf, Department of Botany, Faculty of Science, Cairo, University and comparison with herbarium specimens. Voucher Specimens were kept in herbarium, Department of Botany, Faculty of Science, Cairo, University (Boulos 1999). Extraction and isolation compounds Air-dried ground pods of A. nilotica (1.5 kg) were defatted with petroleum ether (40–60 ◦ C), and extracted three times at room temperature with CH3 OH:H2 O (7:3). The combined extracts were filtered, evaporated under reduced pressure and lyophilized (180 g). Twenty grams of the dry residue was used for pharmacological studies. Weighed samples of pods of A. nilotica extract were used to prepare the solutions, which were diluted with distilled H2 O to the appropriate concentration for the experiment. The rest of the dry extract was redissolved in 2 l H2 O and extracted with nbutanol (3× 2 l). After evaporation of solvents, the n-butanol extract and the remaining H2 O phase gave dark brown solids 50 and 70 g, respectively. The n-butanol extract was loaded on a polyamide 6S

column chromatography (80 cm × 3 cm). The column was eluted with H2 O, and then H2 O–CH3 OH mixtures of decreasing polarity and 10 fractions (1 l, each) were collected. The major phenolics fractions obtained were combined into five fractions after chromatographic analysis. Fraction 1 (1.5 g) was fractionated by column chromatography on Sephadex LH-20 with aqueous EtOH (0–70%) for elution to give compounds 1 (17 mg) and 4 (25 mg). Fraction 2 (1.6 g) was subjected to column chromatography on cellulose and n-BuOH saturated with H2 O as an eluent to give two major subfractions, then one of them was separately fractionated on a Sephadex LH-20 to yield pure samples 2 (21 mg) and the second was fractionated by column chromatography on Sephadex LH-20 with aqueous EtOH (50%) for elution to give compound 3 (15 mg). Using the same procedure fraction 3 (1.8 g) and fraction 4 (1.4 g) gave chromatographically pure samples 4 (15 mg) and 5 (15 mg). Fraction 5 (1.5 g) was chromatography on Sephadex LH-20 using aqueous acetone (0–25%) for elution to give pure sample 6 (20 mg) and 7 (25 mg). Total phenolic content The concentration of total phenolics of the plant extract was determined according to the method described by Kumar et al. (2008). Gallic acid was used as standard. Briefly, a mixture of 100 ␮l of plant extract (100 ␮g ml–1 ), 500 ␮l of Folin–Ciocalteu reagent and 1.5 ml of Na2 CO3 (20%) was shaken and diluted up to 10 ml with water. After 2 h, the absorbance was measured at 765 nm using a spectrophotometer. All determinations were carried out in triplicate. The total phenolic content value was expressed as the gallic acid equivalents (GAE) in milligrams (mg) per 1 g weight of the extract, using the standard curve generated with the series of gallic acid standard. Total flavonoid content Total flavonoid concentration of plant extract was determined according to the reported procedure by Kumaran and Karunakaran (2007). 100 ␮l of plant extract (10 mg ml−1 ) in methanol was mixed with 100 ␮l of 20% AlCl3 in methanol and a drop of acetic acid, and then diluted to 5 ml with methanol. The absorbance was measured at 415 nm after 40 min against the blank. The blank consisted of all reagents and solvent without AlCl3 . All determinations were carried out in triplicate. The total flavonoid content value was expressed as the rutin equivalents (RE) in milligrams (mg) per 1 g weight of the extract, using the standard curve generated with the series of rutin standard. Analytical HPLC Analysis was performed on an Agilent 1200 Series HPLC coupled with a diode array detector (DAD). The analytical column was an Agilent ZORBAX Eclipse Plus C18 column (4.6 mm × 150 mm, 5 ␮m, PN 883952-702), was used at 30 ◦ C. Two solvents were used with a constant flow rate of 1.0 ml/min. Solvent A consisted of 0.5 acetic acid/H2 O, solvent B methanol. All the solvents used were of HPLC grade. For the elution program, delivered at a flow rate of 1.0 ml per min as the following proportions of solvent B were used: 0–15 min, 20% B; 15– 30 min, 40% B; 30–45 min, 60% B; 45–55 min, 80% B; 55–60 min, 90% B. Drugs and chemicals Streptozotocin and diagnostic kits were purchased from Sigma (St. Louis, MO, USA). Glibenclamide was obtained from Sanofiaventis Deutchland GmbH, Germany. The powder was dissolved in distilled water and orally administrated at dose 0.03 mg/kg b.wt. for

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60 days. This dose equals the therapeutic dose for human (4 mg/kg) (Abd El-Rahim et al., 2010). Animals Male mature Sprague Dawley rats purchased from Animal House Colony, National Research Centre, Cairo, Egypt (weighing: 120–150 g). Animals were divided into seven equal groups (six rats each) housed under standard environmental conditions (23 ± 1 ◦ C, 55 ± 5% humidity and a 12-h light:12-h dark cycle) and maintained on a standard laboratory diet ad libitum with free access to water and they were housed in polycarbonated clean cages. Animal care and the experimental protocols were approved by the National Research Centre Animal Care and Use Committee was in accordance with the guidelines of the International Association for the Study of Pain Committee for Research and Ethical Issues (Zimmermann 1983). Experimental design Rats were divided into two main groups: normal non-diabetic groups; group 1: was normal control, groups 2 and 3 were administered A. nilotica extract 150 mg and 300 mg/kg b.wt., respectively. Diabetic groups were injected intraperitoneally with single dose of streptozotocin (60 mg/kg b.wt. dissolved in 10 mM citrate buffer pH 4.5), 7 days post injection, fasting blood sugar was determined. Rats with blood glucose level above 200 mg/dl were included in the

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experiment. The diabetic groups: group 4, was control diabetic and received equivalent volume of saline; groups 5, and 6 were orally administered 150 and 300 mg/kg b.wt., respectively. While group 7, was given the reference drug glibenclamide (25 mg/kg). Treatment schedule continued for 60 days. Biochemical analysis Blood samples were drawn after overnight fast from retroorbital Venus plexus for determination of serum levels of glucose, creatinine (Slot 1965), and urea (Fawcett and Scott 1960). At the end of the experiment, all animals were sacrificed, and then kidneys were removed and a part from the kidney was homogenate and used for determination of: (a) lipid peroxidation (LPO) was determined by estimation of malondialdhyde (MDA) content according to Uchiyama and Mihara (1978); (b) the reduced glutathione (GSH) according to the method of Moron et al. (1979); and (c) nitric oxide (NO) according to Miranda et al. (2001) and superoxide dismutase (SOD) activity using Suttle (1986) method. Histopathological and histochemical studies A part from the rat kidney from all groups was removed and immediately fixed in 10% neutral buffered formalin, dehydrated in gradual ethanol (50–100%), cleared in xylene and embedded in paraffin. 4–5 ␮m thick sections were prepared and stained with Hematoxylin and Eosin (H&E) for photomicroscopic observation

Fig. 1. (A) Chemical structures of the isolated compounds. (B) HPLC chromatogram of A. nilotica pods extract. Identification of compounds: catechin (1); catechin-7-O-gallate (2); catechin-4 -O-gallate (3); catechin-3 -O-gallate (4); gallic acid (5); quercetin 3-O-glucoside (6) and quercetin (7).

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Srum glucose

(Drury and Wallington 1980). The periodic acid Scfiff’s technique (PAS) was used to demonstrate the presence of polysaccharides (glycogen) in the kidney (Mac-Manus and Cason 1950).

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Morphometry measurement was achieved using computerized image analyzer (Leica Qwin 500 image) in Image Analyzer Unit, Pathology Department, National Research Center, and Cairo, Egypt. Image processing and analysis system was used for interactive automatic measurement of Renal morphometry was studied by measuring: diameter of glomeruli and total area damage on slides stained with H&E by 15 random fields per slide. Mean score of diameter of glomeruli and total area damage was calculated in each study group and compared to control.

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Data were statistically analyzed using ANOVA two-way test and least significant difference of the means (LSD) at p < 0.05.

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Fractionation of the extract resulted in isolation and identification of seven phenolic compounds (1–7) (Fig. 1A). The structure of the isolated compounds was established through chromatography as well as conventional chemical and spectroscopic methods of analysis (UV, 1/2D NMR). The HPLC constituent profile of A. nilotica pods recorded at 280 nm and 340 nm can be observed in Fig. 1B. The content of total phenolic compounds was (504.99 ± 1.35 mg GAE/G extract) and flavonoids (205.43 ± 3.58 mg RE/G extract).

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Blood glucose level of diabetic rat (220.5 ± 2.30 mg/dl) was increased significantly (p < 0.05) as compared to the control group (67.8 ± 1.2 mg/dl). Treatment with A. nilotica aqueous alcoholic extract at dose (150 and 300 mg/kg b.wt.) significantly decreased the glucose level of the diabetic groups in dose dependant manner with mean values of 98.3 ± 0.94 and 83.4 ± 2.20 mg/dl in two-dose groups (150 and 300 mg/kg). Glibenclamide-treatment normalized the glucose level (72.3 ± 1.5 mg/dl) as illustrated in Fig. 2. Serum creatinine level significantly elevated in diabetic group (0.78 ± 0.032 mg/dl) when compared with the control values (0.49 ± 0.02 mg/dl). Moreover, groups received A. nilotica extract (150 and 300 mg/kg) or those treated with glibenclamide their creatinine values were within the normal range (0.54 ± 0.014, 0.50 ± 0.025 and 0.56 ± 0.022 mg/dl) whereas these three groups non-significantly different from each other Fig. 2. Urea values also were elevated significantly in the nontreated diabetic group (43.2 ± 1.10 mg/dl) than normal control animals. A. nilotica extract-treatment groups (150 and 300 mg/kg) showed significant decrease in urea serum level to 32.5 ± 1.20 and 28.4 ± 1.60 mg/dl, respectively, dose dependent effect. However, glibenclamide had similar effect to that of the lower dose of A. nilotica (150 mg/kg) to reduce urea concentration to be 35.6 ± 1.30 mg/dl (Fig. 2). Malondialdhyde (MDA) concentration in the renal homogenate significantly elevated (807.33 ± 3.23 nmol/g tissues) in the diabetic group comparing to the control rat (259.17 ± 2.33 nmol/g tissue). A. nilotica extract caused significant reduction in MDA level (369.67 ± 4.5; 352.78 ± 3.16 nmol/g tissue in 150 and 300 mg/kg – treated groups) as shown in Fig. 3.

Fig. 2. Effect of A. nilotica at doses (150 mg and 300 mg/kg) on kidney in control and diabetic rat on blood glucose, creatinin and urea. ANOVA – one way, at p < 0.05. The different capital letters above columns are significantly different.

Reduced glutathione (GSH) content significantly decreased in the renal tissue of diabetic rat (8.43 ± 1.00 mmol/g tissue) compared with control group (17.19 ± 1.10 mmol/g tissue). While treatment either with A. nilotica extract or glibenclamide resulted significant increase in GSH-renal content (A. nilotica 150 mg/kg: 13.60 ± 1.10; A. nilotica 300 mg/kg: 14.37 ± 0.87; and glibenclamide: 16.00 ± 0.09 mmol/g tissue); GSH value non-significantly different than normal control group (Fig. 3).

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Fig. 3. Effect of Acacia nilotica at doses (150 mg and 300 mg/kg) on kidney in control and diabetic rat on MDA, GSH, SOD, and NO. ANOVA – one way, at p < 0.05. The different capital letters above columns are significantly different.

Superoxide dismutase (SOD) in tissue homogenate was significantly inhibited in diabetic rats (27.3 ± 0.39 ␮g/g tissue) comparing with the control group (69.61 ± 0.18 ␮g/g tissue) at p < 0.05. All tested treatments (A. nilotica: 150 and 300 mg/kg and glibenclamide) significantly increased SOD activity (64.33 ± 0.43; 61.17 ± 0.47 and 46.24 ± 0.87 ␮g/g tissue, respectively) comparing to the diabetic non-treated group. Furthermore, this result showed that A. nilotica 150 mg/kg < A. nilotica 300 mg/kg < glibenclamide 25 mg/kg on their effect to increase SOD activity (Fig. 3). Nitric oxide (NO) value significantly reduced in diabetic group when compared with the control and any other treated groups (diabetic: 44.39 ± 2.80; control: 62.40 ± 2.50 ␮M/g tissue). Whereas, all treated diabetic groups had normal NO value as shown in (Fig. 3). Histopathological and histochemical studies The sections from control showed normal histology of the kidney of rats. The proximal convoluted tubules, the distal convoluted tubule and the renal corpuscles with glomerulus and glomerular capsule are very clear and prominent (Fig. 4A). Kidney tissue studies in the groups receiving the extract of A. niotica at dose (150 and 300 mg/kg body weight) in comparison to the control group did not show any abnormal microscopic

findings. The extract did not induce mortality in any of the sequentially treated rats. After 60 days of streptozotocin administration severe changes were observed in kidneys. Histopathological changes in kidney of diabetic rat showed signs of tubular necrosis with loss of their brush border, vacuolar degeneration of proximal tubules and thickened basement membrane. The epithelial lining cells were disrupted with pyknotic nuclei. Hemorrhage and lymphocytic infiltrate in the interstitial area and periglomerular lymphocytic infiltration were noticed. Many tubules contained protein casts (Fig. 4B and C). Most of the glomeruli showed atrophy while remaining glomeruli also showed segmental sclerosis with basement membrane thickening with and spread interstitial fibrosis. Bowman’s capsules were considerable increased in size occupying the whole glomeruli spaces (Fig. 4B). These changes were found to be significantly reduced in kidneys of the experimental group treated with A. nilotica extract. The group of rat’s received low dose of extract (150 mg/kg) showed moderate improvement in the tubular and glomerular morphology (Fig. 4D). Post treatment with the plant extract at 300 mg/kg dose showed considerable improvement in glomeruli and tubules. Bowman’s capsule showed comparatively less hypertrophy. Glomeruli were compact with wide spacing in the Bowman’s capsule. Tubules were organized but debris was seen. Lumens were maintained.

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Fig. 4. Effect of A. nilotica extract (150 and 300 mg/kg/day) for 60 days treatment on kidneys structures of rats with streptozotocin-induced diabetes. (A) Section of the kidney of control rat showing normal histological structure glomerulus (G) and renal tubules (T) (H&E 400×). (B) Section of the kidney of diabetic rat showing vacuolar degeneration of the epithelial cells lining the renal tubules(arrow head), shrinked glomeruli, widened urinary space of the Bowman’s capsule(long arrow) and areas of hemorrhage in interstitial tissue (star) (H&E 400×). (C) Higher magnification of section of the kidney of diabetic rat showing marked degeneration of the epithelial cells lining the renal tubules (long arrow) and pyknotic cells (arrow head) (H&E 1000×). (D) Section of the kidney of diabetic rat treated with A. nilotica at dose (150 mg/kg body weight) showing architecture similar to control, however some areas presented mild vascular degeneration of renal tubules (T), some glomeruli showed lobulation (G), hemorrhage in interstitial tissue (long arrow)and few and pyknotic cells (arrow head) (H&E 400×). (E) Section of the kidney of diabetic rat treated with A. nilotica at dose (300 mg/kg body weight) showing normal glomeruli (G) with normal tubule (T), only few pyknotic cells (H&E 400×). (F) Section of the kidney of diabetic rat treated with Glibenclamide at dose (25 mg/kg body weight) showing tubules more or less normal tubules (T), with slight shrinked glomeruli (G), hemorrhagic area in between the renal tubules (long arrow) and few pyknotic cells (arrow head) (H&E 400×).

Epithelium was intact in most of the tubules hence improved histoarchitecture of kidney (Fig. 4E). The thickening of the walls was decreased by the A. nilotica (150 and 300 mg/kg) treatment (Fig. 4E). Experimental group of the animals injected with streptozotocin and glibenclamide showed mild improvement in of glomeruli and tubules (Fig. 4F). The periodic acid Schiff’s (PAS) technique was used to demonstrate the presence of glycogen in the kidney. The PAS +ve materials were mainly distributed at the brush border and basement membrane of the tubules (Fig. 5A). Treating rats with streptozotocin for 60 days caused reduction of glycogen content in the kidney cells

(Fig. 5B), and absent in the brush borders of proximal convoluted tubules. Also, the glomeruli were less positive than those of the control group. Strong reaction in the basement membrane was also seen (Fig. 5C). Glycogen content in kidney sections of the group treated with A. nilotica (150 and 300 mg/kg) and glibenclamide appeared similar to control group (Fig. 5D–F). However, glomeruli of the diabetic with (150 mg/kg) of A. nilotica group revealed positive PAS reaction and moderate dense positive brush border of the proximal convoluted tubules. The diabetic with (300 mg/kg) of A. nilotica group kidney sections revealed positive PAS reaction and intense brush border of the proximal convoluted tubules.

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Fig. 5. Effect of A. nilotica extracts (150 and 300 mg/kg/day) for 60 days treatment on glycogen content in kidneys tissue of rats with streptozotocin-induced diabetes. (A) Photomicrograph of the kidney of control rats section with PAS positive material in the cytoplasm and brush borders of the proximal tubules and glomeruli were also positive to PAS reaction. (B) Streptozotocin-diabetic rat showing a decreased amount of PAS positive material with strong reaction in the basement membrane and absent in the brush borders (arrow head). (C) Streptozotocin-diabetic rat showing decrease stainability of PAS materials with marked increase in the thick of basement membrane of tubules and glomeruli. Absent in the brush borders were observed (arrow head). (D) Section of the kidney of diabetic rat treated with A. nilotica at dose (150 mg/kg body weight) showing increase stainability of PAS materials of tubules and glomeruli. Moderate preservation of brush borders was observed. (E) Section of the kidney of diabetic rat treated with A. nilotica at dose (300 mg/kg body weight) showing marked increase of amount of PAS positive material in tubules and glomeruli. Preservation of brush borders was observed (arrow). (F) Section of the kidney of diabetic rat treated with glibenclamide at dose (25 mg/kg body weight) showing moderate increase of PAS +ve materials in tubules and glomeruli. Preservation of brush borders was observed (PAS reaction 400×).

Morphometry studies In the morphometric studies, we observed that the quantitative analysis of kidney tissue damage by image analyzer showed that the group treated with A. nilotica at doses (150 and 300 mg/kg/day) and glibenclamide achieved a significantly ameliorated when compared to the diabetic group (p < 0.05). Quantitative analysis of the area of damage showed a dose-relationship in the area of damage in diabetic with A. nilotica at doses (150 and 300 mg/kg/day) and

glibenclamide. The data are showed in (Fig. 6). Also, we observed an atrophy of glomerular size with Bowman’s space dilated in streptozotocin-untreated diabetic rats. Diabetic rats of 60 days duration, exhibited a significant (p < 0.05) atrophy of total area occupied by glomerular capillaries. Thus, mean of the glomeruli size was significantly decreased in the diabetic rats compared to controls. A. nilotica at doses (150 and 300 mg/kg/day) and glibenclamide ameliorated the decreased in mean glomeruli size as compared with diabetic group (Fig. 7).

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Fig. 6. Damaged areas in the renal tissue of control, diabetic and treated groups. × = zero value; no damaged area were found in normal treated groups (mean ± SE of mean, n = 10 fields/slid/rat), ANOVA – one way; the different capital letters are significantly different at p < 0.05.

Glomerular areas Glomerular area (µ2)

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Fig. 7. Glomerular damaged areas in the renal tissue of control, diabetic and treated groups (mean ± SE of mean, n = 10 fields/slid/rat), ANOVA – one way; the different capital letters are significantly different at p < 0.05.

Discussion The present study revealed that diabetic rats in the no treatment group developed sever hyperglycemia, elevated values of serum urea and creatinine. As well as, significant increase in LPO and reduction in SOD, GSH and NO contents in the renal tissue homogenate was detected. Oxidative stress was incriminated as an important mediator in the pathophysiology of diabetic nephropathy (Lee et al. 2003; Kowluru et al. 2004). Both hyperglycemia (Allen et al. 2003) and activation of the renin–angiotensin system (Anjaneyulu and Chopra 2004) play a role in the generation of reactive oxygen species (ROS). Moreover, the oxidative stress is evident in enhanced products of mitochondrial oxidative stress in diabetic vascular complications (Suzuki et al. 1999; Kanauchi et al. 2002; Xu et al. 2004). Also, the increased ROS in the kidney, especially the superoxide radicals; react with NO to form peroxynitrite, which in turn binds to tyrosine and other protein residues, yielding highly cytotoxic compounds such as nitrotyrosine in the renal and other vascular tissues (Pacher et al. 2005; Prabhakar 2007).

Histopathological examinations supported these biochemical results and kidney sections showed progressive damage. Most kidney sections showed lesions similar to human glomerulosclerosis, glomerular membrane thickening, arteriolar hyalinization and widespread tubular necrosis. Progressive glomerulosclerosis and fibrosis associated with decreased kidney function, resulting in end stage renal failure is the major finding in diabetic nephropathy (Striker et al. 1996). O’Donnell et al. (1988) and Harvey et al. (1992) proposed that the glomerular damage in diabetic kidney was due to the increased production of Kallikrein and prostaglandin E2 which caused hyperfiltration and vasodilatation in diabetes. In the present study, tubular necrosis, infiltration of lymphocytes in the interstitial spaces with loss of brush border was observed in most kidney sections. These findings are in agreement with the findings of Ramesh et al. (2007), Kim et al. (2008) and Renno et al. (2008) who showed tubular epithelial changes and enlargement of lining cells of tubules. In the present study, we observed a hypertrophy of glomerular size with Bowman’s space dilated in streptozotocin-untreated diabetic rats. Diabetic glomerular hypertrophy constitutes an early event in the progression of glomerular pathology which occurs in the absence of mesengial expansion. In hyperglycemia there is an increase in the entry of glucosein renal tissue (Belfiore et al. 1986). This has been postulated to cause increased intra-renal glycogen deposition, which leads to glycosylation of basement membrane collagen in the kidney (Anderson and Stowring 1973; Kim et al. 2008). The present study showed that diabetic rats receiving A. nilotica pods extract had reduction of their blood glucose in comparison to diabetic control rats. Our results are agreement with Ahmad et al. (2008) and Asad et al. (2011) who reported that A. nilotica extract leaves decreases the elevated blood glucose levels in diabetic rabbits. Such an effect may be accounted by a decrease in the rate of intestinal glucose absorption, achieved by an extra pancreatic action including the stimulation of peripheral glucose utilization or enhancing glycolytic and glycogenic process with concomitant decrease in glycogenolysis and glyconeogenesis (Luzi and Pozza 1997). However the effect was more significant when compared to standard drug glibenclamide. Our study showed that the treatment with A. nilotica extract had antihyperglycemic effect in dose response manner. A. nilotica ameliorated the increased level of serum urea and creatinine more than glibenclamide treatment group. In renal homogenate, the antioxidant enzyme system (SOD and GSH), NO level and LPO showed better improvement by the studied treatments with A. nilotica extract or glibenclamide, We investigated that A. nilotica pods extract is rich in tannins and polyphenols. Polyphenols decrease the blood glucose levels (Sabu et al. 2002; Tsuneki et al. 2004) and have anti-oxidant properties (Kumar 1983). The mechanism of underlying A. nilotica pods extract effects involved to inhibit the oxidative stress and enhancing the antioxidant enzyme system due to its scavenging property as evidenced by Kalaivani and Mathew (2010) and Singh et al. (2009) in vitro and in vivo studies. In this study, the general morphology of glomeruli and tubular lesions of the diabetic rats with the extract of A. nilotica pods was much improved and seemed quite normal in appearance compared with that of diabetic rats. On the basis of the above evidences it is possible that the presence of flavonoids and tannins are responsible for the observed antidiabetic activity. Conclusion It can be concluded that the aqueous methanol extract of A. niolitica pods showed an antidiabetic effect and diabetic nephropathy complications due to the presence of tannins and polyphenols therein in the extract.

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