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Attenuation of erythrocyte membrane oxidative stress by Sesbania grandiflora in streptozotocin-induced diabetic rats

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Chandrabose Sureka1†, Thiyagarajan Ramesh2†*, Vavamohaideen Hazeena Begum1

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1

613 010, Tamil Nadu, India.

7 8 9

Department of Siddha Medicine, Faculty of Science, Tamil University, Vakaiyur, Thanjavur-

2

Department of Biochemistry, College of Medicine, Prince Sattam Bin Abdulaziz University, AlKharj-11942, Kingdom of Saudi Arabia.

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37

† Both authors are equally contributed to this work.

*Corresponding authors: Dr. T. Ramesh Department of Biochemistry College of Medicine Prince Sattam Bin Abdulaziz University Al-Kharj-11942, Kingdom of Saudi Arabia Tel: +966-556-128-738 Fax: +966-1-5886101 E-mail: [email protected]

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Abstract

43

Sesbania grandiflora flower (SGF) extract on erythrocyte membrane in Streptozotocin (STZ)-

44

induced diabetic rats. Adult male albino rats of Wistar strain, weighing 190–220 g, were made

45

diabetic by an intraperitonial administration of STZ (45 mg/kg). Normal and diabetic rats were

46

treated with SGF and diabetic rats were also treated with glibenclamide as drug control, for 45

47

days. In this study, plasma insulin and haemoglobin levels were decreased and blood glucose,

48

glycosylated haemoglobin, protein oxidation, lipid peroxidation markers and osmotic fragility

49

levels were increased in diabetic rats. Besides, erythrocytes antioxidant enzymes such as

50

superoxide dismutase, catalase, glutathione peroxide, glutathione reductase, glutathione-S-

51

transferase and glucose-6-phosphate dehydrogenase activities and non-enzymatic antioxidants

52

mainly vitamin-C, vitamin-E, reduced glutathione (GSH) and oxidized glutathione (GSSG)

53

levels were altered, similarly the activities of total ATPases, Na+/K+-ATPase, Ca2+-ATPase and

54

Mg2+-ATPase are also decreased in the erythrocytes of diabetic rats. Administration of SGF to

55

STZ-induced diabetic rats reduced the blood glucose and glycosylated haemoglobin level with

56

increased levels of insulin and haemoglobin. Moreover, SGF reversed the protein and lipid

57

peroxidation markers, osmotic fragility, membrane-bound ATPases activities and antioxidant

58

status in STZ-induced diabetic rats. These results suggest that SGF could provide a protective

59

effect on diabetes by decreasing oxidative stress-associated diabetic complications.

The aim of the present study was designed to investigate the protective effects of

60 61 62

Keywords: Streptozotocin diabetes; Sesbania grandiflora flower; Erythrocyte; Antioxidants; Oxidative stress markers; Osmotic fragility; Membrane-bound ATPases

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Introduction

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on epidemiological studies and clinical trials reports hyperglycemia is the principal cause of

69

complications. Undiagnosed or poorly controlled disease may be linked with late complications

70

of diabetes such as enhanced atherosclerosis, blindness, renal insufficiency, stroke, and

71

amputation of extremities. Diabetes is also accompanying with a diminution in life expectancy.

72

Reactive oxygen species (ROS) induced oxidative stress is currently suggested to be a

73

mechanism underlying diabetes and diabetic complications. ROS are generated in biological

74

systems through metabolic processes and through exogenous sources (Martin-Gallan et al. 2013).

75

In normal physiological conditions, several antioxidant defense systems protect against the

76

adverse effects of free radical production in vivo (Martin-Gallan et al. 2013). Hyperglycemia

77

may disturb cellular antioxidant defense systems and damage cells. In diabetes, protein

78

glycation, glycoxidation and glucose oxidation may generate free radicals, which, in turn, cause

79

lipid peroxidation (Pari and Saravanan 2007). Besides, erythrocytes are frequently undergone

80

membrane protein oxidation or carbonylation in diabetes (Dalle-Donne et al. 2003). Hence,

81

protein carbonyls are indicators of oxidative damage to proteins in cells (Dalle-Donne et al.

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2003). Diabetes mellitus also depreciates the components of antioxidant defense systems, such as

83

superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutamate-S-

84

transferase (GST), vitamin-C, vitamin-E and reduced glutathione (GSH) (Pari and Saravanan

85

2007). These decreased antioxidant components accelerates the oxidative attack on erythrocyte

86

membrane lipids, proteins, and cytoskeletal proteins. Besides, the structure and function of

87

erythrocyte membrane lipid bilayer may change, which further damages the erythrocyte

Diabetes is a major health problem; globally it affects about 5% of the population. Based

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membrane as indicated by increased osmotic fragility and modifications in erythrocyte

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morphology (Mohamed et al. 2013).

90 91

In diabetic condition, the life spans of erythrocytes are shown to be decreased. Chronic

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hyperglycemia leads several changes in cell membrane properties such as enhanced rigidity,

93

permeability for cations and transmembrane potential in its absolute magnitude. The biochemical

94

components as well as dynamic prosperities of erythrocyte membranes are considerably

95

transformed in the diabetic state, resulting in debilitated cell function. Hyper polarization was

96

observed in the erythrocyte membrane of diabetic cells as a result of consistent oxidative stress

97

that can be responsible for long term complications in diabetes (Augustyniak et al. 1996;

98

Zavodnik et al. 1997). Alterations in erythrocyte membrane lipid molecules and enzymatic

99

properties of membrane bound enzymes (ATPases) are shown to occur in diabetes. The

100

acceleration of non-enzymatic glycosylation in diabetic extends beyond haemoglobin to the

101

proteins of the erythrocyte membranes. Erythrocytes are highly vulnerable to oxidative damage

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perhaps due to the high content of poly unsaturated fatty acid in their membrane. Thus, the

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reason of ATPases activities in erythrocyte membrane could be serving as simple, safe and

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useful marker of intracellular damage (Ohta et al. 1989). Changes of erythrocyte membrane

105

bound ATPases activity has been found in type 1 and type 2 diabetes (Rizvi and Abu Zeid 1998).

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A decline in membrane bound ATPases activity may play a key role at the cellular level in the

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pathophysiology of numerous late complications of diabetes mellitus like retinopathy,

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neuropathy and nephropathy (Jain and Lim 2000) and in the development of diabetic vascular

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diseases (Jeffcoate 2004). Increased membrane lipid peroxidation (Ramesh and Pugalendi 2007)

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and glycoxidation would also be responsible for the reduction of membrane bound ATPases

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activities during diabetic hyperglycemia. Elevated glycoprotein molecules are associated with

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enhanced glycation of membrane proteins and diabetic hyperlipidemia, which may also be

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responsible for the diminution of membrane bound ATPases activities. Glycation of membrane

114

bound ATPases is also possible during hyperglycemia.

115 116

Sesbania grandiflora (S. grandiflora) is commonly known as ‘agathi’ in Indian system of

117

Ayurvedic and Siddha medicine and reputed in the indigenous medicine. Different parts of this

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plant are used to treat a broad spectrum of diseases including tumors, hepatic disorders,

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bronchitis, anemia, headache, swellings and pains. The bark of this plant is astringent, cooling,

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bitter, tonic, anthelmintic, and febrifuge. The leaves are acrid, bitter, sweet, cooling, aperient,

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tonic, and diuretic. The flowers of S. grandiflora are consumed in India as vegetables and leaves,

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barks and the roots are also used by tribes (Sinha and Lakra 2005). S. grandiflora flower contains

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phenolics, flavonoids, triterpenes, terpenoids, anthocyanin, tannins, alkaloids, saponins, β-

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carotene, carotenoids and ascorbic acid (Andarwulan et al. 2012; Loganayaki et al. 2012; Dethe

125

et al. 2014). Flowers are used as laxative and emollient, juice of the flower is used for the

126

treatment of nasal catarrh and headache, phthisis; snuff given in eye complaints especially in

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weak eye sight and night blindness. In addition, flowers are useful for females suffering from

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amenorrhea.

129 130

The methanol extract of S. grandiflora flowers depicted potential anticancer effect

131

against Ehrich ascites carcinoma bearing Swiss albino mice (Sreelatha et al. 2011). A novel

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protein fraction from S. grandiflora flower illustrated potential anticancer and chemopreventive

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effect, against Daltons lymphoma ascites and colon cancer cells (Laladhas et al. 2010).

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Loganayaki et al (2012) reported that S. grandiflora flower has potential antioxidant, anti-

135

inflammatory, and antinociceptive effect. S. grandiflora flower also exerts its protective action

136

against CCl4 induced hepatotoxicities (Kale et al. 2012). Antimicrobial activity of S. grandiflora

137

flower polyphenol extracts showed on some pathogenic bacteria and growth stimulatory effect

138

on the probiotic organism Lactobacillus acidophilus (China et al. 2012). α-Glucosidase

139

inhibiting properties were found from the flowers of S. grandiflora, this result indicated that

140

S.grandiflora flower has antidiabetic property (Boonmee et al. 2007). However, the mechanisms

141

underlying the antidiabetic effects of SGF on diabetic rats are still unclear. Because the

142

pathogenesis of diabetes is closely associated with oxidative stress, we hypothesized that SGF

143

diminished oxidative stress and hyperglycemia in diabetic rats via its antioxidant activity. To test

144

this hypothesis, we investigated the effect of SGF on blood glucose, insulin, haemoglobin,

145

glycosylated haemoglobin, protein oxidation and lipid peroxidation markers, osmotic fragility,

146

membrane lipids, membrane-bound enzymes activities and antioxidant status in STZ-induced

147

diabetic rats.

148 149 150 151 152 153

Materials and Methods

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All other chemicals and solvents were obtained from Himedia and SD Fine Chemicals (Mumbai,

155

India) and were of the highest purity and analytical grade.

Chemicals and standard drugs The drugs and fine chemicals were purchased from Sigma-Aldrich, St. Louis, MO, USA.

156 157

Animals

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Adult male albino rats of Wistar strain (190–220g) were used for the experiment. The

159

animals were housed in poly propylene cages and maintained in 12-h light/12-h dark cycle, 50% 6

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humidity and 25±2ºC. The animals had free access to standard pellet diet (M/S. Amrut rat feed,

161

Pune, India) and drinking water. This study was carried out according to the animal ethical

162

guidelines suggested by the Institutional Animal Ethics Committee, Tamil University,

163

Thanjavur, Tamil Nadu, India.

164 165 166 167

Induction of experimental diabetes The animals were rendered diabetic by a single intra peritoneal injection of streptozotocin

168

(45 mg/kg body weight) in freshly prepared citrate buffer (0.1 M, pH 4.5) after an overnight fast.

169

STZ-injected animals were given 20% glucose solution for 24 h to prevent initial drug-induced

170

hypoglycemic mortality. Forty-eight hours after STZ administration, rats with moderate diabetes

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having hyperglycemia (i.e with plasma glucose of 200–300 mg/dl) were taken for the

172

experiment.

173 174 175 176

Plant materials Fresh Sesbania grandiflora flowers (SGF) were collected from a local plantation

177

(Thiruvaiyaru, Thanjavur, Tamil Nadu, India). The flowers were washed for any contaminants,

178

dried thoroughly under shade and powdered finely. The powdered flowers of S. grandiflora were

179

used for methanol extraction.

180 181 182 183

Preparation of extracts 500 g of powered SGF material was extracted with 2.5 L of petroleum ether (1:5 w/v)

184

(for disposing lipid and pigments), and then dried in a hot air oven at 40ºC. The residues were

185

further extracted with methanol using Soxhlet apparatus until they became colorless. The solvent

186

was evaporated using a rotary vacuum-evaporator at 50ºC and the remaining water was removed

187

by lyophilization. The freeze-dried extract thus obtained was used for the experiment. The 7

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extract was suspended in 1% sodium carboxy methyl cellulose (SCMC) vehicle solution and fed

189

by intubation.

190 191 192 193

Experimental design

194

SGF and glibenclamide (Glib) were administered orally once in a day in the morning for 45

195

days.

196 197 198 199 200 201 202 203 204 205 206 207

Group I: Normal (1% SCMC only)

208

anaesthetized using ketamine (24 mg/kg/b.wt, intramuscular injection), and sacrificed by cervical

209

decapitation. Blood was collected in tubes with a mixture of potassium oxalate and sodium

210

fluoride for the estimation of blood glucose, haemoglobin and glycosylated haemoglobin. The

211

buffy coat was removed, and the erythrocytes were washed three times with physiological saline.

212

Aliquots of erythrocytes were kept at 4°C until analysis. Plasma was separated for the assay of

213

insulin.

The animals were randomly divided into five groups of six animals each as given below.

Group II: Normal+SGF (250 mg/kg body weight in 1% SCMC) Group III: Diabetic control (1% SCMC only) Group IV: Diabetic+SGF (250 mg/kg body weight in 1% SCMC) Group V: Diabetic+Glib (600 µg/kg body weight in 1% SCMC).

At the end of experimental period, the rats were deprived of food overnight,

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Biochemical Analysis

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Determination of blood glucose and plasma insulin

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(Trinder 1969). Plasma insulin was assayed using an enzyme linked immunosorbent assay

227

(ELISA) kit (Boeheringer, Mannheim, Germany).

228 229 230 231

Blood glucose was estimated colorimetrically using commercial diagnostic kits method

Determination of haemoglobin and glycosylated haemoglobin levels Haemoglobin was estimated by using the cyanmethaemoglobin method described by

232

Drabkin and Austin (1932). Glycosylated haemoglobin was estimated according to the method of

233

Sudhakar Nayak and Pattabiraman (1981) with modifications according to Bannon (1982).

234 235 236 237

Preparation of haemolysate From 2ml of blood, erythrocytes were separated by centrifugation at 1000×g for 10 min

238

at 4◦C. The erythrocyte layer was washed three times with 10 volumes of 10 mmol/l phosphate

239

buffer saline. The washed erythrocytes were suspended in phosphate buffer saline and adjusted

240

to a hematocrit of 5 or 10%. An aliquot of 0.5 ml washed erythrocyte was lysed with 4.5 ml of

241

ice cooled distilled water to prepare haemolysate.

242 243 244 245

Preparation of erythrocyte membrane The erythrocyte membrane (haemoglobin free ghost erythrocyte) was isolated according

246

to the procedure of Dodge et al. (1963) with a change in buffer according to Quist (1980). The

247

packed cells were washed three times with isotonic 310 mM Tris–HCl buffer, pH 7.4. Hemolysis

248

was prepared from red blood cells into 20 mM hypotonic Tris–HCl buffer, pH 7.2. Ghosts were

249

sedimented by using a high-speed refrigerated centrifuge at 20,000×g for 40 min. The

250

haemolysate was decanted carefully and the ghost button was resuspended and the buffer of the 9

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same strength was added to reconstitute the original volume. The ratio of cells to the washing

252

solution was approximately 1:3 (v/v). The procedure was repeated thrice, till the membrane

253

became colourless. The pellets were resuspended in 100 mM Tris–HCl buffers, pH 7.2. Aliquots

254

from this were used for the estimations.

255 256

Determination of protein and lipid oxidative markers and lipid levels

257

Protein oxidation was determined by protein carbonyl (PC) assay by the method of

258

Levine et al (1990). Briefly, 300 µl of haemolysate was added into the tubes, to which 300 µl of

259

10 mM dinitrophenyl hydrazine (DNPH) in 2N HCl was added. The blank tube contained only

260

2N HCl. Samples were then incubated for 1 h at room temperature, stirred every 10 min,

261

precipitated with 10% trichloroacetic acid and centrifuged for 3 min at 16000g. The pellet was

262

washed three times with 1 ml ethanol–ethyl acetate mixture (1 : 1, v/v), then redissolved in 1 ml

263

of 6 M guanidine HCl in 10 mM phosphate trifluoro acetic acid (pH 2.3) and insoluble

264

substances were removed by centrifugation. The difference in absorbance between the DNPH-

265

treated and the HCl-treated samples was determined at 366 nm and using the extinction co-

266

efficient of 22.0/mM/cm for aliphatic hydrazones. The results are expressed as nM/mg protein.

267

Estimation of lipid peroxidation markers like thiobarbituric acid reactive substances

268

(TBARS), lipid hydroperoxides (HP) and conjugated dienes (CD) were done by the methods of

269

Nichans and Samuelson (1968), Jiang et al. (1992) and Rao and Recknagel (1968), respectively.

270

For TBARS estimation 0.1 ml haemolysate was treated with 2ml of TBA–TCA–HCl reagent

271

(1:1:1 ratio) (0.37% thiobarbituric acid, 0.25 M HCl and 15% TCA) placed in water bath for 15

272

min and cooled. The contents were centrifuged at 3500×g for 10 min and the absorbance of clear

273

supernatant was measured against blank at 535 nm. Values were expressed as nM of

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malondialdehyde/mg protein. For the estimation of HP, 0.2 ml of haemolysate was treated with

275

1.8 ml of Fox reagent (88 mg butylated hydroxyl toluene (BHT), 7.6 mg xylenol orange and 9.8

276

mg ammonium sulphate were added to 90 ml of methanol and 10 ml 250 mM sulphuric acid) and

277

incubated at 37°C for 30 min. The contents were centrifuged and the absorbance was read at 540

278

nm. Values were expressed as µM/mg proetin. For estimation of CD, 1.0 ml of haemolysate was

279

mixed thoroughly with 5.0 ml chloroform-methanol reagent (2:1 v/v) and centrifuged for 5 min.

280

To this, 1.5 ml cyclohexane was added and the absorbance was read at 233 nm against a

281

cyclohexane blank. The concentration of conjugated dienes was expressed as nM/mg of protein.

282

The levels of erythrocyte membrane cholesterol and phospholipids were measured by the

283

methods Siedel et al (1983) and Zilversmit and Davis (1950) respectively.

284 285

Osmotic fragility determination

286

Erythrocyte osmotic fragility was determined according to the method described by

287

Dacie and Lewis (1975). Different concentrations of sodium chloride (NaCl) 0.1% to 0.9% in a

288

series of tubes made from appropriate dilutions of 1% NaCl–phosphate buffer, pH 7.4, to a final

289

volume of 5.0 ml. Fresh heparinized blood (20 µl) was pipetted into these tubes containing

290

varying NaCl concentration. The contents were gently mixed and allowed to stand for 30 min at

291

room temperature. At the end, the contents of the tubes were mixed again and centrifuged at

292

500xg for 10 min. Absorbance of the supernatant was measured at 540 nm against water blank.

293

The degree of hemolysis is expressed in percentage, where 100% represents full hemolysis.

294

Median corpuscular fragility is defined as sodium chloride concentration (g/100 ml) bringing

295

about 50% hemolysis.

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Determination of non-enzymic and enzymic antioxidants

299

Vitamin-C was estimated by the methods of Roe and Kuether (1943). 0.5 ml of

300

haemolysate and 1.5 ml of 6% TCA was mixed and centrifuged. To the supernatant, 0.3 g of acid

301

washed norit was added, shaken vigorously and filtered. With 0.5 ml of the filtrate, 0.5 ml of

302

DNPH was added and placed in a water bath at 37 °C for 3 h. Removed, placed in ice-cold water

303

and added 2.5 ml of 85% sulphuric acid. A set of standards containing 20-100 µg of ascorbic

304

acid were taken and processed similarly along with a blank containing 2.0 ml of 4% TCA. The

305

color developed was read at 540 nm. The values are expressed as µg/mg of Hb.

306

Vitamin-E was determined by the method of Baker et al. [37]. 0.1 ml of lipid extract, 1.5

307

ml of ethanol and 2 ml of petroleum ether were added, mixed and centrifuged for 3000xg for 10

308

min. The supernatant was evaporated to dryness at 80°C then 0.2 ml of 2,2-1-dipyridyl solution

309

and 0.2 ml of ferric chloride solution was added and mixed well. This was kept in dark for 5 min

310

and added 2 ml of butanol. Then the absorbance was read at 520 nm. Standards of α-tocopherol

311

in the range of 10–100µg were taken and treated similarly along with blank containing only the

312

reagent. The values are expressed as µg/mg of Hb.

313

Reduced glutathione (GSH) was determined by the method of Ellman (1959). 0.5 ml of

314

haemolysate was precipitated with 2 ml of 5% TCA. 1 ml of the supernatant was taken after

315

centrifugation at 3200g for 20 min and added to it, 0.5 ml of Ellman’s reagent and 3 ml of

316

phosphate buffer (pH 8.0). Then the absorbance was read at 412 nm. A series of standards were

317

treated in a similar manner along with a blank containing 3.5 ml of buffer. The values were

318

expressed as µM of GSH/mg of Hb.

319

Oxidized glutathione (GSSG) was measured according to the method described by Aseni

320

et al. [35] based on the principle of glutathione reductase enzyme reducing GSSG to GSH with 12

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the concomitant oxidation of NADPH to NADP+. To 0.9 ml of 1.75 M/l K3PO4 buffer (pH 7.0)

322

containing 20 mM/l NEM was added 0.05 ml of sample and 0.025 ml of 10 mg/ml of NADPH–

323

Na solution. Absorbance at 340 nm was measured for 30 s immediately after addition of 0.025

324

ml of (10 mg/ml) glutathione reductase to the assay mixture. The redox index was calculated as

325

([GSH] + 2[GSSG]/2[GSSG] ×100).

326

Superoxide dismutase (SOD) activity was assayed by the method of Kakkar et al. (1984).

327

0.5 ml of haemolysate was diluted with 1 ml of water. In this mixture, 2.5 ml of ethanol and 1.5

328

ml of chloroform were added and shaken for 1 min at 4°C and then centrifuged. The enzyme

329

activity in the supernatant was determined. The assay mixture contained 1.2 ml of sodium

330

pyrophosphate buffer (0.025 M, pH 8.3), 0.1 ml of 186 µM phenazine methosulphate, 0.3 ml of

331

30 µM nitroblue tetrazolium, 0.2 ml of 780 µM NADH, appropriately diluted enzyme

332

preparation and water in a total volume of 3 ml. Reaction was started by the addition of NADH.

333

After incubation at 30°C for 90 s, the reaction was stopped by the addition of 1 ml glacial acetic

334

acid. The reaction mixture was stirred vigorously and shaken with 4 ml of n-butanol. The

335

intensity of the chromogen in the butanol layer was measured at 560 nm against butanol blank. A

336

system devoid of enzyme served as control. One unit of the enzyme activity is defined as the

337

enzyme reaction, which gave 50% inhibition of nitroblue tetrazolium reduction in one minute

338

under the assay conditions.

339

Catalase (CAT) activity was estimated by the method of Sinha (1972). 1.5 ml of reaction

340

mixture contained 1.0 ml of 0.01 M phosphate buffer (pH 7.0), 0.1 ml of haemolysate and 0.4 ml

341

of 2M hydrogen peroxide (H2O2). The reaction was stopped by the addition of 2.0 ml of

342

dichromate-acetic acid reagent (5% potassium dichromate and glacial acetic acid were mixed in

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1:3 ratio). Then the absorbance was read at 620 nm; catalase activity was expressed as µM of

344

H2O2 consumed/min/mg of Hb.

345

Glutathione peroxidase (GPx) activity was measured by the method of Rotruck et al.

346

(1973). Briefly, the reaction mixture contained 0.2 ml of 0.4 M phosphate buffer (pH 7.0), 0.1 ml

347

of 10 mM sodium azide, 0.2 ml of haemolysate in 0.4 M phosphate buffer (pH 7.0), 0.2 ml of

348

glutathione, and 0.1 ml of 0.2 mM H2O2. The contents were incubated for 10 min at 37°C, 0.4 ml

349

of 10% TCA was added to stop the reaction and centrifuged at 3200g for 20 min. The

350

supernatant was assayed for glutathione content using Ellman’s reagent (19.8 mg 5,5´-

351

dithiobisnitrobenzoic acid in 100 ml of 0.1% sodium nitrate).The activities were expressed as

352

µM of GSH consumed/min/mg of Hb.

353

Glutathione reductase (GR) activity was assayed by the method of Horn and Burns [33].

354

The reaction mixture containing 1 ml of phosphate buffer, 0.5 ml of EDTA, 0.5 ml of GSSG and

355

0.2 ml of NADPH was made up to 3 ml with water. After the addition of 0.1 ml of haemolysate,

356

the change in optical density at 340 nm was monitored for 2 min at 30 s intervals. Values were

357

expressed as nM of NADPH oxidized/min/mg of Hb.

358

Glutathione-S-transferase (GST) activity was determined spectrophotometrically by the

359

method of Habig et al. (1974). The reaction mixture contained 1.0 ml of 100 mM phosphate

360

buffer (pH6.5), 0.1 ml of 30 mM 1-chloro-2,4-dinitrobenzene (CDNB), and 0.7 ml of double

361

distilled water. After pre-incubating the reaction mixture for 5 min at 37°C, the reaction was

362

started by the addition of 0.1 ml of haemolysate and 0.1 ml of glutathione as substrate. After 5

363

min, the absorbance was read at 340 nm. Reaction mixture without the enzyme was used as a

364

blank. The activity of GST was expressed as µM of GSH conjugated with CDNB /min/mg of Hb.

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Glucose 6-phosphate dehydrogenase (G6PDH) activity was assayed by the method of

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Ellis and Kirkman (1961). The incubation mixture contained 1 ml of Tris-HCl buffer (0.05 M,

367

pH 7.5), 0.1 ml of magnesium chloride, 0.1 ml of NADP+, 0.5 ml of phenazine methosulphate,

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0.4 ml of 2,6-dichlorophenol indo phenol dye solution and 0.5 ml of haemolysate. The contents

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were incubated at 37°C for 10 min. The reaction was initiated by the addition of 0.5 ml of

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glucose 6-phosphate. The absorbance was read spectrophotometrically at 640 nm against water

371

blank at 1-min intervals for 3 to 5 min. G6PDH activity was expressed as µM of NADPH

372

liberated/min/mg of Hb.

373 374

Determination of membrane bound enzymes

375

The activities were indirectly measured by estimating the phosphorous liberated after the

376

incubation of erythrocyte membrane in a reaction mixture containing the substrate ATP. Total

377

ATPase activity was measured by the method of Evans (1969). The ATPase activity in 0.1 ml

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erythrocyte membrane were measured in a final volume of 2 ml containing 0.1 ml of 100 mM

379

Tris–HCl (pH 7.4), 0.1 ml of 100 mM NaCl, 0.1 ml of 100 mM MgCl2, 1.5 ml of 100 mM KCl,

380

and 0.1 ml of 10 mM ATP. The reaction was stopped at 20 min by the addition of 1 ml of 10%

381

TCA and then centrifuged (1500×g for 10 min), and the inorganic phosphorus (Pi) liberated was

382

estimated in the protein free supernatant. Then the amount of liberated Pi was estimated

383

according to the method of Fiske and Subbarow (1925) using commercial diagnostic kit.

384 385

The activity of Na+/K+-ATPase was determined by the method of Bonting (1970). In this

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assay, 2.2 ml of total volume containing 0.2 ml of erythrocyte membrane, 1 ml of 184 mM Tris–

387

HCl buffer (pH 7.5), 0.2 ml of 50 mM MgSO4, 0.2 ml of 50 mM KCl, 0.2 ml of 600 mM NaCl,

388

0.2 ml of 1 mM EDTA and 0.2 ml of 10 mM ATP and incubated for 15 min at 37◦C. The 15

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Page 16 of 43

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reaction was arrested by the addition of 1 ml of ice cold 10% TCA. Then the amount of Pi

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liberated was estimated in protein free supernatant.

391 392

The activity of Ca2+-ATPase was assayed according to the method of Hjerken and Pan

393

(1983). The reaction mixture of total volume of 0.4 containing 0.1 ml of erythrocyte membrane,

394

0.1 ml of 125 mM Tris–HCl buffer (pH 8), 0.1 ml of 50 mM CaCl2 and 0.1 ml of 10 mM ATP.

395

The contents were incubated at 37◦C for 15 min. The reaction was then arrested by the addition

396

of 0.5 ml of ice cold 10% TCA and centrifuged. The amount of Pi liberated was estimated in

397

supernatant.

398 399

The activity of Mg2+-ATPase was assayed by the method of Ohinishi et al (1982). The

400

incubation mixture of total volume 0.5 ml containing 0.1 ml of 375 mM Tris–HCl buffer (pH

401

7.6), 0.1 ml of 25 mM MgCl2, 0.1 ml of 10 mM ATP, 0.1 ml water and 0.1 ml of erythrocyte

402

membrane. The contents were incubated for 15 min at 37◦C and the reaction was arrested by

403

adding 0.5 ml of 10% TCA. The Pi liberated was then estimated in protein free supernatant.

404 405 406 407

Statistical analysis The data obtained were subjected to one way analysis of variance (ANOVA) and a post

408

hoc test was performed for inter-group comparisons using Tukey's multiple comparison with the

409

Graphpad prism software package (Version 5.0) for Windows. Values are expressed as the

410

means±standard deviation (SD) for 6 animals in each group. The level of significance was fixed

411

at P

Attenuation of erythrocyte membrane oxidative stress by Sesbania grandiflora in streptozotocin-induced diabetic rats.

The aim of the present study was to investigate the protective effects of Sesbania grandiflora flower (SGF) extract on erythrocyte membrane in Strepto...
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