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Attenuation of erythrocyte membrane oxidative stress by Sesbania grandiflora in streptozotocin-induced diabetic rats
3 4
Chandrabose Sureka1†, Thiyagarajan Ramesh2†*, Vavamohaideen Hazeena Begum1
5 6
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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63 64 65 66 67
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.
82
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
92
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
102
perhaps due to the high content of poly unsaturated fatty acid in their membrane. Thus, the
103
reason of ATPases activities in erythrocyte membrane could be serving as simple, safe and
104
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).
106
A decline in membrane bound ATPases activity may play a key role at the cellular level in the
107
pathophysiology of numerous late complications of diabetes mellitus like retinopathy,
108
neuropathy and nephropathy (Jain and Lim 2000) and in the development of diabetic vascular
109
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
113
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
118
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,
120
bitter, tonic, anthelmintic, and febrifuge. The leaves are acrid, bitter, sweet, cooling, aperient,
121
tonic, and diuretic. The flowers of S. grandiflora are consumed in India as vegetables and leaves,
122
barks and the roots are also used by tribes (Sinha and Lakra 2005). S. grandiflora flower contains
123
phenolics, flavonoids, triterpenes, terpenoids, anthocyanin, tannins, alkaloids, saponins, β-
124
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
127
weak eye sight and night blindness. In addition, flowers are useful for females suffering from
128
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
132
protein fraction from S. grandiflora flower illustrated potential anticancer and chemopreventive
133
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
154
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
158
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
171
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
221 222 223 224 225
Determination of blood glucose and plasma insulin
226
(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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321
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
13
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Page 14 of 43
343
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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Page 15 of 43
365
Glucose 6-phosphate dehydrogenase (G6PDH) activity was assayed by the method of
366
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,
368
0.4 ml of 2,6-dichlorophenol indo phenol dye solution and 0.5 ml of haemolysate. The contents
369
were incubated at 37°C for 10 min. The reaction was initiated by the addition of 0.5 ml of
370
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
378
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
386
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
389
reaction was arrested by the addition of 1 ml of ice cold 10% TCA. Then the amount of Pi
390
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
Page 1 of 43
1 2
Attenuation of erythrocyte membrane oxidative stress by Sesbania grandiflora in streptozotocin-induced diabetic rats
3 4
Chandrabose Sureka1†, Thiyagarajan Ramesh2†*, Vavamohaideen Hazeena Begum1
5 6
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]
1
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Page 2 of 43
38 39 40 41 42
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
2
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63 64 65 66 67
Introduction
68
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.
82
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
3
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Page 4 of 43
88
membrane as indicated by increased osmotic fragility and modifications in erythrocyte
89
morphology (Mohamed et al. 2013).
90 91
In diabetic condition, the life spans of erythrocytes are shown to be decreased. Chronic
92
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
102
perhaps due to the high content of poly unsaturated fatty acid in their membrane. Thus, the
103
reason of ATPases activities in erythrocyte membrane could be serving as simple, safe and
104
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).
106
A decline in membrane bound ATPases activity may play a key role at the cellular level in the
107
pathophysiology of numerous late complications of diabetes mellitus like retinopathy,
108
neuropathy and nephropathy (Jain and Lim 2000) and in the development of diabetic vascular
109
diseases (Jeffcoate 2004). Increased membrane lipid peroxidation (Ramesh and Pugalendi 2007)
110
and glycoxidation would also be responsible for the reduction of membrane bound ATPases
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111
activities during diabetic hyperglycemia. Elevated glycoprotein molecules are associated with
112
enhanced glycation of membrane proteins and diabetic hyperlipidemia, which may also be
113
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
118
plant are used to treat a broad spectrum of diseases including tumors, hepatic disorders,
119
bronchitis, anemia, headache, swellings and pains. The bark of this plant is astringent, cooling,
120
bitter, tonic, anthelmintic, and febrifuge. The leaves are acrid, bitter, sweet, cooling, aperient,
121
tonic, and diuretic. The flowers of S. grandiflora are consumed in India as vegetables and leaves,
122
barks and the roots are also used by tribes (Sinha and Lakra 2005). S. grandiflora flower contains
123
phenolics, flavonoids, triterpenes, terpenoids, anthocyanin, tannins, alkaloids, saponins, β-
124
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
127
weak eye sight and night blindness. In addition, flowers are useful for females suffering from
128
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
132
protein fraction from S. grandiflora flower illustrated potential anticancer and chemopreventive
133
effect, against Daltons lymphoma ascites and colon cancer cells (Laladhas et al. 2010).
5
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134
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
154
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
158
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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160
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
171
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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Page 8 of 43
188
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,
214 215 216 217 218 219 220 8
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Biochemical Analysis
221 222 223 224 225
Determination of blood glucose and plasma insulin
226
(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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251
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
10
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Page 11 of 43
274
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.
296 297 11
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Page 12 of 43
298
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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Page 13 of 43
321
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
13
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Page 14 of 43
343
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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Page 15 of 43
365
Glucose 6-phosphate dehydrogenase (G6PDH) activity was assayed by the method of
366
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,
368
0.4 ml of 2,6-dichlorophenol indo phenol dye solution and 0.5 ml of haemolysate. The contents
369
were incubated at 37°C for 10 min. The reaction was initiated by the addition of 0.5 ml of
370
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
378
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
386
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
389
reaction was arrested by the addition of 1 ml of ice cold 10% TCA. Then the amount of Pi
390
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
