Drug Chemical Toxicology

Introduction

Diabetes mellitus, a disease of metabolic disorders, is associated with a number of chronic complications, such as nephropathy, neuropathy, retinopathy, and cardiovascular diseases (Mahdi et al., 2003; Chehade and Mooradian, 2000), characterized by hyperglyc-emic, glucoseurea, and negative nitrogen balance, mainly due to lack of insulin secretion in beta cells of the pancreas and desensitization of insulin recep-tors for insulin. It is the most prevalent disease in the world, affecting 25% of the population (150 million people) and is projected to rise to 300 million by 2025 (Vats et al., 2000).

Hyperglycemia has been found to play a key role in reactive oxygen species (ROS)-generated damage (Ha and Kim, 1999; Maritim et al., 2003; Ugochukwu et al., 2003). Oxygen free radicals are formed dispro-portionately in diabetes by glucose oxidation, nonen-zymatic glycation of proteins, and the subsequent oxidative degradation of glycated proteins (Maritim et al., 2003). Scientific reports indicate that diabetic complications are associated with overproduction of free radicals and accumulation of lipid peroxidation (LPO) by-products (Palanduz et al., 2001).

Recently, the search for appropriate hypogly-cemic agents has focused mainly on plants and natural products, which are used in traditional

Drug and Chemical Toxicology, 2010; 33(2): 120–130

R E S E A R C H A R T I C L E

Biochemical evaluation of the protective effect of Aegle marmelos (L.), Corr. leaf extract on tissue antioxidant defense system and histological changes of pancreatic β-cells in streptozotocin-induced diabetic rats

Ramasamy Thangavelu Narendhirakannan1, and Sorimuthu Subramanian2

1Department of Biotechnology, School of Biotechnology, Karunya University, Karunaya Nagar, Coimbatore, India, and 2Department of Biochemistry, University of Madras, Guindy Campus, Chennai, India

AbstractOxidative stress is produced under diabetic conditions and is likely involved in the progression of pancreatic damage found in diabetes. This study was undertaken to evaluate the protective effect of Aegle marmelos leaf extract, a medicinal plant, on the tissue antioxidant defense system and lipid peroxidative status in streptozotocin-induced diabetic rats. Ethanol extract of A. marmelos was administered orally for 30 days (150 mg/kg body weight/day) to diabetic rats. Activity of the three primary scavenger enzymes (superoxide dismutase, catalase, and glutathione peroxidase), levels of glutathione, and lipid peroxidation were esti-mated in plasma and pancreas of diabetic rats and compared to the reference drug, glibenclamide (600 µg/kg body weight/day). A significant increase in the levels of plasma glucose, vitamin E, ceruloplasmin, lipid peroxides, and a concomitant decrease in the levels of vitamin C and reduced glutathione were observed in diabetic rats. The activities of antioxidant enzymes were altered in diabetic rats. These alterations were reverted back to near normal levels after treatment with A. marmelos and glibenclamide. Histopathological studies also revealed the protective effect of A. marmelos on pancreatic β-cells. The present study indicates that extract of A. marmelos modulates the activity of enzymic and nonenzymic antioxidants and enhances the defense against reactive oxygen species–generated damage in diabetic rats.

Keywords: Aegle marmelos; antioxidants; free radical; ROS; diabetes; medicinal plants; streptozotocin

Address for Correspondance: Ramasamy Thangavelu Narendhirakannan, Department of Biotechnology, School of Biotechnology, Karunya University, Karunaya Nagar, Coimbatore 641 114, India; Fax: +91- 422- 2615615; E-mail: [email protected]

(Received 11 May 2009; revised 14 July 2009; accepted 23 July 2009)

ISSN 0148-0545 print/ISSN 1525-6014 online © 2010 Informa UK LtdDOI: 10.3109/01480540903203984 http://www.informahealthcare.com/dct

Drug and Chemical Toxicology

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Antioxidative defense system of A. Marmelos in diabetic rats 121

medicines (Rates, 2001). Diabetes is still not completely curable by the present antidiabetic agents. Insulin therapy is the only satisfactory approach in diabetic mellitus even though it has several drawbacks, such as insulin resistance, ano-rexia, brain atrophy, and fatty liver during chronic treatment (Piedrola et al., 2001).

A number of medicinal plants named rasayana and traditionally used for over 1,000 years are present in herbal preparations of Indian traditional health care systems (Scartezzini and Sproni, 2000). The World Health Organization (WHO) has listed 21,000 plants that are used for medicinal purposes around the world. Among these, 2,500 species are from India, of which 150 species are used commercially on a fairly large scale.

The major advantages of herbal medicines are their efficacy, low incidence of side effects, and low cost. Several herbal drugs in different formula-tions have been tested to find an effective treatment for diabetes with certain curing claims on record. We have already reported the effect of inorganic constituents and preliminary antidiabetic properties of four medicinal plants (in the form of ash contain-ing many mineral elements) (Narendhirakannan et al., 2005).

The current article focuses on the free radical–scavenging activity of A. marmelos leaf extract and its interaction on the protective effect of streptozotocin-induced diabetes during the treatment of diabetes mellitus. Reports of our previous preliminary studies show the antidiabetic potential of Aegle marmelos (Narendhirakannan et al., 2006); therefore, it was our aim to screen the antidiabetic potential along with antioxidant ability of A. marmelos on plasma and pancreas of streptozotocin-induced diabetic animals.

Materials and methods

Chemicals

Tolbutamide and streptozotocin (STZ) were obtained from Hoechst Pharmaceuticals (Mumbai, India) and Sigma Chemical Company (St. Louis, Missouri, USA), respectively. All other chemicals and reagents used were of analytical grade and were obtained from commercial sources: thiobarbi-turic acid, nitro blue tetrazolium (NBT), and nico-tinamide adenine dinucleotide (NADH) were from Loba Chemie (Mumbai, India), whereas 5,5dithio bis-2-nitro benzoic acid (DTNB) and reduced glu-tathione (GSH) were from SISCO Research Lab (Bombay, India).

Collection and identification of plant material

Fresh A. marmelos leaves were collected from a rural region of Dindigul district, Tamil Nadu, India, and the plant was identified by Prof. V. Kaviyarasan, M.Sc.Ph.D. (Center for Advanced Studies in Botany, University of Madras, Chennai, India). The leaves were dried under shade. The voucher specimen of the plant is being retained in the department herbarium. Plant material was shade-dried, powdered, and passed through sieve number 40 to obtain uniform powder and packed in air-tight sealed envelopes for further studies.

Preparation of plant extracts

The shade-dried leaf powder samples of the A. marmelos were subjected to extraction with 95% eth-anol, using a Soxhlet apparatus (Borosil Glass Works, Mumbai, India) for 15 hours. After filtration through cotton wool, the filtrate was concentrated at 65°C by a rotavapor (Laborota 4000; Heidolph Instruments, Schwabach, Germany). The concentrate was then freeze-dried to yield 20–30% of the dried powder and stored in a refrigerator maintained at 5°C until further use.

Animals

Male albino rats of the Wistar strain, weighing approx-imately 160–180 g, were procured from Tamilnadu Veterinary and Animal Sciences University (Chennai, India) after obtaining approval from the ethics com-mittee, and the experiments were conducted as per the Institutional Animal Ethical Committee, India (IAEC) guidelines (IAEC no. 01/032/04). Rats were housed in an air-conditioned room at 22 ± 1°C with a lighting schedule of 12 hours light and 12 hours dark. Animals were acclimatized to animal house conditions. Rats were fed with a commercial rat diet (Hindustan Lever, Mumbai, India) and water ad libitum.

Induction of experimental diabetes

Animals were allowed to fast for 12 hours and were administered freshly prepared STZ (55 mg/kg body weight, intraperitoneally; i.p.) in 0.1 mol/L of cold citrate buffer (pH 4.5) (Rakieten et al., 1963). The STZ-treated animals were allowed to drink 5% glucose solu-tion overnight to overcome drug-induced hypoglyc-emia. After 1 week, to allow for the development and

122 R.T. Narendhirakannan, and S. Subramanian

aggravation of diabetes, rats with moderate diabetes and having persistent glycosuria and hyperglycemia (blood glucose, >250 mg/dL) were considered dia-betic and were used for further experimentation.

Experimental design

Animals were divided into four groups, consisting of a minimum of 6 animals each, as follows: (1) group 1, control rats receiving 0.1 mol/L citrate buffer (pH 4.5); (2) group 2, diabetic controls; (3) group 3, diabetic rats administered 150 mg/kg per day of A. marmelos etha-nol extract orally for 30 days; and (4) group 4, diabetic rats given glibenclamide (600 μg/kg body weight) in 1 mL of aqueous solution daily, using an intragastric tube for 30 days (Pari and Uma Maheswari, 2000). The body-weight gain, fasting blood glucose, and urine sugar of all groups were determined at regular inter-vals during the experimental period.

Biochemical assays

Determination of blood glucose, hemoglobin, and glycosylated hemoglobinAfter 30 days, 16-hour–fasted rats were killed by cervi-cal dislocation. Blood was collected in tubes contain-ing heparin. Plasma was separated and used for the estimation of glucose, while hemoglobin and gly-cosylated hemoglobin were performed. The pancreas was dissected out, washed immediately in ice-cold saline, and homogenized in Tris-HCl buffer (pH 7.4; 0.1 mol/L) with a Teflon homogenizer.

Blood glucose was estimated by o-toluidine method of Sasaki et al. (1972). First, 0.1 mL of the blood was mixed with 1.9 mL of 10% TCA solution to precipitate proteins and then centrifuged. Next, 1.0 mL of super-natant was mixed with 4.0 mL of o-toluidine reagent and was kept in boiling water bath for 15 minutes and cooled. The absorbance was read at 620 nm. Data were expressed as mg/dL.

Hemoglobin was estimated by using the cyan-methemoglobin method described by Drabkin and Austin (1932). The glycosylated hemoglobin in vitro was measured colorimetrically, as suggested by Nayak and Pattabiraman (1981). Then, 5g/mL in 0.01 M of phosphate buffer (pH 7.4) was incubated for 72 hour in the presence of 2 g/100 mL concentration of glu-cose in order to find out the best condition for hemo-globin glycosylation. The assay was performed by adding 1 mL of glucose solution, 1 mL of hemoglobin solution, and 1 mL of gentamicin (20 mg/100 mL) in 0.01 M of phosphate buffer (pH 7.4). The mixture was incubated in the dark at room temperature. The

degree of glycosylation of hemoglobin in the presence of different concentrations of the extract and their absence was measured colorimetrically at 440 nm. α-tocopherol was used as the standard.

Determination of in vivo antioxidant activity

Thiobarbituric acid reactive substances (TBARS)Lipid peroxides (TBARS) in plasma and tissue homogenate were estimated by using TBARS by the method of Okhawa et al. (1979). To 0.2 mL of tis-sue homogenate or plasma, 0.2 mL of 8.1% sodium dodecyl sulfate (SDS), 1.5 mL of 20% acetic acid, and 1.5 mL of 0.8% tert-butyl alcohol (TBA) were added. The mixture was made up to 4 mL with water and then heated in a water bath at 95°C for 60 minutes, using a glass ball as a condenser. After cooling, 1 mL of water and 5 mL of n-butanol/pyridine mixture were added and shaken vigorously. After centrifugation at 4,000 rpm for 10 minutes, the organic layer was taken and its absorbance at 532 nm was measured. 1,1,3,3-tetramethoxypropane was used as the standard. The level of lipid peroxides was expressed as mM of TBA reactants/100 g of wet tissue or n moles/mL of plasma.

Measurement of glutathione (GSH)

Reduced glutathione (GSH) was estimated by the method of Ellman (1959). First, 0.1 mL of tissue homogenate or plasma was precipitated with 5% TCA. The contents were mixed well for complete pre-cipitation of proteins and centrifuged. To an aliquot of clear supernatant, we added 2.0 mL of 0.6-mM DTNB reagent and 0.2 M of phosphate buffer (pH 8.6) to a final volume of 4.0 mL. The absorbance was read at 412 nm against a blank containing 5% TCA instead of sample. A series of standards treated in a similar way was also run to determine the GSH content. The amount of GSH is expressed as mg/100g wet tissue or mg/dL of plasma.

Measurement of superoxide dismutase (SOD) (EC 1.15.1.1)

SOD was assayed by the method of Misra and Fridovich (1972). First, 0.1 mL of tissue homogenate was added to tubes containing 0.75 mL of ethanol and 0.15 mL of chloroform (chilled in ice) and cen-trifuged. To 0.5 mL of supernatant, we added 0.5 mL of 0.6-mM ethylene diamine tetraacetic acid (EDTA) solution and 1 mL of 0.1-M carbonate-bicarbonate


buffer (pH 10.2). The reaction was initiated by the addition of 0.5 mL of 1.8-mM epinephrine, and the increase in absorbance at 480 nm was measured in a Shimadzu ultraviolet (UV) spectrophotometer, University of Madras, Chennai. The enzyme activity is expressed as 50% inhibition of epinephrine auto-oxidation/min.

Measurement of glutathione peroxidase (GPx) (EC 1.11.1.9)

GPx was assayed by the method of Rotruck et al. (1973). The reaction mixture consisted of 0.2 mL of 0.8-mM EDTA, 0.1 mL of 10-mM sodium azide, 0.1 mL of 2.5-mM H

2O

2, 0.2 mL of 4-mm GSH, 0.4 mL

of 0.4-M phosphate buffer (pH 7.0), and 0.2 mL of homogenate was incubated at 37°C for 10 min-utes. The reaction was arrested by the addition of 0.5 mL of 10% TCA, and the tubes were centrifuged at 2,000 rpm. To the supernatant, 3 mL of 0.3-M disodium hydrogen phosphate and 1.0 mL of 0.04% DTNB were added and the color developed was read at 420 nm immediately. The activity of GPx was expressed as moles of glutathione oxidized/min/mg protein.

Measurement of catalase (CAT ) (EC 1.11.1.6)

CAT was assayed by the method of Takahara et al. (1960). To 1.2 mL of 50-mM phosphate buffer (pH 7.0), 0.2 mL of the tissue homogenate was added and the enzyme reaction was started by the addition of 1.0 mL of 30-mM H

2O

2 solution. The decrease in absorbance

was measured at 240 nm at 30-second intervals for 3 minutes. The enzyme blank was run simultaneously with 1.0 mL of distilled water instead of hydrogen peroxide. The enzyme activity is expressed as moles of H

2O

2 decomposed/min/mg protein.

Estimation of vitamin E

Vitamin E was estimated by the method of Desai (1984). To 1.0 mL of plasma, 1.0 mL of ethanol was added and thoroughly mixed. Then, 3 mL of petroleum ether was added, shaken rapidly, and centrifuged. Next, 2 mL of supernatant was taken and evaporated to dryness. To this, 0.2 mL of 0.2% bathophenanthoraline was added. The assay mix-ture was protected from light and 0.2 mL of 0.001 M ferric chloride was added, followed by 0.2 mL of 0.001-M o-phosphoric acid. The total volume was made up to 3 mL with ethanol. The color developed

was read at 530 nm. The level of vitamin E was expressed as mg/dL of plasma.

Estimation of vitamin C

Vitamin C was determined by the method of Omaye et al. (1971). To 0.5 mL of plasma, 0.5 mL of water and 1 mL of 5% TCA were added, mixed thoroughly, and centrifuged. To 1 mL of the supernatant, 0.2 mL of dithiocarbamate (DTC) reagent was added and incubated at 37°C for 3 hours. Then, 1.5 mL of 65% sulphuric acid was added, mixed well, and the solu-tion was allowed to stand at room temperature for another 30 minutes. The color developed was read at 520 nm. The level of vitamin C was expressed as mg/dL of plasma.

Estimation of ceruloplasmin

Ceruloplasmin was estimated by the method of Ravin (1961). First, 0.1 mL of plasma was taken into three 15-mL test tubes, one for control and two for tests, respectively. One milliter of 0.02% sodium azide was added to the control tube only. Then, 8.0 mL of 0.1-M acetate buffer (pH 5.2) was added to all the tubes, followed by the addition of 1 mL of 0.1% p-phenylene diamine. The solution was mixed thor-oughly and placed in a water bath at 37°C for 1 hour. After incubation, the tubes were removed and 1 mL of 0.02% sodium azide was added to each of the tubes containing test solution. The contents were shaken well and cooled at 4°C for 30 minutes. The intensity of the color developed was measured at 530 nm. The ceruloplasmin content was expressed as mg/dL of plasma.

Estimation of protein

Protein was estimated by the method of Lowry et al. (1951). To 0.1 mL of tissue homogenate, 0.9 mL of water and 4.5 mL of alkaline copper reagent were added and kept at room temperature for 10 min-utes. Then, 0.5 mL of Folin’s reagent was added and the color developed was read after 20 minutes at 640 nm. The level of protein was expressed as mg/g of tissue.

Histopathological studies

For histopathological studies, rats from control and experimental groups were perfused with 10%


neutral formalin solution. Liver, kidney, and brain were removed immediately from the rats, then paraf-fin sections of 5-μm thickness were made and stained by hematoxylin-eosin (H&E) stain. After staining, the sections were observed under a light microscope and photographs were taken.

Statistical analysis

All the grouped data were statistically evaluated with SPSS/10 software (SPSS, Inc., Chicago, Illinois, USA). Hypothesis testing methods included one-way analy-sis of variance (ANOVA), followed by the least signifi-cant difference (LSD) test. P-values of less than 0.05 were considered to indicate statistical significance. All the results were expressed as mean ± standard deviation for 6 animals in each group.

Results

Table 1 shows the level of blood glucose, total hemo-globin, and glycosylated hemoglobin in normal and experimental rats. A significant (P < 0.05) increase in the level of blood glucose and glycosylated

hemoglobin with the concomitant decrease in the level of total hemoglobin (group 2) were observed in diabetic rats, when compared to control rats (group 1). Administration of A. marmelos (group 3) and glibenclamide (group 4) to diabetic rats signifi-cantly (P < 0.05) reverted to near normal level.

Table 2 presents the concentration of TBARS in plasma and tissues of control and experimental groups of rats. There was a significant elevation in the level of TBARS in plasma and pancreas of diabetic rats (group 2). A. marmelos– (group 3) and gliben-clamide- (group 4) treated diabetic rats showed a significant decrease (P < 0.05) in the level of TBARS, when compared to diabetic rats.

Figure 1 illustrates the activity of SOD in pancreatic tissues of control and experimental groups of rats. A significant (P < 0.05) increase was observed in the

Table 1. Blood glucose, hemoglobin, and glycosylated hemoglobin levels of normal and experimental animals.

GroupsBlood Glucose

(mg/dL)Hemoglobin

(g/dL)Glycosylated

hemoglobin(%Hb)

Group 1 98.3 ± 4.2 14.01 ± 0.8 6.1 ± 0.7Group 2 238.5 ± 12.7*,a 11.86 ± 0.7*,a 11.9 ± 1.1*,a

Group 3 112.5 ± 7.3*,b 12.59 ± 0.7*,b 7.2 ± 0.8*,b

Group 4 121.7 ± 8.1*,b 13.01 ± 0.6*,b 7.1 ± 0.9*,b

Values are given as mean ± standard deviation for groups of 6 animals each.aComparison made with groups 1 and 2.bGroups 3 and 4 compare with group 2.*Values are statistically significant at P < 0.05.

Table 2. Levels of TBARS in plasma and pancreas of control and experimental groups of rats.

GroupsPlasma TBARS

(n mol/mL)Pancreas TBARS

(mM/100 g wet tissue)Group 1 3.21 ± 0.31 43.88 ± 2.87Group 2 8.01 ± 0.92*,a 72.76 ± 6.22*,a

Group 3 3.93 ± 0.39*,b 52.99 ± 4.71*,b

Group 4 4.26 ± 0.57*,b 50.75 ± 4.01*,b

Values are given as mean ± standard deviation for groups of 6 animals each.aComparison made with groups 1 and 2.bGroups 3 and 4 compare with group 2.*Values are statistically significant at P < 0.05.

Group I

50%

of i

nhib

ition

of e

pine

phrin

eau

to o

xida

tion

per m

in

Group II Group III Group IV

8

7

6

5

4

3

2

1

0

SOD*a

*b *b

Figure 1. Activities of superoxide dismutase in pancreas of con-trol and experimental groups of rats. Units: 50% of inhibition of epinephrine auto-oxidation per minute. Values are given as mean ± standard deviation for groups of 6 animals each. aComparison made with groups 1 and 2; bgroups 3 and 4 compare with group 2; *values are statistically significant at P < 0.05. (See colour version of this figure online at www.informahealthcare.com/dct)

Group I

µ m

oles

of h

ydro

gen

pero

xide

deco

mpo

sed

per m

in p

er m

g pr

otei

n


35

30

25

20

15

10

5

0

Catalase

*a

*b *b

Figure 2. Activity of CAT in pancreas of control and experimental groups of rats. Values are given as mean ± standard deviation for groups of 6 animals each. aComparison made with groups 1 and 2; bgroups 3 and 4 compare with group 2; *values are statistically significant at P < 0.05. (See colour version of this figure online at www.informahealthcare.com/dct)


activities of SOD in pancreatic tissue of diabetic rats (group 2). Administration of A. marmelos and gliben-clamide (groups 3 and 4) tended to bring the activity of this enzyme to near normal (P < 0.05) in diabetic rats.

Figure 2 shows the activity of catalase in the tis-sues of control and experimental groups of rats. Activity of catalase was increased in diabetic rats (group 2) and reverted signficantly (P < 0.001) to nor-mal in A. marmelos– and glibenclamide-administered rats (groups 3 and 4).

A significant (P < 0.05) increase in the activ-ity of GPx was found in STZ-induced diabetic rats (group 2), when compared with normal rats (group 1). This increased activity significantly (P < 0.05) reverted to normal in A. marmelos– and glibenclamide-treated rats (Figure 3).

Figures 4 and 5 reveal the levels of GSH in plasma and pancreas of control and experimental groups of rats, respectively. A significant decrease (P < 0.05) observed in the concentration of GSH in plasma and pancreas of diabetic rats approached (P < 0.05) near

normal levels in diabetic rats treated with A. marme-los and glibenclamide.

A significant increase (P < 0.05) in the level of vita-min E and a concomitant decrease in the levels of ceruloplasmin were found in diabetic rats (group 2). Diabetic rats treated with A. marmelos (group 3) and glibenclamide (group 4) tended to bring these levels to near normal (Figure 6).

Figure 7 shows the levels of vitamin C in plasma of control and experimental groups of rats. There was a significant elevation (P < 0.05) in the level of vitamin C noticed in diabetic rats (group 2). Administration of A. marmelos and glibenclamide brought the levels of vitamin C to near normal levels (groups 3 and 4).

The pancreas of control rat is presented showing normal islets (Plate 1A). In the diabetic group, degen-eration and necrosis of the islets were observed in the pancreas (Plate 1B). The cytoplasm of periacinar pan-creatic cells showed either a single large or multiple small, round, empty vacuoles that distended the cell

Group I

mol

e of

glu

tath

ione

oxi

dize

dpe

r min

per

mg

prot

ein


12

10

8

6

4

2

0

GPx

*a

*b *b

Figure 3. Activity of GPx in pancreas of control and experimental groups of rats. Values are given as mean ± standard deviation for groups of 6 animals each. aComparison made with groups 1 and group 2; bgroups 3 and 4 compare with group 2; *values are statis-tically significant at P < 0.05.

Group I

mg/

dl


*a

*b *b 35

30

25

20

15

10

5

0

Figure 4. Levels of reduced glutathione (GSH) in plasma of con-trol and experimental groups of rats. Values are given as mean ± standard deviation for groups of 6 animals each. aComparison made with groups 1 and 2; bgroups 3 and 4 compare with group 2; *values are statistically significant at P < 0.05.

Group I

mg/

100g

wet

tiss

ue

Group II

Pancreas

Group III Group IV

*a

*b *b30

25

20

15

10

5

0

Figure 5. Levels of reduced glutathione (GSH) in pancreas of control and experimental groups of rats. Values are given as mean ± standard deviation for groups of 6 animals each. aComparison made with groups 1 and 2; bgroups 3 and 4 compare with group 2; *values are statistically significant at P < 0.05.

Group I

mg/

dl


*a

*b

*b

*b

*b

4.54

3.53

2.52

1.5

0.51

0

*a

Vitamin E Ceruloplasmin

Figure 6. Levels of vitamin E and ceruloplasmin in plasma of control and experimental groups of rats. Values are given as mean ± standard deviation for groups of 6 animals each. aComparison made with groups 1 and 2; bgroups 3 and 4 compare with group 2; *values are statistically significant at P < 0.05. (See colour version of this figure online at www.informahealthcare.com/dct)


cytoplasm and displaced the nucleus to the periphery in histological sections of pancreas stained with H&E. These degenerative changes were also seen, to a lesser extent, in the midzonal regions. Parenchymatous degeneration was observed in the peripheral regions. A. marmelos–treated diabetic pancreas (Plate 1C) showed initial stages of regenerating islets and an increase in the islets, as compared to the diabetic pancreas.

Discussion

The global term, reactive oxygen species (ROS), includes both oxygen radicals, such as superoxide (O

2˙¯), alkoxyl (RO˙), peroxyl (ROO˙), and hydroxyl

radicals (OH˙), plus nonradical derivatives of oxygen, namely hydrogen peroxide (H

2O

2) and ozone (O

3).

ROS is continuously generated in physiological condi-tions and effectively eliminated by several intra- and

extracellular antioxidative systems. When the genera-tion of ROS exceeds cellular defences, these unstable ROS will interact with essential biological cellular macromolecules, such as lipids, proteins, and DNA, which leads to histologic changes as well as functional abnormalities. The shift of balance between pro- and antioxidant activity in favor of the former results in potential damage, which is defined as oxidative stress (Sies, 1991). Hyperglycemia, a well-recognized patho-genetic factor of long-term complications in diabetes mellitus (Reichard et al., 1993), not only generates more ROS, but also attenuates antioxidative mecha-nisms through glycation of the scavenging enzymes. Therefore, oxidative stress has been considered to be a common pathogenic factor of diabetic complica-tions (Wolff, 1993).

Various mechanisms have been suggested to con-tribute in the formation of these reactive oxygen–free radicals. Glucose oxidation is believed to be the main source of free radicals. In its enediol form, glucose is oxidized in a transition-metal–dependent reaction to an enediol radical anion that is converted into reac-tive keto aldehydes and to superoxide anion radicals. The superoxide anion radicals undergo dismutation to hydrogen peroxide, and if not, it will be degraded by CAT or GPx. In the presence of transition metals, this can lead to the production of extremely reactive hydroxyl radicals (Jiang et al., 1990). Hyperglycemia is also found to promote LPO of low-density lipoprotein (LDL) by a superoxide-dependent pathway, resulting in the generation of free radicals (Kawamura et al., 1994).

During diabetes, the excess glucose present in the blood reacts with hemoglobin to form glycosylated hemoglobin. It has been reported that various pro-teins, including hemoglobin, albumin, collagen, LDL, or crystalline proteins, undergo nonenzymatic

Group I

mg/

dl

Group II

Vitamin C

Group III Group IV

*a

*b *b

35

30

25

20

15

10

5

0

Figure 7. Levels of vitamin C in plasma of control and experi-mental groups of rats. Values are given as mean ± standard devia-tion for groups of 6 animals each. aComparison made with groups 1 and 2; bgroups 3 and 4 compare with group 2; *values are statisti-cally significant at P < 0.05.

A B C

Plate 1. Histological examinations of 30 days on experimental rat pancreas (10X magnification). (A) Pancreatic sections of normal rat shows cells with normal architecture and well-preserved cytoplasm and nucleus. (B) In the pancreatic sections of STZ-intoxicated rats, the cells are irregular, show vascular degeneration, are not well defined, and there are defects in the cell membrane. Necrosis of the cells is very clear. (C) A. Marmelos treatment was improved and showing initial stages of regeneration also restored the altered histopathologi-cal changes. (See colour version of this figure online at www.informahealthcare.com/dct)


glycation in diabetes (Klein, 1995). The rate of glycation is proportional to the concentration of blood glucose (Das et al., 2000). In uncontrolled or poorly controlled diabetes, there is an increased glycation of a number of proteins, including hemoglobin and a crystalline of lens. Glycated hemoglobin (HbA

1C) was found to

increase in patients with diabetes mellitus and the amount of increase is directly proportional to the fasting blood glucose level (Alberti and Press, 1982). Further, the level of glycated hemoglobin is consid-ered as one of the markers of degree of oxidative stress in diabetes mellitus. Treatment with A. marmelos showed a significant decrease in the glycosylated hemoglobin levels in the diabetic rats that could have been due to an improvement in glycemic status.

LPO-mediated damage has been observed in the development of both type I and II diabetes mellitus (Walsh and Pek, 1984). Induction of diabetes in rats with STZ uniformly results in an increase in LPO (TBARS), an indirect evidence of intensified free radical production (Maritim et al., 2003). Most of the tissue damage is considered to be mediated by these free radicals by attacking membranes through per-oxidation of unsaturated fatty acids (Stringer et al., 1989). In the present study, the concentration of lipid peroxide was found to be increased in diabetic rats, which indicates an increase in the generation of free radicals. The increase in the level of lipid peroxides in plasma is thought to be the consequence of increased production and liberation into the circulation of tis-sue lipid peroxides due to the pathological changes (Selvam and Anuradha, 1990). An observed increase in the level of TBARS in the pancreas may have been due to the increased susceptibility of the tissue of dia-betic rats to LPO (Matkovics et al., 1998). The present finding indicates a significant increase in the LPO of rats exposed to STZ and its attenuation by A. marme-los treatment. This shows the protective role of A. marmelos leaves, which could be due to the antioxi-dative effect of flavonoids (Sahare et al., 2008) present in the leaves that act as a strong superoxide radical and singlet oxygen quenchers.

The increase in the superoxide radical in diabetes may inhibit the activity of CAT (Kono and Fridovich, 1982) and GPx (Blun and Fridovich, 1985). SOD scavenges the superoxide radical by converting it to H

2O

2 and molecular oxygen (McCrod et al., 1976). In

the present study, the increased CAT, SOD, and GPx activities were observed in STZ-induced diabetic rats. The increase in the CAT activity may be a compen-satory response to an increase in endogenous H

2O

2

production in diabetic pancreas. Insulin deficiency promotes the β-oxidation of fatty acids with result-ing H

2O

2 formation (Horie et al., 1981). Increase in

the SOD activity could be due to its induction by the increased production of superoxide, which has been implicated in cell dysfunction. H

2O

2 has been

reported to act as an inducer of tissue SOD, and hence, an increase in pancreatic SOD was observed, which correlates with our findings. Increased activ-ity of GPx in the pancreas of diabetic rats has been reported (Wohaieb and Godin, 1987). However, in this study, we found that A. Marmelos maintains pancreatic activity of SOD and CAT at the control level. The altered SOD and CAT activity in diabetic rats treated with A. Marmelos indicates an adaptive mechanism in response to oxidative stress.

Glutathione peroxidase (GPx) and reductase (GR) are the two enzymes that are found in the cytoplasm, mitochondria, and nucleus. GPx metabolizes hydro-gen peroxide to water by using GSH as a hydrogen donor (Santini et al., 1997). Glutathione disulfide is recycled back to GSH by GR, using the cofactor, NADPH, generated by glucose-6-phosphate dehydro-genase (G6PD). GSH reacts with free radicals and is a crucial substrate for GPx and glutathione transferase (GST), which takes part in the cellular defense mech-anisms against intermediate oxygenated products of metabolism. The observed decrease in GSH level in the diabetic groups of rats represents increased utili-zation due to oxidative stress (Gregus et al., 1996).

The effects of A. Marmelos on plasma glucose and pancreatic LPO produced by STZ may be related to the significant rise in pancreatic and plasma GSH induced by this extract. Paolisso et al. (1993) have pro-posed that the ratio of GSH/GSSG (oxidized glutath-ione) plays a crucial role in the glucose homeostasis of diabetes. It has been suggested that thiol groups are important in the intracellular and membrane redox state of the secretory function of β-pancreatic cells. In the A. Marmelos extract administered to rats, the GSH content in plasma and pancreas was found to be increased, which may enhance the GSH/GSSG ratio and, therefore, improve plasma-glucose regulation. The pancreas has been reported as the organ with the lowest levels of antioxidant enzymes, and pancreatic β-cells are considered to be exceptionally vulner-able to the cytotoxic actions of oxygen free radicals because of their relatively low levels of antioxidant enzymes (Tiedge et al., 1997).

Vitamins A, C, and E are diet-derived and detoxify free radicals directly. They also interact in recycling processes to generate reduced forms of the vitamins. These vitamins also foster toxicity by producing pro-oxidants under some conditions. Vitamin E, a component of the total peroxyl radical-trapping antioxidant system (Weber et al., 1997), reacts directly with the peroxyl and superoxide radicals,


and singlet oxygen, which protects the membranes from LPO. The deficiency of vitamin E is concurrent with increased peroxides and aldehydes in many tissues. There have been conflicting reports about vitamin E levels in diabetic animals and human subjects. Plasma and/or tissue levels of vitamin E are reported to be unaltered (Martinoli et al., 1993), increased (Asayama et al., 1994), or decreased (Cinar et al., 2001) by diabetes.

GSH may contribute to antioxidant defense by networking with other major antioxidants, such as vitamins E and C. Vitamin E can transfer its phenolic hydrogen to a peroxyl free radical of a peroxidized polyunsaturated fatty acids (PUFAs), thereby break-ing the radical chain reaction and preventing the per-oxidation of PUFAs in cellular and subcellular mem-brane phospholipids. As a reducing agent, vitamin C reacts with a vitamin E radical to yield a vitamin C radical while regenerating vitamin E. A vitamin C radical is converted back to vitamin C by GSH. These vitamins also directly scavenge ROS and upregulate the activities of antioxidant enzymes (Fang et al., 2002).

Vitamin E is used in combating free radicals, and if vitamin C is present, vitamin E levels are preserved. This increase may also be due to the decreased level of vitamin C or the storage of vitamin E by diabetic rats, when compared with the controls (Jain and Levine, 1995). The plasma protein, ceruloplasmin, is a pow-erful free radical scavenger that oxidizes iron from the ferrous to ferric state. The generation of oxygen products, such as superoxide radical and hydrogen peroxides, lead to the increase in the levels of cerulo-plasmin. The decreased level of ceruloplasmin found in diabetic rats treated with A. marmelos in our study may be a protective response to the decrease in circu-lating unbound Fe2+, which may act as an inhibitor for further free radical–induced lipid peroxidation.

Histopathological studies revealed that alterations occurred in the architecture of pancreatic tissue in STZ-induced diabetic rats. The pancreas of normal rats did not show any pathological alteration. The degenerative changes were seen in the midzonal regions. Parenchymatous degeneration was also observed in the peripheral regions. It is interesting to note that these alterations are corrected to near normal by A. marmelos leaf extract in STZ-induced diabetes mellitus. From the previous reports, we found that compounds such as triterpenoid, lupeol, and alkaloidal-amide Aegeline are present in the leaf extract of A. marmelos (Papi Reddy et al., 2009; Narender et al., 2007). In our present study, the free radical–scavenging activity of A. marmelos leaf extract in the diabetic rats may be due to the presence of the above compounds..

Conclusions

From the results obtained, it could be concluded that A. marmelos improves the antioxidant status in the tissues of diabetic rats by possessing the struc-tural features of an antioxidant. A morphological assessment also shows that the damage caused by STZ to the tissues was also markedly reduced by the administration of A. marmelos. The hypogly-cemic activity of A. marmelos was compared with glibenclamide, a standard hypoglycemic drug. Sulfonylureas, such as glibenclamide, have been used for many years to treat diabetes and to stimu-late insulin secretion from pancreatic β-cells. From the results of the present study, it may be suggested that the mechanism of action of A. marmelos seems to be similar to glibenclamide action. But, further mechanistic studies are needed to elucidate the exact mechanism of this antioxidant effect and to develop A. marmelos as a potent oral hypoglycemic drug.

Acknowledgments

The authors thank Prof. M. Kandaswamy, M. Sc., M.Phil., Ph.D (Head, Department of Inorganic Chemistry, School of Chemical Sciences, University of Madras, Chennai, India) for the contribution of advice and institutional resources. Also, the first author would like to express his appreciation to Dr. Paul Dinakaran, M.B.A., Ph.D, Chancellor, Dr. Paul. P. Appasamy, M.S (Chemical English)., M.B.A., Ph.D, Vice-Chancellor, and Dr. Anne Mary Fernandez, M.A.,M.Phil.,Ph.D, Registrar, of Karunya University (Coimbatore, India) for providing the necessary facilities to carry out the experiments and Dr. Rashmi Chandran, M.Sc., H.N.C.,Ph.D, (Postdoctoral Researcher, Faculty of Science, Masaryk University, Brno, Czech Republic) for her support in the study and preparation of the manuscript for this article.

Declaration of interest: There is no conflict of interest.

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