Asiatic acid prevents lipid peroxidation and improves antioxidant status in rats with...

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Asiatic acid prevents lipid peroxidation and improvesantioxidant status in rats with streptozotocin-induceddiabetes

Vinayagam Ramachandran, Ramalingam Saravanan*

Department of Biochemistry and Biotechnology, Faculty of Science, Annamalai University, Annamalainagar 608 002, Tamil Nadu, India

A R T I C L E I N F O A B S T R A C T

Article history:

Received 28 October 2012

Received in revised form

1 March 2013

Accepted 4 March 2013

Available online xxxx

Keywords:

Asiatic acid

Streptozotocin

Antidiabetic

Antioxidant

Oxidative stress

1756-4646/$ - see front matter Published byhttp://dx.doi.org/10.1016/j.jff.2013.03.003

* Corresponding author. Tel.: +91 4144 238343E-mail address: [email protected]

Please cite this article in press as: Ramachandrain rats with streptozotocin-induced diabetes, Jo

Oxidative stress is a common pathogenesis of diabetes mellitus and asiatic acid (AA) plays

an important role in ameliorating those difficulties. The present study was designed the

protective effects of AA on altered lipid peroxidation products, enzymic and nonenzymic

antioxidants in streptozotocin (STZ)-induced diabetic rats. Diabetes was induced in exper-

imental rats by single dose STZ (40 mg/kg b.w.) injection. Diabetic rats showed significantly

increased levels of plasma glucose, thiobarbituric acid reactive substances, lipid hydroper-

oxides, aspartate aminotransferase, alanine aminotransferase, bilirubin, creatine kinase,

urea, uric acid, creatinine and decreased levels of plasma insulin. The activities of enzy-

matic antioxidants such as superoxide dismutase, catalase, glutathione peroxidase and

glutathione-S-transferase and the levels of non-enzymatic antioxidants such as vitamin

C, vitamin E and reduced glutathione were decreased in diabetic rats. Oral treatment with

AA (20 mg/kg b.w.) showed near normalized levels of plasma glucose, insulin, lipid perox-

idation products, enzymatic and nonenzymatic markers in diabetic rats. The results dem-

onstrate that AA possesses potent antioxidant effect comparable with glibenclamide in

improving antihyperglycemia and attenuating antioxidant status in diabetic rats.

Published by Elsevier Ltd.

1. Introduction

Diabetes mellitus (DM) is a heterogeneous metabolic disorder

characterized by common feature of chronic hyperglycemia

with disturbance in carbohydrate, fat and protein metabo-

lism. The World Health Organization estimates that more

than 346 million people Worldwide have diabetes mellitus.

Without intervention, this number is likely to increase more

than twofold by 2030 (WHO, 2012). Various studies have

shown that diabetes mellitus is associated with oxidative

stress, leading to an increased production of reactive oxygen

species (ROS), including superoxide radical (O��2 ), hydrogen

peroxide (H2O2), and hydroxyl radical (�OH) or reduction of

antioxidant defense system (Sklavos et al., 2010). Oxidative

Elsevier Ltd.

; fax: +91 4144 239141.(R. Saravanan).

n, V., & Saravanan, R., Aurnal of Functional Foods

stress results from an imbalance between radical-generating

and radical-scavenging systems that are, increased free radi-

cal production or reduced activity of antioxidant defenses or

both these phenomena. In diabetes, protein glycation and

glucose autoxidation may generate free radicals, which in

turn catalyze lipid peroxidation (Baynes, 1991). The cellular

antioxidant status determines the susceptibility to oxidative

damage and is usually altered in response to oxidative stress.

Accordingly, interest has recently grown in the role and usage

of natural antioxidants as a means to prevent oxidative dam-

age in diabetes with high oxidative stress. The antioxidants

such as Vitamins C and E, enzymes superoxide dismutase

(SOD), catalase (CAT), glutathione peroxidase (GPx) have been

shown to protect the cells against lipid peroxidation, the

siatic acid prevents lipid peroxidation and improves antioxidant status(2013), http://dx.doi.org/10.1016/j.jff.2013.03.003

http://dx.doi.org/10.1016/j.jff.2013.03.003

mailto:[email protected]




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Fig. 1 – Structure of asiatic acid.

2 J O U R N A L O F F U N C T I O N A L F O O D S x x x ( 2 0 1 3 ) x x x – x x x

initial step of many pathological processes (Pandey & Rizvi,

2010).

Hypoglycemic sulphonylureas such as glibenclamide can

increase pancreatic insulin secretion from the existing b-cells

in STZ-induced diabetes by membrane depolarization, and

stimulation of Ca2+ influx, an initial key step in insulin secre-

tion. Moreover, glibenclamide has shown a protection effect

against oxidative stress in diabetes (Elmai, Altan, & Bukan,

2004). Glibenclamide is often used as a reference drug in

STZ-induced moderate diabetic model. Though sulphonylure-

as are valuable in treatment of diabetes, their use is restricted

by their limited action and side effects (hypoglycaemia and li-

ver problems) (Rajalakasmi, Eliza, Cecilia, Nirmala, & Daisy,

2009). The focus has been shifted to treat the various ailments

through dietary fruits, green leaves, vegetables and natural

plant-derived drugs due to their safety, efficacy, and lesser

side effects (Khanal, Howard, Wilkes, Rogers, & Prior, 2010).

Triterpenoids receiving considerable attention across the

world for the potential health benefits in relation to many dis-

eases including diabetic disorders (Szakiel, Paczkowski, Pen-

sec, & Bertsch., 2012). Thus, antioxidant therapy may be a

promising therapeutic approach for controlling diabetes or

diabetic complications.

Triterpenes are widely available in dietary fruits and vege-

tables, and are major components in many medicinal plants

used in Asian countries. Asiatic acid (AA; 2a, 3b, 23-tri-

hydroxyurs-12-en-28-oic acid; Fig. 1), is a pentacyclic triterpe-

noid that contributes to the waxy coats on apples, other

fruits, and many herbs, including some folkloric herbal med-

icines for diabetes. Various reports have demonstrated that

AA has antioxidant (Lee, Jin, Beak, Lee, & Kim, 2003), hepato-

protective (Ma Zhang, Zhu, & Lou, 2009), anticancer (Liu,

Duan, Pan, Zhang, & Yao, 2006), antiinflammation (Huang

et al., 2011), neurotoxicity activity (Jew et al., 2000). Recently,

we reported that AA administration significantly improved

glucose homeostasis through improved activities of key car-

bohydrate metabolizing enzymes in STZ-induced diabetic

rats (Ramachandran & Saravanan, 2013). As yet there is no

published study on the role of AA on oxidative stress in STZ

induced diabetic rats. Hence, the present study aimed to

investigate the ameliorative potential of AA on hyperglycemia

mediated oxidative stress in STZ induced diabetic rats and

the effect of AA was compared with glibenclamide, an oral

antihyperglycemic drug with antioxidant potential.

2. Materials and methods

2.1. Chemicals

Asiatic acid, streptozotocin and glibenclamide (99%) were pur-

chased from Sigma–Aldrich (St. Louis, MO, USA). All other

Please cite this article in press as: Ramachandran, V., & Saravanan, R., Ain rats with streptozotocin-induced diabetes, Journal of Functional Foods

chemicals used were purchased from standard commercial

suppliers and were of analytical grade.

2.2. Animals

Adult Male albino Wistar rats (9 weeks old; 180–200 g) were ob-

tained from Central Animal House, Department of Experimen-

tal Medicine, Rajah Muthiah Medical College and Hospital,

Annamalai University, India, Tamil Nadu and were housed in

clean, sterile, polypropylene cages under standard vivarium

conditions (12 h light/dark cycle) with ad libitum access to

standard rat chow and water. The whole experiment was car-

ried out according to the guidelines of the Committee for the

Purpose of Control and Supervision of Experiments on Ani-

mals, New Delhi, India and approved by the Animal Ethics

Committee of Annamalai University, India, Tamil Nadu, Anna-

malainagar (Reg No.: 160/1999/CPCSEA, Proposal No.: 848).

2.3. Induction of experimental diabetes

Experimental diabetes was induced in 12 h fasted rats by sin-

gle i.p. injection of streptozotocin (40 mg/kg b.w.) dissolved in

cold citrate buffer (0.1 M, pH 4.5). STZ-injected animals were

given 20% glucose solution for 24 h to prevent initial drug-in-

duced hypoglycaemia. STZ-injected animals exhibited hyper-

glycemia within a few days. Diabetic rats were confirmed by

measuring the elevated plasma glucose (by glucose oxidase

method) 72 h after injection with STZ. The animals with glu-

cose above 235 mg/dL were selected for the experiment.

2.4. Experimental design

The rats were randomly segregated into five groups of six rats

in each group. AA were dissolved in 5% dimethyl sulfoxide

and glibenclamide was diluted in water and administered or-

ally to experimental groups using intragastric tube daily for a

period of 45 days:

Group 1: normal rats.

Group 2: normal + AA (20 mg/kg b.w.).

Group 3: diabetic control.

Group 4: diabetic + AA (20 mg/kg b.w.).

Group 5: diabetic + glibenclamide (600 lg/kg b.w.).

At the end of the treatment period, the rats were fasted

overnight, anesthetized (ketamine, 24 mg/kg b.w. i.p.) and

sacrificed by cervical decapitation on 46th day morning and

tissues dissected out, washed, weighed, homogenized and

centrifuged. The blood was collected with or without antico-

agulant for plasma and serum separation, respectively.

2.5. Biochemical analysis

2.5.1. Measurement of plasma glucose and insulinPlasma glucose was estimated using a commercial kit (Sigma

Diagnostics Pvt. Ltd., Baroda, India) by the method of Trinder

(1969). Insulin in the rat plasma was assayed by the solid

phase system amplified sensitivity immunoassay using

reagent kits obtained from Medgenix INS-ELISA, Biosource,



J O U R N A L O F F U N C T I O N A L F O O D S x x x ( 2 0 1 3 ) x x x – x x x 3

Europe S.A., Nivelles, Belgium by the method of Burgi, Briner,

Franken, and Kessler (1988).

2.5.2. Estimation of lipid peroxidation in liver and kidneyLipid hydroperoxides as evidenced by formation of thiobarbi-

turic acid reactive substances (TBARS) and lipid hydroperox-

ides (LOOH) were measured by the method of Fraga,

Leibovitz, and Tappel (1988) and Jiang, Hunt, and Wolff

(1992), respectively. In brief, 0.1 ml of tissue homogenate

(Tris–HCl buffer, pH 7.5) was treated with 2.0 ml of (1:1:1, v/

v/v) TBA–TCA–HCl reagent (0.37%, Thiobarbituric acid,

0.25 M HCl and 15% TCA) and mixed thoroughly. The mixture

placed in water bath for 15 min, cooled and centrifuged at

room temperature for 10 min at 1000g. The absorbance of

clear supernatant was measured against reference blank at

535 nm.

For hydroperoxides, 0.1 ml of tissue homogenate was trea-

ted with 0.9 ml of Fox reagent (88 mg butylated hydroxytoluene

(BHT), 7.6 mg xylenol orange and 9.8 ml of methanol and 10 ml

250 mM sulphuric acid) and incubated at 37 �C for 30 min. the

color developed was read at 560 nm calorimetrically.

2.5.3. Activity of enzymatic antioxidantsSOD was determined by the method of Kakkar, Das, and

Viswanathan (1984). A single unite of enzyme was expressed

as 50% inhibition of NBT (nitroblue tetrazolium) reduction/

min/mg protein. CAT was assayed colorimetrically at 620 nm

and expressed as l mol of H2O2 consumed/min/mg protein

as described by Sinha (1972). The reaction mixture (1.5 ml)

contained 1.0 ml of 0.01 M pH 7.0 phosphate buffer, 0.1 ml of

tissue homogenate and 0.4 ml of 2 M H2O2. The reaction

supped by the addition of 2.0 ml of dichromate-acetic acid re-

agent (5% potassium dichromate and glacial acetic acid were

mixed at a 1:3 ratio).

GPx activity was measured by the method described by

Rotruck, Pope, Ganther, and Swason (1973). Briefly, reaction

mixture contained 0.2 ml of 0.4 M phosphate buffer pH 7.0,

0.1 ml of 10 mM sodium azide, 0.2 ml of tissue homogenate

(homogenate on 0.4 M phosphate buffer, pH 7.0), and 0.2 ml

glutathione, 0.1 of 0.2 mM H2O2. The content was incubated

at 37 �C for 10 min. The reaction was arrested by 0.4 ml of

10% TCA and centrifuged. Supernatant was assayed for gluta-

thione content by using Ellmans reagent.

Glutathione-S-transferase (GST) activity was determined

spectrophotometrically by the method of Habig, Pabst, and

Jakpoly (1974). The reaction mixture contained 1.0 ml of

100 mM phosphate buffer (pH 6.5), 0.1 ml of 30 mM 1-chloro-

2,4-dinitrobenzene, and 0.7 ml of double distilled water. After

pre-incubating the reaction mixture for 5 min at 37 �C, the

reaction was started by the addition of 0.1 ml of tissue

homogenate and 0.1 ml of glutathione as substrate. After

5 min, the absorbance was read at 340 nm.

2.5.4. Estimation of nonenzymatic antioxidantsVitamin E was determined by the method of Baker, Frank, De

Angelis, and Feingod (1980). A portion of the sample (0.1 ml),

1.5 ml of ethanol and 2 ml of petroleum ether were added,

mixed and centrifuged for 3000g for 10 min. The supernatant

was evaporated to dryness at 80 �C then 0.2 ml of 2,2 0-dipyri-

dyl solution and 0.2 ml of ferric chloride solution was added


and mixed well. This was kept in dark for 5 min and added

2 ml of butanol. Then the absorbance was read at 520 nm.

Ascorbic acid in the tissues was estimated by the method

of Omaye, Turbull, and Sauberlich (1979). To 0.5 ml of sample,

1.5 ml of 6% TCA was added and allowed to stand for 5 min

and centrifuged. To the supernatant, 0.3 g of acid washed nor-

it was added, shaken vigorously and filtered. This converts

ascorbic acid to dehydroascorbic acid. 0.5 ml of the filtrate

was taken and 0.5 ml of DNPH was added, stoppered and

placed in a water bath at 37 �C for exactly 3 h. Removed,

placed in ice-cold water and added 2.5 ml of 85% sulphuric

acid drop by drop. The contents of the tubes were mixed well

and allowed to stand at room temperature for 30 min. A set of

standards containing 20–100 lg of ascorbic acid were taken

and processed similarly along with a blank containing

2.0 ml of 4% TCA. The color developed was read at 540 nm.

Reduced glutathione (GSH) was determined by the method

of Ellman (1959). To the homogenate added 10% TCA, centri-

fuged. One millilitre of the supernatant was treated with

0.5 ml of Ellmans reagents 19.8 mg of 5,5 0-dithiobis-(2-nitro-

benzoic acid) in 100 ml of 0.1% sodium nitrate) and 3.0 ml of

phosphate buffer (0.2 M, pH 8.0). The absorbance was read

at 412 nm.

2.5.5. Activities of serum aspartate transaminase (AST) andalanine transaminase (ALT)Activities of AST and ALTwere assayed by the method of Reit-

man and Frankel (1957). 0.2 ml aliquot of serum with 1 ml of

substrate (aspartate and a-ketoglutarate for AST: alanine

and a-ketoglutarate for ALT) in phosphate buffer (pH 7.4)

was incubated for 1 h for AST and 30 min for ALT. One millili-

tre aliquot of DNPH solution was added to arrest the reaction

and kept for 20 min at room temperature. After incubation,

1 ml of 0.4 M NaOH was added and the absorbance was read

at 540 nm.

2.5.6. Estimation of bilirubinSerum bilirubin was estimated by the method of Malloy and

Evelyn (1937). Diazotised sulphonilic acid (0.5 ml) reacts with

bilirubin in diluted serum (0.2 ml serum + 1.8 ml distilled

water) to form a purple-colored azobilirubin, which was mea-

sured at 540 nm.

2.5.7. Estimation of creatine kinaseThe activity of creatine kinase was estimated by the method

of Okinaka et al. (1961). This reaction involves the conversion

of creatine to creatine phosphate. The amount of phospho-

rous liberated was estimated at 640 nm.

2.5.8. Estimation of ureaUrea in the plasma was estimated by using the diagnostic kit

based on the method of Fawcett and Scott (1960). One millili-

tre of buffered enzyme (phosphate buffer, urease, sodium

nitroprusside and ethylenediaminetetraacetic acid), 10 ll of

sample added, mixed well and kept at 37 �C for 5 min. Ten

microlitres of standard and 10 ll of distilled water (blank)

were also processed simultaneously. To all the tubes, 1.0 ml

of color developing reagent was added and mixed will. Exactly

after 5 min of incubation at 37 �C, 1.0 ml of distilled water was

added and the color developed was read at 600 nm.




2.5.9. Estimation of uric acidUric acid in the plasma was estimated by using the diagnostic

kit based on the enzymic method described by Caraway

(1955). To 1 ml of the enzyme reagent, 25 ll of sample were

added and mixed by inversion. Twenty-five microlitres of

standard and 25 ll of distilled water (blank) were also pro-

cessed simultaneously. The tubes were incubated at 37 �Cfor 5 min and the color developed was read at 510 nm.

2.5.10. Estimation of creatinineCreatinine in the plasma was estimated using the diagnostic

kit based on the method of Tietz (1987) using Jaffe (1886) color

reaction. A portion of the sample (0.1 ml) was added to a re-

agent mixture containing 0.5 ml picric acid solution and

0.5 ml of sodium hydroxide. The tubes were mixed well and

incubated for 20 s. With the spectrophotometer adjusted to

zero absorbance with distilled water, reading was taken at

510 nm.

2.6. Statistical analysis

The results were expressed as mean ± SD of six rats per group

and statistical significance was evaluated by one-way ANOVA

using SPSS (version 16.0) program followed by the post hoc

test, least significant difference. Values were considered sta-

tistically significant when p < 0.05.

3. Results

Fig. 2 depicts the values of the initial and final body weights of

the normal and experimental rats. Body weight significantly

(p < 0.05) decreased in diabetic rats compared to normal con-

trol rats. Oral administration of AA to diabetic rats protects

the loss of body weight compared to diabetic control rats.

Table 1 epitomizes the levels of plasma glucose and insulin

in normal and experimental rats. Diabetic rats were signifi-

cantly (p < 0.05) increase in the level of plasma glucose and

a significantly (p < 0.05) decrease in plasma insulin in diabetic

rats compared with control rats. Oral administration of AA as

well as glibenclamide to diabetic rats significantly (p < 0.05)

normalized the altered levels of plasma glucose and plasma

insulin when compared with diabetic rats.

Fig. 3 shows the levels of TBARS and LOOH in liver and kid-

ney of normal and experimental rats. Diabetic rats exhibited

increased levels of TBARS and LOOH when compared to nor-

mal control. Administration of AA and glibenclamide to dia-

betic rats significantly decreased lipid peroxidation markers

in liver and kidney when compared to diabetic rats.

Table 2 represents the activities of antioxidant enzymes

(SOD, CAT, GPx and GST) in the liver and kidney of normal

and experimental rats. A fall in the activities of antioxidants

enzymes was observed in diabetic rats when compared to

normal control. AA and glibenclamide administration to dia-

betic rats significantly improved the activities of the above

enzymes.

The levels of liver and kidney non-enzymatic antioxidants

such as vitamin C, vitamin E and GSH are represented in Ta-

ble 3. Diabetic rats showed a significantly (p < 0.05) decrease

in these levels when compared with control rats. Conversely,


administration of AA as well as glibenclamide to diabetic rats

significantly (p < 0.05) increased the levels to near control

values.

The levels of serum ALT, AST, bilirubin, CK, urea, uric acid

and creatinine in normal and experimental rats are repre-

sented in Table 4. The activities of ALT, AST, bilirubin, CK,

urea, uric acid and creatinine were significantly (p < 0.05) in-

creased in diabetic rats. These values were brought back to

near normal levels after treatment with AA and

glibenclamide.

4. Discussion

In the present study, we evaluated the antidiabetic and anti-

oxidant effects of AA in STZ diabetic rats. In the past few

years, natural substances have been shown to have the po-

tential to treat DM. Attention has been notably focused on

plants rich in triterpenoid, which generally have shown anti-

oxidant and antidiabetic effects (Ardiles et al., 2012; Manna,

Sinha, & Sil, 2009). The plant Centella asiatica contains huge

amount of AA and its alcoholic extracts in humans predicts

a detectable concentration of AA in plasma. AA which is a

metabolite of asiaticoside and by the hydrolytic cleavage of

the sugar moiety it becomes AA which is responsible for the

therapeutic effects and it clearly delineates the pharmacoki-

netic nature of AA (Grimaldi et al., 1990; Nair, Menon, & Sha-

ilajan, 2012). Moreover, AA is a non-toxic compound with a

LD50 value of 980 mg/kg when administered to rats. AA

administration 5, 10 and 20 mg/kg body weight gave signifi-

cant reduction of plasma glucose in STZ-diabetic rats. Since

AA at 20 mg dose gave a maximum improvement on body

weight, and decreased plasma glucose level, it was fixed as

the optimum dose (Ramachandran & Saravanan, 2013). From

the results of the present study, it is evident that the AA, a

triterpenoids significantly attenuated hyperglycemia and im-

proved antioxidant in experimentally induced diabetic rats.

STZ causes depletion in the secretion of insulin by partial

destroying pancreatic b-cells (Frode & Medeiros, 2008). Reduc-

tion in insulin production results in enhancement of blood

glucose level that inturn causes protein glycosylation. Oxida-

tion of enhanced glucose triggers overproduction in ROS that

leads to diabetic complications. Current antidiabetic treat-

ment strictly focuses on the management of glycaemia along

with reduction of associated diabetic complications. Some

bioactive compounds isolated from plants like terpenoids

was reported to stimulate insulin secretion with numerous

mechanisms such as exertion distal to K+-ATP channels and

Ca2+ channels (Hoa et al., 2007). Since oxidative stress and

free radicals injure or destroy pancreatic b-cells in diabetes,

AA is able to increase the secretion of insulin via its antioxi-

dant actions.

The level of LPO is a measure of membrane damage and

alterations in structure and function of cellular membranes.

The level of thiobarbituric acid reactive substance is an indi-

rect measurement of lipid peroxidation (Halliwell, Aeschbach,

Loligger, & Aruoma, 1995). Free radical-induced lipid peroxi-

dation has been associated with a number of disease pro-

cesses including diabetes mellitus. Increased endogenous

peroxides may initiate uncontrolled lipid peroxidation, thus



Fig. 2 – Effect of AA on body weight in control and experimental rats. NC – normal control; AA – asiatic acid; DC – diabetic

control. Values are means ± SD for six rats. Values not sharing a common marking (a, b, c) differ significantly at p < 0.05

(DMRT).

Table 1 – Effect of AA on plasma glucose and insulin in normal and experimental rats.

Groups Plasma glucose (mg/dL) Insulin (lU/mL)

Normal control 80.02 ± 5.24a 14.02 ± 1.01a

Normal + AA (20 mg/kg b.w.) 82.39 ± 6.21a 14.54 ± 1.05a

Diabetic control 248.36 ± 12.47b 6.74 ± 0.47b

Diabetic + AA (20 mg/kg b.w.) 105.11 ± 7.89c 12.35 ± 1.22c

Diabetic + glibenclamide (600 lg/kg b.w.) 98.74 ± 5.76c 13.87 ± 1.43c

AA – asiatic acid.

Values are means ± SD for six rats.

Values not sharing a common marking (a, b, c) differ significantly at p < 0.05 (DMRT).


leading to cellular in filtration and islet cell damage (Pasu-

pathi, Chandrasekar, & Senthil kiumar, 2009). In diabetes, it

is thought that hypoinsulinemia increases the activity of the

enzyme, fatty acyl coenzyme A oxidase, which initiates

beta-oxidation of fatty acids, resulting in LPO (Rahimi, Nikfar,

Larijani, & Abdollahi, 2005). Increased LPO impairs membrane

function by decreasing membrane fluidity and changing the

activities of membrane-bound enzymes and receptors. STZ-

induced diabetic rats showed an increased concentration of

lipid peroxidation products such as TBARS and LOOH in the

tissues, an indirect evidence of intensified free radical pro-

duction (Maritim, Sanders, & Watkins, 2003). The accumula-

tion of free radical observed in diabetic rats is attributed to


chronic hyperglycemia that alters antioxidant defense system

as demonstrated by previous studies (Hong et al., 2004). Free

radicals may also be formed via the auto-oxidation of unsat-

urated lipids in plasma and membrane lipids. They may react

with polyunsaturated fatty acids in cell membrane leading to

lipid peroxidation (Lery, Zaltzber, Ben-Amotz, Kanter, & Avi-

ram, 1999). Recent studies have shown that the supplementa-

tion of food triterpenoid with antioxidant potential is

significantly associated with a reduction in the level of lipid

peroxidation (Manna, Ghosh, Das, & Si, 2010). Oral treatment

of AA to diabetic rats prevented the lipid peroxidation mark-

ers enzymes to near normal levels which could be as a result

of improved glycemic control and antioxidants status.



Fig. 3 – Effect of AA on TBARS and hydroperoxides in normal and experimental rats. NC – normal control; AA – asiatic acid; DC

– diabetic control. Values are means ± SD for six rats. Values not sharing a common marking (a, b, c) differ significantly at

p < 0.05 (DMRT).


Cytosolic free radicals are either removed non-enzymati-

cally or by antioxidant enzymes such as SOD, CAT. SOD one

of the first antioxidant enzymes in the line of defense against

the deleterious effects of oxygen radicals in the cells, scav-

enges ROS by catalyzing the dismutation of superoxide to

H2O2 (McCord, Keele, & Fridovich, 1976), while CAT is an enzy-

mic antioxidant, which decomposes hydroxyl radicals and is

widely distributed in all animal tissues with the highest activ-

ity in the red blood cells and liver (Maritim et al., 2003). Reduc-

tion in these enzyme activities results in various deleterious

effects due to accumulation of superoxide and hydroxyl rad-

icals. In the present study, a reduced activity of CAT has been

observed. In diabetic conditions, the uncontrolled production


of hydrogen peroxide due to the auto-oxidation of glucose,

protein glycation and lipid oxidation led to a marked decline

in the CAT activity (Rajasekaran, Sivagnanam, & Subramani-

an, 2005).

GPx, a selenium containing tetrameric glycoprotein, pres-

ent in significant concentrations, detoxifies hydrogen perox-

ide into water and molecular oxygen through the oxidation

of reduced glutathione (Ewis & Abdel-Rahman, 1995). GPx

has been shown to be an important adaptive response to con-

dition of increased peroxidative stress. During diabetic condi-

tions, the activity of glutathione peroxidase is decreased as a

result of radical-induced inactivation and glycation of the en-

zyme (Zhang & Tan, 2000). The low activity of GPx could be



Table 3 – Effect of AA on nonenzymatic antioxidant in normal and experimental rats.

Groups Normal control Normal + AA (20 mg/kg b.w.) Diabetic control Diabetic + AA (20 mg/kg b.w.) Diabetic + glibenclamide (600 lg/kg b.w.)

Liver (lg/mg of protein) Vitamin C 0.91 ± 0.05a 0.95 ± 0.04a 0.51 ± 0.03b 0.74 ± 0.06c 0.79 ± 0.05c

Vitamin E 0.73 ± 0.06a 0.76 ± 0.04a 0.29 ± 0.02b 0.48 ± 0.03c 0.53 ± 0.03c

GSH 13.68 ± 10.98a 13.93 ± 1.21a 6.84 ± 0.38b 9.38 ± 0.75c 10.12 ± 0.91c

Kidney (lg/mg of protein) Vitamin C 0.85 ± 0.07a 0.88 ± 0.05a 0.54 ± 0.03b 0.68 ± 0.04c 0.71 ± 0.05c

Vitamin E 0.65 ± 0.040a 0.63 ± 0.05a 0.25 ± 0.02b 0.39 ± 0.02c 0.42 ± 0.03c

GSH 12.84 ± 1.12a 12.90 ± 0.87a 7.46 ± 0.34b 10.12 ± 0.48c 11.25 ± 0.95c




Table 2 – Effect of AA on enzymatic antioxidant in normal and experimental rats.

Groups Normal control Normal + AA (20 mg/kg b.w.) Diabetic control Diabetic + AA (20 mg/kg b.w.) Diabetic + glibenclamide (600 lg/kg b.w.)

SOD (U*/mg of protein) Liver 8.73 ± 0.51a 8.95 ± 0.68a 4.65 ± 0.27b 7.40 ± 0.59c 7.93 ± 0.66c

Kidney 13.91 ± 0.96a 14.15 ± 1.06a 7.21 ± 0.48b 11.75 ± 1.00c 12.46 ± 1.01c

CAT (U**/mg of protein) Liver 82.36 ± 6.18a 85.73 ± 6.02a 47.91 ± 3.67b 68.91 ± 4.95c 71.24 ± 4.81c

Kidney 41.71 ± 3.10a 44.86 ± 2.93a 20.99 ± 1.36b 34.15 ± 2.74c 36.48 ± 3.42c

GPx (U@/mg of protein) Liver 10.86 ± 0.82a 10.68 ± 0.74a 5.12 ± 0.25b 8.64 ± 0.68c 9.52 ± 0.55c

Kidney 7.93 ± 0.68a 8.06 ± 0.57a 4.61 ± 0.37b 5.98 ± 0.41c 6.34 ± 0.57c

GST (U$/mg of protein) Liver 7.05 ± 0.52a 7.23 ± 0.61a 3.59 ± 0.24b 5.81 ± 0.47c 5.21 ± 0.38c

Kidney 5.71 ± 0.48a 5.79 ± 0.51a 3.15 ± 0.7b 4.68 ± 0.39c 5.18 ± 0.394c




U* = enzyme concentration required to inhibit the chromogen produced by 50% in 1 m in under standard condition.

U** = lmol of hydrogen peroxide decomposed/min.

U@ = lmol of GSH utilized/min.

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Please cite this article in press as: Ramachandran, V., & Saravanan, R., Asiin rats with streptozotocin-induced diabetes, Journal of Functional Foods (2

directly explained by the low content of glutathione found in

diabetic state, since glutathione is a substrate and cofactor

of GPx. AA augmented the activities of antioxidant enzymes

in STZ-treated rats by inhibiting lipid peroxidation. Hence, a

compound that could prevent the generation of these oxy-

gen free radicals or increase the free radical scavenging en-

zymes may be effective in STZ-diabetes. In our study the

enzymatic antioxidant activities such as SOD, CAT and GPx

decreased in diabetic rats, and diabetic rats treated with

AA possess free radical scavenging activity.

GST, a glutathione-dependent enzyme, protects cell s

from ROS by utilizing a wide variety of products of oxidative

stress as substrates. The present investigation revealed sig-

nificant decrease in liver GPx and GST activities in the

STZ-induced diabetic rats, as compared to the control group.

In accordance with our results, Schettler et al. (1994) demon-

strated that the reduced antioxidant production may be due

to the increase in oxygen metabolites that causes a decrease

in the activity of the antioxidant defense system. Moreover,

Kennedy and Baynes (1984) reported that the decrease in

antioxidant enzyme activity in diabetes mellitus may be

due to non-enzymatic glycosylation of the enzymes. Accord-

ing by Al-Wabel, Mousa, Omer, and Abdel-Salam (2008) sug-

gested that the depletion of GSH content also may lower GST

enzyme, because GSH is required as a substrate for GST

activity. In this context, other workers also reported a dimin-

ished activity of enzymatic antioxidants in diabetic rats

(Karthikesan, Pari, & Menon, 2010). However, oral adminis-

tration of AA to diabetic rats significantly ameliorates the

activities of enzymatic antioxidants, which in turn reflects

the antioxidant property of AA.

Earlier research has shown that diabetics have low levels

of vitamin C, vitamin E and GSH. Vitamin E supplementation

can help prevent the development of diabetes. Ascorbic acid

is a major antioxidant that is essential for the scavenging of

toxic free radicals in both blood and tissues. Vitamin E, a lipo-

philic antioxidant, transfers its phenolic hydrogen to a per-

oxyl free radical of peroxidized polyunsaturated fatty acids,

thereby breaking the radical chain reaction and averting

the peroxidation of membrane lipids (Opara, 2002). GSH is a

tripeptide (L-c-glutamyl cysteinyl glycine), an antioxidant

and a powerful nucleophile, critical for cellular protection,

such as detoxification of ROS, conjugation and excretion of

toxic molecules and control of inflammatory cytokine cas-

cade (Brown, Harris, Ping, & Gauthier, 2004). Depletion of tis-

sues GSH levels demonstrated among diabetic rats clearly

suggests the increased utilization by the hepatic cells which

could be the result of decreased synthesis or increased degra-

dation of GSH by oxidative stress in diabetes (Furfaro et al.,

2012). In earlier published reports, which shows that the

GSH concentration decreases in the diabetic rats (Sayed,

2012). It has been observed that oral treatment of AA and gli-

benclaimaide significantly elevates the vitamin C, vitamin E

and GSH levels in diabetic rats. AA may act by reducing

hyperglycemia-mediated oxidative stress probably by

decreasing the consumption of free radical scavengers.

STZ induced DM results in abnormal values for kidney

and liver enzymes. This phenomenon is attributed to free

radical production that causes membrane damage especially

in the liver and kidney tissues. In DM, raised activities of

atic acid prevents lipid peroxidation and improves antioxidant status013), http://dx.doi.org/10.1016/j.jff.2013.03.003



transaminase enzymes (ALTand AST) are employed as the evi-

dence of liver injury. These enzymes pour out of liver cells in

greater quantities when liver is damaged, thus their levels in

the blood were raised. The serum urea, creatinine, creatinine

kinase and bilirubin levels are elevated in the diabetics due

to increased protein catabolism, glomerular injury and renal

dysfunction (Prangthip et al., 2012). The damage to the renal

cells is mainly due to glucose mediated osmotic diuresis, reac-

tive oxygen species and glucose overload. Uric acid is the end

product of purine catabolism, a potent antioxidant in blood.

The increased concentration of serum uric acid in diabetes is

associated with accumulation of purine bases due to oxidative

stress induced cellular necrosis. Renal dysfunction, insulin

resistance and increased cellular turnover also lead to in-

creased production of uric acid (Anwar & Meki, 2003). The

reversal of ALT, AST, bilirubin, creatinine kinase, urea, uric

acid and creatinine activities in AA treated diabetic rats to-

wards near normalcy indicate the liver and kidney protective

nature. These results are in agreement with, Ma Zhang et al.

(2009) who reported that AA improved hepatic enzymes in

hepatotoxicity.

AA is a natural triterpenoid that is also derived from

medicinal plants, fruits, green leaves and vegetables possess-

ing a C2a-OH function exhibited more potent glycogen phos-

phorylase inhibitory activity, making it an interesting

compound for the treatment of diseases caused by abnormal-

ities in glycogen metabolism, such as diabetes (Zhang et al.,

2009). AA has a powerful antioxidant property mainly pos-

sessing a number of hydroxyl groups at a position of C2a,

C3b, C23-trihydroxyl within their structure, which is favorable

for the antioxidative, anti-inflammatory (Lee et al., 2003) and

ester formation with the C(28) carboxylic acid, are relatively

important to enhance the wound healing activity. C-2 posi-

tion on AA showed the most potent hepatoprotective activity

against carbon tetrachloride-induced hepatotoxicity. AA has

the ability to trigger the proinsulin synthesis and also insulin

release, which might be helpful to reduce the plasma glucose

and increase insulin during diabetes (Liu et al., 2010). Like-

wise, the antioxidant potential of AA are due to the hydroxyl

groups present at C2, C3 and C28th positions and thus the oral

administration of AA is an essential trigger for the liver and

kidney to revert its normal homeostasis during experimental

diabetes.

5. Conclusion

In conclusion, the current results indicate that AA has the

ability to ameliorate oxidative stress in tissues of STZ-

induced diabetic rats as evidenced by improved glycemic and

antioxidant status along with decreased lipid peroxidation.

Therefore, further studies are necessary to elucidate the exact

mechanism by which AA elicits its modulatory effects.

Acknowledgement

The authors thank the Indian Council of Medical Research

(ICMR), New Delhi, India for providing financial support for

this research project, in the form of Senior Research Fellow-

ship (SRF) to Mr. V. Ramachandran.


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