JOURNAL OF FUNCTIONAL FOODS 5 ( 20 1 3 ) 10 7 710 8 7

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Asiatic acid prevents lipid peroxidation and improves

antioxidant status in rats with streptozotocin-induced
diabetes

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: Oxidative stress is a common pathogenesis of diabetes mellitus and asiatic acid (AA) plays
Received 28 October 2012 an important role in ameliorating those difficulties. The present study was designed the
Received in revised form protective effects of AA on altered lipid peroxidation products, enzymic and nonenzymic
1 March 2013 antioxidants in streptozotocin (STZ)-induced diabetic rats. Diabetes was induced in exper-
Accepted 4 March 2013 imental rats by single dose STZ (40 mg/kg b.w.) injection. Diabetic rats showed significantly
Available online 29 March 2013 increased levels of plasma glucose, thiobarbituric acid reactive substances, lipid hydroper-
oxides, aspartate aminotransferase, alanine aminotransferase, bilirubin, creatine kinase,
Keywords: urea, uric acid, creatinine and decreased levels of plasma insulin. The activities of enzy-
Asiatic acid matic antioxidants such as superoxide dismutase, catalase, glutathione peroxidase and
Streptozotocin glutathione-S-transferase and the levels of non-enzymatic antioxidants such as vitamin
Antidiabetic
C, vitamin E and reduced glutathione were decreased in diabetic rats. Oral treatment with
Antioxidant
AA (20 mg/kg b.w.) showed near normalized levels of plasma glucose, insulin, lipid perox-
Oxidative stress
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 stress results from an imbalance between radical-generating

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 (O2 ), hydrogen
peroxide (H2O2), and hydroxyl radical (OH) or reduction of
antioxidant defense system (Sklavos et al., 2010). Oxidative
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

* Corresponding author. Tel.: +91 4144 238343; fax: +91 4144 239141.
E-mail address: jayamsaranbio@gmail.com (R. Saravanan).
1756-4646/$ - see front matter Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.jff.2013.03.003
1078 JOURNAL OF FUNCTIONAL FOODS 5 ( 2 0 1 3 ) 1 0 7 7 1 0 8 7

chemicals used were purchased from standard commercial

suppliers and were of analytical grade.

2.2. Animals

Adult Male albino Wistar rats (9 weeks old; 180200 g) were ob-
Fig. 1 Structure of asiatic acid. 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
initial step of many pathological processes (Pandey & Rizvi,
clean, sterile, polypropylene cages under standard vivarium
2010).
conditions (12 h light/dark cycle) with ad libitum access to
Hypoglycemic sulphonylureas such as glibenclamide can
standard rat chow and water. The whole experiment was car-
increase pancreatic insulin secretion from the existing b-cells
ried out according to the guidelines of the Committee for the
in STZ-induced diabetes by membrane depolarization, and
Purpose of Control and Supervision of Experiments on Ani-
stimulation of Ca2+ influx, an initial key step in insulin secre-
mals, New Delhi, India and approved by the Animal Ethics
tion. Moreover, glibenclamide has shown a protection effect
Committee of Annamalai University, India, Tamil Nadu, Anna-
against oxidative stress in diabetes (Elmai, Altan, & Bukan,
malainagar (Reg No.: 160/1999/CPCSEA, Proposal No.: 848).
2004). Glibenclamide is often used as a reference drug in
STZ-induced moderate diabetic model. Though sulphonylure-
2.3. Induction of experimental diabetes
as are valuable in treatment of diabetes, their use is restricted
by their limited action and side effects (hypoglycaemia and li-
Experimental diabetes was induced in 12 h fasted rats by sin-
ver problems) (Rajalakasmi, Eliza, Cecilia, Nirmala, & Daisy,
gle i.p. injection of streptozotocin (40 mg/kg b.w.) dissolved in
2009). The focus has been shifted to treat the various ailments
cold citrate buffer (0.1 M, pH 4.5). STZ-injected animals were
through dietary fruits, green leaves, vegetables and natural
given 20% glucose solution for 24 h to prevent initial drug-in-
plant-derived drugs due to their safety, efficacy, and lesser
duced hypoglycaemia. STZ-injected animals exhibited hyper-
side effects (Khanal, Howard, Wilkes, Rogers, & Prior, 2010).
glycemia within a few days. Diabetic rats were confirmed by
Triterpenoids receiving considerable attention across the
measuring the elevated plasma glucose (by glucose oxidase
world for the potential health benefits in relation to many dis-
method) 72 h after injection with STZ. The animals with glu-
eases including diabetic disorders (Szakiel, Paczkowski, Pen-
cose above 235 mg/dL were selected for the experiment.
sec, & Bertsch., 2012). Thus, antioxidant therapy may be a
promising therapeutic approach for controlling diabetes or
2.4. Experimental design
diabetic complications.
Triterpenes are widely available in dietary fruits and vege-
The rats were randomly segregated into five groups of six rats
tables, and are major components in many medicinal plants
in each group. AA were dissolved in 5% dimethyl sulfoxide
used in Asian countries. Asiatic acid (AA; 2a, 3b, 23-tri-
and glibenclamide was diluted in water and administered or-
hydroxyurs-12-en-28-oic acid; Fig. 1), is a pentacyclic triterpe-
ally to experimental groups using intragastric tube daily for a
noid that contributes to the waxy coats on apples, other
period of 45 days:
fruits, and many herbs, including some folkloric herbal med-
icines for diabetes. Various reports have demonstrated that
Group 1: normal rats.
AA has antioxidant (Lee, Jin, Beak, Lee, & Kim, 2003), hepato-
Group 2: normal + AA (20 mg/kg b.w.).
protective (Ma Zhang, Zhu, & Lou, 2009), anticancer (Liu,
Group 3: diabetic control.
Duan, Pan, Zhang, & Yao, 2006), antiinflammation (Huang
Group 4: diabetic + AA (20 mg/kg b.w.).
et al., 2011), neurotoxicity activity (Jew et al., 2000). Recently,
Group 5: diabetic + glibenclamide (600 lg/kg b.w.).
we reported that AA administration significantly improved
glucose homeostasis through improved activities of key car-
At the end of the treatment period, the rats were fasted
bohydrate metabolizing enzymes in STZ-induced diabetic
overnight, anesthetized (ketamine, 24 mg/kg b.w. i.p.) and
rats (Ramachandran & Saravanan, 2013). As yet there is no
sacrificed by cervical decapitation on 46th day morning and
published study on the role of AA on oxidative stress in STZ
tissues dissected out, washed, weighed, homogenized and
induced diabetic rats. Hence, the present study aimed to
centrifuged. The blood was collected with or without antico-
investigate the ameliorative potential of AA on hyperglycemia
agulant for plasma and serum separation, respectively.
mediated oxidative stress in STZ induced diabetic rats and
the effect of AA was compared with glibenclamide, an oral
antihyperglycemic drug with antioxidant potential. 2.5. Biochemical analysis

2. Materials and methods 2.5.1. Measurement of plasma glucose and insulin

Plasma glucose was estimated using a commercial kit (Sigma
2.1. Chemicals Diagnostics Pvt. Ltd., Baroda, India) by the method of Trinder
(1969). Insulin in the rat plasma was assayed by the solid
Asiatic acid, streptozotocin and glibenclamide (99%) were pur- phase system amplified sensitivity immunoassay using
chased from SigmaAldrich (St. Louis, MO, USA). All other reagent kits obtained from Medgenix INS-ELISA, Biosource,
 JOURNAL OF FUNCTIONAL FOODS
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 kidney
Lipid 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
(TrisHCl buffer, pH 7.5) was treated with 2.0 ml of (1:1:1, v/
v/v) TBATCAHCl 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 antioxidants
SOD 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 antioxidants
Vitamin 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
5 ( 2 01 3 ) 10 7 710 8 7 1079
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 20100 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) and
alanine transaminase (ALT)
Activities of AST and ALT were 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 bilirubin
Serum 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 kinase
The 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 urea
Urea 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.
1080 JOURNAL OF FUNCTIONAL FOODS
2.5.9. Estimation of uric acid
Uric 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 C
for 5 min and the color developed was read at 510 nm.
2.5.10. Estimation of creatinine
Creatinine 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,
5 ( 2 0 1 3 ) 1 0 7 7 1 0 8 7
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
 JOURNAL OF FUNCTIONAL FOODS 5 ( 2 01 3 ) 10 7 710 8 7 1081

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)
a
Normal control 80.02 5.24 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.
1082 JOURNAL OF FUNCTIONAL FOODS 5 ( 2 0 1 3 ) 1 0 7 7 1 0 8 7

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 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

JOURNAL OF FUNCTIONAL FOODS

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).
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.
U$ = lg of CDNB conjugate formed/min.

5 ( 2 01 3 ) 10 7 710 8 7
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
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).

1083
1084 JOURNAL OF FUNCTIONAL FOODS 5 ( 2 0 1 3 ) 1 0 7 7 1 0 8 7

Diabetic + glibenclamide (600 lg/kg b.w.) 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-
160.13 6.21c
29.45 1.36c
81.28 7.03c

28.35 2.24c
zymes may be effective in STZ-diabetes. In our study the
0.61 0.03c

1.57 0.09c
0.98 0.05c
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
Diabetic + AA (20 mg/kg b.w.)

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
165.68 7.18c
32.17 1.45c
84.01 4.97c

30.23 2.61c
Table 4 Effect of AA on ALT, AST, bilirubin, CK, urea, uric acid and creatinine in normal and experimental rats.

0.65 0.04c

1.65 0.15c
1.13 0.08c

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-
Diabetic control

tration of AA to diabetic rats significantly ameliorates the

215.12 13.73b
115.87 9.06b
58.33 4.26b

37.08 2.48b

activities of enzymatic antioxidants, which in turn reflects

1.18 0.08b

2.30 0.16b
2.10 0.11b

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

is a major antioxidant that is essential for the scavenging of

Normal + AA (20 mg/kg b.w.)

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,
139.79 10.06a

thereby breaking the radical chain reaction and averting

22.71 1.25a
72.07 4.12a

23.86 1.45a
0.49 0.02a

1.35 0.07a
0.80 0.06a

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
Normal control

140.11 9.12a

could be the result of decreased synthesis or increased degra-

25.02 1.40a
75.05 5.01a

24.41 1.87a
0.52 0.02a

1.38 0.08a
0.85 0.04a

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

Values are means SD for six rats.

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
Creatinine (mg/dL)
Uric acid (mg/dL)

AA asiatic acid.
Bilirubin (mg/dL)

hyperglycemia-mediated oxidative stress probably by

decreasing the consumption of free radical scavengers.
Urea (mg/dL)
AST (IU/L)

STZ induced DM results in abnormal values for kidney

ALT (IU/L)

CK (IU/L)
Groups

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
 JOURNAL OF FUNCTIONAL FOODS
transaminase enzymes (ALT and 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.
5 ( 2 01 3 ) 10 7 710 8 7 1085
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