PHYTOTHERAPY RESEARCH

Phytother. Res. 18, 310–314 (2004)

310 Published online in Wiley InterScience (www.interscience.wiley.com).
M. SRI BALASUBASHINIDOI: 10.1002/ptr.1440
ET AL.

Ferulic Acid Alleviates Lipid Peroxidation

in Diabetic Rats

M. Sri Balasubashini1, R. Rukkumani1, P. Viswanathan2 and Venugopal P. Menon1*

1
Department of Biochemistry, Faculty of Science, Annamalai University, Annamalai Nagar-608 002, Tamil Nadu, India
2
Department of Pathology, RMMCH, Annamalai University, Annamalai Nagar-608 002, Tamil Nadu, India

Diabetes mellitus is a metabolic disorder associated with increased formation of free radicals. The objective of
our study was to determine whether ferulic acid (FA), a phenolic acid, has any role to play in diabetes induced
free radical formation. Diabetes was induced with streptozotocin. The levels of blood glucose, thiobarbituric
acid reactive substances (TBARS), hydroperoxides and free fatty acids (FFA) increased in the liver of diabetic
animals. The activities of glutathione peroxidase (GPx), superoxide dismutase (SOD) and catalase (CAT)
decreased in the liver. Histopathology of pancreas also shows shrunken islets. Supplementation of FA to the
diabetic rats resulted in a decrease in the levels of glucose, TBARS, hydroperoxides, FFA and an increase in
reduced glutathione (GSH). FA also resulted in increased activities of SOD, CAT, GPx and expansion
of pancreatic islets. The effect was much pronounced with lower dose treatment. Thus our study shows
that administration of ferulic acid helps in enhancing the antioxidant capacity of these diabetic animals by
neutralizing the free radicals formed thereby reducing the intensity of diabetes. Copyright © 2004 John Wiley
& Sons, Ltd.

Keywords: ferulic acid; diabetes; lipid peroxidation; antioxidants; blood glucose.

Since oxidative stress is associated with diabetes we

INTRODUCTION
Diabetes is the most common endocrine disorder
characterized by hyperglycemia. Hyperglycemia in dia-
betes may result from: (a) decreased entry of glucose
into cells; (b) decreased utilization of glucose by vari-
ous tissues and (c) increased production of glucose
(gluconeogenesis) by liver.
Hyperglycemia causes over production of free radi-
cals thereby creating oxidative stress (Arango et al.,
2000). Oxidative stress is defined as an imbalance
between the levels of prooxidants and antioxidant in
the biological systems, leading to cellular injury (Villa-
Caballero et al., 2000). According to Randle’s glucose-
fatty acid cycle, increased lipolysis during diabetes
and hence elevated FFA levels in tissues leads to
hyperglycemia and also causes the production of free
radicals (Efrat, 2001).
A most successful strategy for preventing diabetic
complication is intensive treatment to prevent hyper-
glycemia and thereby oxidative stress. Current therapy
for diabetes includes a modification of lifestyle, such as
diet and exercise, and the use of a variety of pharmaco-
logical agents. These agents target increased insulin
secretion, decreased hepatic glucose production and
increased sensitivity to insulin (Kelly and Mandarino,
2000). Many of the synthetic drugs like sulphonylureas
and biguanides can produce side effects including
hematological, cutaneous and gastrointestinal reactions
and disturbances in liver and kidney (Larner et al., 1985).
used a natural antioxidant – ferulic acid, a phenolic acid,
which finds wide distribution in the plant kingdom, and
is more bioavailable than other dietary flavonoid and
monophenolics studied (Graf, 2000). Ferulic acid has
been proved to be a potent antioxidant, reported to
terminate free radical chain reaction and reduces the
risk for coronary heart diseases (Bourne et al., 2000).
Ferulic acid also has a protective effect in liver toxicity
induced by drugs and is used as anti-inflammatory drug
in Japanese oriental medicine (Wu et al., 1995).
Previous studies have shown that turmeric and
curcumin have antidiabetic properties (Arun and Nalini,
2000). Turmeric is a spice, which is consumed in many
parts of our country. One of the active principles of
turmeric is curcumin. Curcumin is broken down into
ferulic acid and vanillin. Since we had studied the
effect of curcumin under different diseased conditions
such as liver toxicity (Rukkumani et al., 2002), colon
cancer (Devasena et al., 2002) and diabetes, we thought
that ferulic acid, one of the components may play some
role in offering protection. This phenolic compound is
found not only in curcumin but also in other dietary
sources like vegetables, fruits, cereals etc. (Bourne and
Rice-Evans, 1998). Hence in our study we have concen-
trated on both the antihyperglycemic and antioxidant
effect of ferulic acid in diabetes.
RESEARCH DESIGN AND METHOD

Materials
* Correspondence to: Dr V. P. Menon, Department of Biochemistry,
Faculty of Science, Annamalai University, Annamalai Nagar-608 002,
Tamil Nadu, India. Streptozotocin (STZ) was purchased from the Sigma
E-mail: cmrana@sify.com chemical company; (St. Louis, MO, USA). Ferulic acid
Copyright © 2004 John Wiley & Sons, Ltd. Received
Phytother. Res. 9 April(2004)
18, 310–314 2002
Accepted 23 July 2003
Copyright © 2004 John Wiley & Sons, Ltd.
 FERULIC ACID ALLEVIATES LIPID PEROXIDATION IN DIABETIC RATS
was purchased from Fluka Chemika (Steinheim,
Switzerland). All other chemicals and reagents used
were of analytical grade.
Animals
Experiments were performed on adult female rats of
Wistar strain obtained from the Central Animal House,
Rajah Muthiah Medical College, body weight in the
range of 160–170 g. The rats were fed a standard
pellet diet (Karnataka State Agro Corporation (P) Ltd,
Agro Feeds Division, Bangalore, India) and were
given water ad libitum. The animals used in the pre-
sent study were cared as per the principles and guide-
lines of the Ethical Committee of Animal Care of
Annamalai University in accordance with the Indian
National Law on animal care and use.
Diabetes was induced by a single intraperitoneal
injection of streptozotocin (40 mg/kg in citrate buffer
of pH 4.0). Blood glucose concentration and changes
in body weight were monitored regularly. Only those
diabetic rats that exhibited a blood glucose concentra-
tion ≥ 200 mg/dL were divided into four groups of eight
rats each and included in the study along with a normal
group. The different groups are:
Group 1 – Normal rats received 1 ml water by intra-
gastric intubation.
Group 2 – Diabetic control received 1 ml water.
Group 3 – Diabetic rats treated orally with a high dose
of ferulic acid – 40 mg/kg (HD) in 1 ml water
as suspension.
Group 4 – Diabetic rat treated orally with a low dose of
ferulic acid – 10 mg/kg (LD), in 1 ml water
as suspension.
Group 5 – Diabetic rats treated orally with the refer-
ence drug glibenclamide – 0.6 mg/kg (GB)
in 1 ml water.
After 45 days of treatment, the animals were fasted
overnight, anaesthetized with ketamine Hcl and sacri-
ficed. The blood was collected in heparinized tubes and
liver tissue in ice-cold containers and used for various
biochemical estimations.
Biochemical estimations
Estimation of blood glucose. Glucose was estimated
in blood (Sasaki et al., 1972) using O-toluidine in glacial
acetic acid, which, when heated with glucose, pro-
duces a colored product. The aldehydes group of
glucose apparently condenses with the reagent to form
glycosylamine and an schiff’s base, which gives the
colored product.
Estimation of lipid peroxidation and antioxidants. The
concentration of thiobarbituric acid reactive substances
(TBARS) was estimated in the tissues by the method
of Ohkawa et al. (1979). The pink coloured chromogen
formed by the reaction of 2-thiobarbituric acid with
breakdown products of lipid peroxidation was meas-
ured. Hydroperoxides was estimated (Jiang et al., 1992)
using Fox reagent, based on the oxidation of ferrous
ion under the acidic condition in the presence of xylenol
orange.
Copyright © 2004 John Wiley & Sons, Ltd.
311
Superoxide dismutase (SOD) activity in the tissues
was assayed (Kakkar et al., 1984) based on the inhibi-
tion of formation of NADH-phenazine methosulphate-
nitro blue tetrazolium complex. Catalase (CAT) activity
was assayed (Sinha, 1972) by quantifying the hydrogen
peroxide after reacting with dichromate in acetic acid.
The activity of glutathione peroxidase (GPx) was as-
sayed by the method of Rotruck et al. (1973). A known
amount of enzyme preparation was allowed to react
with hydrogen peroxide (H2O2) and glutathione (GSH)
for a specified time period. Then the GSH content re-
maining after the reaction was measured (Ellman, 1959).
Total reduced glutathione content (GSH) was meas-
ured by the method of Ellman. This method is based
on the development of a yellow colour when 5′ 5′ dithio
bisnitro benzoic acid (DTNB) is added to compounds
containing sulphydryl groups.
Estimation of free fatty acids. Free fatty acids (Falholt
et al., 1973) were extracted with chloroform-heptane-
methanol mixture to eliminate interference from
phospholipids and the extract was shaken with a high-
density copper reagent at pH 8.1. The copper soap
remained in the upper organic layer. An aliquot from
this was removed and the copper content determined
colorimetrically by treating with diphenyl carbazide.
Histopathological studies
For hisotpathological study, the rats were perfused
with 10% formalin. Pancreas was removed and was
then embedded in paraffin, thinly sectioned using a
microtome, stained with Hematoxylin and Eosin (H &
E) and mounted in neutral DPX medium and examined
using light microscope.
Statistical Analysis
The results were statistically analysed by one-way ana-
lysis of variance (ANOVA) followed by least significant
difference (LSD). The significance was set at p < 0.001.
RESULTS
Effect on blood glucose and body weight changes
(Table 1)
The changes in the levels of blood glucose and body
weight before and after treatment of diabetic rats are
presented in Table 1. Treatment with ferulic acid was
found to decrease the blood glucose levels and increase
the body weight. The effect was more pronounced
with the low dose than that of high dose of ferulic
acid and was comparable with that of reference drug
glibenclamide (p < 0.001).
Effect on FFA and lipid peroxidation in liver
(Table 2)
Free fatty acid, TBARS and hydroperoxides con-
centration were found to be elevated in diabetic rats.
Phytother. Res. 18, 310–314 (2004)
312 M. SRI BALASUBASHINI ET AL.

Table 1. Changes in body weight and blood glucose of control and experimental rats (values are mean ± SD, n = 6)

Blood glucose (mg/100 ml) Body weight (g)

Groups Before treatment After treatment Initial Final

1. Normal 81.71 ± 2.54 82.00 ± 1.63 158.33 ± 10.27 192.50 ± 8.53

2. Diabetic control 248.33 ± 23.74 281.66 ± 18.04a 120.00 ± 2.88 93.33 ± 5.52a
3. Diabetic + FA (LD) 236.50 ± 13.75 113.83 ± 4.30a,b 116.60 ± 5.52 150.00 ± 7.63b
4. Diabetic + FA (HD) 247.33 ± 13.54 168.00 ± 14.81a,b 127.08 ± 10.09 141.42 ± 7.42a,b
5. Diabetic + GB 253.42 ± 17.75 115.00 ± 5.57a,b 120.83 ± 5.33 156.66 ± 5.52b

ANOVA followed by LSD.

a
2,3,4 and 5th groups are compared with 1, significant at p ≤ 0.001.
b
3,4 and 5th groups are compared with 2, significant at p ≤ 0.001.

Table 2. Levels of TBARS, hydroperoxides and FFA in liver (values are mean ± SD, n = 6)

TBARS Hydroperoxides FFA

Groups (mM/100 g tissue) (mM/100 g tissue) (mM/100 g tissue)

1. Normal 0.79 ± 0.09 69.46 ± 5.16 755.83 ± 47.16

2. Diabetic control 3.86 ± 0.25a 161.22 ± 7.01a 1361.98 ± 136.74a
3. Diabetic + FA (LD) 1.44 ± 0.17a,b 85.00 ± 9.09a,b 847.50 ± 54.01a,b
4. Diabetic + FA (HD) 2.73 ± 0.26a,b 116.67 ± 7.50a,b 983.33 ± 79.66a,b
5. Diabetic + GB 1.15 ± 0.11a,b 77.33 ± 5.68a,b 800.50 ± 31.58a,b

ANOVA followed by LSD.

a
2,3,4 and 5th groups are compared with 1, significant at p < 0.001.
b
3,4 and 5th groups are compared with 2, significant at p < 0.001.

Treatment with ferulic acid (HD) resulted in significant Effect on pancreas

decrease in the levels of TBARS, hydroperoxides and
FFA in liver (p < 0.001), whereas treatment with ferulic
acid (LD) showed a better reduction than that the high
dose of ferulic acid.
Effect on antioxidant status in liver (Table 3)
Pancreas of STZ diabetic rats showed shrunken islets
and fat accumulation (Fig. 2), when compared to normal
(Fig. 1). All treated group rats showed expansion of
islets (Figs 3, 4 and 5), when compared to the pancreas
of diabetic rats. However the low dose of ferulic acid
showed the best with little fatty infiltration (Fig. 4).

The changes in the levels of GSH, GPX, SOD, and CAT

in the liver are presented in Table 3. The antioxidant
status was found to decrease in diabetic animals and
was improved significantly after ferulic acid treatment
(p < 0.001). Treatment with ferulic acid (LD) was found
to be more effective in restoring the antioxidant status
than those treated with ferulic acid (HD).
DISCUSSION
Ferulic acid, a ubiquitous phenolic acid, with antioxidant
property, reduced the effect produced by STZ-induced
diabetic rats. In our study diabetic rats (Group 2)

Table 3. Activities of SOD, CAT, GPx and levels of GSH in liver (values are mean ± SD, n = 6)

SOD CAT GPx GSH

Groups (Units/mg protein) (Units*/mg protein) (Units**/mg protein) (mg/100 g tissue)

1. Normal 15.01 ± 1.48 74.85 ± 5.98 12.23 ± 0.09 157.68 ± 7.95

2. Diabetic control 4.43 ± 0.39a 44.42 ± 4.31a 4.82 ± 0.61a 42.43 ± 4.01a
3. Diabetic + FA (LD) 9.12 ± 0.72a,b 62.82 ± 3.92a,b 9.89 ± 0.91a,b 121.32 ± 7.26a,b
4. Diabetic + FA (HD) 7.60 ± 0.73a,b 58.09 ± 6.02a,b 7.12 ± 0.44a,b 82.80 ± 2.20a,b
5. Diabetic + GB 8.51 ± 0.89a,b 60.57 ± 4.94a,b 7.26 ± 0.88a,b 133.08 ± 5.98a,b

Units – Enzyme reaction, which gives 50% inhibition of NBT reduction/min.

Units * – µmoles of H2O2 liberated/min.
Units ** – µmoles of glutathione utilised/min.
ANOVA followed by LSD.
a
2,3,4 and 5th groups are compared with 1, significant at p < 0.001.
b
3,4 and 5th groups are compared with 2, significant at p < 0.001.

Copyright © 2004 John Wiley & Sons, Ltd. Phytother. Res. 18, 310–314 (2004)
 FERULIC ACID ALLEVIATES LIPID PEROXIDATION IN DIABETIC RATS
Figure 1. Pancreas of normal rat shows islets of Langerhans: H
& E × 20.
Figure 3. Pancreas of diabetic rat treated with ferulic acid (HD),
shows expansion of islets along with fatty infiltration: H & E ×
20.
Figure 5. Pancreas of diabetic rat treated with glibenclamide
shows expansion of islets: H & E × 20.
showed a significant increase in blood glucose level and
significant decrease in body weight compared to the
normal rats (Group 1). The elevation in glucose level
may be due to an oxidative stress created on pancreas
by STZ, producing single strand breaks in DNA of the
pancreatic islets (Omamoto et al., 1981). Group 3 and 4
rats exhibited a reduction in the level of blood glucose
and an increase in body weight, due to the antihyper-
glycemic effect of ferulic acid.
Copyright © 2004 John Wiley & Sons, Ltd.
313
Figure 2. Pancreas of STZ-induced diabetic rat, shows shrink-
age and fatty infiltration in islets: H & E × 20.
Figure 4. Pancreas of diabetic rat treated with ferulic acid (LD)
shows expansion of islets with reduction in fatty infiltration: H
& E × 20.
Ferulic acid, which has been shown to have anti-
oxidant properties (Graf, 2000), helps to neutralize the
free radicals produced by STZ in the pancreas and
thereby decrease the toxicity of STZ. This decreased
oxidative stress/toxicity on the pancreas may help the
beta cells to proliferate and secrete more insulin, which
may have been reduced due to STZ treatment. This
increased insulin secretion can cause increased utiliza-
tion of glucose by the extra hepatic tissues and thereby
decrease the blood glucose level. Dose dependent
study shows that treatment with ferulic acid (LD) was
found to decrease the blood glucose level better than
ferulic acid (HD); which was similar to that of the
glibenclamide group.
Experimental studies indicate that the oxidative stress
is implicated in aging and pathogenesis of diabetic com-
plications and long-term complications still represents
the main cause of morbidity and mortality (Mezzatti
et al., 2000).
Hyperglycemia is a well-known cause for elevated
free radical concentration and this can leads to incre-
ased lipid peroxidation (TBARS and hydroperoxides).
According to Randle’s glucose-fatty acid hypothesis,
excessive free fatty acid released from the adipose
tissue for oxidation causes the production of meta-
bolites that inhibit glucose utilization by the tissues.
These metabolites of fatty acid oxidation, which are
Phytother. Res. 18, 310–314 (2004)
314 M. SRI BALASUBASHINI ET AL.
implicated in the glucose-fatty acid cycle, are reactive
oxygen species and hydrogen peroxide. These sub-
stances may cause damage to cellular structures and
impair glucose metabolism.
Elevated free radical concentration and lipid
peroxidation decreases the antioxidant defense in
biological systems. The important antioxidants are:
(a) GPx, which catalyses the removal of hydrogen
peroxide to non-toxic products by utilizing the re-
duced glutathione, GSH (Amdur et al., 1991); (b) SOD,
which protects the tissues against oxygen free radicals,
and converts these super oxides to hydrogen peroxide
and thereby prevents any damage to the membrane
and biological system (Halliwell and Gutteridge, 1999)
and (c) Catalase is a major enzyme in detoxification
of hydrogen peroxide formed from SOD (Li et al.,
1997).
Our studies show that ferulic acid decreases the
oxidative stress caused during diabetes. This decrease
in oxidative stress correlates with the reduction in
levels of TBARS, hydroperoxides and FFA in liver.
The levels of GSH and activities of antioxidant enzymes
like GPx, SOD and CAT were elevated in liver and
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