Acta Diabetol (2003) 40:118–122
DOI 10.1007/s00592-003-0099-6
ORIGINAL
M. Sri Balasubashini • R. Rukkumani • V.P. Menon
Protective effects of ferulic acid on hyperlipidemic diabetic rats
Received: 21 March 2002 / Accepted in revised form: 19 February 2003
Abstract Diabetes, when uncontrolled, causes dyslipidemia
often followed by atherogenic abnormalities. The present
study was focused to determine whether ferulic acid (FA), a
flavonoid, has any role to play in diabetes-induced dyslipi-
demia. Diabetes in rats was induced with streptozotocin.
The levels of blood glucose and plasma triglycerides (TG),
free fatty acids (FFA), cholesterol and phospholipids were
elevated during diabetes. Treatment with FA significantly
reduced the elevated plasma lipid and blood glucose levels;
a more pronounced effect was found with low-dose ferulic
acid than with high dose. Thus, our study demonstrates that
ferulic acid lowers the lipid levels in diabetic rats and hence
prevents further complications.
Key words Diabetes • Hyperlipidemia • Ferulic acid
Flavonoids
M. Sri Balasubashini • R. Rukkumani • V.P. Menon ()
Department of Biochemistry
Faculty of Science
Annamalai University
Annamalai Nagar, 608 002 Tamil Nadu, India
© Springer-Verlag 2003
Introduction
Changes in human behavior and lifestyle over the last century
have resulted in a dramatic increase in the incidence of dia-
betes. The past two decades have seen an explosive increase in
the number of people diagnosed with diabetes worldwide [1].
Diabetes consists of a group of disorders involving distinct
pathogenic mechanisms with hyperglycemia as the common
denominator, due to the impaired metabolism of glucose and
other energy-yielding fuels such as lipids and proteins.
Diabetics are at an increased risk of developing chronic
complications related to ophthalmic, renal, neurological, cere-
brovascular, cardiovascular and peripheral vascular diseases.
Consequently people with diabetes are more likely than those
• without the disease to have cardiac arrest, stroke, amputation,
kidney failure and blindness [2]. Typical dyslipidemia associ-
ated with diabetes is highly atherogenic and plays a key role in
pathogenesis of other complications. Therefore prevention of
complications is a key issue because of the huge premature
morbidity and mortality associated with the disease [3].
In the past decade, several major studies have focused
attention on the need for strict control of glycemia to prevent
and reduce the risk of both specific microvascular and the less
specific macrovascular complications [4]. However, the con-
tinuous use of synthetic drugs like sulphonlyureas and
biguanides is known to produce serious side effects [5].
There is a wealth of in vitro evidence for the powerful
antioxidant properties of flavonoid components of the diet.
However, few studies have examined the in vitro antioxidant
potential of hydroxycinnamates, major constituents of fruits
(e.g. orange), some vegetables (e.g. tomato, carrot), beverages
(e.g. beer) and grains (e.g. rice bran, wheat bran) [6]. Hence,
we tested the natural antioxidant ferulic acid (3-methoxy 4-
hydroxycinnamic acid), which is more bioavailable than other
dietary flavonoids and monophenolics so far studied [7].
Ferulic acid has a protective effect in liver toxicity
induced by drugs and is used as an anti-inflammatory drug
in Japanese oriental medicine [8]. Ferulic acid is also report-
M. Sri Balasubashini et al.: Ferulic acid and hyperlipidemia
ed to terminate free radical chain reactions and reduce the
risk of coronary artery diseases [9]. Therefore, in our study
we focused on the effects of ferulic acid on lipid levels in
experimentally induced diabetes.
Materials and methods
Streptozotocin (STZ) was purchased from the Sigma Chemical (St.
Louis, USA). Ferulic acid was purchased from Fluka Chemika,
Switzerland. All other chemicals and reagents used were of analyt-
ical grade.
Animals
Experiments were performed on adult female rats of Wistar strain
obtained from the Central Animal House, Rajah Muthiah Medical
College, of body weight 160–170 g. The rats were fed a standard
pellet diet (Karnataka State Agro, Agro Feeds Division, Bangalore,
India) and were given water ad libitum. The animals were cared for
according to the principles and guidelines 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, pH 4.0). Blood glucose
concentration and changes in body weight were monitored regu-
larly. Only those diabetic rats that exhibited a blood glucose con-
centration ≥200 mg/dl were divided into 4 groups of 8 rats each
and included in the study along with a normal group. The different
groups were treated as follows:
- Group 1. Normal rats received 1 ml water by intragastic intu-
bation.
- Group 2. Diabetic controls received 1 ml water.
- Group 3. Diabetic rats were treated orally with a low dose (LD)
of ferulic acid (10 mg/kg body weight, in 1 ml water as sus-
pension).
- Group 4. Diabetic rats were treated orally with a high dose
(HD) of ferulic acid (40 mg/kg in 1 ml water as suspension).
- Group 5. Diabetic rats were treated orally with the reference
drug glibenclamide (GB; 0.6 mg/kg in 1 ml water).
After 45 days of treatment, the smear from each animal was
examined and the diestrus phase was identified. The animals were
fasted overnight, anesthetized with ketamine hydrochloride and
sacrificed. Blood was collected in heparinized tubes, processed for
plasma and used for various biochemical estimations.
Biochemical methods
Glucose was estimated using O-toluidine reagent by the method of
Dubowski modified by Sasaki et al. [10]. Briefly, 0.2 ml blood was
treated with 1.8 ml 10% trichloroacetic acid (TCA) to precipitate
protein, which was removed by centrifugation. To 1.0 ml of the
supernatant liquid, 4 ml O-toluidine reagent was added. The tubes
were heated in a boiling water bath for 8 min. The color developed
was read at 640 nm.
119
Plasma lipids were extracted according to the method of Folch
et al. [11]. Briefly, 0.2 ml plasma was mixed with 9.8 ml chloro-
form:methanol mixture (2:1), shaken vigorously and allowed to
stand for 30 min. After centrifugation, 4 ml lipid extract was added
to tubes containing 8.0 ml saturated saline solution. The tubes were
stoppered, shaken vigorously, allowed to stand for 1 h and cen-
trifuged. This yielded a two-phase system. The upper aqueous
layer was discarded and the chloroform layer containing the lipid
was filtered into a dry tube.
Cholesterol was estimated by the method of Zlatkis et al. [12].
Briefly, 0.1 ml extract was evaporated to dryness and 5.0 ml ferric
chloride-acetic acid reagent was added. Then, 3.0 ml concentrated
sulfuric acid was added and the absorbance was read after 20 min
at 560 nm against a reagent blank.
Phospholipids were estimated by the method of Zilversmit and
Davis [13]. An aliquot of the lipid extract was pipetted into a
Kjeldahl flask and evaporated to dryness. Then, 1.0 ml 5 N H2SO4
was added and the sample waas digested in a digestion rack until
the appearance of light brown color; 2–3 drops of concentrated
nitric acid were added and the digestion was continued until it
became colorless. The Kjeldahl flask was cooled, 1.0 ml water was
added and the flask was heated in a boiling water bath for about 5
min. Then, 1.0 ml 2.5% ammonium molybdate and 0.1 ml ANSA
were added. The volume was then made up to 5 ml with distilled
water and the absorbance was measured at 660 nm within 10 min.
Free fatty acids were estimated by the method of Falholt et al. [14].
Briefly, 0.1 ml lipid extract or 0.1 ml plasma was evaporated to dry-
ness. Then, 1.0 ml phosphate buffer, 6.0 ml extraction solvent and 2.5
ml copper reagent were added. All the tubes were shaken vigorously
for 90 s and were kept aside for 15 min, centrifuged and 3.0 ml of the
upper layer was transferred to another tube containing 0.5 ml diphenyl-
carbazide solution and mixed carefully. The absorbance was read at
550 nm after 15 min; 1.0 ml phosphate buffer was treated as blank.
Triglycerides were estimated by the method of Foster and
Dunn [15]. To an aliquot of dried lipid extract, 4 ml isopropanol
was added and mixed well and then 400 mg alumina was added.
The tubes were placed in a mechanical rotor for 15 min and then
centrifuged. To 2 ml supernatant fluid, 0.6 ml potassium hydrox-
ide was added, stoppered and incubated at 60°–70° C for 15 min.
The tubes were then cooled; 1 ml metaperiodate solution and 0.5
ml acetylacetone reagent were added. The samples were mixed,
stoppered and incubated at 50° C for 30 min. The tubes were then
cooled and read at 405 nm against a reagent blank.
Statistical methods
The results were statistically analyzed by one-way analysis of vari-
ance (ANOVA) followed by least significant difference (LSD). The
significance was set at p<0.001. SPSS tools (7.5 version) were
used for the statistical analysis.
Results
A drastic increase was found in the levels of blood glucose
of diabetic rats (Table 1). Low-dose ferulic acid decreased
blood glucose in a way similar to that of the reference drug,
120 M. Sri Balasubashini et al.: Ferulic acid and hyperlipidemia

Table 1 Blood glucose concentrations in normal and diabetic rats before and after treatment, by study group. Values are mean (SD) for 6
rats in each group

Blood glucose, mg/dl

Group Before After

Normal 81.71 1(2.54) 82.00 1(1.63)*

Diabetic
Control 248.33 (23.74) 281.66 1(18.04)*
Low-dose FA 236.50 (13.75) 113.83 1(4.30)*
High-dose FA 247.33 (13.54) 168.00 (14.81)*
Glibenclamide 253.42 (17.75) 115.00 1(5.57)*

ANOVA followed by LSD:  p<0.001 vs. normal rats; * p<0.001 vs. diabetic control rats
FA, ferulic acid

glibenclamide (p<0.001). Although the treatment with high-
dose ferulic acid significantly decreased (p<0.001) the blood
glucose level, the decrease was not comparable to that of the
reference drug.
Free fatty acid (FFA) and triglyceride (TG) concentra-
tions were elevated in plasma of diabetic rats when com-
pared to normal rats (Table 2). Treatment with ferulic acid
resulted in a significant decrease in the levels of both TG
and FFA in plasma (p<0.001). Treatment with low-dose fer-
ulic acid showed a better reduction than that with high dose.
Similar to FFA and TG, cholesterol and phospholipids
levels were also elevated in the diabetic rats and decreased
significantly on treatment with ferulic acid (p<0.001).
Treatment with low-dose ferulic acid was better than with
high-dose ferulic acid.
Discussion
In this study, rats treated with streptozotocin (STZ) showed
elevated levels of blood glucose when compared to normal
rats. The elevation was due to the action of STZ, which cre-
ates an oxidative stress on the pancreas, producing single-

Table 2 Plasma free fatty acid and triglyceride concentrations in normal (control) rats and diabetic rats after treatment, by study group.
Values are mean (SD) for 6 rats in each group

Group Free fatty acids, mg/dl Triglycerides, mg/dl

Normal 80.04 (5.45)* 86.08 (8.02)*

Diabetic
Control 150.00 (8.89)* 178.50 (9.07)*
Low-dose FA 96.50 (4.84)* 106.00 (7.04)*
High-dose FA 115.33 (7.55)* 136.50 (6.86)*
Glibenclamide 83.66 (4.17)* 92.16 (5.26)*

ANOVA followed by LSD: p<0.001 vs. normal rats; *p<0.001 vs. diabetic control rats
FA, ferulic acid

Table 3 Cholesterol and phospholipid concentrations in plasma of normal and diabetic rats, by study group. Values are mean (SD) for 6
rats in each group

Group Cholesterol, mg/dl Phospholipids, mg/dl

Normal 80.07 (5.76) 89.64 (4.87)

Diabetic
Control 129.77 (8.40) 142.18 (6.73)
Low-dose FA 93.18 (5.29)* 114.83 (3.97)*
High-dose FA 108.49 (5.62)* 123.00 (7.37)*
Glibenclamide 90.50 (4.03)* 106.66 (9.45)*

ANOVA followed by LSD:  p<0.001 vs. normal rats; * p<0.001 vs. diabetic control rats
FA, ferulic acid
M. Sri Balasubashini et al.: Ferulic acid and hyperlipidemia
strand breaks in pancreatic islet DNA. This ultimately leads
to the impaired secretion of insulin, which paves the way for
the decreased utilization of glucose by the tissues [16].
Apart from the regulation of carbohydrate metabolism,
insulin also plays an important role in the metabolism of
lipids. Insulin is a potent inhibitor of lipolysis, since it
inhibits the activity of the hormone-sensitive lipases in adi-
pose tissue and suppresses the release of FFA [17]. During
diabetes, enhanced activity of this enzyme increases lipoly-
sis and releases more FFA into the circulation [18].
Increased FFA concentration also increases the β-oxida-
tion of fatty acids, producing more acetyl-CoA and choles-
terol during diabetes. In normal conditions, insulin increas-
es the receptor-mediated removal of LDL-cholesterol;
reduction in insulin level during diabetes causes hypercho-
lesterolemia. Glycosylation of lipoproteins in long-term
diabetes may also decrease cholesterol degradation and
plays a chief role in the genesis of atherosclerosis of the
vital arteries [19].
Hypertriglyceridemia is due to the defective removal of
TG, decreased lipoprotein lipase activity and overproduc-
tion of TG during diabetes [20]. Under normal conditions,
insulin activates lipoprotein lipase (LPL) which hydrolyzes
TG [21]. Elevation in the levels of TG is also due to the
enhanced reesterification of FFA, released in circulation.
This leads to an increased risk of ischemic heart diseases.
Enhanced lipolysis during diabetes also increases the release
of glycerol and hence increases the synthesis of phospho-
lipids [22]. Thus, the levels of lipids were increased in dia-
betic rats.
Ferulic acid, which has been shown to have antioxidant
properties [9], helps to neutralize the free radicals produced
by STZ in the pancreas and thereby decreases the toxicity of
STZ. This may help the pancreatic beta cells to proliferate
and secrete more insulin. The increased insulin secretion can
cause increased utilization of glucose by extrahepatic tissues
and thereby decreases the blood glucose level. Our dose-
dependent study showed that treatment with low-dose fer-
ulic acid decreased the blood glucose level better than high-
dose ferulic acid.
Supplementation with ferulic acid decreased the levels
of FFA, TG, cholesterol and phospholipids in plasma. In our
study hypolipidemic effect of low-dose ferulic acid was
found to be better than that of the high dose. The exact
mechanism by which FA lowers lipid levels is not known,
however, studies have shown that γ-oryzanol, a mixture of
FA can decrease cholesterol level in blood and lower the
incidence of coronary heart disease [23]. FA is a potent
antioxidant and prevents LDL oxidation induced by copper
ions, hence it facilitates the uptake and degradation of cho-
lesterol by the liver [24]. In this context, FA was also found to
be effective in treating ischemic stroke in China [25].
Curcumin (active principle of turmeric) on photo-irra-
diation produces vanillin and ferulic acid [26].
Hypocholesterolemic effect of curcumin is due to the
121
increased HDL formation, which transports the excess
cholesterol from extrahepatic tissues to liver, where it is
catabolized [27]. Curcumin also decreases the absorption of
cholesterol [28]. One reports suggested that curcumin
increases the activity of 7α-hydroxylase, the main enzyme
involved in the conversion of cholesterol to bile acid and
thus facilitates biliary cholesterol excretion [29]. Photo-irra-
diated curcumin more effectively reduced lipid levels in
alcohol- and PUFA-induced hyperlipidemia than that of cur-
cumin; the suggested mechanism of action was due to the
potent antioxidants vanillin and ferulic acid present in the
photo-irradiated curcumin [30].
Thus, our study suggests that ferulic acid has antihyper-
lipidemic effect and normalizes the dyslipidemia caused
during diabetes by an unknown mechanism. Even at high
dose, FA did not produce any harmful effects such as gas-
trointestinal disturbances or hypoglycemia, thus proving
itself to be a safer drug for the treatment of diabetes. Hence
ferulic acid protects the tissues and organs from atherogen-
esis and further complications.
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