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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.
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 311
was purchased from Fluka Chemika (Steinheim, Superoxide dismutase (SOD) activity in the tissues
Switzerland). All other chemicals and reagents used was assayed (Kakkar et al., 1984) based on the inhibi-
were of analytical grade. tion of formation of NADH-phenazine methosulphate-
nitro blue tetrazolium complex. Catalase (CAT) activity
was assayed (Sinha, 1972) by quantifying the hydrogen
Animals peroxide after reacting with dichromate in acetic acid.
The activity of glutathione peroxidase (GPx) was as-
Experiments were performed on adult female rats of sayed by the method of Rotruck et al. (1973). A known
Wistar strain obtained from the Central Animal House, amount of enzyme preparation was allowed to react
Rajah Muthiah Medical College, body weight in the with hydrogen peroxide (H2O2) and glutathione (GSH)
range of 160–170 g. The rats were fed a standard for a specified time period. Then the GSH content re-
pellet diet (Karnataka State Agro Corporation (P) Ltd, maining after the reaction was measured (Ellman, 1959).
Agro Feeds Division, Bangalore, India) and were Total reduced glutathione content (GSH) was meas-
given water ad libitum. The animals used in the pre- ured by the method of Ellman. This method is based
sent study were cared as per the principles and guide- on the development of a yellow colour when 5′ 5′ dithio
lines of the Ethical Committee of Animal Care of bisnitro benzoic acid (DTNB) is added to compounds
Annamalai University in accordance with the Indian containing sulphydryl groups.
National Law on animal care and use.
Diabetes was induced by a single intraperitoneal Estimation of free fatty acids. Free fatty acids (Falholt
injection of streptozotocin (40 mg/kg in citrate buffer et al., 1973) were extracted with chloroform-heptane-
of pH 4.0). Blood glucose concentration and changes methanol mixture to eliminate interference from
in body weight were monitored regularly. Only those phospholipids and the extract was shaken with a high-
diabetic rats that exhibited a blood glucose concentra- density copper reagent at pH 8.1. The copper soap
tion ≥ 200 mg/dL were divided into four groups of eight remained in the upper organic layer. An aliquot from
rats each and included in the study along with a normal this was removed and the copper content determined
group. The different groups are: colorimetrically by treating with diphenyl carbazide.
Group 1 – Normal rats received 1 ml water by intra-
gastric intubation.
Histopathological studies
Group 2 – Diabetic control received 1 ml water.
Group 3 – Diabetic rats treated orally with a high dose
For hisotpathological study, the rats were perfused
of ferulic acid – 40 mg/kg (HD) in 1 ml water
with 10% formalin. Pancreas was removed and was
as suspension.
then embedded in paraffin, thinly sectioned using a
Group 4 – Diabetic rat treated orally with a low dose of
microtome, stained with Hematoxylin and Eosin (H &
ferulic acid – 10 mg/kg (LD), in 1 ml water
E) and mounted in neutral DPX medium and examined
as suspension.
using light microscope.
Group 5 – Diabetic rats treated orally with the refer-
ence drug glibenclamide – 0.6 mg/kg (GB)
in 1 ml water.
Statistical Analysis
After 45 days of treatment, the animals were fasted
overnight, anaesthetized with ketamine Hcl and sacri- The results were statistically analysed by one-way ana-
ficed. The blood was collected in heparinized tubes and lysis of variance (ANOVA) followed by least significant
liver tissue in ice-cold containers and used for various difference (LSD). The significance was set at p < 0.001.
biochemical estimations.
Estimation of blood glucose. Glucose was estimated Effect on blood glucose and body weight changes
in blood (Sasaki et al., 1972) using O-toluidine in glacial (Table 1)
acetic acid, which, when heated with glucose, pro-
duces a colored product. The aldehydes group of The changes in the levels of blood glucose and body
glucose apparently condenses with the reagent to form weight before and after treatment of diabetic rats are
glycosylamine and an schiff’s base, which gives the presented in Table 1. Treatment with ferulic acid was
colored product. found to decrease the blood glucose levels and increase
the body weight. The effect was more pronounced
Estimation of lipid peroxidation and antioxidants. The with the low dose than that of high dose of ferulic
concentration of thiobarbituric acid reactive substances acid and was comparable with that of reference drug
(TBARS) was estimated in the tissues by the method glibenclamide (p < 0.001).
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- Effect on FFA and lipid peroxidation in liver
ured. Hydroperoxides was estimated (Jiang et al., 1992) (Table 2)
using Fox reagent, based on the oxidation of ferrous
ion under the acidic condition in the presence of xylenol Free fatty acid, TBARS and hydroperoxides con-
orange. centration were found to be elevated in diabetic rats.
Copyright © 2004 John Wiley & Sons, Ltd. 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)
Table 2. Levels of TBARS, hydroperoxides and FFA in liver (values are mean ± SD, n = 6)
Table 3. Activities of SOD, CAT, GPx and levels of GSH in liver (values are mean ± SD, n = 6)
Copyright © 2004 John Wiley & Sons, Ltd. Phytother. Res. 18, 310–314 (2004)
FERULIC ACID ALLEVIATES LIPID PEROXIDATION IN DIABETIC RATS 313
Figure 1. Pancreas of normal rat shows islets of Langerhans: H Figure 2. Pancreas of STZ-induced diabetic rat, shows shrink-
& E × 20. age and fatty infiltration in islets: H & E × 20.
implicated in the glucose-fatty acid cycle, are reactive the effect was more pronounced with the low dose of
oxygen species and hydrogen peroxide. These sub- ferulic acid than the high dose. In this context it was
stances may cause damage to cellular structures and observed that ferulic acid and its derivatives like me-
impair glucose metabolism. thyl esters were found to increase the activity of SOD
Elevated free radical concentration and lipid in blood vessel injury during thrombosis (Kayahara
peroxidation decreases the antioxidant defense in et al., 1999).
biological systems. The important antioxidants are: Histopathological study shows that ferulic acid has
(a) GPx, which catalyses the removal of hydrogen the capacity to increase islet cells mass. However the
peroxide to non-toxic products by utilizing the re- expansion was better with the low dose than with the
duced glutathione, GSH (Amdur et al., 1991); (b) SOD, high dose. The increased β -cell mass would increase
which protects the tissues against oxygen free radicals, the secretion of insulin, which may increase the periph-
and converts these super oxides to hydrogen peroxide eral utilization of glucose (Vonner-Wier, 2000). Hence
and thereby prevents any damage to the membrane the observed antihyperglycemic activity is due to the
and biological system (Halliwell and Gutteridge, 1999) islets retuning to near-normal size and activity.
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 CONCLUSION
oxidative stress caused during diabetes. This decrease
in oxidative stress correlates with the reduction in The results presented here suggest that ferulic acid
levels of TBARS, hydroperoxides and FFA in liver. has both antihyperglycemic and antioxidant properties
The levels of GSH and activities of antioxidant enzymes and also reduces the intensity of diabetes and prevents
like GPx, SOD and CAT were elevated in liver and further complications.
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Copyright © 2004 John Wiley & Sons, Ltd. Phytother. Res. 18, 310–314 (2004)