Ferulic acid alleviates lipid peroxidation in diabetic rats

310 M. SRI BALASUBASHINI ET AL.

Copyright © 2004 John Wiley & Sons, Ltd. Phytother. Res. 18, 310–314 (2004)

Copyright © 2004 John Wiley & Sons, Ltd.

Received 9 April 2002Accepted 23 July 2003

PHYTOTHERAPY RESEARCHPhytother. Res. 18, 310–314 (2004)Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/ptr.1440

Ferulic Acid Alleviates Lipid Peroxidationin Diabetic Rats

M. Sri Balasubashini1, R. Rukkumani1, P. Viswanathan2 and Venugopal P. Menon1*1Department of Biochemistry, Faculty of Science, Annamalai University, Annamalai Nagar-608 002, Tamil Nadu, India2Department 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 ofour study was to determine whether ferulic acid (FA), a phenolic acid, has any role to play in diabetes inducedfree radical formation. Diabetes was induced with streptozotocin. The levels of blood glucose, thiobarbituricacid reactive substances (TBARS), hydroperoxides and free fatty acids (FFA) increased in the liver of diabeticanimals. 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 thediabetic rats resulted in a decrease in the levels of glucose, TBARS, hydroperoxides, FFA and an increase inreduced glutathione (GSH). FA also resulted in increased activities of SOD, CAT, GPx and expansionof pancreatic islets. The effect was much pronounced with lower dose treatment. Thus our study showsthat administration of ferulic acid helps in enhancing the antioxidant capacity of these diabetic animals byneutralizing 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.

* Correspondence to: Dr V. P. Menon, Department of Biochemistry,Faculty of Science, Annamalai University, Annamalai Nagar-608 002,Tamil Nadu, India.E-mail: [email protected]

Since oxidative stress is associated with diabetes weused a natural antioxidant – ferulic acid, a phenolic acid,which finds wide distribution in the plant kingdom, andis more bioavailable than other dietary flavonoid andmonophenolics studied (Graf, 2000). Ferulic acid hasbeen proved to be a potent antioxidant, reported toterminate free radical chain reaction and reduces therisk for coronary heart diseases (Bourne et al., 2000).Ferulic acid also has a protective effect in liver toxicityinduced by drugs and is used as anti-inflammatory drugin Japanese oriental medicine (Wu et al., 1995).

Previous studies have shown that turmeric andcurcumin have antidiabetic properties (Arun and Nalini,2000). Turmeric is a spice, which is consumed in manyparts of our country. One of the active principles ofturmeric is curcumin. Curcumin is broken down intoferulic acid and vanillin. Since we had studied theeffect of curcumin under different diseased conditionssuch as liver toxicity (Rukkumani et al., 2002), coloncancer (Devasena et al., 2002) and diabetes, we thoughtthat ferulic acid, one of the components may play somerole in offering protection. This phenolic compound isfound not only in curcumin but also in other dietarysources like vegetables, fruits, cereals etc. (Bourne andRice-Evans, 1998). Hence in our study we have concen-trated on both the antihyperglycemic and antioxidanteffect of ferulic acid in diabetes.

RESEARCH DESIGN AND METHOD

Materials

Streptozotocin (STZ) was purchased from the Sigmachemical company; (St. Louis, MO, USA). Ferulic acid

INTRODUCTION

Diabetes is the most common endocrine disordercharacterized by hyperglycemia. Hyperglycemia in dia-betes may result from: (a) decreased entry of glucoseinto 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 imbalancebetween the levels of prooxidants and antioxidant inthe biological systems, leading to cellular injury (Villa-Caballero et al., 2000). According to Randle’s glucose-fatty acid cycle, increased lipolysis during diabetesand hence elevated FFA levels in tissues leads tohyperglycemia and also causes the production of freeradicals (Efrat, 2001).

A most successful strategy for preventing diabeticcomplication is intensive treatment to prevent hyper-glycemia and thereby oxidative stress. Current therapyfor diabetes includes a modification of lifestyle, such asdiet and exercise, and the use of a variety of pharmaco-logical agents. These agents target increased insulinsecretion, decreased hepatic glucose production andincreased sensitivity to insulin (Kelly and Mandarino,2000). Many of the synthetic drugs like sulphonylureasand biguanides can produce side effects includinghematological, cutaneous and gastrointestinal reactionsand disturbances in liver and kidney (Larner et al., 1985).

FERULIC ACID ALLEVIATES LIPID PEROXIDATION IN DIABETIC RATS 311


was purchased from Fluka Chemika (Steinheim,Switzerland). All other chemicals and reagents usedwere of analytical grade.

Animals

Experiments were performed on adult female rats ofWistar strain obtained from the Central Animal House,Rajah Muthiah Medical College, body weight in therange of 160–170 g. The rats were fed a standardpellet diet (Karnataka State Agro Corporation (P) Ltd,Agro Feeds Division, Bangalore, India) and weregiven 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 ofAnnamalai University in accordance with the IndianNational Law on animal care and use.

Diabetes was induced by a single intraperitonealinjection of streptozotocin (40 mg/kg in citrate bufferof pH 4.0). Blood glucose concentration and changesin body weight were monitored regularly. Only thosediabetic rats that exhibited a blood glucose concentra-tion ≥ 200 mg/dL were divided into four groups of eightrats each and included in the study along with a normalgroup. 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 wateras suspension.

Group 4 – Diabetic rat treated orally with a low dose offerulic acid – 10 mg/kg (LD), in 1 ml wateras 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 fastedovernight, anaesthetized with ketamine Hcl and sacri-ficed. The blood was collected in heparinized tubes andliver tissue in ice-cold containers and used for variousbiochemical estimations.

Biochemical estimations

Estimation of blood glucose. Glucose was estimatedin blood (Sasaki et al., 1972) using O-toluidine in glacialacetic acid, which, when heated with glucose, pro-duces a colored product. The aldehydes group ofglucose apparently condenses with the reagent to formglycosylamine and an schiff’s base, which gives thecolored product.

Estimation of lipid peroxidation and antioxidants. Theconcentration of thiobarbituric acid reactive substances(TBARS) was estimated in the tissues by the methodof Ohkawa et al. (1979). The pink coloured chromogenformed by the reaction of 2-thiobarbituric acid withbreakdown products of lipid peroxidation was meas-ured. Hydroperoxides was estimated (Jiang et al., 1992)using Fox reagent, based on the oxidation of ferrousion under the acidic condition in the presence of xylenolorange.

Superoxide dismutase (SOD) activity in the tissueswas assayed (Kakkar et al., 1984) based on the inhibi-tion of formation of NADH-phenazine methosulphate-nitro blue tetrazolium complex. Catalase (CAT) activitywas assayed (Sinha, 1972) by quantifying the hydrogenperoxide 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 knownamount of enzyme preparation was allowed to reactwith 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 basedon the development of a yellow colour when 5′ 5′ dithiobisnitro benzoic acid (DTNB) is added to compoundscontaining sulphydryl groups.

Estimation of free fatty acids. Free fatty acids (Falholtet al., 1973) were extracted with chloroform-heptane-methanol mixture to eliminate interference fromphospholipids and the extract was shaken with a high-density copper reagent at pH 8.1. The copper soapremained in the upper organic layer. An aliquot fromthis was removed and the copper content determinedcolorimetrically by treating with diphenyl carbazide.

Histopathological studies

For hisotpathological study, the rats were perfusedwith 10% formalin. Pancreas was removed and wasthen embedded in paraffin, thinly sectioned using amicrotome, stained with Hematoxylin and Eosin (H &E) and mounted in neutral DPX medium and examinedusing light microscope.

Statistical Analysis

The results were statistically analysed by one-way ana-lysis of variance (ANOVA) followed by least significantdifference (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 bodyweight before and after treatment of diabetic rats arepresented in Table 1. Treatment with ferulic acid wasfound to decrease the blood glucose levels and increasethe body weight. The effect was more pronouncedwith the low dose than that of high dose of ferulicacid and was comparable with that of reference drugglibenclamide (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.



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 ± 8.532. 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 FFAGroups (mM/100 g tissue) (mM/100 g tissue) (mM/100 g tissue)

1. Normal 0.79 ± 0.09 69.46 ± 5.16 755.83 ± 47.162. 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.

Effect on pancreas

Pancreas of STZ diabetic rats showed shrunken isletsand fat accumulation (Fig. 2), when compared to normal(Fig. 1). All treated group rats showed expansion ofislets (Figs 3, 4 and 5), when compared to the pancreasof diabetic rats. However the low dose of ferulic acidshowed the best with little fatty infiltration (Fig. 4).

DISCUSSION

Ferulic acid, a ubiquitous phenolic acid, with antioxidantproperty, reduced the effect produced by STZ-induceddiabetic 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 GSHGroups (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 ± 7.952. 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.

Treatment with ferulic acid (HD) resulted in significantdecrease in the levels of TBARS, hydroperoxides andFFA in liver (p < 0.001), whereas treatment with ferulicacid (LD) showed a better reduction than that the highdose of ferulic acid.

Effect on antioxidant status in liver (Table 3)

The changes in the levels of GSH, GPX, SOD, and CATin the liver are presented in Table 3. The antioxidantstatus was found to decrease in diabetic animals andwas improved significantly after ferulic acid treatment(p < 0.001). Treatment with ferulic acid (LD) was foundto be more effective in restoring the antioxidant statusthan those treated with ferulic acid (HD).

FERULIC ACID ALLEVIATES LIPID PEROXIDATION IN DIABETIC RATS 313


Figure 1. Pancreas of normal rat shows islets of Langerhans: H& E × 20.

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.

Figure 3. Pancreas of diabetic rat treated with ferulic acid (HD),shows expansion of islets along with fatty infiltration: H & E ×20.

Ferulic acid, which has been shown to have anti-oxidant properties (Graf, 2000), helps to neutralize thefree radicals produced by STZ in the pancreas andthereby decrease the toxicity of STZ. This decreasedoxidative stress/toxicity on the pancreas may help thebeta cells to proliferate and secrete more insulin, whichmay have been reduced due to STZ treatment. Thisincreased insulin secretion can cause increased utiliza-tion of glucose by the extra hepatic tissues and therebydecrease the blood glucose level. Dose dependentstudy shows that treatment with ferulic acid (LD) wasfound to decrease the blood glucose level better thanferulic acid (HD); which was similar to that of theglibenclamide group.

Experimental studies indicate that the oxidative stressis implicated in aging and pathogenesis of diabetic com-plications and long-term complications still representsthe main cause of morbidity and mortality (Mezzattiet al., 2000).

Hyperglycemia is a well-known cause for elevatedfree 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 adiposetissue for oxidation causes the production of meta-bolites that inhibit glucose utilization by the tissues.These metabolites of fatty acid oxidation, which are

showed a significant increase in blood glucose level andsignificant decrease in body weight compared to thenormal rats (Group 1). The elevation in glucose levelmay be due to an oxidative stress created on pancreasby STZ, producing single strand breaks in DNA of thepancreatic islets (Omamoto et al., 1981). Group 3 and 4rats exhibited a reduction in the level of blood glucoseand an increase in body weight, due to the antihyper-glycemic effect of ferulic acid.

Figure 5. Pancreas of diabetic rat treated with glibenclamideshows expansion of islets: H & E × 20.



implicated in the glucose-fatty acid cycle, are reactiveoxygen species and hydrogen peroxide. These sub-stances may cause damage to cellular structures andimpair glucose metabolism.

Elevated free radical concentration and lipidperoxidation decreases the antioxidant defense inbiological systems. The important antioxidants are:(a) GPx, which catalyses the removal of hydrogenperoxide 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 peroxideand thereby prevents any damage to the membraneand biological system (Halliwell and Gutteridge, 1999)and (c) Catalase is a major enzyme in detoxificationof hydrogen peroxide formed from SOD (Li et al.,1997).

Our studies show that ferulic acid decreases theoxidative stress caused during diabetes. This decreasein oxidative stress correlates with the reduction inlevels of TBARS, hydroperoxides and FFA in liver.The levels of GSH and activities of antioxidant enzymeslike GPx, SOD and CAT were elevated in liver and

the effect was more pronounced with the low dose offerulic acid than the high dose. In this context it wasobserved that ferulic acid and its derivatives like me-thyl esters were found to increase the activity of SODin blood vessel injury during thrombosis (Kayaharaet al., 1999).

Histopathological study shows that ferulic acid hasthe capacity to increase islet cells mass. However theexpansion was better with the low dose than with thehigh dose. The increased β-cell mass would increasethe secretion of insulin, which may increase the periph-eral utilization of glucose (Vonner-Wier, 2000). Hencethe observed antihyperglycemic activity is due to theislets retuning to near-normal size and activity.

CONCLUSION

The results presented here suggest that ferulic acidhas both antihyperglycemic and antioxidant propertiesand also reduces the intensity of diabetes and preventsfurther complications.

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Ferulic acid alleviates lipid peroxidation in diabetic rats