Abstract Diabetes, when uncontrolled, causes dyslipidemiaoften followed by atherogenic abnormalities. The presentstudy was focused to determine whether ferulic acid (FA), aflavonoid, 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 wereelevated during diabetes. Treatment with FA significantlyreduced the elevated plasma lipid and blood glucose levels;a more pronounced effect was found with low-dose ferulicacid than with high dose. Thus, our study demonstrates thatferulic acid lowers the lipid levels in diabetic rats and henceprevents further complications.

Key words Diabetes • Hyperlipidemia • Ferulic acid •

Flavonoids

Introduction

Changes in human behavior and lifestyle over the last centuryhave resulted in a dramatic increase in the incidence of dia-betes. The past two decades have seen an explosive increase inthe number of people diagnosed with diabetes worldwide [1].Diabetes consists of a group of disorders involving distinctpathogenic mechanisms with hyperglycemia as the commondenominator, due to the impaired metabolism of glucose andother energy-yielding fuels such as lipids and proteins.

Diabetics are at an increased risk of developing chroniccomplications related to ophthalmic, renal, neurological, cere-brovascular, cardiovascular and peripheral vascular diseases.Consequently people with diabetes are more likely than thosewithout 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 inpathogenesis of other complications. Therefore prevention ofcomplications is a key issue because of the huge prematuremorbidity and mortality associated with the disease [3].

In the past decade, several major studies have focusedattention on the need for strict control of glycemia to preventand reduce the risk of both specific microvascular and the lessspecific macrovascular complications [4]. However, the con-tinuous use of synthetic drugs like sulphonlyureas andbiguanides is known to produce serious side effects [5].

There is a wealth of in vitro evidence for the powerfulantioxidant properties of flavonoid components of the diet.However, few studies have examined the in vitro antioxidantpotential 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 otherdietary flavonoids and monophenolics so far studied [7].

Ferulic acid has a protective effect in liver toxicityinduced by drugs and is used as an anti-inflammatory drugin Japanese oriental medicine [8]. Ferulic acid is also report-

Acta Diabetol (2003) 40:118–122DOI 10.1007/s00592-003-0099-6 © Springer-Verlag 2003

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

O R I G I N A L

M. Sri Balasubashini • R. Rukkumani • V.P. Menon (�)Department of BiochemistryFaculty of ScienceAnnamalai UniversityAnnamalai Nagar, 608 002 Tamil Nadu, India

M. Sri Balasubashini et al.: Ferulic acid and hyperlipidemia 119

ed to terminate free radical chain reactions and reduce therisk of coronary artery diseases [9]. Therefore, in our studywe focused on the effects of ferulic acid on lipid levels inexperimentally 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 strainobtained from the Central Animal House, Rajah Muthiah MedicalCollege, of body weight 160–170 g. The rats were fed a standardpellet diet (Karnataka State Agro, Agro Feeds Division, Bangalore,India) and were given water ad libitum. The animals were cared foraccording to the principles and guidelines of the EthicalCommittee of Animal Care of Annamalai University in accordancewith the Indian National Law on animal care and use.

Diabetes was induced by a single intraperitoneal injection ofstreptozotocin (40 mg/kg in citrate buffer, pH 4.0). Blood glucoseconcentration 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 eachand included in the study along with a normal group. The differentgroups 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 referencedrug 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 werefasted overnight, anesthetized with ketamine hydrochloride andsacrificed. Blood was collected in heparinized tubes, processed forplasma and used for various biochemical estimations.

Biochemical methods

Glucose was estimated using O-toluidine reagent by the method ofDubowski modified by Sasaki et al. [10]. Briefly, 0.2 ml blood wastreated with 1.8 ml 10% trichloroacetic acid (TCA) to precipitateprotein, which was removed by centrifugation. To 1.0 ml of thesupernatant liquid, 4 ml O-toluidine reagent was added. The tubeswere heated in a boiling water bath for 8 min. The color developedwas read at 640 nm.

Plasma lipids were extracted according to the method of Folchet al. [11]. Briefly, 0.2 ml plasma was mixed with 9.8 ml chloro-form:methanol mixture (2:1), shaken vigorously and allowed tostand for 30 min. After centrifugation, 4 ml lipid extract was addedto tubes containing 8.0 ml saturated saline solution. The tubes werestoppered, shaken vigorously, allowed to stand for 1 h and cen-trifuged. This yielded a two-phase system. The upper aqueouslayer was discarded and the chloroform layer containing the lipidwas 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 ferricchloride-acetic acid reagent was added. Then, 3.0 ml concentratedsulfuric acid was added and the absorbance was read after 20 minat 560 nm against a reagent blank.

Phospholipids were estimated by the method of Zilversmit andDavis [13]. An aliquot of the lipid extract was pipetted into aKjeldahl flask and evaporated to dryness. Then, 1.0 ml 5 N H2SO4

was added and the sample waas digested in a digestion rack untilthe appearance of light brown color; 2–3 drops of concentratednitric acid were added and the digestion was continued until itbecame colorless. The Kjeldahl flask was cooled, 1.0 ml water wasadded and the flask was heated in a boiling water bath for about 5min. Then, 1.0 ml 2.5% ammonium molybdate and 0.1 ml ANSAwere added. The volume was then made up to 5 ml with distilledwater 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 2.5ml copper reagent were added. All the tubes were shaken vigorouslyfor 90 s and were kept aside for 15 min, centrifuged and 3.0 ml of theupper layer was transferred to another tube containing 0.5 ml diphenyl-carbazide solution and mixed carefully. The absorbance was read at550 nm after 15 min; 1.0 ml phosphate buffer was treated as blank.

Triglycerides were estimated by the method of Foster andDunn [15]. To an aliquot of dried lipid extract, 4 ml isopropanolwas added and mixed well and then 400 mg alumina was added.The tubes were placed in a mechanical rotor for 15 min and thencentrifuged. 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 0.5ml acetylacetone reagent were added. The samples were mixed,stoppered and incubated at 50° C for 30 min. The tubes were thencooled 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). Thesignificance was set at p<0.001. SPSS tools (7.5 version) wereused for the statistical analysis.

Results

A drastic increase was found in the levels of blood glucoseof diabetic rats (Table 1). Low-dose ferulic acid decreasedblood glucose in a way similar to that of the reference drug,

120 M. Sri Balasubashini et al.: Ferulic acid and hyperlipidemia

glibenclamide (p<0.001). Although the treatment with high-dose ferulic acid significantly decreased (p<0.001) the bloodglucose level, the decrease was not comparable to that of thereference 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 acidresulted in a significant decrease in the levels of both TGand 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 phospholipidslevels 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 withhigh-dose ferulic acid.

Discussion

In this study, rats treated with streptozotocin (STZ) showedelevated levels of blood glucose when compared to normalrats. The elevation was due to the action of STZ, which cre-ates an oxidative stress on the pancreas, producing single-

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

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 ratsFA, ferulic acid

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

M. Sri Balasubashini et al.: Ferulic acid and hyperlipidemia 121

strand breaks in pancreatic islet DNA. This ultimately leadsto the impaired secretion of insulin, which paves the way forthe decreased utilization of glucose by the tissues [16].

Apart from the regulation of carbohydrate metabolism,insulin also plays an important role in the metabolism oflipids. Insulin is a potent inhibitor of lipolysis, since itinhibits the activity of the hormone-sensitive lipases in adi-pose tissue and suppresses the release of FFA [17]. Duringdiabetes, 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-termdiabetes may also decrease cholesterol degradation andplays a chief role in the genesis of atherosclerosis of thevital arteries [19].

Hypertriglyceridemia is due to the defective removal ofTG, decreased lipoprotein lipase activity and overproduc-tion of TG during diabetes [20]. Under normal conditions,insulin activates lipoprotein lipase (LPL) which hydrolyzesTG [21]. Elevation in the levels of TG is also due to theenhanced reesterification of FFA, released in circulation.This leads to an increased risk of ischemic heart diseases.Enhanced lipolysis during diabetes also increases the releaseof 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 antioxidantproperties [9], helps to neutralize the free radicals producedby STZ in the pancreas and thereby decreases the toxicity ofSTZ. This may help the pancreatic beta cells to proliferateand secrete more insulin. The increased insulin secretion cancause increased utilization of glucose by extrahepatic tissuesand 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 levelsof FFA, TG, cholesterol and phospholipids in plasma. In ourstudy hypolipidemic effect of low-dose ferulic acid wasfound to be better than that of the high dose. The exactmechanism by which FA lowers lipid levels is not known,however, studies have shown that γ-oryzanol, a mixture ofFA can decrease cholesterol level in blood and lower theincidence of coronary heart disease [23]. FA is a potentantioxidant and prevents LDL oxidation induced by copperions, hence it facilitates the uptake and degradation of cho-lesterol by the liver [24]. In this context, FA was also found tobe 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

increased HDL formation, which transports the excesscholesterol from extrahepatic tissues to liver, where it iscatabolized [27]. Curcumin also decreases the absorption ofcholesterol [28]. One reports suggested that curcuminincreases the activity of 7α-hydroxylase, the main enzymeinvolved in the conversion of cholesterol to bile acid andthus facilitates biliary cholesterol excretion [29]. Photo-irra-diated curcumin more effectively reduced lipid levels inalcohol- and PUFA-induced hyperlipidemia than that of cur-cumin; the suggested mechanism of action was due to thepotent antioxidants vanillin and ferulic acid present in thephoto-irradiated curcumin [30].

Thus, our study suggests that ferulic acid has antihyper-lipidemic effect and normalizes the dyslipidemia causedduring diabetes by an unknown mechanism. Even at highdose, FA did not produce any harmful effects such as gas-trointestinal disturbances or hypoglycemia, thus provingitself to be a safer drug for the treatment of diabetes. Henceferulic acid protects the tissues and organs from atherogen-esis and further complications.

References

1. Zimmet P, Alberti KGMM, Shaw J (2002) Global and societalimplications of the diabetes epidemic. Nature 414:782–787

2. Hirany S, O’Byrne D, Devaraj S, Jialal I (2000) Remnant-like particle-cholesterol concentrations in patients with type 2diabetes mellitus and end-stage renal disease. Clin Chem46(5):667–672

3. Zimmet P (2000) Globalization, coca-colonization and thechronic disease epidemic: can the doomsday scenario beaverted? J Intern Med 247:301–310

4. Zimmet P (1999) Diabetes epidemology as a trigger to dia-betes research. Diabetologia 42:499–518

5. Moller DE (2001) New drug targets for type 2 diabetes andthe metabolic syndrome. Nature 414:821–826

6. Bourne LC, Rice-Evans C (1998) Bioavailability of ferulicacid. Biochem Biophys Res Commun 253(2):222–227

7. Graf E (2000) Antioxidant potential of ferulic acid. FreeRadic Biol Med 28(8):1249–1256

8. Wu DF, Peng RX, Wang H (1995) Sodium ferulate alleviatesprednisolone induced liver toxicity in mice. Yao HsuehHsueh Pao 30(11):801–805

9. Bourne LC, Panganga G, Baxter D, Hughers P, Rice Evans C(2000) Absorption of ferulic acid from low alcohol beer. FreeRadic Res 32(3):273–280

10. Sasaki T, Matsui S, Sonae A (1972) Effect of acetic acid con-centration on the colour reaction in the O-toluidine-boric acidmethod for blood glucose estimation. Rinshokagaku1:346–353

11. Folch J, Lee M, Sloane-Stanley GH (1957) A simple methodfor isolation and purification of total lipids from animal tis-sues. J Biol Chem 226:497–509

12. Zlatkis A, Zak B, Boyle AJ (1953) A new method for thedirect determination of serum cholesterol. J Lab Clin Med45:486

122 M. Sri Balasubashini et al.: Ferulic acid and hyperlipidemia

13. Zilversmit DB, Davis AK (1950) Microdetermination of plas-ma phospholipids by trichloroacetic acid precipitation. J LabClin Invest 35:155

14. Falholt K, Falholt W, Lund B (1973) An easy calorimetricmethod for routine determination of free fatty acids in plas-ma. Clin Chem Acta 46:105

15. Foster CS, Dunn O (1973) Stable reagents for determinationof serum triglyceride by a colorimetric Hantzsch condensa-tion method. Clin Chem 19:338–340

16. Omamoto H, Uchigata Y, Hiroskitckan (1981) STZ and allox-an induces DNA strand breaks and poly(ADP ribose) syn-thatase in pancreatic islets. Nature 294(19):284–286

17. Loci AS, Shahba M, Khazraji AL, Husni A, Twaija A (1994)Hypoglycemic effect of a valuable extract of Artemicisia herbalba II. Effect of a valuable extract on some blood parametersin diabetic animals. J Ethnopharmacol 43:167–171

18. Agardh CD, Bjorgell P, Nilsson EP (1999) The effect oftolbutamide on lipoproteins, lipoprotein lipase and hormonesensitive lipase Diabetes Res Clin Pract 46(2):99–108

19. Durrington PN (1989) Secondary hyperlipidaemia. In:Hyperlipidaemia diagnosis and management. Butterworth,UK, pp 219–276

20. Ganda OMP (1985) Pathogenesis of macrovascular diseaseincluding the influence of lipids. In: Marble A, Krall LP,Bradley RF, Christlieb AR, Soeldner JS (eds) Joslin’s dia-betes mellitus, 12th edn. Lea and Feiber Warghese, Bombay(Indian edn), pp 217–250

21. Taskinen MR, Beltz WF, Harper I (1986) Effect of non-insulin dependent diabetes mellitus on VLDL, triglycerides

and apolipoprotein B metabolism. Studies before and aftersulfonylurea therapy. Diabetes 35:1268–1277

22. Khosla P, Gupta D, Nagpal RK (1995) Effect of Trigonellafoenum graecum (fenugreek) on serum lipids in normal anddiabetic rats. Ind J Pharmacol 27:89–93

23. Scavariello EM, Ardlano DB (1998) g-Oryzanol: an impor-tant component in rice bran oil. Arch Latinoam Nutr 48:7–12

24. Bourne LC, Rice ECA (1997) The effect of the phenolicantioxidant ferulic acid on the oxidation of low-densitylipoprotein depends on the pro-oxidant used. Free Radic Res27(3):337–344

25. Chen KJ, Chen K (1992) Ischemic stroke treated withLigusticum chuanxiong. Chin Med J 105(10):870–873

26. Dahl TA, Bilski P, Reszka K, Chignell CF (1994)Photocytotoxicity of curcumin. Photochem Photobiol59:290–294

27. Soudamini KK, Unnikrishnan MC, Soni KB, Kuttan R(1992) Inhibition of lipid peroxidation and cholesterol levelsin mice by curcumin. Ind J Physiol Pharmacol 36:239–243

28. Rao DS, Chandrasekhara N, Sathyanarayana MN, SrinivasanM (1970) Effect of curcumin on serum and liver cholesterollevels in rat. J Nutr 100:1307–1315

29. Babu PS, Srinivasan K (1997) Hypolipidemic action of curcum-in, the active principle of turmeric (Curcuma longa) in strepto-zotocin induced diabetic rats. Mol Cell Biochem 166:169–175

30. Rukkumani R, Sri Balasubashini M, Vishwanathan P, MenonVP (2003) Comparative effects of curcumin and photo-irra-diated curcumin on alcohol- and polyunsaturated fatty acid-induced hyperlipidemia. Pharmacol Res (in press)