Hepatoprotective role of ferulic acid-a dose dependent study

JOURNAL OF MEDICINAL FOODJ Med Food 7 (4) 2004, 456–461© Mary Ann Liebert, Inc. and Korean Society of Food Science and Nutrition

Hepatoprotective Role of Ferulic Acid: A Dose-Dependent Study

Rajagopalan Rukkumani, Kode Aruna, Penumathsa Suresh Varma, and Venugopal Padmanabhan Menon

Department of Biochemistry, Faculty of Science, Annamalai University, Annamalainagar, Tamilnadu, India

ABSTRACT Alcohol use is contributing to an unprecedented decline in life expectancy. Damage to the liver after ethanoladministration is a well-known phenomenon. Free radical mechanisms have been proposed to play a part in ethanol-inducedliver toxicity. Ingestion of diets rich in polyunsaturated fatty acids (PUFAs) along with alcohol is known to result in enhancedliver damage. The present work is aimed at evaluating the protective role of ferulic acid, a naturally occurring plant compo-nent, on alcohol- and PUFA-induced liver toxicity. Three different doses of ferulic acid (10, 20, and 40 mg/kg of body weight)were administered to rats given alcohol, heated PUFA (�PUFA), and alcohol � �PUFA. Influence of ferulic acid on alco-hol- and PUFA-induced liver damage was evaluated by analyzing the activities of the liver marker enzymes alkaline phos-phatase, �-glutamyl transferase, alanine transaminase, and aspartate transaminase. The activities of these liver marker enzymeswere increased in the alcohol, �PUFA, and alcohol � �PUFA groups but were decreased significantly on treatment with fer-ulic acid. The low dose (10 mg/kg of body weight) was not effective, but both 20 mg and 40 mg/kg of body weight werefound to be effective. The 20 mg/kg of body weight dose was found to be more effective than 40 mg/kg of body weight (thehigh dose). The administration of ferulic acid to normal rats did not produce any harmful effects. Thus our results show thatferulic acid is an effective anti-hepatotoxic agent without side effects and may be a good candidate in the current search fora natural hepatoprotective agent.

KEY WORDS: • alcohol • ferulic acid • hepatotoxicity • liver marker enzymes • polyunsaturated fatty acids

INTRODUCTION

ALCOHOL’S CONTRIBUTION to the global burden of diseaseis significant and growing. Chronic alcohol consump-

tion is associated with an increased risk of cirrhosis, livercancer, and premature death.1 The liver is the major targetorgan for the toxic effects of ethanol in the human body be-cause of the extraordinary metabolic machinery containedin hepatocytes. The hepatotoxicity of alcohol results fromthe oxidative metabolism, which involves alcohol dehydro-genase-mediated excessive generation of nicotinamide adenine dinucleotide and acetaldehyde, the main oxidativemetabolite of ethanol.2

Alcohol, which is itself a direct hepatotoxin,3,4 is alsoknown to enhance the acute toxicity, when given along witha fat-rich diet. Fat is an important dietary component, whichaffects both growth and health. It is widely accepted that ahigh fat diet is detrimental to health. Replacing the tradi-tional cooking fats, considered to be atherogenic, with re-fined vegetable oils, promoted as “heart friendly” becauseof their polyunsaturated fatty acid (PUFA) content, has re-

sulted in increased prevalence of heart disease in India. Cur-rent data on dietary fats indicate that it is not just the pres-ence of PUFAs but the type of PUFA that is important. Ahigh PUFA n-6 content and a high n-6/n-3 ratio in dietaryfats is considered to be dangerous.5 The newer heart-friendlyoils like sunflower oil possess this undesirable PUFA con-tent, and thus excess intake of these vegetable oils is actu-ally detrimental to health.

Alcoholics usually, after a heavy binge of alcohol, takefried food items normally made up of PUFAs. Previous re-ports found that the intake of PUFAs along with alcohol aggravates hepatotoxicity, especially when the PUFA isheated.6,7

We have probably all been told at some point to “eat ourvegetables” because they are good for health. There is nowmuch work to suggest that intake of vegetables is benefi-cial. Fresh fruits and vegetables are good sources of an-tioxidants, and it is these antioxidants that are now thoughtto help in the prevention of illness.

Ferulic acid (FA) is a phytochemical commonly found infruits and vegetables such as tomatoes, sweet corn, rice bran,etc.8 It is the predominant bound phenolic acid in sweet corn,which constitutes about 78% of the total phenolic acids. FAis a strong membrane antioxidant and is believed to protectagainst cancer, colds, influenza, skin aging, and musclewasting. The health benefits of FA are now receiving muchattention in the research world, but its hepatoprotective ac-

Manuscript received 6 November 2003. Revision accepted 17 February 2004.

Address reprint requests to: Dr. Venugopal P. Menon, Department of Biochemistry, Fac-ulty of Science, Annamalai University, Annamalainagar-608 002, Tamil Nadu, India, E-mail: [email protected]

456

HEPATOPROTECTIVE ROLE OF FERULIC ACID 457

tion has not yet been entirely proven. So the present workconcentrates on the influence of FA in alcohol- and heatedPUFA (�PUFA)-induced liver toxicity.

MATERIALS AND METHODS

Male Albino rats, Wistar strain, weighing 140–160 g andbred in the Central Animal House, Rajah Muthiah MedicalCollege, Chidambaram, Tamilnadu, India, fed on pellet diet(Agro Corp. Private Ltd., Bangalore, India) were used forthe study. Water was given ad libitum. The standard pelletdiet comprised 21% protein, 5% lipids, 4% crude fiber, 8%ash, 1% calcium, 0.6% phosphorus, 3.4% glucose, 2% vit-amin, and 55% nitrogen-free extract (carbohydrates). It pro-vides metabolizable energy of 3,600 kcal.

The animals were housed in plastic cages under controlledconditions of 12-hour light/dark cycle, 50% humidity, and30 � 2°C. The animals used in the present study were main-tained in accordance with the guidelines of the National In-stitute of Nutrition, Indian Council of Medical Research, Hyderabad, India and approved by the Animal Ethical Com-mittee of Annamalai University.

Chemicals

Absolute ethanol was obtained from Hayman Ltd.(Witham, UK). To obtain �PUFA, sunflower oil was heatedat 180°C for 30 minutes, twice. FA was obtained from SigmaAldrich Private Ltd. (Bangalore).

Experimental design

The animals were divided into 16 groups of six ratseach: Group 1 (Control), control rats; Group 2 (Alcohol),

rats given 20% ethanol (7.9 g/kg of body weight)9 orallyusing an intragastric tube; Group 3 (�PUFA), rats givenhigh fat diet (15% heated sunflower oil) mixed with thediet; Group 4 (Alcohol � �PUFA), rats given 20% ethanol� 15% heated sunflower oil; Group 5 [Alcohol � FA (1)],rats given 20% ethanol � FA (10 mg/kg of body weight)dissolved in ethanol; Group 6 [Alcohol � FA (2)], ratsgiven 20% ethanol � FA (40 mg/kg of body weight);Group 7 [Alcohol � FA (3)], rats given 20% ethanol �FA (20 mg/kg of body weight); Group 8 [�PUFA � FA(1)], rats given 15% heated sunflower oil � FA (10 mg/kgof body weight) dissolved in distilled water; Group 9 [�P-UFA � FA (2)], given 15% heated sunflower oil � FA(40 mg/kg of body weight); Group 10 [�PUFA � FA (3)],rats given 15% heated sunflower oil � FA (20 mg/kg bodyof weight); Group 11 [Alcohol � �PUFA � FA (1)], ratsgiven 20% ethanol � 15% heated sunflower oil � FA (10mg/kg of body weight] dissolved in ethanol; Group 12[Alcohol � �PUFA � FA (2)], rats given 20% ethanol� 15% heated sunflower oil � FA (40 mg/kg of bodyweight); Group 13 [Alcohol � �PUFA � FA (3)], ratsgiven 20% ethanol � 15% heated sunflower oil � FA (20mg/kg of body weight); Group 14 [FA (1); low dose], ratsgiven FA (10 mg/kg of body weight) dissolved in distilledwater orally using an intragastric tube; Group 15 [FA (2);high dose], rats given FA (40 mg/kg of body weight) dis-solved in distilled water orally using an intragastric tube;and Group 16 [FA (3); medium dose], rats given FA (20mg/kg of body weight) dissolved in distilled water orallyusing an intragastric tube.

At the end of the experimental period of 45 days, the ratswere sacrificed, and the blood was collected for various bio-chemical analyses.

TABLE 1. ACTIVITIES OF GGT IN PLASMA

Study number Group GGT (IU/L)

1 Normal 0.525 � 0.04a,n,o,p

2 Alcohol 2.042 � 0.17b,c,l

3 �PUFA 2.125 � 0.24c,b

4 Alcohol � �PUFA 2.708 � 0.17d

5 Alcohol � FA (1) 1.875 � 0.08e,l,m

6 Alcohol � FA (2) 1.200 � 0.13f,i

7 Alcohol � FA (3) 0.980 � 0.09g,i,j

8 �PUFA � FA (1) 1.708 � 0.08h,m

9 �PUFA � FA (2) 1.083 � 0.13i,f,g

10 �PUFA � FA (3) 0.845 � 0.07j,g

11 Alcohol � �PUFA � FA (1) 2.450 � 0.12k,b,c

12 Alcohol � �PUFA � FA (2) 1.942 � 0.19l,b,e

13 Alcohol � �PUFA � FA (3) 1.740 � 0.18m,e,h

14 FA (1) 0.553 � 0.02n,a,o,p

15 FA (2) 0.537 � 0.06o,a,h,p

16 FA (3) 0.542 � 0.04p,a,n,o

Data are mean � SD values from six rats in each group.Differences were analyzed by analysis of variance followed by Duncan’s multiple

range test. Values not sharing a common superscript differ significantly at P � .05.

Biochemical parameters

To assess the membrane protective role of FA, the activ-ities of the following liver marker enzymes were estimated:alkaline phosphatase (ALP) by the method of King and Arm-strong,10 �-glutamyl transferase (GGT) by the fixed timemethod of Orlowski and Meister,11 alanine transaminase(ALT) by the method of Reitman and Frankel12 using thereagent kit, and aspartate transaminase (AST) by the methodof Reitman and Frankel.12

RESULTS

The activities of the liver marker enzymes GGT (Table1), ALP (Table 2), ALT (Table 3), and AST (Table 4) weresignificantly increased in the Alcohol, �PUFA, and Alco-hol � �PUFA groups. On treatment with FA (medium doseand high dose) there was a significant reduction in their ac-tivities. The decrease was more significant with FA (mediumdose) than with FA (high dose). Treatment with FA (lowdose) did not produce any significant effect.

458 RUKKUMANI ET AL.

TABLE 2. ACTIVITIES OF ALP IN PLASMA

Study number Group ALP (IU/L)

1 Normal 76.000 � 8.27a,n,o,p

2 Alcohol 177.667 � 16.13b,c

3 �PUFA 173.500 � 12.49c,b,l

4 Alcohol � �PUFA 242.00 � 20.97d

5 Alcohol � FA (1) 157.000 � 8.89e,h,l,m

6 Alcohol � FA (2) 121.667 � 10.46f,i

7 Alcohol � FA (3) 103.667 � 10.31g,j

8 �PUFA � FA (1) 150.667 � 7.84h,e,l,m

9 �PUFA � FA (2) 117.000 � 11.21i,f

10 �PUFA � FA (3) 93.833 � 7.03j,g,n,o,p

11 Alcohol � �PUFA � FA (1) 218.833 � 11.92k

12 Alcohol � �PUFA � FA (2) 163.000 � 14.85l,c,e,h,m

13 Alcohol � �PUFA � FA (3) 152.667 � 13.43m,e,h,l

14 FA (1) 83.500 � 2.43n,a,j,o,p

15 FA (2) 82.333 � 7.31o,a,j,n,p

16 FA (3) 83.667 � 8.64p,a,j,n,o



TABLE 3. ACTIVITIES OF ALT IN PLASMA

Study number Group ALT (IU/L)

1 Normal 28.667 � 2.16a,n,o,p

2 Alcohol 89.167 � 3.60b

3 �PUFA 80.500 � 4.04c,e,l

4 Alcohol � �PUFA 110.167 � 4.07d

5 Alcohol � FA (1) 82.000 � 4.29e,c,l

6 Alcohol � FA (2) 62.833 � 2.86f,m

7 Alcohol � FA (3) 48.000 � 3.16g

8 �PUFA � FA (1) 75.167 � 2.86h,l

9 �PUFA � FA (2) 60.167 � 2.48i,f,m

10 �PUFA � FA (3) 43.500 � 2.43j

11 Alcohol � �PUFA � FA (1) 100.333 � 3.88k

12 Alcohol � �PUFA � FA (2) 78.667 � 4.32l,c,e,h

13 Alcohol � �PUFA � FA (3) 59.000 � 3.03m,f,i

14 FA (1) 30.333 � 2.73n,a,o,p

15 FA (2) 29.000 � 3.35o,a,n,p

16 FA (3) 28.833 � 2.86p,a,n,o



DISCUSSION

The activities of the liver marker enzymes were increasedin the Alcohol, �PUFA, and Alcohol � �PUFA groups. In-creased ingestion of alcohol increases its metabolism via thenicotinamide adenine dinucleotide phosphate-dependentCYP2E1 oxidizing pathway, which results in the excessivegeneration of reactive oxygen species. These reactive oxy-gen species are able to induce several damaging events inliver, including the peroxidation of cell membrane phos-pholipids. This plays a pivotal role in the pathogenesis of alcoholic liver injury.13 Ethanol induces microsomalCYP2E1 up to 10-fold normal levels in humans and in ratmodels of chronic alcohol administration. This leads to thelipid peroxidation phenomenon within the liver and the for-mation of protein adducts with reactive aldehyde end prod-ucts of lipid peroxidation.14 Thus CYP2E1 plays a key rolein the generation of lipid peroxidative products duringethanol ingestion and causes membrane damage.

Edible oils, which are polyunsaturated, are prone to lipidperoxidation and can cause damage to cellular structure.15

It has been demonstrated that the fatty acid composition ofthe membranes can be affected by dietary variation of sat-urated and unsaturated fat.16 Increased intake of PUFAs in-creases the degree of unsaturation of the membrane andmakes them more susceptible to lipid peroxidation.17 More-over, wide utilization of fats, which are highly susceptibleto oxidation during cooking and frying, may alter physio-logical effects of their PUFA content and generate lipid per-oxides that cause membrane damage and increase lipid in-filtration.18 Thus, the increased ingestion of �PUFA resultsin increased membrane damage.

Reports have shown that intake of �PUFA along with al-

cohol results in a severalfold increase in the induction ofCYP2E1.19 Thus the combined ingestion of alcohol and �PUFA results in increased generation of free radicals andenhanced liver damage.

Because of the damage produced by alcohol, �PUFA, andalcohol � �PUFA, many important cell functions such astransport processes are affected.20 Because of severe dam-age, the hepatocyte membranes become leaky, and livermarker enzymes such as GGT, ALP, ALT, and AST are re-leased into the circulation. Thus their activities increase inthe plasma during these conditions.


TABLE 4. ACTIVITIES OF AST IN PLASMA

Study number Group AST (IU/L)

1 Normal 75.000 � 4.94a,j,n,o,p

2 Alcohol 135.500 � 13.11b,c,e,k,l

3 �PUFA 135.333 � 13.65c,b,e,k,l

4 Alcohol � �PUFA 157.833 � 14.33d

5 Alcohol � FA (1) 125.000 � 5.48e,b,c,h,l

6 Alcohol � FA (2) 110.833 � 11.51f,h,i,m

7 Alcohol � FA (3) 87.333 � 5.72g,j

8 �PUFA � FA (1) 115.333 � 4.80h,f,m

9 �PUFA � FA (2) 103.000 � 7.87i,f

10 �PUFA � FA (3) 83.000 � 6.07j,a,n,o,p

11 Alcohol � �PUFA � FA (1) 142.000 � 6.16k,b,c,d,l

12 Alcohol � �PUFA � FA (2) 135.833 � 12.59l,b,c,d,e,k

13 Alcohol � �PUFA � FA (3) 114.000 � 11.15m,f

14 FA (1) 72.833 � 5.91n,a,j,o,p

15 FA (2) 75.500 � 4.14o,a,j,n,p

16 FA (3) 74.000 � 4.73p,a,j,n,o

Data are mean � SD values from six rats in each group.Differences were analyzed by analysis of variance followed by Duncan’s multiple range

test. Values not sharing a common superscript differ significantly at P � .05.

FIG. 1. Chemical structure of FA.

Treatment with FA decreased the damage to the liver.This may be because of the effective antioxidant sparing ac-tion of FA.

The use of antioxidants has been recognized as an im-portant countermeasure against conditions in which oxida-tive stress is implicated. Among the many classes of com-pounds, naturally occurring phenolics have been givenattention.21 Explicitly, FA has been shown to possess someactivity towards peroxynitrite22 and oxidized low-densitylipoprotein in vitro.23

Normally the phenolic compounds act by scavenging freeradicals24 and quenching the lipid peroxidative chain. Thehydroxy and phenoxy groups of phenolic compounds do-nate their electron to the free radicals and quench them. Inturn, the phenolic radical forms a quinone methide inter-mediate, which is stable and excreted via bile.25 Thus FAbeing a phenolic compound might have inhibited lipid per-oxidation in our study.

As seen in Figure 1, FA possesses distinct structural mo-tifs that can possibly contribute to the free radical scaveng-ing capability of this compound. The presence of electrondonating groups on the benzene ring (3-methoxy and moreimportantly 4-hydroxyl) of FA gives the additional propertyof terminating free radical chain reactions. The next func-tionality—the carboxylic acid group in FA with an adjacentunsaturated C–C double bond—can provide additional at-tack sites for free radicals and thus prevent them from at-tacking the membrane. In addition, this carboxylic acidgroup also acts as an anchor of FA by which it binds to thelipid bilayer, providing some protection against lipid per-oxidation. Clearly, the presence of electron-donating sub-stituents enhances the antioxidant properties of FA.26

FA effectively quenches the free radicals, preventingthem from attacking the membrane and protecting the mem-brane. Thus FA inhibits the leakage of liver markers intocirculation.

Although it has been recognized that the use of antioxi-dant is an important preventive method to minimize thepathological and toxic effects associated with oxidativestress, there are several key considerations that need to beaddressed in evaluating a potential antioxidant. The fore-most issue deals with the inherent toxicity of the target com-pound. If it exerts harmful effects on cells, its usefulness isnot justified. In our present study, all three doses of FA usedhere did not produce any damage to the system. The lowdose (10 mg/kg of body weight) was not effective, becauseits concentration might not have been enough to quench allfree radicals generated. The high dose (40 mg/kg of bodyweight) was not as effective as the medium dose (20 mg/kgof body weight), which suggests that an increased concen-tration may result in the formation of undesirable by-prod-ucts, which in turn might have interfered with the antioxi-dant capacity of FA.

The second property of an antioxidant is that it should beefficient and protective at low concentrations, preferably ca-pable of inhibiting the lipid peroxidation process, wheremost of the oxidative damage occurs. As presented here, FA

is proved to be a good antioxidant against alcohol- andPUFA-induced toxicity at a comparatively low dose (20mg/kg of body weight).

Thus from the results obtained in this work, we concludethat FA, a naturally occurring nutritional compound, is apromising hepatoprotective agent. Further considerationshould be given for making FA a target initial structure for de-veloping a potent drug for the treatment of hepatotoxicity.

ACKNOWLEDGMENTS

The scholarship awarded by the Jawaharlal Nehru Memo-rial Fund, New Delhi, India, for the above study is grate-fully acknowledged.

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Hepatoprotective role of ferulic acid-a dose dependent study