Protective effects of curcumin and photoirradiated curcumin on circulatory lipids and lipid peroxidation products in alcohol and polyunsaturated fatty acid-induced toxicity

8/8/2019 Protective effects of curcumin and photoirradiated curcumin on circulatory lipids and lipid peroxidation products in a

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EFFECTS OF CURCUMIN AND IC ON CIRCULATORY LIPIDS AND LPO 925

Copyright 2003 John Wiley & Sons, Ltd. Phytother. Res. 17, 925929 (2003)

Copyright 2003 John Wiley & Sons, Ltd.

Received 9 April 2002

Accepted 5 August 2002

PHYTOTHERAPY RESEARCHPhytother. Res.17, 925929 (2003)Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/ptr.1254

Protective Effects of Curcumin and Photo-Irradiated Curcumin on Circulatory Lipids andLipid Peroxidation Products in Alcohol and

Polyunsaturated Fatty Acid-induced Toxicity

R. Rukkumani, M. Sri Balasubashini and Venugopal P. MenonDepartment of Biochemistry, Faculty of Science, Annamalai University, Annamalainagar, Tamil Nadu, India

Alcohol is a neurotoxin associated with significant morbidity and mortality. Ethanol is found to induce a dose

dependent increase in lipid peroxidation (LPO). The elevation in lipid peroxidative products and the loss of

antioxidant defense potential are enhanced when alcohol is taken along with polyunsaturated fatty acid (PUFA)

or heated PUFA. The present study was undertaken to evaluate the effects of curcumin and photo-irradiated

curcumin on alcohol and PUFA induced LPO and lipid profiles in plasma. The levels of vitamin C and E were

decreased significantly in alcohol +++++ raw as well as heated PUFA groups. The treatment with curcumin and

photo-irradiated curcumin (IC) increased their levels significantly. The increase was more significant in the IC

group than the curcumin group. The levels of cholesterol, phospholipids (PL), triglycerides (TG), free fatty

acids (FFA), thiobarbituric acid reactive substances (TBARS) and hydroperoxides (HP) were increased signi-

ficantly in alcohol +++++ raw as well as heated PUFA groups and the treatment with curcumin and IC, brought back

the levels. But the IC reduced the levels more significantly than curcumin. Thus, our results indicate that IC

is a more potent antioxidant than curcumin. Copyright 2003 John Wiley & Sons, Ltd.

Keywords: Alcohol; PUFA; curcumin; photo-irradiated curcumin; lipid peroxidation; circulatory lipids.

INTRODUCTION

It is well-established that alcoholic patients and experi-mental animals exposed to ethanol display biochemicalsigns of oxidative damage. The primary mechanismsof oxidative damage include aldehyde derived proteinmodifications resulting from ethanol metabolism andlipid peroxidation. Fat is a dietary component and theactual lipid content, especially the fatty acid composi-tions of diets, are significant. In recent years there hasbeen an increased focus on replacing some of the indi-vidual fat intake with unsaturated fat. Current data in-dicates that the newer heart-friendly oils like sunfloweroil posses a high PUFA n-6 content and high n-6/n-3ratio, which are actually detrimental to health (Sircar

and Kansra, 1998). Moreover heating of edible fats isknown to alter their nutritional properties, especiallywhen fat is rich in PUFA. During deep fat frying manyvolatile and non-volatile products are produced, someof which are toxic, depending on the level of intake(Alexander, 1981). Thus, it is evident that the lipidperoxidation is more prominent when alcohol is takenalong with PUFA and heated PUFA.

Curcumin (Diferuloyl methane), the active ingredi-ent of turmeric (Curcuma longa), inhibits LPO in vitro

(Sharma, 1976). Moreover, curcumin also decreasesserum cholesterol levels in hyperlipidemic rats (Raoet al., 1970). When curcumin is irradiated, stable pho-toproducts like vanillin and ferulic acid (FA) are pro-duced (Dahl et al., 1994). In the present investigationthe efficiency of curcumin in inhibiting peroxidationof lipids under the influence of known free radicalinducing alcohol + PUFA as well as heated PUFA hasbeen studied and compared with the efficiency of irra-diated curcumin, whose antioxidant property is not wellestablished.

MATERIALS AND METHODS

Female Albino rats, Wistar strain of body weightranging 140150 g bred in the central Animal House,Rajah Muthiah Medical College, were fed on pellet diet(Agro Corporation Private Ltd, Bangalore, India) andwater, ad libitum. The standard pellet diet comprised21% protein, 5% lipids, 4% crude fibre, 8% ash, 1%calcium, 0.6% phosphorus, 3.4% glucose, 2% Vitaminsand 55% Carbohydrates and provides metabolisableenergy of 3600 Kcal/Kg. The animals were housed inplastic cages under controlled conditions of 12 h light/dark cycle, 50% humidity and at 30 3 C.

Materials Used.

Sunflower Oil: Sunflower oil marketed by Gold Win-ner was purchased from local market,Chidambaram, Tamil Nadu, India.

* Correspondence to: Dr V. P. Menon, Department of Biochemistry,

Faculty of Science, Annamalai University, Annamalai Nagar-608 002,Tamil Nadu, India. Tel: 0091 4144 38343 (0). Fax: 0091 4144 38343.E-mail: [email protected]


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926 R. RUKKUMANI ET AL.


Curcumin (C): Curcumin was obtained from centraldrug house private limited, Mumbai,India.

Heated PUFA: Sunflower oil was subjected to heatingat 180 C for 30 min, twice.

IrradiatedCurcumin (IC): Curcumin was subjected to photo-

oxidation by exposing curcumin tobright sunlight for 5 h continuously.

(All other chemicals and solvents used were of ana-lytical grade)

Animals and Treatment.Group 1: Control rats (Rats given standard pellet diet

and glucose solution isocalorific to ethanoland high fat diet).

Group 2: Rats given 20% ethanol (Rajakrishnan et al.,1997) and a high fat diet (15%) [Raw Sun-flower oil].

Group 3: Rats given 20% ethanol and a high fatdiet (15%) [Thermally oxidised sunflower

oil].Group 4: Rats given curcumin (80 mg Kg1 body

weight) (Rajakrishnan et al., 2000), dissolvedin 20% ethanol (7.9 g Kg1 body weight) anda high fat diet [15% raw sunflower oil].

Group 5: Rats given curcumin (80 mg Kg1 bodyweight) dissolved in 20% ethanol (7.9 g Kg1

body weight) and a high fat diet [15% heatedsunflower oil].

Group 6: Rats given photo-irradiated curcumin(80 mg Kg1 body weight) dissolved in 20%ethanol (7.9 g Kg1 body weight) and a highfat diet [15% heated sunflower oil].

At the end of the experimental period (45 days), therats were sacrificed by decapitation after an overnightfast. Blood was collected in heparinised tubes and(Table 1) plasma was separated for various analyses.

Biochemical Estimations. The activities of Alkalinephosphatase (ALP) (E.C.3.1.3.1) by p-nitro phenylphosphate (PNPP) method (King and Armstrong,1988) using a reagent kit and -glutamyl transferase bythe fixed time method of Orlowski and Meister (Fialaet al., 1972) and the levels of TBARS by Thiobarbituricassay method (Niehaus and Samuelson, 1968), lipid

hydroperoxides (Jiang et al., 1992), ascorbic acid (Roeand Kuether, 1943), -tocopherol (Baker et al., 1980),Plasma cholesterol (Allain et al., 1974) using a reagentkit, plasma triglycerides (TG) (Foster and Dunn, 1973),Phospholipids (Zilversmit and Davis, 1950) and Freefatty acids (Falholt et al., 1973) were analyzed.

Statistical Analysis. The data given in the tables are

average values standard deviation (SD). Data wereanalyzed statistically by analysis of variance (ANOVA)and groups were compared by least significant differ-ence (LSD).

RESULTS

In this study, the levels of liver markers (ALP, GGT)(Table 2), lipid peroxidative end products (TBARS,HP) (Table 3) and lipids (Cholesterol, PL, TG, FFA)(Table 4), were increased significantly in the plasma ofboth alcohol + raw as well as heated PUFA groups.The treatment with curcumin significantly decreased thelevels, but photo-irradiated curcumin treatment wasfound to be more effective than curcumin. Similarly,the antioxidant levels (Vitamin C and Vitamin E)(Table 3), were decreased significantly in alcohol + rawas well as heated PUFA groups, which improvedsignificantly on curcumin treatment. The improvementwas more significant in IC group compared to curcumin.

DISCUSSION

Chronic ethanol intoxication may be related to delete-rious changes that occur at the cellular level. The inter-action of ethanol with biological membranes altersmembrane fluidity, modulates fatty acid compositionand thus alters its function. Moreover, poly unsaturatededible oils are more prone to lipid peroxidation andfurther damages the cellular structures (Ibrahim et al.,1998) and makes the membrane leaky to liver markerswhen given along with alcohol. The lipid peroxidesgenerated during cooking and frying of oils can causemembrane damage and increase lipid infiltration(Jethmalani et al., 1989), so the hepatic injury caused

Table 1. Experimental design

Group

Normal (N)

Alcohol + raw PUFA (A + R)

Alcohol + Heated PUFA (A + H)

Alcohol + raw PUFA + curcumin

(A + R + C)

Alcohol + heated PUFA+ curcumin

(A + H + C)

Alcohol + heated PUFA +

photo-irradiated curcumin

(A + H + I.C.)

Average food intake/day: 15 g/150 g rat.

Total calories/day: 508 Kcal/150 g rat.

No. of rats

6

6

6

6

6

6

Treatment

Glucose

20% alcohol + high fat diet

20% alcohol + high fat diet

20% alcohol + high fat

diet + curcumin


diet + curcumin


diet + irradiated curcumin

Dose administered

36.2 g Kg1 body weight (b.w.)

7.9 g Kg1 b.w. ethanol + 15% fat (raw

sunflower oil)

7.9 g Kg1 b.w. ethanol + 15% fat (heated

sunflower oil)

7.9 g Kg1 b.w. ethanol + 15% fat (raw

sunflower oil) + 80 mg Kg1 b.w. curcumin


sunflower oil) + 80 mg Kg1 b.w. curcumin


sunflower oil) + 80 mg Kg1 b.w.

photo-irradiated curcumin


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Table 2. Activities of marker enzymes in plasma [Values are mean SD from 6 rats in each

group]

Groups GGT (IU/L) ALP (IU/L)

1. Normal 0.597 0.06 85.880 7.00

2. Alcohol + raw PUFA 1.658 0.16a 174.020 15.85a

3. Alcohol + heated PUFA 2.508 0.14a 239.560 22.14a

4. Alcohol+

raw PUFA+

curcumin 0.783

0.13

bd

119.780

10.21

ad

5. Alcohol + heated PUFA + curcumin 1.433 0.12ag 177.410 16.28ag

6. Alcohol + heated PUFA + 1.150 0.16agj 149.147 12.13agk

irradiated curcumin

F ratio 160.764 77.259

ANOVA followed by LSD

Groups 2,3,4,5,6 are compared with group 1; a =p 0.001; b =p 0.01; c =p 0.05;

Group 4 is compared with group 2; d =p 0.001; e =p 0.01; f =p 0.05;

Groups 5,6 are compared with group 3; g =p 0.001; h =p 0.01; i =p 0.05;

Group 6 is compared with group 5; j =p 0.001; k =p 0.01; l =p 0.05;n= no significance.

Table 4. Lipid levels in plasma [Values are mean SD from 6 rats in each group]

Cholesterol Triglycerides Phospholipids Free fatty acids

Groups mg/100 ml Plasma mg/100 ml Plasma mg/100 ml Plasma mg/100 ml Plasma

1. Normal 89.813 4.18 81.333 7.87 92.500 9.08 72.622 4.13

2. Alcohol + raw PUFA 165.747 12.86a 136.000 4.73a 165.000 10.75a 136.867 10.23a

3. Alcohol + heated PUFA 207.402 15.64a 170.667 4.13a 223.833 17.22a 172.190 9.48a

4. Alcohol + raw PUFA + curcumin 114.802 12.01bd 101.333 6.53ad 116.250 7.87bd 87.822 6.14cd

5. Alcohol + heated PUFA + curcumin 158.500 17.70ag 137.333 11.15ag 166.250 12.92ag 129.166 14.50ag

6. Alcohol + heated PUFA + 129.342 6.78agj 121.667 9.91agk 142.500 10.60agj 105.483 10.27agj

irradiated curcumin

F ratio 67.305 333.115 89.882 83.323


Units Enzyme reaction, which gives 50% inhibition of NBT reduction/min.





by alcohol and heated PUFA may disorganize mem-brane and release enzymes from the cell.

The hike in TBARS and HP is due to the induction

of hepatic cytochrome p450 monooxygenases activityby ethanol (Wisniewska and Wronska, 1994), whichleads to the production of free radical species that cause

membrane damage through lipid peroxidation pro-ducts. The changes in the composition of erythrocytes,with increased erythrocyte deformability exvivo have

been reported with increased intake of PUFA (Nordeyand Goodnight, 1990). The increase in dietary unsat-urated fat increases the degree of unsaturation in the

Table 3. Levels of nonenzymic antioxidants and extent of lipid peroxidation in plasma [Values are mean SD from 6 rats in each group]

Vitamin C Vitamin E TBARS Hydroperoxidesmg/100 ml mg/100 ml mM/100 ml 105 mM/100 ml

Groups plasma plasma plasma plasma

1. Control 1.850 0.11 1.621 0.07 0.130 0.02 9.933 1.00

2. Alcohol + raw PUFA 1.167 0.13a 0.898 0.39a 0.370 0.04a 19.477 1.76a

3. Alcohol + heated PUFA 0.767 0.05a 0.533 0.06a 0.415 0.02a 25.915 2.05a

4. Alcohol + raw PUFA + curcumin 1.383 0.25ad 1.412 0.07nd 0.183 0.02ad 13.115 0.68bd

5. Alcohol + heated PUFA + curcumin 1.100 0.09ag 1.018 0.07ag 0.228 0.02ag 18.962 1.92ag

6. Alcohol + heated PUFA + 1.233 0.08agl 1.249 0.12bgl 0.200 0.01agl 16.777 1.20agl

irradiated curcumin

F ratio 76.088 29.078 173.789 79.387







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928 R. RUKKUMANI ET AL.


membranes (Jaya et al., 1993), since unsaturated bondsare more susceptible to lipid peroxidation, in alcohol +raw PUFA group, the TBARS and HP levels are higher.It has been reported that heated PUFA contains detec-table amounts of TBARS and HP (Hageman et al.,1991), which aggravates damage when given along withalcohol. Thus, the largest increase in lipid peroxidativeindices were seen in alcohol + heated PUFA group.

There appears to be an inverse correlation betweenalcohol + PUFA (raw and heated) mediated oxidativestress and antioxidant defenses. The alcohol and PUFA(raw and heated) actually challenge the Vitamin C andVitamin E, by increasing the susceptibility of tissuesto free radical mediated oxidative damage. In vitroexperiments have shown that Vitamin C at higher levelsact as an effective antioxidant (Steinberg and workshopparticipants, 1992) and Vitamin E is known to pro-vide protection by directly terminating lipid peroxidat-ive side chain (Cohly et al., 1998). Thus their counterbalancing action against lipid peroxidative productsdecreases their levels.

Marked alterations in lipid metabolism have beenreported in chronic ethanol feeding (Day et al., 1993).Reports show that chronic ethanol ingestion resultsin moderate hypercholesterolemia and hypertrigly-ceridemia (Antonenkov et al., 1983). Chronic admin-istration of ethanol increases tissue cholesterol and FFA.Increase in cholesterol may be due to the accumula-tion of fat in tissues and excess cholesterol leaking outinto the blood. High blood cholesterol concentrationshave been reported in diets rich in PUFA (Hocquetteand Bauchart, 1999). Moreover reports show thatthe higher plasma cholesterol has been observed inheated oil fed rats (Narasimhamurthy and Raina, 1999).Thus both in alcohol + raw as well as heated PUFAgroups the cholesterol levels were higher.

Fielding et al. have found increased plasma TG con-centration after acute ethanol ingestion (Fielding et al.,2000).In vitro alcohol has been shown to exert a directeffect on lipid metabolism, causing an increase in fattyacid esterification and a decrease in oxidation, thusaccumulating TG. The increased TG levels after PUFAingestion may be due to the increased availability ofsubstrate, FFA for esterification. Since the availabilityof FFA is more in heated PUFA group, the TG levelsare also comparatively higher.

Phospholipids are the vital components of bio mem-branes. They are the primary targets of peroxidationand can be altered by ethanol (Yamada et al., 1985).

The increased levels of PL may be due to the increasedavailability of FFA. Since PUFA is a component of PL,the increased PUFA intake may increase the levels ofPL in plasma.

The free fatty acid levels are enhanced in alcohol fedgroup, which may be attributed to the increased forma-tion of acetate, which in turn forms FFA. The increasedNADH/NAD+ ratio due to ethanol ingestion also fav-ours fatty acid synthesis. Increased FFA in alcohol +PUFA (raw + heated) may also be due to increasedlipid breakdown and increased dietary PUFA, also mayeventually increase the FFA levels.

Curcumin by scavenging or neutralizing free radicals(Soudamini et al., 1992), interacting with oxidative

cascade (Unnikrishnan and Rao, 1992), quenchingoxygen, inhibiting oxidative enzymes like cytochromeP450 (Soudamini et al., 1992), and by chelating metal

ions like Fe2+ (Sreejavan and Rao, 1994), inhibits lipidperoxidation and thus reduces TBARS and HP, sta-bilizes membrane and thus prevents the hike of ALPand GGT. Its effective antioxidant property decreasesthe utilization of Vitamin C and Vitamin E and thusmaintains their levels.

The decrease in cholesterol level by curcumin is dueto the induction of 7-hydroxylase activity by curcumin,

which facilitates biliary excretion of cholesterol (Babuand Srinivasan, 1997). The spices are known to affectbile acid excretion and thus curcumin also influenceother lipid levels. The decreased levels of PL and TGmay also be due to the decreased FFA synthesis bycurcumin, which may suppress the enzymes involved inFFA synthesis.

In our study, IC was found to be more effectivethan curcumin in protecting the membrane againstlipid peroxidation and improving antioxidant status.Flavonoids and monophenolic compounds have beenwell described over recent years for their properties asantioxidants and scavengers of reactive oxygen speciesand nitrogen species (Bourne et al., 2000). Ferulic acidis a flavonoid and both ferulic acid and vanillin aremonophenolics and thus both are effective antioxidants.Reports show that ferulic acid is effective on lipidperoxidative systems induced by Fe2+ [Uchinda et al.,1996]. UV absorption of ferulic acid catalyses stablephenoxy radical formation and thereby potentiatesits ability to terminate free radical chain reaction. Byvirtue of effectively scavenging deleterious radicalsand suppressing oxidative reactions, FA is shown as animportant antioxidant (Graf, 1992). Reports have shownthat FA is absorbed as Ferulic acid -glucuronide, andthe antioxidant property of ferulic acid is not only dueto the hydrophobic FA but also due to the hydrophilicsugar moiety (Ohta et al., 1997). Thus FA is proved tobe an effective antioxidant.

Reports say that the supplementation of phenoliccompounds leads to increased antioxidant activity inplasma and a significant increase in Vitamin E content(Carbonneau et al., 1997). The presence of phenolicgroup in vanillin may contribute to the antioxidantproperty. It has been proposed that hydroxy andhydroperoxy radicals initiate hydrogen abstraction froma free phenolic substrate to form a phenoxy radicalthat can rearrange to a quinone methide radical inter-mediate (Pan et al., 1999) and thus reduce free radicals.Thus the combined effects of FA and vanillin in ICeffectively decrease the TBARS and HP and effectively

modulate the antioxidant status.The levels of lipids were significantly reduced inphoto-irradiated curcumin treatment groups comparedto curcumin. The vanillin and ferulic acid, the compon-ents of IC, due to their effective antioxidant property,protects membrane and prevents FFA release. FFAbeing a substrate for other lipids, its decrease may re-flect on the levels of lipids. Recent reports show that -oryzanol, a mixture of ferulic acid, can lower cholesterollevel in blood and lower the incidence of coronary heartdisease. In Japan it has been used as a natural anti-oxidant in foods, beverages and cosmetics (Scavarielloand Ardlano, 1998). Thus, the vanillin and ferulic acideffectively reduce lipids by an unknown mechanism.

We therefore conclude from our results that IC ismore effective in controlling lipid peroxidation and lipidlevels in circulation than curcumin.


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Protective effects of curcumin and photoirradiated curcumin on circulatory lipids and lipid peroxidation products in alcohol and polyunsaturated fatty acid-induced toxicity