International Journal of Pharmacy and Pharmaceutical Sciences

ISSN- 0975-1491 Vol 2, Suppl 4, 2010

ResearchArticle

EFFECTOFPHYLLANTHUSNIRURIONALCOHOLANDPOLYUNSATURATEDFATTYACID INDUCEDOXIDATIVESTRESSINLIVER

MAGESWARIBASKARAN,LATHAPERIYASAMYANDRUKKUMANIRAJAGOPALAN*
DepartmentofBiochemistryandMolecularBiology,PondicherryUniversity,Kalapet,Tamilnadu,India605014 Email:ruks2k2@yahoo.co.in Received:18May2010,RevisedandAccepted:21Jun2010 ABSTRACT Alcoholisatoxinassociatedwithsignificantmorbidityandmortality.Itinducesoxidativestresswhentakenathigherconcentrations.Theelevation in lipid peroxidative products and the loss of antioxidant defense potential are enhanced when alcohol is consumed along with polyunsaturated fattyacid(PUFA).ThebiologicaldisturbancesareintensifiedwhenthePUFAisthermallyoxidized.Henceinourstudyweinvestigatedtheeffectsof the crude extract of Phyllanthus niruri (PN), a traditional medicinal herb used in India and China, on alcohol and heated PUFA induced hepatotoxicity. Male Albino Wistar rats were used for the study. The liver marker enzymes Gammaglutamyl transferase (GGT), Alkaline phosphatase(ALP),LipidperoxidativeindicesThiobarbituricacidreactivesubstances(TBARS),Lipidhydroperoxides(LH);Antioxidantsvitamin C,vitaminE,reducedglutathione(GSH),superoxidedismutase(SOD),catalase(CAT),glutathioneperoxidase(GPx)wereassayedforestimatingthe antioxidantpotentialof P.niruri.Histopathologywasdonetoconfirmtheresults.Ourresultssuggestedthatthecrudeextractof P.nirurieffectively decreasedtheoxidativestressandassociateddamage. Keywords:Alcohol,Polyunsaturatedfattyacid,Liver,Antioxidants,Lipidperoxidation,Livermarkerenzymes,Phyllanthusniruri INTRODUCTION The liver is the most important organ governing essential biochemical activities in the human body. It has great capacity to detoxify and synthesize useful substances, and therefore damage to the liver has grave consequences 1. Ethanolinduced oxidative stress is knownto play a major role in causing liver injury. Many pathways have been suggested to contribute to the ethanol induced oxidative stress. Some of these include redox state changes,productionofthereactiveproductacetaldehyde,damage to mitochondria, direct or membrane effects caused by hydrophobic ethanol, ethanolinduced hypoxia, effects on the immunesystem,alteredcytokineproduction,inductionofCYP2E1 andmobilizationofiron2. Theadverseeffectofethanolisfurtheraggravatedbyintakeofdiet rich in polyunsaturated fatty acids (PUFA). Present day reports on dietaryfatsindicatethatincreasedintakeofPUFAisdetrimentalto health. A high PUFA n6 content and a high n6/n3 ratio in the newer heart friendly oils like sunflower oil are considered to be dangerous3.Moreovertherepeatedheatingoftheseoilschangesthe oilcharacteristicsthroughthermaloxidation.Duringdeepfatfrying, many volatile and nonvolatile products are produced, some of which are toxic, depending on the level of intake4. Consumption of such oils has devastating effects on the functions of many organs especially liver5. Many alcoholics as a custom take fried food items afteraheavybingeofalcohol.ThiscombinationofalcoholandPUFA canaggravatethetoxiceffectsespeciallybyincreasingtheoxidative stress6. Phyllanthus niruri (family Euphorbiaceae) is a plant possessing severalpharmacologicalproperties.Itisanayurvedicmedicineused for treating gastric lesion7, urolithiasis8, urinary infections and diuretics9.Thisherbisusedsinceancienttimesforthetreatmentof jaundice and other liver related disorders. It is also known to possessantiinflammatorypropertybydecreasingtheinflammation causedbyseveralpathways10.Theadministrationoftheextracthas been found to increase the life span of rats with hepatocellular carcinoma11. It is shown to have effective hepatoprotective actions against paracetamol12, carbon tetrachloride13, galactosamine14 and thioacetamideinducedtoxicity15. Hence in the present study we tested the effect of crude extract of Phyllanthus niruri on alcohol and PUFA induced oxidative stress. MATERIALSANDMETHODS Animals

Male albino rats of Wistar strain of body weight ranging 140160g bredinCentralAnimalHouse,RajahMuthiahMedicalCollege,Tamil Nadu, India, fed on standard pellet diet (Agro Corporation Private Limited, Bangalore, India) were used for the study and water was given ad libitum. The standard pellet diet comprised 21% protein, 5%lipids,4%crudefibre,8%ash,1%calcium,0.6%phosphorous, 3.4% glucose, 2% vitamin and 55% nitrogen free extract (carbohydrates).Itprovidesmetabolisableenergyof3600Kcal. The animals were housed in plastic cages under controlled conditionof12hlight/12hdarkcycles,50%humidityandat30 2C. The animals used in the present study were maintained in accordance with the guidelines of the National Institute of nutrition, Indian Council for Medical Research, Hyderabad, India and approved by the Animal Ethical Committee, Pondicherry University. Materials Ethanol Absolute ethanol (AR) was obtained from Private Limited, England. Thermally oxidized Sunfloweroil(Gold Winner)wassubjectedto heating at PUFA (PUFA) 180 C twice to produce thermally oxidizedPUFA. Phyllanthus niruri (PN) The fresh leaves and stems of the young plantswerehomogenizedin50mMsodiumphosphatebuffer,pH7.2 at 40C and centrifuged at 12,000 g for 30 minutes to remove unwanted debris. 3 ml of the supernatant was used for the treatment. All other chemicals and solvents used in this experiment were purchasedfromHimediaPvtLtd,India. Experimentaldesign Theanimalsweredividedinto4groupsof6ratseach. At the end of the experimental period (45 days), the rats were sacrificedafteranovernightfastbycervicaldislocationandtheliver was collected, cleared off blood and immediately transferred to ice cold containers containing 0.9% NaCl for various biochemical estimations.

Rajagopalanetal. IntJPharmPharmSci,Vol2,Suppl4,5862 Table1:Treatmentschedule Groups Group1 (Normal) Group2 (Alcohol+PUFA) Group3 (Alcohol+PUFA+PN) Group4 (Phyllanthusniruri) Preparationofplasma Bloodwascollectedinheparinisedtubesandplasmawasseparated bycentrifugationat1000gfor15minutesfortheestimationofGGTand ALP. Preparationofliverhomogenate Known amount of the liver tissue was weighed and homogenized in appropriate buffer for the estimation of lipid peroxidative indices and enzymicandnonenzymicantioxidants. Biochemicalinvestigations The activity of plasma Gammaglutamyl transferase (GGT) was assayed by the method of Fiala et al.,16. The activity of alkaline phosphatase (ALP) was assayed by the method of King and Armstrong using a reagent kit17. The concentration of Thiobarbituric acid reactive substances (TBARS) was estimated by themethodofNiehausandSamuelsson18andLipidhydroperoxides (LH)bythemethodofJiangetal.,19.Ascorbicacidwasestimatedby the method of Roe and Kuether20 and vitamin E by the method of BakerandFrank21.Reducedglutathione(GSH)contentinthetissue was determined by the method of Ellman22. The activity of
b

Dietfollowed Standardpelletdiet 20%ethanolorallyusingintragastrictube+15%heatedsunfloweroilmixedwiththediet 20%ethanol+15%heatedsunfloweroilmixedwiththediet+3mlofcrudeextractofPhyllanthusniruri 3mlofcrudeextractofPhyllanthusniruri

glutathioneperoxidase(GPx)wasassayedbythemethodofRotruck etal.,23,superoxidedismutase(SOD)bythemethodofkakkaretal., 24 andcatalase(CAT)bythemethodofSinha25. Forhistopathologicalstudy,twoanimalsfromeachgroupwereperfused withformalin(10%)andthetissueswereseparatedandstoredin10% formalin.Theywerelattersectionedusingamicrotome,dehydratedin graded alcohol, embedded in paraffin section, and stained with hemotoxylinandEosin(H&E). Statisticalanalysis Statistical analysis was done by analysis of variance (ANOVA) followed by Tukeys test. The values were considered statistically significantwhenp0.05. RESULTS Figure 1 and 2 show the changes in the activities of GGT and ALP. The activities of these liver marker enzymes were increased significantlyinplasmaofalcohol+ PUFAgroupwhencomparedto normal.CoadministrationofPNsignificantlyreducedtheiractivity. PN control group showed no significant change in the activities whencomparedtonormal.

a a a

Fig1:ChangesintheactivitiesofGGTinplasma.valuesaremean S.D(n=6)Valuessharingacommonsuperscriptdonotdiffer significantlyatP0.05 Figure 3 and 4 show the changes in the levels of TBARS and lipid hydroperoxidesinliver.ThelevelsofTBARSandHPwereincreased significantly in alcohol + PUFA group when compared to normal. Treatment with PN significantly decreased their levels. PN control group did not show any significant change in the levels when comparedtonormal. ThechangesinthelevelsofvitaminC,vitaminEandGSHinliverare given in Figure 5, 6 and 7 respectively. There was a significant decrease in the levels of vitamin C, vitamin E and GSH in alcohol + Fig2:ChangesintheactivitiesofALPinplasma.Valuesaremean S.D(n=6)Valuessharingacommonsuperscriptdonotdiffer significantlyatP0.05

PUFA groups when compared to normal. The levels were significantly increased in PN treated groups. PN control group showed no significant change in their levels when compared to normal The activities SOD (Figure 8), CAT (Figure 9), GPx (Figure 10) showed a significant decrease in liver of alcohol + PUFA group. Treatment with PN significantly increased their activities. Administration of PN alone did not alter the activities of these enzymicantioxidants.

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Rajagopalanetal. IntJPharmPharmSci,Vol2,Suppl4,5862

b b

a a a

Fig.3:LevelsofTBARSinlivertissue.ValuesaremeanS.D(n=6). Valuessharingacommonsuperscriptdonotdiffersignificantlyat P0.05

Fig.4:LevelsofHydroperoxidesinlivertissue.Valuesaremean S.D(n=6)Valuessharingacommonsuperscriptdonotdiffer significantlyatP0.05

a a

a a

Fig.5:LevelsofVitaminCinlivertissue.ValuesaremeanS.D (n=6)Valuessharingacommonsuperscriptdonotdiffer significantlyatP0.05 a a

Fig.6:LevelsofVitaminEinlivertissue.ValuesaremeanS.D (n=6)Valuessharingacommonsuperscriptdonotdiffer significantlyatP0.05 a a c

b b

Fig.7:LevelsofReducedGlutathioneinlivertissue.Valuesare meanS.D(n=6)Valuessharingacommonsuperscriptdonot differsignificantlyatP0.05 a a a a

Fig.8:ActivitiesofSuperoxideDismutaseinlivertissue.Valuesare meanS.D(n=6)Valuessharingacommonsuperscriptdonot differsignificantlyatP0.05 *enzymereactionwhichgives50%inhibitionofNBTreduction/minute

Fig.9:ActivitiesofCatalaseinlivertissue.ValuesaremeanS.D (n=6)Valuessharingacommonsuperscriptdonotdiffer significantlyatP0.05 *mofH2O2liberated/minute.

Fig.10:ActivitiesofGlutathionePeroxidaseinlivertissue.Values aremeanS.D(n=6)Valuessharingacommonsuperscriptdonot differsignificantlyatP0.05 *mmolesofglutathioneliberated/minute.

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Rajagopalanetal. IntJPharmPharmSci,Vol2,Suppl4,5862 In histopathological observations (Figure 11), the liver of alcohol + heated PUFA treated rats showed fibrosis (Fig B), and Phyllanthus niruri treated rats showed only sinusoidal dilatation (Fig C). The histology of liver was not affected in both normal (Fig A) and Phyllanthusniruricontrol(FigD)rats.

Fig.11:Histopathologicalobservations DISCUSSION Oxidativestressisdefinedastheimbalancebetweentheprooxidant and antioxidant status. It results primarily due to increased production of reactive oxygen species or decreased levels of antioxidant defense in several tissues and organs. This may finally leadtoirreversibleorganandtissuedamagewhentheproductionof thesefreeradicalsisoutsized.Theliveristhemajorsitefortoxicity ofxenobioticsanddrugssincemostoftheorallyingesteddrugsand chemicalsaremetabolizedintotoxicintermediates.Henceincreased ingestionofalcohol,mainlythroughitsmetabolism,producessevere injury to the liver. Due to this injury, the biomembranes of the hepatocytes are damaged. This causes the increase in the activities oflivermarkerenzymesintheplasma. Moreover, the membranes are mostly made up of lipid conjugates. The polyunsaturated fatty acids are highly susceptible to lipid peroxidation. The increased intake of dietary polyunsaturated fatty acidincreasesthedegreeofunsaturationofbiomembranesandthus the lipid peroxidation is intensified. Hence the membrane becomes leaky to the liver marker enzymes, which is evidenced by the increasein thelevels ofGGT andALPinour study. The increase in the lipid peroxidation which is implied by the increase in the measured TBARS and hydroperoxides in the alcohol and PUFA is alsoindicativeofthehepaticdamage. Theconstantproductionofthesefreeradicalsmustbecounteracted by the enzymic and nonenzymic antioxidants. But the uncontrollable increase in the free radicals directs the increased exploitation of the antioxidants leading to their depletion. In our study, the levels of nonenzymic antioxidants reduced glutathione, vitamin C and vitamin E were significantly reduced in the alcohol andPUFAtreatedratsbecauseoftheircompleteutilizationdueto oxidative stress. The activities of enzymic antioxidants like glutathione peroxidase, catalase and superoxide dismutase were also decreased in our study. The high amount of H2O2 and lipid hydroperoxides, O2, 1hydroxy ethyl radical, produced during alcohol and PUFA ingestion, might have contributed to their inactivationanddepletion.

Phyllanthus niruri, a widely distributed herb is in use in herbal medicine systems and in clinical research over years. It is effective against toxicity caused by several hepatotoxicants. It is used for treating liver, brain and kidney diseases due to iron overload. P. niruri can potentially inhibit the transcription of pro inflammatory cytokines(TNF)produced bythe kupffer cells.Thusit could have actedasanantiinflammatoryagent,curingtheinflammationcaused byalcoholandPUFA.ThehepatoprotectivenatureofP.niruricanbe attributedtothe presence of several bioactive compounds such as lignans,alkaloids,terpenoidsandtanninsespeciallyphyllanthinand hypophyllanthin, quercetin, astragalin, gallic acid, ellagic acid and corilagin. It also possesses glycosides, flavonoids, flavonols, polyphenols, phenylpropanoids, which are known anti oxidants26,27,28,29.Thepresenceofallthesephenoliccompoundsmay contributeforthehighfreeradicalscavengingactivityof P. niruri30. Phenolic compounds, by virtue of their hydrogen donating ability, formingaryloxylradicals,actasfreeradicalscavengersandquench thelipidperoxidation.Moreover P. niruriextracthasbeenfoundto inhibitmanydrugsmetabolizingP450enzymes31.Thiscouldalsobe one of the reasons for the increased protection of hepatocytes duringP.niruritreatment. The protective effect is further confirmed by the histopathological analysis,inwhichtheseverepathologicalchangefibrosis,whichwas observed in alcohol+ heated PUFA rats, was altered by Phyllanthus niruri treatment. The Phyllanthus niruri treated rats showed only sinusoidal dilatation which is the preliminary state of damage. Administrationof Phyllanthus niruri tonormalratsdidnotalterthe histologyoftheliver. Administrationofthecrudeextractof P.niruritargetedadropinthe lipid peroxidation, enhanced the antioxidant status and thereby preventedthedamagetotheliverandleakageofenzymesGGTand ALP. Previous studies have shown that the crude extract has very high potency in scavenging DPPH radical than all other extracts 32. This is indicative of its high radical scavenging property and hepatoprotectiveactivity.Suchahighantioxidantpropertyhasbeen shownonlybyfewotherplants33.AllthesepropertiesmakeP.niruri, anovelherbfortreatingoxidativestressandassociatedtoxicity.

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