Studies on antidiarrhoeal activity of Jatropha curcus root extract in albino mice

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In vitro Antioxidant andHepatoprotective Effect of the WholePlant of Glossocardia bosvallea (L. f.) D.C. against CCl4-Induced Oxidative Stressin Liver Slice Culture ModelAnagha A. Rajopadhye a & Anuradha S. Upadhye aa Plant Science Division, Agharkar Research Institute, Pune, India

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Journal of Herbs, Spices & Medicinal Plants, 18:274–286, 2012Copyright © Taylor & Francis Group, LLCISSN: 1049-6475 print/1540-3580 onlineDOI: 10.1080/10496475.2012.690363

In vitro Antioxidant and HepatoprotectiveEffect of the Whole Plant of Glossocardiabosvallea (L. f.) D. C. against CCl4-Induced

Oxidative Stress in Liver Slice Culture Model

ANAGHA A. RAJOPADHYE and ANURADHA S. UPADHYEPlant Science Division, Agharkar Research Institute, Pune, India

Glossocardia boswallea (Pittapapda or Parpat) is used in theIndian system of traditional medicine. The present study inves-tigated the antioxidant and hepatoprotective activity of hexane(GH), ethanol (GE), and water extract (GW) of the whole plant.Results showed that GH and GE inhibited 1,1-diphenyl 2-picrylhydrazyl (DPPH), nitric oxide (NO) and superoxide dismutase(SOD) radicals in a dose-dependent manner. The total antioxidantcapacities of lipid-soluble substances in GH, GE and GW were14.342, 12.656, and 9.890 nmol g−1 trolox equivalent, respec-tively. The trade of phenol content was GW <GH <GE. Thehepatoprotective effect was assayed in CCl4-induced cytotoxicityin a liver slice culture model. Depletion was observed in lactatedehydrogenase, lipid peroxidation and antioxidative enzymes onadministration of GH and GE or ascorbic acid as standard inCCl4-induced cytotoxicity in the liver. GH and GE significantlyprevented oxidative liver damage.

KEYWORDS Photochemiluminescence, spectrophotometry

Received July 22, 2011.The authors are greatly thankful to the director, Agharkar Research Institute, Pune, India,

for providing facilities and encouragement throughout the work. The authors are also thankfulto Dr. A. M. Mujumdar, ex-head, Plant Science Division, and Dr. (Mrs.) V. S. Ghate, in-chargeBotany, ARI, Pune for their valuable suggestions throughout the work.

Address correspondence to Anuradha S. Upadhye, Plant Science Division, AgharkarResearch Institute, G.G. Agarkar Road, Pune 411 004, India. E-mail: [email protected]

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Hepatoprotective Effect of Glossocardia bosvallea 275

INTRODUCTION

Glossocardia boswallea (Asteraceae) known as “Pittapapda” or “Parpat” inSanskrit is distributed over the greater parts of India as a weed, by road-sides and on hills ascending up to 9,000 feet. The herb is annual, oftendiffuse or scandent, and has multifid compound leaves and yellow flow-ers. Ethnobotanical reports and ayurvedic literature report that the wholeplant as being used as blood purifier, diuretic, laxative, stomachic, and tonicand to treat abdominal cramps, diarrhea, fever, and jaundice (7,15). It is amajor constituent of many household and ayurvedic medicinal preparationsincluding Parpatadi-kwath, Parpatadi-arishta, and Parpatadi-arka (28).

Studies reported diastereoisomeric monoterpene mixtures from chloro-form: methanol extract of this plant (2) and the antimicrobial activity ofessential oil evaluated (20,26). This study investigated the in vitro antioxidantand hepatoprotective activity of the whole plant extracts of G. boswal-lea against CCl4-induced oxidative stress in liver slice culture model byphotochemiluminescence and spectrophotometric methods.

MATERIALS AND METHODS

Collection of Plant Material and Extraction

The plant material was collected in bulk quantity from Baramati, Pune(Maharashtra, India) during October 2007. The plant sample was identifiedand authenticated, and a voucher specimen (AHMA- 24858) was depositedin Agharkar Herbarium of Maharashtra Association of Cultivation Science,Agharkar Research Institute, Pune, India.

Whole plant materials were shade-dried, coarsely powdered, and storedin an airtight container at 25◦C ± 4◦C. Powdered material (75 g) was extractedsuccessively with hexane, ethanol, and water using an ASE 100 acceler-ated solvent extractor (Dionex, Vienna, Austria). Extraction was performed at100 bar and temperature at 60◦C for 20 min in five-replicate cycles and con-centrated under vacuum using a rotary evaporator. Yields of hexane (GH),ethanol (GE), and water extract (GW) were 5.83%, 10.08%, and 12.06%,respectively.

Chemical and Reagents

1,1-diphenyl 2-picryl hydrazyl, (DPPH), griess reagent, 2,2’-azinobis(3-ethylbenzothiazoline-6-sulfonate) (ABTS), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), potassium superoxide,pyrocatechol, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES),β-hydroxy butarate, lactate dehydrogenase (LDH), nicotinamide adeninedinucleotide (NADH), pyrogallol, catalase (CAT), bradford reagent, and

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276 A. A. Rajopadhye and A. S. Upadhye

glutathione reductase (GR) were obtained from Sigma Aldrich, USA. Ferroussulphate (FeSO4), trichloroacetic acid (TCA), thiobarbituric acid (TBA),acetic acid, ethylene diaminetetraacetic acid, sodium nitroprusside (SNP),sodium phosphate, dimethyl sulfoxide (DMSO), nitrobluetetrazolium (NBT),sodium chloride (NaCl), potassium chloride (KCl), calcium chloride (CaCl2),monopotassium phosphate (KH2PO4), magnesium sulfate (MgSO4), carbontetrachloride (CCl4), ascorbic acid, hydrochloric acid, potassium phosphate,glucose, and folin-ciocalteu were purchased from s. d. fine chemicals,Mumbai, India.

Antioxidant Activity

RADICAL-SCAVENGING EFFECT OF EXTRACTS IN DPPH RADICALS

DPPH radical-scavenging ability was assessed as described earlier (14).Briefly, to a methanolic solution of DPPH (60 mM, 2 mL), 50 µL of test extractdissolved in methanol at different concentrations was added. Absorbancemeasurements commenced immediately at 515 nm. Decrease in absorbancewas determined after 70 min when the absorbance stabilized. Absorbance ofthe DPPH radical without extracts and the control was measured. Ascorbicacid was used as reference antioxidant. Percent inhibition of DPPH radicalin the samples was calculated as

% Inhibition = [(AC(0)−AA(t)

)/AC(0)

] × 100

where AC(0) is the absorbance of the control at t = 0 min and AA(t) is theabsorbance in presence of antioxidant at t = 70 min.

NITRIC OXIDE–SCAVENGING ACTIVITY

Nitric oxide–scavenging activity was measured as given earlier (17). Sodiumnitroprusside (5 mM) in phosphate-buffered saline was mixed with testextracts and incubated at 25◦C for 30 min, and 1.5 mL of the incubated solu-tion was diluted with 1.5 mL of Griess’s reagent. Absorbance of chromophoreformed during diazotization of nitrite with sulfanilamide and subsequent cou-pling with naphthylethylene diamine was measured at 546 nm along withcontrol, using ascorbic acid reference antioxidant. Percentage inhibition ofnitric oxide generated was measured by comparing absorbance values ofcontrol and test samples.

SUPEROXIDE-SCAVENGING ACTIVITY

Superoxide-scavenging activity was carried out by using the alkaline DMSOmethod (12). Solid potassium superoxide was allowed to stand in contact

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with dry DMSO for at least 24 h, and the solution was filtered immediatelybefore use. Filtrate (200 mL) was added to 2.8 mL of an aqueous solutioncontaining nitroblue tetrazolium (56 mM), ethylenediaminetetraacetic acid(10 mM), and potassium phosphate buffer (10 mM, pH 7.4). Test extracts(1 mL) in water were added, and absorbance was recorded at 560 nm againstcontrol in pure DMSO.

Trolox Equivalent Antioxidant Capacity Assay

The total antioxidant activity was measured using trolox equivalentantioxidant capacity (TEAC) assay (21) with minor modifications. TEAC valuebased on the ability of antioxidant to scavenge blue-green 2,2’-azinobis(3-ethylbenzothiazoline-6-sulfonate (ABTS+) radical cation relative to ABTS+

scavenging ability of the water-soluble vitamin E analogue, Trolox. ABTS+

radical cation were generated by interaction of ABTS+ (100 µM), H2O2

(50 µM), and peroxidase (4.4 unit mL−1). To measure antioxidant capac-ity, 0.25 mL of each extract concentration was mixed with an equal volumeof ABTS+, H2O2, peroxidase and deionized water. Absorbance was moni-tored at 734 nm for 10 min. Decrease in absorbance at 734 nm after additionof reactant was used to calculate the TEAC value and expressed as milimo-lar concentration of trolox solution having an antioxidant equivalent to a1,000 ppm solution of the sample under investigation.

Photochemiluminescence Assay

Photochemiluminescence (PCL) was used for determination of integralantioxidative capacity (AC) of lipid-soluble and water-soluble substances inextracts. Apparatus used included Photochem with standard kit; antioxidantcapacity of water-soluble (ACW); and antioxidant capacity of lipid-soluble(ACL; Analitik jena AG), where luminol played a double role of pho-tosensitizer and the radical-detecting agent (8). Each extract was mea-sured at 10 µg mL−1 concentration. A standard curve was plotted, andthe results were calculated for lipid-soluble substance in trolox equiva-lents (nmol.g−1) and water-soluble substance in ascorbic acid equivalents(nmol.g−1).

Total Phenolic Content

Total phenolic content in each extract was determined with folin-ciocalteureagent (27) using pyrocatechol as a reference standard; 2 mL of 2% Na2CO3

was added to 0.1 mL extract and mixed thoroughly. After 5 min of incubation,0.1 mL of 50% folin-ciocalteu reagent was added and allowed to stand for

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2 h with intermittent shaking. Absorbance was measured as micrograms ofpyrocatechol equivalent (PCE) from the standard graph.

In vitro Hepatoprotective Activity

ANIMALS

Adult albino mice (6 to 8 weeks old) of either gender and bred in the ani-mal house at Agharkar Research Institute, Pune, India were used for thepreparation of liver slices. Approval for using animals was obtained from theInstitutional Animal Ethical Committee.

LIVER SLICE CULTURE

Liver slice culture was maintained as per Wormser and Ben (29) and Invittoxprotocol No. 42 (13). The mice were dissected open after cervical disloca-tion, and liver lobes were removed and transferred to pre-warmed Kred’sRinger Hepes (KRH; 2.5 mM Hepes, pH 7.4, 11 8 mM NaCl, 2.85 mM KCl,2.5 mM CaCl2, 1.5 mM KH2PO4, 1.18 mM MgSO4, 5 mM β-hydroxy butarate,and 4.0 mM glucose). The liver was cut into thin slices using sharp blade, andslices weighing between 4 and 6 mg were used for the study. Each exper-imental system contained 20 to 22 slices weighing 100 to 120 mg. Theseslices were washed with 10 mL KRH medium every 10 min over a periodof 1 h and pre-incubated for 60 min in small plugged beakers containing2 mL KRH on a shaker water bath at 37◦C. At the end of pre-incubation,the medium was replaced by 2 mL of fresh KRH and incubated for 2 hat 37◦C.

Experiment Design

The liver slices were further divided into individual cultures for furtherrespective treatments. Set 1, Control, Set 2, 15.5 mM CCl4; Set 3, 50 µg mL−1

GH; Set 4, 50 µg mL−1 GE; Set 5, 50 µg mL−1 GW; Set 6, 15.5 mM CCl4+ 10, 25, or 50 µg mL−1 of GH; Set 7, 15.5 mM CCl4 + 10, 25, or 50µg mL−1 of GE; Set 8, 15.5 mM CCl4 + 10, 25, or 50 µg mL−1 of GW;Set 9, 15.5 mM CCl4 + 10 mM ascorbic acid; and Set 10, 10 mM ascorbicacid. After respective treatments, all the cultures were incubated in constant-temperature water bath at 37◦C for 2 h. At the end of incubation, each groupof slices was homogenized in appropriate volume of chilled potassium phos-phate buffer (100 mM, pH 7.8) in an ice bath to give a tissue concentrationof 100 mg mL−1. The culture medium was collected and used for estima-tion of lactate dehydrogenase (LDH) employed as a cytotoxicity marker.The homogenates were centrifuged at 10,000 rpm for 10 min at 4◦C, and

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supernatants were assayed for LDH, catalase, peroxidase, and superoxidedismutase using ascorbic acid as standard.

Measurement of Lactate Dehydrogenase Activity

LDH (EC 1.1.1.27) activity was measured at 340 nm (24). Each unit of enzymewas calculated as 1µmol of nicotinamide adenine phosphate (NAD) reducedper minute. Commercially available LDH was used as the standard.

Measurement of Lipid Peroxidation

Lipid peroxidation was estimated in terms of thiobarbaturic acid reactivesubstances formed in liver tissue homogenate, with some modifications (19).Tissue homogenate was prepared in 5% TCA. To 1 mL homogenate, 4 mL of0.5% thiobarbituric acid in 20% TCA was added and incubated at 95◦C for30 min. The mixture was immediately cooled on ice, during which time thecolor changed from orange to pink. Mixture was centrifuged at 4,000 rpm for10 min. Estimation of µmoles of malondialdehyde (MDA) formed was doneby calculating the difference in the absorbance of the supernatant at 532 nm(specific) and 600 nm (nonspecific).

Measurement of Antioxidant Enzymes

The superoxide dismutase (SOD) activity was assayed spectrophotometri-cally by means of inhibition of pyrogallol autooxidation (18). The extentto which the enzyme decreases reduction of NBT by superoxide radicalgenerated by riboflavin in the presence of light was monitored at 560 nm.One unit of enzyme was defined as the amount of enzyme causing 50%reduction in formazan formation under specified conditions. Catalase (CAT;EC 1.11.1.6) assay was carried out after the method of Aebi (1). One unitwas defined as that amount of enzyme that converts 1 µmol H2O2 towater in 1 min. Glutathione reductase (GR; EC 1.6.4.2) was estimated asgiven by Ellman (5) and Bulaj et al. (4). One unit was defined as theamount of enzyme required for oxidization of 1 µmol of nicotinamide ade-nine dinucleotide phosphate (NADPH) to nicotinamide adenine dinucleotidephosphate (NAD) per minute.

Estimation of Proteins

Protein in tissue homogenates were estimated as given by Bradford (3).

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STATISTICAL ANALYSIS

Data were expressed as mean ± standard deviation for three parallel experi-ments. The results of treatment effects were analyzed by a one-way analysisof variance test (Graphpad Prism 4; p < 0.05).

RESULTS

Antioxidant Activity

Antioxidant activity was evaluated in terms of scavenging of DPPH, nitricoxide, SOD radical, TEAC, and photochemiluminescence in vitro systems(Figure 1 and Table 1). GH and GE showed high antioxidant activity followedby GW, which decreased the DPPH, NO, and superoxide radical in a dose-dependent manner compared to standard. IC50 values of GH, GE, GW, andascorbic acid for DPPH were 31.55 ± 1.45, 34.67 ± 1.67, 38.90 ± 1.34 and 4.1µg mL−1, respectively; for NO 35.33 ± 0.85, 34.66 ± 1.23, 43.34 ± 1.67 and3.9 µg mL−1, respectively; and for superoxide radical 36.33 ± 0.85, 35.66 ±1.23, 42.34 ± 1.67 and 5.3 µg mL−1, respectively. The trend of TEAC valueswas as GH > GE > GW (see Table 1). Integral antioxidant capacity (IAC)represented the antioxidant capacity of hydrophilic and lipophilic antiox-idants, calculated as nanomoles equivalents in activity of Trolox/ascorbicacid. Sample extracts differed in ACW and ACL values. GH and GE had highIAC than GW (see Table 1). Total phenol content, expressed as milligrams

FIGURE 1 Free radical–scavenging capacity of G. boswallea extracts in hexane (GH), ethanol(GE), and water (GW). Values are mean ± SD of three experiments; DPPH-1,1-diphenyl2-picryl hydrazyl; NO = nitric oxide; SOD = superoxide dismutase.

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TABLE 1 Free Radical–Scavenging Capacity of G. boswallea Extracts in Hexane(GH), Ethanol (GE), and Water (GW)

Samples TEAC (mM)

PhotochemiluminescenceACW in ascorbic acidequivalent (nmol g−1)

PhotochemiluminescenceACL in trolox equivalent

(nmo g−1)

GH 3.47 ± 0.06 2.213 14.342GE 2.89 ± 0.19 3.141 12.656GW 1.58 ± 0.193 3.541 9.890AA 3.89 ± 0.23 — —

TEAC = trolox equivalent antioxidant capacity; ACW = antioxidant capacity of water soluble;ACL = antioxidant capacity of lipid soluble.Note: Values are mean ± SD of three experiments.

pyrocatechol equivalent (PCE) g−1 extracts, was affected by the extractingsolvents with the following order from high to low: GW (112 mg PCE g−1) <

GH (172 mg PCE. g−1) < GE (202 mg PCE. g−1). No correlation was foundbetween the yield of extract and the total phenol content. GW gave a higheryield than GH although GH showed higher total phenol contents.

In vitro Hepatoprotective Activity

Release of LDH in the liver slice culture medium was used as a cytotoxicitymarker. The GH, GE, and GW were nontoxic at 50 µg mL−1 as it showedpercentage release of LDH in the medium similar to that of control untreatedslices. CCl4 was highly toxic to the treated cells, which increased LDH con-centration five times higher (42.33 ± 1.63 units 100 mg tissue wet wt.−1) ascompared to control. The amount of LDH release in medium reduced afteraddition of GH and GE than GW along with CCl4 cytotoxicant, and the activ-ity was dose-dependent. The activity was comparable with standard ascorbicacid (Table 2).

The liver tissue treated only with CCl4 continuously released LDH (50%)in 2.0 h, whereas the LDH release by the control (untreated) was constant(below 10%; see Table 2). However, the extract-treated liver tissue showed20% LDH release up to 1.0 h incubation and further decreased equivalentto the control level at 2.0 h incubation. CCl4 is known to generate oxidativestress in cells, which can be measured from the extent of lipid peroxidationin liver tissue.

Lipid peroxidation levels in the liver slice culture medium were assessedby thiobarbaturic acid reactive substances assay. Lipid peroxidation was mea-sured in terms of thiobarbituric acid reactive substances and was expressedas µmoles of malondialdehyde formed per 100 mg tissue. The amount oflipid peroxidation increased in CCl4-treated liver cells compared to respec-tive control (Figure 2). Extent of lipid peroxidation was reduced to nearcontrol levels when liver cells were treated either with GH and GE than

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TABLE 2 Effect of Extracts in Protecting Liver Cells from CCl4-Induced Cytotoxicity byAmeliorating Oxidative Stress

Treatments

LDH Units100 mg tissue

wet wt.−1

SOD Units100 mg tissue

wet wt.−1

CAT Units100 mg tissue

wet wt.−1

GR Units100 mg tissue

wet wt.−1

Control 8.83 ± 1.16 16.83 ± 1.47 14.00 ± 1.41 0.141 ± 0.01215.5 mM CCl4 42.33 ± 1.63 55.33 ± 2.25 86.5 ± 1.04 0.488 ± 0.008AA 8.53 ± 1.47 16.5 ± 2.14 14.33 ± 1.75 0.140 ± 0.007GH 8.00 ± 2.09 16.43 ± 1.72 14.65 ± 1.37 0.146 ± 0.003GE 8.33 ± 1.16 16.23 ± 1.47 14.83 ± 1.22 0.149 ± 0.011GW 8.17 ± 1.47 17.0 ± 1.41 14.67 ± 1.67 0.148 ± 0.00615.5 mM CCl4 + GH

10 µg mL−127.83 ± 0.75∗ 29.83 ± 0.75∗ 39.5 ± 1.37∗ 0.272 ± 0.008∗

15.5 mM CCl4 + GH25 µg mL−1

25.33 ± 2.48∗∗ 24.5 ± 0.54∗∗ 28.24 ± 1.09∗∗ 0.236 ± 0.008∗∗

15.5 mM CCl4 + GH50 µg mL−1

18.83 ± 1.21∗ 18.17 ± 1.16∗ 22.74 ± 2.28∗ 0.206 ± 0.008∗

15.5 mM CCl4 + GE10 µg mL−1

33.33 ± 1.04∗ 31.0 ± 0.89∗ 43.67 ± 1.03∗ 0.312 ± 0.008∗


26.5 ± 1.09∗∗ 25.33 ± 1.96∗ 33.5 ± 1.04∗∗ 0.284 ± 0.005∗∗


20.33 ± 1.36∗∗ 19.67 ± 0.81∗∗ 25.17 ± 2.13∗∗ 0.251 ± 0.007∗

15.5 mM CCl4 +GW10 µg mL−1

37.33 ± 0.81∗ 47.67 ± 0.81∗ 43.83 ± 0.75∗ 0.346 ± 0.055∗


30.0 ± 0.89∗ 43.0 ± 0.89∗ 38.5 ± 0.54∗ 0.326 ± 0.005∗


26.0 ± 0.89∗∗ 40.17 ± 1.94∗∗ 35.67 ± 0.81∗∗ 0.288 ± 0.008∗∗

15.5 mM CCl4 + 50 mM AA 13.67 ± 1.03∗∗ 15.33 ± 1.63∗∗ 19.83 ± 1.83∗∗ 0.189 ± 0.018∗∗

Note: Cytotoxicity was assessed in terms of % lactate dehydrogenase (LDH) released, and the responseto oxidative stress was measured in terms of antioxidant enzymes SOD; superoxide dismutase; CAT,Catalase; GR, glutathione reductase activity. Ascorbic acid was used as a standard. Values representmeans of at least three experiments and their standard deviation.∗p < 0.05; ∗∗p < 0.001.

with GW along with CCl4 (see Figure 2). Time course of lipid peroxidationwas assessed in the presence of a cytotoxic agent alone and together withdifferent extracts. CCl4-treated cells showed increase in lipid peroxidationparalleled with the increase in LDH release by the cells. However, in thepresence of GH and GW along with a cytotoxic agent, the lipid peroxidation,like the LDH release, returned to the control levels (see Figure 2).

CCl4-induced oxidative stress in the cells by generation of reactiveoxygen species (ROS). Antioxidant enzymes are known to be induced inresponse to ROS and play a role in detoxifying these ROS. In the case ofliver slices treated with CCl4, the amount of all three antioxidant enzymes(SOD, CAT, and GR) were increased (see Table 2). Activities of SOD, CAT,and GR were increased in the liver tissue treated with CCl4. The liver tis-sue treated with GH and GE along with CCl4 showed reduced antioxidantenzyme activities when added with the toxicants to the culture (see Table 2).

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FIGURE 2 Percentage release of lactate dehydrogenase (LDH) and extent of lipidperoxidation (LPO) in liver slice culture in CCl4-induced cytotoxicity. Values are mean ofthree experiments; CCl4 + carbon tetrachloride; AA = standard ascorbic acid, at 50 mMconcentration.

DISCUSSION

Liver slice is a microcosm of the intact liver consisting of a highly organizedcellular community in which different cell types are subject to mutual contact.Such cultures offer study of hepatotoxicity as it provides desirable complex-ity of structurally and functionally intact cells (6,23). Development of hepaticdisease in response to CCl4 exposure associated with metabolic imbalancein the liver led to the formation of ROS. Inadequate removal of ROS maycause cell damage by attacking membrane lipids and proteins and inacti-vating antioxidant enzymes, thus mediating several forms of tissue damage(9–11,16,22,25).

Lipid peroxidation in terms of MDA formation was increased withincrease of CCl4 exposure up to 2.0 h in the liver tissue treated with ethanol.However, the administration of GH and GE significantly decreased MDA

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formation in the liver tissue than in GW. These results would indicate thatthe GH and GE extracts could hinder their interaction with polyester fattyacids and could abolish the enhancement of lipid peroxidation process lead-ing to MDA formation (23). On application of GH and GE, the cytotoxiceffect was substantially lowered, probably through reduction of oxidativestress.

Activities of three antioxidant enzymes were measured to assess theoxidative stress in cells. Oxidative SOD and CAT are known to prevent dam-age by directly scavenging the harmful active oxygen species. With GH orGB added along with these pro-oxidants, the activity of all three enzymesdecreased to levels comparable with those seen either in untreated culturesor in cultures treated with the known antioxidant.

The key role of phenol compounds is to scavenge free radicals and ROSsuch as singlet oxygen, superoxide free radical, and hydroxyl radicals (11).GH and GE showed higher phenol content. A significant correlation wasshown by total phenol content and free radical–scavenging activities of allextracts. The results confirmed that greater antioxidant activity of GH andGE was probably due to their higher amount of phenol compounds.

The results in this study revealed that significant depletion was observedin lipid peroxidation; antioxidative enzymes SOD, CAT, and GR on adminis-tration of GH and GE; or ascorbic acid in the CCl4-induced toxicity in liver.This would indicate that GH and GW have antioxidant and hepatoprotectivepotentials.

In conclusion, the results of this study suggest that GH and GE couldprevent oxidative liver damage. Further comprehensive pharmacologicalinvestigations will be needed to validate the ethnobotanical claims and toelucidate the mechanism of this hepatoprotective effect.

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