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Journal of Ethnopharmacology 131 (2010) 276–281

Contents lists available at ScienceDirect

Journal of Ethnopharmacology

journa l homepage: www.e lsev ier .com/ locate / je thpharm

epatoprotective and antioxidant effects of the methanolicxtract from Halenia elliptica

o Huang, Xiaoquan Ban, Jingsheng He, Hong Zeng, Peng Zhang, Youwei Wang ∗

nstitute of the TCM and Natural Products, College of Pharmacy, Wuhan University, Donghu Road 185, Wuhan 430071, Hubei, People’s Republic of China

r t i c l e i n f o

rticle history:eceived 23 December 2009eceived in revised form 12 June 2010ccepted 19 June 2010vailable online 25 June 2010

eywords:alenia ellipticaepatoprotective activityarbon tetrachlorideadical scavenging

a b s t r a c t

Aims of study: Halenia elliptica, a medicinal herb of Tibetan origin, was commonly used in folk medicine totreat hepatitis. The aim of the present study is to evaluate the hepatoprotective and antioxidant activityof Halenia elliptica against experimentally induced liver injury.Materials and methods: The antioxidant property of methanolic extract (ME) of Halenia elliptica was inves-tigated by employing various established in vitro systems. The ME of Halenia elliptica was studied here forits hepatoprotective effects against CCl4-induced liver toxicity in rats. Activity was measured by moni-toring the levels of alanine aminotransferase (ALT), aspartate transaminase (AST), alkaline phosphatase(ALP), and total bilirubin.Results: The ME possessed strong antioxidant activity in vitro. The results of CCl4-induced liver toxicityexperiment showed that rats treated with the ME of Halenia elliptica (100 mg/kg and 200 mg/kg), and

ntioxidant activity also the standard treatment, silymarin (50 mg/kg), showed a significant decrease in ALT, AST, ALP, andtotal bilirubin levels, which were all elevated in the CCl4 group (p < 0.01). The results observed afteradministration of 100 mg/kg ME were comparable to those of silymarin at 50 mg/kg (p > 0.05). The MEdid not show any mortality at doses up to 2000 g/kg body weight.Conclusion: These results seem to support the traditional use of Halenia elliptica in pathologies involvinghepatotoxicity, and the possible mechanism of this activity may be due to strong free radical-scavengingand antioxidant activities of ME.

. Introduction

Despite tremendous advances in modern medicine, hepaticisease remains a worldwide health problem; thus the searchor new medicines is still ongoing. Numerous formulations of

edicinal plants are used to treat liver disorders in Chinesethnomedical practice and traditional medicine. Many of thesereatments act as radical scavengers, whereas others are enzymenhibitors or mitogens (Fadhel and Amran, 2002; He et al., 2002).

any drugs and plant extracts have been evaluated for hepato-rotective and antioxidant effects against chemical-induced liveramage in a carbon tetrachloride-induced hepatotoxicity modelBhathal et al., 1983). Carbon tetrachloride accumulates in hep-tic parenchyma cells and is metabolized to CCl3• radicals by

iver cytochrome P450-dependent monooxygenases (Recknagel,983).

The genus Halenia (family Gentianaceae) has approximately 100pecies distributed mainly in America but with a few species in

∗ Corresponding author. Tel.: +86 27 68759323; fax: +86 27 68759010.E-mail address: wyw@whu.edu.cn (Y. Wang).

378-8741/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.jep.2010.06.029

© 2010 Elsevier Ireland Ltd. All rights reserved.

Asia. Halenia elliptica is one of two species distributed in Qinghai-Tibet Plateau (Ji et al., 2004). Halenia elliptica is a medicinal herbof Tibetan origin, and in China, was commonly used for clearingthe “fire and heat of the liver and gall bladder” in the treatment ofhepatitis (Luo, 1997). Previous reports have shown that this planthas other biological activities, such as involvement in vasodilation(Wang et al., 2009), antioxidant activity (Gao et al., 2004), antibac-terial and antitumor activity (Dai et al., 2002). Halenia ellipticacontains several phenolic compounds, such as xanthones (Zhanget al., 1983, 1987; Dhasmana, 1989; Zhang et al., 2003; Gao et al.,2004; Liu et al., 2009; Wang et al., 2009) and flavonoids (Yang etal., 2006).

Research shows that hepatoprotective effects have been associ-ated with plant extracts that are rich in phenolic compounds (Deet al., 1996; Shanmugasundaram and Venkataraman, 2006; Jain etal., 2008; Sabir and Rocha, 2008; Huang et al., 2010; Srivastava andShivanandappa, 2009; Zeashan et al., 2009). However, to the best

of our knowledge, the hepatoprotective effects of Halenia elliptica,against CCl4-induced liver injury in rats has not been demonstrated.The present study focused on evaluating the potential hepato-protective effects of methanolic extracts from Halenia elliptica onCCl4-induced liver injury in rats.

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. Materials and methods

.1. Chemicals

�,�-Diphenyl-�-picrylhydrazyl (DPPH), Folin-Ciocalteau’seagent and silymarin was purchased from Sigma–Aldrich (St.ouis, MO). 2,2′-Azinobis(3-ethylbenzthiazoline-6-sulphonic acidABTS) was purchased from Fluka (Menlo Park, CA). Gallic acid,utin, and butylated hydroxytoluene (BHT) were purchased fromhe National Institute for the Control of Pharmaceutical and Biolog-cal Products (Beijing, China). All other chemicals used for analysis

ere AnalaR grade and obtained from China Medicine (Group)hanghai Chemical Reagent Corporation (Shanghai, China).

.2. Plant materials

Halenia elliptica (whole plant) was purchased in Tibetan Tradi-ional Medicine Pharmaceutical Factory, Lhasa city, Tibet, P.R. ofhina, and authenticated under references and authoritative booksWang et al., 1997; Editor Committee of National Chinese Medical

anage Bureau, 1999; Li et al., 2008) by Prof. Qing Wang, Wuhanotanical garden, Chinese Academy of Sciences, Wuhan, China.

.3. Preparation of plant extracts

The powder (50 g, size was less than 0.25 mm) of Halenia ellipticaas extracted with 70% methanol at room temperature for 24 h.

olvent was evaporated by rotary evaporation at 35 ◦C. The residueas lyophilized and the resulting dry powder was stored at 4 ◦C. The

ield of methanolic extract (ME) from Halenia elliptica was 10.67%elative to dry starting material.

.4. Determinations of total phenolic contents

Total phenolic content of ME was determined using the Folin-iocalteau assay according to the method previously describedSabir and Rocha, 2008). Total phenolic content was calculated fromhe calibration curve of a gallic acid standard solution. Results werexpressed as gallic acid equivalents, in mg/g dry extract.

.5. DPPH radical scavenging assay

The free-radical-scavenging activity of ME was measured by ourreviously published method (Huang et al., 2010). Extract solutiont a range of concentrations (0.3 mL) was mixed with a solution of.2 mM DPPH in methanol (2.7 mL). The mixture was shaken vig-rously and allowed to stand for 1 h before the absorbance waseasured at 517 nm. Radical scavenging activity was calculated

s the following percentage: [(As − Ai/As)] × 100 (As = absorbancef DPPH alone, Ai = absorbance of DPPH in the presence of variousxtracts). A concentration of BHT that was identical to the experi-ental samples was used as reference.

.5.1. ABTS radical scavenging assayThe ability of ME to scavenge the ABTS radical cation was

etermined by our previously published method (Huang et al.,010). A solution of ABTS radical cation (ABTS+) was preparedy the reaction of 7 mM ABTS and 2.45 mM potassium persul-ate at room temperature in the dark for 16 h. The ABTS+ solutionas then diluted with 80% ethanol to obtain an absorbance of

.700 ± 0.005 at 734 nm. Extract solution at a range of concentra-ions (0.3 mL) was mixed with ABTS+ solution (2.7 mL), the reaction

ixture was allowed to stand at 30 ◦C for exactly 30 min, andbsorbance at 734 nm was recorde. BHT at the same concentra-ion as the samples was used as reference. The level of radical

macology 131 (2010) 276–281 277

scavenging was calculated using the equation described above forDPPH.

2.5.2. Scavenging of •OH radicalsThe ability of the ME to scavenge •OH was determined as

described previously (Kaur et al., 2008) with slight modifications.The reaction mixture contained 500 mL of 2-deoxyribose (2.8 mM)in potassium phosphate buffer (50 mM, pH 7.4), 200 mL of pre-mixed ferric chloride (100 mM) and EDTA (100 mM) solution (1:1,v/v), 100 mL of H2O2 (200 mM) without or with the extract solu-tion (100 mL). The reaction was initiated by the addition 100 mL of300 mM ascorbate and incubated for 1 h at 37 ◦C. A solution of thio-barbituric acid (TBA) in 1 mL (1%, w/v) of 50 mM NaOH and 1 mL of2.8% (w/v, aqueous solution) trichloroacetic acid (TCA) was added.The mixture was heated for 15 min in a boiling water bath and thencooled. The absorbance was measured at 532 nm. The absorbance ofthe control was determined by replacing the sample with methanol.BHT at the same concentration as the samples was used as refer-ence. Radical scavenging activity was calculated using the equationdescribed above for DPPH.

2.5.3. Superoxide radical scavenging assayThe capacity of ME to scavenge superoxide radicals was exam-

ined by a pyrogallol auto-oxidation system (Xiang and Ning, 2008)with slight modifications. Briefly, reaction mixtures containing testextracts (0.2 mg/mL) in Tris–HCl buffer (4.50 mL, 50 mM, pH 8.2)were incubated for 10 min at 25 ◦C, and then 150 �L of pyrogallicacid (3 mM, prepared in 10 mM HCl) was added. The absorbanceof the reaction mixture at 325 nm was measured immediately, andthen at 30 s intervals thereafter. The auto-oxidation rate constant(Kb) of pyrogallic acid was calculated from the curve of A325 nmvs. time. The control did not contain test extracts and a concen-tration of BHT identical to the samples was used as a reference.The inhibitory actions of test extracts on the auto-oxidation rate ofpyrogallic acid correlated with their ability to scavenge superoxideradicals.

2.5.4. ˇ-Carotene bleaching assayAntioxidant activity of ME was evaluated according to the

�-carotene bleaching method (Shon et al., 2003) with slight mod-ifications. A solution of �-carotene was prepared by dissolving6 mg of �-carotene in 20 mL of chloroform. Four millilitres of thissolution was pipetted into a 500 mL round-bottom flask. After chlo-roform was removed under vacuum, 80 mg of purified linoleic acid,800 mg of Tween 80 emulsifier, and 200 mL of aerated distilledwater were added to the flask with vigorous shaking. Aliquots(3.0 mL) of this emulsion were transferred into different test tubescontaining 0.2 mL solution of ME (500 mg/mL). BHT was used forcomparative purposes. As soon as the emulsion was added toeach tube, the zero time absorbance was measured at 470 nm. Thetubes were placed at 50 ◦C in a water bath. Absorbance readingswere then recorded at 30-min intervals in 2 h. Lipid peroxidation(LPO) inhibition was calculated using the following equation: LPOinhibition = [(As − Ai)/As] × 100 (As = absorbance of control of assay;Ai = absorbance of 2 h later of assay).

2.5.5. Antioxidant activity in a linoleic acid system usingferrothiocyanate (FTC)

The FTC method was adapted from Osawa and Namaki (1981).ME (400 �g) in ethanol (4 mL) was mixed with 2.5% linoleic acidin ethanol (4 mL), phosphate buffer (8 mL, 50 mM, pH 7.0), and

distilled water (4 mL). The mixtures were incubated at 40 ◦C inscrew-cap tubes in the dark. Aliquots (0.1 mL) were withdrawn andmixed with 75% ethanol (9.7 mL) and 30% ammonium thiocyanate(0.1 mL). Ferrous chloride (0.1 mL, 20 mM in 3.5% HCl) was addedto each aliquot, and precisely 3 min later, absorbance at 500 nm

2 opharmacology 131 (2010) 276–281

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Table 1Antioxidant activity of various extracts of Halenia elliptica.

Sample IC50 values LPO

DPPH (�g/mL) ABTS (�g/mL) •OH (mg/mL) �-Carotene (%)

ME 354.40 ± 17.49 13.00 ± 1.20 0.641 ± 0.011 39.24 ± 0.19BHT 20.76 ± 0.95 2.80 ± 0.09 1.774 ± 0.025 44.64 ± 0.22

IC value was determined to be the effective concentrations at which radicals were

can significantly slow down the auto-oxidation reaction of pyro-gallic acid (Xiang and Ning, 2008). As indicated in Table 2, thescavenging effect of ME on the superoxide anion radical was moreefficient than BHT. These results suggest that the extracts of Halenia

Table 2

78 B. Huang et al. / Journal of Ethn

as measured. Aliquots were withdrawn and assayed in an iden-ical fashion at 24 h intervals until a constant maximum value waseached. Controls without extract and standard containing BHT inlace of extract were subjected to the same procedure.

.6. Test animals

Sprague–Dawley rats (220 ± 20 g) and KM mice (20 ± 2 g) of bothexes were purchased from the Laboratory Animal Center of Wuhanniversity (Wuhan, China). They were housed on a 12 h light–darkycle at 25 ± 2 ◦C, and in a relative humidity of 30–60%. Animalsere fed ad libitum on a diet of standard pellets and water. The

nimals were allowed to acclimate to the housing conditions for 3ays prior to experimentation. The study received clearance fromhe Institutional Animal Ethical Committee (IAEC) of the Commit-ee for the Purpose of Control and Supervision of Experiments onnimals (CPCSEA), Wuhan University, Wuhan, China.

.7. Acute toxicity studies

After fasting overnight, mice were administered ME in gradedoses of up to 2000 mg/kg body weight. Animals were observed forymptoms of toxicity and mortality for 72 h.

.8. Carbon tetrachloride-induced oxidative toxicity

Rats were divided into six groups consisting of six animals inach group. Rats in group I received distilled water containing 0.3%odium carboxymethylcellulose (CMC-Na) (1 mL/kg body weight,.o.) for 5 days, and olive oil (1 mL/kg body weight, s.c.) on daysand 3 (Jain et al., 2008). Group II (CCl4) received 0.3% CMC-Na

1 mL/kg body weight, p.o.) for 5 days, and a 1:1 mixture of CCl4nd olive oil (2 mL/kg body weight, s.c.) on days 2 and 3. GroupII was treated with the standard drug silymarin (50 mg/kg body

eight, p.o.) daily for 5 days, and also received the CCl4–olive oilixture (1:1, 2 mL/kg body weight, s.c.) on days 2 and 3, 30 min

fter administration of silymarin. Groups IV–VI (test group animals)ere administered a dose of 50, 100, and 200 mg/kg body weight ofE (p.o.) for 5 days. Additionally, 30 min after administration of ME,

hey received a dose of the CCl4–olive oil mixture (1:1, 2 mL/kg, s.c.)n days 2 and 3. On day 6, animals were anaesthetized by inhala-ion of ethylether, blood was collected, allowed to clot, and serumas separated for assessment of enzyme activity. The rats were

hen sacrificed by bleeding, the livers were carefully dissected andleaned of excess tissue. Part of the liver tissue was immediatelyransferred into 10% formalin for histopathological investigation.

.9. Biochemical determinations

Biochemical parameters were assayed according to standardethods. Activity of the following serum enzymes was measured:

lutamate pyruvate transaminase (ALT), aspartate aminotrans-erase (AST), and alkaline phosphatase (ALP) using the method ofing (1965). Total bilirubin was measured by the method of Malloynd Evelyn (1937). Assay kits were obtained from the Institute ofiological Engineering of Nanjing Jianchen (Nanjing, China).

.10. Histopathological studies

Liver tissues were fixed in 10% formalin for at least 24 h, embed-ed in paraffin, and cut into 5 �m-thick sections using a rotaryicrotome. The sections were stained with Haematoxylin–Eosin

ye and observed under a microscope (IX51, Olympus, Japan) tobserve histopathological changes in the liver.

50

inhibited by 50%. LPO, lipid peroxidation inhibition; ME, 70% methanol extract; BHT,butylated hydroxytoluene. Each value was presented as the mean ± S.E.M. of threereplicate measurements.

2.11. Statistical analysis

All experiments were done in triplicate and results werereported as mean ± S.E.M. (n = 6). The data were analyzed byone-way ANOVA, and statistically significant effects were furtheranalyzed by means comparison using Duncan’s multiple range test.Statistical significance was determined at p < 0.05.

3. Results and discussion

Phenolic compounds have been reported to be responsible forthe antioxidant activity (Moreno et al., 2000; Huang et al., 2010;Jain et al., 2008; Kaur et al., 2008; Sabir and Rocha, 2008). The totalphenolic content of ME was determined to be 358.99 ± 6.21 mg/ggallic acid equivalent. The antioxidant activity of ME was deter-mined by its capacity to scavenge the stable free radicals DPPH andABTS, which have been widely used to test free-radical-scavengingactivity, and the results were compared with the scavenging abil-ity of control samples of BHT (Table 1). In the DPPH scavengingassay, the IC50 (the concentration required to scavenge 50% ofradical) values of ME and BHT were 354.40 ± 17.49 �g/mL and20.76 ± 0.95 �g/mL, respectively. In the ABTS assay, the IC50 val-ues of ME and BHT were 13.00 ± 1.20 �g/mL and 2.80 ± 0.09 �g/mL,respectively.

The •OH-radical-generating system containing EDTA, FeCl3,H2O2, and ascorbic acid (Fenton reaction model system) was usedto generate •OH radicals. Ascorbic acid increases the rate of •OHgeneration by reducing iron and maintaining a supply of Fe2+-EDTA. The generated •OH radical can degrade deoxyribose into aseries of fragments that react with TBA upon heating and at lowpH. This reaction forms a pink color that absorbs 532 nm light.When added to the reaction mixture, the test compounds com-pete with deoxyribose for •OH radicals and inhibit deoxyribosedegradation (Table 1). The ME showed greater radical-scavengingactivity (IC50 0.641 ± 0.011 mg/mL) than the synthetic antioxidantBHT (IC50 1.774 ± 0.025 mg/mL).

The superoxide anion radical (•O2−) is the most common free

radical generated in vivo. Pyrogallic acid can auto-oxidize underalkaline conditions to produce •O2

− directly, and the rate constantof this auto-oxidation reaction is dependent on the •O2

− concentra-tion. Due to its ability to scavenge •O2

− radicals, the test compound

Inhibition of pyrogallic acid auto-oxidation by the methanol extract of Haleniaelliptica.

Sample Control ME BHT

Kb value (×10−4) 14.68 ± 0.01 10.87 ± 0.02 13.69 ± 0.01

ME, 70% methanol extract; BHT, butylated hydroxytoluene. Each value was pre-sented as the mean ± S.E.M. of three replicate measurements.

B. Huang et al. / Journal of Ethnophar

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ig. 1. Antioxidant activity monitored daily using the FTC method. Low absorbancendicates strong antioxidant activity. ME, 70% methanol extract; BHT, butylatedydroxytoluene.

lliptica are capable of scavenging superoxide radicals, and couldelp prevent or ameliorate oxidative damage.

The antioxidant activity of carotenoids is based on the radi-al adducts of carotenoid with free radicals from linoleic acid. Theinoleic acid free radical attacks the highly unsaturated �-carotene

odels (Shon et al., 2003). The results of �-carotene bleaching assayhowed that ME was as effective as BHT as an antioxidant (Table 1),nd the LPO of ME and BHT was 39.24 ± 0.19% and 44.64 ± 0.22%,espectively.

The antioxidant activity exhibited by ME according to the FTCethods was shown in Fig. 1. The FTC method provides a mea-

ure of the amount of peroxide generated at the initial stage ofipid peroxidation. Peroxides formed during linoleic acid oxida-ion convert Fe2+ to Fe3+, and the Fe3+ ions are determined bybsorbance at 500 nm following complexation with SCN− (Osawand Namaki, 1981). Thus antioxidant activity is detected as a reduc-ion in absorbance at 500 nm. The results in Fig. 1 showed that MEad strong antioxidant activity, but less efficient than BHT.

In acute toxicity studies, animals injected with ME did nothow any symptoms of toxicity or mortality when dosed up to000 mg/kg body weight.

The hepatoprotective effect of ME was assessed by measur-

ng liver-related biochemical parameters following CCl4-inducedepatotoxicity in vivo. The values of liver-related biochemicalarameters observed in the control group (I), CCl4 group (II), sily-arin group (III) and ME-treated groups (IV–VI) are presented in

able 3. The activity of the enzymes ALT, AST, ALP, and the total

able 3ffect of 70% methanolic extract from Halenia elliptica on biochemical parameters of CCl4

Group ALT (IU/L) AST (IU/

Group I control 35.40 ± 2.00 134.26Group II CCl4 286.90 ± 23.22 745.08Group III silymarin 50 (mg/kg) 150.22 ± 19.77** 381.12Group IV ME 50 (mg/kg) 234.20 ± 20.89* 560.50Group V ME 100 (mg/kg) 119.91 ± 13.21*** 440.96Group VI ME 200 (mg/kg) 120.50 ± 24.48*** 318.52

alues are mean ± S.E.M., n = 6 animals in each group. Symbols represent statistical signifiE, 70% methanolic extract of Halenia elliptica; ALT, glutamate pyruvate transaminase; A* p < 0.05, as compared to CCl4-intoxicated group.

** p < 0.01, as compared to CCl4-intoxicated group.*** p < 0.001, as compared to CCl4-intoxicated group.

macology 131 (2010) 276–281 279

bilirubin value were significantly increased in the CCl4 group com-pared to the control group (p < 0.001). The rats treated with ME(50 mg/kg, group IV) showed a significant decrease in ALT and ALPcompared to the CCl4 group (p < 0.05), but the AST and total biliru-bin values were comparable to those of CCl4 group (p > 0.05). Therats treated with ME (100 mg/kg, group V and 200 mg/kg, groupVI), and also the standard treatment silymarin (50 mg/kg, groupIII), showed a significant decrease in all of the parameters that wereelevated in the CCl4 group (p < 0.01). Overall, the results observedafter administration of 100 mg/kg ME were comparable to thoseof silymarin at 50 mg/kg (p > 0.05). Although silymarin and higherdosages of ME showed a strong hepatoprotective effect againstCCl4-induced liver injury, the levels of liver-related biochemicalparameters were still significantly higher than those of the controlgroup which did not receive CCl4 (p < 0.01).

The CCl4-induced hepatotoxicity model is extensively used toevaluate the hepatoprotective effects of drugs and plant extracts(Bhathal et al., 1983). Carbon tetrachloride is accumulated inhepatic parenchyma cells and metabolized to the CCl3 radicalby cytochrome P450-dependent monooxygenases in the liver(Recknagel, 1983). The CCl3 radical reacts very rapidly with oxygento yield a highly reactive trichloromethyl peroxyl radical, CCl3OO•.These radicals react with proteins and lipids. They facilitate theremoval of hydrogen atoms from unsaturated lipids and lead tolipid peroxidation (Recknagel et al., 1989). It has been reportedthat one of the principal causes of CCl4-induced liver injury islipid peroxidation, which is induced and accelerated by, free radi-cal derivatives of CCl4 (Maling et al., 1974). In this study, the freeradical scavenging activity of ME was determined using severalradicals, DPPH, ABTS, hydroxy radical and superoxide anion rad-ical. Our results showed that LLE had strong scavenging activitycompared with synthetic antioxidants, indicating that it was alsoa potent antioxidant. It has been reported that Halenia elliptica isrich in xanthones and flavonoids (Dhasmana, 1989; Yang et al.,2006), which are known as free radical scavengers (Cakir et al.,2003; Balasundari et al., 2005; Conforti et al., 2008). Therefore, thehepatoprotective activity of ME may be due to the presence of phe-nolic compounds in the extracts, but detailed information on thefree-radical-scavenging activity of Halenia elliptica is still unknownand requires further investigation.

Carbon tetrachloride can lead to hepatic damage that histologi-cally resembles viral hepatitis. Loss of metabolic enzymes that arelocated in intracellular structures results from changes in the endo-plasmic reticulum (Recknagel, 1983). Therefore, hepatoprotectiveactivity was evaluated by measuring total bilirubin, measuringactivity of the enzymes AST, ALT, and ALP, and by histopathological

examination. In our studies, CCl4-damaged rats that were previ-ously treated with ME showed a significant decrease in serum ASTand ALT. This is evidence that both stabilization of the plasma mem-brane and repair of CC14-induced hepatic tissue damage. Hepaticinjury by CCl4 results in raised ALP and total bilirubin levels, which

-damaged livers in rats.

L) ALP (IU/L) Total bilirubin (mg/dL)

± 11.48 114.00 ± 1.60 0.76 ± 0.08± 104.35 248.83 ± 13.89 3.13 ± 0.28± 42.67* 125.86 ± 9.03*** 1.61 ± 0.15***

± 63.71 194.30 ± 12.72* 2.40 ± 0.15± 51.76** 136.66 ± 8.99*** 2.03 ± 0.09**

± 42.33** 151.78 ± 7.84*** 1.67 ± 0.14***

cance.ST, aspartate aminotransferase; ALP, alkaline phosphatase.

280 B. Huang et al. / Journal of Ethnophar

Fig. 2. Liver sections from CCl4-treated rats showing the ventral vein (CV) and hep-atic cells (hematoxylin–eosin staining, 100×). (A) Control group, (B) CCl4-olive oil(1:1, 2 mL/kg), (C) CCl4 + 70% methanolic extracts of Halenia elliptica (100 mg/kg).

macology 131 (2010) 276–281

could reflect a pathological alteration in biliary flow. ME treatmentreduced the level of both, suggesting that it was able to stabilizeCCl4-induced biliary dysfunction in the rat liver. Histological obser-vations also supported the results from the serum assays. To a largeextent, ME administration reversed the hepatic lesions caused byCCl4 toxicity.

Histopathological observations of liver sections from the controlgroup showed normal cellular architecture with distinct hepaticcells, sinusoidal spaces and a central vein (Fig. 2A). In contrast, theCCl4 group exhibited the most severe damage of any of the groups.The liver sections in this group showed massive fatty changes,necrosis, ballooning degeneration, broad infiltration of lympho-cytes, and the loss of cellular boundaries (Fig. 2B). The liver sectionsof the rats treated with ME (Fig. 2C) showed a relatively normallobular pattern with a mild degree of fatty change, necrosis, andlymphocyte infiltration that was more similar to the control group(Fig. 2A).

4. Conclusion

Based on these results, it can be concluded that the methanolicextracts of Halenia elliptica seems to have hepatoprotective effectsin rats. No toxic symptoms or mortality was observed after 72 hin mice. These results support the traditional use of this plant inhepatotoxic disorders.

Acknowledgements

This work was supported by the Commonweal SpecializedResearch Fund of China Agriculture (No. 3-21), the ImportantNational Science & Technology Specific Projects (No. 2009ZX09301-14) and the Fundamental Research Funds for the CentralUniversities (20103010101000175).

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