In vitro and in vivo hepatoprotective and antioxidant activity of ethanolic extract from Meconopsis integrifolia (Maxim.) Franch

In vitro and in vivo hepatoprotective and antioxidant activity of ethanolic extract from Meconopsis integrifolia (Maxim.) Franch

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In vitro and in vivo hepatoprotective and antioxidant activity of ethanolic extract from Meconopsis integrifolia (Maxim.) Franch Gao Zhou, Yuxin Chen, Song Liu, Xingcheng Yao, Youwei Wang n Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (Wuhan University), Ministry of Education, and Institute of TCM & Natural Products, School of Pharmaceutical Sciences, Wuhan University, Wuhan 430071, PR China

art ic l e i nf o

a b s t r a c t

Article history: Received 14 October 2012 Received in revised form 25 February 2013 Accepted 15 May 2013

Ethnopharmacological relevance: Meconopsis integrifolia (Maxim.) Franch is a high mountain endemic species used as a traditional Tibetan and Mongolian herb to treat hepatitis, pneumonia, and edema. This study aims to investigate the hepatoprotective and antioxidant effects of Meconopsis integrifolia ethanolic extract (MIE) in vitro and in vivo. Materials and methods: The in vitro antioxidant property of MIE was investigated by employing various established systems. Rats with carbon tetrachloride (CCl4)-induced liver injury were used to assess the hepatoprotective and antioxidant effect of MIE in vivo. The level or activity of alkaline phosphatase (ALP), glutamate pyruvate transaminase (ALT), aspartate aminotransferase (AST), and total bilirubin (TB) in the blood serum and thiobarbituric acid reactive substances (TBARS), superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH) in the liver and kidney of the rats were assayed using standard procedures. Results: MIE exhibited strong antioxidant ability in vitro. In the rats with CCl4-induced liver injury, the groups treated with MIE and silymarin showed significantly lower levels of ALT, AST, ALP, and TB. MIE demonstrated good antioxidant activities in both the liver and kidney of the rats in vivo. Conclusions: MIE exhibits excellent hepatoprotective effects and antioxidant activities in vitro and in vivo, supporting the traditional use of Meconopsis integrifolia in the treatment of hepatitis. & 2013 Elsevier Ireland Ltd. All rights reserved.

Keywords: Antioxidant activity Carbon tetrachloride Hepatoprotective activity In vitro In vivo Meconopsis integrifolia

1. Introduction Liver diseases have become a health burden worldwide because of unhealthy dietary habits, environment pollution, and virus infections. The geographical environment in the Tibetan plateaus limits the agricultural development in the area. Therefore, traditional Tibetans have maintained a diet with high in saturated fat and low in fruits and vegetables (Moreno, et al., 2000; Owen and Johns, 2002), thereby resulting in a high incidence of liver and gallbladder diseases among the aboriginal adult people (Fang, 2009). The development and exchange of culture since the ancient times have greatly contributed to the preservation and dissemination of knowledge on traditional Tibetan medicine. The traditional Tibetan herbal medicines, which include numerous prescriptions used to treat liver diseases, are still widely used by the Tibetans. Several herbs, such as Halenia elliptica and Swertia mussotii, have been proven to possess good hepatoprotective activities based on modern pharmacology experiment method (Huang et al., 2010b; Lv et al., 2010). The earliest record of the application of Meconopsis integrifolia (Maxim.) Franch can be traced back to the eighth century

n

Corresponding author. Tel.: +86 276 875 9323; fax: +86 27 68759010. E-mail addresses: [email protected], [email protected] (Y. Wang).

(Yue Wang Yao Zhen, eighth century A.D.). Meconopsis integrifolia has been utilized as oubei to treat hepatitis, pneumonia, and edema. Meconopsis integrifolia, as one of the most widely distributed species of the genus, is a flagship species of the alpine scree in the Qinghai–Tibetan Plateau. The bright yellow flowers and leaf blade margins of the species make it distinguishable from other species (Yang et al., 2012). Meconopsis punicea, Meconopsis quintuplinervia, Meconopsis torquata, and Meconopsis lancifolia in the same genus were also used as oubei. Together with Meconopsis integrifolia, these herbs were believed to contain therapeutic effects in the treatment of hepatitis (Luo et al., 1984; Du et al., 2011; Wu et al., 2011). Modern pharmacological research on oubei mainly focuses on Meconopsis quintuplinervia. The ethanolic extract of Meconopsis quintuplinervia has significant hepatoprotective effects on rats with carbon tetrachloride (CCl4)-induced liver damage (Ding and Li, 2007). He et al. (2012) showed that the ethanolic extract of Meconopsis quintuplinervia exhibited strong in vitro and in vivo antioxidant activity. Zhou et al. (2009) compared the main alkaloids in six Meconopsis species. Their results showed that Meconopsis integrifolia contained less alkaloid compared with other species. Antioxidant effects have an important function in liver protection (Fraschini et al., 2002). This study aims to evaluate the in vitro and in vivo hepatoprotective and antioxidant activities of the Meconopsis integrifolia (Maxim.) Franch ethanolic extract (MIE).

0378-8741/$ - see front matter & 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jep.2013.05.027

iPlease cite this article as: Zhou, G., et. al., In vitro and in vivo hepatoprotective and antioxidant activity of ethanolic extract from Meconopsis integrifolia (Maxim.) Franch Journal Of Ethnopharmacology (2013), http://dx.doi.org/10.1016/j.jep.2013.05.027

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2. Materials and methods 2.1. Chemicals 2,2′-Azinobis-(3-ethylbenzthiazoline-6-sulfonate) (ABTS), α,αDiphenyl-β-picrylhy-drazyl (DPPH), and Folin–Ciocalteau's reagent were purchased from Sigma-Aldrich (St. Louis, MO). β-carotene was purchased from Fluka (Menlo Park, CA). Linoleic acid was purchased from Aladdintm. Ascorbic acid (Vc), gallic acid, rutin, and butylated hydroxytoluene (BHT) were purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). All other chemicals used in the analysis were of analytical grade and were obtained from the China Medicine (Group) Shanghai Chemical Reagent Corporation (Shanghai, China).

2.2. Plant materials Meconopsis integrifolia (whole plant) was purchased from the Tibetan Traditional Medicine Pharmaceutical Factory (Lhasa City, Tibet, China) and was authenticated under references and authoritative books (Flora of China, Pharmacopoeia Committee of Ministry of Health of China, 1995) by the corresponding author (Wuhan University, Wuhan, China). A specimen (No. 592) was deposited at the Institute of Traditional Chinese Medicine and Natural Products, School of Pharmaceutical Sciences, Wuhan University.

2.3. Preparation of plant extracts The powder (100 g, gradient size of less than 0.25 mm) of Meconopsis integrifolia was extracted under supersonic washer using 70% ethanol (1:10) at room temperature for 45 min. The solvent was evaporated via rotary evaporation at 35 1C. The residue was lyophilized, and the resulting dry powder was stored at 4 1C. The ethanol extract yield from Meconopsis integrifolia was 16.04% relative to the dry starting material. 2.4. Determination of total phenolic and flavonoid content The total phenolic content of the extract was determined using the Folin–Ciocalteau assay (Sabir and Rocha, 2008). The results were expressed in mg of gallic acid equivalents per g of dry extract. The total flavonoid content was determined using a colorimetric assay (Zheng et al., 2011), with rutin as standard. The results were expressed in mg of rutin equivalents per g of dry extract.

2.5. In vitro antioxidant activity 2.5.1. DPPH radical scavenging assay The free-radical-scavenging activity of MIE was measured using an improved DPPH assay (Huang et al., 2010a). The extract solution with concentration of 0.3 mL was mixed with a solution of 0.2 mmol/L DPPH in methanol (2.7 mL). The mixture was mixed vigorously, and then left to stand for 1 h at room temperature before measuring the absorbance value at 517 nm. Radical scavenging activity was calculated using the following equation: Percent inhibition rate ¼ ½ðAs−Ai=AsÞ  100; where As is the absorbance of DPPH alone, and Ai is the absorbance of DPPH in the presence of various extracts. The concentrations of BHT and Vc identical to the experimental samples were used as reference.

2.5.2. ABTS radical scavenging assay The ability of the extract to scavenge ABTS radical was determined according to a previously published method (Huang et al., 2011). ABTS was dissolved in deionized water at 7 mmol/L concentration, and potassium persulfate with a concentration of 2.45 mmol/L was added afterward. The reaction mixture was kept in the dark at room temperature for 16 h. The mixture was then diluted with 80% ethanol to obtain an absorbance value of 0.700 70.005 at 734 nm. Test substances (0.3 mL) at various concentrations were incubated with ABTS+ solution (2.7 mL) in a 30 1C water bath for 30 min in the dark. The absorbance at 734 nm was immediately recorded. Samples of BHT and Vc at the same concentrations were used as references. The level of radical scavenging was calculated using the aforementioned equation for DPPH.

2.5.3. Super oxide radical scavenging assay The capacity of MIE to scavenge superoxide radicals was examined using a pyrogallol auto-oxidation system (Xiang and Ning, 2008), with slight modifications. Reaction mixtures that contain test extracts (0.2 mg/mL) in Tris–HCl buffer (4.50 mL, 50 mmol/L, pH 8.2) were incubated for 10 min at 25 1C. Subsequently, 0.15 mL of pyrogallol (3 mmol/L, prepared in 10 mmol/L HCl) was added. The absorbance of the reaction mixture at 325 nm was measured immediately and at 30 s intervals thereafter. The auto-oxidation rate constant (Kb) of pyrogallic acid was calculated from the curve of 325 nm vs. time. The control did not contain test extracts, and concentrations of BHT and Vc identical to the samples were used as reference. The inhibitory actions of the test extracts on the auto-oxidation rate of pyrogallic acid correlated with their ability to scavenge superoxide radicals.

2.5.4. Reducing power assay The reducing power of the extracts was estimated using the method by Oyaizu (1986). Various concentrations of 0.2 mL extracts were mixed with 2.5 mL of phosphate buffer (0.2 mol/L, pH 6.6) and 2.5 mL of potassium ferricyanide (1%). After incubation at 50 1C for 20 min, 2.5 mL of trichloroacetic acid (10%) was added; each mixture was centrifuged at 1000 rpm for 10 min. Subsequently, 2.5 mL of the supernate was collected and mixed with 2.5 mL of deionized water and 0.5 mL of ferric chloride (0.1%). The absorbance was measured at 700 nm. The increased absorbance of the reaction mixture indicates an increase in reducing power. BHT and Vc were used as standards for comparison.

2.5.5. β-Carotene bleaching assay The antioxidant activity of MIE was evaluated according to the β-carotene bleaching method (Shon et al., 2003), with slight modifications. A solution of β-carotene was prepared by dissolving 6 mg of β-carotene in 20 mL of chloroform. Approximately 4 mL of the β-carotene solution, 80 mg of purified linoleic acid, and 800 mg of Tween80 emulsifier were then pipetted into a 500 mL round-bottom flask. After the mixture was thoroughly incorporated, chloroform was removed via vacuum, and 200 mL of aerated distilled water was then added to the flask with vigorous shaking. Aliquots (3.0 mL) of the resulting emulsion were transferred into different test tubes containing 0.2 mL solution of MIE (0.2 mg/mL), and were incubated in a water bath at 50 1C. Absorbance readings were recorded at 30-min intervals for 2 h. BHT was used as standards for comparison. Lipid peroxidation (LPO) inhibition was calculated as follows: LPO inhibition ¼[(As−Ai)/As]  100, where As is the initial absorbance of the assay and Ai is the absorbance of the assay after 2 h.

iPlease cite this article as: Zhou, G., et. al., In vitro and in vivo hepatoprotective and antioxidant activity of ethanolic extract from Meconopsis integrifolia (Maxim.) Franch Journal Of Ethnopharmacology (2013), http://dx.doi.org/10.1016/j.jep.2013.05.027

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2.5.6. Antioxidant activity in a linoleic acid system using ferrothiocyanate (FTC) and thiobarbituric acid (TBA) The ferric thiocyanate (FTC) test was conducted as described by Osawa and Namiki (1981), with slight modifications. Extracts (0.1 mg) in 1 mL of ethanol, 1 mL of 2.5% linolenic acid in ethanol, 2 mL of 50 mmol/L phosphate buffer (pH 7.0), and 1 mL of distilled water were mixed in a clean Eppendorf tube. The mixed solution was placed in a dark oven at 40 1C. Exactly 0.1 mL of the solution, 0.1 mL of 0.02 mol/L ammonium ferrous chloride in 3.5% hydrochloric acid, and 0.1 mL of 30% ammonium thiocyanate were added to 9.7 mL of 75% ethanol and 0.1 mL of 30% ammonium thiocyanate. After 3 min, the absorbance of the mixture was recorded at 500 nm. The solution replaced by 50% ethanol was used as blank absorption. The absorbance was determined at 24-h intervals until a constant maximum was reached. A mixture without the extract sample was used as control. BHT and Vc served as reference antioxidants. The thiobarbituric acid (TBA) value of the extract was assayed using the method by Kikuzaki and Nakatani (1993), with slight modifications. In this experiment, 0.67% TBA (1 mL) and 20% trichloroacetic acid (1 mL) were mixed thoroughly with 0.5 mL of the extract solution, and then placed in a boiling water bath for 10 min. As the mixture cooled, 4 mL of water saturation n-buOH extract was added to each sample via vortex blending, and then centrifuged at 3000 rpm for 10 min. The absorbance of the colored organic phase was measured at 532 nm. The inhibition rate was calculated using the following equation: [(Ac−As)/Ac]  100%, where Ac is the absorbance of the control and As is the absorbance of the sample. 2.6. In vivo hepatoprotective and antioxidant activity 2.6.1. Test animals Sprague–Dawley rats (220 720 g) of both sexes were purchased from the Experimental Animal Department of Tongji Medical College, Huazhong Science and Technology University. The rats were kept in a controlled environment at 25 72 1C and 30% to 60% relative humidity with a 12-h light and dark cycle. The animals were fed with standard rodent pellet diet and water ad libitum. The study received clearance from the Institutional Animal Ethical Committee of the Committee for the Purpose of Control and Supervision of Experiments on Animals, Wuhan University (Wuhan, China). 2.6.2. Carbon tetrachloride (CCl4)-induced oxidative toxicity The rats were randomly divided into six groups based on their body weight; each group contains six animals. Group I was given a gavage of distilled water with 0.3% sodium carboxymethylcellulose (CMC-Na) (1 mL/kg body weight, p.o.) daily for 5 d and olive oil on days 2 and 3 via intraperitoneal injection. Group II (CCl4 control) was given 0.3% CMC-Na (1 mL/kg bodyweight, p.o.) solution for 5 d and a 1:1 mixture of CCl4 and olive oil (2 mL/kg bodyweight, s.c.) on days 2 and 3. Group III (positive control) was given silymarin (100 mg/kg body weight, p.o.) daily for 5 d and a 1:1 mixture of CCl4 and olive oil (2 mL/kg bodyweight, s.c.) on days 2 and 3. Groups IV, V, and VI (test group) were given 100, 200, and 400 mg/ kg body weight of MIE, respectively, for 5 d and a 1:1 mixture of CCl4 and olive oil (2 mL/kg bodyweight, s.c.) on days 2 and 3. At the end of the experiment (day 6), the animals were anaesthetized by inhalation of ethyl ether. Blood was collected and allowed to clot. The serum was separated for the assessment of enzyme activity. The rats were then sacrificed by cervical dislocation; the liver and kidney samples were dissected, cleaned of blood with ice-cold saline, and immediately stored in a refrigerator for biochemical index determination.

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2.6.3. Biochemical determinations Biochemical parameters were assayed according to standard methods. Samples of the supernatants were withdrawn and analyzed for remaining protein by Bradford method (Bradford, 1976) and the calibration curve was prepared using solutions of bovine serum albumin (BSA). The protein concentration was calculated from calibration curve: C¼ 190.38A-12.40 (C: concentration of protein; A: absorbance at 595 nm, R2 ¼ 0.9992). The activities of the following serum index were measured: glutamatepyruvic transaminase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and total bilirubin (TB). Liver and kidney homogenates (10% w/v) were prepared in ice-cold physiological saline, and the resulting suspension was centrifuged at 1000 rpm for 10 min at 4 1C. The clear supernatant was used for the determination of superoxide dismutase (SOD), catalase (CAT), thiobarbituric acid reactive substances (TBARS), and glutathione (GSH). The assay was conducted using the kits obtained from the Institute of Biological Engineering of Nanjing Jianchen, (Nanjing, China) according to the protocol of the manufacturer; the results were expressed in mg per g protein. 2.7. Statistical analysis All experiments were done in triplicate, and the results were reported as mean 7 SD. Data were analyzed with one-way ANOVA. Statistically significant effects were further analyzed, and the means were compared using Duncan's multiple range test. Statistical significance was determined at p o0.05. 3. Results and discussion 3.1. Total phenolic and flavonoid content Phenolic and flavonoid compounds are recognized as material base of the antioxidant activity of plant extract (Kahkonen et al., 1999; Pietta, 2000). The total phenolic content of the extract from Meconopsis integrifolia was 141.78 74.86 mg/g gallic acid equivalent, and the total flavonoid content was 61.45 70.48 mg/g rutin equivalent. The results show that Meconopsis integrifolia has relatively high flavonoid content. 3.2. In vitro assays 3.2.1. DPPH and ABTS radical scavenging activity DPPH and ABTS are two relatively stable free radical compounds widely used to test free-radical-scavenging activity (Sanchez-Moreno, 2002). The radical scavenging activities of DPPH and ABTS in different concentrations of MIE are presented in Fig. 1. In the DPPH scavenging assay, the IC50 (the concentration required to scavenge 50% of radical) values of MIE, BHT, and Vc were 35.55 71.21 μg/mL, 18.72 70.48 μg/mL, and 5.40 70.05 μg/mL, respectively. In the ABTS scavenging assay, the IC50 values of MIE, BHT, and Vc were 5.57 70.39 μg/mL, 1.0770.06 μg/mL, and 1.36 7 0.05 μg/mL, respectively. 3.2.2. Super oxide radical scavenging assay The superoxide anion radical (dO2−) is the most common free radical generated in vivo. Pyrogallic acid can auto-oxidize under alkaline conditions to produce dO2− directly, and the rate constant of this auto-oxidation reaction is dependent on the dO2−concentration. The test compound can significantly slow down the autooxidation reaction of pyrogallic acid because of its ability to scavenge dO2− radicals (Xiang and Ning, 2008). The Kb value (  10−4 A/s) of the control, MIE, BHT, and Vc samples were 7.30 7 0.88, 3.937 0.30, 6.557 0.22, and 0.09 70.00, respectively.

iPlease cite this article as: Zhou, G., et. al., In vitro and in vivo hepatoprotective and antioxidant activity of ethanolic extract from Meconopsis integrifolia (Maxim.) Franch Journal Of Ethnopharmacology (2013), http://dx.doi.org/10.1016/j.jep.2013.05.027

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Fig. 3. Antioxidant activity of Meconopsis integrifolia extract in the β-carotene bleaching assay system. High absorbance indicates a strong antioxidant activity. All samples were measured at the same concentration of 0.2 mg/mL. MIE, Meconopsis integrifolia extract; BHT, butylated hydroxytoluene.

Fig. 1. Antioxidant activity of Meconopsis integrifolia ethanolic extract as determined by the (A) DPPH and (B) ABTS radical scavenging assay. MIE, Meconopsis integrifolia extract; BHT, butylated hydroxytoluene; Vc, ascorbic acid.

Fig. 2. Reducing power of the Meconopsis integrifolia extracts. MIE, Meconopsis integrifolia extract; BHT, butylated hydroxytoluene; Vc, ascorbic acid.

A smaller Kb corresponds to a stronger dO2− scavenging ability; Vc has the best dO2− scavenging ability. The dO2− scavenging ability of MIE is weaker than that of Vc, but better than that of BHT.

Fig. 4. Antioxidant activity of Meconopsis integrifolia extract assayed using the (A) FTC and (B) TBA methods. Low absorbance indicates a strong antioxidant activity. All samples were measured at the same concentration of 0.1 mg/mL. MIE, Meconopsis integrifolia extract; Vc, ascorbic acid; BHT, butylated hydroxytoluene. Statistically significant differences are indicated by asterisks (***p o 0. 001, **p o 0.01, compared with the control group).

iPlease cite this article as: Zhou, G., et. al., In vitro and in vivo hepatoprotective and antioxidant activity of ethanolic extract from Meconopsis integrifolia (Maxim.) Franch Journal Of Ethnopharmacology (2013), http://dx.doi.org/10.1016/j.jep.2013.05.027

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3.2.3. Reducing power The reducing power assay is a direct embodiment of the metal ion reductive ability. Increased absorbance is correlated with the reducing power of the extract. Fig. 2 shows that the dosedependent reducing capacities of the sample indicate that the order of reductive potential is Vc 4BHT 4MIE. 3.2.4. β-Carotene bleaching assay β-Carotene is a yellow or orange-red fat-soluble pigment that can be bleached by the auto-oxidation products of linoleic acid. Fig. 3 shows that BHT has the best activity, and the absorbance of the control group fell sharply with increased time. The LPO inhibition values of BHT and MIE were 79.5 7 1.6% and 35.274.5%. 3.2.5. Antioxidant activity in a linoleic acid system using ferrothiocyanate (FTC) and thiobarbituric acid (TBA) The auto-oxidation products of linoleic acid can oxidize Fe2+ into Fe3+. Fe3+ ions form a thiocyanate complex with SCN−, which has a maximum absorbance at 500 nm (Liu and Yao, 2007). The final products of linoleic acid oxidation, which includes TBARS, can react with TBA to form a red complex that can be determined at 532 nm. Fig. 4(A) shows that the absorbance of the control increases over time and reaches its peak value on day 8. MIE and BHT exhibited strong and comparative activities. Fig. 4 (B) shows that MIE and BHT also exhibited excellent antioxidant activities, and the inhibition rates of Vc, MIE, and BHT were 34.1 73.6%, 92.3 71.7%, and 96.2 7 3.6%. Based on our antioxidant experiment results in vitro, MIE exhibited obviously good antioxidant activity. Oxidative stress plays an important role in the pathogenesis of toxic liver diseases

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and other hepatic alterations (Feher et al., 1998). Reactive oxygen species (ROS) are produced by metabolism of normal cells. However, in liver diseases, ROS are increased and further lead to peroxidation of lipids, DNA, and proteins. Polyunsaturated lipids are essential to the entire supporting system of cells, including cell membranes, endoplasm-micreticulum, and mitochondria (Muriel, 2009). Lipid peroxidation is closely related to human diseases including liver injury (Jaeschke et al., 2002). Antioxidant is an important pathway to realize hepatoprotective effects (Fraschini et al., 2002). As a consequence, antioxidants have been proposed as an adjunct therapy for various liver diseases (Muriel, 2009). Some natural products with antioxidant activity have been proved to have good hepatoprotective effects (Lin et al., 2009; Huang et al., 2010a). There are two types of assays comprised by anti-lipid peroxidation and radical scavenging widely used for investigating antioxidant activity. In the present study, MIE showed good radical scavenging activity, which was even better than the positive control BHT in the super oxide radical scavenging assay. In the assays associated with anti-lipid peroxidation, MIE exhibited excellent anti-lipid peroxidation activity better than the positive control Vc and only slightly weaker than the positive control BHT. These suggest that MIE may have antioxidant and hepatoprotective activity in vivo. 3.3. In vivo assays 3.3.1. Result of biochemical determinations Hepatic injury through CCl4-induced lipid peroxidation is widely known and has been extensively used in experimental models to understand the cellular mechanisms behind oxidative damage and evaluate the therapeutic potential of drugs and

Fig. 5. Effect of Meconopsis integrifolia extracts on the biochemical parameters of CCl4-damaged livers of rats. (A) AST, aspartate aminotransferase; (B) ALT, glutamate pyruvate transaminase; (C) ALP, alkaline phosphatase; (D) TB, total bilirubin. Statistically significant differences are indicated by asterisks (***p o 0.001, **p o 0.01, *p o0.05, compared with the CCl4-intoxicated group). Group I: control; Group II: CCl4; Group III: silymarin 100 (mg/kg); Group IV: MIE 100 (mg/kg); Group V: MIE 200 (mg/kg); Group VI: MIE 400 (mg/kg).

iPlease cite this article as: Zhou, G., et. al., In vitro and in vivo hepatoprotective and antioxidant activity of ethanolic extract from Meconopsis integrifolia (Maxim.) Franch Journal Of Ethnopharmacology (2013), http://dx.doi.org/10.1016/j.jep.2013.05.027

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Fig. 6. The SOD, CAT, and GSH activities, and the level of TBARS in the liver and kidney of CCl4-intoxicated rats supplemented with silymarin or Meconopsis integrifolia extract. The values are in mean 7 SD, with n¼ 6 animals in each group. Statistically significant differences are indicated by asterisks (***p o 0.001, **p o 0.01, *p o 0.05 compared with CCl4-intoxicated group). (A) T-SOD, total superoxide dismutase; (B) CAT, catalase; (C) GSH, glutathione; and (D) TBARS, thiobarbituric acid reactive substances. Group I: control; Group II: CCl4; Group III: silymarin 100 (mg/kg); Group IV: MIE (100 mg/kg); Group V: MIE (200 mg/kg); and Group VI: MIE (400 mg/kg).

dietary antioxidants (Basu, 2003). In CCl4-induced toxicity, CCl4 is metabolized through the cytochrome P450 monooxygenase system to produce the trichloromethyl radical, which then reacts with oxygen to form the trichloromethyl-peroxyl radical (Shenoy et al., 2001). These radicals further attack cellular macromolecules, such as proteins or lipids, thereby leading to lipid peroxidation and cell necrosis in parts of the liver. ALT, AST, and ALP, which are important metabolic enzymes in liver cells, are released into the blood when the liver cells are damaged. TB is an index of normal hepatic metabolism. The TB levels in the blood serum can reflect the extent of liver injury. Fig. 5 shows that the ALT, AST, ALP, and TB levels are higher in the CCl4 group (II) than that in the control group (I) (p o0.01). Compared with the CCl4 group, the corresponding indicators of the rats treated with silymarin and MIE (III– IV) are significantly lower (p o0.05). The low concentration group (100 mg/kg body weight) of MIE showed the comparable effects with the positive control group (silymarin, 100 mg/kg body weight). MIE did not reveal a significant dose effect because the level of the indicators approximates that of the control group. The CCl4-induced hepatotoxicity model is extensively utilized in the evaluation of the antioxidant effects of drugs and plant extracts (Bhathal et al., 1983; Weber et al., 2003; Awaad et al., 2006). Both SOD and CAT have important functions in defense mechanisms against the harmful effects of reactive oxygen species and free radicals in biological systems. GSH content is another important parameter that reveals oxidative damage in liver and kidney. TBARS are cytotoxic products that are hallmarks of lipid peroxidation (Huang et al., 2010a). Determinations of the in vivo antioxidant activities of MIE in liver and kidney are presented in

Fig. 6. Compared with the control group, the CCl4-intoxicated rats exhibited a significant decrease in total SOD (liver and kidney), CAT (liver), and significant increase in the level of TBARS (in liver and kidney) (p o0.001). These changes were significantly reversed after treatment with MIE and silymarin. The CAT in the kidney and the GSH in both liver and kidney did not exhibit a significant decrease (p4 0.05); however, MIE showed an increasing trend. The identity of the operation will also have an impact to the experimental results.

4. Conclusions Based on the results, the ethanolic extract of Meconopsis integrifolia exhibited excellent hepatoprotective effects and good in vitro and in vivo antioxidant activity in rats with CCl4-induced liver injury, supporting the traditional use of this plant to treat hepatitis. However, further research on the pharmacological activities of this plant is still needed.

Acknowledgment This work was supported by the National Mega Project on Major Drug Development (2011ZX09401-302), the Commonwealth Specialized Research Fund of China Agriculture (201103016), the Key Program of Natural Science Foundation of Hubei Province, China (2010CBB02301).

iPlease cite this article as: Zhou, G., et. al., In vitro and in vivo hepatoprotective and antioxidant activity of ethanolic extract from Meconopsis integrifolia (Maxim.) Franch Journal Of Ethnopharmacology (2013), http://dx.doi.org/10.1016/j.jep.2013.05.027

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iPlease cite this article as: Zhou, G., et. al., In vitro and in vivo hepatoprotective and antioxidant activity of ethanolic extract from Meconopsis integrifolia (Maxim.) Franch Journal Of Ethnopharmacology (2013), http://dx.doi.org/10.1016/j.jep.2013.05.027