Research Article Received: 6 April 2013
Revised: 2 September 2014
Accepted article published: 16 September 2014
Published online in Wiley Online Library: 14 October 2014
(wileyonlinelibrary.com) DOI 10.1002/jsfa.6904
Protective effects of Cholestin on ethanol induced oxidative stress in rats You-Liang Hsieh,a Yen-Hung Yeh,b,c* Ya-Ting Leed and Chih-Yang Huanga,e,f Abstract BACKGROUND: Male Wistar rats were divided into seven groups as follows: group A, basal diet; group B, basal diet with Cholestin at 0.1667 g kg−1 body weight (BW); groups C–F, oral feeding of ethanol at 7.9 g kg−1 BW; groups D–F, Cholestin in diet at 0.1667, 0.3333 and 0.5 g kg−1 BW respectively; group G, silymarin in diet at 200 mg kg−1 BW. RESULTS: The results showed that treatment with Cholestin for 8 weeks reduced the impact of ethanol toxicity on serum markers of liver damage: aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP). The antioxidant system was significantly enhanced: plasma and hepatic thiobarbituric acid-reactive substance (TBARS) levels were lowered while hepatic superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), ethanol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) activities and non-enzymatic antioxidants (vitamin E, vitamin C and GSH) were elevated. CONCLUSION: Cholestin shows a protective effect against hepatotoxicity indices in ethanol-fed rats comparable to that of silymarin, as supported by the evaluation of liver histopathology. The data suggest that Cholestin exerts its hepatoprotective effect by decreasing lipid peroxidation and improving antioxidants status, thus proving itself as an effective antioxidant in ethanol-induced oxidative damage in rats. © 2014 Society of Chemical Industry Keywords: Cholestin; antioxidant activity; ethanol; hepatoprotective; hepatotoxicity
INTRODUCTION
J Sci Food Agric 2015; 95: 799–808
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Correspondence to: Yen-Hung Yeh, School of Health Diet and Industry Management, Chung Shan Medical University, Taichung, Taiwan, ROC. E-mail: [email protected]
a Department of Health and Nutrition Biotechnology, Asia University, Taichung, Taiwan, ROC b School of Health Diet and Industry Management, Chung Shan Medical University, Taichung, Taiwan, ROC c Department of Nutrition, Chung Shan Medical University Hospital, Taichung, Taiwan, ROC d Department of Beauty Science, National Taichung University of Science and Technology, Taichung, Taiwan, ROC e Graduate Institute of Basic Medical Science, China Medical University, Taichung, Taiwan, ROC f Department of Chinese Medicine, China Medical University Hospital, Taichung, Taiwan, ROC
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Of the various health problems suffered by people in Taiwan, liver diseases, including hepatocellular carcinoma, fibrosis, cirrhosis and hepatitis, appear to be among the most serious.1 Reactive oxygen species (ROS) are continuously produced in biological systems by the action of the mitochondrial electron transport system and nicotinamide adenine dinucleotide phosphate (NADP) oxidase. These ROS are cellular renegades and wreak havoc in biological systems by causing tissue damage, altering biochemical compounds, corroding cell membranes and killing outright. Intake of ethanol results in excessive generation of free radicals, which alter biomembranes and cause damage. Cells have several antioxidant enzymes, including catalase (CAT), glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD). Many studies have reported that natural antioxidants are efficacious in preventing oxidative stress-related liver pathologies via particular interactions and synergisms.2,3 ROS production is linked with oxidative stress, which is defined as an imbalance between oxidant generation and antioxidant defense.4 Regarding the central role of ROS in liver disease and pathology, antioxidants might prevent hepatic damage through scavenger activity and increase the activity of intracellular antioxidant enzymes such as SOD, GSH-Px and CAT. There is evidence indicating that natural substances from edible and medicinal plants exhibit strong antioxidant activity that could act against hepatic toxicity caused by various toxicants.5,6 A major defense mechanism involves antioxidant enzymes such as SOD, CAT and GSH-Px that convert active oxygen molecules into
non-toxic compounds. One such candidate is Cholestin, which was chosen for the present study. Cholestin is the fermented product of rice on which red yeast (Monascus purpureus) has been grown and is a dietary staple in many Asian countries, with typical daily consumption in the range 0.5–2 oz (∼14–56 g) per person.7 This product has been used as a food preservative for maintaining taste and color in fish and meat and also as a functional medicine.8 The medicinal properties of
www.soci.org red yeast extract were described by pharmacologists of the Ming Dynasty (1368–1644), as mentioned by Mei.9 Increased levels of cholesterol and triglycerides are known to be risk factors for developing coronary artery diseases. Lipid-lowering agents that inhibit 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase are now prominent among the drugs of choice for treating hypercholesterolemia. Another effective way to control cholesterol levels is with diet and food supplements.10 Cholestin also contains 20–60 g kg−1 fatty acids, including palmitic, linoleic, oleic and stearic acids.11 Some of these have been shown to have the ability to reduce blood lipid levels in animal models and humans.12 Indeed, diets enriched with Cholestin were effective in reducing cholesterol in high-cholesterol rabbits and rats.13 Recently, Cholestin played an important role in reducing the toxic effects of vitamin A, oxidized cholesterol and oxidized fish oil in rats.14,15 One medicinal property of Cholestin is that it favorably impacts lipid profiles of hypercholesterolemic patients,16,17 and it is used as a colorant in the food industry. Many secondary metabolites are produced in Cholestin. It contains various pigments, mainly yellow (ankaflavin and monascin), orange (monascorubrin and rubropunctanin) and red (monascorubramine and rubropunctamine) pigments. The antihypercholesterolemic agent monacolin K, the hypertensive agent 𝛾-aminobutyric acid (GABA) and antioxidants are also present.18,19 Its total antioxidant capacity may include the activities of most of the antioxidants possibly present in Cholestin, such as flavonoids, polyphenols, carotenoids, alkaloids, vitamins, etc. These compounds are very important for health and as components of health foods or functional foods. Therefore in this study we investigated the activity of Cholestin against ethanol-induced oxidative stress and hepatotoxicity in rats for 8 weeks. Hepatic GSH-Px and thiobarbituric acid-reactive substance (TBARS) levels as well as aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) activities in serum and CAT, SOD, GSH, GSH-Px, ethanol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) activities in liver tissues were measured to monitor liver injury and gene expression of liver CYP2E1. The extent of ethanol-induced liver injury was also analyzed through histopathological examination.
MATERIALS AND METHODS Materials Ethanol, olive oil and silymarin were purchased from Sigma Chemical Co. (St Louis, MO, USA). All other chemicals used were in the purest form available commercially.
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Preparation of Cholestin Cholestin (red yeast rice) is described as the fermented product of rice on which red yeast (M. purpureus) has been grown. Monascus purpureus strain BCRC 31498 was purchased from the Bioresources Collection and Research Center (Food Industry Research and Development Institute, Hsinchu, Taiwan). The fungus was maintained on malt extract broth agar containing 4 g yeast extract, 20 g malt extract, 20 g glucose and 20 g agar (pH 7) L−1 . Freshly inoculated cultures were incubated at 28 ∘ C for 5 days, after which stock cultures were kept at 4 ∘ C and transferred to fresh medium monthly. Monascus purpureus strain BCRC 31498 was grown in liquid medium by inoculating one loop of stock culture into a 500 mL Erlenmeyer flask containing 50 mL of malt extract broth I (Blakeslee’s formula) growth medium (20 g malt extract, 20 g
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glucose and 1 g peptone L−1 distilled water, pH 4.7) and incubating the culture at 30 ∘ C on a rotary shaker at 220 × g. Lovastatin esterase activity was induced by the addition of lovastatin ammonium salt to the flask to a final concentration of 0.5 mg mL−1 . The culture was then allowed to incubate for another day before it was harvested. Dried red yeast rice was extracted with boiling water at 100 ∘ C for 4 h. The extract was then filtered through a Büchner funnel and freeze-dried red yeast rice was stored at −20 ∘ C until use. Animals Male Wistar rats weighing 210–220 g were purchased from the National Laboratory Animal Center (Taipei, Taiwan) and housed individually in stainless steel wire bottom cages under a controlled environment (25 ∘ C, 50–60% humidity, 12 h light daily) for 2 weeks of acclimatization. During this time, the animals were fed standard Purina Rodent Chow 5001 (Labdiet®, Richmond, IN, USA). Chow consumption was 70 ± 3.2 g kg−1 day−1 and water consumption was 130 ± 6.3 g kg−1 day−1 . All animals were fasted for 12 h before the experiment started. Our Institutional Animal Care and Use Committee approved the protocols for the animal study, and the animals were cared for in accordance with institutional ethical guidelines. Treatments After 2 weeks, the animals were randomly divided into seven groups, each consisting of eight rats. Group A was fed a basal diet (without the addition of Cholestin) formulated according to the American Institute of Nutrition20 (AIN-76 diet). Group B was fed the basal diet with the addition of Cholestin at 1% (0.1667 g kg−1 body weight (BW)). To induce hepatotoxicity, animals of groups C–F were orally administered ethanol at 20% (3.95 g kg−1 BW twice a day, i.e. 7.9 g kg−1 BW day−1 ). Groups D–F were given Cholestin in the diet at 1% (0.1667 g kg−1 BW), 2% (0.3333 g kg−1 BW) and 3% (0.5 g kg−1 BW) respectively. Group G was given silymarin in the diet at 12% (200 mg kg−1 BW). The components of all group diets are listed in Table 1. On weeks 2, 4 and 8, blood was obtained by tail vein puncture. Blood samples were centrifuged at 3000 × g for 15 min and then stored at −80 ∘ C for subsequent analyses. The activities of serum blood urea nitrogen (BUN), creatinine, AST, ALT and ALP were assayed spectrophotometrically according to standard procedures using commercially available diagnostic kits (Merck, Darmstadt, Germany). Livers were stored at −40 ∘ C for GSH-Px and TBARS determinations. Antioxidant activities Appropriate liver tissues were dissected, weighed and immersed in liquid N2 within 60 s of death and kept frozen at −70 ∘ C. Prior to enzyme determinations, thawed tissue samples were homogenized in 20 volumes of ice-cold 50 mmol L−1 phosphate buffer (pH 7.4) and centrifuged at 3200 × g for 20 min at 5 ∘ C. The supernatants were used for antioxidant enzyme determinations.21 CAT activity The mitochondria pellet was dissolved in 1 mL of 0.25 mol L−1 sucrose buffer. Then 10 μL of the mitochondria solution was added to a cuvette containing 2.89 mL of 50 mmol L−1 potassium phosphate buffer (pH 7.4), and the reaction was initiated
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Table 1. Composition of experimental diet in each group for test of Cholestin extract and ethanol Diets
Ingredient (%) Sucrose Casein Corn starch Cellulose Corn oil Methionine Choline AIN mineral mixa AIN vitamin mixb Cholestin Silymarin
Control (A) 20 35 30 5 5 0.3 0.2 3.5 1 0 0
Cholestin (B) 20 35 29 5 5 0.3 0.2 3.5 1 1 0
Ethanol (C) 20 35 30 5 5 0.3 0.2 3.5 1 0 0
Cholestin + ethanol (D) 20 35 29 5 5 0.3 0.2 3.5 1 1 0
Cholestin + ethanol (E) 20 35 28 5 5 0.3 0.2 3.5 1 2 0
Cholestin + ethanol (F) 20 35 27 5 5 0.3 0.2 3.5 1 3 0
Silymarin + ethanol (G) 20 35 18 5 5 0.3 0.2 3.5 1 0 12
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Minerals per 100 g diet: NaCl 7.4 g, K2 C6 H5 O7 · H2 O 22 g, K2 SO4 5.2 g, CaHPO4 50 g, MgO 2.4 g, FeC6 H5 O7 · 5H2 O 0.6 g, MnCO3 0.35 g, CuCO3 30 mg, CrK(SO4 )2 · 12H2 O 55 mg, CoCl2 · 6H2 O 10 mg, KI 1 mg, ZnCO3 160 mg. b Vitamins per 100 g diet: thiamine 100 mg, riboflavin 150 mg, pyridoxine-HCl 100 mg, nicotinamide 1000 mg, D-panthenate 500 mg, folic acid 50 mg, vitamin B12 0.1 mg, vitamin A 2.5 × 105 IU, vitamin E 100 mg, calciferol 2 × 104 IU, vitamin C 3.7 × 103 mg.
by adding 0.1 mL of 30 mmol L−1 H2 O2 to make a final volume of 3 mL at 25 ∘ C. The decomposition rate of H2 O2 was measured at 240 nm for 5 min on a spectrophotometer. A molar extinction coefficient of 0.041 L mol−1 cm−1 was used to determine CAT activity, expressed as μmol H2 O2 decrease min−1 mg−1 protein.
of formation of TNB is followed spectrophotometrically and the assay is calibrated using standards. If the sample is reacted with 2-vinylpyridine, GSH is derivatized and only GSSG is detected during subsequent assay.23
TBARS concentration Lipid peroxide concentrations in hepatic subcellular fractions and serum were determined by measuring TBARS according to the method of Ohkawa et al.24 The reaction mixture contained the hepatic homogenate solution, the subcellular fractions and thiobarbituric acid (TBA) and was incubated in boiling water for 30 min. After centrifugation at 1000 × g for 10 min, the absorbance of the upper layer was measured at 532 nm on a fluorescence detector (Hitachi, Japan). TBARS concentration was expressed as nmol malondialdehyde (MDA) g−1 liver or mL−1 serum.
GSH-Px level GSH-Px levels were measured using a glutathione assay kit (Calbiochem, San Diego, CA, USA). An equal volume of ice-cold 100 mL L−1 metaphosphoric acid was added to the liver preparations. Supernatants were collected after centrifugation at 1000 × g for 10 min and analyzed for GSH-Px as per the manufacturer’s instructions. Total GSH-Px in the samples was normalized with protein.23
Ascorbic acid level To 0.5 mL of sample, 1.5 mL of 60 mL L−1 trichloroacetic acid and 0.5 mL of DNPH reagent (20 mL L−1 2,4-dinitrophenylhydrazine and 40 mL L−1 thiourea in 9 mol L−1 H2 SO4 ) were added and the mixture was incubated for 3 h at room temperature. After incubation, 2.5 mL of 850 mL L−1 H2 SO4 was added and the color developed was read at 530 nm after 30 min.25
GSH level GSH of glutathione reacts non-enzymatically with 5,5′ dithiobis(2-nitrobenzoic acid) (DTNB) to yield glutathione disulfide (GSSG) and 2-nitro-5-thiobenzoic acid (TNB). GSSG is then reduced enzymatically by NADPH and glutathione reductase (GR) to regenerate GSH. Concentrations of DNTB, NADPH and GR are chosen such that the rate of the overall reaction is linearly proportional to the concentration of total glutathione. The rate
Vitamin E level To 0.5 mL of lipid extract, 1.5 mL of ethanol and 2 mL of petroleum ether were added and the mixture was centrifuged. The supernatant was evaporated to dryness at 80 ∘ C, then 0.2 mL of 20 mL L−1 2,2′ -dipyridyl and 0.2 mL of 5 mL L−1 ferric chloride were added, the mixture was kept in the dark for 5 min and finally 4 mL of butanol was added. The color developed was read at 520 nm.
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SOD activity SOD activity was measured according to the method of Marklund and Marklund,22 with slight modification. A 100 μL aliquot of the cytosol supernatant was mixed with 1.5 mL of Tris-EDTA-HCl buffer (pH 8.5) and 100 μL of 15 mmol L−1 pyrogallol and incubated at 25 ∘ C for 10 min. The reaction was terminated by adding 50 μL of 1 mol L−1 HCl. SOD activity was measured at 440 nm. One unit of activity was defined as the amount of enzyme that inhibited the oxidation of pyrogallol by 50%. SOD activity was expressed as unit mg−1 protein.
www.soci.org Hepatic ADH and ALDH activities For ADH activity measurement, two reaction tubes were prepared. One contained 50 μL of liver homogenate, 50 μL of phosphate-buffered saline (PBS, pH 7) and 1 mL of 0.1 mol L−1 glycine-NaOH buffer (pH 10.8) containing 10 mmol L−1 NAD+ . The other contained 50 μL of liver homogenate, 50 μL of PBS (pH 7) and 1 mL of 0.1 mol L−1 glycine-NaOH buffer (pH 10.8) containing 10 mmol L−1 NAD+ and 0.016 mol L−1 ethanol. After a 4 min reaction, the difference in absorbance between the two reaction tubes was measured at 340 nm. Hepatic ADH activity was calculated by taking the extinction coefficient of NADH to be 6.22 × 103 L μmol−1 cm−1 . One unit of ADH was expressed as the amount of enzyme that produced 1 mol NADH min−1 at 25 ∘ C.26 For measurement of ALDH activity, two reaction tubes were prepared. One contained 100 μL of liver homogenate and 1 mL of 50 mmol L−1 sodium pyrophosphate buffer (pH 8.8) containing 1 mmol L−1 NAD+ , 0.2 mmol L−1 4-methylpyrazole, 1 mmol L−1 MgCl2 , 2 μmol L−1 rotenone and 10 mL L−1 Triton X-100. The other contained 100 μL of liver homogenate and 1 mL of 50 mmol L−1 sodium pyrophosphate buffer (pH 8.8) containing 1 mmol L−1 NAD+ , 0.2 mmol L−1 4-methylpyrazole, 1 mmol L−1 MgCl2 , 2 μmol L−1 rotenone, 10 mL L−1 Triton X-100 and 5 mmol L−1 acetaldehyde. After a 30 min reaction, the difference in absorbance between the two reaction tubes was measured at 340 nm. Hepatic ALDH activity was calculated by taking the extinction coefficient of NADH to be 6.22 × 103 L μmol−−1 cm−1 . One unit of ALDH was expressed as the amount of enzyme that produced 1 mol NADH min−1 at 25 ∘ C.26 Gene expression of liver CYP2E1 Total RNA was isolated from the stored frozen liver tissues using an E.Z.N.A.® total RNA kit (Omega Bio-Tek, Inc., Norcross, GA, USA) according to the manufacturer’s protocol. Reverse transcription was carried out with 2 μg of total RNA, 10 μL of reaction buffer, 1 μL of dNTPs, 2.5 μL of 10 μmol L−1 oligo-dT and 0.5 μL of RTase (Improm II™, Promega, Madison, WI, USA) with diethyl pyrocarbonate and water in a final volume of 50 μL at 42 ∘ C for 1 h. After heat inactivation, 1 μL of cDNA product was used for polymerase chain reaction (PCR) amplification. The appropriate primers of target genes that were designed for rat cytochrome P450 2E1 (CYP2E1, GenBank No. AF061442.1) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH, GenBank No. DQ403053.1) were as follows: CYP2E1: sense 5′ -CTCCTCGTCATATCCATCTG-3′ , antisense 5′ -GCAGCCAATCACAAATGTGG-3′ ; GAPDH: sense 5′ -GACCCCTTCATTGACCTCAAC-3′ , antisense 5′ -GGAGATGATGAC CCTTTTGGC-3′ . The sizes of reaction products were 474 bp for CYP2E1 and 264 bp for GAPDH. PCR amplification was performed using a DNA thermal cycler (2720 Thermal Cycler, Applied Biosystems/Life Technologies, Foster City, CA, USA) under the following conditions: CYP2E1: 35 cycles of 94 ∘ C for 1 min, 54 ∘ C for 1 min and 72 ∘ C for 2 min, followed by 10 min at 72 ∘ C; GAPDH: 25 cycles of 94 ∘ C for 1 min, 52 ∘ C for 1 min and 72 ∘ C for 2 min, followed by 10 min at 72 ∘ C. The final products were subjected to electrophoresis on 20 g L−1 agarose gel and detected by ethidium bromide staining and UV light. The relative expression levels of the mRNAs of the target genes were normalized using GAPDH internal standard.27
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Histopathological examination The livers were preserved in 100 mL−1 buffered formalin for at least 24 h, dehydrated with a sequence of ethanol solutions and
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processed for embedding in paraffin. Sections 5–6 mm thick were cut, deparaffinized, rehydrated, stained with hematoxylin and eosin (H&E) for the estimation of hepatocyte necrosis and vacuolization, as well as with Masson trichrome stain and Sirius red stain for the estimation of hepatocyte fibrosis, and subjected to photomicroscopic examination. The histological scoring of hepatic damage and fibrosis was expressed using the following score system: 0, no histopathologic change; ≤1, mild histopathologic change; ≤2, moderate histopathologic change; ≤3, severe histopathologic change.23 Statistical analysis All data were subjected to two-way analysis of variance (ANOVA).28 Duncan’s new multiple range test was applied to revolve differences among treatment means. All statistical analyses were performed using the statistical software SPSS 11.0 (SPSS UK Ltd, Woking, UK). A P value below 0.05 was considered statistically significant. Ratio values were not sin−1 transformed before statistical analysis.
RESULTS Effect of Cholestin on ethanol-induced liver injury in rats The effect of various doses of Cholestin on serum biochemical markers in ethanol-intoxicated rats was studied (Fig. 1). After injection of ethanol, serum activities of AST, ALT and ALP enzymes in the ethanol-treated groups (groups C–G) were significantly increased (P < 0.05) compared with the normal control group (group A). Treatment of animals with different doses of Cholestin significantly reduced the activities of serum AST, ALT and ALP compared with the group treated with ethanol alone. The positive control drug silymarin at a dose of 200 mg kg−1 also reduced the levels of serum AST, ALT and ALP. Effect on rat kidney There was no significant difference (P > 0.05) in plasma concentrations of BUN and creatinine among the various groups. Effect of ethanol metabolism enzymes on gene expression of liver CYP2E1 Higher CYP2E1 gene expression was observed in the ethanol group than in the other groups (Fig. 2). Hence the acceleration of ethanol elimination by Cholestin is highly correlated with increased ADH and ALDH in ethanol-fed rats (Fig. 2). Effect of Cholestin on non-enzymatic antioxidants Figure 2 shows the levels of non-enzymatic antioxidants (vitamin E, vitamin C and GSH) in tissues. The levels of vitamin E, vitamin C and GSH were significantly (P < 0.05) reduced in ethanol-treated rats compared with control rats. Administration of Cholestin (groups D–F) and silymarin (group G) significantly (P < 0.05) restored the levels of non-enzymatic antioxidants in tissues. Effect of Cholestin on antioxidant enzymes The hepatic antioxidant enzyme activities of SOD and CAT were decreased in the liver of rats treated with ethanol; however, their activities were restored by Cholestin (groups D–F). As shown in Fig. 3, the hepatic GSH-Px level was markedly lower in ethanol-intoxicated rats. However, the GSH-Px level was significantly increased by Cholestin (groups D–F) treatment compared
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Figure 1. Effects of ethanol and Cholestin extract on BUN, creatinine, AST, ALT and ALP levels of rats. For each parameter, values in the same week with different letters are significantly different (P < 0.05). Group diets A–G are shown in Table 1.
Table 2. When compared with the normal liver tissues of vehicle controls, liver tissues in rats treated with ethanol revealed extensive liver injuries characterized by moderate to severe hepatocellular hydropic degeneration and necrosis around the central vein, lipidosis, hepatic fibrosis and cholangiocyte hyperplasia. However, the histopathological hepatic lesions induced by administration of ethanol were only markedly ameliorated in central lobular necrosis, hepatic lipidosis, and hepatic fibrosis by treatment with Cholestin and silymarin.
Histopathological examination The histological observations supported the results obtained from serum enzyme assays. Liver sections from control rats showed normal lobular architecture and hepatic cells with well-preserved cytoplasm and well-defined nucleus and nucleoli (Fig. 4). The results of hepatic histopathological examination are shown in
DISCUSSION
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In the present study the capability of Cholestin to protect against ethanol toxicity and oxidative stress was investigated. AST and ALT are reliable markers for liver function. It is established that AST can be found in the liver, cardiac muscle, skeletal muscle, kidney, brain, pancreas, lungs, leukocytes and erythrocytes, while
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with the ethanol group. Administration of silymarin (group G) significantly increased (P < 0.05) the activities of SOD, CAT and GSH-Px compared with the ethanol-treated group. The expected increases in hepatic and serum lipid peroxidative indices in the ethanol-treated group confirmed that oxidative damage had been induced (Fig. 3). Treatment with various doses of Cholestin (groups D–F) led to significantly lower levels of TBARS in the liver and plasma compared with the ethanol-treated group. Silymarin (group G) also inhibited the elevation of TBARS levels caused by ethanol administration.
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Figure 2. Effects of ethanol and Cholestin extract on vitamin C, vitamin E and GSH levels and ADH and ALDH activities in liver and CYP2E1 gene expression of rats. For each parameter, values with different letters are significantly different (P < 0.05). Group diets A–G are shown in Table 1.
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ALT is present in the liver.29 Increased levels of serum enzyme such as AST and ALT indicate increased permeability and damage and/or necrosis of hepatocytes.30 In our study we found that chronic ethanol consumption caused a significant increase in the activities of AST, ALT and ALP, which could cause severe damage to tissue membranes. The decreased activities of these enzymes in Cholestin-administered rats indicate the hepatoprotective effect of Cholestin. We studied further other underlying mechanisms responsible for this hepatoprotective action of Cholestin in rats. The protective effect of Cholestin was accompanied by partial prevention of GSH-Px depletion in liver tissue. It is considered that hepatic GSH-Px represents an enzyme reserve of the liver that is responsible for reducing hepatotoxicity induced by active metabolites of ethanol toxicity. As GSH-Px is also a crucial determinant of tissue susceptibility to oxidative damage,31 the partial protection of
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GSH-Px reserves by Cholestin provides an additional action not only to remove active metabolites of ethanol toxicity but also to scavenge free radicals that are involved in lipid peroxidation. Ethanol toxicity also increased lipid peroxidation, as a result of which the hepatic TBARS level was elevated.32 Cholestin treatment prevented this effect, indicating that Cholestin was able to attenuate lipid peroxidation induced by ethanol toxicity. Non-enzymatic antioxidants such as GSH, vitamin C and vitamin E are closely interlinked with each other and play a vital role in protecting cells from lipid peroxidation. In addition to GSH, we also observed a decrease in the levels of antioxidants such as vitamin C and vitamin E in tissue of ethanol-treated rats. Supplementation of Cholestin to ethanol-treated rats restored non-enzymatic antioxidant levels in the liver. The animal groups treated with Cholestin showed an increase in the levels of SOD and CAT, indicating the antioxidant activity of Cholestin.
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Figure 3. Effects of ethanol and Cholestin extract on SOD, CAT and GSH-Px activities in liver and plasma and hepatic TBARS concentrations of rats. For each parameter, values with different letters are significantly different (P < 0.05). Group diets A–G are shown in Table 1.
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toxicity-induced liver injury is the formation of lipid peroxides by free radical derivatives of ethanol toxicity. Thus the antioxidant activity or the inhibition of free radical generation is important in the protection against ethanol toxicity-induced hepatopathy. The body has an effective defense mechanism to prevent and neutralize free radical-induced damage. This is provided by a set of endogenous antioxidant enzymes such as SOD and CAT. These enzymes constitute a mutually supportive team of defense against ROS.2 Lipid peroxidation, an ROS-mediated mechanism, has been implicated in the pathogenesis of various liver injuries and subsequent liver fibrogenesis in experimental animals. The significant non-dose-dependent decrease in hepatic lipid hydroperoxides confirmed that treatment with Cholestin could effectively protect against hepatic lipid peroxidation induced by ethanol toxicity. Hence it is possible that the mechanism of hepatoprotection of
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Hepatocellular necrosis leads to the elevation of serum AST, ALT and ALP activities and increased incidence and severity of histopathological hepatic lesions in rats. The present study revealed a significant increase in the activities of AST, ALT and ALP on exposure to ethanol toxicity, indicating considerable hepatocellular injury. Administration of Cholestin attenuated the increased levels of serum enzymes (AST, ALT and ALP) induced by ethanol toxicity and caused a subsequent recovery towards normalization comparable to the control group, with the hepatoprotective effects being comparable to those of silymarin. The effect of Cholestin was further confirmed by histopathological examinations. A higher dose of Cholestin offers hepatoprotection effective in central lobular necrosis, hepatic lipidosis and cholangiocyte hyperplasia compared with lower doses in rats. It has been hypothesized that one of the principal causes of ethanol
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Figure 4. Microscopic cross-sections of liver lobules in rats after 8 weeks (×400, H&E). Bar represents 0.01 mm. Group diets A–G are shown in Table 1.
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Cholestin may be due to its antioxidant activity. Moreover, the reduced activities of SOD and CAT observed point out the hepatic damage in rats administered with ethanol toxicity. The animal groups treated with Cholestin showed an increase in the levels of SOD and CAT, indicating the antioxidant activity of Cholestin. GSH-Px acts as an enzymatic antioxidant both intracellularly and extracellularly in conjunction with various enzymatic processes that reduce hydrogen peroxide and hydroperoxides. The depletion of hepatic GSH-Px has been shown to be associated with enhanced toxicity due to chemicals, including ethanol toxicity.33 In the present study a decrease in hepatic tissue GSH-Px level was observed in the ethanol-treated groups. The increase in hepatic
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GSH-Px level in rats treated with Cholestin may be due to de novo GSH-Px synthesis and/or GSH-Px regeneration. Administration of Cholestin significantly decreased the TBARS level compared with ethanol-treated rats, which may be due to the scavenging of free radicals generated by ethanol. Silymarin, an antioxidant flavonoid complex isolated from the seed of milk thistle (Silybum marianum, Compositae), has been used to treat hepatotoxicity diseases in clinical practice for at least two decades. Silymarin has powerful free radical-scavenging properties and regulates intracellular GSH levels.34 The way in which silymarin works is by preventing lipid peroxidation and hepatotoxicity, by acting as a chain-breaking antioxidant for
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Table 2. Effects of Cholestin extract on hepatic histopathology of liver damage in rats treated with ethanol Treatment groups
Parameter
Control (A)
Hepatocellular hydropic degeneration Central lobular necrosis Hepatic lipidosis Hepatic fibrosis Cholangiocyte hyperplasia
0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0
Cholestin (B) 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0
Ethanol (C) 2.7 ± 0.3* 2.6 ± 0.5* 2.5 ± 0.5* 2.6 ± 0.2* 2.5 ± 0.3*
Cholestin + ethanol (D) 2.1 ± 0.3* 2.2 ± 0.2* 2.1 ± 0.2* 2.3 ± 0.3* 2.2 ± 0.2*
Cholestin + ethanol (E) 1.6 ± 0.3* 1.5 ± 0.2* 1.7 ± 0.2* 1.8 ± 0.3* 1.5 ± 0.2*
Cholestin + ethanol (F)
Silymarin + ethanol (G)
0.5 ± 0.2 0.5 ± 0.1 0.3 ± 0.2 0.2 ± 0.2 0.1 ± 0.2
0.5 ± 0.2 0.6 ± 0.3 0.5 ± 0.1 0.3 ± 0.2 0.2 ± 0.1
Values are expressed as mean ± SD (n = 8) in each group. Scores: 0, no histopathologic change; ≤1, mild histopathologic change; ≤2, moderate histopathologic change; ≤3, severe histopathologic change. * P < 0.05, significantly different from control.
scavenging free radicals or by a combination of these effects.35 In fact, a considerable body of experimental work in animal models has shown that silymarin, as a positive control, reduced ethanol-induced hepatotoxic effects by preventing lipid peroxidation. In the present study, silymarin acted as an effective positive control as evidenced by it decreasing AST, ALT and ALP levels and increasing SOD, CAT and GSH-Px activities and GSH levels in the liver, while decreasing TBARS levels. Ethanol is mainly metabolized by ADH in cytosols, CYP2E1 in the endoplasmic reticulum and CAT in peroxisomes to form acetaldehyde, and is further catabolized to acetic acid by ALDH.36 However, during alcohol metabolism by CYP2E1, ROS are also generated and increase lipid peroxidation, i.e. MDA, in the liver.25,37,38 However, the level of TBARS in the plasma is an additional indicator of liver injury. The level of TBARS in the plasma of rats subjected to ethanol toxicity was significantly reduced when they were supplemented with Cholestin. This result is in agreement with previous reports.14,39 – 42 Therefore it is reasonable to assume that Cholestin may act as a good scavenger in reducing lipid peroxidation induced by oxidized fish oil,39 oxidized cholesterol,40 carbon tetrachloride41 and vitamin A.14 The results of functional tests together with histological observations suggest that ethanol leads to serious changes in liver histology. The increased formation of lipid peroxides and associated ROS leads to membrane integrity damage and other pathological changes in the liver. The efficacy of any protective drug is essentially dependent on its capacity to reduce the harmful effects of toxins and maintain the normal physiology of cells and tissues. The membrane-protective properties and antioxidant nature of Cholestin might help in alleviating pathological changes caused by ethanol in the liver. Our data indicate that Cholestin has a protective action against ethanol-induced toxicity as evidenced by the lowered tissue lipid peroxidation and elevated levels of enzymatic and non-enzymatic antioxidants in the liver. Hence our study suggests that Cholestin can play a beneficial role in the treatment of ethanol-induced tissue damage, which could be one of its therapeutic values.
ACKNOWLEDGEMENTS
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1 Wu J, Danielsson A and Zern MA, Toxicity of hepatotoxins: new insights into mechanisms and therapy. Expert Opin Investig Drugs 8:585–607 (1999). 2 Vitaglione P, Morisco F, Caporaso N and Fogliano V, Dietary antioxidant compounds and liver health. Crit Rev Food Sci Nutr 44:575–586 (2004). 3 Cheng F, Chen W, Huang Y, Zhang C, Shen Y, Hai H, et al., Protective effect of areca inflorescence extract on hydrogen peroxide-induced oxidative damage to human serum albumin. Food Res Int 44:98–102 (2011). 4 Cogger VC, Muller M, Fraser R, McLean AJ, Khan J and Le Couteur DG, The effects of oxidative stress on the liver sieve. J Hepatol 41:370–376 (2004). 5 Zeashan H, Amresh G, Singh S and Rao CV, Hepatoprotective activity of Amaranthus spinosus in experimental animals. Food Chem Toxicol 46:3417–3421 (2008). 6 Madureira AR, Amorim M, Gomes AM, Pintado ME and Malcata FX, Protective effect of whey cheese matrix on probiotic strains exposed to simulated gastrointestinal conditions. Food Res Int 44:465–470 (2011). 7 Stuart GA, Chinese Materia Medica – Vegetable Kingdom. Southern Material Center, Taipei (1979). 8 Ma J, Li Y, Ye Q, Li J, Hua Y, Ju D, et al., Constituents of red yeast rice, a traditional Chinese food and medicine. J Agric Food Chem 48:5220–5225 (2000). 9 Mei F, Fang Mei’s Illustrated Cookbook of Regional Chinese Cuisine. Guangxi National Press, Guang xi, pp. 177–188 (1990). 10 Brosche T, Kral C, Summa JD and Platt D, Effective lovastatin therapy in elderly hypercholesterolemic patients – an antioxidative impact? Arch Gerontol Geriatr 22:207–221 (1996). 11 Zhang M and Duan ZXS, Active components of xuezhikang. Chin J New Drugs 7:213–214 (1998). 12 Su CF, Liu IM and Cheng JT, Improvement of insulin resistance by Hon-Chi in fructose-rich chow-fed rats. Food Chem 104:45–52 (2007). 13 Wang IK, Lin-Shiau SY, Chen PC and Lin JK, Hypotriglyceridemic effect of Anka (a fermented rice product of Monascus sp.) in rats. J Agric Food Chem 48:3183–3189 (2000). 14 Yeh YH, Lee YT and Hsieh HS, Effect of Cholestin on toxicity of vitamin A in rats. Food Chem 132:311–318 (2012). 15 Yeh YH, Lee YT, Hsieh HS and Hwang DF, Effect of red yeast rice on toxicity of oxidized cholesterol and oxidized fish oil in rats. e-SPEN 5:e230–e237 (2010). 16 Li YG, Liu H and Wang ZT, A validated stability-indicating HPLC with photodiode array detector (PDA) method for the stress tests of Monascus purpureus fermented rice, red yeast rice. J Pharmaceut Biomed Anal 39:82–90 (2003). 17 Segura B, Red yeast rice: an easy way to lower cholesterol. Nutr Bytes 9(1) (2003). 18 Su YC, Wang JJ, Lin TT and Pan TM, Production of the secondary metabolism 𝛽-aminobutyric acid and monacolin K by Monascus. J Ind Microbiol Biotechnol 30:41–46 (2003).
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This study was supported by Asia University (100-asia-18) and National Science Council (MOST 103-2622-B-040-001-CC3).
REFERENCES
www.soci.org 19 Wang JJ, Lee CL and Pan TM, Modified mutation method for screening low citrinin-producing strains of Monascus purpureus on rice culture. J Agric Food Chem 52:6977–6982 (2004). 20 American Institute of Nutrition (AIN), Report of the American Institute of Nutrition Ad Hoc Committee on Standards for Nutritional Studies. J Nutr 107:1340–1348 (1977). 21 Hsieh YL, Yeh YH, Lee YT and Hsieh CH, Ameliorative effect of Pracparatum mungo extract on high cholesterol diets in hamsters. Food Funct 5:149–157 (2014). 22 Marklund S and Marklund G, Involvement of the superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase. Eur J Biochem 47:469–474 (1974). 23 Yeh YH, Hsieh YL and Lee YT, Effects of yam peel extract against carbon tetrachloride-induced hepatotoxicity in rats. J Agric Food Chem 61:7387–7396 (2013). 24 Ohkawa H, Ohishi N and Yagi K, Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 95:351–358 (1979). 25 Pari L and Suresh A, Effect of grape (Vitis vinifera L.) leaf extract on alcohol induced oxidative stress in rats. Food Chem Toxicol 46:1627–1634 (2008). 26 Hsieh YL, Yeh YH, Lee YT and Huang CY, Effect of taurine in chronic alcoholic patients. Food Funct 5:1529–1535 (2014). 27 Fang YJ, Chiu CH, Chang YY, Chou CH, Lin HW, Chen MF, et al., Taurine ameliorates alcoholic steatohepatitis via enhancing selfantioxidant capacity and alcohol metabolism. Food Res Int 44:3105–3110 (2011) 28 Puri SC and Mullen K, Applied Statistics for Food Agricultural Scientists. G.K. Hall Medical Publishers, Boston, MA, pp. 146–183 (1980). 29 Rej R, Aspartate aminotransferase activity and isoenzyme proportions in human liver tissues. Clin Chem 24:1971–1979 (1978). 30 Goldberg DM and Watts C, Serum enzyme changes as evidence of liver reaction to oral alcohol. Gastroenterology 49:256–261 (1965).
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31 Ischiropoulos H, Zhu L and Beckman JS, Peroxynitrite formation from macrophage-derived nitric oxide. Arch Biochem Biophys 298:446–451 (1992). 32 Ruf JC, Berger JL and Renaud S, Platelet rebound effect of alcohol withdrawal and wine drinking in rats. Relation to tannins and lipid oxidation. Arterioscler Thromb Vasc Biol 15:140–144 (1995). 33 Abraham P, Wilfred G and Ramakrishna B, Oxidative damage to the hepatocellular proteins after chronic ethanol intake in the rat. Clin Chim Acta 325:117–125 (2002). 34 Shaker E, Mahmoud H and Mnaa S, Silymarin, the antioxidant component and Silybum marianum extracts prevent liver damage. Food Chem Toxicol 48:803–806 (2010). 35 Letteron P, Labbe G, Degott C, Berson A, Fromenty B, Delaforge M, et al., Mechanism for the protective effects of silymarin against carbon tetrachloride-induced lipid peroxidation and hepatotoxicity in mice. Biochem Pharmcol 39:2027–2034 (1990). 36 Lieber CS, Biochemical factors in alcoholic liver disease. Semin Liver Dis 13:136–153 (1993). 37 Gill K, Amit Z and Smith BR, The regulation of alcohol consumption in rats: the role of alcohol-metabolising enzymes – catalase and aldehyde dehydrogenase. Alcohol 13:347–353 (1996). 38 Song Z, Zhou Z, Chen T, Hill D, Kang J, Barve S, et al., S-adenosylmethionine (SAMe) protects against acute alcohol induced hepatotoxicity in mice. J Nutr Biochem 14:591–594 (2003). 39 Yeh YH, Lee YT, Hsieh YL and Hwang DF, Effect of red yeast rice on toxicity of oxidized cholesterol and oxidized fish oil in rats. e-SPEN 5:e230–e237 (2010). 40 Yeh YH, Lee YT, Hsieh YL and Hwang DF, Dietary Cholestin (red yeast rice) reduces toxicity of oxidized cholesterol in rats. J Food Drug Anal 18:211–220 (2010). 41 Yeh YH, Hsieh YL, Lee YT and Hsieh CH, Protective effects of Cholestin against carbon tetrachloride-induced hepatotoxicity in rats. e-SPEN 6:e264–e271 (2011). 42 Yeh YH, Hsieh YL and Lee YT, Effect of RYR, MFA and MFB on serum lipids and antioxidant activity in rats. e-SPEN 7:e176–e181 (2012).
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J Sci Food Agric 2015; 95: 799–808
Revised: 2 September 2014
Accepted article published: 16 September 2014
Published online in Wiley Online Library: 14 October 2014
(wileyonlinelibrary.com) DOI 10.1002/jsfa.6904
Protective effects of Cholestin on ethanol induced oxidative stress in rats You-Liang Hsieh,a Yen-Hung Yeh,b,c* Ya-Ting Leed and Chih-Yang Huanga,e,f Abstract BACKGROUND: Male Wistar rats were divided into seven groups as follows: group A, basal diet; group B, basal diet with Cholestin at 0.1667 g kg−1 body weight (BW); groups C–F, oral feeding of ethanol at 7.9 g kg−1 BW; groups D–F, Cholestin in diet at 0.1667, 0.3333 and 0.5 g kg−1 BW respectively; group G, silymarin in diet at 200 mg kg−1 BW. RESULTS: The results showed that treatment with Cholestin for 8 weeks reduced the impact of ethanol toxicity on serum markers of liver damage: aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP). The antioxidant system was significantly enhanced: plasma and hepatic thiobarbituric acid-reactive substance (TBARS) levels were lowered while hepatic superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), ethanol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) activities and non-enzymatic antioxidants (vitamin E, vitamin C and GSH) were elevated. CONCLUSION: Cholestin shows a protective effect against hepatotoxicity indices in ethanol-fed rats comparable to that of silymarin, as supported by the evaluation of liver histopathology. The data suggest that Cholestin exerts its hepatoprotective effect by decreasing lipid peroxidation and improving antioxidants status, thus proving itself as an effective antioxidant in ethanol-induced oxidative damage in rats. © 2014 Society of Chemical Industry Keywords: Cholestin; antioxidant activity; ethanol; hepatoprotective; hepatotoxicity
INTRODUCTION
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∗
Correspondence to: Yen-Hung Yeh, School of Health Diet and Industry Management, Chung Shan Medical University, Taichung, Taiwan, ROC. E-mail: [email protected]
a Department of Health and Nutrition Biotechnology, Asia University, Taichung, Taiwan, ROC b School of Health Diet and Industry Management, Chung Shan Medical University, Taichung, Taiwan, ROC c Department of Nutrition, Chung Shan Medical University Hospital, Taichung, Taiwan, ROC d Department of Beauty Science, National Taichung University of Science and Technology, Taichung, Taiwan, ROC e Graduate Institute of Basic Medical Science, China Medical University, Taichung, Taiwan, ROC f Department of Chinese Medicine, China Medical University Hospital, Taichung, Taiwan, ROC
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Of the various health problems suffered by people in Taiwan, liver diseases, including hepatocellular carcinoma, fibrosis, cirrhosis and hepatitis, appear to be among the most serious.1 Reactive oxygen species (ROS) are continuously produced in biological systems by the action of the mitochondrial electron transport system and nicotinamide adenine dinucleotide phosphate (NADP) oxidase. These ROS are cellular renegades and wreak havoc in biological systems by causing tissue damage, altering biochemical compounds, corroding cell membranes and killing outright. Intake of ethanol results in excessive generation of free radicals, which alter biomembranes and cause damage. Cells have several antioxidant enzymes, including catalase (CAT), glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD). Many studies have reported that natural antioxidants are efficacious in preventing oxidative stress-related liver pathologies via particular interactions and synergisms.2,3 ROS production is linked with oxidative stress, which is defined as an imbalance between oxidant generation and antioxidant defense.4 Regarding the central role of ROS in liver disease and pathology, antioxidants might prevent hepatic damage through scavenger activity and increase the activity of intracellular antioxidant enzymes such as SOD, GSH-Px and CAT. There is evidence indicating that natural substances from edible and medicinal plants exhibit strong antioxidant activity that could act against hepatic toxicity caused by various toxicants.5,6 A major defense mechanism involves antioxidant enzymes such as SOD, CAT and GSH-Px that convert active oxygen molecules into
non-toxic compounds. One such candidate is Cholestin, which was chosen for the present study. Cholestin is the fermented product of rice on which red yeast (Monascus purpureus) has been grown and is a dietary staple in many Asian countries, with typical daily consumption in the range 0.5–2 oz (∼14–56 g) per person.7 This product has been used as a food preservative for maintaining taste and color in fish and meat and also as a functional medicine.8 The medicinal properties of
www.soci.org red yeast extract were described by pharmacologists of the Ming Dynasty (1368–1644), as mentioned by Mei.9 Increased levels of cholesterol and triglycerides are known to be risk factors for developing coronary artery diseases. Lipid-lowering agents that inhibit 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase are now prominent among the drugs of choice for treating hypercholesterolemia. Another effective way to control cholesterol levels is with diet and food supplements.10 Cholestin also contains 20–60 g kg−1 fatty acids, including palmitic, linoleic, oleic and stearic acids.11 Some of these have been shown to have the ability to reduce blood lipid levels in animal models and humans.12 Indeed, diets enriched with Cholestin were effective in reducing cholesterol in high-cholesterol rabbits and rats.13 Recently, Cholestin played an important role in reducing the toxic effects of vitamin A, oxidized cholesterol and oxidized fish oil in rats.14,15 One medicinal property of Cholestin is that it favorably impacts lipid profiles of hypercholesterolemic patients,16,17 and it is used as a colorant in the food industry. Many secondary metabolites are produced in Cholestin. It contains various pigments, mainly yellow (ankaflavin and monascin), orange (monascorubrin and rubropunctanin) and red (monascorubramine and rubropunctamine) pigments. The antihypercholesterolemic agent monacolin K, the hypertensive agent 𝛾-aminobutyric acid (GABA) and antioxidants are also present.18,19 Its total antioxidant capacity may include the activities of most of the antioxidants possibly present in Cholestin, such as flavonoids, polyphenols, carotenoids, alkaloids, vitamins, etc. These compounds are very important for health and as components of health foods or functional foods. Therefore in this study we investigated the activity of Cholestin against ethanol-induced oxidative stress and hepatotoxicity in rats for 8 weeks. Hepatic GSH-Px and thiobarbituric acid-reactive substance (TBARS) levels as well as aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) activities in serum and CAT, SOD, GSH, GSH-Px, ethanol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) activities in liver tissues were measured to monitor liver injury and gene expression of liver CYP2E1. The extent of ethanol-induced liver injury was also analyzed through histopathological examination.
MATERIALS AND METHODS Materials Ethanol, olive oil and silymarin were purchased from Sigma Chemical Co. (St Louis, MO, USA). All other chemicals used were in the purest form available commercially.
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Preparation of Cholestin Cholestin (red yeast rice) is described as the fermented product of rice on which red yeast (M. purpureus) has been grown. Monascus purpureus strain BCRC 31498 was purchased from the Bioresources Collection and Research Center (Food Industry Research and Development Institute, Hsinchu, Taiwan). The fungus was maintained on malt extract broth agar containing 4 g yeast extract, 20 g malt extract, 20 g glucose and 20 g agar (pH 7) L−1 . Freshly inoculated cultures were incubated at 28 ∘ C for 5 days, after which stock cultures were kept at 4 ∘ C and transferred to fresh medium monthly. Monascus purpureus strain BCRC 31498 was grown in liquid medium by inoculating one loop of stock culture into a 500 mL Erlenmeyer flask containing 50 mL of malt extract broth I (Blakeslee’s formula) growth medium (20 g malt extract, 20 g
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glucose and 1 g peptone L−1 distilled water, pH 4.7) and incubating the culture at 30 ∘ C on a rotary shaker at 220 × g. Lovastatin esterase activity was induced by the addition of lovastatin ammonium salt to the flask to a final concentration of 0.5 mg mL−1 . The culture was then allowed to incubate for another day before it was harvested. Dried red yeast rice was extracted with boiling water at 100 ∘ C for 4 h. The extract was then filtered through a Büchner funnel and freeze-dried red yeast rice was stored at −20 ∘ C until use. Animals Male Wistar rats weighing 210–220 g were purchased from the National Laboratory Animal Center (Taipei, Taiwan) and housed individually in stainless steel wire bottom cages under a controlled environment (25 ∘ C, 50–60% humidity, 12 h light daily) for 2 weeks of acclimatization. During this time, the animals were fed standard Purina Rodent Chow 5001 (Labdiet®, Richmond, IN, USA). Chow consumption was 70 ± 3.2 g kg−1 day−1 and water consumption was 130 ± 6.3 g kg−1 day−1 . All animals were fasted for 12 h before the experiment started. Our Institutional Animal Care and Use Committee approved the protocols for the animal study, and the animals were cared for in accordance with institutional ethical guidelines. Treatments After 2 weeks, the animals were randomly divided into seven groups, each consisting of eight rats. Group A was fed a basal diet (without the addition of Cholestin) formulated according to the American Institute of Nutrition20 (AIN-76 diet). Group B was fed the basal diet with the addition of Cholestin at 1% (0.1667 g kg−1 body weight (BW)). To induce hepatotoxicity, animals of groups C–F were orally administered ethanol at 20% (3.95 g kg−1 BW twice a day, i.e. 7.9 g kg−1 BW day−1 ). Groups D–F were given Cholestin in the diet at 1% (0.1667 g kg−1 BW), 2% (0.3333 g kg−1 BW) and 3% (0.5 g kg−1 BW) respectively. Group G was given silymarin in the diet at 12% (200 mg kg−1 BW). The components of all group diets are listed in Table 1. On weeks 2, 4 and 8, blood was obtained by tail vein puncture. Blood samples were centrifuged at 3000 × g for 15 min and then stored at −80 ∘ C for subsequent analyses. The activities of serum blood urea nitrogen (BUN), creatinine, AST, ALT and ALP were assayed spectrophotometrically according to standard procedures using commercially available diagnostic kits (Merck, Darmstadt, Germany). Livers were stored at −40 ∘ C for GSH-Px and TBARS determinations. Antioxidant activities Appropriate liver tissues were dissected, weighed and immersed in liquid N2 within 60 s of death and kept frozen at −70 ∘ C. Prior to enzyme determinations, thawed tissue samples were homogenized in 20 volumes of ice-cold 50 mmol L−1 phosphate buffer (pH 7.4) and centrifuged at 3200 × g for 20 min at 5 ∘ C. The supernatants were used for antioxidant enzyme determinations.21 CAT activity The mitochondria pellet was dissolved in 1 mL of 0.25 mol L−1 sucrose buffer. Then 10 μL of the mitochondria solution was added to a cuvette containing 2.89 mL of 50 mmol L−1 potassium phosphate buffer (pH 7.4), and the reaction was initiated
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Table 1. Composition of experimental diet in each group for test of Cholestin extract and ethanol Diets
Ingredient (%) Sucrose Casein Corn starch Cellulose Corn oil Methionine Choline AIN mineral mixa AIN vitamin mixb Cholestin Silymarin
Control (A) 20 35 30 5 5 0.3 0.2 3.5 1 0 0
Cholestin (B) 20 35 29 5 5 0.3 0.2 3.5 1 1 0
Ethanol (C) 20 35 30 5 5 0.3 0.2 3.5 1 0 0
Cholestin + ethanol (D) 20 35 29 5 5 0.3 0.2 3.5 1 1 0
Cholestin + ethanol (E) 20 35 28 5 5 0.3 0.2 3.5 1 2 0
Cholestin + ethanol (F) 20 35 27 5 5 0.3 0.2 3.5 1 3 0
Silymarin + ethanol (G) 20 35 18 5 5 0.3 0.2 3.5 1 0 12
a
Minerals per 100 g diet: NaCl 7.4 g, K2 C6 H5 O7 · H2 O 22 g, K2 SO4 5.2 g, CaHPO4 50 g, MgO 2.4 g, FeC6 H5 O7 · 5H2 O 0.6 g, MnCO3 0.35 g, CuCO3 30 mg, CrK(SO4 )2 · 12H2 O 55 mg, CoCl2 · 6H2 O 10 mg, KI 1 mg, ZnCO3 160 mg. b Vitamins per 100 g diet: thiamine 100 mg, riboflavin 150 mg, pyridoxine-HCl 100 mg, nicotinamide 1000 mg, D-panthenate 500 mg, folic acid 50 mg, vitamin B12 0.1 mg, vitamin A 2.5 × 105 IU, vitamin E 100 mg, calciferol 2 × 104 IU, vitamin C 3.7 × 103 mg.
by adding 0.1 mL of 30 mmol L−1 H2 O2 to make a final volume of 3 mL at 25 ∘ C. The decomposition rate of H2 O2 was measured at 240 nm for 5 min on a spectrophotometer. A molar extinction coefficient of 0.041 L mol−1 cm−1 was used to determine CAT activity, expressed as μmol H2 O2 decrease min−1 mg−1 protein.
of formation of TNB is followed spectrophotometrically and the assay is calibrated using standards. If the sample is reacted with 2-vinylpyridine, GSH is derivatized and only GSSG is detected during subsequent assay.23
TBARS concentration Lipid peroxide concentrations in hepatic subcellular fractions and serum were determined by measuring TBARS according to the method of Ohkawa et al.24 The reaction mixture contained the hepatic homogenate solution, the subcellular fractions and thiobarbituric acid (TBA) and was incubated in boiling water for 30 min. After centrifugation at 1000 × g for 10 min, the absorbance of the upper layer was measured at 532 nm on a fluorescence detector (Hitachi, Japan). TBARS concentration was expressed as nmol malondialdehyde (MDA) g−1 liver or mL−1 serum.
GSH-Px level GSH-Px levels were measured using a glutathione assay kit (Calbiochem, San Diego, CA, USA). An equal volume of ice-cold 100 mL L−1 metaphosphoric acid was added to the liver preparations. Supernatants were collected after centrifugation at 1000 × g for 10 min and analyzed for GSH-Px as per the manufacturer’s instructions. Total GSH-Px in the samples was normalized with protein.23
Ascorbic acid level To 0.5 mL of sample, 1.5 mL of 60 mL L−1 trichloroacetic acid and 0.5 mL of DNPH reagent (20 mL L−1 2,4-dinitrophenylhydrazine and 40 mL L−1 thiourea in 9 mol L−1 H2 SO4 ) were added and the mixture was incubated for 3 h at room temperature. After incubation, 2.5 mL of 850 mL L−1 H2 SO4 was added and the color developed was read at 530 nm after 30 min.25
GSH level GSH of glutathione reacts non-enzymatically with 5,5′ dithiobis(2-nitrobenzoic acid) (DTNB) to yield glutathione disulfide (GSSG) and 2-nitro-5-thiobenzoic acid (TNB). GSSG is then reduced enzymatically by NADPH and glutathione reductase (GR) to regenerate GSH. Concentrations of DNTB, NADPH and GR are chosen such that the rate of the overall reaction is linearly proportional to the concentration of total glutathione. The rate
Vitamin E level To 0.5 mL of lipid extract, 1.5 mL of ethanol and 2 mL of petroleum ether were added and the mixture was centrifuged. The supernatant was evaporated to dryness at 80 ∘ C, then 0.2 mL of 20 mL L−1 2,2′ -dipyridyl and 0.2 mL of 5 mL L−1 ferric chloride were added, the mixture was kept in the dark for 5 min and finally 4 mL of butanol was added. The color developed was read at 520 nm.
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SOD activity SOD activity was measured according to the method of Marklund and Marklund,22 with slight modification. A 100 μL aliquot of the cytosol supernatant was mixed with 1.5 mL of Tris-EDTA-HCl buffer (pH 8.5) and 100 μL of 15 mmol L−1 pyrogallol and incubated at 25 ∘ C for 10 min. The reaction was terminated by adding 50 μL of 1 mol L−1 HCl. SOD activity was measured at 440 nm. One unit of activity was defined as the amount of enzyme that inhibited the oxidation of pyrogallol by 50%. SOD activity was expressed as unit mg−1 protein.
www.soci.org Hepatic ADH and ALDH activities For ADH activity measurement, two reaction tubes were prepared. One contained 50 μL of liver homogenate, 50 μL of phosphate-buffered saline (PBS, pH 7) and 1 mL of 0.1 mol L−1 glycine-NaOH buffer (pH 10.8) containing 10 mmol L−1 NAD+ . The other contained 50 μL of liver homogenate, 50 μL of PBS (pH 7) and 1 mL of 0.1 mol L−1 glycine-NaOH buffer (pH 10.8) containing 10 mmol L−1 NAD+ and 0.016 mol L−1 ethanol. After a 4 min reaction, the difference in absorbance between the two reaction tubes was measured at 340 nm. Hepatic ADH activity was calculated by taking the extinction coefficient of NADH to be 6.22 × 103 L μmol−1 cm−1 . One unit of ADH was expressed as the amount of enzyme that produced 1 mol NADH min−1 at 25 ∘ C.26 For measurement of ALDH activity, two reaction tubes were prepared. One contained 100 μL of liver homogenate and 1 mL of 50 mmol L−1 sodium pyrophosphate buffer (pH 8.8) containing 1 mmol L−1 NAD+ , 0.2 mmol L−1 4-methylpyrazole, 1 mmol L−1 MgCl2 , 2 μmol L−1 rotenone and 10 mL L−1 Triton X-100. The other contained 100 μL of liver homogenate and 1 mL of 50 mmol L−1 sodium pyrophosphate buffer (pH 8.8) containing 1 mmol L−1 NAD+ , 0.2 mmol L−1 4-methylpyrazole, 1 mmol L−1 MgCl2 , 2 μmol L−1 rotenone, 10 mL L−1 Triton X-100 and 5 mmol L−1 acetaldehyde. After a 30 min reaction, the difference in absorbance between the two reaction tubes was measured at 340 nm. Hepatic ALDH activity was calculated by taking the extinction coefficient of NADH to be 6.22 × 103 L μmol−−1 cm−1 . One unit of ALDH was expressed as the amount of enzyme that produced 1 mol NADH min−1 at 25 ∘ C.26 Gene expression of liver CYP2E1 Total RNA was isolated from the stored frozen liver tissues using an E.Z.N.A.® total RNA kit (Omega Bio-Tek, Inc., Norcross, GA, USA) according to the manufacturer’s protocol. Reverse transcription was carried out with 2 μg of total RNA, 10 μL of reaction buffer, 1 μL of dNTPs, 2.5 μL of 10 μmol L−1 oligo-dT and 0.5 μL of RTase (Improm II™, Promega, Madison, WI, USA) with diethyl pyrocarbonate and water in a final volume of 50 μL at 42 ∘ C for 1 h. After heat inactivation, 1 μL of cDNA product was used for polymerase chain reaction (PCR) amplification. The appropriate primers of target genes that were designed for rat cytochrome P450 2E1 (CYP2E1, GenBank No. AF061442.1) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH, GenBank No. DQ403053.1) were as follows: CYP2E1: sense 5′ -CTCCTCGTCATATCCATCTG-3′ , antisense 5′ -GCAGCCAATCACAAATGTGG-3′ ; GAPDH: sense 5′ -GACCCCTTCATTGACCTCAAC-3′ , antisense 5′ -GGAGATGATGAC CCTTTTGGC-3′ . The sizes of reaction products were 474 bp for CYP2E1 and 264 bp for GAPDH. PCR amplification was performed using a DNA thermal cycler (2720 Thermal Cycler, Applied Biosystems/Life Technologies, Foster City, CA, USA) under the following conditions: CYP2E1: 35 cycles of 94 ∘ C for 1 min, 54 ∘ C for 1 min and 72 ∘ C for 2 min, followed by 10 min at 72 ∘ C; GAPDH: 25 cycles of 94 ∘ C for 1 min, 52 ∘ C for 1 min and 72 ∘ C for 2 min, followed by 10 min at 72 ∘ C. The final products were subjected to electrophoresis on 20 g L−1 agarose gel and detected by ethidium bromide staining and UV light. The relative expression levels of the mRNAs of the target genes were normalized using GAPDH internal standard.27
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Histopathological examination The livers were preserved in 100 mL−1 buffered formalin for at least 24 h, dehydrated with a sequence of ethanol solutions and
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processed for embedding in paraffin. Sections 5–6 mm thick were cut, deparaffinized, rehydrated, stained with hematoxylin and eosin (H&E) for the estimation of hepatocyte necrosis and vacuolization, as well as with Masson trichrome stain and Sirius red stain for the estimation of hepatocyte fibrosis, and subjected to photomicroscopic examination. The histological scoring of hepatic damage and fibrosis was expressed using the following score system: 0, no histopathologic change; ≤1, mild histopathologic change; ≤2, moderate histopathologic change; ≤3, severe histopathologic change.23 Statistical analysis All data were subjected to two-way analysis of variance (ANOVA).28 Duncan’s new multiple range test was applied to revolve differences among treatment means. All statistical analyses were performed using the statistical software SPSS 11.0 (SPSS UK Ltd, Woking, UK). A P value below 0.05 was considered statistically significant. Ratio values were not sin−1 transformed before statistical analysis.
RESULTS Effect of Cholestin on ethanol-induced liver injury in rats The effect of various doses of Cholestin on serum biochemical markers in ethanol-intoxicated rats was studied (Fig. 1). After injection of ethanol, serum activities of AST, ALT and ALP enzymes in the ethanol-treated groups (groups C–G) were significantly increased (P < 0.05) compared with the normal control group (group A). Treatment of animals with different doses of Cholestin significantly reduced the activities of serum AST, ALT and ALP compared with the group treated with ethanol alone. The positive control drug silymarin at a dose of 200 mg kg−1 also reduced the levels of serum AST, ALT and ALP. Effect on rat kidney There was no significant difference (P > 0.05) in plasma concentrations of BUN and creatinine among the various groups. Effect of ethanol metabolism enzymes on gene expression of liver CYP2E1 Higher CYP2E1 gene expression was observed in the ethanol group than in the other groups (Fig. 2). Hence the acceleration of ethanol elimination by Cholestin is highly correlated with increased ADH and ALDH in ethanol-fed rats (Fig. 2). Effect of Cholestin on non-enzymatic antioxidants Figure 2 shows the levels of non-enzymatic antioxidants (vitamin E, vitamin C and GSH) in tissues. The levels of vitamin E, vitamin C and GSH were significantly (P < 0.05) reduced in ethanol-treated rats compared with control rats. Administration of Cholestin (groups D–F) and silymarin (group G) significantly (P < 0.05) restored the levels of non-enzymatic antioxidants in tissues. Effect of Cholestin on antioxidant enzymes The hepatic antioxidant enzyme activities of SOD and CAT were decreased in the liver of rats treated with ethanol; however, their activities were restored by Cholestin (groups D–F). As shown in Fig. 3, the hepatic GSH-Px level was markedly lower in ethanol-intoxicated rats. However, the GSH-Px level was significantly increased by Cholestin (groups D–F) treatment compared
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Table 2. When compared with the normal liver tissues of vehicle controls, liver tissues in rats treated with ethanol revealed extensive liver injuries characterized by moderate to severe hepatocellular hydropic degeneration and necrosis around the central vein, lipidosis, hepatic fibrosis and cholangiocyte hyperplasia. However, the histopathological hepatic lesions induced by administration of ethanol were only markedly ameliorated in central lobular necrosis, hepatic lipidosis, and hepatic fibrosis by treatment with Cholestin and silymarin.
Histopathological examination The histological observations supported the results obtained from serum enzyme assays. Liver sections from control rats showed normal lobular architecture and hepatic cells with well-preserved cytoplasm and well-defined nucleus and nucleoli (Fig. 4). The results of hepatic histopathological examination are shown in
DISCUSSION
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In the present study the capability of Cholestin to protect against ethanol toxicity and oxidative stress was investigated. AST and ALT are reliable markers for liver function. It is established that AST can be found in the liver, cardiac muscle, skeletal muscle, kidney, brain, pancreas, lungs, leukocytes and erythrocytes, while
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with the ethanol group. Administration of silymarin (group G) significantly increased (P < 0.05) the activities of SOD, CAT and GSH-Px compared with the ethanol-treated group. The expected increases in hepatic and serum lipid peroxidative indices in the ethanol-treated group confirmed that oxidative damage had been induced (Fig. 3). Treatment with various doses of Cholestin (groups D–F) led to significantly lower levels of TBARS in the liver and plasma compared with the ethanol-treated group. Silymarin (group G) also inhibited the elevation of TBARS levels caused by ethanol administration.
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Figure 2. Effects of ethanol and Cholestin extract on vitamin C, vitamin E and GSH levels and ADH and ALDH activities in liver and CYP2E1 gene expression of rats. For each parameter, values with different letters are significantly different (P < 0.05). Group diets A–G are shown in Table 1.
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ALT is present in the liver.29 Increased levels of serum enzyme such as AST and ALT indicate increased permeability and damage and/or necrosis of hepatocytes.30 In our study we found that chronic ethanol consumption caused a significant increase in the activities of AST, ALT and ALP, which could cause severe damage to tissue membranes. The decreased activities of these enzymes in Cholestin-administered rats indicate the hepatoprotective effect of Cholestin. We studied further other underlying mechanisms responsible for this hepatoprotective action of Cholestin in rats. The protective effect of Cholestin was accompanied by partial prevention of GSH-Px depletion in liver tissue. It is considered that hepatic GSH-Px represents an enzyme reserve of the liver that is responsible for reducing hepatotoxicity induced by active metabolites of ethanol toxicity. As GSH-Px is also a crucial determinant of tissue susceptibility to oxidative damage,31 the partial protection of
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GSH-Px reserves by Cholestin provides an additional action not only to remove active metabolites of ethanol toxicity but also to scavenge free radicals that are involved in lipid peroxidation. Ethanol toxicity also increased lipid peroxidation, as a result of which the hepatic TBARS level was elevated.32 Cholestin treatment prevented this effect, indicating that Cholestin was able to attenuate lipid peroxidation induced by ethanol toxicity. Non-enzymatic antioxidants such as GSH, vitamin C and vitamin E are closely interlinked with each other and play a vital role in protecting cells from lipid peroxidation. In addition to GSH, we also observed a decrease in the levels of antioxidants such as vitamin C and vitamin E in tissue of ethanol-treated rats. Supplementation of Cholestin to ethanol-treated rats restored non-enzymatic antioxidant levels in the liver. The animal groups treated with Cholestin showed an increase in the levels of SOD and CAT, indicating the antioxidant activity of Cholestin.
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Figure 3. Effects of ethanol and Cholestin extract on SOD, CAT and GSH-Px activities in liver and plasma and hepatic TBARS concentrations of rats. For each parameter, values with different letters are significantly different (P < 0.05). Group diets A–G are shown in Table 1.
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toxicity-induced liver injury is the formation of lipid peroxides by free radical derivatives of ethanol toxicity. Thus the antioxidant activity or the inhibition of free radical generation is important in the protection against ethanol toxicity-induced hepatopathy. The body has an effective defense mechanism to prevent and neutralize free radical-induced damage. This is provided by a set of endogenous antioxidant enzymes such as SOD and CAT. These enzymes constitute a mutually supportive team of defense against ROS.2 Lipid peroxidation, an ROS-mediated mechanism, has been implicated in the pathogenesis of various liver injuries and subsequent liver fibrogenesis in experimental animals. The significant non-dose-dependent decrease in hepatic lipid hydroperoxides confirmed that treatment with Cholestin could effectively protect against hepatic lipid peroxidation induced by ethanol toxicity. Hence it is possible that the mechanism of hepatoprotection of
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Hepatocellular necrosis leads to the elevation of serum AST, ALT and ALP activities and increased incidence and severity of histopathological hepatic lesions in rats. The present study revealed a significant increase in the activities of AST, ALT and ALP on exposure to ethanol toxicity, indicating considerable hepatocellular injury. Administration of Cholestin attenuated the increased levels of serum enzymes (AST, ALT and ALP) induced by ethanol toxicity and caused a subsequent recovery towards normalization comparable to the control group, with the hepatoprotective effects being comparable to those of silymarin. The effect of Cholestin was further confirmed by histopathological examinations. A higher dose of Cholestin offers hepatoprotection effective in central lobular necrosis, hepatic lipidosis and cholangiocyte hyperplasia compared with lower doses in rats. It has been hypothesized that one of the principal causes of ethanol
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Figure 4. Microscopic cross-sections of liver lobules in rats after 8 weeks (×400, H&E). Bar represents 0.01 mm. Group diets A–G are shown in Table 1.
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Cholestin may be due to its antioxidant activity. Moreover, the reduced activities of SOD and CAT observed point out the hepatic damage in rats administered with ethanol toxicity. The animal groups treated with Cholestin showed an increase in the levels of SOD and CAT, indicating the antioxidant activity of Cholestin. GSH-Px acts as an enzymatic antioxidant both intracellularly and extracellularly in conjunction with various enzymatic processes that reduce hydrogen peroxide and hydroperoxides. The depletion of hepatic GSH-Px has been shown to be associated with enhanced toxicity due to chemicals, including ethanol toxicity.33 In the present study a decrease in hepatic tissue GSH-Px level was observed in the ethanol-treated groups. The increase in hepatic
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GSH-Px level in rats treated with Cholestin may be due to de novo GSH-Px synthesis and/or GSH-Px regeneration. Administration of Cholestin significantly decreased the TBARS level compared with ethanol-treated rats, which may be due to the scavenging of free radicals generated by ethanol. Silymarin, an antioxidant flavonoid complex isolated from the seed of milk thistle (Silybum marianum, Compositae), has been used to treat hepatotoxicity diseases in clinical practice for at least two decades. Silymarin has powerful free radical-scavenging properties and regulates intracellular GSH levels.34 The way in which silymarin works is by preventing lipid peroxidation and hepatotoxicity, by acting as a chain-breaking antioxidant for
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Table 2. Effects of Cholestin extract on hepatic histopathology of liver damage in rats treated with ethanol Treatment groups
Parameter
Control (A)
Hepatocellular hydropic degeneration Central lobular necrosis Hepatic lipidosis Hepatic fibrosis Cholangiocyte hyperplasia
0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0
Cholestin (B) 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0
Ethanol (C) 2.7 ± 0.3* 2.6 ± 0.5* 2.5 ± 0.5* 2.6 ± 0.2* 2.5 ± 0.3*
Cholestin + ethanol (D) 2.1 ± 0.3* 2.2 ± 0.2* 2.1 ± 0.2* 2.3 ± 0.3* 2.2 ± 0.2*
Cholestin + ethanol (E) 1.6 ± 0.3* 1.5 ± 0.2* 1.7 ± 0.2* 1.8 ± 0.3* 1.5 ± 0.2*
Cholestin + ethanol (F)
Silymarin + ethanol (G)
0.5 ± 0.2 0.5 ± 0.1 0.3 ± 0.2 0.2 ± 0.2 0.1 ± 0.2
0.5 ± 0.2 0.6 ± 0.3 0.5 ± 0.1 0.3 ± 0.2 0.2 ± 0.1
Values are expressed as mean ± SD (n = 8) in each group. Scores: 0, no histopathologic change; ≤1, mild histopathologic change; ≤2, moderate histopathologic change; ≤3, severe histopathologic change. * P < 0.05, significantly different from control.
scavenging free radicals or by a combination of these effects.35 In fact, a considerable body of experimental work in animal models has shown that silymarin, as a positive control, reduced ethanol-induced hepatotoxic effects by preventing lipid peroxidation. In the present study, silymarin acted as an effective positive control as evidenced by it decreasing AST, ALT and ALP levels and increasing SOD, CAT and GSH-Px activities and GSH levels in the liver, while decreasing TBARS levels. Ethanol is mainly metabolized by ADH in cytosols, CYP2E1 in the endoplasmic reticulum and CAT in peroxisomes to form acetaldehyde, and is further catabolized to acetic acid by ALDH.36 However, during alcohol metabolism by CYP2E1, ROS are also generated and increase lipid peroxidation, i.e. MDA, in the liver.25,37,38 However, the level of TBARS in the plasma is an additional indicator of liver injury. The level of TBARS in the plasma of rats subjected to ethanol toxicity was significantly reduced when they were supplemented with Cholestin. This result is in agreement with previous reports.14,39 – 42 Therefore it is reasonable to assume that Cholestin may act as a good scavenger in reducing lipid peroxidation induced by oxidized fish oil,39 oxidized cholesterol,40 carbon tetrachloride41 and vitamin A.14 The results of functional tests together with histological observations suggest that ethanol leads to serious changes in liver histology. The increased formation of lipid peroxides and associated ROS leads to membrane integrity damage and other pathological changes in the liver. The efficacy of any protective drug is essentially dependent on its capacity to reduce the harmful effects of toxins and maintain the normal physiology of cells and tissues. The membrane-protective properties and antioxidant nature of Cholestin might help in alleviating pathological changes caused by ethanol in the liver. Our data indicate that Cholestin has a protective action against ethanol-induced toxicity as evidenced by the lowered tissue lipid peroxidation and elevated levels of enzymatic and non-enzymatic antioxidants in the liver. Hence our study suggests that Cholestin can play a beneficial role in the treatment of ethanol-induced tissue damage, which could be one of its therapeutic values.
ACKNOWLEDGEMENTS
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1 Wu J, Danielsson A and Zern MA, Toxicity of hepatotoxins: new insights into mechanisms and therapy. Expert Opin Investig Drugs 8:585–607 (1999). 2 Vitaglione P, Morisco F, Caporaso N and Fogliano V, Dietary antioxidant compounds and liver health. Crit Rev Food Sci Nutr 44:575–586 (2004). 3 Cheng F, Chen W, Huang Y, Zhang C, Shen Y, Hai H, et al., Protective effect of areca inflorescence extract on hydrogen peroxide-induced oxidative damage to human serum albumin. Food Res Int 44:98–102 (2011). 4 Cogger VC, Muller M, Fraser R, McLean AJ, Khan J and Le Couteur DG, The effects of oxidative stress on the liver sieve. J Hepatol 41:370–376 (2004). 5 Zeashan H, Amresh G, Singh S and Rao CV, Hepatoprotective activity of Amaranthus spinosus in experimental animals. Food Chem Toxicol 46:3417–3421 (2008). 6 Madureira AR, Amorim M, Gomes AM, Pintado ME and Malcata FX, Protective effect of whey cheese matrix on probiotic strains exposed to simulated gastrointestinal conditions. Food Res Int 44:465–470 (2011). 7 Stuart GA, Chinese Materia Medica – Vegetable Kingdom. Southern Material Center, Taipei (1979). 8 Ma J, Li Y, Ye Q, Li J, Hua Y, Ju D, et al., Constituents of red yeast rice, a traditional Chinese food and medicine. J Agric Food Chem 48:5220–5225 (2000). 9 Mei F, Fang Mei’s Illustrated Cookbook of Regional Chinese Cuisine. Guangxi National Press, Guang xi, pp. 177–188 (1990). 10 Brosche T, Kral C, Summa JD and Platt D, Effective lovastatin therapy in elderly hypercholesterolemic patients – an antioxidative impact? Arch Gerontol Geriatr 22:207–221 (1996). 11 Zhang M and Duan ZXS, Active components of xuezhikang. Chin J New Drugs 7:213–214 (1998). 12 Su CF, Liu IM and Cheng JT, Improvement of insulin resistance by Hon-Chi in fructose-rich chow-fed rats. Food Chem 104:45–52 (2007). 13 Wang IK, Lin-Shiau SY, Chen PC and Lin JK, Hypotriglyceridemic effect of Anka (a fermented rice product of Monascus sp.) in rats. J Agric Food Chem 48:3183–3189 (2000). 14 Yeh YH, Lee YT and Hsieh HS, Effect of Cholestin on toxicity of vitamin A in rats. Food Chem 132:311–318 (2012). 15 Yeh YH, Lee YT, Hsieh HS and Hwang DF, Effect of red yeast rice on toxicity of oxidized cholesterol and oxidized fish oil in rats. e-SPEN 5:e230–e237 (2010). 16 Li YG, Liu H and Wang ZT, A validated stability-indicating HPLC with photodiode array detector (PDA) method for the stress tests of Monascus purpureus fermented rice, red yeast rice. J Pharmaceut Biomed Anal 39:82–90 (2003). 17 Segura B, Red yeast rice: an easy way to lower cholesterol. Nutr Bytes 9(1) (2003). 18 Su YC, Wang JJ, Lin TT and Pan TM, Production of the secondary metabolism 𝛽-aminobutyric acid and monacolin K by Monascus. J Ind Microbiol Biotechnol 30:41–46 (2003).
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This study was supported by Asia University (100-asia-18) and National Science Council (MOST 103-2622-B-040-001-CC3).
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