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Corn oligopeptides protect against early alcoholic liver injury in rats
Food and Chemical Toxicology 50 (2012) 2149–2154
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Corn oligopeptides protect against early alcoholic liver injury in rats Feng Zhang, Jiali Zhang, Yong Li ⇑ Department of Nutrition and Food Hygiene, School of Public Health, Peking University, Xueyuan Road 38, Haidian District, Beijing 100191, PR China Beijing Key Laboratory of Toxicological Research and Risk Assessment for Food Safety, Peking University, Xueyuan Road 38, Haidian District, Beijing 100191, PR China
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Article history: Received 15 February 2012 Accepted 31 March 2012 Available online 13 April 2012 Keywords: Corn oligopeptides Alcoholic liver injury Antioxidation Lipid metabolism
a b s t r a c t Background: The present study aimed to investigate the effects of corn oligopeptides (COPs) on early alcoholic liver injury in rats. A total of 70 Wistar rats were randomly assigned to 7 groups, including a normal control group, 3 alcohol control groups (2.0, 4.0 and 6.0 g/kg/BW ethanol), and 3 COP intervention groups (2.0, 4.0 and 6.0 g/kg/BW ethanol, with 900 mg/kg/BW COPs). The study duration lasted for 4 weeks. Serum markers were assayed, and a histopathological examination was conducted. Results: We found that the COP treatment prevented the elevation of serum aminotransferase and alleviated the hepatic histological damage that was induced by alcohol. In addition, the COPs counteracted the changes in the SOD activity and the MDA content in serum. Furthermore, the COPs ameliorated the abnormal lipid metabolism. Conclusion: These findings suggest that COPs have a significant protective effect on early alcoholic liver injury in rats. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Alcoholism remains a serious health problem worldwide, and it is reported that there are approximately 2 billion alcohol consumers worldwide and 76.3 million with diagnosable alcohol use disorders (WHO, 2004). The excessive and chronic intake of alcohol can lead to alcoholic liver disease (ALD) of which alcoholic fatty liver, alcoholic hepatitis and alcoholic cirrhosis are the most widely diagnosed forms (O’Shea et al., 2010). There are more than 2 million ALD patients estimated in the United States (McClain et al., 2004); in addition, it is reported that patients with alcoholic hepatitis and cirrhosis had greater than 60% mortality over a 4-year period in the Veterans Administration Cooperative Studies (Arteel et al., 2003). Several mechanisms have been identified to contribute to ALD, such as oxidative stress, mitochondrial damage and inflammatory factors (Albano, 2008; Tilg and Diehl, 2000), and increasing evidence now supports the notion that oxidative stress plays a pivotal role in the progression of liver damage (Arteel, 2003; Yuan et al., 2007). ALD has been well recognized a major cause of morbidity and mortality in the world; yet, other than abstinence, few satisfactory treatments for ALD have been described. Although incomprehensive, it has been reported that several bioactive peptides, including Ganoderma lucidum peptides (Shi et al., 2008) and shark hepatic stimulator substance (Lu et al., 2004), have protective effects against the liver injury induced by D-galactosamine or acetaminophen. ⇑ Corresponding author. Address: Department of Nutrition and Food Hygiene, School of Public Health, Peking University, Xueyuan Road 38, Haidian District, Beijing 100191, PR China. Tel./fax: +86 10 82801177. E-mail address: [email protected] (Y. Li). 0278-6915/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fct.2012.03.083
Indeed, these hepatoprotective agents found both in plants and animals are currently attracting much attention. Corn gluten meal (CGM) is a byproduct of the starch industry and contains approximately 60% (w/w) protein (Lin et al., 2011); corn oligopeptides (COPs) are low molecular weight peptides derived from CGM by enzymatic hydrolysis. The various multifunctional properties of COPs have been described previously, such as the inhibition of angiotensin I-converting enzyme (Lin et al., 2011), the alleviation of fatigue (Chang, 2004), resistance to lipid peroxidation (Xu et al., 2002) and the facilitation of alcohol metabolism (Yamaguchi et al., 1997). However, the effect of COPs on alcoholic liver injury has not yet been elucidated. Therefore, in the present study, the effect of COPs on early alcoholic liver injury was investigated in rats. Liver injury was evaluated by both biochemical parameters and histopathological changes. In addition, as indicators of hepatic lipid peroxidation, we evaluated the levels of MDA and SOD to determine the possible mechanisms. 2. Materials and methods 2.1. Materials Commercial kits used for the determination of T-SOD and MDA in serum were purchased from the Jiancheng Institute of Biotechnology (Nanjing, China). Ethanol and the other chemicals used were all of analytical grade and purchased from Beijing Chemical Co. (Beijing, China). 2.2. Preparation and identification of COPs The COPs, which were prepared from CGM, were donated by CF Haishi Biotechnology Ltd. Co. (Beijing, China). Briefly, after being ground through a 60-mesh sieve, the CGM was suspended in distilled water (1:10, w/w) and then hydrolyzed at pH
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11.0 and 90 °C for 1 h. The suspension was neutralized and centrifuged to recover the insoluble protein precipitate, which was then resuspended and subjected to the same procedure above to produce the wet corn protein isolate (CPI). The wet CPI was resuspended to a concentration of 6% (w/w) and subjected to a two-step enzymatic hydrolysis. The first step, with crude alkaline proteinases, was performed at pH 8.5 and 55 °C for 3 h. The second step, with crude neutral proteinases, was performed at pH 7.0 and 45 °C for 2 h. The resulting hydrolysates were centrifuged to remove the impurities, and the supernatant was filtered successively through 10 and 1 kDa MWCO ceramic membranes. A procedure of nanofiltration was then performed to remove the mineral salts, and the purified liquid was condensed by cryoconcentration under a vacuum at 70 °C with an evaporation rate of 500 kg/h. When the concentration was almost 30 Baume degrees, it was decolored with 12% active carbon at 75 °C for 1 h, and then the carbon was removed by filtration. Most of the water was removed by spray drying with a pressure of 20 MPa. This scheme produced the COP powder that was used in the following procedures. The COP sample contained approximately 91.61% hydrolyzed protein, 5.56% ash, 0.82% carbohydrate, 1.49% water and 0.52% fat. The molecular weight distributions of the COPs were determined using an HPLC system (LC-20AD, Shimadzu, Kyoto, Japan) and a TSK-gel 2000 SWXL column (7.8 300 mm, Tosoh, Tokyo, Japan), according to the method of Kong et al. (2008). The HPLC was performed with the mobile phase (45% acetonitrile with 0.1% TFA, v/v) at a flow rate of 0.5 ml/min. A molecular weight calibration curve was prepared from the average retention times of the following standards (Sigma, St. Louis, MO, USA): cytochrome C (12.5 kDa), aprotinin (6.5 kDa), bacitracin (1450 Da), tetrapeptide GGYR (451 Da) and tripeptide GGG (189 Da). In addition, a total amino acid analysis was conducted using an amino acid analyser (835–50, Hitachi, Tokyo, Japan), according to the method of Yang et al. (2007). The results indicated that 96.77% of the peptides in the COPs were distributed below 1000 Da and that the average molecular weight in the COP mixture was 363 Da. The average molecular weight of the amino acids was 137 Da, and the mean peptide length was approximately 2.7 residues. The composition of amino acids is shown in Table 1.
group, which received an equivalent of distilled water. After 1 h, the rats of the three intervention groups were intragastrically administered COPs, whereas the others received an equal volume of vehicle as a control. The study lasted for 4 weeks. The animal body weights were obtained weekly to determine the effects of the COPs on the body weight and to adjust the injection volumes. At 12 h after the last alcohol treatment, the rats were anesthetized by CO2 inhalation and then sacrificed. The blood was obtained, and the serum was separated by centrifugation at 3000 rpm for 10 min; portions of the liver for histopathological examination were also obtained. 2.5. Histological study Portions of tissues from the same lobe of the liver in each rat were immediately prepared for frozen sections (10 lm) and Oil Red-O staining and were then observed at 400 magnification under a light microscope (Nikon Y-FL light microscope, Tokyo, Japan). 2.6. Biochemical assays The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total cholesterol (TC), triglycerides (TG), low-density lipoprotein-cholesterol (LDLC) and high-density lipoprotein-cholesterol (HDL-C) in the serum were detected using an automatic biochemistry analyser (Hitachi 7020, Tokyo, Japan). The SOD activity and the MDA contents in the serum were determined using detection kits, according to the manufacturer’s respective protocols. 2.7. Statistical analysis All of the values were expressed as the means ± SD. Statistical analyses were performed using SPSS for Windows, version 17.0 and the analysis of variance (ANOVA) and post hoc LSD tests. p < 0.05 was considered statistically significant.
2.3. Animals A total of 70 male Wistar rats, weighing 180–200 g, were obtained from the Animal Service of Health Science Center, Peking University and adapted to the vivarium for 1 week before the treatment began. The conditions consisted of a filter-protected air-conditioned room, with a controlled temperature (25 ± 28 °C), relative humidity (60 ± 5%) and 12-h light/dark cycles (light on between 07:30 and 19:30 h). The animal treatment and maintenance were conducted in complete accordance with the Principle of Laboratory Animal Care (NIH Publication No. 85–23, revised 1985) and the guidelines of the Peking University Animal Research Committee.
2.4. Experimental procedure All of the rats were fed with a model AIN-93M rodent diet (Vital River Limited Company, Beijing, China), with casein as the main protein source. The animals were randomly assigned to 7 groups, including a normal control group (defined as NC group), 3 alcohol control groups (2.0, 4.0 and 6.0 g/kg/BW ethanol, designated as the LA, MA and HA groups), and 3 COP intervention groups (2.0, 4.0 and 6.0 g/kg/ BW ethanol, with 900 mg/kg/BW COP, designated as the CLA, CMA and CHA groups). COP intervention started on the same day when the rats received alcohol. The rats were intragastrically administered alcohol, except for the rats in the NC
Table 1 Amino acid composition of corn oligopeptides. Amino acid
No. of residues/100 residues
Alanine Aspartic acid Arginine Cysteine Glutamic acid Glycine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Proline Serine Threonine Tryptophan Tyrosine Valine
9.68 5.28 1.79 0.36 24.21 1.61 1.36 3.49 18.27 0.25 2.51 5.82 8.29 4.83 2.84 0.26 5.51 3.63
3. Results 3.1. Histopathological observation The hepatoprotective effect of COP was confirmed by histopathological examination (Fig. 1). Morphological changes in the liver were observed using the Oil Red-O staining method. There were no pathological changes in the livers in the NC group (Fig. 1a). Alcohol intoxication (Fig. 1b–d) induced the assembly of lipid droplets in the hepatocytes, especially in the HA group (Fig. 1d), and the COP treatment effectively reduced these changes (Fig. 1e–g). 3.2. Effects of COPs on body weight During the first week, there was no significant difference observed among the different groups. After the second week, the body weights of the CMA and CHA groups had declined significantly (p < 0.05), compared to the NC group. From the third week, the body weights of the HA group had reduced markedly (p < 0.05), and the COP treatment did not change the body weight notably in comparison to the rats administered alcohol alone (Table 2). 3.3. Serum ALT and AST levels The serum levels of ALT and AST were used to evaluate the early liver injury induced by the alcohol. The rats intoxicated with alcohol (the MA and HA groups) developed hepatocellular injuries, with a significant change (p < 0.05) in the serum ALT activities, which increased by 19.6% and 21.4%, respectively, compared to the NC group. The COP treatment demonstrated protection against alcohol-induced injury by lowering the elevation of the ALT level to the normal level. The ALT level decreased by 15.8% in the CMA group compared to the MA group (p < 0.05), whereas a 13.6% reduction was found in the CHA group compared to the HA group (p < 0.05), which was similar to the NC group. In contrast, neither alcohol administration nor the COP treatment resulted in significant changes in the serum AST activities compared to the NC group (Table 3).
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Fig. 1. Representative histopathological changes in the liver of different groups (400). Seventy male Wistar rats weighing 180–200 g were randomly assigned to 7 groups, including normal control group (defined as NC group), 3 alcohol control groups (2.0, 4.0 and 6.0 g/kg/BW ethanol, defined as LA, MA and HA groups), and 3 COP intervention groups (2.0, 4.0 and 6.0 g/kg/BW ethanol, with 900 mg/kg/BW COP, respectively, defined as CLA, CMA and CHA groups). After 4 weeks, rats were sacrificed and portions of tissues from the same lobe of liver in each rat were immediately prepared for further histological study. Morphological change in liver was observed using Oil Red-O staining method (400). There were no pathological changes observed in NC group (a). Alcohol intoxication (b–d) could induce lipid droplets assembled in hepatocytes, especially in HA group (d). COP treatment reduced these changes effectively (e–g). (a) NC group; (b) LA group; (c) MA group; (d) HA group; (e) CLA group; (f) CMA group; (g) CHA group. NC Group, normal control group; LA Group, ethanol 2.0 g/kg/BW; MA Group, ethanol 4.0 g/kg/BW; HA Group, ethanol 6.0 g/kg/BW CLA Group; COP 900 mg/kg/BW + ethanol 2.0 g/kg/BW; CMA Group, COP 900 mg/kg/BW + ethanol 4.0 g/kg/BW; CHA Group, COP 900 mg/kg/BW + ethanol 6.0 g/kg/BW.
3.4. Serum lipid levels The serum TC levels increased notably in the MA and HA groups (p < 0.05) by 21.1% and 34.0%, respectively, compared to the NC group. The COP treatment restored this change in the CHA group compared with the HA group (p < 0.05). Furthermore, the HDL-C levels in the MA and HA groups showed prominent changes com-
pared to the NC group (p < 0.05), and these alterations returned to normal after the COP treatment. In addition, the LDL-C levels in the MA and HA groups were significantly elevated compared to the NC group (p < 0.05). The COP treatment significantly lowered this change in the CHA group (p < 0.05) compared to the HA group. However, the serum triglyceride (TG) levels showed no changes among the different groups (Table 4).
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Table 2 Effect of COP on body weights. Group
NC LA MA HA CLA CMA CHA
Table 5 Effect of COP on serum SOD and MDA.
Body weights(g)a W1
W2
W3
W4
231.4 ± 10.4 232.6 ± 8.0 231.1 ± 8.7 231.2 ± 7.7 230.8 ± 8.9 231.7 ± 9.5 230.8 ± 8.2
298.9 ± 10.3 296.4 ± 11.8 291.7 ± 17.3 290.1 ± 15.5 292.2 ± 16.9 284.1 ± 18.2* 282.1 ± 22.0*
342.0 ± 24.6 340.2 ± 15.3 334.6 ± 21.8 316.9 ± 27.5* 333.2 ± 23.9 323.0 ± 21.7* 313.3 ± 38.3*
382.1 ± 29.3 379.5 ± 17.8 373.4 ± 37.9 349.8 ± 33.4* 376.9 ± 27.3 363.1 ± 25.1* 347.9 ± 34.3*
NC Group, normal control group; LA Group, ethanol 2.0 g/kg/BW; MA Group, ethanol 4.0 g/kg/BW; HA Group, ethanol 6.0 g/kg/BW; CLA Group, COP 900 mg/kg/ BW + ethanol 2.0 g/kg/BW; CMA Group, COP 900 mg/kg/BW + ethanol 4.0 g/kg/BW; CHA Group, COP 900 mg/kg/BW + ethanol 6.0 g/kg/BW. a Values are means ± SD, n = 10. * Statistical significance: p < 0.05, compared with NC group.
Table 3 Effects of COPs on serum ALT and AST levels. Group
ALT(IU/L)a
AST(IU/L)a
NC LA MA HA CLA CMA CHA
35.37 ± 3.26 37.10 ± 2.67 42.30 ± 5.18* 42.93 ± 6.65* 36.13 ± 5.92 35.62 ± 4.54# 37.10 ± 5.04##
157.82 ± 20.01 150.53 ± 27.17 160.67 ± 13.43 157.17 ± 14.41 148.63 ± 32.49 151.47 ± 14.09 152.37 ± 18.83
NC Group, normal control group; LA Group, ethanol 2.0 g/kg/BW; MA Group, ethanol 4.0 g/kg/BW; HA Group, ethanol 6.0 g/kg/BW; CLA Group, COP 900 mg/kg/ BW + ethanol 2.0 g/kg/BW; CMA Group, COP 900 mg/kg/BW + ethanol 4.0 g/kg/BW; CHA Group, COP 900 mg/kg/BW + ethanol 6.0 g/kg/BW. a Values are means ± SD, n = 10. * Statistical significance: p < 0.05, compared with NC group. # Statistical significance: p < 0.05, compared with MA group. ## Statistical significance: p < 0.05, compared with HA group.
Table 4 Effect of COP on serum lipid levels. Group
HDL-C (mmol/L)a
LDL-C (mmol/ L)a
TG (mmol/ L)a
TC (mmol/L)a
NC LA MA HA CLA CMA CHA
1.76 ± 0.20 1.92 ± 0.19 2.14 ± 0.13* 2.34 ± 0.37* 1.91 ± 0.10 1.84 ± 0.13# 1.92 ± 0.39##
0.38 ± 0.06 0.46 ± 0.10 0.51 ± 0.11* 0.66 ± 0.13* 0.49 ± 0.07 0.45 ± 0.10 0.51 ± 0.12*,##
1.35 ± 0.43 1.36 ± 0.51 1.46 ± 0.39 1.34 ± 0.45 1.15 ± 0.40 1.16 ± 0.26 1.15 ± 0.29
1.94 ± 0.20 2.23 ± 0.39 2.35 ± 0.22* 2.60 ± 0.19* 2.22 ± 0.22 2.08 ± 0.22 2.11 ± 0.23##
NC Group, normal control group; LA Group, ethanol 2.0 g/kg/BW; MA Group, ethanol 4.0 g/kg/BW; HA Group, ethanol 6.0 g/kg/BW; CLA Group, COP 900 mg/kg/ BW + ethanol 2.0 g/kg/BW; CMA Group, COP 900 mg/kg/BW + ethanol 4.0 g/kg/BW; CHA Group, COP 900 mg/kg/BW + ethanol 6.0 g/kg/BW. a Values are means ± SD, n = 10. * Statistical significance: p < 0.05, compared with NC group. # Statistical significance: p < 0.05, compared with MA group. ## Statistical significance: p < 0.05, compared with HA group.
3.5. Serum MDA and SOD levels The liver injury induced by alcohol administration caused a significant reduction in the SOD activities (p < 0.05) in the sera of the MA and HA groups compared to the NC group: the SOD activities decreased by 20.4% and 36.5%, respectively. In addition, a remarkable reduction in SOD was also observed in the CHA group compared to the NC group (p < 0.05). The activities of SOD were
Group
SOD(U/mL)a
MDA(nmol/L)a
NC LA MA HA CLA CMA CHA
154.1 ± 17.4 147.3 ± 13.2 122.7 ± 12.1* 97.9 ± 4.0* 148.5 ± 10.5 146.2 ± 6.2# 136.8 ± 10.7*,##
7.1 ± 4.2 11.8 ± 1.4 17.2 ± 4.7* 23.7 ± 3.9* 11.4 ± 3.6 12.9 ± 5.7 18.7 ± 3.6*,##
NC Group, normal control group; LA Group, ethanol 2.0 g/kg/BW; MA Group, ethanol 4.0 g/kg/BW; HA Group, ethanol 6.0 g/kg/BW; CLA Group, COP 900 mg/kg/ BW + ethanol 2.0 g/kg/BW; CMA Group, COP 900 mg/kg/BW + ethanol 4.0 g/kg/BW; CHA Group, COP 900 mg/kg/BW + ethanol 6.0 g/kg/BW. a Values are means ± SD, n = 10. * Statistical significance: p < 0.05, compared with NC group. # Statistical significance: p < 0.05, compared with MA group. ## Statistical significance: p < 0.05, compared with HA group.
markedly increased by 16.2% (p < 0.05) by the COP treatment in the CMA group compared to the MA group. The same effect was also observed in the CHA group, with a 39.7% increase in comparison to the HA group (Table 5). Compared to the NC group, the serum MDA content in the MA and HA groups increased by 142.3% and 233.8%, respectively, which was significant (p < 0.05). The MDA content in the CHA group rose markedly compared with the NC group (p < 0.05), and the COP treatment attenuated this change in the CHA group compared with the HA group (p < 0.05) (Table 5). 4. Discussion ALD is one of the most serious health problems globally (Arteel et al., 2003; McClain et al., 2004). In spite of the tremendous strides in modern medicine, few drugs are available for liver disorders (Bhandarkar and Khan, 2004). Recently, much attention has been paid to bioactive peptides from foods for multiple physiological activities. COP derived from CGM that has been used in food processing was reported to possess a lot of beneficial properties (Chang, 2004; Lin et al., 2011; Xu et al., 2002; Yamaguchi et al., 1997). Our analysis of the low-molecular-weight distribution confirmed that the main composition of the COPs was small peptides, which were rich in Glu, Leu, Ala, Pro, Phe and Tyr. This study was designed to examine the effect of COP on early alcoholic liver injury, and various methods have been developed for alcoholic liver injury models to validate hepatoprotective agents. A dose of 6 g/ kg/BW ethanol was administered orally to rats for 8 days to induce liver injury (Wang et al., 2009). In another study, a dose of 10 g/kg/ BW ethanol was used for 4 weeks (Kono et al., 2001). In the present study, a variety of doses of alcohol were adopted to show the spectrum of alcohol-induced toxic effects, and 900 mg/kg/BW COPs was administered for the intervention based on our previous studies (unpublished). According to the reversion of the respective parameters, the results of both the biochemical measurements and the liver histopathological observations clearly demonstrated that the COPs exhibited beneficial effects. The effects of alcohol are multi-factorial, and various hypotheses regarding the mechanism of alcohol-induced hepatocyte injury have been suggested (Beier and McClain, 2010; Beier et al., 2011). Increasing evidence now supports the notion that, of the many factors that contribute to the pathogenesis of ALD, oxidative stress plays an important role in the pathogenesis of alcoholic liver disease (Bjelakovic et al., 2011; Gao et al., 2011; Sakaguchi et al., 2011). As a major by-product resulting from lipid peroxidation, MDA is an indicator of oxidative stress (Vaca et al., 1988). In addition, SOD is an enzyme for which reduced activity is associated
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with the accumulation of reactive free radicals, leading to deleterious effects (Jayakumar et al., 2006). Our results showed that the rats in the MA and HA groups had higher MDA levels and lower SOD activities in their sera than the control rats. The present study indicated that, to some extent, the COP treatment exhibited a beneficial effect on the above results, including the reversal of the MDA content in the CHA groups and the SOD activity in the CMA and CHA groups. Our findings are in accordance with several recent reports (Li et al., 2007; Guo et al., 2009; Xu et al., 2002) in which COP treatment had similar effects on oxidative stress. In other words, the protection conferred by COP against oxidative stress may be involved in the mechanism of its action to ameliorate the impairment of liver function. Aberrant lipid metabolism can be found in numerous patients with ALD (Lieber, 1974), and lipid peroxidation of the hepatocellular membrane is also stimulated by alcohol. Although the most common disturbance of lipid metabolism that is induced by alcohol abuse is the excessive accumulation of lipids in the liver, the mechanism of abnormal lipid metabolism induced by alcohol remains unclear. In our study, we observed that the TC and HDL-C levels were increased in the sera of the MA and HA groups and that the COP treatment could significantly ameliorate this change. Our findings that the TC and HDL-C levels had positive correlations with alcohol intake are in accordance with several studies (Castelli et al., 1977; Veenstra et al., 1990). It is reported that LDL-C showed a weaker negative correlation in humans (Castelli et al., 1977); however, this change was not observed in the present study, and there is no precise explanation for this phenomenon. The AST and ALT serum marker enzymes are usually cytoplasmic and leak into the blood upon liver injury due to the altered permeability of the membranes (Jayakumar et al., 2006). Our results indicated a significant elevation of the serum levels of ALT in the MA and HA groups, and the effects were markedly reduced when the rats were treated with the COPs. In the present study, instead of H&E staining, Oil-Red O staining was performed for histopathological examination, which is more suitable for displaying the fat droplets clearly in liver cells. The histopathological observations further confirmed the protective effect of the COPs in the alcohol-challenged rats, and the hepatocyte steatosis induced by alcohol was largely prevented by the COP treatment. It should also be noted that alcohol-induced liver disorder has dose- and time-dependent properties (Isayama et al., 2003). The present study showed that there was no biochemical or histopathological difference between the LA group and NC groups, which was in accordance with Holmberg’s study (Holmberg et al., 1986). The other negative results reported above could be attributed to the short duration of the study or the low dose of alcohol administered. The design of the present study was to build a model of early liver injury caused by alcohol; thus, some of the severe changes reported by other investigators were not observed.
5. Conclusion In summary, our current investigation demonstrated that COP could inhibit early alcoholic liver injury. The potential mechanisms of the hepatoprotective effects might be based on its antioxidant properties. COPs may potentially be beneficial as a functional food; however further studies, such as a long-term design and human trial, are needed.
Conflict of Interest The authors declare that there are no conflict of interest.
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Acknowledgements The authors gratefully thank CF Haishi Biotechnology Ltd. for providing the samples used in this study. This research was supported (Grant 2006BAD27B08) by the Foundation from the Ministry of Science and Technology of the People’s Republic of China.
References Albano, E., 2008. Oxidative mechanisms in the pathogenesis of alcoholic liver disease. Mol. Aspects Med. 29, 9–16. Arteel, G.E., 2003. Oxidants and antioxidants in alcohol-induced liver disease. Gastroenterology 124, 778–790. Arteel, G., Marsano, L., Mendez, C., Bentley, F., McClain, C.J., 2003. Advances in alcoholic liver disease. Best Pract. Res. Clin. Gastroenterol. 17, 625–647. Beier, J.I., McClain, C.J., 2010. Mechanisms and cell signaling in alcoholic liver disease. Biol. Chem. 391, 1249–1264. Beier, J.I., Arteel, G.E., McClain, C.J., 2011. Advances in alcoholic liver disease. Curr. Gastroenterol. Rep. 13, 56–64. Bhandarkar, M.R., Khan, A., 2004. Antihepatotoxic effect of Nymphaea stellata willd., against carbon tetrachloride-induced hepatic damage in albino rats. J. Ethnopharmacol. 91, 61–64. Bjelakovic, G., Gluud, L.L., Nikolova, D., Bjelakovic, M., Nagorni, A., Gluud, C., 2011. Antioxidant supplements for liver diseases. Aliment Pharmacol. Ther. 32, 356– 367. Castelli, W.P., Doyle, J.T., Gordon, T., Hames, C.G., Hjortland, M.C., Hulley, S.B., Kagan, A., Zukel, W.J., 1977. Alcohol and blood lipids. The cooperative lipoprotein phenotyping study. Lancet 2, 153–155. Chang, Y., 2004. Study on anti-fatigue effect of corn-peptide. Food Sci. 9, 173–178. Gao, B., Seki, E., Brenner, D.A., Friedman, S., Cohen, J.I., Nagy, L., Szabo, G., Zakhari, S., 2011. Innate immunity in alcoholic liver disease. Am. J. Physiol. Gastrointest. Liver Physiol. 300, G516–G525. Guo, H., Sun, J., He, H., Yu, G.C., Du, J., 2009. Antihepatotoxic effect of corn peptides against Bacillus calmette-guerin/lipopolysaccharide-induced liver injury in mice. Food Chem. Toxicol. 47, 2431–2435. Holmberg, B., Kronevi, T., Ekner, A., 1986. Subchronic Toxicity Investigation of Ethyl Alcohol: A Test for Lowest Effective Dose (LED) to be used in a Long-Term Bioassay for Carcinogenicity. National Board of Occupational safety and Health, Solna, Sweden. Isayama, F., Froh, M., Bradford, B.U., McKim, S.E., Kadiiska, M.B., Connor, H.D., Mason, R.P., Koop, D.R., Wheeler, M.D., Arteel, G.E., 2003. The CYP inhibitor 1aminobenzotriazole does not prevent oxidative stress associated with alcoholinduced liver injury in rats and mice. Free Radic. Biol. Med. 35, 1568–1581. Jayakumar, T., Ramesh, E., Geraldine, P., 2006. Antioxidant activity of the oyster mushroom, Pleurotus ostreatus, on CCl(4)-induced liver injury in rats. Food Chem. Toxicol. 44, 1989–1996. Kong, X., Guo, M., Hua, Y., Cao, D., Zhang, C., 2008. Enzymatic preparation of immunomodulating hydrolysates from soy proteins. Bioresour. Technol. 99, 8873–8879. Kono, H., Arteel, G.E., Rusyn, I., Sies, H., Thurman, R.G., 2001. Ebselen prevents early alcohol-induced liver injury in rats. Free Radic. Biol. Med. 30, 403–411. Li, H.M., Guo, P., Hu, X., Xu, L., Zhang, X.Z., 2007. Preparation of corn (Zea mays) peptides and their protective effect against alcohol-induced acute hepatic injury in NH mice. Biotechnol. Appl. Biochem. 47, 169–174. Lieber, C.S., 1974. Effects of ethanol upon lipid metabolism. Lipids 9, 103–116. Lin, F., Chen, L.A., Liang, R., Zhang, Z.F., Wang, J.B., Cai, M.Y., Li, Y., 2011. Pilot-scale production of low molecular weight peptides from corn wet milling byproducts and the antihypertensive effects in vivo and in vitro. Food Chem. 124, 801–807. Lu, Z.B., Li, Q., Ye, B.P., Bian, S., Wang, Y., Ruan, Q.P., Wu, W.T., 2004. Protective effects of shark hepatic stimulator substance against acute hepatic injury induced by acetaminophen in mice. Yao Xue Xue Bao 39, 17–21. McClain, C.J., Song, Z., Barve, S.S., Hill, D.B., Deaciuc, I., 2004. Recent advances in alcoholic liver disease. IV. Dysregulated cytokine metabolism in alcoholic liver disease. Am. J. Physiol. Gastrointest. Liver Physiol. 287, G497–G502. O’Shea, R.S., Dasarathy, S., McCullough, A.J., 2010. Alcoholic liver disease. Hepatology 51, 307–328. Sakaguchi, S., Takahashi, S., Sasaki, T., Kumagai, T., Nagata, K., 2011. Progression of alcoholic and non-alcoholic steatohepatitis: common metabolic aspects of innate immune system and oxidative stress. Drug Metab. Pharmacokinet. 26, 30–46. Shi, Y., Sun, J., He, H., Guo, H., Zhang, S., 2008. Hepatoprotective effects of Ganoderma lucidum peptides against D-galactosamine-induced liver injury in mice. J. Ethnopharmacol. 117, 415–419. Tilg, H., Diehl, A.M., 2000. Cytokines in alcoholic and nonalcoholic steatohepatitis. N. Engl. J. Med. 343, 1467–1476. Vaca, C.E., Wilhelm, J., Harms-Ringdahl, M., 1988. Interaction of lipid peroxidation products with DNA. A Rev. Mutat. Res. 195, 137–149. Veenstra, J., Ockhuizen, T., van de Pol, H., Wedel, M., Schaafsma, G., 1990. Effects of a moderate dose of alcohol on blood lipids and lipoproteins postprandially and in the fasting state. Alcohol Alcohol. 25, 371–377.
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F. Zhang et al. / Food and Chemical Toxicology 50 (2012) 2149–2154
Wang, H., Feng, F., Zhuang, B.Y., Sun, Y., 2009. Evaluation of hepatoprotective effect of Zhi-Zi-Da-Huang decoction and its two fractions against acute alcoholinduced liver injury in rats. J. Ethnopharmacol. 126, 273–279. WHO, 2004. Global Status Report on Alcohol 2004. World Health Organization, Geneva. Xu, L., Liu, L., Li, X., Qiao, Y., Wu, X., Zhang, X., 2002. Study on the preparation of corn functional oligopeptides and its SOD-like activity. Chin. J. Biochem. Pharm. 23, 78–80.
Yamaguchi, M., Nishikiori, F., Ito, M., Furukawa, Y., 1997. The effects of corn peptide ingestion on facilitating alcohol metabolism in healthy men. Biosci. Biotechnol. Biochem. 61, 1474–1481. Yang, Y., Tao, G., Liu, P., Liu, J., 2007. Peptide with angiotensin I-converting enzyme inhibitory activity from hydrolyzed corn gluten meal. J. Agric. Food. Chem. 55, 7891–7895. Yuan, G., Gong, Z., Li, J., Li, X., 2007. Ginkgo biloba extract protects against alcoholinduced liver injury in rats. Phytother. Res. 21, 234–238.
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Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox
Corn oligopeptides protect against early alcoholic liver injury in rats Feng Zhang, Jiali Zhang, Yong Li ⇑ Department of Nutrition and Food Hygiene, School of Public Health, Peking University, Xueyuan Road 38, Haidian District, Beijing 100191, PR China Beijing Key Laboratory of Toxicological Research and Risk Assessment for Food Safety, Peking University, Xueyuan Road 38, Haidian District, Beijing 100191, PR China
a r t i c l e
i n f o
Article history: Received 15 February 2012 Accepted 31 March 2012 Available online 13 April 2012 Keywords: Corn oligopeptides Alcoholic liver injury Antioxidation Lipid metabolism
a b s t r a c t Background: The present study aimed to investigate the effects of corn oligopeptides (COPs) on early alcoholic liver injury in rats. A total of 70 Wistar rats were randomly assigned to 7 groups, including a normal control group, 3 alcohol control groups (2.0, 4.0 and 6.0 g/kg/BW ethanol), and 3 COP intervention groups (2.0, 4.0 and 6.0 g/kg/BW ethanol, with 900 mg/kg/BW COPs). The study duration lasted for 4 weeks. Serum markers were assayed, and a histopathological examination was conducted. Results: We found that the COP treatment prevented the elevation of serum aminotransferase and alleviated the hepatic histological damage that was induced by alcohol. In addition, the COPs counteracted the changes in the SOD activity and the MDA content in serum. Furthermore, the COPs ameliorated the abnormal lipid metabolism. Conclusion: These findings suggest that COPs have a significant protective effect on early alcoholic liver injury in rats. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Alcoholism remains a serious health problem worldwide, and it is reported that there are approximately 2 billion alcohol consumers worldwide and 76.3 million with diagnosable alcohol use disorders (WHO, 2004). The excessive and chronic intake of alcohol can lead to alcoholic liver disease (ALD) of which alcoholic fatty liver, alcoholic hepatitis and alcoholic cirrhosis are the most widely diagnosed forms (O’Shea et al., 2010). There are more than 2 million ALD patients estimated in the United States (McClain et al., 2004); in addition, it is reported that patients with alcoholic hepatitis and cirrhosis had greater than 60% mortality over a 4-year period in the Veterans Administration Cooperative Studies (Arteel et al., 2003). Several mechanisms have been identified to contribute to ALD, such as oxidative stress, mitochondrial damage and inflammatory factors (Albano, 2008; Tilg and Diehl, 2000), and increasing evidence now supports the notion that oxidative stress plays a pivotal role in the progression of liver damage (Arteel, 2003; Yuan et al., 2007). ALD has been well recognized a major cause of morbidity and mortality in the world; yet, other than abstinence, few satisfactory treatments for ALD have been described. Although incomprehensive, it has been reported that several bioactive peptides, including Ganoderma lucidum peptides (Shi et al., 2008) and shark hepatic stimulator substance (Lu et al., 2004), have protective effects against the liver injury induced by D-galactosamine or acetaminophen. ⇑ Corresponding author. Address: Department of Nutrition and Food Hygiene, School of Public Health, Peking University, Xueyuan Road 38, Haidian District, Beijing 100191, PR China. Tel./fax: +86 10 82801177. E-mail address: [email protected] (Y. Li). 0278-6915/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fct.2012.03.083
Indeed, these hepatoprotective agents found both in plants and animals are currently attracting much attention. Corn gluten meal (CGM) is a byproduct of the starch industry and contains approximately 60% (w/w) protein (Lin et al., 2011); corn oligopeptides (COPs) are low molecular weight peptides derived from CGM by enzymatic hydrolysis. The various multifunctional properties of COPs have been described previously, such as the inhibition of angiotensin I-converting enzyme (Lin et al., 2011), the alleviation of fatigue (Chang, 2004), resistance to lipid peroxidation (Xu et al., 2002) and the facilitation of alcohol metabolism (Yamaguchi et al., 1997). However, the effect of COPs on alcoholic liver injury has not yet been elucidated. Therefore, in the present study, the effect of COPs on early alcoholic liver injury was investigated in rats. Liver injury was evaluated by both biochemical parameters and histopathological changes. In addition, as indicators of hepatic lipid peroxidation, we evaluated the levels of MDA and SOD to determine the possible mechanisms. 2. Materials and methods 2.1. Materials Commercial kits used for the determination of T-SOD and MDA in serum were purchased from the Jiancheng Institute of Biotechnology (Nanjing, China). Ethanol and the other chemicals used were all of analytical grade and purchased from Beijing Chemical Co. (Beijing, China). 2.2. Preparation and identification of COPs The COPs, which were prepared from CGM, were donated by CF Haishi Biotechnology Ltd. Co. (Beijing, China). Briefly, after being ground through a 60-mesh sieve, the CGM was suspended in distilled water (1:10, w/w) and then hydrolyzed at pH
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11.0 and 90 °C for 1 h. The suspension was neutralized and centrifuged to recover the insoluble protein precipitate, which was then resuspended and subjected to the same procedure above to produce the wet corn protein isolate (CPI). The wet CPI was resuspended to a concentration of 6% (w/w) and subjected to a two-step enzymatic hydrolysis. The first step, with crude alkaline proteinases, was performed at pH 8.5 and 55 °C for 3 h. The second step, with crude neutral proteinases, was performed at pH 7.0 and 45 °C for 2 h. The resulting hydrolysates were centrifuged to remove the impurities, and the supernatant was filtered successively through 10 and 1 kDa MWCO ceramic membranes. A procedure of nanofiltration was then performed to remove the mineral salts, and the purified liquid was condensed by cryoconcentration under a vacuum at 70 °C with an evaporation rate of 500 kg/h. When the concentration was almost 30 Baume degrees, it was decolored with 12% active carbon at 75 °C for 1 h, and then the carbon was removed by filtration. Most of the water was removed by spray drying with a pressure of 20 MPa. This scheme produced the COP powder that was used in the following procedures. The COP sample contained approximately 91.61% hydrolyzed protein, 5.56% ash, 0.82% carbohydrate, 1.49% water and 0.52% fat. The molecular weight distributions of the COPs were determined using an HPLC system (LC-20AD, Shimadzu, Kyoto, Japan) and a TSK-gel 2000 SWXL column (7.8 300 mm, Tosoh, Tokyo, Japan), according to the method of Kong et al. (2008). The HPLC was performed with the mobile phase (45% acetonitrile with 0.1% TFA, v/v) at a flow rate of 0.5 ml/min. A molecular weight calibration curve was prepared from the average retention times of the following standards (Sigma, St. Louis, MO, USA): cytochrome C (12.5 kDa), aprotinin (6.5 kDa), bacitracin (1450 Da), tetrapeptide GGYR (451 Da) and tripeptide GGG (189 Da). In addition, a total amino acid analysis was conducted using an amino acid analyser (835–50, Hitachi, Tokyo, Japan), according to the method of Yang et al. (2007). The results indicated that 96.77% of the peptides in the COPs were distributed below 1000 Da and that the average molecular weight in the COP mixture was 363 Da. The average molecular weight of the amino acids was 137 Da, and the mean peptide length was approximately 2.7 residues. The composition of amino acids is shown in Table 1.
group, which received an equivalent of distilled water. After 1 h, the rats of the three intervention groups were intragastrically administered COPs, whereas the others received an equal volume of vehicle as a control. The study lasted for 4 weeks. The animal body weights were obtained weekly to determine the effects of the COPs on the body weight and to adjust the injection volumes. At 12 h after the last alcohol treatment, the rats were anesthetized by CO2 inhalation and then sacrificed. The blood was obtained, and the serum was separated by centrifugation at 3000 rpm for 10 min; portions of the liver for histopathological examination were also obtained. 2.5. Histological study Portions of tissues from the same lobe of the liver in each rat were immediately prepared for frozen sections (10 lm) and Oil Red-O staining and were then observed at 400 magnification under a light microscope (Nikon Y-FL light microscope, Tokyo, Japan). 2.6. Biochemical assays The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total cholesterol (TC), triglycerides (TG), low-density lipoprotein-cholesterol (LDLC) and high-density lipoprotein-cholesterol (HDL-C) in the serum were detected using an automatic biochemistry analyser (Hitachi 7020, Tokyo, Japan). The SOD activity and the MDA contents in the serum were determined using detection kits, according to the manufacturer’s respective protocols. 2.7. Statistical analysis All of the values were expressed as the means ± SD. Statistical analyses were performed using SPSS for Windows, version 17.0 and the analysis of variance (ANOVA) and post hoc LSD tests. p < 0.05 was considered statistically significant.
2.3. Animals A total of 70 male Wistar rats, weighing 180–200 g, were obtained from the Animal Service of Health Science Center, Peking University and adapted to the vivarium for 1 week before the treatment began. The conditions consisted of a filter-protected air-conditioned room, with a controlled temperature (25 ± 28 °C), relative humidity (60 ± 5%) and 12-h light/dark cycles (light on between 07:30 and 19:30 h). The animal treatment and maintenance were conducted in complete accordance with the Principle of Laboratory Animal Care (NIH Publication No. 85–23, revised 1985) and the guidelines of the Peking University Animal Research Committee.
2.4. Experimental procedure All of the rats were fed with a model AIN-93M rodent diet (Vital River Limited Company, Beijing, China), with casein as the main protein source. The animals were randomly assigned to 7 groups, including a normal control group (defined as NC group), 3 alcohol control groups (2.0, 4.0 and 6.0 g/kg/BW ethanol, designated as the LA, MA and HA groups), and 3 COP intervention groups (2.0, 4.0 and 6.0 g/kg/ BW ethanol, with 900 mg/kg/BW COP, designated as the CLA, CMA and CHA groups). COP intervention started on the same day when the rats received alcohol. The rats were intragastrically administered alcohol, except for the rats in the NC
Table 1 Amino acid composition of corn oligopeptides. Amino acid
No. of residues/100 residues
Alanine Aspartic acid Arginine Cysteine Glutamic acid Glycine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Proline Serine Threonine Tryptophan Tyrosine Valine
9.68 5.28 1.79 0.36 24.21 1.61 1.36 3.49 18.27 0.25 2.51 5.82 8.29 4.83 2.84 0.26 5.51 3.63
3. Results 3.1. Histopathological observation The hepatoprotective effect of COP was confirmed by histopathological examination (Fig. 1). Morphological changes in the liver were observed using the Oil Red-O staining method. There were no pathological changes in the livers in the NC group (Fig. 1a). Alcohol intoxication (Fig. 1b–d) induced the assembly of lipid droplets in the hepatocytes, especially in the HA group (Fig. 1d), and the COP treatment effectively reduced these changes (Fig. 1e–g). 3.2. Effects of COPs on body weight During the first week, there was no significant difference observed among the different groups. After the second week, the body weights of the CMA and CHA groups had declined significantly (p < 0.05), compared to the NC group. From the third week, the body weights of the HA group had reduced markedly (p < 0.05), and the COP treatment did not change the body weight notably in comparison to the rats administered alcohol alone (Table 2). 3.3. Serum ALT and AST levels The serum levels of ALT and AST were used to evaluate the early liver injury induced by the alcohol. The rats intoxicated with alcohol (the MA and HA groups) developed hepatocellular injuries, with a significant change (p < 0.05) in the serum ALT activities, which increased by 19.6% and 21.4%, respectively, compared to the NC group. The COP treatment demonstrated protection against alcohol-induced injury by lowering the elevation of the ALT level to the normal level. The ALT level decreased by 15.8% in the CMA group compared to the MA group (p < 0.05), whereas a 13.6% reduction was found in the CHA group compared to the HA group (p < 0.05), which was similar to the NC group. In contrast, neither alcohol administration nor the COP treatment resulted in significant changes in the serum AST activities compared to the NC group (Table 3).
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Fig. 1. Representative histopathological changes in the liver of different groups (400). Seventy male Wistar rats weighing 180–200 g were randomly assigned to 7 groups, including normal control group (defined as NC group), 3 alcohol control groups (2.0, 4.0 and 6.0 g/kg/BW ethanol, defined as LA, MA and HA groups), and 3 COP intervention groups (2.0, 4.0 and 6.0 g/kg/BW ethanol, with 900 mg/kg/BW COP, respectively, defined as CLA, CMA and CHA groups). After 4 weeks, rats were sacrificed and portions of tissues from the same lobe of liver in each rat were immediately prepared for further histological study. Morphological change in liver was observed using Oil Red-O staining method (400). There were no pathological changes observed in NC group (a). Alcohol intoxication (b–d) could induce lipid droplets assembled in hepatocytes, especially in HA group (d). COP treatment reduced these changes effectively (e–g). (a) NC group; (b) LA group; (c) MA group; (d) HA group; (e) CLA group; (f) CMA group; (g) CHA group. NC Group, normal control group; LA Group, ethanol 2.0 g/kg/BW; MA Group, ethanol 4.0 g/kg/BW; HA Group, ethanol 6.0 g/kg/BW CLA Group; COP 900 mg/kg/BW + ethanol 2.0 g/kg/BW; CMA Group, COP 900 mg/kg/BW + ethanol 4.0 g/kg/BW; CHA Group, COP 900 mg/kg/BW + ethanol 6.0 g/kg/BW.
3.4. Serum lipid levels The serum TC levels increased notably in the MA and HA groups (p < 0.05) by 21.1% and 34.0%, respectively, compared to the NC group. The COP treatment restored this change in the CHA group compared with the HA group (p < 0.05). Furthermore, the HDL-C levels in the MA and HA groups showed prominent changes com-
pared to the NC group (p < 0.05), and these alterations returned to normal after the COP treatment. In addition, the LDL-C levels in the MA and HA groups were significantly elevated compared to the NC group (p < 0.05). The COP treatment significantly lowered this change in the CHA group (p < 0.05) compared to the HA group. However, the serum triglyceride (TG) levels showed no changes among the different groups (Table 4).
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Table 2 Effect of COP on body weights. Group
NC LA MA HA CLA CMA CHA
Table 5 Effect of COP on serum SOD and MDA.
Body weights(g)a W1
W2
W3
W4
231.4 ± 10.4 232.6 ± 8.0 231.1 ± 8.7 231.2 ± 7.7 230.8 ± 8.9 231.7 ± 9.5 230.8 ± 8.2
298.9 ± 10.3 296.4 ± 11.8 291.7 ± 17.3 290.1 ± 15.5 292.2 ± 16.9 284.1 ± 18.2* 282.1 ± 22.0*
342.0 ± 24.6 340.2 ± 15.3 334.6 ± 21.8 316.9 ± 27.5* 333.2 ± 23.9 323.0 ± 21.7* 313.3 ± 38.3*
382.1 ± 29.3 379.5 ± 17.8 373.4 ± 37.9 349.8 ± 33.4* 376.9 ± 27.3 363.1 ± 25.1* 347.9 ± 34.3*
NC Group, normal control group; LA Group, ethanol 2.0 g/kg/BW; MA Group, ethanol 4.0 g/kg/BW; HA Group, ethanol 6.0 g/kg/BW; CLA Group, COP 900 mg/kg/ BW + ethanol 2.0 g/kg/BW; CMA Group, COP 900 mg/kg/BW + ethanol 4.0 g/kg/BW; CHA Group, COP 900 mg/kg/BW + ethanol 6.0 g/kg/BW. a Values are means ± SD, n = 10. * Statistical significance: p < 0.05, compared with NC group.
Table 3 Effects of COPs on serum ALT and AST levels. Group
ALT(IU/L)a
AST(IU/L)a
NC LA MA HA CLA CMA CHA
35.37 ± 3.26 37.10 ± 2.67 42.30 ± 5.18* 42.93 ± 6.65* 36.13 ± 5.92 35.62 ± 4.54# 37.10 ± 5.04##
157.82 ± 20.01 150.53 ± 27.17 160.67 ± 13.43 157.17 ± 14.41 148.63 ± 32.49 151.47 ± 14.09 152.37 ± 18.83
NC Group, normal control group; LA Group, ethanol 2.0 g/kg/BW; MA Group, ethanol 4.0 g/kg/BW; HA Group, ethanol 6.0 g/kg/BW; CLA Group, COP 900 mg/kg/ BW + ethanol 2.0 g/kg/BW; CMA Group, COP 900 mg/kg/BW + ethanol 4.0 g/kg/BW; CHA Group, COP 900 mg/kg/BW + ethanol 6.0 g/kg/BW. a Values are means ± SD, n = 10. * Statistical significance: p < 0.05, compared with NC group. # Statistical significance: p < 0.05, compared with MA group. ## Statistical significance: p < 0.05, compared with HA group.
Table 4 Effect of COP on serum lipid levels. Group
HDL-C (mmol/L)a
LDL-C (mmol/ L)a
TG (mmol/ L)a
TC (mmol/L)a
NC LA MA HA CLA CMA CHA
1.76 ± 0.20 1.92 ± 0.19 2.14 ± 0.13* 2.34 ± 0.37* 1.91 ± 0.10 1.84 ± 0.13# 1.92 ± 0.39##
0.38 ± 0.06 0.46 ± 0.10 0.51 ± 0.11* 0.66 ± 0.13* 0.49 ± 0.07 0.45 ± 0.10 0.51 ± 0.12*,##
1.35 ± 0.43 1.36 ± 0.51 1.46 ± 0.39 1.34 ± 0.45 1.15 ± 0.40 1.16 ± 0.26 1.15 ± 0.29
1.94 ± 0.20 2.23 ± 0.39 2.35 ± 0.22* 2.60 ± 0.19* 2.22 ± 0.22 2.08 ± 0.22 2.11 ± 0.23##
NC Group, normal control group; LA Group, ethanol 2.0 g/kg/BW; MA Group, ethanol 4.0 g/kg/BW; HA Group, ethanol 6.0 g/kg/BW; CLA Group, COP 900 mg/kg/ BW + ethanol 2.0 g/kg/BW; CMA Group, COP 900 mg/kg/BW + ethanol 4.0 g/kg/BW; CHA Group, COP 900 mg/kg/BW + ethanol 6.0 g/kg/BW. a Values are means ± SD, n = 10. * Statistical significance: p < 0.05, compared with NC group. # Statistical significance: p < 0.05, compared with MA group. ## Statistical significance: p < 0.05, compared with HA group.
3.5. Serum MDA and SOD levels The liver injury induced by alcohol administration caused a significant reduction in the SOD activities (p < 0.05) in the sera of the MA and HA groups compared to the NC group: the SOD activities decreased by 20.4% and 36.5%, respectively. In addition, a remarkable reduction in SOD was also observed in the CHA group compared to the NC group (p < 0.05). The activities of SOD were
Group
SOD(U/mL)a
MDA(nmol/L)a
NC LA MA HA CLA CMA CHA
154.1 ± 17.4 147.3 ± 13.2 122.7 ± 12.1* 97.9 ± 4.0* 148.5 ± 10.5 146.2 ± 6.2# 136.8 ± 10.7*,##
7.1 ± 4.2 11.8 ± 1.4 17.2 ± 4.7* 23.7 ± 3.9* 11.4 ± 3.6 12.9 ± 5.7 18.7 ± 3.6*,##
NC Group, normal control group; LA Group, ethanol 2.0 g/kg/BW; MA Group, ethanol 4.0 g/kg/BW; HA Group, ethanol 6.0 g/kg/BW; CLA Group, COP 900 mg/kg/ BW + ethanol 2.0 g/kg/BW; CMA Group, COP 900 mg/kg/BW + ethanol 4.0 g/kg/BW; CHA Group, COP 900 mg/kg/BW + ethanol 6.0 g/kg/BW. a Values are means ± SD, n = 10. * Statistical significance: p < 0.05, compared with NC group. # Statistical significance: p < 0.05, compared with MA group. ## Statistical significance: p < 0.05, compared with HA group.
markedly increased by 16.2% (p < 0.05) by the COP treatment in the CMA group compared to the MA group. The same effect was also observed in the CHA group, with a 39.7% increase in comparison to the HA group (Table 5). Compared to the NC group, the serum MDA content in the MA and HA groups increased by 142.3% and 233.8%, respectively, which was significant (p < 0.05). The MDA content in the CHA group rose markedly compared with the NC group (p < 0.05), and the COP treatment attenuated this change in the CHA group compared with the HA group (p < 0.05) (Table 5). 4. Discussion ALD is one of the most serious health problems globally (Arteel et al., 2003; McClain et al., 2004). In spite of the tremendous strides in modern medicine, few drugs are available for liver disorders (Bhandarkar and Khan, 2004). Recently, much attention has been paid to bioactive peptides from foods for multiple physiological activities. COP derived from CGM that has been used in food processing was reported to possess a lot of beneficial properties (Chang, 2004; Lin et al., 2011; Xu et al., 2002; Yamaguchi et al., 1997). Our analysis of the low-molecular-weight distribution confirmed that the main composition of the COPs was small peptides, which were rich in Glu, Leu, Ala, Pro, Phe and Tyr. This study was designed to examine the effect of COP on early alcoholic liver injury, and various methods have been developed for alcoholic liver injury models to validate hepatoprotective agents. A dose of 6 g/ kg/BW ethanol was administered orally to rats for 8 days to induce liver injury (Wang et al., 2009). In another study, a dose of 10 g/kg/ BW ethanol was used for 4 weeks (Kono et al., 2001). In the present study, a variety of doses of alcohol were adopted to show the spectrum of alcohol-induced toxic effects, and 900 mg/kg/BW COPs was administered for the intervention based on our previous studies (unpublished). According to the reversion of the respective parameters, the results of both the biochemical measurements and the liver histopathological observations clearly demonstrated that the COPs exhibited beneficial effects. The effects of alcohol are multi-factorial, and various hypotheses regarding the mechanism of alcohol-induced hepatocyte injury have been suggested (Beier and McClain, 2010; Beier et al., 2011). Increasing evidence now supports the notion that, of the many factors that contribute to the pathogenesis of ALD, oxidative stress plays an important role in the pathogenesis of alcoholic liver disease (Bjelakovic et al., 2011; Gao et al., 2011; Sakaguchi et al., 2011). As a major by-product resulting from lipid peroxidation, MDA is an indicator of oxidative stress (Vaca et al., 1988). In addition, SOD is an enzyme for which reduced activity is associated
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with the accumulation of reactive free radicals, leading to deleterious effects (Jayakumar et al., 2006). Our results showed that the rats in the MA and HA groups had higher MDA levels and lower SOD activities in their sera than the control rats. The present study indicated that, to some extent, the COP treatment exhibited a beneficial effect on the above results, including the reversal of the MDA content in the CHA groups and the SOD activity in the CMA and CHA groups. Our findings are in accordance with several recent reports (Li et al., 2007; Guo et al., 2009; Xu et al., 2002) in which COP treatment had similar effects on oxidative stress. In other words, the protection conferred by COP against oxidative stress may be involved in the mechanism of its action to ameliorate the impairment of liver function. Aberrant lipid metabolism can be found in numerous patients with ALD (Lieber, 1974), and lipid peroxidation of the hepatocellular membrane is also stimulated by alcohol. Although the most common disturbance of lipid metabolism that is induced by alcohol abuse is the excessive accumulation of lipids in the liver, the mechanism of abnormal lipid metabolism induced by alcohol remains unclear. In our study, we observed that the TC and HDL-C levels were increased in the sera of the MA and HA groups and that the COP treatment could significantly ameliorate this change. Our findings that the TC and HDL-C levels had positive correlations with alcohol intake are in accordance with several studies (Castelli et al., 1977; Veenstra et al., 1990). It is reported that LDL-C showed a weaker negative correlation in humans (Castelli et al., 1977); however, this change was not observed in the present study, and there is no precise explanation for this phenomenon. The AST and ALT serum marker enzymes are usually cytoplasmic and leak into the blood upon liver injury due to the altered permeability of the membranes (Jayakumar et al., 2006). Our results indicated a significant elevation of the serum levels of ALT in the MA and HA groups, and the effects were markedly reduced when the rats were treated with the COPs. In the present study, instead of H&E staining, Oil-Red O staining was performed for histopathological examination, which is more suitable for displaying the fat droplets clearly in liver cells. The histopathological observations further confirmed the protective effect of the COPs in the alcohol-challenged rats, and the hepatocyte steatosis induced by alcohol was largely prevented by the COP treatment. It should also be noted that alcohol-induced liver disorder has dose- and time-dependent properties (Isayama et al., 2003). The present study showed that there was no biochemical or histopathological difference between the LA group and NC groups, which was in accordance with Holmberg’s study (Holmberg et al., 1986). The other negative results reported above could be attributed to the short duration of the study or the low dose of alcohol administered. The design of the present study was to build a model of early liver injury caused by alcohol; thus, some of the severe changes reported by other investigators were not observed.
5. Conclusion In summary, our current investigation demonstrated that COP could inhibit early alcoholic liver injury. The potential mechanisms of the hepatoprotective effects might be based on its antioxidant properties. COPs may potentially be beneficial as a functional food; however further studies, such as a long-term design and human trial, are needed.
Conflict of Interest The authors declare that there are no conflict of interest.
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Acknowledgements The authors gratefully thank CF Haishi Biotechnology Ltd. for providing the samples used in this study. This research was supported (Grant 2006BAD27B08) by the Foundation from the Ministry of Science and Technology of the People’s Republic of China.
References Albano, E., 2008. Oxidative mechanisms in the pathogenesis of alcoholic liver disease. Mol. Aspects Med. 29, 9–16. Arteel, G.E., 2003. Oxidants and antioxidants in alcohol-induced liver disease. Gastroenterology 124, 778–790. Arteel, G., Marsano, L., Mendez, C., Bentley, F., McClain, C.J., 2003. Advances in alcoholic liver disease. Best Pract. Res. Clin. Gastroenterol. 17, 625–647. Beier, J.I., McClain, C.J., 2010. Mechanisms and cell signaling in alcoholic liver disease. Biol. Chem. 391, 1249–1264. Beier, J.I., Arteel, G.E., McClain, C.J., 2011. Advances in alcoholic liver disease. Curr. Gastroenterol. Rep. 13, 56–64. Bhandarkar, M.R., Khan, A., 2004. Antihepatotoxic effect of Nymphaea stellata willd., against carbon tetrachloride-induced hepatic damage in albino rats. J. Ethnopharmacol. 91, 61–64. Bjelakovic, G., Gluud, L.L., Nikolova, D., Bjelakovic, M., Nagorni, A., Gluud, C., 2011. Antioxidant supplements for liver diseases. Aliment Pharmacol. Ther. 32, 356– 367. Castelli, W.P., Doyle, J.T., Gordon, T., Hames, C.G., Hjortland, M.C., Hulley, S.B., Kagan, A., Zukel, W.J., 1977. Alcohol and blood lipids. The cooperative lipoprotein phenotyping study. Lancet 2, 153–155. Chang, Y., 2004. Study on anti-fatigue effect of corn-peptide. Food Sci. 9, 173–178. Gao, B., Seki, E., Brenner, D.A., Friedman, S., Cohen, J.I., Nagy, L., Szabo, G., Zakhari, S., 2011. Innate immunity in alcoholic liver disease. Am. J. Physiol. Gastrointest. Liver Physiol. 300, G516–G525. Guo, H., Sun, J., He, H., Yu, G.C., Du, J., 2009. Antihepatotoxic effect of corn peptides against Bacillus calmette-guerin/lipopolysaccharide-induced liver injury in mice. Food Chem. Toxicol. 47, 2431–2435. Holmberg, B., Kronevi, T., Ekner, A., 1986. Subchronic Toxicity Investigation of Ethyl Alcohol: A Test for Lowest Effective Dose (LED) to be used in a Long-Term Bioassay for Carcinogenicity. National Board of Occupational safety and Health, Solna, Sweden. Isayama, F., Froh, M., Bradford, B.U., McKim, S.E., Kadiiska, M.B., Connor, H.D., Mason, R.P., Koop, D.R., Wheeler, M.D., Arteel, G.E., 2003. The CYP inhibitor 1aminobenzotriazole does not prevent oxidative stress associated with alcoholinduced liver injury in rats and mice. Free Radic. Biol. Med. 35, 1568–1581. Jayakumar, T., Ramesh, E., Geraldine, P., 2006. Antioxidant activity of the oyster mushroom, Pleurotus ostreatus, on CCl(4)-induced liver injury in rats. Food Chem. Toxicol. 44, 1989–1996. Kong, X., Guo, M., Hua, Y., Cao, D., Zhang, C., 2008. Enzymatic preparation of immunomodulating hydrolysates from soy proteins. Bioresour. Technol. 99, 8873–8879. Kono, H., Arteel, G.E., Rusyn, I., Sies, H., Thurman, R.G., 2001. Ebselen prevents early alcohol-induced liver injury in rats. Free Radic. Biol. Med. 30, 403–411. Li, H.M., Guo, P., Hu, X., Xu, L., Zhang, X.Z., 2007. Preparation of corn (Zea mays) peptides and their protective effect against alcohol-induced acute hepatic injury in NH mice. Biotechnol. Appl. Biochem. 47, 169–174. Lieber, C.S., 1974. Effects of ethanol upon lipid metabolism. Lipids 9, 103–116. Lin, F., Chen, L.A., Liang, R., Zhang, Z.F., Wang, J.B., Cai, M.Y., Li, Y., 2011. Pilot-scale production of low molecular weight peptides from corn wet milling byproducts and the antihypertensive effects in vivo and in vitro. Food Chem. 124, 801–807. Lu, Z.B., Li, Q., Ye, B.P., Bian, S., Wang, Y., Ruan, Q.P., Wu, W.T., 2004. Protective effects of shark hepatic stimulator substance against acute hepatic injury induced by acetaminophen in mice. Yao Xue Xue Bao 39, 17–21. McClain, C.J., Song, Z., Barve, S.S., Hill, D.B., Deaciuc, I., 2004. Recent advances in alcoholic liver disease. IV. Dysregulated cytokine metabolism in alcoholic liver disease. Am. J. Physiol. Gastrointest. Liver Physiol. 287, G497–G502. O’Shea, R.S., Dasarathy, S., McCullough, A.J., 2010. Alcoholic liver disease. Hepatology 51, 307–328. Sakaguchi, S., Takahashi, S., Sasaki, T., Kumagai, T., Nagata, K., 2011. Progression of alcoholic and non-alcoholic steatohepatitis: common metabolic aspects of innate immune system and oxidative stress. Drug Metab. Pharmacokinet. 26, 30–46. Shi, Y., Sun, J., He, H., Guo, H., Zhang, S., 2008. Hepatoprotective effects of Ganoderma lucidum peptides against D-galactosamine-induced liver injury in mice. J. Ethnopharmacol. 117, 415–419. Tilg, H., Diehl, A.M., 2000. Cytokines in alcoholic and nonalcoholic steatohepatitis. N. Engl. J. Med. 343, 1467–1476. Vaca, C.E., Wilhelm, J., Harms-Ringdahl, M., 1988. Interaction of lipid peroxidation products with DNA. A Rev. Mutat. Res. 195, 137–149. Veenstra, J., Ockhuizen, T., van de Pol, H., Wedel, M., Schaafsma, G., 1990. Effects of a moderate dose of alcohol on blood lipids and lipoproteins postprandially and in the fasting state. Alcohol Alcohol. 25, 371–377.
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F. Zhang et al. / Food and Chemical Toxicology 50 (2012) 2149–2154
Wang, H., Feng, F., Zhuang, B.Y., Sun, Y., 2009. Evaluation of hepatoprotective effect of Zhi-Zi-Da-Huang decoction and its two fractions against acute alcoholinduced liver injury in rats. J. Ethnopharmacol. 126, 273–279. WHO, 2004. Global Status Report on Alcohol 2004. World Health Organization, Geneva. Xu, L., Liu, L., Li, X., Qiao, Y., Wu, X., Zhang, X., 2002. Study on the preparation of corn functional oligopeptides and its SOD-like activity. Chin. J. Biochem. Pharm. 23, 78–80.
Yamaguchi, M., Nishikiori, F., Ito, M., Furukawa, Y., 1997. The effects of corn peptide ingestion on facilitating alcohol metabolism in healthy men. Biosci. Biotechnol. Biochem. 61, 1474–1481. Yang, Y., Tao, G., Liu, P., Liu, J., 2007. Peptide with angiotensin I-converting enzyme inhibitory activity from hydrolyzed corn gluten meal. J. Agric. Food. Chem. 55, 7891–7895. Yuan, G., Gong, Z., Li, J., Li, X., 2007. Ginkgo biloba extract protects against alcoholinduced liver injury in rats. Phytother. Res. 21, 234–238.