Research Communication In vivo protective effects of dietary curcumin and capsaicin against alcohol-induced oxidative stress
Chang-Won Pyun1 Ji-Han Kim1 Kyu-Ho Han2 Go-Eun Hong1 Chi-Ho Lee1*
1
Department of Food Science and Biotechnology of Animal Resources, Konkuk University, Seoul, 143-701, Republic of Korea
2
Department of Food Science, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-8555, Japan
Abstract BALB/c mice were exposed to chronic alcohol-induced oxidative stress by intragastric administration of excessive ethanol (5 g/kg body weight) during the 24-week period. Curcumin (0.016%) or capsaicin (0.014%) containing diets were fed with or without ethanol treatment in four groups. There was no statistically significant difference in the behavioral test between all groups during the experimental period. Only one alcoholtreated mouse fed a normal diet showed a behavioral disorder and died before the raising period was completed. There were
no effects on the activity of catalase and superoxide dismutase in the brain. However, curcumin or capsaicin treatment prevented alcohol-induced decline in brain weight. Furthermore, the levels of malondialdehyde and phosphatidylcholine hydroperoxide were significantly reduced in the brain tissue extract. The findings of this study demonstrated and confirmed the antioxidant effect of curcumin and capsaicin against alcoholinduced oxidative stress, and they suggest a direction for furC 2014 BioFactors, 00(00):000–000, 2014 ther studies. V
Keywords: ethanol; curcumin; capsaicin; brain; oxidative stress; phosphatidylcholine hydroperoxide; malondialdehyde
1. Introduction Chronic exposure to alcohol-induced oxidative stress leads to development of many diseases, directly or indirectly [1–3]. Especially, alcoholic neuropathy, the later stage of alcoholrelated diseases, is caused by various factors and complex mechanisms [1, 4]. Alcohol-related nutritional problems, such as thiamine deficiency, are indirect causes of alcoholic
C 2014 International Union of Biochemistry and Molecular Biology V
Volume 00, Number 00, Month/Month 2014, Pages 00–00 *Address for correspondence: Prof. Chi-Ho Lee, Ph.D.; Department of Food Science and Biotechnology of Animal Resources, Konkuk University, Seoul, 143–701, Korea. Tel.: 18224 503 681; Fax: 18224 53 19 48; E-mail: [email protected]. Disclosure: There is no conflict of interest. The first author (C. W. Pyun) contributed mostly to the experimental part of this work. Received 14 April 2014; accepted 22 May 2014 DOI 10.1002/biof.1172 Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com)
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neuropathy [5]. Cytotoxic proteins are generated by the surplus acetaldehydes, which are the metabolites induced by excessive consumption of ethanol (EtOH). These proteins negatively affect the neuronal cells [6]. Also, the calorie intake, which is induced by alcohol consumption, disturbs nutrient absorption [7]. Despite the efforts made to investigate the mechanism of alcohol-induced neuropathy in many studies, it has not yet been clearly identified. When the mechanism of alcohol-induced neuropathy was studied, the scholars focused on the Wernicke–Korsakoff syndrome, which occurs due to thiamine deficiency [5, 8]. In earlier days, it was thought that the main cause of alcoholinduced neuronal damage is the disturbance in thiamine metabolism, which is caused by damage to the digestive organs. Since the role of cytochrome P450 2E1 (CYP2E1) has been investigated, it has been receiving attention as the main cause of alcohol-induced neuronal damage [9, 10]. The activation of CYP2E1 induces injury to several organ tissues, including neuronal tissues, by catalyzing the production of ROS and influencing the carcinogenic precursors, such as nitrosamines and azo [11].
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Hence, the establishment of precautions against alcoholinduced oxidative stress is essential in an aging society. Actually, many biological or physiological studies were performed and reported in food science, immunology, and pharmacology journals for preventing alcohol-induced neuropathy. Capsicum spp. And Curcuma longa L. are rich in capsaicin and curcumin, respectively. These compounds increase the bioactivity in vivo under different situations and mechanisms. Capsaicin is the pungent component of capsicum spp., and its antiinflammatory or anticarcinogenic effect is more formally known [12–15]. Prevention of oxidative stress was also demonstrated in several studies [16–18]. The modulating effect of capsaicin on lipopolysaccharide-induced oxidative stress in rat organs has been reported recently [19]. Capsaicin protects gastric mucosa of rats against alcohol-induced oxidative stress injury [20, 21]. Curcumin is an antioxidant obtained from Curcuma longa rhizome, the most popular food source in South East Asia, and many researches to assess its antioxidative activity have been conducted globally [4, 22–24]. A recent study showed that proper consumption (20–80 mg/kg body weight) of curcumin prevents alcoholic neuropathy-induced behavioral aberration or alcoholic liver diseases in rodents [25, 26].
2. Aim of Work In this study, we focused on the oxidative stress indicators to determine the effect of curcumin and capsaicin against brain damage caused by alcohol-induced oxidative stress. Behavioral research, malondialdehyde level, phosphatidylcholine hydroperoxide level, catalase activity, and superoxide dismutase activity in the mouse brain tissue were analyzed.
3. Materials and Methods 3.1. Animal Experimental Plan A total of 45 BALB/c mice (7 weeks old) were purchased from the Central Animal Science (Seoul, Korea). Mice were fed for 7 days with sufficient amount of normal diet and water for adaptation to the new conditions. After the refining period, mice were trained for performing the water maze task, and they were divided into six groups, depending on their behavioral tendency. The detailed explanations for the six groups are as follows: (EtOH, ethanol; NaCl, sodium chloride): CON (n 5 7); normal diet 1 2 mL/kg of saline (0.85% NaCl); ALC (n 5 8); normal diet 1 5 g EtOH/2 mL/kg body weight; CUR (n 5 7); curcumin containing diet (0.016%) 1 2 mL/kg of saline (0.85% NaCl); CUR1ALC (n 5 8); curcumin containing diet (0.016%) 1 5 g EtOH/2 mL/kg body weight; CAP (n 5 7); capsaicin containing diet (0.014%) 1 2 mL/kg of saline (0.85% NaCl); and CAP 1 ALC (n 5 8); capsaicin containing diet (0.014%) 1 5 g EtOH/2 mL/kg body weight.
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The diet composition was designed by the previous research [23, 24, 27–29]. Saline solution and ethanol were intragastrically administered using an oral feeding needle.
3.2. Water Maze Test Recording of the escape latency time in the modified Morris water maze task was used for behavioral investigations [30]. The behavioral research was discontinued after 24 weeks.
3.3. Preparation of Phosphatidylcholine Hydroperoxide Standard Solution PC–OOH standard solution was prepared by photo-irradiation of phosphatidylcholine (PC) [31]. PC (from bovine heart, Sigma, Saint Louis, MO) was dissolved in methanol containing 0.01 mM methylene blue and was exposed to a UV lamp (50 W) for 8 hr. Oxidant was cleaned up using Supelclean LC-Si SPE tube (Supelco, Bellefonte, PA) to remove the methylene blue. The amount of peroxide in the standard solution was determined by a method of the American Oil Chemist’s Society [32]. As a result, the hydroperoxide concentration of the photo-oxidized PC was 19.1 lmol hydroperoxide-O2 per g of PC.
3.4. Determination of Brain Phosphatidylcholine Hydroperoxide Level Excessive ethanol consumption induced–tissue injury causes an increase in the PC–OOH level in the liver or brain tissue [33]. Therefore, this indicator was used to diagnose the occurrence of brain tissue injury. Total lipids in the brains were extracted by a modified Folch method [34], and the PC–OOH level in the brain extract was determined by the CL–HPLC system [31, 35]. The brain extracts were dissolved in chloroform–methanol (2:1, v/v) solution and injected into the CL–HPLC system, the detailed conditions for which are described below; for separating analytical samples, a Zorbax NH2 (4.6 3 250 mm, 5 lm, Agilent Technologies, Wilmington, DE) column was used as the stationary phase, and 2propanol-methanol-water (135:45:20, v/v/v, flow rate: 1 mL/min) as the mobile phase. The luminescence reagent was prepared by dissolving 10 mg/L of cytochrome c (from bovine heart, Sigma, Saint Louis, MO) and 2 mg/L of luminol (for chemiluminescence analysis, Wako Pure Chemicals, Osaka, Japan) in 50 mM borate buffer (pH 10.0). The luminesence reagent was inserted into the sample chamber (40 C) of the chemiluminescence analyzer (CLD1100, Tohoku Electronic Industries Co., Sendai, Japan) using a peristaltic pump at a flow rate of 1.0 mL/min [31, 35].
3.5. Quantitative Analysis of Brain Malondialdehyde Level MDA level in the brains was calculated using a colorimetric assay kit (Cell Biolabs, San Diego, CA). The tissue lysate production and assay protocol were performed as described by the manufacturer.
3.6. Measuring the Antioxidant Capacity The activity of antioxidative enzymes was determined for measuring the antioxidative capacity of the brain tissue samples. The activities of superoxide dismutase (SOD) and catalase among the antioxidative enzymes were determined. These
Curcumin and Capsaicin Protect Alcoholic Oxidation
FIG 1
Side-effect of chronic curcumin consumption with ethanol treatment.
factors were analyzed using colorimetric assay kits (Cell Biolabs, San Diego, CA) and accomplished by following the manufacturer’s instructions.
3.7. Statistical Analysis All data are presented as the mean 6 standard error of the mean (SEM.). A significant difference between the groups was calculated by a Tukey’s honestly significant difference test (P < 0.05). All statistical analyses were performed using the Statistical Analysis Software (Statistics Analytical System Institute, Cary, NC).
4. Results The alcohol-treated groups (ALC, CUR 1 ALC, and CAP 1 ALC) showed a higher death rate (50%) than the nontreated groups
TABLE 1
(CON, CUR, and CAP, 14.2%). This result indicates that curcumin and capsaicin consumption could not reduce the increased alcohol-related death rate in the present study. The exact causes of death were not investigated, although external symptoms were different in the treatment groups. The peculiar finding was that two of the mice in the CUR 1 ALC group had shown priapism symptoms approximately 2 weeks before their death (Fig. 1). There were some previous reports of heavy alcohol intake-related priapism [36, 37]. However, in this study we did not observe these symptoms in the mice of the ALC group. The relationship between curcumin consumption and priapism has not been investigated in previous studies. Therefore, for analyzing this side-effect, further studies may be needed before using curcumin as a food ingredient or preventive medicine. The maze test did not show any statistically significant difference in the escape latency time between the six groups during the rearing experiment because of its large deviation (data not shown). However, one of the mice in the ALC group showed a behavioral disorder and increased escape latency time before death.
4.1. Biological Effects of Curcumin and Capsaicin on Ethanol-Treated Mice The body weight and the weights of major organs (liver, spleen, kidney, and brain) are presented in Table 1. The spleen weight was not affected by alcohol, curcumin, and capsaicin consumption. The body and liver weights were significantly lower (P < 0.05) in the ALC group than the other 5 groups. The kidney weight was higher in the ALC group than the other groups (P < 0.05). Chronic alcohol treatment reduced the brain weight, and curcumin or capsaicin consumption prevented the alcohol-induced decline in brain weight (P < 0.05). Curcumin and capsaicin consumption prevented the decline in body and liver weights, which was induced by
Biological effects of curcumin and capsaicin on chronic alcohol-treated mice
CON
ALC
CUR
CUR 1 ALC
CAP
CAP 1 ALC
2.46 6 0.09b
2.44 6 0.09b
2.53 6 0.13b
2.53 6 0.13ab
2.88 6 0.11a
2.47 6 0.12b
Body weight (g)
21.72 6 1.19a
16.65 6 1.03b
21.80 6 1.17a
18.4 6 0.49ab
20.77 6 1.30a
18.33 6 0.31ab
Liver (g/kg body weight)
35.69 6 0.76ab
29.24 6 1.12c
37.86 6 1.16a
36.40 6 1.82ab
34.91 6 0.49b
35.83 6 0.68ab
Kidney (g/kg body weight)
15.86 6 0.47b
19.73 6 1.13a
15.70 6 0.76b
16.68 6 0.52b
16.33 6 1.01b
17.49 6 0.45ab
Spleen (g/kg body weight)
2.77 6 0.15
2.31 6 0.20
3.15 6 0.26
2.06 6 0.22
2.84 6 0.38
2.87 6 0.13
Food intake (g/day)
Brain (mg)
bc
383.3 6 7.2
c
367.3 6 8.9
ab
400.2 6 11.4
bc
376.5 6 7.3
a
405.8 6 8.0
384.0 6 8.9abc
All values are presented as means 6 SEM. The treatment groups are denoted as follows: CON, normal diet with saline treatment; ALC, normal diet with alcohol treatment; CUR, curcumin– containing normal diet with saline treatment; CUR 1 ALC, curcumin–containing normal diet with alcohol treatment; CAP, capsaicin-containing normal diet with saline treatment; and CAP 1 ALC, capsaicin-containing normal diet with alcohol treatment. a–c Significant differences between values in the same row (P < 0.05).
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4.3. Brain PC–OOH Level
FIG 2
Effect of curcumin and capsaicin on the brain MDA level in chronic alcohol-treated mice. All values are presented as mean 6 SEM. The treatment groups are denoted as follows: CON, normal diet with saline treatment; ALC, normal diet with alcohol treatment; CUR, curcumin-containing normal diet with saline treatment; CUR 1 ALC, curcumin-containing normal diet with alcohol treatment; CAP, capsaicincontaining normal diet with saline treatment; and CAP 1 ALC, capsaicin-containing normal diet with alcohol treatment. a,bSignificant differences between the values in the same row (P < 0.05).
alcohol treatment. The loss of body weight in the ALC group was caused by the prevention of decline in the plasma leptin [38]. Decreased loss of body and liver weights in the curcumin and capsaicin-treated groups indicated that these compounds may protect the body against alcohol consumption damage. This result confirms the results of previous studies, which demonstrated the preventive effect of curcumin against alcohol-induced liver damage in rodents [39]. Capsaicin also affected the liver and body weights in alcohol-treated mice, but there were no data on this finding in the earlier reports. There was no previous information on the effect of curcumin and capsaicin on brain weight. However, the data on brain weight showed the protective effect of curcumin and capsaicin against alcohol-induced brain damage. This finding indicates that curcumin and capsaicin may have a positive effect on the ethanol-treated mouse brain. The symptoms of enlarged kidneys, induced by the alcoholic liver damage [40], were observed in the ALC group. These symptoms were improved by curcumin and capsaicin treatment. This finding confirms the protective effect of curcumin and capsaicin on the liver.
4.2. Brain MDA Level The brain MDA level was increased by ethanol treatment (Fig. 2), as was suggested in previous studies [41]. Thus, the brains of mice in the ALC group were negatively affected by chronic exposure to ethanol-induced oxidative stress. Curcumin– and capsaicin–treated groups exhibited significantly lower MDA levels (P < 0.05). The reduced brain MDA levels were observed in 21% of mice in the CUR 1 ALC group and in 15% of mice in the CAP 1 ALC group compared to mice in the ALC group.
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The trend for the PC–OOH levels was similar to the trend observed for the MDA levels, that is, PC–OOH levels in mice of the ALC group were significantly higher than the PC–OOH levels in mice of the CON group (P < 0.05), and the corresponding levels in mice of the CUR 1 ALC and CAP 1 ALC groups were reduced (59.0 and 30.1% of the PC–OOH levels in mice of the ALC group, respectively; Fig. 3). The data on the MDA level and PC–OOH level suggest that alcohol consumption negatively affected the brain. In contrast, curcumin and capsaicin consumption reduced the effect of alcohol-induced oxidative stress on the brain. This antioxidative phenomenon in the brain was similar to that reported previously [19, 42–44], and the antioxidative effect against alcohol–induced oxidative stress in the brain was primary detected in this study.
4.4. Activity of Antioxidant Enzymes in the Brain Tissue Lysate There was no significant difference in the antioxidative enzyme activity in mice of each group, but the mean antioxidative enzyme activity in mice of the ALC group was slightly higher than that in mice of the other groups (P < 0.05; Fig. 4). Therefore, we supposed that the major mechanism of this antioxidative effect is the repression of superoxide formation, which is catalyzed by CYP2E1 [9]. The other possibility is that the activity of other antioxidants such as glutathione is affected by the consumption of these compounds.
FIG 3
Effect of curcumin and capsaicin consumption on the brain PC-OOH level in chronic alcohol-treated mice. All values are presented as mean 6 SEM. The treatment groups are denoted as follows: CON, normal diet with saline treatment; ALC, normal diet with alcohol treatment; CUR, curcumin-containing normal diet with saline treatment; CUR 1 ALC, curcumincontaining normal diet with alcohol treatment; CAP, capsaicin-containing normal diet with saline treatment; and CAP 1 ALC, capsaicin-containing normal diet with alcohol treatment. a,bSignificant differences between the values in the same row (P < 0.05).
Curcumin and Capsaicin Protect Alcoholic Oxidation
FIG 4
Antioxidative enzyme activities in mouse brain tissue lysate. (a) SOD activity, (b) catalase activity. All values are presented as mean 6 SEM. The treatment groups are denoted as follows: CON, normal diet with saline treatment; ALC, normal diet with alcohol treatment; CUR, curcumin-containing normal diet with saline treatment; CUR 1 ALC, curcumincontaining normal diet with alcohol treatment; CAP, capsaicin-containing normal diet with saline treatment; and CAP 1 ALC, capsaicin-containing normal diet with alcohol treatment. No significant difference was reported between the six groups (P < 0.05).
5. Discussion The body, liver, and brain weights were significantly reduced in chronic ethanol-treated mice. This result suggests that chronic ethanol consumption induced organ tissue injury and abnormal hormone secretion [38]. The present study confirmed the protective effect of curcumin and capsaicin against these phenomena. These results support the previous researches [20, 21, 25]. In addition, one of the chronic alcoholtreated mice (ALC) fed a normal diet showed an increased escape latency time on the maze task and finally died, while mice in the curcumin or capsaicin-treated groups did not show any behavioral problems. The catalase and superoxide dismutase activities were not affected by ethanol treatment in this study. This finding was different from those in previous studies [45], in which the effect of short-term ethanol treatment was investigated. Based
on this finding, we assumed that the superoxide dismutase and catalase activities were recovered during the long-term chronic ethanol consumption. This result and those in previous researches suggest that long-term treatment with ethanol or antioxidative compounds does not influence the enzyme activity [46]. The levels of malondialdehyde and phosphatidylcholine hydroperoxide in ethanol-treated brain tissue were significantly higher than those in nontreated group. This indicates that the mice were continuously exposed to oxidative stress due to chronic ethanol consumption [47]. The curcumin or capsaicin containing diets were useful for decreasing these levels. Based on previous studies, we assumed that curcumin decreased alcohol-induced superoxide production by repressing the CYP2E1 activity, as well as the capsaicin scavenged the generated superoxide [48]. However, the curcumin containing diet induced an increase in susceptibility to alcohol; two mice in the group showed a side-effect of priapism, which is known as an excessive chronic alcohol intake-related disease [36, 37]. This phenomenon suggests that curcumin consumption caused suppression of increased activity of CYP2E1 and disturbed the alcohol metabolism. This mechanism was beneficial in vivo against alcohol-induced oxidative stress because the superoxide production was relatively reduced, but the risk of immediate effect of alcohol was increased. Therefore, we assumed that direct exposure to alcohol induced priapism. The mice in the capsaicin containing diet group did not show any side-effects. This suggests that the mechanism of in vivo antioxidation effect is different between capsaicin and curcumin. Based on the previous studies, we could assume that the capsaicin containing diet increases the antioxidant bioactivity [49]. One of the important factors is the pungent taste of the capsaicin containing diet, which leads to a longer time period for adjusting to the diet, and also some of the mice may not have consumed the spicy diet. The concentration of capsaicin used in this study (0.014%) can also be intolerable to humans. Therefore, the optimum concentration of capsaicin for therapeutic use should be investigated in a further study, as well as the other antioxidative mechanisms and pathways should be studied.
6. Conclusion This study demonstrated the protective effect of curcumin and capsaicin against alcohol-induced brain damage, as well as confirmed their effect in preventing alcohol-induced liver injury. Long-term consumption of curcumin or capsaicin without alcohol treatment induced an increase in brain weight. We also suggest that the aim of future research should be to investigate the antioxidative mechanism of these compounds. If the antioxidative mechanisms of curcumin and capsaicin involve inhibition of the generation of reactive oxygen species, as previously reported, it is also likely that these compounds will affect the
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metabolic pathway of ethanol. This poses a further health risk because these compounds decrease the rate of ethanol metabolism, and thus the detoxification of ethanol is also inhibited. Therefore, it is necessary that side effects such as priapism that were noted in this study, as well as the influence of curcumin on ethanol metabolism, should be investigated further. The investigation of relationship between the various daily intake amounts and their effects is also necessary in order to use these compounds for industrial or therapeutic purposes.
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not affect plasma leptin, corticosterone, and insulin levels in pubertal rats. Metabolism 47, 1269–1273. [39] El-Deen, N. A. and Eid, M. (2010) Efficacy of curcumin to reduce hepatic damage induced by alcohol and thermally treated oil in rats. Vet. Ital. 46, 83–92. [40] Laube, H., Norris, H. T., and Robbins, S. L. (1967) The nephromegaly of chronic alcoholics with liver disease. Arch. Pathol. 84, 290–294. [41] Ibrahim, M., Hassan, W., Meinerz, D. F., Leite, Gde. O., Noqueira, C. W., et al. (2012) Ethanol-induced oxidative stress: the role of binaphthyldiselenide as a potent antioxidant. Biol. Trace Elem. Res. 147, 309–314. [42] Canales-Aguirre, A. A., Gomez-Pinedo, U. A., Luquin, S., Ramırez-Herrera, ~ a, M. L., et al. (2012) Curcumin protects against the M. A., Mendoza-Magan oxidative damage induced by the pesticide parathion in the hippocampus of the rat brain. Nutr. Neurosci. 15, 62–69. [43] Dairam, A., Fogel, R., Daya, S., and Limson, J. L. (2008) Antioxidant and ironbinding properties of curcumin, capsaicin, and S-allylcysteine reduce oxidative stress in rat brain homogenate. J. Agric. Food Chem. 56, 3350–3356.
[44] Sankar, P., Telang, A. G., and Manimaran, A. (2012) Protective effect of curcumin on cypermethrin-induced oxidative stress in Wistar rats. Exp. Toxicol. Pathol. 64, 487–493. [45] Manjunatha, H. and Srinivasan, K. (2007) Hypolipidemic and antioxidant effects of dietary curcumin and capsaicin in induced hypercholesterolemic rats. Lipids 42, 1133–1142. [46] De, A. K., and Ghosh, J. J. (1989) Short- and long-term effects of capsaicin on the pulmonary anti-oxidant enzyme system in rats. Phytother. Res. 3, 184–187. [47] Altura, B. M., Gebrewold, A., Zhang, A., and Altura, B. T. (2002) Ethanol induces lipid peroxidation and activation of nuclear factor-kappa B in cerebral vascular smooth muscle: relation to alcohol-induced brain injury in rats. Neurosci. Lett. 325, 95–98. [48] De, A. K. and Ghosh, J. J. (1989) Capsaicin pretreatment protects free radical induced rat lung damage on exposure to gaseous chemical lung irritants. Phytother. Res. 3, 159–161. [49] Zhuang, Y., Chen, L., Sun, L., and Cao, J. (2012) Bioactive characteristics and antioxidant activities of nine peppers. J. Funct. Foods 4, 331–338.
7
Chang-Won Pyun1 Ji-Han Kim1 Kyu-Ho Han2 Go-Eun Hong1 Chi-Ho Lee1*
1
Department of Food Science and Biotechnology of Animal Resources, Konkuk University, Seoul, 143-701, Republic of Korea
2
Department of Food Science, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-8555, Japan
Abstract BALB/c mice were exposed to chronic alcohol-induced oxidative stress by intragastric administration of excessive ethanol (5 g/kg body weight) during the 24-week period. Curcumin (0.016%) or capsaicin (0.014%) containing diets were fed with or without ethanol treatment in four groups. There was no statistically significant difference in the behavioral test between all groups during the experimental period. Only one alcoholtreated mouse fed a normal diet showed a behavioral disorder and died before the raising period was completed. There were
no effects on the activity of catalase and superoxide dismutase in the brain. However, curcumin or capsaicin treatment prevented alcohol-induced decline in brain weight. Furthermore, the levels of malondialdehyde and phosphatidylcholine hydroperoxide were significantly reduced in the brain tissue extract. The findings of this study demonstrated and confirmed the antioxidant effect of curcumin and capsaicin against alcoholinduced oxidative stress, and they suggest a direction for furC 2014 BioFactors, 00(00):000–000, 2014 ther studies. V
Keywords: ethanol; curcumin; capsaicin; brain; oxidative stress; phosphatidylcholine hydroperoxide; malondialdehyde
1. Introduction Chronic exposure to alcohol-induced oxidative stress leads to development of many diseases, directly or indirectly [1–3]. Especially, alcoholic neuropathy, the later stage of alcoholrelated diseases, is caused by various factors and complex mechanisms [1, 4]. Alcohol-related nutritional problems, such as thiamine deficiency, are indirect causes of alcoholic
C 2014 International Union of Biochemistry and Molecular Biology V
Volume 00, Number 00, Month/Month 2014, Pages 00–00 *Address for correspondence: Prof. Chi-Ho Lee, Ph.D.; Department of Food Science and Biotechnology of Animal Resources, Konkuk University, Seoul, 143–701, Korea. Tel.: 18224 503 681; Fax: 18224 53 19 48; E-mail: [email protected]. Disclosure: There is no conflict of interest. The first author (C. W. Pyun) contributed mostly to the experimental part of this work. Received 14 April 2014; accepted 22 May 2014 DOI 10.1002/biof.1172 Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com)
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neuropathy [5]. Cytotoxic proteins are generated by the surplus acetaldehydes, which are the metabolites induced by excessive consumption of ethanol (EtOH). These proteins negatively affect the neuronal cells [6]. Also, the calorie intake, which is induced by alcohol consumption, disturbs nutrient absorption [7]. Despite the efforts made to investigate the mechanism of alcohol-induced neuropathy in many studies, it has not yet been clearly identified. When the mechanism of alcohol-induced neuropathy was studied, the scholars focused on the Wernicke–Korsakoff syndrome, which occurs due to thiamine deficiency [5, 8]. In earlier days, it was thought that the main cause of alcoholinduced neuronal damage is the disturbance in thiamine metabolism, which is caused by damage to the digestive organs. Since the role of cytochrome P450 2E1 (CYP2E1) has been investigated, it has been receiving attention as the main cause of alcohol-induced neuronal damage [9, 10]. The activation of CYP2E1 induces injury to several organ tissues, including neuronal tissues, by catalyzing the production of ROS and influencing the carcinogenic precursors, such as nitrosamines and azo [11].
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Hence, the establishment of precautions against alcoholinduced oxidative stress is essential in an aging society. Actually, many biological or physiological studies were performed and reported in food science, immunology, and pharmacology journals for preventing alcohol-induced neuropathy. Capsicum spp. And Curcuma longa L. are rich in capsaicin and curcumin, respectively. These compounds increase the bioactivity in vivo under different situations and mechanisms. Capsaicin is the pungent component of capsicum spp., and its antiinflammatory or anticarcinogenic effect is more formally known [12–15]. Prevention of oxidative stress was also demonstrated in several studies [16–18]. The modulating effect of capsaicin on lipopolysaccharide-induced oxidative stress in rat organs has been reported recently [19]. Capsaicin protects gastric mucosa of rats against alcohol-induced oxidative stress injury [20, 21]. Curcumin is an antioxidant obtained from Curcuma longa rhizome, the most popular food source in South East Asia, and many researches to assess its antioxidative activity have been conducted globally [4, 22–24]. A recent study showed that proper consumption (20–80 mg/kg body weight) of curcumin prevents alcoholic neuropathy-induced behavioral aberration or alcoholic liver diseases in rodents [25, 26].
2. Aim of Work In this study, we focused on the oxidative stress indicators to determine the effect of curcumin and capsaicin against brain damage caused by alcohol-induced oxidative stress. Behavioral research, malondialdehyde level, phosphatidylcholine hydroperoxide level, catalase activity, and superoxide dismutase activity in the mouse brain tissue were analyzed.
3. Materials and Methods 3.1. Animal Experimental Plan A total of 45 BALB/c mice (7 weeks old) were purchased from the Central Animal Science (Seoul, Korea). Mice were fed for 7 days with sufficient amount of normal diet and water for adaptation to the new conditions. After the refining period, mice were trained for performing the water maze task, and they were divided into six groups, depending on their behavioral tendency. The detailed explanations for the six groups are as follows: (EtOH, ethanol; NaCl, sodium chloride): CON (n 5 7); normal diet 1 2 mL/kg of saline (0.85% NaCl); ALC (n 5 8); normal diet 1 5 g EtOH/2 mL/kg body weight; CUR (n 5 7); curcumin containing diet (0.016%) 1 2 mL/kg of saline (0.85% NaCl); CUR1ALC (n 5 8); curcumin containing diet (0.016%) 1 5 g EtOH/2 mL/kg body weight; CAP (n 5 7); capsaicin containing diet (0.014%) 1 2 mL/kg of saline (0.85% NaCl); and CAP 1 ALC (n 5 8); capsaicin containing diet (0.014%) 1 5 g EtOH/2 mL/kg body weight.
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The diet composition was designed by the previous research [23, 24, 27–29]. Saline solution and ethanol were intragastrically administered using an oral feeding needle.
3.2. Water Maze Test Recording of the escape latency time in the modified Morris water maze task was used for behavioral investigations [30]. The behavioral research was discontinued after 24 weeks.
3.3. Preparation of Phosphatidylcholine Hydroperoxide Standard Solution PC–OOH standard solution was prepared by photo-irradiation of phosphatidylcholine (PC) [31]. PC (from bovine heart, Sigma, Saint Louis, MO) was dissolved in methanol containing 0.01 mM methylene blue and was exposed to a UV lamp (50 W) for 8 hr. Oxidant was cleaned up using Supelclean LC-Si SPE tube (Supelco, Bellefonte, PA) to remove the methylene blue. The amount of peroxide in the standard solution was determined by a method of the American Oil Chemist’s Society [32]. As a result, the hydroperoxide concentration of the photo-oxidized PC was 19.1 lmol hydroperoxide-O2 per g of PC.
3.4. Determination of Brain Phosphatidylcholine Hydroperoxide Level Excessive ethanol consumption induced–tissue injury causes an increase in the PC–OOH level in the liver or brain tissue [33]. Therefore, this indicator was used to diagnose the occurrence of brain tissue injury. Total lipids in the brains were extracted by a modified Folch method [34], and the PC–OOH level in the brain extract was determined by the CL–HPLC system [31, 35]. The brain extracts were dissolved in chloroform–methanol (2:1, v/v) solution and injected into the CL–HPLC system, the detailed conditions for which are described below; for separating analytical samples, a Zorbax NH2 (4.6 3 250 mm, 5 lm, Agilent Technologies, Wilmington, DE) column was used as the stationary phase, and 2propanol-methanol-water (135:45:20, v/v/v, flow rate: 1 mL/min) as the mobile phase. The luminescence reagent was prepared by dissolving 10 mg/L of cytochrome c (from bovine heart, Sigma, Saint Louis, MO) and 2 mg/L of luminol (for chemiluminescence analysis, Wako Pure Chemicals, Osaka, Japan) in 50 mM borate buffer (pH 10.0). The luminesence reagent was inserted into the sample chamber (40 C) of the chemiluminescence analyzer (CLD1100, Tohoku Electronic Industries Co., Sendai, Japan) using a peristaltic pump at a flow rate of 1.0 mL/min [31, 35].
3.5. Quantitative Analysis of Brain Malondialdehyde Level MDA level in the brains was calculated using a colorimetric assay kit (Cell Biolabs, San Diego, CA). The tissue lysate production and assay protocol were performed as described by the manufacturer.
3.6. Measuring the Antioxidant Capacity The activity of antioxidative enzymes was determined for measuring the antioxidative capacity of the brain tissue samples. The activities of superoxide dismutase (SOD) and catalase among the antioxidative enzymes were determined. These
Curcumin and Capsaicin Protect Alcoholic Oxidation
FIG 1
Side-effect of chronic curcumin consumption with ethanol treatment.
factors were analyzed using colorimetric assay kits (Cell Biolabs, San Diego, CA) and accomplished by following the manufacturer’s instructions.
3.7. Statistical Analysis All data are presented as the mean 6 standard error of the mean (SEM.). A significant difference between the groups was calculated by a Tukey’s honestly significant difference test (P < 0.05). All statistical analyses were performed using the Statistical Analysis Software (Statistics Analytical System Institute, Cary, NC).
4. Results The alcohol-treated groups (ALC, CUR 1 ALC, and CAP 1 ALC) showed a higher death rate (50%) than the nontreated groups
TABLE 1
(CON, CUR, and CAP, 14.2%). This result indicates that curcumin and capsaicin consumption could not reduce the increased alcohol-related death rate in the present study. The exact causes of death were not investigated, although external symptoms were different in the treatment groups. The peculiar finding was that two of the mice in the CUR 1 ALC group had shown priapism symptoms approximately 2 weeks before their death (Fig. 1). There were some previous reports of heavy alcohol intake-related priapism [36, 37]. However, in this study we did not observe these symptoms in the mice of the ALC group. The relationship between curcumin consumption and priapism has not been investigated in previous studies. Therefore, for analyzing this side-effect, further studies may be needed before using curcumin as a food ingredient or preventive medicine. The maze test did not show any statistically significant difference in the escape latency time between the six groups during the rearing experiment because of its large deviation (data not shown). However, one of the mice in the ALC group showed a behavioral disorder and increased escape latency time before death.
4.1. Biological Effects of Curcumin and Capsaicin on Ethanol-Treated Mice The body weight and the weights of major organs (liver, spleen, kidney, and brain) are presented in Table 1. The spleen weight was not affected by alcohol, curcumin, and capsaicin consumption. The body and liver weights were significantly lower (P < 0.05) in the ALC group than the other 5 groups. The kidney weight was higher in the ALC group than the other groups (P < 0.05). Chronic alcohol treatment reduced the brain weight, and curcumin or capsaicin consumption prevented the alcohol-induced decline in brain weight (P < 0.05). Curcumin and capsaicin consumption prevented the decline in body and liver weights, which was induced by
Biological effects of curcumin and capsaicin on chronic alcohol-treated mice
CON
ALC
CUR
CUR 1 ALC
CAP
CAP 1 ALC
2.46 6 0.09b
2.44 6 0.09b
2.53 6 0.13b
2.53 6 0.13ab
2.88 6 0.11a
2.47 6 0.12b
Body weight (g)
21.72 6 1.19a
16.65 6 1.03b
21.80 6 1.17a
18.4 6 0.49ab
20.77 6 1.30a
18.33 6 0.31ab
Liver (g/kg body weight)
35.69 6 0.76ab
29.24 6 1.12c
37.86 6 1.16a
36.40 6 1.82ab
34.91 6 0.49b
35.83 6 0.68ab
Kidney (g/kg body weight)
15.86 6 0.47b
19.73 6 1.13a
15.70 6 0.76b
16.68 6 0.52b
16.33 6 1.01b
17.49 6 0.45ab
Spleen (g/kg body weight)
2.77 6 0.15
2.31 6 0.20
3.15 6 0.26
2.06 6 0.22
2.84 6 0.38
2.87 6 0.13
Food intake (g/day)
Brain (mg)
bc
383.3 6 7.2
c
367.3 6 8.9
ab
400.2 6 11.4
bc
376.5 6 7.3
a
405.8 6 8.0
384.0 6 8.9abc
All values are presented as means 6 SEM. The treatment groups are denoted as follows: CON, normal diet with saline treatment; ALC, normal diet with alcohol treatment; CUR, curcumin– containing normal diet with saline treatment; CUR 1 ALC, curcumin–containing normal diet with alcohol treatment; CAP, capsaicin-containing normal diet with saline treatment; and CAP 1 ALC, capsaicin-containing normal diet with alcohol treatment. a–c Significant differences between values in the same row (P < 0.05).
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4.3. Brain PC–OOH Level
FIG 2
Effect of curcumin and capsaicin on the brain MDA level in chronic alcohol-treated mice. All values are presented as mean 6 SEM. The treatment groups are denoted as follows: CON, normal diet with saline treatment; ALC, normal diet with alcohol treatment; CUR, curcumin-containing normal diet with saline treatment; CUR 1 ALC, curcumin-containing normal diet with alcohol treatment; CAP, capsaicincontaining normal diet with saline treatment; and CAP 1 ALC, capsaicin-containing normal diet with alcohol treatment. a,bSignificant differences between the values in the same row (P < 0.05).
alcohol treatment. The loss of body weight in the ALC group was caused by the prevention of decline in the plasma leptin [38]. Decreased loss of body and liver weights in the curcumin and capsaicin-treated groups indicated that these compounds may protect the body against alcohol consumption damage. This result confirms the results of previous studies, which demonstrated the preventive effect of curcumin against alcohol-induced liver damage in rodents [39]. Capsaicin also affected the liver and body weights in alcohol-treated mice, but there were no data on this finding in the earlier reports. There was no previous information on the effect of curcumin and capsaicin on brain weight. However, the data on brain weight showed the protective effect of curcumin and capsaicin against alcohol-induced brain damage. This finding indicates that curcumin and capsaicin may have a positive effect on the ethanol-treated mouse brain. The symptoms of enlarged kidneys, induced by the alcoholic liver damage [40], were observed in the ALC group. These symptoms were improved by curcumin and capsaicin treatment. This finding confirms the protective effect of curcumin and capsaicin on the liver.
4.2. Brain MDA Level The brain MDA level was increased by ethanol treatment (Fig. 2), as was suggested in previous studies [41]. Thus, the brains of mice in the ALC group were negatively affected by chronic exposure to ethanol-induced oxidative stress. Curcumin– and capsaicin–treated groups exhibited significantly lower MDA levels (P < 0.05). The reduced brain MDA levels were observed in 21% of mice in the CUR 1 ALC group and in 15% of mice in the CAP 1 ALC group compared to mice in the ALC group.
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The trend for the PC–OOH levels was similar to the trend observed for the MDA levels, that is, PC–OOH levels in mice of the ALC group were significantly higher than the PC–OOH levels in mice of the CON group (P < 0.05), and the corresponding levels in mice of the CUR 1 ALC and CAP 1 ALC groups were reduced (59.0 and 30.1% of the PC–OOH levels in mice of the ALC group, respectively; Fig. 3). The data on the MDA level and PC–OOH level suggest that alcohol consumption negatively affected the brain. In contrast, curcumin and capsaicin consumption reduced the effect of alcohol-induced oxidative stress on the brain. This antioxidative phenomenon in the brain was similar to that reported previously [19, 42–44], and the antioxidative effect against alcohol–induced oxidative stress in the brain was primary detected in this study.
4.4. Activity of Antioxidant Enzymes in the Brain Tissue Lysate There was no significant difference in the antioxidative enzyme activity in mice of each group, but the mean antioxidative enzyme activity in mice of the ALC group was slightly higher than that in mice of the other groups (P < 0.05; Fig. 4). Therefore, we supposed that the major mechanism of this antioxidative effect is the repression of superoxide formation, which is catalyzed by CYP2E1 [9]. The other possibility is that the activity of other antioxidants such as glutathione is affected by the consumption of these compounds.
FIG 3
Effect of curcumin and capsaicin consumption on the brain PC-OOH level in chronic alcohol-treated mice. All values are presented as mean 6 SEM. The treatment groups are denoted as follows: CON, normal diet with saline treatment; ALC, normal diet with alcohol treatment; CUR, curcumin-containing normal diet with saline treatment; CUR 1 ALC, curcumincontaining normal diet with alcohol treatment; CAP, capsaicin-containing normal diet with saline treatment; and CAP 1 ALC, capsaicin-containing normal diet with alcohol treatment. a,bSignificant differences between the values in the same row (P < 0.05).
Curcumin and Capsaicin Protect Alcoholic Oxidation
FIG 4
Antioxidative enzyme activities in mouse brain tissue lysate. (a) SOD activity, (b) catalase activity. All values are presented as mean 6 SEM. The treatment groups are denoted as follows: CON, normal diet with saline treatment; ALC, normal diet with alcohol treatment; CUR, curcumin-containing normal diet with saline treatment; CUR 1 ALC, curcumincontaining normal diet with alcohol treatment; CAP, capsaicin-containing normal diet with saline treatment; and CAP 1 ALC, capsaicin-containing normal diet with alcohol treatment. No significant difference was reported between the six groups (P < 0.05).
5. Discussion The body, liver, and brain weights were significantly reduced in chronic ethanol-treated mice. This result suggests that chronic ethanol consumption induced organ tissue injury and abnormal hormone secretion [38]. The present study confirmed the protective effect of curcumin and capsaicin against these phenomena. These results support the previous researches [20, 21, 25]. In addition, one of the chronic alcoholtreated mice (ALC) fed a normal diet showed an increased escape latency time on the maze task and finally died, while mice in the curcumin or capsaicin-treated groups did not show any behavioral problems. The catalase and superoxide dismutase activities were not affected by ethanol treatment in this study. This finding was different from those in previous studies [45], in which the effect of short-term ethanol treatment was investigated. Based
on this finding, we assumed that the superoxide dismutase and catalase activities were recovered during the long-term chronic ethanol consumption. This result and those in previous researches suggest that long-term treatment with ethanol or antioxidative compounds does not influence the enzyme activity [46]. The levels of malondialdehyde and phosphatidylcholine hydroperoxide in ethanol-treated brain tissue were significantly higher than those in nontreated group. This indicates that the mice were continuously exposed to oxidative stress due to chronic ethanol consumption [47]. The curcumin or capsaicin containing diets were useful for decreasing these levels. Based on previous studies, we assumed that curcumin decreased alcohol-induced superoxide production by repressing the CYP2E1 activity, as well as the capsaicin scavenged the generated superoxide [48]. However, the curcumin containing diet induced an increase in susceptibility to alcohol; two mice in the group showed a side-effect of priapism, which is known as an excessive chronic alcohol intake-related disease [36, 37]. This phenomenon suggests that curcumin consumption caused suppression of increased activity of CYP2E1 and disturbed the alcohol metabolism. This mechanism was beneficial in vivo against alcohol-induced oxidative stress because the superoxide production was relatively reduced, but the risk of immediate effect of alcohol was increased. Therefore, we assumed that direct exposure to alcohol induced priapism. The mice in the capsaicin containing diet group did not show any side-effects. This suggests that the mechanism of in vivo antioxidation effect is different between capsaicin and curcumin. Based on the previous studies, we could assume that the capsaicin containing diet increases the antioxidant bioactivity [49]. One of the important factors is the pungent taste of the capsaicin containing diet, which leads to a longer time period for adjusting to the diet, and also some of the mice may not have consumed the spicy diet. The concentration of capsaicin used in this study (0.014%) can also be intolerable to humans. Therefore, the optimum concentration of capsaicin for therapeutic use should be investigated in a further study, as well as the other antioxidative mechanisms and pathways should be studied.
6. Conclusion This study demonstrated the protective effect of curcumin and capsaicin against alcohol-induced brain damage, as well as confirmed their effect in preventing alcohol-induced liver injury. Long-term consumption of curcumin or capsaicin without alcohol treatment induced an increase in brain weight. We also suggest that the aim of future research should be to investigate the antioxidative mechanism of these compounds. If the antioxidative mechanisms of curcumin and capsaicin involve inhibition of the generation of reactive oxygen species, as previously reported, it is also likely that these compounds will affect the
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BioFactors
metabolic pathway of ethanol. This poses a further health risk because these compounds decrease the rate of ethanol metabolism, and thus the detoxification of ethanol is also inhibited. Therefore, it is necessary that side effects such as priapism that were noted in this study, as well as the influence of curcumin on ethanol metabolism, should be investigated further. The investigation of relationship between the various daily intake amounts and their effects is also necessary in order to use these compounds for industrial or therapeutic purposes.
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