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Protective effect of green tea (-)-epigallocatechin-3-gallate against the monoamine oxidase B enzyme activity increase in adult rat brains
Nutrition 26 (2010) 1195–1200
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Basic nutritional investigation
Protective effect of green tea (-)-epigallocatechin-3-gallate against the monoamine oxidase B enzyme activity increase in adult rat brains Shyh-Mirn Lin Ph.D. a, b, Shih-Wei Wang M.S. b, Su-Chen Ho Ph.D. c, Ya-Li Tang Ph.D. a, b, * a
Department of Food and Nutrition, Chung Hwa University of Medical Technology, Taiwan, ROC Graduate Institute of Biological Science and Technology, Chung Hwa University of Medical Technology, Taiwan, ROC c Department of Food Science, Yuanpei University, HsinChu, Taiwan, ROC b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 March 2009 Accepted 16 November 2009
Objective: Monoamine oxidase B (MAO-B) levels were observed increasing during aging in rat brains. (-)-Epigallocatechin-3-gallate (EGCG) is the major polyphenolic constituent in green tea. The objective of the present study was to investigate the EGCG compound for its effect on preventing an increase in MAO-B activity in rat brains. The total antioxidant capacity and lipid peroxidation of rats were also assessed. Methods: Rats were assigned to three groups: Control, VE (a-tocopherol), and EGCG. Twenty-four male Long-Evans rats were fed normal diets for a total of 11 wk and test diets for a total of 12 wk. The serum analysis, serum total antioxidant capacity, tissue lipid peroxidation, and monoamine oxidase B enzyme activity were measured. The differences between the groups and between the control and experimental groups were analyzed. The correlation among the experimental results was also analyzed. Results: The serum total antioxidant capacity of the EGCG group was higher than that observed in the Control and VE groups. In rat brains and livers, the lipid peroxidation levels were lower in the VE and EGCG groups compared with Control groups. EGCG and VE groups showed lower MAO-B enzyme activity in rat brains compared with Control groups. In contrast to the brain findings, there were no significant differences in the MAO-B enzyme activity among groups in rat livers. Conclusion: The present study first indicates that EGCG supplementation was able to execute a tissue-selective decrease in the brain MAO-B enzyme activity in adult rats, in which it was actualized by way of preventing physiological peroxidation. Ó 2010 Elsevier Inc. All rights reserved.
Keywords: Polyphenol EGCG Monoamine oxidase B MAO-B Brain Antioxidation
Introduction The oxidative deamination, which is performed by monoamine oxidase (MAO, EC1.4.3.4), is the most important degradative pathway for many bioactive amines. MAO is an enzyme that has two isoenzymes that are coded by two related, but separate, genes: type A and type B [1]. It is widely distributed in tissues including the nerves, kidneys, liver, and gastrointestinal tract. The enzyme catalyzes the metabolism of biologically active amine compounds and participates in the oxidative deamination reaction of an amine neurotransmitters’ variety, such as dopamine, adrenaline, and serotonin [2]. Based on the observation that the MAO-B levels increase during aging [3,4], the relationship between MAO-B and aging-related diseases was extensively studied. Several neurodegenerative diseases, such as Parkinson’s * Corresponding author. Tel.: þ886 6 2605779; fax: þ886 6 2605779. E-mail address: [email protected] (Y.-L. Tang). 0899-9007/$ – see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.nut.2009.11.022
and Alzheimer’s diseases, show high MAO-B in brains, but there is no difference in the MAO-A [3,5,6]. Thus, selective MAO-B inhibitors have been employed in neurodegeneration patients, in which improvements have been observed [7]. Some MAO inhibitors also showed further characteristics of tissue selectivity, namely, that they inhibited MAO enzymes in the brain, but caused little inhibition of the enzymes in the liver [8]. Despite these benefits, the side effects (including cheese reaction, hepatotoxicity, orthostatic hypotension, sleep disturbances, anxiety, headache, nausea, etc.) that are caused by MAO inhibitor agents are still unavoidable [9]. Polyphenolic compounds have been well investigated in green tea, tomatoes, red wine, and other foods. Recently, polyphenols have been discussed as dietary supplements because many studies support evidence of the beneficial action of polyphenols on health [10]. Several polyphenolic compounds, known as catechins, are found in green tea, for example, (-)-epigallocatechin-3-gallate (EGCG), (-)-epicatechin-3-gallate,
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(-)-epigallocatechin, (-)-epicatechin, and (þ)-catechin. EGCG is the major polyphenolic constituent in green tea. More than 65% of the total catechin content and more than 10% of the extract dry weight of green tea are composed of EGCG [11]. A number of publications assume that EGCG is probably responsible for the majority of the potential health benefits attributed to the consumption of green tea [12]. EGCG is best known for its antioxidant properties, which have led to their evaluation in many diseases that are associated with reactive oxygen species. Among age-associated pathologies and neurodegenerative diseases, green tea polyphenols have been demonstrated to afford significant protection against Parkinson’s disease, Alzheimer’s disease, and ischemic damage [13,14]. Despite the suggested neurodegenerative preventive effects of EGCG, more studies are needed to understand the extensive mechanisms that are involved in EGCG-modulated neuronal functions. EGCG prevents striatal dopamine depletion and substantia nigra dopaminergic neuronal loss that is induced by 1methyl-4-phenyl-1,2,3,6-tetrahydropyridine, while it is a rather poor inhibitor of MAO-B enzyme in vitro [15,16]. Nonetheless, it has been suggested that food constituents, such as catechin, EGCG, and curcumin, attenuate MAO-B activity in C6 cells [17]. Because the in vivo influences of EGCG on MAO-B are still unknown, the purpose of the present study was to evaluate the protective effect of EGCG against MAO-B activity increases in adult rat organs. Moreover, the physiological antioxidation status and serum parameters were assessed to elucidate the mechanism of these EGCG effects. Materials and methods
Table 1 Composition of the test diets Diet ingredients (g/kg diet)
Control
VE
EGCG
Casein Methionine Sucrose Corn starch Alphacel Soybean oil Choline Mineral mix (AIN 76 mineral mix*) Vitamin mix (AIN 76 vitamin mix*) Vitamin E EGCG
200 3 325 325 50 50 2 35
200 3 325 325 50 50 2 35
200 3 325 325 50 50 2 35
10
10
10
*
d d
450 IU d
d 240 mg
AIN 76 mineral mix, AIN 76 vitamin mix [42].
At the end of the 12 wk, the rats fasted for 12 h and then were sacrificed by carbon dioxide inhalation. Their blood was collected into tubes followed by centrifugation (3000 g, 20 min, 4 C) to separate the serum. The rat organs were removed and stored at 80 C for the experiments described below. Serum analysis The concentrations of serum glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), uric acid, blood urea nitrogen (BUN), total cholesterol, triglyceride (TG), and glucose in the experimental rats were measured by the VITROS 950 Chemistry System (Johnson & Johnson, New Brunswick, NJ, USA).
Chemicals The benzylamine, 2,2-azobis (2-amidinopropane) dihydrochloride, b-phycoerythrin, Trolox, 2,6-ditertbutyl-4-methylphenol, thiobarbituric acid, trichloroacetic acid, and a-tocopherol were all purchased from Sigma Chemical Co. (St. Louis, MO, USA). AIN 76-based diets were purchased from ICN Biomedicals (Los Angeles, CA, USA). EGCG was applied via the product of YoCome-purified EGCG capsules (purified green tea EGCG 90%; Yung Shin Pharm. Ind. Co., Ltd., Taichung, Taiwan). Animals and diets The experimental design was approved by the Animal Experiment Committee of Chung Hwa University of Medical Technology. Experiments were carried out with 24 male LongEvans rats (7 wk of age) that were purchased from the National Laboratory Animal Center. The rats were housed individually in stainless steel wire-bottomed cages and were maintained in a room with a controlled temperature and 12-h light and dark cycles. Food and distilled water were provided ad libitum. They were fed on chow diets for 10 wk and then were fed on diets based on AIN-76 for 1 wk. Next, the rats were divided into the three following groups: The control group (Control, eight rats), Vitamin E group (VE, positive control, eight rats), and EGCG group (EGCG, eight rats). The assigned procedure was conducted randomly by body weight to equalize the mean body weight of the rats in each group. The composition of the diets for the three groups is shown in Table 1. The administration of EGCG in the fortified test diets was adjusted to 240 mg/kg diet in the EGCG group. The food intake amounts and body weight were recorded every 3 d.
Serum oxygen-radical absorbance capacity (ORAC) assay The total antioxidant capacity in the serum of rats was determined by using the ORAC assay [18]. The 0.01 mL diluted rat’s serum contained 2,2-azobis(2-amidinopropane) dihydrochloride (75 mM, 0.01 mL), b-phycoerythrin (0.4 mM, 0.015 mL), and sodium phosphate buffer to a 0.25 mL final volume. The fluorescence of the assay mixture was measured at an excitation wavelength of 492 nm, and an emission wavelength 565 nm for 200 min at 37 C (FLUOstar OPTIMA microplate reader, BMG LABTECH Inc., Cary, NC, USA). The Trolox was used for the standard curve and antioxidant equivalent calculations. The final results were calculated by using the differences of the areas under the fluorescence curves for 200 min, in which they were expressed as mM Trolox equivalents. Lipid peroxidation assay The modified thiobarbituric acid reactive substances (TBARS) method [19] was used to determine the lipid peroxidation levels of rat brains and livers. In brief, 0.5 mL tissue homogenate (in potassium-phosphate buffer, pH 7.4) was mixed with 0.5 mL trichloroacetic acid solution (10%) and centrifuged at 1500 g for 10 min. The clear supernatant was collected and treated with 0.5 mL thiobarbituric acid solution (0.4% in 0.2 N HCl) and 50 mL 2,6-ditertbutyl-4-methylphenol (0.2% in 95% ethanol), and then incubated in a 50 C water bath for 1 h. An amount of 1.5 mL n-butanol was added to the cooled solution and centrifuged at 1500 g for 10 min. The clear supernatant was separated and used for the measurement of the fluorescence at an excitation wavelength of 515 nm and an emission wavelength of 550 nm
The MAO-B enzyme activity in rat tissue was measured by a modified method of the standard techniques assay procedure [20]. The tissues were homogenized in 0.2 M phosphate buffer (pH 7.4) and centrifuged at 1000 g for 10 min and 4 C. The supernatant was collected and further centrifuged at 17000 g for 30 min and 4 C. The pellet was collected and resuspended in a 1 mL phosphate buffer (0.2 M, pH 7.4). The resuspended pellet solution (0.125 mL) was mixed with a benzylamine solution (0.3 mL, 8 mM) and then adjusted to the final volume of 3 mL by a phosphate buffer. The mixture was shaken at 37 C for 3 h. The reaction was stopped by the addition of 0.3 mL 60% perchloric acid. The reaction of the benzylaldehyde product was extracted with 3 mL cyclohexane. The organic layer was separated by centrifugation at 3000 g for 10 min and analyzed for absorbance at 242 nm (Hitachi U-2001 spectrophotometer). The protein concentration assay method was used as described by the Lowry method [21]. For verification purposes, pargyline (MAO-B inhibitor) was used to confirm the type of MAO isoforms. Statistical analysis The data are presented as the means along with their standard deviations from all the sets of independent experiments. The differences between the groups were analyzed by using oneway ANOVA, followed by Duncan’s multiple range test. The differences between the control and experimental groups in terms of all the parameters were analyzed by using the Student’s t test. The difference was considered significant if the P value was 0.05 or less. The correlation among the serum ORAC, brain TBARS, liver TBARS, brain MAO, and liver MAO was analyzed by using the Pearson correlation. The statistical analysis was furthered by using a SAS statistical computer program (Version 13.0.161, SAS Institute Inc., Cary, NC, USA). Results The growth curve of the rats fed on three diets (Control, atocopherol, and EGCG supplements) during the experimental period demonstrated no significant differences between each group throughout the entire period (data not shown). This indicated that the chemical forms of the supplements in these diets had no influence on the rats’ growth. Table 2 shows the Table 2 The serum GOT, GPT, uric acid, BUN, total cholesterol, triglyceride, and glucose of rats fed on the control (Control), tocopherol supplement (VE), or EGCG supplement (EGCG) diets* Control GOT (U/L) GPT (U/L) Uric acid (mg/dL) BUN (mg/dL) TC (mg/dL) TG (mg/dL) Glucose (mg/dL)
169.3 90.9 13.1 12.9 183.6 187.9 601.6
VE 114.4 73.0 1.2 1.8 23.3 56.3 135.5
190.5 93.7 12.6 12.6 150.6 196.0 589.7
EGCG
87.3 40.7 2.4 2.4 19.4y 48.6 187.4
156.3 97.3 10.6 11.0 158.0 179.8 572.2
64.9 47.7 1.4y 2.4 32.7 54.5 136.3
BUN, blood urea nitrogen; GOT, glutamic oxaloacetic transaminase; GPT, glutamic pyruvic transaminase; TC, total cholesterol; TG, triglyceride * Each value represents the means with their standard deviations for 7–8 rats. y Value is significantly different from the control group (Control) by a Student’s t test (P < 0.05).
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0.95
* 0.85
0.75
0.65 Control
VE
EGCG
Fig. 1. Total antioxidant capacity in the serum of rats fed on test diets for 12 wk. The values are the means with their standard deviations for seven to eight rats that are depicted by vertical bars. * Value is significantly different from the control group (Control) when analyzed by Student’s t test (P < 0.05). ORAC, oxygen-radical absorbance capacity.
results of the GOT, GPT, uric acid, BUN, total cholesterol, TG, and glucose analyses of all groups. The groups treated with atocopherol or EGCG did not influence the levels of serum GOT, GPT, BUN, TG, and glucose concentrations. However, EGCG treatment significantly decreased the uric acid level in the EGCG group of rats (Table 2). The total cholesterol level was also significantly decreased in the VE group. After the rats were fed on test diets for 12 wk, the total antioxidant capacity of the EGCG group was significantly (P < 0.05) increased in serum compared to the Control and VE groups (Fig. 1). In addition, the VE and Control groups had no significant differences. The lipid peroxidation status in the brains and livers of the Control, VE, and EGCG groups of the rats was measured by the TBARS method. The TBARS levels of the rats’ brains and livers, which were fed on three test diets for 12 wk, are shown in Figure 2. Treatment with a-tocopherol (VE group) or EGCG
A TBARS (nmol/g brain)
Monoamine oxidase B (MAO-B) activity assay
2.0
Brain 1.5
*
*
VE
EGCG
1.0 0.5 0.0 Control
B 8.0 TBARS (nmol/g liver)
(Hitachi F-4500 Fluorescence Spectrophotometer; Hitachi, HighTechnologies Co., Tokyo, Japan).
ORAC (mM Trolox equivalent)
S.-M. Lin et al. / Nutrition 26 (2010) 1195–1200
Liver 6.0 4.0
*
*
2.0 0.0 Control
VE
EGCG
Fig. 2. Brain (A) and liver (B) TBARS levels of rats fed on test diets for 12 wk. The values are the means with their standard deviations for seven to eight rats, depicted by vertical bars. * Value is significantly different from the control group (Control) when analyzed by Student’s t test (P < 0.05). TBARS, thiobarbituric acid reactive substances.
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MAO activity (U)
A
S.-M. Lin et al. / Nutrition 26 (2010) 1195–1200
Brain
40 30 20
*
*
10 0 Control
VE
EGCG
B 80 Liver MAO activity (U)
60 40 20 0 Control
VE
EGCG
Fig. 3. The brain (A) and liver (B) monoamine oxidase (MAO) activities of rats fed on test diets for 12 wk. The values are the means with their standard deviations for seven to eight rats, depicted by vertical bars. * Value is significantly different from the control group (Control) when analyzed by Student’s t test (P < 0.05). MAO, monoamine oxidase; U, mmol/h/mg protein.
(EGCG group) caused a significant (P < 0.05) decrease in the brain TBARS level, in which the EGCG-fortified diet demonstrated a similar effect with the a-tocopherol-fortified diet (Fig. 2A). Similarly, a significant decrease in the VE and EGCG groups of the rat liver TBARS levels (P < 0.05) was shown (Fig. 2B). It was demonstrated that the EGCG supplement had a similar physiological lipid peroxidation preventive effect as a-tocopherol on rats during age increases. The MAO-B enzyme activities in rat brains and livers are shown in Figure 3. In rat brains, the MAO-B activity showed a significant decrease in the VE and EGCG groups versus Control group (P < 0.05). Despite the significant MAO-B activity decrease, which was observed in the VE and EGCG groups’ brains versus Control group, there were no significant changes that could be noted with respect to the MAO-B enzyme activities in the rat livers among these groups. For confirming the type of MAO isoforms, the rat tissue samples were also treated with MAO-B inhibitor pargyline and showed a decrease in MAO-B enzyme activity. Discussion There were only a few reports that discussed the correlation between dietary supplementation and brain MAO-B enzyme regulation in animals. Our previous work demonstrated that dietary selenium supplements revealed the brain MAO-B activity reduction effect [22]. In the present study, we investigated the protective effect of EGCG against the MAO-B enzyme activity increase in adult rat brains. The rat feeding was designed for dietary uptake and daily EGCG dose was estimated approximately 8.6 mg/kg body weight. The rat serum parameters were analyzed to understand the physiological influences of atocopherol and EGCG. It demonstrated that the a-tocopherol or EGCG supplements did not influence the serum levels of the GOT,
GPT, BUN, TG, and glucose concentrations. We concluded that the administration of doses of a-tocopherol and EGCG did not affect the liver function, lipid metabolism, or glucose metabolism of the experimental rats. However, we observed a decrease in the serum uric acid level in the EGCG group of rats. A recent study also reported that oral pretreatment with EGCG decreased the plasma uric acid levels in isoproterenol-treated rats [23]. It was suggested that EGCG could inhibit the xanthine oxidase enzyme to produce uric acid and reactive oxygen species [24,25]. Therefore, our present study supports the assumption that the decrease in uric acid may have been due to the ability of EGCG to block xanthine oxidase. The advanced mechanisms remain to be investigated. The antioxidant activities of green tea dietary supplements as determined by the ORAC method were well correlated with the total polyphenol content [26]. Furthermore, the method of ORAC was applied in the present study to evaluate the total antioxidant capacity in the serum of rats, in which a significant increase in the EGCG group was observed. An earlier study also indicated that the total antioxidant capacity of human plasma presented a dose-dependent increase after green tea consumption. Therefore, these results can reasonably conclude that EGCG supplementation leads to the increase of the serum total antioxidant capacity of rats. In fact, many of the neuropreventive effects of EGCG are attributable to its antioxidant properties [27]. In contrast to the finding of EGCG supplementation, the result of a-tocopherol supplement (VE group) did not show a significant difference with the control group in terms of rats’ serum. The ORAC method is based on the principle of the absorbance capacity of oxygen radicals by antioxidants. However, the reaction of a-tocopherol is not a counteraction with oxygen, but rather with fatty acid peroxyl radicals, which in turn interrupts the auto-oxidative radical chain reaction process [28]. Thus, it is acceptable that the a-tocopherol supplements (VE group) reveal no effect on the total antioxidant capacity by the ORAC method and due to the result of the insignificant difference with the Control group. Brain and liver tissues are abundant in polyunsaturated fatty acid constituents, which form the toxic lipid peroxidation products during the aging process [29,30]. The physiological functions should be influenced by these lipid peroxidation products in animals. Recently, a report denoted that EGCG supplementation significantly decreased the level of lipid peroxidation in both mitotic (liver) and postmitotic (skeletal muscle) tissues of aged rats [31]. Oral intake of EGCG significantly decreased the TBARS levels in the livers of fatigued animals [32]. The present study further found that EGCG provided protective action for lipid peroxidation in rat brain and liver tissues (Fig. 2). It has been indicated that EGCG augmented the antioxidant-related enzymes and ameliorated the lipid peroxidation status in aged rat brains [33]. These results can be explained by the consequence that EGCG attains a lipid peroxidation decrease effect, at least partially, via the improvement of the physiological anti-oxidation status. Furthermore, the atocopherol supplements (VE group) demonstrated a similar protective action for lipid peroxidation and TBARS level decrease in these tissues. This result also meets the explanation above that a-tocopherol conducts its effect via a reaction with fatty acid peroxyl radicals and interrupts the auto-oxidative radical chain reaction process. It is proven that the MAO-B levels were increased during aging [3,4]. In the present study, all of the groups of rats were fed for a total 30 wk, and the rat brain MAO-B enzyme activity is expected to increase with age. However, the VE and EGCG groups
S.-M. Lin et al. / Nutrition 26 (2010) 1195–1200
exhibited a significant decrease in MAO-B activity versus the Control group in rat brains. In contrast to the findings in brains, there were no significant differences in the MAO-B enzyme activity among the Control, VE, and EGCG groups in the livers of rats. Thus, EGCG seems to possess further characteristics of tissue selectivity because it inhibited the MAO enzymes in brains but also caused rather little inhibition of the enzymes in the liver. This may be consequent to the reason that the MAO enzymes play different roles in brains (amine neurotransmitters transformation) and livers (foreign amine compounds detoxicating) [34]. It was already known that the oral administration of green tea catechins reaches a maximum plasma concentration at around 1 h and is widely distributed into various organs for a longer period [35,36]. However, EGCG may potentially penetrate through the blood-brain barrier at a lower rate and lead to low concentrations of EGCG in the various brain regions [37]. Therefore, EGCG is likely to prevent a brain MAO-B activity increase via physiological antioxidant status improvement, rather than directly affect the enzyme. When the MAO enzyme in brains catalyzes the oxidative deamination reaction of many amine neurotransmitters, a hydrogen peroxide product is generated. Hydrogen peroxide is considered one of the sources of oxidative stress, which induces physiological peroxidation. Therefore, we investigated the correlation between the MAO activities and TBARS levels in rat organs. This showed that brain MAO activity has a positive correlation with the brain and liver’s lipid peroxidation, but also that there is no correlation between the liver’s MAO activity and brain/liver’s lipid peroxidation (Table 3). These results implied the important link between brain MAO enzyme activity and the physiological lipid peroxidation status. In addition, the serum total antioxidant capacity does not have the same correlation with brain MAO activity (Table 3). Therefore, we suggest that lipid peroxidation may play a particular role in brain MAO activity and remain to be further investigated. The serum ORAC showed a negative correlation with brain TBARS. It is known that a decreased serum antioxidant status has been suggested as a risk factor in neurodegenerative diseases [38]. The present study observed a significant increase of the serum total antioxidant capacity in the EGCG group. It was also indicated that EGCG could act synergistically and significantly increase the antioxidant activity of vitamins E and C and then protect lipid peroxidation [39]. So it may be concluded that EGCG supplement improves lipid peroxidation at least partly via increasing serum antioxidant status. However, there are minor correlations between the liver MAO activity, serum total antioxidant capacity, and brain/liver’s lipid peroxidation. These results can explain, at least partly, the Table 3 Correlation among total antioxidant capacity, lipid peroxidation, and monoamine oxidase activity
Serum ORAC (mM Trolox equivalent) Brain TBARS (nmol/g brain) Liver TBARS (nmol/g liver) Brain MAO (mmol/h/mg protein)
TBARS (brain)
TBARS (liver)
MAO (brain)
MAO (liver)
r ¼ 0.45 P ¼ 0.02
r ¼ 0.13 P ¼ 0.58
r ¼ 0.21 P ¼ 0.40
r ¼ 0.03 P ¼ 0.90
r ¼ 0.62 P ¼ 0.003
r ¼ 0.56 P ¼ 0.02 r ¼ 0.75 P ¼ 0.008
r ¼ 0.07 P ¼ 0.77 r ¼ 0.16 P ¼ 0.56 r ¼ 0.45 P ¼ 0.08
MAO, monoamine oxidase, ORAC, oxygen-radical absorbance capacity; TBARS, thiobarbituric acid reactive substances
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diverse prevention effect of EGCG supplements on the brain and liver’s MAO activity variations in rats. It has been reported that both green tea and EGCG are very poor inhibitors of MAO-B enzyme in vitro. This led to the conclusion that MAO inactivation was not involved in the neuroprotection exerted by these compounds [15]. However, EGCG is not stable under most cell culture conditions [40]. Moreover, it has been suggested that the MAO-B inhibitor 7-nitroindazole possesses the neuroprotective effects that partially contribute to antioxidant action in vitro and in vivo [41]. Similarly, EGCG is likely to influence the brain MAO-B activity that is kept from being increased during age increases by preventing physiological peroxidation instead of by directly inhibiting the MAO-B enzyme. Conclusions We first suggest that EGCG supplementations can decrease brain MAO-B enzyme activity in adult rats. EGCG can obtain these effects, at least partly, by improving the physiological antioxidative status. Furthermore, our work can provide for the possible developments of dietary supplements for the tissueselective effect of an MAO-B inhibitor. Acknowledgments This work was partly supported by the National Science Council (NSC91-2320-B-273-001) in Taiwan. There is no conflict of interest to disclose. References [1] Weyler W, Hsu YP, Breakefield XO. Biochemistry and genetics of monoamine oxidase. Pharmacol Ther 1990;47:391–417. [2] Strolin Benedetti M, Dostert P. Monoamine oxidase, brain ageing and degenerative diseases. Biochem Pharmacol 1989;38:555–61. [3] Jossan SS, Gillberg PG, d’Argy R, Aquilonius SM, Langstrom B, Halldin C, et al. Quantitative localization of human brain monoamine oxidase B by large section autoradiography using L-[3H]deprenyl. Brain Res 1991;547:69–76. [4] Nicotra A, Pierucci F, Parvez H, Senatori O. Monoamine oxidase expression during development and aging. Neurotoxicology 2004;25:155–65. [5] Saura J, Luque JM, Cesura AM, Da Prada M, Chan-Palay V, Huber G, et al. Increased monoamine oxidase B activity in plaque-associated astrocytes of Alzheimer brains revealed by quantitative enzyme radioautography. Neuroscience 1994;62:15–30. [6] Sherif F, Gottfries CG, Alafuzoff I, Oreland L. Brain gamma-aminobutyrate aminotransferase (GABA-T) and monoamine oxidase (MAO) in patients with Alzheimer’s disease. J Neural Transm Park Dis Dement Sect 1992;4:227–40. [7] Knoll J. The pharmacological basis of the beneficial effects of (-)deprenyl (selegiline) in Parkinson’s and Alzheimer’s diseases. J Neural Transm Suppl 1993;40:69–91. [8] Nagatsu T, Sawada M. Molecular mechanism of the relation of monoamine oxidase B and its inhibitors to Parkinson’s disease: possible implications of glial cells. J Neural Transm Suppl 2006;71:53–65. [9] Yamada M, Yasuhara H. Clinical pharmacology of MAO inhibitors: safety and future. Neurotoxicology 2004;25:215–21. [10] Williamson G, Holst B. Dietary reference intake (DRI) value for dietary polyphenols: are we heading in the right direction? Br J Nutr 2008;99(Suppl 3):S55–8. [11] Goto T, Yoshida Y, Kiso M, Nagashima H. Simultaneous analysis of individual catechins and caffeine in green tea. J Chromatog A 1996;749:295–9. [12] Nagle DG, Ferreira D, Zhou YD. Epigallocatechin-3-gallate (EGCG): chemical and biomedical perspectives. Phytochemistry 2006;67:1849–55. [13] Mandel S, Youdim MB. Catechin polyphenols: neurodegeneration and neuroprotection in neurodegenerative diseases. Free Radic Biol Med 2004;37:304–17. [14] Weinreb O, Mandel S, Amit T, Youdim MB. Neurological mechanisms of green tea polyphenols in Alzheimer’s and Parkinson’s diseases. J Nutr Biochem 2004;15:506–16. [15] Levites Y, Weinreb O, Maor G, Youdim MB, Mandel S. Green tea polyphenol (-)-epigallocatechin-3-gallate prevents N-methyl-4-phenyl-1,2,3,6-
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[30] Leaden PJ, Catala A. Melatonin and N-acetyl serotonin inhibit selectively enzymatic and non-enzymatic lipid peroxidation of rat liver microsomes. Prostaglandins Leukot Essent Fatty Acids 2007;77:29–35. [31] Senthil Kumaran V, Arulmathi K, Srividhya R, Kalaiselvi P. Repletion of antioxidant status by EGCG and retardation of oxidative damage induced macromolecular anomalies in aged rats. Exp Gerontol 2008;43:176–83. [32] Tanaka M, Baba Y, Kataoka Y, Kinbara N, Sagesaka YM, Kakuda T, et al. Effects of (-)-epigallocatechin gallate in liver of an animal model of combined (physical and mental) fatigue. Nutrition 2008;24:599–603. [33] Srividhya R, Jyothilakshmi V, Arulmathi K, Senthilkumaran V, Kalaiselvi P. Attenuation of senescence-induced oxidative exacerbations in aged rat brain by (-)-epigallocatechin-3-gallate. Int J Dev Neurosci 2008;26:217–23. [34] Raciti G, Mazzone P, Raudino A, Mazzone G, Cambria A. Inhibition of rat liver mitochondrial monoamine oxidase by hydrazine-thiazole derivatives: structure-activity relationships. Bioorg Med Chem 1995;3:1485–91. [35] Suganuma M, Okabe S, Oniyama M, Tada Y, Ito H, Fujiki H. Wide distribution of [3H](-)-epigallocatechin gallate, a cancer preventive tea polyphenol, in mouse tissue. Carcinogenesis 1998;19:1771–6. [36] Mata-Bilbao Mde L, Andres-Lacueva C, Roura E, Jauregui O, Escribano E, Torre C, et al. Absorption and pharmacokinetics of green tea catechins in beagles. Br J Nutr 2008;100:496–502. [37] Lin LC, Wang MN, Tseng TY, Sung JS, Tsai TH. Pharmacokinetics of (-)-epigallocatechin-3-gallate in conscious and freely moving rats and its brain regional distribution. J Agric Food Chem 2007;55:1517–24. [38] Marlatt MW, Lucassen PJ, Perry G, Smith MA, Zhu X. Alzheimer’s disease: cerebrovascular dysfunction, oxidative stress, and advanced clinical therapies. J Alzheimers Dis 2008;15:199–210. [39] Intra J, Kuo SM. Physiological levels of tea catechins increase cellular lipid antioxidant activity of vitamin C and vitamin E in human intestinal caco-2 cells. Chem Biol Interact 2007;169:91–9. [40] Sang S, Yang I, Buckley B, Ho CT, Yang CS. Autoxidative quinone formation in vitro and metabolite formation in vivo from tea polyphenol (-)-epigallocatechin-3-gallate: studied by real-time mass spectrometry combined with tandem mass ion mapping. Free Radic Biol Med 2007;43:362–71. [41] Thomas B, Saravanan KS, Mohanakumar KP. In vitro and in vivo evidences that antioxidant action contributes to the neuroprotective effects of the neuronal nitric oxide synthase and monoamine oxidase-B inhibitor, 7nitroindazole. Neurochem Int 2008;52:990–1001. [42] American Institute of Nutrition. Report of the American Institute of Nutrition ad hoc Committee on Standards for Nutritional Studies. J Nutr 1977;107:1340–8.
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Basic nutritional investigation
Protective effect of green tea (-)-epigallocatechin-3-gallate against the monoamine oxidase B enzyme activity increase in adult rat brains Shyh-Mirn Lin Ph.D. a, b, Shih-Wei Wang M.S. b, Su-Chen Ho Ph.D. c, Ya-Li Tang Ph.D. a, b, * a
Department of Food and Nutrition, Chung Hwa University of Medical Technology, Taiwan, ROC Graduate Institute of Biological Science and Technology, Chung Hwa University of Medical Technology, Taiwan, ROC c Department of Food Science, Yuanpei University, HsinChu, Taiwan, ROC b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 March 2009 Accepted 16 November 2009
Objective: Monoamine oxidase B (MAO-B) levels were observed increasing during aging in rat brains. (-)-Epigallocatechin-3-gallate (EGCG) is the major polyphenolic constituent in green tea. The objective of the present study was to investigate the EGCG compound for its effect on preventing an increase in MAO-B activity in rat brains. The total antioxidant capacity and lipid peroxidation of rats were also assessed. Methods: Rats were assigned to three groups: Control, VE (a-tocopherol), and EGCG. Twenty-four male Long-Evans rats were fed normal diets for a total of 11 wk and test diets for a total of 12 wk. The serum analysis, serum total antioxidant capacity, tissue lipid peroxidation, and monoamine oxidase B enzyme activity were measured. The differences between the groups and between the control and experimental groups were analyzed. The correlation among the experimental results was also analyzed. Results: The serum total antioxidant capacity of the EGCG group was higher than that observed in the Control and VE groups. In rat brains and livers, the lipid peroxidation levels were lower in the VE and EGCG groups compared with Control groups. EGCG and VE groups showed lower MAO-B enzyme activity in rat brains compared with Control groups. In contrast to the brain findings, there were no significant differences in the MAO-B enzyme activity among groups in rat livers. Conclusion: The present study first indicates that EGCG supplementation was able to execute a tissue-selective decrease in the brain MAO-B enzyme activity in adult rats, in which it was actualized by way of preventing physiological peroxidation. Ó 2010 Elsevier Inc. All rights reserved.
Keywords: Polyphenol EGCG Monoamine oxidase B MAO-B Brain Antioxidation
Introduction The oxidative deamination, which is performed by monoamine oxidase (MAO, EC1.4.3.4), is the most important degradative pathway for many bioactive amines. MAO is an enzyme that has two isoenzymes that are coded by two related, but separate, genes: type A and type B [1]. It is widely distributed in tissues including the nerves, kidneys, liver, and gastrointestinal tract. The enzyme catalyzes the metabolism of biologically active amine compounds and participates in the oxidative deamination reaction of an amine neurotransmitters’ variety, such as dopamine, adrenaline, and serotonin [2]. Based on the observation that the MAO-B levels increase during aging [3,4], the relationship between MAO-B and aging-related diseases was extensively studied. Several neurodegenerative diseases, such as Parkinson’s * Corresponding author. Tel.: þ886 6 2605779; fax: þ886 6 2605779. E-mail address: [email protected] (Y.-L. Tang). 0899-9007/$ – see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.nut.2009.11.022
and Alzheimer’s diseases, show high MAO-B in brains, but there is no difference in the MAO-A [3,5,6]. Thus, selective MAO-B inhibitors have been employed in neurodegeneration patients, in which improvements have been observed [7]. Some MAO inhibitors also showed further characteristics of tissue selectivity, namely, that they inhibited MAO enzymes in the brain, but caused little inhibition of the enzymes in the liver [8]. Despite these benefits, the side effects (including cheese reaction, hepatotoxicity, orthostatic hypotension, sleep disturbances, anxiety, headache, nausea, etc.) that are caused by MAO inhibitor agents are still unavoidable [9]. Polyphenolic compounds have been well investigated in green tea, tomatoes, red wine, and other foods. Recently, polyphenols have been discussed as dietary supplements because many studies support evidence of the beneficial action of polyphenols on health [10]. Several polyphenolic compounds, known as catechins, are found in green tea, for example, (-)-epigallocatechin-3-gallate (EGCG), (-)-epicatechin-3-gallate,
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(-)-epigallocatechin, (-)-epicatechin, and (þ)-catechin. EGCG is the major polyphenolic constituent in green tea. More than 65% of the total catechin content and more than 10% of the extract dry weight of green tea are composed of EGCG [11]. A number of publications assume that EGCG is probably responsible for the majority of the potential health benefits attributed to the consumption of green tea [12]. EGCG is best known for its antioxidant properties, which have led to their evaluation in many diseases that are associated with reactive oxygen species. Among age-associated pathologies and neurodegenerative diseases, green tea polyphenols have been demonstrated to afford significant protection against Parkinson’s disease, Alzheimer’s disease, and ischemic damage [13,14]. Despite the suggested neurodegenerative preventive effects of EGCG, more studies are needed to understand the extensive mechanisms that are involved in EGCG-modulated neuronal functions. EGCG prevents striatal dopamine depletion and substantia nigra dopaminergic neuronal loss that is induced by 1methyl-4-phenyl-1,2,3,6-tetrahydropyridine, while it is a rather poor inhibitor of MAO-B enzyme in vitro [15,16]. Nonetheless, it has been suggested that food constituents, such as catechin, EGCG, and curcumin, attenuate MAO-B activity in C6 cells [17]. Because the in vivo influences of EGCG on MAO-B are still unknown, the purpose of the present study was to evaluate the protective effect of EGCG against MAO-B activity increases in adult rat organs. Moreover, the physiological antioxidation status and serum parameters were assessed to elucidate the mechanism of these EGCG effects. Materials and methods
Table 1 Composition of the test diets Diet ingredients (g/kg diet)
Control
VE
EGCG
Casein Methionine Sucrose Corn starch Alphacel Soybean oil Choline Mineral mix (AIN 76 mineral mix*) Vitamin mix (AIN 76 vitamin mix*) Vitamin E EGCG
200 3 325 325 50 50 2 35
200 3 325 325 50 50 2 35
200 3 325 325 50 50 2 35
10
10
10
*
d d
450 IU d
d 240 mg
AIN 76 mineral mix, AIN 76 vitamin mix [42].
At the end of the 12 wk, the rats fasted for 12 h and then were sacrificed by carbon dioxide inhalation. Their blood was collected into tubes followed by centrifugation (3000 g, 20 min, 4 C) to separate the serum. The rat organs were removed and stored at 80 C for the experiments described below. Serum analysis The concentrations of serum glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), uric acid, blood urea nitrogen (BUN), total cholesterol, triglyceride (TG), and glucose in the experimental rats were measured by the VITROS 950 Chemistry System (Johnson & Johnson, New Brunswick, NJ, USA).
Chemicals The benzylamine, 2,2-azobis (2-amidinopropane) dihydrochloride, b-phycoerythrin, Trolox, 2,6-ditertbutyl-4-methylphenol, thiobarbituric acid, trichloroacetic acid, and a-tocopherol were all purchased from Sigma Chemical Co. (St. Louis, MO, USA). AIN 76-based diets were purchased from ICN Biomedicals (Los Angeles, CA, USA). EGCG was applied via the product of YoCome-purified EGCG capsules (purified green tea EGCG 90%; Yung Shin Pharm. Ind. Co., Ltd., Taichung, Taiwan). Animals and diets The experimental design was approved by the Animal Experiment Committee of Chung Hwa University of Medical Technology. Experiments were carried out with 24 male LongEvans rats (7 wk of age) that were purchased from the National Laboratory Animal Center. The rats were housed individually in stainless steel wire-bottomed cages and were maintained in a room with a controlled temperature and 12-h light and dark cycles. Food and distilled water were provided ad libitum. They were fed on chow diets for 10 wk and then were fed on diets based on AIN-76 for 1 wk. Next, the rats were divided into the three following groups: The control group (Control, eight rats), Vitamin E group (VE, positive control, eight rats), and EGCG group (EGCG, eight rats). The assigned procedure was conducted randomly by body weight to equalize the mean body weight of the rats in each group. The composition of the diets for the three groups is shown in Table 1. The administration of EGCG in the fortified test diets was adjusted to 240 mg/kg diet in the EGCG group. The food intake amounts and body weight were recorded every 3 d.
Serum oxygen-radical absorbance capacity (ORAC) assay The total antioxidant capacity in the serum of rats was determined by using the ORAC assay [18]. The 0.01 mL diluted rat’s serum contained 2,2-azobis(2-amidinopropane) dihydrochloride (75 mM, 0.01 mL), b-phycoerythrin (0.4 mM, 0.015 mL), and sodium phosphate buffer to a 0.25 mL final volume. The fluorescence of the assay mixture was measured at an excitation wavelength of 492 nm, and an emission wavelength 565 nm for 200 min at 37 C (FLUOstar OPTIMA microplate reader, BMG LABTECH Inc., Cary, NC, USA). The Trolox was used for the standard curve and antioxidant equivalent calculations. The final results were calculated by using the differences of the areas under the fluorescence curves for 200 min, in which they were expressed as mM Trolox equivalents. Lipid peroxidation assay The modified thiobarbituric acid reactive substances (TBARS) method [19] was used to determine the lipid peroxidation levels of rat brains and livers. In brief, 0.5 mL tissue homogenate (in potassium-phosphate buffer, pH 7.4) was mixed with 0.5 mL trichloroacetic acid solution (10%) and centrifuged at 1500 g for 10 min. The clear supernatant was collected and treated with 0.5 mL thiobarbituric acid solution (0.4% in 0.2 N HCl) and 50 mL 2,6-ditertbutyl-4-methylphenol (0.2% in 95% ethanol), and then incubated in a 50 C water bath for 1 h. An amount of 1.5 mL n-butanol was added to the cooled solution and centrifuged at 1500 g for 10 min. The clear supernatant was separated and used for the measurement of the fluorescence at an excitation wavelength of 515 nm and an emission wavelength of 550 nm
The MAO-B enzyme activity in rat tissue was measured by a modified method of the standard techniques assay procedure [20]. The tissues were homogenized in 0.2 M phosphate buffer (pH 7.4) and centrifuged at 1000 g for 10 min and 4 C. The supernatant was collected and further centrifuged at 17000 g for 30 min and 4 C. The pellet was collected and resuspended in a 1 mL phosphate buffer (0.2 M, pH 7.4). The resuspended pellet solution (0.125 mL) was mixed with a benzylamine solution (0.3 mL, 8 mM) and then adjusted to the final volume of 3 mL by a phosphate buffer. The mixture was shaken at 37 C for 3 h. The reaction was stopped by the addition of 0.3 mL 60% perchloric acid. The reaction of the benzylaldehyde product was extracted with 3 mL cyclohexane. The organic layer was separated by centrifugation at 3000 g for 10 min and analyzed for absorbance at 242 nm (Hitachi U-2001 spectrophotometer). The protein concentration assay method was used as described by the Lowry method [21]. For verification purposes, pargyline (MAO-B inhibitor) was used to confirm the type of MAO isoforms. Statistical analysis The data are presented as the means along with their standard deviations from all the sets of independent experiments. The differences between the groups were analyzed by using oneway ANOVA, followed by Duncan’s multiple range test. The differences between the control and experimental groups in terms of all the parameters were analyzed by using the Student’s t test. The difference was considered significant if the P value was 0.05 or less. The correlation among the serum ORAC, brain TBARS, liver TBARS, brain MAO, and liver MAO was analyzed by using the Pearson correlation. The statistical analysis was furthered by using a SAS statistical computer program (Version 13.0.161, SAS Institute Inc., Cary, NC, USA). Results The growth curve of the rats fed on three diets (Control, atocopherol, and EGCG supplements) during the experimental period demonstrated no significant differences between each group throughout the entire period (data not shown). This indicated that the chemical forms of the supplements in these diets had no influence on the rats’ growth. Table 2 shows the Table 2 The serum GOT, GPT, uric acid, BUN, total cholesterol, triglyceride, and glucose of rats fed on the control (Control), tocopherol supplement (VE), or EGCG supplement (EGCG) diets* Control GOT (U/L) GPT (U/L) Uric acid (mg/dL) BUN (mg/dL) TC (mg/dL) TG (mg/dL) Glucose (mg/dL)
169.3 90.9 13.1 12.9 183.6 187.9 601.6
VE 114.4 73.0 1.2 1.8 23.3 56.3 135.5
190.5 93.7 12.6 12.6 150.6 196.0 589.7
EGCG
87.3 40.7 2.4 2.4 19.4y 48.6 187.4
156.3 97.3 10.6 11.0 158.0 179.8 572.2
64.9 47.7 1.4y 2.4 32.7 54.5 136.3
BUN, blood urea nitrogen; GOT, glutamic oxaloacetic transaminase; GPT, glutamic pyruvic transaminase; TC, total cholesterol; TG, triglyceride * Each value represents the means with their standard deviations for 7–8 rats. y Value is significantly different from the control group (Control) by a Student’s t test (P < 0.05).
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0.95
* 0.85
0.75
0.65 Control
VE
EGCG
Fig. 1. Total antioxidant capacity in the serum of rats fed on test diets for 12 wk. The values are the means with their standard deviations for seven to eight rats that are depicted by vertical bars. * Value is significantly different from the control group (Control) when analyzed by Student’s t test (P < 0.05). ORAC, oxygen-radical absorbance capacity.
results of the GOT, GPT, uric acid, BUN, total cholesterol, TG, and glucose analyses of all groups. The groups treated with atocopherol or EGCG did not influence the levels of serum GOT, GPT, BUN, TG, and glucose concentrations. However, EGCG treatment significantly decreased the uric acid level in the EGCG group of rats (Table 2). The total cholesterol level was also significantly decreased in the VE group. After the rats were fed on test diets for 12 wk, the total antioxidant capacity of the EGCG group was significantly (P < 0.05) increased in serum compared to the Control and VE groups (Fig. 1). In addition, the VE and Control groups had no significant differences. The lipid peroxidation status in the brains and livers of the Control, VE, and EGCG groups of the rats was measured by the TBARS method. The TBARS levels of the rats’ brains and livers, which were fed on three test diets for 12 wk, are shown in Figure 2. Treatment with a-tocopherol (VE group) or EGCG
A TBARS (nmol/g brain)
Monoamine oxidase B (MAO-B) activity assay
2.0
Brain 1.5
*
*
VE
EGCG
1.0 0.5 0.0 Control
B 8.0 TBARS (nmol/g liver)
(Hitachi F-4500 Fluorescence Spectrophotometer; Hitachi, HighTechnologies Co., Tokyo, Japan).
ORAC (mM Trolox equivalent)
S.-M. Lin et al. / Nutrition 26 (2010) 1195–1200
Liver 6.0 4.0
*
*
2.0 0.0 Control
VE
EGCG
Fig. 2. Brain (A) and liver (B) TBARS levels of rats fed on test diets for 12 wk. The values are the means with their standard deviations for seven to eight rats, depicted by vertical bars. * Value is significantly different from the control group (Control) when analyzed by Student’s t test (P < 0.05). TBARS, thiobarbituric acid reactive substances.
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MAO activity (U)
A
S.-M. Lin et al. / Nutrition 26 (2010) 1195–1200
Brain
40 30 20
*
*
10 0 Control
VE
EGCG
B 80 Liver MAO activity (U)
60 40 20 0 Control
VE
EGCG
Fig. 3. The brain (A) and liver (B) monoamine oxidase (MAO) activities of rats fed on test diets for 12 wk. The values are the means with their standard deviations for seven to eight rats, depicted by vertical bars. * Value is significantly different from the control group (Control) when analyzed by Student’s t test (P < 0.05). MAO, monoamine oxidase; U, mmol/h/mg protein.
(EGCG group) caused a significant (P < 0.05) decrease in the brain TBARS level, in which the EGCG-fortified diet demonstrated a similar effect with the a-tocopherol-fortified diet (Fig. 2A). Similarly, a significant decrease in the VE and EGCG groups of the rat liver TBARS levels (P < 0.05) was shown (Fig. 2B). It was demonstrated that the EGCG supplement had a similar physiological lipid peroxidation preventive effect as a-tocopherol on rats during age increases. The MAO-B enzyme activities in rat brains and livers are shown in Figure 3. In rat brains, the MAO-B activity showed a significant decrease in the VE and EGCG groups versus Control group (P < 0.05). Despite the significant MAO-B activity decrease, which was observed in the VE and EGCG groups’ brains versus Control group, there were no significant changes that could be noted with respect to the MAO-B enzyme activities in the rat livers among these groups. For confirming the type of MAO isoforms, the rat tissue samples were also treated with MAO-B inhibitor pargyline and showed a decrease in MAO-B enzyme activity. Discussion There were only a few reports that discussed the correlation between dietary supplementation and brain MAO-B enzyme regulation in animals. Our previous work demonstrated that dietary selenium supplements revealed the brain MAO-B activity reduction effect [22]. In the present study, we investigated the protective effect of EGCG against the MAO-B enzyme activity increase in adult rat brains. The rat feeding was designed for dietary uptake and daily EGCG dose was estimated approximately 8.6 mg/kg body weight. The rat serum parameters were analyzed to understand the physiological influences of atocopherol and EGCG. It demonstrated that the a-tocopherol or EGCG supplements did not influence the serum levels of the GOT,
GPT, BUN, TG, and glucose concentrations. We concluded that the administration of doses of a-tocopherol and EGCG did not affect the liver function, lipid metabolism, or glucose metabolism of the experimental rats. However, we observed a decrease in the serum uric acid level in the EGCG group of rats. A recent study also reported that oral pretreatment with EGCG decreased the plasma uric acid levels in isoproterenol-treated rats [23]. It was suggested that EGCG could inhibit the xanthine oxidase enzyme to produce uric acid and reactive oxygen species [24,25]. Therefore, our present study supports the assumption that the decrease in uric acid may have been due to the ability of EGCG to block xanthine oxidase. The advanced mechanisms remain to be investigated. The antioxidant activities of green tea dietary supplements as determined by the ORAC method were well correlated with the total polyphenol content [26]. Furthermore, the method of ORAC was applied in the present study to evaluate the total antioxidant capacity in the serum of rats, in which a significant increase in the EGCG group was observed. An earlier study also indicated that the total antioxidant capacity of human plasma presented a dose-dependent increase after green tea consumption. Therefore, these results can reasonably conclude that EGCG supplementation leads to the increase of the serum total antioxidant capacity of rats. In fact, many of the neuropreventive effects of EGCG are attributable to its antioxidant properties [27]. In contrast to the finding of EGCG supplementation, the result of a-tocopherol supplement (VE group) did not show a significant difference with the control group in terms of rats’ serum. The ORAC method is based on the principle of the absorbance capacity of oxygen radicals by antioxidants. However, the reaction of a-tocopherol is not a counteraction with oxygen, but rather with fatty acid peroxyl radicals, which in turn interrupts the auto-oxidative radical chain reaction process [28]. Thus, it is acceptable that the a-tocopherol supplements (VE group) reveal no effect on the total antioxidant capacity by the ORAC method and due to the result of the insignificant difference with the Control group. Brain and liver tissues are abundant in polyunsaturated fatty acid constituents, which form the toxic lipid peroxidation products during the aging process [29,30]. The physiological functions should be influenced by these lipid peroxidation products in animals. Recently, a report denoted that EGCG supplementation significantly decreased the level of lipid peroxidation in both mitotic (liver) and postmitotic (skeletal muscle) tissues of aged rats [31]. Oral intake of EGCG significantly decreased the TBARS levels in the livers of fatigued animals [32]. The present study further found that EGCG provided protective action for lipid peroxidation in rat brain and liver tissues (Fig. 2). It has been indicated that EGCG augmented the antioxidant-related enzymes and ameliorated the lipid peroxidation status in aged rat brains [33]. These results can be explained by the consequence that EGCG attains a lipid peroxidation decrease effect, at least partially, via the improvement of the physiological anti-oxidation status. Furthermore, the atocopherol supplements (VE group) demonstrated a similar protective action for lipid peroxidation and TBARS level decrease in these tissues. This result also meets the explanation above that a-tocopherol conducts its effect via a reaction with fatty acid peroxyl radicals and interrupts the auto-oxidative radical chain reaction process. It is proven that the MAO-B levels were increased during aging [3,4]. In the present study, all of the groups of rats were fed for a total 30 wk, and the rat brain MAO-B enzyme activity is expected to increase with age. However, the VE and EGCG groups
S.-M. Lin et al. / Nutrition 26 (2010) 1195–1200
exhibited a significant decrease in MAO-B activity versus the Control group in rat brains. In contrast to the findings in brains, there were no significant differences in the MAO-B enzyme activity among the Control, VE, and EGCG groups in the livers of rats. Thus, EGCG seems to possess further characteristics of tissue selectivity because it inhibited the MAO enzymes in brains but also caused rather little inhibition of the enzymes in the liver. This may be consequent to the reason that the MAO enzymes play different roles in brains (amine neurotransmitters transformation) and livers (foreign amine compounds detoxicating) [34]. It was already known that the oral administration of green tea catechins reaches a maximum plasma concentration at around 1 h and is widely distributed into various organs for a longer period [35,36]. However, EGCG may potentially penetrate through the blood-brain barrier at a lower rate and lead to low concentrations of EGCG in the various brain regions [37]. Therefore, EGCG is likely to prevent a brain MAO-B activity increase via physiological antioxidant status improvement, rather than directly affect the enzyme. When the MAO enzyme in brains catalyzes the oxidative deamination reaction of many amine neurotransmitters, a hydrogen peroxide product is generated. Hydrogen peroxide is considered one of the sources of oxidative stress, which induces physiological peroxidation. Therefore, we investigated the correlation between the MAO activities and TBARS levels in rat organs. This showed that brain MAO activity has a positive correlation with the brain and liver’s lipid peroxidation, but also that there is no correlation between the liver’s MAO activity and brain/liver’s lipid peroxidation (Table 3). These results implied the important link between brain MAO enzyme activity and the physiological lipid peroxidation status. In addition, the serum total antioxidant capacity does not have the same correlation with brain MAO activity (Table 3). Therefore, we suggest that lipid peroxidation may play a particular role in brain MAO activity and remain to be further investigated. The serum ORAC showed a negative correlation with brain TBARS. It is known that a decreased serum antioxidant status has been suggested as a risk factor in neurodegenerative diseases [38]. The present study observed a significant increase of the serum total antioxidant capacity in the EGCG group. It was also indicated that EGCG could act synergistically and significantly increase the antioxidant activity of vitamins E and C and then protect lipid peroxidation [39]. So it may be concluded that EGCG supplement improves lipid peroxidation at least partly via increasing serum antioxidant status. However, there are minor correlations between the liver MAO activity, serum total antioxidant capacity, and brain/liver’s lipid peroxidation. These results can explain, at least partly, the Table 3 Correlation among total antioxidant capacity, lipid peroxidation, and monoamine oxidase activity
Serum ORAC (mM Trolox equivalent) Brain TBARS (nmol/g brain) Liver TBARS (nmol/g liver) Brain MAO (mmol/h/mg protein)
TBARS (brain)
TBARS (liver)
MAO (brain)
MAO (liver)
r ¼ 0.45 P ¼ 0.02
r ¼ 0.13 P ¼ 0.58
r ¼ 0.21 P ¼ 0.40
r ¼ 0.03 P ¼ 0.90
r ¼ 0.62 P ¼ 0.003
r ¼ 0.56 P ¼ 0.02 r ¼ 0.75 P ¼ 0.008
r ¼ 0.07 P ¼ 0.77 r ¼ 0.16 P ¼ 0.56 r ¼ 0.45 P ¼ 0.08
MAO, monoamine oxidase, ORAC, oxygen-radical absorbance capacity; TBARS, thiobarbituric acid reactive substances
1199
diverse prevention effect of EGCG supplements on the brain and liver’s MAO activity variations in rats. It has been reported that both green tea and EGCG are very poor inhibitors of MAO-B enzyme in vitro. This led to the conclusion that MAO inactivation was not involved in the neuroprotection exerted by these compounds [15]. However, EGCG is not stable under most cell culture conditions [40]. Moreover, it has been suggested that the MAO-B inhibitor 7-nitroindazole possesses the neuroprotective effects that partially contribute to antioxidant action in vitro and in vivo [41]. Similarly, EGCG is likely to influence the brain MAO-B activity that is kept from being increased during age increases by preventing physiological peroxidation instead of by directly inhibiting the MAO-B enzyme. Conclusions We first suggest that EGCG supplementations can decrease brain MAO-B enzyme activity in adult rats. EGCG can obtain these effects, at least partly, by improving the physiological antioxidative status. Furthermore, our work can provide for the possible developments of dietary supplements for the tissueselective effect of an MAO-B inhibitor. Acknowledgments This work was partly supported by the National Science Council (NSC91-2320-B-273-001) in Taiwan. There is no conflict of interest to disclose. References [1] Weyler W, Hsu YP, Breakefield XO. Biochemistry and genetics of monoamine oxidase. Pharmacol Ther 1990;47:391–417. [2] Strolin Benedetti M, Dostert P. Monoamine oxidase, brain ageing and degenerative diseases. Biochem Pharmacol 1989;38:555–61. [3] Jossan SS, Gillberg PG, d’Argy R, Aquilonius SM, Langstrom B, Halldin C, et al. Quantitative localization of human brain monoamine oxidase B by large section autoradiography using L-[3H]deprenyl. Brain Res 1991;547:69–76. [4] Nicotra A, Pierucci F, Parvez H, Senatori O. Monoamine oxidase expression during development and aging. Neurotoxicology 2004;25:155–65. [5] Saura J, Luque JM, Cesura AM, Da Prada M, Chan-Palay V, Huber G, et al. Increased monoamine oxidase B activity in plaque-associated astrocytes of Alzheimer brains revealed by quantitative enzyme radioautography. Neuroscience 1994;62:15–30. [6] Sherif F, Gottfries CG, Alafuzoff I, Oreland L. Brain gamma-aminobutyrate aminotransferase (GABA-T) and monoamine oxidase (MAO) in patients with Alzheimer’s disease. J Neural Transm Park Dis Dement Sect 1992;4:227–40. [7] Knoll J. The pharmacological basis of the beneficial effects of (-)deprenyl (selegiline) in Parkinson’s and Alzheimer’s diseases. J Neural Transm Suppl 1993;40:69–91. [8] Nagatsu T, Sawada M. Molecular mechanism of the relation of monoamine oxidase B and its inhibitors to Parkinson’s disease: possible implications of glial cells. J Neural Transm Suppl 2006;71:53–65. [9] Yamada M, Yasuhara H. Clinical pharmacology of MAO inhibitors: safety and future. Neurotoxicology 2004;25:215–21. [10] Williamson G, Holst B. Dietary reference intake (DRI) value for dietary polyphenols: are we heading in the right direction? Br J Nutr 2008;99(Suppl 3):S55–8. [11] Goto T, Yoshida Y, Kiso M, Nagashima H. Simultaneous analysis of individual catechins and caffeine in green tea. J Chromatog A 1996;749:295–9. [12] Nagle DG, Ferreira D, Zhou YD. Epigallocatechin-3-gallate (EGCG): chemical and biomedical perspectives. Phytochemistry 2006;67:1849–55. [13] Mandel S, Youdim MB. Catechin polyphenols: neurodegeneration and neuroprotection in neurodegenerative diseases. Free Radic Biol Med 2004;37:304–17. [14] Weinreb O, Mandel S, Amit T, Youdim MB. Neurological mechanisms of green tea polyphenols in Alzheimer’s and Parkinson’s diseases. J Nutr Biochem 2004;15:506–16. [15] Levites Y, Weinreb O, Maor G, Youdim MB, Mandel S. Green tea polyphenol (-)-epigallocatechin-3-gallate prevents N-methyl-4-phenyl-1,2,3,6-
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