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Studies about Monoamine Oxidase Inhibitory Activities of Korean Green Tea (Teae sinensis L.) Harvested from Different Time and Location
Studies about Monoamine Oxidase Inhibitory Activities of Korean Green Tea (Teae sinensis L.) Harvested from Different Time and Location
Natural Product Sciences. 2013. Dec, 19(4): 281-285
Copyright © 2013, The Korean Society of Pharmacognosy
  • Received : August 01, 2013
  • Accepted : August 28, 2013
  • Published : December 31, 2013
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About the Authors
You Jin Choi
Plant Resource Research Institute, Duksung Women’s University, 132-714, Seoul, Korea
Han-Soo Chong
Department of Forest Products and Biotechnology, Kookmin University, 77, Jeongneung-ro, Seoul, 136-702, Korea
Young-kyoon Kim
Department of Forest Products and Biotechnology, Kookmin University, 77, Jeongneung-ro, Seoul, 136-702, Korea
Keum Hee Hwang
Plant Resource Research Institute, Duksung Women’s University, 132-714, Seoul, Korea
hwangkh@duksung.ac.kr

Abstract
This study was designed to investigate the nervous sedative effects of green tea. The sedative effect was evaluated by examination of Monoamine oxidases (MAOs) inhibitory activity in vitro in the brain and liver of rat fed on green tea cultivated and harvested from the different regions and periods. It showed that methanol extracts of green tea inhibited significantly the brain MAO-A activity. Especially late harvested green tea extracts showed potential inhibitory activity. The liver MAO-B activity was also inhibited by all of the green tea extracts with strong intensity. This study confirmed that major compounds of green tea such as catechin, epigallocatechin-3-gallate (EGCG) and L-theanine, which were well known for the main bioactive components in the tea plants, were not associated with the MAO inhibitory activities of green tea. These results suggested that a MAO inhibition activity comes from other minor tea components we have to search in the future.
Keywords
Introduction
The green tea means the processed young leaves of Teae sinensis L. (Theaceae), which harvested in early spring. Various bioactivities of green tea have been reported. Anticancer (Wang ., 2002 , Yang ., 2002 , Hsu ., 2001) , antioxidant (Lau ., 2002 , Liebler ., 2001 Nagai ., 2002 , Cai ., 2002) , and antimutagenic activities (Gupta ., 2002) , protective effects on UVA- and UVB- induced skin damage (Tobi ., 2002) , neuroprotective effects (Kakuda, ., 2002 , Pan, ., 2003) , anti-inflammatory activities (Das ., 2002 , Tedeschi ., 2002) , induction of apoptosis (Vergote ., 2002) were studied with isolated compounds as well as crude extracts of green tea. Catechins from green tea were extensively studied about the activities of anticarcinogenic (Yang ., 2002) , antioxidant (Liebler ., 2001) . EGCG and (–)-epigallocatechin (EGC), other two major components of green tea, were also reported for the anticancer activities (Wang ., 2002) , antioxidative effects (Liebler ., 2001) , protective effects on UV light-induced skin damages (Tobi ., 2002) , apoptosis activities (Vergote ., 2002) . Polyphenols of green tea were also reported for antiinflammatory activities (Tedeschi ., 2002) , chemopreventive activities (Hsu ., 2001) , antioxidative effects (Cai ., 2002) , and brain cell preventive effects (Choi ., 2002) . Green tea extracts has been studied as the therapeutic purpose of inflammation (Das ., 2002) , brain protection (Pan, ., 2003) , and cardiovascular disease. However, studies about antihypertension, nervous sedative effect and dementia treatment (Choi ., 2002) were rare. Monoamine oxidase (MAO; EC1.4.3.4) is the most extensively studied enzyme associated with central monoamine transmitter systems. Pharmacologically, MAO can be divided into two forms, termed MAO-A and MAO-B. Monoamine oxidases (MAOs) play a central role in the metabolism of many amines including the neurotransmitter monoamines. MAOs are flavorproteins found exclusively in the mitochondrial outer membrane, occurring in MAOA and MAO-B subtypes. MAO-A delaminates serotonin and norepinephrine, whereas MAO-B prefers phenylethylamine and benzylamine as substrates. MAO inhibitors have been used for the purpose of therapeutics of Parkinson’s disease, depressant and hypertension (Blaschko ., 1974 , Cooper ., 1996) . In this study we determined the inhibitory activity of Korean green tea obtained from the different regions and harvested periods on MAO activity in the brain and the liver of rat by in vitro system.
Experimental
Plant materials and reagents − The green teas harvested and processed in the different periods and different regions in Korea were purchased. The voucher specimens (#KM120925) were kept in the research laboratory in Kookmin university (Seoul, Korea). Sprague-Dawley male rats were purchased from Bio Genomics, Ind., which was licensed by Charles River Technology Experimental Animal Co. (Seoul, Korea). Serotonin, benzylamine, (+)-catechin, (−)-catechin, L-theanine, (−)-epigallocatechin-3-gallate [(−)-EGCG] and Amberlite CG-50 (H + form) were obtained from Sigma Co. (St. Louis, U.S.A.). This research was conducted in accordance with the internationally accepted principles for laboratory animal use and care as found in for the US guidelines (NIH publication #85-23, revised in 1985). The green tea samples were classified according to harvested period, in April moderate (Apr-M), in April terminal (Apr-T), in May early (May-E), and in May moderate (May-M). Each 10 g of eight dried green tea was minced by domestic mixer and added 10 parts of 80% methanol. Following stood in room temperature for 7 days, methanol extracts were filtered and concentrated by vacuum pump evaporator on 45℃ water bath. Each extract was examined for the inhibitory activities on MAO-A and MAO-B.
Preparation and assay of MAO-A − Enzyme sources were prepared from brain of Sprague-Dawley male rat by the routine procedures (Hwang ., 2003) . The rats were anaesthetized with ethyl-ether and were lost blood with 3.13% sodium citrated syringe from heart. The brain tissue was obtained from decapitated brain immediately. The brain was washed with 0.01 M phosphate buffered saline (PBS, pH 7.0), and homogenate at 4℃ for 1 minute followed by added cold 0.25 M sucrose by 9 parts of wet weight of tissue. Centrifuged at 700 g in 4℃ for 20 min. Supernatant was centrifuged at 18,000 g for 20 min immediately. Pellet was suspended in 5 parts of PBS, and used for crude enzyme preparation.
Prepared crude MAO-A (0.5 mL) was added to test tubes with 1.0 mL of green tea extracts. It was incubated in shaking incubator at 37.5℃ for 15 min. As a substrate, 0.5 mL of 1.0 mM serotonin was added and incubated at 37.5℃ for 90 min. To terminate the enzyme action, test tubes were heated at 95℃ water bath for 3 min. and centrifuged at 700 g for 20min. immediately. Supernatants were poured in prepared Amberlite CG-50 (H + form) column (0.6 × 4 cm). After washed with distilled water thoroughly (over 40 mL), eluted with 3 mL of 4 N acetic acid, elute was determined of absorbance at 277 nm. Instead of samples, same volumes of distilled water were added in control. In the sample controls, the substrates were added on the time of activity termination instead of initiation of action. Each group was performed with duplicated and calculated for the inhibition percentages of samples by proper expression.
Preparation and assay of MAO-B − Enzyme sources were prepared from liver of Sprague-Dawley male rat by the routine procedures. The rat was anaesthetized with ethyl-ether and was lost blood with 3.13% sodium citrated syringe from heart. Obtained liver tissue was washed with 0.01 M phosphate buffered saline (PBS, pH 7.0), and homogenate at 4℃ for 1 minute followed by added cold 0.25 M sucrose by 5 parts of wet weight of tissue and centrifuged at 700 g in 4℃ for 20 min. Supernatant was centrifuged at 18,000 g for 20 min immediately. Pellet was suspended in 5 parts of PBS, and preserved at freezer before the treatment of samples. Enzyme assay methods were performed by McEwen’s methods (Lyketsos ., 2002) . Prepared crude MAO-B 0.5 mL was added to test tubes with 1.0 mL of green tea extracts. It was incubated at 37.5℃ for 15 min in shaking incubator. As a substrate, 0.5 mL of 4.0 mM benzylamine was added and incubated at 37.5℃ for 90 min. To terminate the enzyme action, added 0.2 mL of 60% perchloric acid and added 4 mL of cyclohexane, simultaneously. Mixed immediately with vortex mixer and centrifuged at 700 g for 20 min to precipitate the protein. Cyclohexane layer was determined of absorbance at 242 nm. In the same manner as in MAOA, instead of samples, same volumes of distilled water were added in control. In the test controls, the substrates were added on the time of activity termination instead of initiation. Each group was performed with duplicates and calculated for the inhibition percentages of samples by proper expression.
The inhibitory activity of Green tea on the MAO − The methanol extracts of Korean green tea harvested from the different region and period were measured about inhibitory activities on MAO-A and MAO-B. At the same time, EGCG, (−)-catechin and (+)-catechin, which were well known the major components of green tea, were also examined. Enzyme sources were prepared from brain and liver of Sprague-Dawley male rats by the routine procedure and enzyme assay methods were performed by previous report (Hwang ., 2003) . Since the methanol extract of 〈region A May-E〉 green tea showed potent inhibitory activity against MOA-A, the TLC patterns of the two groups, one is green tea extract group and another is already known pure compound group such as EGCG and catechin, were examined weather or not the MAO activities were from the known compounds.
Statistical analysis − All experiments were conducted in independent triplicate (n = 3) and data were expressed as mean ± SD. Statistical significance was evaluated by one-way analysis of variance using SPSS Win program (Version 19.0, Cary, NC), and individual comparisons were determined using Duncan’s multiple range tests at the p < 0.05 level.
Results and Discussion
The methanol extracts of green tea from two different locations and four different harvesting times were examined on the inhibitory activity of MAO-A and MAO-B. All of the green tea extracts showed inhibitory activities about MAO-A and MAO-B. In general, it is recognized that early harvested green tea was thought to have high product value in markets. But it did not show strong inhibitory activity on MAOs. We found that the inhibitory activity of green tea on MAOs gradually increased with harvest period until May early, and decreased after ( Fig. 1. ). All of them, May-E extract showed the most potent inhibitory activity in both enzymes. For the purpose of determining the MAO inhibitory activities of the green tea, harvested in different period, examined the inhibitory activities in various concentrations (0.05, 0.50 and 5.0 mg/ml). A (region A) and B (region B) green teas were also compared. Region A green tea showed a potent inhibitory activity in dosedependent manner. All of them, May-E green tea showed the most potent inhibitory activity on MAO-A and MAO-B ( Fig. 2. ). And the methanol extract of green tea extract especially from 〈region A May-E〉 also showed inhibitory activity in a dose dependent manner in MAO-A and MAO-B ( Fig. 2. ). In order to study the active compound of green tea, the activity guided isolation was considered. First of all, the activities of methanol extract of 〈region A May-E〉 green tea and well known tea compounds, such as EGCG and catechins, were compared. Brain MAO-A was inhibited by methanol extracts of green tea. And the liver MAO-B was also inhibited with strong intensity. Especially 〈A May-E〉 showed potent inhibitory activities on both enzymes. The IC 50 values on MAO-A and MAO-B were 0.33 and 0.33 mg/ml, respectively. But they showed weak inhibitory activities on MAO-A as much as methanol extract of 〈region A May-E〉. L-theanine did not show inhibitory activity on any of enzymes ( Table 1 ). Based on the TLC patterns of above three compounds with methanol extracts of 〈region A May-E〉 green tea, it turned out there were many spots methanol extract besides standard major compounds in our chromatographic solvent system. These results suggested that a MAO inhibition activity comes from other minor tea components we have to search in the future. And we will continue to study active compounds of green tea on MAO inhibition. Psychiatric disturbances affect as many as 90% of patients with Alzheimer’s disease (AD) and are a major focus of treatment. Depression is one of the most frequent psychiatric complications of AD, affecting as many as 50% of patients (Lyketsos ., 2002 , Lyketsos ., 2003 , Olin ., 2002) . In recent studies, MAO inhibitor is focused as the drug on the treatment of depression associated with Alzheimer disease. Irie et al . have reported in a recent communication that eugenol exhibits an antidepressant-like activity in mice comparable to that of imipramine, the classical tricyclic antidepressant (Irie ., 2004) . Tao et al. have also reported eugenol isolated from the botanical Rhizoma acori graminei for inhibitory activity toward the MAO-A and MAO-B activities in a rat brain mitochondrial fraction. They discussed on their results that the antidepressant-like action of eugenol could be mediated by its MAO-A inhibitory activity. Their findings provide for the first time a scientific rationale for the traditional use of RAG for the treatment of one of the most prevalent neuropsychiatric comorbidities of AD: depression (Tao ., 2005) . Fusar-Poli et al . reported psychologic therapy using MAOIs offers further advantages after heart transplantation (Fusar ., 2006) . Traditionally, MAO inhibitors were used for treatment of Parkinson's disease (Gutschow ., 2006 , Olanow ., 2006 , Siderowf ., 2006) .
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Inhibitory activities of green tea harvested in different region and period on MAO. Data represent the means ± S.D. Different subscript letters indicate a significant difference at the p < 0.05 by Duncan’s multiple range tests.
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Inhibitory activities of green tea harvested in different period on MAO-A and MAO-B. Prepared crude MAO-A (0.5 mL) was added to test tubes with 1.0 mL of green tea extracts. It was incubated in shaking incubator at 37.5℃ for 15 min. As a substrate, 0.5 mL of 1.0 mM serotonin was added and incubated at 37.5℃ for 90 min. Prepared crude MAO-B 0.5 mL was added to test tubes with 1.0 mL of green tea extracts. It was incubated at 37.5 ℃ for 15 min in shaking incubator.
IC50value and specific activity of green teas on MAO
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IC50 value and specific activity of green teas on MAO
Acknowledgements
This research was supported by Basic Science Research Program through the National Research Foundation of korea (NRF) Funded by the Ministry of Education, Science and Technology (2010-0021753).
References
Blaschko H. (1974) The natural history of amine oxidase. Rev. Physiol. Biochem. Pharmacol. 70 83 - 148
Cai Y.J. , Ma L.P. , Hou L.F. , Zhou B. , Yang L. , Liu Z.L. (2002) Antioxidant effects of green tea polyphenols on free radical initiated peroxidation of rat liver microsomes. Chem. Phys Lipids 120 109 - 117    DOI : 10.1016/S0009-3084(02)00110-X
Cho,i J.Y. , Park C.S. , Kim D.J. , Cho M.H. , Jin B.K , Pie J.E. , Chung W.G. (2002) Prevention of nitricoxide-mediated1-methyl-4-phenyl-1,2,3,6-tetrahydroPyridine-inducedParkinson's disease in mice by tea phenolic epigallocatechin 3-gallate. Neurotoxicology 23 367 - 374    DOI : 10.1016/S0161-813X(02)00079-7
Cooper J.R. , Bloom F.E. , Roth R.H. (1996) The biochemical basis of neuropharmacology. Oxford University Press New York, USA
Das M. , Sur P. , Gomes A. , Vedasiromoni J.R. , Ganguly D.K. (2002) Inhibition of tumor growth and inflammation by consumption of tea. Phytother. Res. 16 S40 - S44    DOI : 10.1002/ptr.797
Fusar, Poli P. , Picchioni M. , Martinelli V. , Bhattacharyya S. , Cortesi M. , Barale F. , Politi P. (2006) Anti-depressive therapies after heart transplantation. J. Heart Lung Transplant. 7 785 - 793    DOI : 10.1016/j.healun.2006.03.019
Gupta S. , Chaudhuri T. , Seth P. , Ganguly D.K. , Giri A.K. (2002) Antimutagenic effects of black tea (World Blend) and its two active polyphenols theaflavins and thearubigins in Salmonella assays. Phytother. Res. 16 655 - 661    DOI : 10.1002/ptr.1038
Gutschow M. , Meusel M. (2006) Enzyme inhibitors in Parkinson treatment. Pharm. Unserer Zeit. 35 218 - 225    DOI : 10.1002/pauz.200600169
Hsu S. , Lewis J.B. , Borke J.L. , Singh B. , Dickinson D.P. , Caughman G.B. , Athar M. , Drake L. , Aiken A.C. , Huynh C.T. , Das B.R. , Osaki T. , Schuster G.S. (2001) Chemopreventive effects of green tea polyphenols correlate with reversible induction of p57 expression. Anticancer Res. 21 3743 - 3748
Hwang K.H. (2003) Monoamine oxidase inhibitory activities of Korean medicinal plants classified to cold drugs by the theory of KIMI. Food Sci. Biotechnol. 12 238 - 241
Irie Y. , Itokazu N. , Anjiki N. , Ishige A. , Watanabe K. , Keung W.M. (2004) Eugenol exhibits antidepressant-like activity in mice and induces expression of metallothionein-III in the hippocampus. Brain Research 1011 243 - 246    DOI : 10.1016/j.brainres.2004.03.040
Kakuda T. (2002) Neuroprotective effects of the green tea components theanine and catechins. Biol. Pharm. Bull. 25 1513 - 1518    DOI : 10.1248/bpb.25.1513
Lau K.M. , He Z.D. , Dong H. , Fung K.P. , But P.P. (2002) Anti-oxidative, anti-inflammatory and hepato-protective effects of Ligustrum robustum. J. Ethnopharmacol. 83 63 - 71    DOI : 10.1016/S0378-8741(02)00192-7
Liebler D.C. , Valcic S. , Arora A. , Burr J.A. , Cornejo S. , Nair M.G. , Timmerman B.N. (2001) Antioxidant reactions of green tea catechins and soy isoflavones. Adv. Exp. Med. Biol. 500 191 - 197
Lyketsos C.G. , DelCampo L. , Steinberg M. , Miles Q. , Steele C.D. , Munro V. , Baker A.S. , Sheppard J.M.E. , Frangakis C. , Brandt J. , Rabins P.V. (2003) Treating depression in Alzheimer disease. Efficacy and safety of sertraline therapy, and the benefits of depression reduction. The DIADS. Arch. Gen. Psychiatry. 60 737 - 746    DOI : 10.1001/archpsyc.60.7.737
Lyketsos C.G. , Olin J. (2002) Depression in Alzheimer’s disease: overview and treatment. Biol. Psychiatry 52 243 - 252    DOI : 10.1016/S0006-3223(02)01348-3
Nagai K. , Jiang M.H. , Hada J. , Nagata T. , Yajima Y. , Yamamoto S. , Nishizaki T. (2002) (−)-Epigallocatechin gallate protects against NO stress-induced neuronal damageafter ischemia by acting as an antioxidant. Brain Res. 956 319 - 322    DOI : 10.1016/S0006-8993(02)03564-3
Olanow C.W. (2006) Rationale for considering that propargylamines might be neuroprotective in Parkinson's disease. Neurology 66 S69 - S79    DOI : 10.1212/WNL.66.10_suppl_4.S69
Olin J.T. , Katz I.R. , Meyers B.S. , Schneider L.S. , Lebowitz B.D. (2002) Provisional diagnostic criteria for depression of Alzheimer disease. Am. J. Geriatr. Psychiatry 10 129 - 141    DOI : 10.1097/00019442-200203000-00004
Pan T. , Fei J. , Zhou X. , Jankovic J. , Le W. (2003) Effects of green tea polyphenols on dopamine uptake and on MPP-(+)-induced dopamine neuron injury. Life Sci. 72 1073 - 1083    DOI : 10.1016/S0024-3205(02)02347-0
Siderowf A. , Stern M. (2006) Clinical trials with rasagiline: evidence for short-term and long-term effects. Neurology 66 S80 - S88    DOI : 10.1212/WNL.66.10_suppl_4.S80
Tao G. , Irie Y. , Li D.J. , Keung W.M. (2005) Eugenol and its structural analogs inhibit monoamine oxidase A and exhibit antidepressant-like activity. Bioorganic & Medicinal Chemistry 13 4777 - 4788    DOI : 10.1016/j.bmc.2005.04.081
Tedeschi E. , Suzuki H. , Menegazzi M. (2002) Antiinflammatory cction of EGCG, the Main component of green tea, through STAT-1 inhibition. Ann. N. Y. Acad. Sci. 973 435 - 437    DOI : 10.1111/j.1749-6632.2002.tb04678.x
Tobi S.E. , Gilbert M. , Paul N. , McMillan T.J. (2002) The green tea polyphenol, epigallocatechin-3-gallate, protects against the oxidative cellular and genotoxic damage of UVA radiation. Int. J. Cancer. 102 439 - 444    DOI : 10.1002/ijc.10730
Vergote D. , Cren-Olive C. , Chopin V. , Toillon R.A. , Rolando C. , Hondermarck H. , Le Bourhis X. (2002) (−)-Epigallocatechin (EGC) of green tea induces apoptosis of human breast cancer cells but not of their normal counterparts. Breast Cancer Res. Treat. 76 195 - 201    DOI : 10.1023/A:1020833410523
Wang Y.C. , Bachrach U. (2002) The specific anti-cancer activity of green tea (−) epigallocatechin-3-gallate (EGCG). Amino Acids 22 131 - 143    DOI : 10.1007/s007260200002
Yang CS , Maliakal P , Meng X. (2002) Inhibition of carcinogenesis by tea. Annu.Rev.Pharmacol Toxicol. 42 25 - 54    DOI : 10.1146/annurev.pharmtox.42.082101.154309
Yang C.S. , Maliakal P. , Meng X. (2002) Inhibition of carcinogenesis by tea. Annu. Rev. Pharmacol Toxicol. 42 25 - 54    DOI : 10.1146/annurev.pharmtox.42.082101.154309