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1 Lab. Anim. Res. 2010: 26(1), Anxiolytic Effect of a Combination of Green Tea Extract and L-theanine Won Kyung Lee 1,#, Tae Il Kim 2,#, Sang-ki Park 1, Hyoung Kook Park 1 and Jin Tae Hong 2, * 1 LG Household & Health Care Co., Ltd., Daejeon, Korea 2 College of Pharmacy, Chungbuk National University, Cheongju, Korea The purpose of this study is to investigate whether the combination of green tea extract (GTE) and L- theanine has an anxiolytic effect by oral administration through behavioral tests and neurtransmitters (or hormone) anaylses. Four week oral administration of GTE (24 mg/kg), L-theanine (4 mg/kg) or their combination showed anxiety-reducing effects determined by increasing numbers of head-dips in a hole board test and reducing retention time in a rota-rod test without changing spontaneous locomotor activity. Biochemical analyses indicated that the test materials decreased dopamine (DA), noradrenaline (NA), corticosterone (CS) and increased serotonin (5-HT) levels in brain cortex, hippocampus, and striatum, which suggests a possible mechanism of previous behavioral tests. Although the synergistic effects of GTE and L-theanine combination were not observed on the behavioral test, its effects on neurotransmitters (NA, CS) were synergistic and comparable to diazepam (2 mg/kg i.p.) with much less muscle relaxation side effect. Therefore, a combination of GTE and L-theanine may be useful as a functional food ingredient having an anxiolytic effect. Key words: Green tea extract, L-theanine, rota rod test, hole board test, neurotransmitters (Received 17 April 2009; Revised version received 14 January 2010; Accepted 15 March 2010) x z ƒ š 10~30% ùküš (Wittchen and Hoyer, 2001). w z, ƒ y w e (Greenberg et al., 1999),»k e w w» w. x ƒ š benzodiazepine m diazepam, lorazepam, clonazepam, alprazolam š w (sedation), (myorelaxation), (amnesia), (dependency)» š. w w e š p w ƒ w (Rex et al., 2002; Seo et al., 2007). x w q x (Hole board test), z # These authors equally contributed to this study *Corresponding author: Jin Tae Hong, College of Pharmacy, Chungbuk National University, 410 Seongbong-ro, Heungdukgu, Cheongju-shi, Chungbuk , Korea Tel: Fax: jinthong@chungbuk.ac.kr x (Rota rod test), w y x (Locomotor activity test) sƒw ƒ e sƒw x, sƒw. Diazepam t w x n z e w w z y (Silva et al., 2007). w ü y l p ƒw, w y mw dopamine, serotonin, noradrenaline k,, x x ƒ y w. l (L-theanine) 1~2% wš γ-glutamylethylamide ƒ š. l LDL y wwš, x w w z ƒ. w p w qq ƒ» š š(rao et al., 2007), yww n z p ƒ w y 63

2 64 Won Kyung Lee et al. y (Park et al., 2009). x s ƒw w sƒ ( q x, z x, w y x ) mw l w n z e w w x w sƒwš y y d w w»» t ƒ y wš w. lò z 8 f ICR mouse x ( ) l w, 1 y j w š, d w w w w. x w x k GLP» wš,, n, x w. x» x f 3 w š, y ü 23±3 o C, 55±10%, 12 ( 7 - z 7 ), lux. x w w š, l n w» w 1 s³ d w. x ( )LG y œ w. (Tiantai ming yuan tea products, China) w w elk 20%, caffeine 5%» t ww» ƒ L-l (Taiyo Kagaku, Japan) 98%, k yw t w. l x s³ ³ w wš ƒ 12 w 1 š w 24 mg/kg l 4mg/kg ƒ ƒ yww 4 n w. Diazepam n w š 2mg/kg w x 30 1z n w. kò l q x(hole board test): q x(hole board test) ƒ cm v 3cm 16 ƒ e e w. ƒ ü š, ƒ w (head dipping) w. kƒ w head dipping w, 5 d w hole ƒ head dipping w hole ü e ƒ w z d w x w z sƒw. z x(rota rod test): w w w w t, d w» w, z x w. 1 10z w 3 cm rota-rod z x z w w ƒ 5 d w d w w x ƒ w q w. w y x(locomotor activity test): w w x w y k wy ƒ w w», ƒ x w d w w y x w. Tilting type ambulometer w d w, e 20 cm, ¾ 18 cm basket basket, z d w. 10 x k z 5 d w. ž w x z, l brain w v (cortex) w (hippocampus), (striatum) w w ¾ 20 C o w.» 200~500 µl extraction buffer (PRO-PREP protein extraction solution (C/T), p lj, û, w ) ³ y k z vƒ 1mL extraction buffer ƒw yww 2 4 C o lysis g.» óù 1.5 ml tube 3 5 vortex wš 4 o C, 15,000 rpm 15 w z w. mj ü dopamine (DA) noradrenaline (NA) Norepinephrine/Dopamine ELISA (Cat# BA , LND GmbH & Co. KG, Nordhom, Germany), serotonin (5-HT) serotonin ultra sensitive ELISA (Cat# BA , LND GmbH & Co. KG, Nordhom, Germany), corticosterone (CS) Corticosterone EIA kit (Cat# , Assay Designs, Inc. Ann Arbor, MI, USA) w œw x w.

3 j e one-way ANOVA w n q wš mw post hoc test Dunnett s test w w. s³±standard error (S.E) ùkü š P<0.05 w (*P<0.05, **P<0.01). d l j k n l w 4 n w q x w head dipping z ƒ ƒw w t sƒw. n (44.5±3.2z, P<0.01), l n (46.7±1.3z, P<0.01) l w n (44.0±2.6z, P<0.01) (29.2±2.3 z) w w head dipping z ƒ ƒw x ùkû y w. l w w w head dipping z ƒ ƒw w z ƒ y w Anxiolytic effect of GTE and L-theanine combination 65 ù, l z ùkü w z y w (Figure 1). m l j n x sƒw» w x z š ¾ d w ùkü sƒw. (245.9±15.9 ) w n (108.8±17.6, P<0.01), l n (90.0±10.6, p<0.01), l w n (71.0±21.1, P<0.01) z y w x w ùkû y w ù, diazepam (18.1±3.8 ) w z ƒ x û ùkû y w (Figure 2). k mò l w x w w z w y Figure 1. Green tea extract (GTE) and L-theanine combination shows anxiolytic activity by increasing number of head dips in hole board test. Diazepam (DZP) was administered intraperitoneally 30 mins before test. The results are represented as mean±se. **P<0.01 compared with non-treated control. Figure 2. Green tea extract (GTE) and L-theanine combination shows muscle relaxant activity by rota rod test. Diazepam (DZP) was administered intraperitoneally 30 mins before test. The results are represented as mean±se. **P<0.001 compared with nontreated control.

4 66 Won Kyung Lee et al. Figure 3. Green tea extract (GTE) and L-theanine combination shows no effect on spontaneous locomotor activity. Diazepam (DZP) was administered intraperitoneally 30 mins before test. The results are represented as mean±se. Figure 4. Green tea extract (GTE) and L-theanine combination shows anxiolytic activity by modulating neurotransmitters. Diazepam (DZP) was administered intraperitoneally 30 mins before test. (a) dopamine (DA), (b) noradrenaline (NA), (c) serotonin (5-HT), (d) corticosterone (CS) concentrations in various brain areas (cortex, hippocampus and striatum). The results are represented as mean±se. ** P<0.01. ƒ w w» w w y x w. x 40z 63z r ƒ ù w q x w z w y ƒ ƒ (Figure 3). º m w x z x l w v, w, l DA, NA, 5-HT, CS w y d w. DA, n (71.3±5.0 ng/mg protein, P<0.01), l n (74.9±1.3 ng/mg protein, P<0.05) l w n (69.5±0.8 ng/mg protein, P<0.01) (84±1.9 ng/mg protein) w w y w š yƒ (Figure 4a). NA v l w n ( v 45.2±1.1 ng/mg protein, P<0.01; 32.6±1.3 ng/mg protein, P<0.01) ( v 60.8±1.6 ng/mg protein; 44.7± 1.0 ng/mg protein) w ùkü, w l n (68.8±3.3 ng/mg protein, P<0.01) l w n (63.4±2.7

5 ng/mg protein, P<0.01) (86.1±0.7 ng/mg protein) w w w. p v n z ƒ ùkù ù l w n w w w z ùk ü š, w l w n z ùkü (Figure 4b). 5-HT w ƒ v ù kû, w l n (57.1±1.4 ng/mg protein, P<0.01) l w n (58.1±0.2 ng/mg protein, P<0.01), š n (46.5±0.7 ng/mg protein, P<0.01) l n (48.5±0.3 ng/mg protein, P<0.01), l w n (52.0±0.4 ng/mg protein, P<0.01) (w 44.7±0.6 ng/mg protein; 41.0±1.6 ng/mg protein) w ƒw (Figure 4c). v CS l w n (56.2±6.2 pg/mg protein, P<0.01) (162.4 ±9.3 pg/mg protein, P<0.01) w, l n (128.3±19.0 pg/mg protein) l w n (87.5±12.3 pg/mg protein, P<0.01) (298.1±26.8 pg/mg protein) w ùkü. v l ƒƒ n w w ù l w n w w w w z ùkü (Figure 4d). Camellia sinensis, caffeine, polyphenol, theanine w» pw w š. w q(α)q ƒ g ¼, w w šƒ (Yoon and Kim, 1997; Kim and Yoon, 1998), l w p t z š (Yokogoshi et al., 1998; Yamada et al., 2005). l x yw n w ª l w w z wš w. 4 x n w z l w e x k Anxiolytic effect of GTE and L-theanine combination 67 z w d w z sƒw., /l w w w z y w. q x l n (4 mg/kg) l w n ( 24 mg/kg, l 4mg/kg) w w w z ƒ w y w. w ƒƒ n û z ù w w z ƒw. z x l n w n w w ùkù x, mw ¼ y mw w /w p z ù ký d. ù diazepam w l w x w y w. x w y w w sƒ w, w z x w y w (false positive) ƒ w w» w w y x w., x w w w z x w y. l w q w, l w ¼ y mw w z ùkü diazepam z x û w w š. w x z y l w, DA, NA, CS l w n w w w š, 5-HT ƒw y w. p, DA, NA, 5-HT l w n 88.5~105.1% z ùkü š, NA CS l w w z. l w w x ƒ n w z ü w ùkù wù caffeine w ƒ. ü caffeine w 4% x w n w 1mg/kg. x w caffeine š n (10 mg/kg ) p ƒ jš, ( 1mg/kg) ¼ tƒ k(δ) q jš ƒ, ùkü k(β) q ƒ j š š (Kakuda et al,

6 68 Won Kyung Lee et al. 2000). w x Rogers et al., (2008) caffeine l wn w, caffeine l, j ƒ j w w šwš. l w ƒƒ n w w z caffeine ƒ, z w l ¼ z ƒ ù l. ù» benzodiazepine ú w w» yw w ¼ y w n w z û z w w w š q. l w (Yokogoshi et al., 1998) l ü p NA, DA, 5-HTƒ yw šw, p w l w n w y w t w w w z y w. l w n x j z ƒ w» t ƒ w ƒ. q x LG y w ww. k Greenberg, P.E., Sisitsky, T., Kessler, R.C., Finkelstein, S.N., Berndt, E.R., Davidson, J.R., Ballenger, J.C. and Fryer, A.J. (1999) The economic burden of anxiety disorders in the 1990s. J. Clin. Psychiat. 60(7), Kakuda, T., Nozawa, A., Unno, T., Okamura, N. and Okai, O. (2000) Inhibiting effects of theanine on caffeine stimulation evaluated by EEG in the rat. Biosci. Biotechnol. Biochem. 64(2), Kim, S. and Yoon, S.W. (1998) Analytical study for the effects of green tea with GABA about the brain wave and short term memory. J. Kor. Jungshin. Sci. Soc. 2(1), Park, S., Kim, T.I., Lee, W.K., Park, H.K. and Hong J.T. (2009) Combination of green tea extract and L-theanine alleviates electric foot shock induced stress by modulating neurotransmitters in mice. Yakhak Hoeji 53(5), Rao, T.P., Ozeki, M. and Juneja, L.R. (2007) Suntheanine: A pure and safe L-theanine dietary supplement for relaxation and stress relief. Neutra Cos. 1-2, Rex, A., Morgenstern, E. and Fink, H. (2002) Anxiolytic-like effects of Kava-Kava in the elevated plus maze test- a comparison with diazepam. Prog. Neuropsychopharmacol. Biol. Psychiatry 26(5), Rogers, P.J., Smith, J.E., Heatherley, S.V. and Pleydell-Pearce, C.W. (2008) Time for tea: mood, blood pressure and cognitive performance effects of caffeine and theanine administered alone and together. Psychopharmacology (berl). 195(4), Seo, J.J., Lee, S., Lee, Y., Kwon, B., Ma, Y., Hwang, B., Hong, J. and Oh, K. (2007) Anxiolytic-like effects of obovatol isolated from Magnolia obovata: Involvement of GABA/benzodiazepine receptors complex. Prog. Neuropsychopharmacol. Biol. Psychiatry 31(7), Silva, M.I.G., de Aquino Neto, M.R., Neto, P.F.T., Moura, B.A.M., do Amaral J.F., de Sousa, D.P., Vasconcelos, S.M.M. and de Sousa, F.C.F. (2007) Central nervous system activity of acute administration of isopulegol in mice. Phamacol. Biochem. Behav. 88, Wittchen, H.U. and Hoyer, J. (2001) Generalized anxiety disorders: nature and course. J. Clin. Psychiatry 62(suppl. 11), Yamada, T., Terashima, T., Okubo, T., Juneja, L.R. and Tokogoshi, H. (2005) Effects of theanine, gamma-glutamylethylamide, on neurotransmitter release and its relationship with glutamic acid neurotransmission. Nutr. Neurosci. 8(4), Yokogoshi, H., Kobayashi, M., Mochizuki, M. and Terashima, T. (1998) Effect of theanine, r-glutamylethylamide, on brain monoamines and striatal dopamine release in conscious rats. Neurochem. Res. 23, Yoon, S.W. and Kim, S. (1997) Analytical study for the effect of Sun-α containing drink on the brain wave and memory. J. Kor. Jungshin. Sci. Soc. 1(2),