Lab. Anim. Res. 2010: 26(1), 63-68 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 361-763, Korea Tel: +82-43-261-2813 Fax: +82-43-268-2732 E-mail: 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
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 ), 150-200 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 50 50 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-10-5500, LND GmbH & Co. KG, Nordhom, Germany), serotonin (5-HT) serotonin ultra sensitive ELISA (Cat# BA-10-5900, LND GmbH & Co. KG, Nordhom, Germany), corticosterone (CS) Corticosterone EIA kit (Cat# 900-097, Assay Designs, Inc. Ann Arbor, MI, USA) w œw x w.
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.
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
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,
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