Effects of Polyphenol Rich Herbal Medicine - ACS Publications

Chapter 37

Effects of Polyphenol Rich Herbal Medicine, Ginkgo biloba Extracts on Neurotransmitter Levels in Rat Brain

Downloaded by CORNELL UNIV on October 9, 2016 | http://pubs.acs.org Publication Date: September 19, 2008 | doi: 10.1021/bk-2008-0993.ch037

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Hiroyuki Sakakibara , Yuki Izawa , Jun-ichiro Nakajima , Shujiro Seo Toshiaki Tamaki , Yoshichika Kawai , and Junji Terao 3

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Department of Food Science, Institute of Health Biosciences, The University of Tokushima Graduate School, Kuramoto-cho 3-18-15, Tokushima 770-8503, Japan Department of Pharmacology, Institute of Health Biosciences, The University of Tokushima Graduate School, Kuramoto-cho 3-18-15, Tokushima 770-8503, Japan Tokiwa phytochemical Company, Ltd., 158, Kinoko, Sakura-shi, Chiba 285-0801, Japan Current address: Institute of Environmental Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan 2

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This study was aimed to evaluate the antidepressant mechanism of polyphenol rich extracts of Ginkgo biloba (EGB) from the aspect of neurotransmitter regulation in rat brain. Forced swimming stress for 5 min significantly reduced norepinephrine (NE) level in the rat hypothalamus region of vehicle group from 18.8 to 13.5 nmol/g tissue. However, daily oral treatment of EGB (10 mg/kg body weight/day) for 14 days, which were the effective amount and days for antidepressant action as described in our previous study, maintained NE amount at basal level when forced swimming stress was loaded. Consequently, severe changes of neurotransmitter levels in the hypothalamus at the stress loading are considered one of the possible factors to provoke depression. Daily consumption of some ingredients, such as EGB, that can maintain the neurotransmitter levels at the stress loading, may be helpful to prevent depressive illness.

© 2008 American Chemical Society Shibamoto et al.; Functional Food and Health ACS Symposium Series; American Chemical Society: Washington, DC, 2008.

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430 Depression represents a major public disease affecting nearly 2-5% of the world population in recent years (7). Therefore understanding how to prevent and treat depression is an urgent subject. Chronic stress has been reported to be a main trigger for this disease (Y), and therefore to regulate stress signals in brain probably contributes to the prevention of the depressive illness. The Ginkgo biloba tree has been used as a traditional Chinese herbal medicine for thousands of years (5). Several research groups have shown that extracts from the green leaves of the Ginkgo biloba tree (EGB) possess diverse effects on the improvement of mood and cognitive performance, protection of memory deficits and so on (4, 5). Recently, we reported that EGB significantly shortened the immobility time in the rat forced swimming test (FST) after repeated oral treatment of 10 mg/kg body weight/day for 14 days without change of motor dysfunction in the open field test (6). Therefore, these results indicated EGB might possess an antidepressant activity. The mechanism of antidepressant action of antidepressants including EGB has not been cleared. However, dopaminagic (norepinephrine, NE and dopamine, DA) and serotonergic (serotonin, 5-HT) system in the brain are suggested to play an important role in the etiology of depression. Actually, therapeutic effects of antidepressants, for example selective serotonin reuptake inhibitors, are believed to be caused by the regulation of central dopaminagic or serotonergic systems. Here we aimed to evaluate the effects of EGB on the levels of NE, DA and 5HT and their metabolites in the rat brain after repeated treatment using FST as a stress loading model.

Materials and Methods Male CD rats (4 weeks) from Charles River Japan Inc., Yokohama, Japan, were housed in a 12 h light/dark cycle at a constant temperature of 23 ± 1 °C with free access to food and water. A l l experiments were conducted in accordance with the animal experimental guidelines of the University of Tokushima. Each group (n = 5) was orally administered distilled water, anti depressant drug imipramine (15 mg/kg body weight/day) or EGB (Ginkgolon-24 from Tokiwa Phytochemical Co., Ltd., Chiba, Japan). Administered amount of EGB was 10 mg/kg/day, which indicated remarkable antidepressant effects in the rat forced swimming test (FST) (6). After repeated oral treatment for 14 days, each group was subjected to forced swimming stress for 5 min according to the method of Porsolt et al (7), and then immediately sacrificed by decapitation to collect a hypothalamus region. The groups without forced swimming stress were also subjected a dissection. Neurotransmitter levels in the hypothalamus region were analyzed using high performance liquid chromatography (HPLC) equipped with coulometric

Shibamoto et al.; Functional Food and Health ACS Symposium Series; American Chemical Society: Washington, DC, 2008.


431 array system by the method of Lakshmana and Raju (8) and Vaarmann et al (9) with some modifications. Briefly, the hypothalamus (50 mg) was mixed with 0.5 mL of 0.12 M perchloric acid containing 0.1% cysteine, and 0.5 nmol of isoproterenol was added as an internal standard. The sample mixture was homogenized, and then centrifiiged at 12,000 g for 15 min at 4 °C. The supernatant was filtrated with 0.2 pm membrane filter Millex-LG (Millipore Co., Bedford, MA). An aliquot of 10 pL of the filtrate was subjected to HPLC with a Capcell Pak CI8 MGII column (250 x 4.6 mm I.D., Shiseido Co., Ltd., Tokyo, Japan) equipped with a guard column (10 x 4.0 mm I.D.). The columns were maintained at 25°C. The mobile phase was acetonitrile/55 mM tartaric acid and 45 mM sodium acetate containing 0.65 mM 1-octanesulfonic acid sodium salt (10:90, v/v) at flow rate of 1.0 mL/min. Neurotransmitter and their related compounds were detected with a coulometric array detector (Model 5600A CoulArray System, ESA, Inc., Chemsford, MA) at + 200 mV for DA, NE, 5-HT, 3,4-dihydroxyphenylalanine (DOPA), epinephrine (E), 3,4-dihydroxy-phenylacetic acid (DOPAC), homovanillic acid (HVA), 3-methoxytyramine (3-MT) and 5-hydroxytryptophan (5-HTP), and at +550 mV for tyrosine, trypto-phan and 5-hydroxyindoleacetic acid (5-HIAA). Data analysis was performed by analysis of variance with Fisher-PLSD post hoc test for multiple comparisons. The results were considered significant if the probability of error was less than 5%.

Results and Discussion Figure 1 shows a typical HPLC profile with 12 standard compounds, which were neurotransmitters (DA, NE, E, 5-HT), their precursors (tyrosine, tryptophan, DOPA, 5-HTP), and major biological metabolites (HVA, 3-MT, DOPAC, 5-HIAA) at concentration of 10 pmol each, indicating that every compound could be analyzed simultaneously within 30 min. Rats were orally administered antidepressant imipramine (15 mg/kg body weight/day), EGB (10 mg/kg/day), or vehicle (distilled water) for 14 days. The animals were then subjected to the forced swimming stress for 5 min, which was the same condition for screening model of antidepressants, forced swimming test (FST). Neurotransmitter levels in the hypothalamus region of both with and without swimming stress were analyzed using HPLC-coulometric array system as shown in Table I. Daily treatment of imipramine, which is a tricyclic-type antidepressant acting as monoamine reuptake inhibitor, produced significant reduction in NE level from 18.8 nmol/g tissue to 10.4 nmol/g tissue, as suggested by Butterweck et al (10). Similar reductive pattern of NE, but not


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Figure 1. Typical HPLC profile for neurotransmitters. Numbers show the following standard chemicals: J, 3,4-dihydroxyphenylalanine (DOPA); 2, tyrosine 3, norepinephrine (NE); 4, epinephrine (E); 5, 5-hydroxytryptophan (5-HTP); 6, dopamine (DA); 7, 3,4-dihydroxyphenylacetic acid (DOPAC); 8, 5-hydroxyindoleacetic acid (5-HIAA), 9, 3-methoxytyramine (3-MT), 10, 5-hydroxytryptamine (5-HT); 11, tryptophane; and 12, homovanillic acid (HVA).

significantly, was obtained in the group administered EGB. Forced swimming stress for 5 min, which is suggested to induce a depressive state in rats, also reduced NE level remarkably in vehicle group from 18.8 nmol/g tissue to 13.5 nmol/g tissue. However, NE amounts in the both groups administered imipramine and EGB were maintained at the same range. Similar alteration was suggested in DA and 5-HT, but not significant. Consequently, severe changes of neurotransmitter (dopaminagic and/or serotonergic) levels in the hypothalamus at a stress loading are considered one of the factors to provoke depression. Therefore, daily consumption of some ingredients, such as EGB, that can maintain the neurotransmitter levels when the stress is loaded may be helpful to prevent depressive illness.



65.8 ± 4 . 8 6.4 ± 0 . 7 3.9 ± 0 . 9 13.5 ±0.7* u.d. 4.1 ± 0 . 6 0 u.d. 0.61 ± 0 . 0 7

+Stress 63.8 ± 3 . 7 3.9 3.3 ± 1 . 0 10.4 ±1.1* u.d. 3.9 ±0.65 u.d. 0.52 ± 0 . 1 5

Imipramine -Stress 54.2 ± 0 . 6 4.3 ± 1 . 4 4.0 ± 0 . 6 11.0 ±1.5* u.d. 4.6 ±0.93 u.d. 0.62 ±0.13

+Stress 70.4 ± 4 . 9 4.6 ± 0 . 9 2.7 ± 0 . 6 13.9 ± 1 . 2 u.d. 3.7 ± 1 . 0 u.d. 0.67 ± 0 . 1 9

EGB -Stress

55.9 ± 5 . 8 4.3 ± 1 . 6 3.1 ± 1.0 14.8 ± 2 . 9 u.d. 3.1 ± 0 . 8 4 u.d. 0.56 ±0.15

+Stress

Serotonagic compounds Tryptophane 26.1 ± 1.4 26.3 ± 1.9 27.8 ± 0.7 27.4 ± 0 27.8 ± 1.1 25.7 ± 2.5 5-HTP 0.19 ± 0 . 0 3 0.25 ± 0 . 0 2 0.19 ± 0 . 0 4 0.25 ±0.03 0.23 ± 0 . 0 3 0.22 ± 0.02 5-HT 10.1 ± 1 . 0 13.0±0.5 11.4±2.4 11.6 =b 1.1 10.8±1.0 11.3 ± 2 . 7 5-HIAA 9.0 ± 0 . 3 10.3 ± 0 . 9 8.1 ± 0 . 4 8.0 ± 0 . 6 10.3 ± 0 . 5 9.6 ± 1 . 3 Values are mean ± S.E. expressed as nmol/g wet tissues of 5 animals in each group. Rats were administered vehicle (distilled water), imipramine (15 mg/kg body weight/day), and EGB (10 mg/kg body weight/day) for 14 days, and then subjected to measurement of neurotransmitter levels in the hypothalamus as described in the Materials and Methods. */?

Effects of Polyphenol Rich Herbal Medicine - ACS Publications

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