Daidzein Activates Choline Acetyltransferase from MC-IXC Cells and Improves Drug-Induced Amnesia

Biosci. Biotechnol. Biochem., 70 (1), 107–111, 2006

Ho Jin HEO,1;2 Young-Min SUH,2 Mi-Jeong KIM,2 Soo-Jung CHOI,2 Nam Shik MUN,3 Hye-Kyung KIM,4 Eunki KIM,5 Chang-Ju KIM,6 y Hong-Yon CHO,2 Young Jun KIM,2 and Dong-Hoon SHIN2;

1Jeonnam Innovation Agency, #100 Namak-ri, Samhyang-myeon, Muan-county, Jeonnam 534-700, Korea 2Graduate School of Biotechnology, Korea University, Seoul 136-701, Korea 3Department of Psychiatry, College of Medicine, Chosun University, Gwangju 501-759, Korea 4Department of Food Biotechnology, Hanseo University, Seosan 356-706, Korea 5Department of Biological Engineering, Inha University, Incheon 402-751, Korea 6Department of Physiology, Kyung Hee University, Seoul 130-701, Korea

Received June 21, 2005; Accepted October 14, 2005

The choline acetyltransferase (ChAT) activator, is known to be related both to the degree of dementia which enhances cholinergic transmission via an aug- and to the severity of the neuropathological hallmarks of mentation of the enzymatic production of acetylcholine AD. ChAT activity was significantly lower in AD (ACh), is an important factor in the treatment of patients than in age-matched control subjects in the Alzheimer’s disease (AD). Methanolic extracts from frontal cortex, temporal cortex, hippocampus, and Pueraria thunbergiana exhibited an activation effect cerebellum.1) The most consistent biochemical finding (46%) on ChAT in vitro. Via the sequential isolation of in AD patients is a decrease in ACh levels within the Pueraria thunbergiana, the active component was ulti- basal forebrain.2) This enzyme is a single-strand globular mately identified as daidzein (40,7-dihydroxy-isofla- protein, which is synthesized in the perikarion and then vone). In order to investigate the effects of daidzein transported to the nerve terminals, probably via both from Pueraria thunbergiana on scopolamine-induced slow and rapid axoplasmic flow.3) ChAT is known to be impairments of learning and memory, we conducted present in abundance in the cytosol of the cholinergic a series of in vivo tests. Administration of daidzein cell bodies. Approximately 80–90% of the ChAT (4.5 mg/kg body weight) to mice was shown significantly located in the nerve terminals exists as a cytosolic to reverse scopolamine-induced amnesia, according to protein, a portion of which may be ionically associated the results of a Y-maze test. Injections of scopolamine with the synaptic membranes, whereas the remaining into mice resulted in impaired performance on Y-maze 10–20% of the total enzyme appears to be nonionically tests (a 37% decreases in alternation behavior). By way bound to the plasma membrane.4) Despite the key role of of contrast, mice treated with daidzein prior to the ChAT in neurotransmission, the extremely low amounts scopolamine injections were noticeably protected from and instability of the enzyme in the nervous tissues are this performance impairment (an approximately 12%– factors that hinder biochemical and structural analysis.5) 21% decrease in alternation behavior). These results One strategy aimed toward the amelioration of AD indicate that daidzein might play a role in acetylcholine symptoms is the restitution of near-normal acetylcholine biosynthesis as a ChAT activator, and that it also concentrations in the synaptic cleft, which might ameliorates scopolamine-induced amnesia. improve cholinergic neurotransmission. ChAT activa- tors have been shown to effect an increase in ACh Key words: amnesia; choline acetyltransferase; daidzein synthesis, elevating the endogenous ACh levels in the brain and thereby enhancing cholinergic neurotransmis- The etiological events that lead to Alzheimer’s sion, resulting in an improvement of cognitive function disease (AD) remain somewhat obscure, but highly in cases of mild to moderate AD. In an attempt to typical neuropathological changes have been observed identify a natural active constituent which can signifi- in the brains of AD patients. A reduction in choline cantly increase ChAT levels, we screened 90 Korean acetyltransferase (ChAT, EC 2.3.1.6), a synthetic en- traditional tea plants. We identified a daidzein (40,7- zyme of acetylcholine (ACh) in the cholinergic neurons, dihydroxy isoflavone) of Pueraria thunbergiana,a

y To whom correspondence should be addressed. Tel: + 82-2-923-8732; Fax: +82-2-3290-3429; E-mail: [email protected] Abbreviations: AD, Alzheimer’s disease; ACh, acetylcholine; AChE, acetylcholinesterase; ChAT, choline acetyltransferase; SCOP, scopolamine 108 H. J. HEO et al. member of the Leguminosae family. We then assessed (22–24), 20:80 (25–27), 10:90 (28–30), 0:100 (31–33); daidzein’s possible effects with regard to the reversal or v/v] respectively (flow rate, 2.5 ml/min). The activation amelioration of scopolamine-induced learning and of enzyme activity for each of the fractions was memory deficits. calculated in comparison with the control values. The sample concentration was 500 mg/ml. The values repre- Materials and Methods sent the means ðn ¼ 3ÞSD. Thirty-three fractions were evaporated for tests. Among these, the no. 5 sub- Materials. The dried plants were purchased in a local fraction (90:10) activated ChAT to a level 80% over market for Oriental medicines in Seoul, Korea in 2000, control levels. The No. 5 sub-fraction was evaporated and were ground until the resulting powder could be to dryness (74 mg) under reduced pressure and dissolved passed through a fine screen (about 1 mm). The plant in chloroform (1 ml). It was then fractionated by a materials were authenticated by the Institute of Bio- second silica gel open column chromatography. It technology at Korea University, in which voucher was then subjected to a chromatographic open column specimens are maintained. Minimum Essential Medium (1:5 30 cm) packed with silica gel suspended in (MEM), penicillin, and streptomycin were acquired chloroform. The column was then washed with 200 ml from Gibco BRL (Grand Island, NY, U.S.A.). Acetyl of chloroform and eluted with 10 ml of a chloroform– coenzyme A (acetyl-1-14C) was provided by NEN methanol mixture, in the following ratio: [100:0 (1–3), (Boston, MA, U.S.A.). All of the other chemicals used 96:4 (4–6), 92:8 (7–9), 90:10 (10–12), 88:12 (13–15), were purchased from the Sigma Chemical (St. Louis, 85:15 (16–18), 82:20 (19–21), 0:100 (22–24); v/v] MO, U.S.A.). respectively (flow rate, 1.0 ml/min). The degree to which enzyme activity had been activated for each Isolation of daidzein from Pueraria thunbergiana. We fraction was calculated in comparison with the control screened 90 Korean traditional tea plants in our search values. The sample concentration was 500 mg/ml. for a ChAT activator that can be obtained from natural Values represent the means ðn ¼ 3ÞSD. Among these resources. Among these edible tea plants, methanol 24 fractions, sub-fractions 16–17 (85:15) exerted a extracts from the powdered roots of Pueraria thun- substantial activating effect on ChAT activity, elevating bergiana (1 kg) exhibited the most profound effects on the activity level to more than 135% above control ChAT activity (Table 1). An evaporated methanol values. The active fraction was then purified via HPLC. extract of Pueraria thunbergiana was dissolved in The condition of HPLC for purification was equal to that distilled water (120 ml). This dissolved extract was of analytical HPLC. Analytical HPLC was conducted then partitioned with hexane (360 ml, 3), chloroform with a Waters 2690 (C18 -bondapak reverse column: (360 ml, 3) and ethyl acetate (360 ml, 3), respective- 3:9 300 mm, mobile phase, 80 min gradient of 0– ly, for 24 h. Each layer was then concentrated using a 100% methanol in water, flow rate, 1 ml/min, detector: rotary evaporator. The ethyl acetate fraction exhibited PDA; injection volume, 20 ml). We noted a single significant activity. It was evaporated to dryness (3.5 g) significant peak at 39 min. The data were collected over under reduced pressure, then dissolved in chloroform a range of 200–800 nm, and detection was conducted at (3 ml). The sample was added to a chromatographic 248 nm (Millennium Manager System Ver. 2.15; Wa- open column (3:0 40 cm) packed with silica gel ters, Milford, MA, U.S.A.). The structure of the resulting suspended in chloroform. The column was then washed compound was then analyzed by means of an Avance with 200 ml of chloroform, and eluted with 100 ml of DRX-600 1H-NMR (Bruker, Billerica, MA, U.S.A.) for chloroform–methanol mixtures in the following ratios: chemical shift and electron impact mass spectrometry [100:0 (1–3), 90:10 (4–6), 80:20 (7–9), 70:30 (10–12), (EI-MS: Hewlett-Packard 5890-JMS AX505WA, Palo 60:40 (13–15), 50:50 (16–18), 40:60 (19–21), 30:70 Alto, CA).

Cell culture. MC-IXC cells were cultured and main- Table 1. Eﬀects of Extracts from Various Edible Plants on ChAT tained according to the previously described methods.6) Activity The cells, which were obtained from a human neuro- Edible tea plants used Activation eﬀect blastoma, were grown in Minimum Essential Medium for screening on ChAT activity (%) (MEM) containing 15% fetal bovine serum (FBS). The

1 a MC-IXC cells were grown in the presence of penicillin Pueraria thunbergiana 46.1 Pueraria thunbergiana1-1 52.0a (100 U/ml) and streptomycin (0.1 mg/ml) at 37 C, in an Citrus junos1 25.4a atmosphere containing 5% CO2. They were passaged Fiatoua villosa1 30.1a when the culture had achieved 80–90% conﬂuence. The

1Extracts with methanol. 1-1Extracts with ethyl acetate. cells were dislodged from the surfaces of the culture ChAT activity (%): The percentage of enzyme activity values for each of dishes (100 mm) and then dispersed into a single-cell the samples was calculated in comparison with the control activity. The final suspension by repeated and forceful trituration of the concentration of each sample in the assay mixture was 500 mg/ml. aData were significantly different, with P values of < 0:05, when compared culture medium directly onto them. They were then with those of the control. subcultured once per week, at a split ratio of 1:3. Daidzein Activates Choline Acetyltransferase 109 Choline acetyltransferase activity measurement. Statistical analysis. All data were expressed as Whole cellular extracts were constructed from the MC- mean SD. Statistical analysis was conducted using IXC cells. The ChAT activity in the MC-IXC cells was Student’s t tests, with Sigma Plot software (SPSS, measured by a modification of the method described by Chicago, IL, U.S.A.). Statistical comparisons within the Fonnum.7) In the preparation of the enzyme source, the same group were performed for paired observations. A P MC-IXC cells were homogenized using a Glass-Col value of < 0:05 was considered to be significant. The homogenizer (Terre Haute, IN, U.S.A.) with 5 volumes data obtained in the in vivo test were analyzed using of homogenizing buffer [20 mM Tris–HCl (pH 7.2), con- Duncan’s multiple range tests in SAS (Cary, NC, taining 150 mM NaCl, 10 mM MgCl2, and 0.5% Triton X- U.S.A.). The statistical significance of differences 100],6) and centrifuged for 30 min at 10;000 g. The result- between groups was evaluated via one-way ANOVA. ing supernatant was then utilized as an enzyme source. All extraction steps were conducted at 4 C. Protein con- Results and Discussion centrations were measured with a BCA kit (Bicincho- ninic acid; Sigma, St. Louis, MO, U.S.A.), using bovine Alzheimer’s disease has been associated with a loss of serum albumin (BSA) as the protein standard. The cholinergic neurons within the basal forebrain. Research enzymatic reaction contained 0.5% Triton X-100, 200 into the cholinergic system’s role in cognitive function mM NaCl, 1 mM EDTA, 8 mM choline chloride, 0.1 mM has, as a result of this, been greatly expanded in recent eserine, and [14C] acetyl-CoA 5 mCi. The ACh formed as years.11–14) The most consistent biochemical finding a result of this was then extracted with sodium tetraphen- associated with cases of AD is a decrease in the levels of ylborate in the assay. Radioactivity was evaluated with ACh in the cholinergic neurons in the basal forebrain, a Beckman scintillation counter (Fullerton, CA, U.S.A.). coupled with profound neuronal degeneration.2) In an attempt to locate a synthesis enzyme of Ach that Y-maze task. Male ICR mice (Samtaco BioKorea, functions as a ChAT activator, we screened a total of 90 Seoul, Korea), weighing 25–30 g, were used in passive Korean traditional tea plants (data not shown). Among avoidance and Y-maze tests after a one-week adaptation the screened plants, the methanol extracts of ground period (20–23 C; 12 h light cycle from 09:00 to 21:00; Pueraria thunbergiana, which exhibited an activation food, Agribrand Purina Korea, and water ad libitum). effect on ChAT generated in MC-IXC cells, was The experimental procedures were approved by the concentrated to dryness and dissolved in water, then guidelines established by the Animal Care and Use successively partitioned with hexane, chloroform, and Committee at Korea University. The mice were permit- ethyl acetate. Of these fractions, the ethyl acetate ted free access to normal feed, or feed mixed with fraction was determined to exert the most profound daidzein, for 4 weeks. Scopolamine, a muscarinic activation effect on ChAT activity (Table 1). This antagonist, impairs both learning and memory in rodents fraction was then chromatographed on the first and and humans, and particularly affects the processes of second silica gel columns. The fractions obtained from learning and short-term memory.8,9) Hence it has been the second silica gel column were then purified by used in many previous studies in models for the HPLC. The purified compound was identified using EI- screening of anti-amnesic drugs. In this study, amnesia MS and 1H-NMR spectra, and was directly compared was induced in mice via subcutaneous administrations of with an authentic sample. We ultimately concluded that scopolamine (1 mg/kg body weight). Behavioral tests the ChAT activator was daidzein, (40,7-dihydroxy iso- were initiated 30 min after injection. flavone) which is structurally an isoflavone. Recording of spontaneous alternation behavior in a Y- Intraperitoneal injection of scopolamine hydrobro- maze was used to assess immediate working memory mide, a muscarinic cholinergic receptor blocker, has performance.6,10) The Y-maze task was conducted after been shown to result in memory deficits and a decrease 4 weeks of daidzein administration. The maze used in in cholinergic activity and hence in behavioral perform- this study was constructed of black-painted wood, and ance.15) The injection method of scopolamine admin- each of the arms of the maze was 25 cm long, 14 cm istration is valid for the construction of an in vivo AD high, and 5 cm wide. The arms were positioned at equal model. In this experiment, the mice were treated with angles to one another. The mice were positioned at the purified daidzein in drinking water, at various concen- end of one arm and allowed to move freely through the trations, for up to 4 weeks prior to scopolamine maze for 8-min sessions. The sequence of entries by the administration. The average water intake per mouse mice into each arm was visually recorded, and arm entry per d was approximately 8–10 ml. Therefore, as the was considered to be complete when the hind paws of drinking water contained between 0.1–0.45 mg/100 ml the mouse had all entered completely into a given arm. daidzein, the amount of daidzein consumed by the mice Alternation was defined as successive entry into the ranged between 1.5 and 4.5 mg/kg. All mice treated three arms, on overlapping triplet sets. The percentage with daidzein gained body weight in a normal fashion alternation was calculated as the ratio of actual to (39:3 1:2 g for control mice vs. 37:8 1:4 g for mice possible alternations (the total number of arm entries treated with daidzein for 4 weeks), and showed no signs minus two), multiplied by 100. of toxicity throughout the experiment (data not shown). 110 H. J. HEO et al. Table 2. Effect of Pueraria thunbergiana on Spontaneous Alterna- Previous neuroendocrine studies have determined that tion Behavior in Scopolamine (SCOP)-Treated Mice gonadal hormones affect the activities of both ChAT and Experimental Alternation Number of acetylcholinesterase (AChE) in the horizontal/vertical group behavior (%) arm entries nuclei of the diagonal bands, and in the medial preoptic 26,27) Control 66:78 5:66a 38:6 2:3 nucleus. The amygdala also exhibits increases in 28) SCOP1 42:11 1:17c 34:6 3:0 ChAT activity after administration of estrogen. In- Daidzein (1.5 mg/kg) + creased ChAT levels have been recorded in the medial 59:04 4:98ab 32:6 4:1 SCOP1 septum of the horizontal nucleus of the diagonal band 24 Daidzein (3.0 mg/kg) + 54:99 9:16b 35:6 2:9 and 72 h after administration of estrogen.27) AD has been SCOP1 Daidzein (4.5 mg/kg) + associated with a loss of cholinergic neurons within the 53:28 7:84b 34:2 3:1 SCOP1 basal forebrain. It remains unclear, however, whether the improvements noted were the result of a direct action After scopolamine (SCOP) injection, each of the behavioral tests was conducted. Spontaneous alternation behavior and the number of arm entries of daidzein at cholinergic or other sites, or of some sort were both recorded in the Y-maze test, which was conducted in 8-min of combined action occurring at several sites. Although sessions. Duncan’s multiple range tests, in SAS, revealed significant daidzein has been clearly shown in previous studies to differences (P < 0:01). The data are expressed as means SD mean (n ¼ 10). The control mice were injected with 0.85% (w/v) saline (100 ml). activate ChAT, the mechanism by which it exerts its SCOP1: SCOP (1 mg/kg) treated mice with water. P < 0:05 vs. saline- neuroprotective action remains obscure. The precise treated control. P < 0:01 vs. saline-treated control. molecular mechanisms by which it manifests its influ- ence on ChAT activation, as discovered in this study, We then evaluated the effects of daidzein on learning remain largely unclear, but, the information uncovered and memory in vivo, using a scopolamine (SCOP)- in this study might provide a basis for further detailed induced amnesia model.16) Spontaneous alternation studies of these mechanisms. behavior, which is considered a measure of spatial In conclusion, our results indicate that the daidzein of memory,10) was assessed using the Y-maze test. Mice Pueraria thunbergiana might perform a ChAT-activa- injected with SCOP exhibited significantly impaired tion function in the biosynthesis of ACh, and also that spatial working memory (37% decreases in alternation this compound clearly causes improvements in both behavior). Pretreatment with daidzein attenuated the memory and cognition after the induction of scopol- SCOP-induced decrease in alternation behavior, and a amine-induced deficits. Therefore, we suggest that the protective effect was clearly observed in mice that had daidzein contained in Pueraria thunbergiana might been pretreated with daidzein for 4 weeks (a 12–21% prove immensely useful in attempts to ameliorate or decreases in alternation behavior) (Table 2). By way of prevent neurodegenerative diseases, most notably AD. contrast, the numbers of arm entries were similar among all the experimental groups, and general locomotor Acknowledgment activity did not appear to have been affected by the SCOP treatment (Table 2). This work was supported by the Technology Devel- In this study, we have determined that daidzein (40,7- opment Program of the Ministry of Agriculture and dihydroxy isoflavone) is a natural isoflavone found in Forestry of the Republic of Korea. 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