Catechin and epicatechin from Smilacis chinae rhizome protect cultured rat cortical neurons against amyloid β protein (25–35)-induced neurotoxicity through inhibition of cytosolic calcium elevation

Life Sciences 79 (2006) 2251 – 2259 www.elsevier.com/locate/lifescie

Catechin and epicatechin from Smilacis chinae rhizome protect cultured rat cortical neurons against amyloid β protein (25–35)-induced neurotoxicity through inhibition of cytosolic calcium elevation Ju Yeon Ban a,1 , So-Young Jeon b,1 , KiWhan Bae c , Kyung-Sik Song b , Yeon Hee Seong a,⁎ a

College of Veterinary Medicine and Research Institute of Herbal Medicine, Chungbuk National University, Cheongju, Chungbuk 361-763, South Korea b College of Agriculture and Life-Sciences, Kyungpook National University, Daegu, 702-701, South Korea c College of Pharmacy, Chungnam National University, Taejon, 305-764, South Korea Received 14 April 2006; accepted 24 July 2006

Abstract We previously reported that the Smilacis chinae rhizome inhibits amyloid β protein (25–35) (Aβ (25–35))-induced neurotoxicity in cultured rat cortical neurons. Here, we isolated catechin and epicatechin from S. chinae rhizome and also studied their neuroprotective effects on Aβ (25–35)induced neurotoxicity in cultured rat cortical neurons. Catechin and epicatechin inhibited 10 μM Aβ (25–35)-induced neuronal cell death at a concentration of 10 μM, which was measured by a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay and Hoechst 33342 staining. Catechin and epicatechin inhibited 10 μM Aβ (25–35)-induced elevation of cytosolic calcium concentration ([Ca2+]c), which was measured by a fluorescent dye, Fluo-4 AM. Catechin and epicatechin also inhibited glutamate release into medium induced by 10 μM Aβ (25–35), which was measured by HPLC, generation of reactive oxygen species (ROS) and activation of caspase-3. These results suggest that catechin and epicatechin prevent Aβ (25–35)-induced neuronal cell damage by interfering with the increase of [Ca2+]c, and then by inhibiting glutamate release, generation of ROS and caspase-3 activity. Furthermore, these effects of catechin and epicatechin may be associated with the neuroprotective effect of the S. chinae rhizome. © 2006 Elsevier Inc. All rights reserved. Keywords: Catechin; Epicatechin; Smilax chinae rhizome; Neuroprotection; Amyloid β protein; Cortical neurons

Introduction Alzheimer's disease (AD) is characterized by neuronal loss and extracellular senile plaque, whose major constituent is βamyloid protein (Aβ), a 39–43 amino acid peptide derived from amyloid precursor protein (Ivins et al., 1999). Both in vitro (Iversen et al., 1995) and in vivo (Chen et al., 1994) studies have reported the toxic effects of Aβ or Aβ peptide fragments suggesting an important role for Aβ in the pathogenesis of AD. In cultures, Aβ can directly induce neuronal cell death (Ueda et al., 1994) and can render neurons vulnerable to excitotoxicity (Koh et al., 1990) and oxidative insults (Goodman and Mattson, 1994). The mechanisms underlying Aβ-neurotoxicity are ⁎ Correspoding author. Tel.: +82 43 261 2968; fax: +82 43 267 3150. E-mail address: [email protected] (Y.H. Seong). 1 Both authors contributed equally to this study. 0024-3205/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2006.07.021

complex but may involve N-methyl-D-aspartate (NMDA) receptor, a glutamate receptor subtype, modulation induced by glutamate release, sustained elevations of intracellular Ca2+ concentration ([Ca2+]i), and oxidative stresses (Forloni, 1993; Gray and Patel, 1995; Ueda et al., 1997; Ekinci et al., 2000). NMDA receptor acts either as a selective substrate of Aβ binding or as a mediator of Aβ-triggered glutamate excitotocixity (Harkany et al., 1999). NMDA receptor is a ligand-gated/ voltage-sensitive cation channel, especially highly permeable to Ca2+. Extensive elevation of the [Ca2+]i may lead directly to cellular dysfunction, overexcitation or death (Horn et al., 1999). Therefore, Ca2+ influx through NMDA receptor activation by Aβ exposure may be a critical role in Aβ-induced neurotoxicity, as proved by a report that the neurotoxic effect of Aβ was reduced by an NMDA receptor antagonist, (5R,10S)-(+)-5Methyl-10,11-dihydro-5H-dibenzo [a,d] cyclohepten-5,10imine (MK-801) (Tibor et al., 1999). However, accumulating

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evidence suggests that oxidative stress is involved in the mechanism of Aβ-induced neurotoxicity. Neurotoxic effects of Aβ are at least in part mediated by free radicals and some antioxidants such as vitamin E and melatonin have been proved to rescue cells from Aβ-mediated apoptosis (Pike et al., 1997; Huang et al., 1999; Pereira and Oliveira, 2000; Miranda et al., 2000). Catechins, which comprise epigallocatechin-3-gallate (EGCG), epicatechin, epigallocatechin (EGC), epicatechin-3gallate (ECG), and catechin, are members of the flavonoid family of natural plant polyphenols. Catechins have attracted significant attention recently, because of their potential neuroprotective activities with powerful antioxidant properties. Catechins, primarily EGCG known as the major polyphenol component of green tea, have been regarded as a good candidate for the prevention of aging-related neurodegenerative diseases associated with reactive oxygen species (ROS) (Komatsu and Hiramatsu, 2000; Levites et al., 2001; Lee et al., 2004; Zaveri, 2006). Several studies in animals and cell culture models suggest that catechins may affect several potential targets associated with AD progression (Mandel et al., 2004; Marambaud et al., 2005; Rezai-Zadeh et al., 2005). It has been also reported that catechins prevent Aβ-induced cellular damage in cultured neuronal cells (Choi et al., 2001; Conte et al., 2003; Heo and Lee, 2005). However, these reports have focused on the antioxidant effect of catechins, as a major contributable property for the neuroprotection. In a recent study, we reported that methanol extract of Smilacis chinae rhizome protected Aβ (25–35)induced neuronal cell damage in cultured rat cortical neurons (Ban et al., 2006). We isolated various active components including 3,4-dihydroxybenzoic acid, oxyresveratrol, catechin and epicatechin, to which S. chinae rhizome-induced neuroprotection might be attributable. Among them, catechin and epicatechin showed the most potent protective effect against Aβ (25–35)-induced neurotoxicity in cultured rat cortical neurons. Here we show that catechin and epicatechin from S. chinae rhizome reduce Aβ (25–35)-induced neuronal damage mainly through the inhibition of cytosolic Ca2+([Ca2+]c) elevation, followed by inhibition of ROS generation, glutamate release and then apoptosis in primarily cultured rat cortical neurons. Materials and methods Plant material, extraction, isolation and instrumental analyses Plant material S. chinae rhizome was purchased at an herbal medicine market in Daegu, Korea and identified by Dr. Jong-Hwan Kwak at Sungkyunkwan University, Suwon, Korea. A voucher specimen (No. SC-0401) has been deposited at The Innovative Research Laboratory of Natural Products Medicine, Kyungpook National University, Daegu, Korea. Extraction and isolation The dried S. chinae rhizome (1.8 kg) was refluxed in MeOH (2000 ml × 3) and the extract was evaporated to dryness. The MeOH extract (100.0 g) was suspended in water and the sus-

pension was consecutively partitioned with methylene chloride (CH2Cl2, 700 ml × 3) and ethyl acetate (EtOAc, 700 ml × 3). The EtOAc soluble fraction (6.9 g) was chromatographed on a Sephadex LH-20 column (4.0 × 68.0 cm, stepwise gradient of 50 to 100% MeOH) to yield nineteen fractions (Fr. 1–19). HPLC (μ-Bondapak C18, 7.8 × 300 mm, Waters, 10–100% MeOH for compound 1 and 1% HOAc in 18% MeOH for compound 2, 1.5 ml min− 1) of Fr. 3 afforded 10.5 mg of compound 1 and 16.1 mg of compound 2, respectively. Compound 1 Catechin: 1H NMR (400 MHz, CD3OD): δ = 6.83 (1H, s, H2′), 6.76 (1H, d, J = 8.0 Hz, H-6′), 6.71 (1H, d, J = 8.0 Hz, H-5′), 5.92 (1H, d, J = 1.4 Hz, H-8), 5.85 (1H, d, J = 1.4 Hz, H-6), 4.56 (1H, d, J = 7.4 Hz, H-2), 3.97 (1H, m, H-3), 2.84 (1H, dd, J = 16.1 and 5.4 Hz H-4a), 2.50 (1H, d, J = 16.1 and 8.1 Hz, H-4b); 13C NMR (100 MHz, CD3OD): δ = 158.2 (C-9), 158.0 (C-7), 157.3 (C-5), 146.6 (C-3′, 4′), 132.6 (C-1′), 120.4 (C-6′), 116.5 (C-5′), 115.6 (C-2′), 101.2 (C-10), 96.7 (C-6), 95.9 (C-8), 83.3 (C-2), 69.2 (C-3), 28.1 (C-4). Compound 2 Epicatechin: 1H NMR (400 MHz, CD3OD): δ = 6.97 (1H, s, H-2′), 6.79 (1H, d, J = 8.1 Hz, H-5′), 6.75 (1H, d, J = 8.1 Hz, H6′), 5.94 (1H, d, J = 1.3 Hz, H-8), 5.91 (1H, d, J = 1.3 Hz, H-6), 4.81 (1H, s, H-2), 4.17 (1H, brs, H-3), 2.86 (1H, dd, J = 16.7 and 4.1 Hz, H-4a), 2.73 (1H, d, J = 16.7 Hz, H-4b); 13C NMR (100 MHz, CD3OD): δ = 158.4 (C-9), 158.1 (C-7), 157.8 (C-5), 146.3 (C-3′), 146.2 (C-4′), 132.7 (C-1′), 119.8 (C-6′), 116.3 (C5′), 115.7 (C-2′), 100.5 (C-10), 96.8 (C-6), 96.3 (C-8), 80.3 (C2), 67.9 (C-3), 29.7 (C-4). (Tripetch et al., 2002). Chemicals (reagents), physiological solution and treatment Aβ (25–35) was purchased from Bachem (Bubendorf, Switzerland). 2-Mercaptoethanol, 3-[4,5-dimethylthiazol-2-yl]-2,5diphenyl-tetrazolium bromide (MTT), o-phthaldialdehyde (OPA), trypsin (from bovine pancreas), Dulbecco's modified Eagle's medium (DMEM), Joklik-modified MEM, poly-L-lysine and amino acids for HPLC standard were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Hoechst 33342 dye, fluo4 AM and 2′,7′-dichlorodihydrofluorescin diacetate (H2DCFDA) were purchased from Molecular Probes Inc. (Eugene, OR, USA). Fetal bovine serum was purchased from JRH Biosciences (Lenexa, KS, USA). PRO-PREP protein extraction solution was purchased from iNtRON Biothechnology Inc. (Seoul, Korea). Western Lightening™ chemiluminescence reagent was purchased from Perkin Elmer Life Sciences Inc. (Boston, MA, USA). Anti-caspase 3 (rabbit polyclonal IgG) and horseradish peroxidase conjugated anti-rabbit IgG were purchased from Upstate Biotechnology (Lake Placid, NY, USA). All other chemicals used were of the highest grade available. Aβ (25–35) stock solution of 2 mM was prepared in sterile distilled water, stored at − 20 °C, and incubated for more than 2 days at 37 °C to aggregate before use. Catechin and epicatechin were dissolved in methanol with the concentration of 50 mM and further diluted with experimental buffers. The final

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concentration of methanol was 0.02%, which did not affect cell viability. Catechin and epicatechin or their vehicle were applied to cells 15 min before the treatment with Aβ (25–35), and were also present in the medium during the incubation period with Aβ (25–35). For some experiments, a HEPES-buffered solution (incubation buffer) containing 8.6 mM HEPES, 154 mM NaCl, 5.6 mM KCl and 2.3 mM CaCl2 at pH 7.4 was used. Experimental animals Specific pathogen-free pregnant Sprague–Dawley (SD) rats (Daehan Biolink Co. Ltd., Chungbuk, Korea) were housed in an environmentally controlled room with temperature of 23 ± 2 °C, relative humidity of 55 ± 5%, and a 12-h light/dark cycle, and food and water were available ad libitum. The procedures involving experimental animals comply with the regulations of the animal ethnical committee of Chungbuk National University for the care and use of laboratory animals. Primary culture of cerebral cortical neurons Primary cortical neuronal cultures were prepared using SD rat fetuses on embryonic days 15 to 16, as described previously (Ban and Seong, 2005). Briefly, fetuses were isolated from a dam anaesthetized with ether. Cortical hemispheres were dissected under sterile conditions and placed into Joklik-modified Eagle's medium containing trypsin (0.25 mg/ml). After slight trituration through a 5-ml pipette five to six times, the cells were incubated for 10 min at 37 °C. Dissociated cells were collected by centrifugation (1500 rpm, 5 min) and resuspended in DMEM supplemented with sodium pyruvate (0.9 mM), L-glutamine (3.64 mM), sodium bicarbonate (44 mM), glucose (22.73 mM), penicillin (40 U/ml), gentamicin (50 μg/ml), KCl (5 mM) and 10% fetal bovine serum at a density of about 2 × 106 cells/ml. Cells were plated onto poly-L-lysine coated 12 well-plates (Corning 3512, NY, USA) for the measurements of cell death and glutamate release, coverslips (Fisher Scientific 12CIR, Pittsburgh, PA, USA) for the measurements of [Ca2+]c, ROS and apoptosis, and 30 mm culture dishes for the measurement of caspase-3 activation. After 2 days incubation, the medium was replaced with a new growth medium in which the concentrations of fetal bovine serum and KCl were changed to 5% and 15 mM, respectively. Cultures were kept at 37 °C in a 5% CO2 atmosphere, changing the medium twice a week. Neurotoxicity experiments were performed on neurons grown for 5–7 days in vitro. Immunochemical staining with anti-microtubule associated protein-2 (MAP-2) antibody and anti-glial fibrillary acidic protein (GFAP) antibody revealed that the culture method used in this study provided cell cultures containing about 90% neurons. Analysis of neuronal viability MTT colorimetric assay This method is based on the reduction of the tetrazolium salt MTT into a crystalline blue formazan product by the cellular oxidoreductase (Lee et al., 2005). Therefore, the amount of

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formazan produced is proportional to the number of viable cells. The culture medium was removed and replaced with serum-free growth medium. Cells were then incubated for 20 min in the medium, and incubated for a further 24 h in the presence of 10 μM Aβ (25–35) at 37 °C to produce neurotoxicity. After completion of incubation with 10 μM Aβ (25–35), the culture medium was replaced by a solution of MTT (0.5 mg/ml) in serum-free growth medium. After a 4 h incubation at 37 °C, this solution was removed, and the resulting blue formazan was solubilized in 0.4 ml of acid-isopropanol (0.04 N HCl in isopropanol), and the optical density was read at 570 nm using microplate reader (Bio-Tek ELX808, Vermont, USA). Serumfree growth medium was used as blank solution. Measurement of apoptotic neuronal death The bis-benzimidazole dye, Hoechst 33342, penetrates the plasma membrane and stains DNA in cells without permeabilization (Ishikawa et al., 1999). In contrast to normal cells, the nuclei of apoptotic cells have highly condensed chromatin that is uniformly stained by Hoechst 33342. These morphological changes in the nuclei of apoptotic cells may be visualized by fluorescence microscopy. Exposed to 10 μM Aβ (25–35) in serum-free growth medium for 24 h as described in MTT assay, neurons on coverslips were fixed in 4% paraformaldehyde at room temperature for 20 min, and then stained with Hoechst 33342 dye at the concentration of 1 μg/ml in the incubation buffer for 15 min. The morphological change was examined under UV illumination using a fluorescence microscope (Olympus IX70-FL, Tokyo, Japan). The dye was excited at 340 nm, and emission was filtered with a 510 nm barrier filter. To quantify the apoptotic process, neurons with fragmented or condensed DNA and normal DNA were counted. Data was shown as apoptotic neurons as a percentage of total neurons. Measurement of [Ca2+ ]c Neurons grown on coverslips were loaded with 3 μM fluo4 AM (dissolved in dimethylsulfoxide (DMSO)) in serum-free growth medium for 45 min at 37 °C in the CO2 incubator, and washed with the incubation buffer. The coverslips containing fluo-4 AM labeled neurons were mounted on a perfusion chamber containing incubation buffer, subjected to a laser scanning confocal microscope (Carl Zeiss LSM 510, Oberkochen, Germany), and then scanned every 3 s with a 488 nm excitation argon laser and a 515 nm longpass emission filter. After the baseline of [Ca2+]c was observed for 30 s, 10 μM Aβ (25–35) was added to the perfusion chamber for the measurement of [Ca2+]c change. In order to test the effects of catechin and epicatechin on Aβ (25–35)induced [Ca2+]c change, neurons were pretreated with the compounds 15 min before the treatment with Aβ (25–35) after being loaded with fluo-4 AM and washed. The compounds were also present in the perfusion chamber during the [Ca2+]c measurement period. All images, about 100 images from the scanning, were processed to analyze changes of [Ca2+]c in a single cell level. The results were expressed as the relative fluorescence intensity (RFI) (Lee et al., 1998).

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Measurement of glutamate concentration

Results

After being washed and equilibrated for 20 min with the incubation buffer, neurons were incubated with the buffer containing 10 μM Aβ (25–35) for 6 h at 37 °C. At the end of the incubation, glutamate secreted into the medium from the treated cells was quantified by high performance liquid chromatography (HPLC) with an electrochemical detector (ECD) (BAS MF series, IN, USA) (Ban and Seong, 2005). Briefly, after a small aliquot was collected from the culture wells, glutamate was separated on an analytical column (ODS2; particle size, 5 μm; 4.6× 100 mm) after pre-derivatization with OPA/2-mercaptoethanol. Derivatives were detected by electrochemistry at 0.1 μA/V, and the reference electrode was set at 0.7 V. The column was eluted with a mobile phase (pH 5.20) containing 0.1 M sodium phosphate buffer with 37% (v/v) HPLC-grade methanol at a flow rate of 0.5 ml/min.

Catechin and epicatechin protect neurons against cell death induced by Aβ (25–35)

Measurement of ROS generation The microfluorescence assay of 2′,7′-dichlorofluorescin (DCF), the fluorescent product of H2DCF-DA, was used to monitor the generation of ROS. Neurons grown on coverslips were washed with phenol red-free DMEM 3 times and incubated with the buffer containing 10 μM Aβ (25–35) at 37 °C for 24 h. The uptake of H2DCF-DA (final concentration, 5 μM) dissolved in DMSO was carried out for the last 10 min of the incubation with 10 μM Aβ (25–35). After being washed, coverslips containing cortical neurons loaded with H2DCF-DA were mounted on the confocal microscope stage, and the neurons were observed by a laser scanning confocal microscope (Bio-Rad, MRC1024ES, Maylands, UK) using 488 nm excitation and 510 nm emission filters. The average pixel intensity of fluorescence was measured within each cell in the field and expressed in the relative units of DCF fluorescence. Values for the average staining intensity per cell were obtained using the image analyzing software supplied by the manufacturer. Challenge of H2DCF-DA and measurement of fluorescence intensity was performed in the dark.

To assess Aβ (25–35)-induced neuronal cell death, the MTT assay was performed. In previous experiments (Ban and Seong, 2005), we have demonstrated that Aβ (25–35) over the concentration range of 5–20 μM produced a concentration-dependent reduction of cell viability in cultured cortical neurons. Therefore, the concentration of 10 μM was used for the determination of Aβ (25–35)-induced neuronal cell damage in the present experiments. Furthermore, the toxicity of 10 μM Aβ (25–35) was very similar to that of 5 μM Aβ (1–42) on cultured cortical neurons. Fig. 1 shows the inhibitory effect of catechin and epicatechin on a 10 μM Aβ (25–35)-induced decrease of MTT reduction. MTT reduction rate decreased to 72.4 ± 2.5% when using 10 μM Aβ (25–35). Catechin (1 and 10 μM) and epicatechin (1 and 10 μM) concentrationdependently reduced Aβ (25–35)-induced decrease of MTT reduction showing 92.1± 3.0% and 106.7 ± 2.4%, respectively, with 10 μM. An additional experiment was performed with Hoechst 33342 staining to assess the neurotoxicity of Aβ (25–35). Cell nuclei stained by Hoechst 33342 enables the occurrence of DNA condensate to be detected, a feature of apoptosis. In neurons treated with 10 μM Aβ (25–35), chromatin condensation and nuclear fragmentation were observed, whereas the control culture had round blue nuclei of viable neurons (Fig. 2A). As shown in Fig. 2B, the proportion of apoptotic neurons was calculated. The treatment of neurons with 10 μM Aβ (25–35) produced apoptosis of 31.6 ± 2.0% of the total population of cultured cortical neurons, as compared with 10.6 ± 1.1% of apoptotic neurons in control cultures. On the other hand, the addition of catechin and epicatechin significantly decreased Aβ

Western blot analysis of caspase-3 Incubated as described in MTT assay, neurons on dishes were washed with PBS and lysed with Proprep buffer. The amount of protein was measured by Lowry's et al. (1951) method. Proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (12% acrylamide) and transferred to nitrocellulose membrane. Transferred to membrane, caspase-3 was detected with a rabbit anti-caspase-3 antiserum. The antigen antibody reaction was visualized by using a secondary antibody conjugated with horseradish peroxidase and enhanced chemiluminescence detection reagents. Statistical analysis Data were expressed as the mean ± SEM and statistical significance was assessed by one-way analysis of variance (ANOVA) with subsequent Turkey's tests. P values of b 0.05 were considered to be significant.

Fig. 1. Inhibitory effects of catechin and epicatechin on Aβ (25–35)-induced cell death in cultured cortical neurons. Neuronal death was measured by the MTT assay. The absorbance of non-treated cells was regarded as 100%. Results are expressed as the mean ± SEM values of the data obtained from at least three independent experiments performed in two to five wells. ## p b 0.01 compared to the control. ⁎⁎ p b 0.01 compared to 10 μM Aβ (25–35).

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Fig. 2. Inhibitory effects of catechin and epicatechin on Aβ (25–35)-induced apoptosis of cultured cortical neurons as measured by Hoechst 33342 staining. Apoptotic cells were counted from 5 to 6 fields per well. (A) Representative photomicrographs of cultured neurons showing Aβ (25–35)-induced apoptosis. The arrows indicate fluorescence typical for apoptotic nuclei. Catechin and epicatechin were treated with the concentration of 10 μM. (B) Results are shown as apoptotic cells as a percentage of total number of cells and expressed as the mean ± SEM values of the data obtained from at least three independent experiments performed in two to five wells. ## p b 0.01 compared to the control. ⁎⁎ p b 0.01 compared to 10 μM Aβ (25–35).

(25–35)-induced apoptotic cell death, showing 12.5 ± 1.0% and 13.3 ± 0.6%, respectively, at the concentration of 10 μM. Catechin and epicatechin did not affect cell viability (data not shown). Catechin and epicatechin inhibit Aβ (25–35)-induced elevation of [Ca2+]c The increase of [Ca 2+ ]c has been postulated to be associated with Aβ-induced cell death in many studies. In cultured cortical neurons, treatment with 10 μM Aβ (25–35)

produced relatively slow and gradual increase of [Ca 2+ ]c. A maximal fluorescence intensity of about 180, compared to a base of 100, with the [Ca2+ ]c elevation was measured about 5 min after the Aβ (25–35) application. After peaking, the fluorescence level was decreased gradually. In contrast, pretreatment with catechin (10 μM) and epicatechin (10 μM) completely inhibited the elevation of [Ca2+ ]c induced by 10 μM Aβ (25–35) throughout the measurement period (Fig. 3). Catechin and epicatechin did not affect basal [Ca2+ ]c (data not shown).

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Fig. 3. Change of [Ca2+]c in response to Aβ (25–35) in the presence or absence of catechin and epicatechin in cultured cerebral cortical neurons. [Ca2+]c was monitored using a laser scanning confocal microscope. All images from the scanning were processed to analyze changes of [Ca2+]c in a single cell level. Results are expressed as the relative fluorescence intensity (RFI). Each trace is a single cell representative from at least three independent experiments.

Catechin and epicatechin inhibit Aβ (25–35)-induced elevation of glutamate release Glutamate released into the extracellular medium by the treatment with 10 μM Aβ (25–35) for 6 h was quantified. As shown in Fig. 4, 10 μM Aβ (25–35) markedly elevated the basal glutamate level from 0.43 ± 0.08 of control neurons to 1.16 ± 0.10 μM. Catechin and epicatechin concentration-dependently blocked Aβ (25–35)-induced elevation of glutamate release showing 0.44 ± 0.06 and 0.48 ± 0.02 μM, respectively, at the concentration of 10 μM.

Fig. 4. Inhibitory effects of catechin and epicatechin on Aβ (25–35)-induced glutamate release in cultured cortical neurons. The amount of released glutamate was measured by HPLC with ECD. Results are expressed as the mean ± SEM values of the data obtained in three independent experiments performed in two or three wells. ## p b 0.01 compared to the control. ⁎ p b 0.05, ⁎⁎ p b 0.01 compared to 10 μM Aβ (25–35).

Fig. 5. Inhibitory effects of catechin and epicatechin on Aβ (25–35)-induced ROS generation in cultured cortical neurons. Values represent mean ± SEM of relative fluorescence intensity obtained from four independent experiments performed in two to four wells. ## p b 0.01 compared to the control. ⁎⁎ p b 0.01 compared to 10 μM Aβ (25–35).

Catechin and epicatechin inhibit Aβ (25–35)-induced ROS generation Aβ (25–35) increased the glutamate release and the concentration of [Ca2+]c. Furthermore, the pathological condition induced by Aβ (25–35) is associated with accelerated formation of ROS. In Aβ (25–35) (10 μM)-treated cells for 24 h, the fluorescence intensity increased about 5 fold to 121.7 ± 7.1 compared to control cells of 24.5 ± 1.9. The Aβ (25–35)-induced

Fig. 6. Inhibitory effects of catechin and epicatechin on Aβ (25–35)-induced caspase-3 expression in cultured cortical neurons. Caspase-3 levels were determined by Western blotting. A representative band obtained from three similar results is shown. Quantified results of density are the mean ± SEM obtained from three independent experiments. 1, control; 2, Aβ (25–35) (10 μM); 3, Aβ (25–35) in the presence of catechin (10 μM); 4, Aβ (25–35) in the presence of epicatechin (10μM). # p b 0.05 compared to the control. ⁎ p b 0.05 compared to 10 μM Aβ (25–35).

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increase of ROS generation was significantly inhibited by catechin and epicatechin (Fig. 5). Catechin and epicatechin did not show direct reaction with H2DCF-DA to generate fluorescence (data not shown). Catechin and epicatechin inhibit Aβ (25–35)-induced caspase-3 protein activation Caspase-3, the 32 kDa protease constitutively expressed by many cell types and tissues, is implicated in apoptosis promoted by different death stimuli (Allen et al., 2001). To elucidate the apoptotic neuronal cell death, caspase-3 immunoreactivity was measured after the treatment with 10 μM Aβ (25–35). In 10 μM Aβ (25–35) treated neurons, caspase-3 activity markedly increased compared to control cultures. Both catechin and epicatechin (10 μM) significantly blocked Aβ (25–35)-induced increase of caspase-3 immunoreactivity (Fig. 6). Discussion The present study provides evidence that Aβ (25–35)induced injury to rat cortical neurons can be prevented by catechin and epicatechin derived from S. chinae rhizome. Catechin and epicatechin were able to reduce the Aβ (25–35)induced [Ca2+]c increase, glutamate release, ROS generation, and, as a result, attenuate apoptotic neuronal death in primarily cultured rat cortical neurons. Aβ-induced neurotoxicity has been attributed in various studies to Ca2+influx, generation of ROS, and activation of caspase-3 (Behl et al., 1994; Arias et al., 1995; Miranda et al., 2000; Harada and Sugimoto, 1999). Our previous studies confirmed that Aβ (25–35) caused neuronal cell death, which was blocked by treatment with MK-801, verapamil, an L-type Ca2+channel blocker, and NG-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase (NOS) inhibitor (Ban and Seong, 2005; Lee et al., 2005; Ban et al., 2006). This result implies the involvement of NMDA-glutamate receptor activation, an increase of Ca2+ influx and generation of ROS in Aβ (25–35)-induced neurotoxicity in cultured cortical neurons, as evidenced in other studies (Gray and Patel, 1995; Ueda et al., 1997; Ekinci et al., 2000). Regardless of the relative contribution of these events to Aβ (25–35)-induced neurotoxicity, the primary event following Aβ (25–35) treatment of cultured neurons has been suggested to be Ca2+ influx, apparently via L-type voltage-dependent Ca2+ channel (L-VDCC), since blockage of this channel and/or Ca2+ chelation prevents all other consequences (Ekinci et al., 1999; Ueda et al., 1997). Furthermore, Aβ (25–35)-induced elevation of [Ca2+]c and neurotoxicity were inhibited by MK-801, suggesting Ca2+ influx through NMDA receptor-coupled VDCC plays a critical role in the neurotoxicity (Tibor et al., 1999; Ban and Seong, 2005; Lee et al., 2005; Ban et al., 2006). In the present study, Aβ (25–35) elicited gradual and significant [Ca2+]c increase, which was blocked by catechin and epicatechin. Catechin and epicatechin also significantly inhibited the Aβ (25–35)-induced glutamate elevation. This result indicates that the sustained inhibition on [Ca2+]c elevation by catechin and epicatechin resulted in the decrease of the Aβ (25–35)-induced glutamate release. On the contrary, the

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inhibition on Aβ (25–35)-induced glutamate release, which, in turn, stimulates the receptors to cause Ca2+ influx, by catechin and epicatechin may lead to the inhibition on [Ca2+]c elevation, since EGCG has been reported to attenuate AMPA-induced [Ca2+]i increase (Bae et al., 2002). ROS generation undoubtedly takes place in glutamate neurotoxicity and is likely due to Ca2+ influx in the cytosol (Pereira and Oliveira, 2000). Many reports demonstrated the involvement of ROS formation in Aβ-induced neurotoxicity (Miranda et al., 2000; Cardoso et al., 2002). Catechin and epicatechin decreased the Aβ (25–35)-induced increase of ROS generation. It has been reported that vitamin-E, an antioxidant, blocked the Aβ-induced generation of ROS, but not Ca2+ influx, and reduction of extracellular Ca2+ inhibited the Aβ-induced increase in intracellular Ca2+ as well as generation of ROS, indicating that ROS generation is a consequence of Ca2+ accumulation (Ekinci et al., 2000). It was also demonstrated that the significant increase of ROS generation took more than 1 h, while the elevation of [Ca2+]c occurred within seconds after the treatment with 10 μM Aβ (25–35) (Ban and Seong, 2005). In addition, L-NAME, a NOS inhibitor, failed to inhibit the Aβ (25–35)-induced increase in [Ca2+]c in the short period of measurement in contrast to the complete inhibition of verapamil on the Aβ (25–35)-induced ROS generation in the previous data (Lee et al., 2005; Ban and Seong, 2005; Ban et al., 2006). Therefore, it is concluded that catechin and epicatechin inhibited the Aβ (25–35)-induced ROS generation via the blockade of [Ca2+]c increase. Many previous reports have also demonstrated that catechin and epicatechin, with the well-characterized EGCG, have protective effect against Aβ-induced neuronal death in cultures (Choi et al., 2001; Conte et al., 2003; Heo and Lee, 2005). It has been concluded that the neuroprotective effect of catechins is attributable to their antioxidant property. However, to our knowledge, there are no reports on the direct modulation of catechin and epicatechin on the Aβ (25–35)induced [Ca2+]c change leading to neuronal injury in cultured cortical neurons. Here we firstly show that the inhibition of [Ca2+ ]c elevation is primarily responsible for the prevention of Aβ (25–35)-induced neuronal cell damage by catechin and epicatechin. Many researchers have demonstrated that Aβ triggered apoptotic degeneration in in vitro neuronal experiment (Harkany et al., 1999; Yan et al., 1999). Cultured cortical neurons exposed to 10 μM Aβ (25–35) for more than 24 h showed increased chromatin condensation, a typical feature of apoptotic cell death in the present work. Catechin and epicatechin protected the neuronal cell against Aβ (25–35)-induced apoptotic death. Caspases are aspartate-specific cysteine proteases, which have been proposed to play a pivotal role in apoptosis (Nicholson, 1997). In the caspase family, which consists of more than 10 homologues, caspase-3 has been suggested to play an important role in Aβinduced apoptosis (Allen et al., 2001; Cardoso et al., 2002). An increase in expression of activated caspase-3 has been detected in AD brains (Su et al., 2001). The activation of caspase-3 may be a downstream event following Ca2+ influx, glutamate release and ROS generation in cortical neurons exposed to Aβ (25–35), since it was blocked by MK-801, verapamil, and L-NAME in the

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previous studies (Ban and Seong, 2005; Lee et al., 2005; Ban et al., 2006). In the present study, catechin and epicatechin also inhibited the activation of caspase-3. It is thus concluded that catechin and epicatechin may prevent the Aβ (25–35)-induced apoptosis of neuronal cell by interfering with the increase of [Ca2+ ]c, and then by inhibiting glutamate release, generation of ROS, and activation of caspase-3. Aβ is believed to play a central role in the pathophysiology of AD (Hsiao et al., 1995; Holcomb et al., 1998). Two proteolytic cleavage events are required to generate Aβ from its precursor, one at the N-terminus by an enzyme termed β-secretase and one at the C-terminus by an enzyme termed γ-secretase. Among the secretases, β-secretase is at present the most attractive target for the inhibition of Aβ production. Catechins from green tea were proposed as good candidates for β-secretase inhibitors (Jeon et al., 2003). Catechin and epicatechin completely blocked Aβ (25– 35)-induced neuronal cell death in the present study. We isolated catechin and epicatechin from the methanolic extract of S. chinae rhizome which was proved to markedly reduce Aβ (25–35)induced neuronal damage in cultured cortical neurons (Ban et al., 2006). These results suggest a further evidence of the possibility of S. chinae rhizome having neuroprotective effect in AD brains with the prevention of the disease progression. In conclusion, catechin and epicatechin could be responsible for the neuroprotective effect of S. chinae rhizome. We provide a mechanistic explanation that catechin and epicatechin protect cultured cortical neurons against Aβ toxicity by firstly interfering with the increase of [Ca2+]c. The protection against Aβ (25–35)-induced neurotoxicity by catechin and epicatechin may help to explain at least their inhibitory actions on the progression of AD, and furthermore provide the pharmacological basis of their clinical usage in treatment of neurodegeneration in AD. Acknowledgements This work was supported by a grant from the BioGreen 21 Program, Rural Development Administration, Republic of Korea and the Brain Korea 21 Project in 2006. References Allen, J.W., Eldadah, B.A., Huang, X., Knoblach, S.M., Faden, A.I., 2001. Multiple caspases are involved in β-amyloid-induced neuronal apoptosis. The Journal of Neuroscience Research 65, 45–53. Arias, C., Arrieta, I., Tapia, R., 1995. β-Amyloid peptide fragment 25–35 potentiates the calcium-dependent release of excitatory amino acids from depolarized hippocampal slices. Journal of Neuroscience Research 41, 561–566. Bae, J.H., Mun, K.C., Park, W.K., Lee, S.R., Suh, S.I., Baek, W.K., Yim, M.B., Kwon, T.K., Song, D.K., 2002. EGCG attenuates AMPA-induced intracellular calcium increase in hippocampal neurons. Biochemical and Biophysical Research Communications 290, 1506–1512. Ban, J.Y., Seong, Y.H., 2005. Blockade of 5-HT3 receptor with MDL72222 and Y25130 reduces β-amyloid protein (25–35)-induced neurotoxicity in cultured rat cortical neurons. European Journal of Pharmacology 520, 12–21. Ban, J.Y., Cho, S.O., Koh, S.B., Song, K.S., Bae, K.W., Seong, Y.H., 2006. Protection of amyloid β protein (25–35)-induced neurotoxicity by methanol extract of Smilacis chinae rhizome in cultured rat cortical neurons. Journal of Ethnopharmacology 106, 230–237. Behl, C., Davis, J.B., Lesley, R., Schubert, D., 1994. Hydrogen peroxide mediates amyloid beta protein toxicity. Cell 77, 817–827.

Cardoso, S.M., Swerdlow, R.H., Oliveira, C.R., 2002. Induction of cytochrome c-mediated apoptosis by amyloid β 25–35 requires functional mitochondria. Brain Research 931, 117–125. Chen, S.Y., Harding, J.W., Barnes, C.D., 1994. Neuropathology of synthetic βamyloid peptide analogs in vivo. Brain Research 715, 44–50. Choi, Y.T., Jung, C.H., Lee, S.R., Bae, J.H., Baek, W.K., Suh, M.H., Park, J.P., Park, C.W., Suh, S.I., 2001. The green tea polyphenol (−)-epigallocatechin gallate attenuates β-amyloid-induced neurotoxicity in cultured hippocampal neurons. Life Sciences 70, 603–614. Conte, A., Pellegrini, S., Tagliazucchi, D., 2003. Synergistic protection of PC12 cells from β-Amyloid toxicity by resveratrol and catechin. Brain Research Bulletin 62, 29–38. Ekinci, F.J., Malik, K.U., Shea, T.B., 1999. Activation of the L voltage-sensitive calcium channel by mitogen-activated protein (MAP) kinase following exposure of neuronal cells to beta-amyloid. MAP kinase mediates βamyloid-induced neurodegeneration. The Journal of Biological Chemistry 274, 30322–30327. Ekinci, F.J., Linsley, M.D., Shea, T.B., 2000. β-Amyloid-induced calcium influx induces apoptosis in culture by oxidative stress rather than tau phosphorylation. Molecular Brain Research 76, 389–395. Forloni, G., 1993. β-amyloid neurotoxicity. Functional Neurology 8, 211–225. Goodman, Y., Mattson, M.P., 1994. Secreted forms of β-amyloid precursor protein protect hippocampal neurons against amyloid β-peptide-induced oxidative injury. Experimental Neurology 128, 1–12. Gray, C.W., Patel, A.J., 1995. Neurodegeneration mediated by glutamate and beta-amyloid peptide: a comparison and possible interaction. Brain Research 691, 169–179. Harada, J., Sugimoto, M., 1999. Activation of caspase-3 in β-amyloid-induced apoptosis of cultured rat cortical neurons. Brain Research 842, 311–323. Harkany, T., Hortobagyi, T., Sasvari, M., Konya, C., Penke, B., Luiten, P.G., Nyakas, C., 1999. Neuroprotective approaches in experimental models of βamyloid neurotoxicity : relevance to Alzheimer's disease. Progress in Neuro-Psychopharmacology & Biological Psychiatry 23, 963–1008. Heo, H.J., Lee, C.Y., 2005. Epicatechin and catechin in cocoa inhibit amyloid beta protein induced apoptosis. Journal of Agricultural and Food Chemistry 53 (5), 1445–1448. Holcomb, L., Gordon, M.N., McGowan, E., Yu, X., Benkovic, S., Kantzen, P., Wright, K., Saad, I., Mueller, R., Morgan, D., Sanders, S., 1998. Accelerated Alzheimer-type phenotype in transgenic mice carrying both mutant amyloid precursor protein and presenilin 1 transgenes. Nature Medicine 4, 97–100. Horn, J., Brouwers, P.J.A.M., Limburg, M., 1999. Disturbances of calcium homeostasis in ischemic stroke: therapeutical implication. CNS Drugs 11, 373–386. Hsiao, K.H., Borchelt, D.R., Olson, K., Johannsdottir, R., Kitt, C., Yunis, W., Xu, S., Eckman, C., Younkin, S., Price, D., Ladecola, C., Clark, H.B., Carlson, G., 1995. Age-related CNS disorder and early death in transgenic FVB/N mice overexpressing Alzheimer amyloid precursor proteins. Neuron 15, 1203–1208. Huang, X., Atwood, C.S., Hartshorn, M.A., Multhaup, G., Goldstein, L.E., Scarpa, R.C., Cuajungco, M.P., Gray, D.N., Lim, J., Moir, R.D., Tanzi, R.E., Bush, A.I., 1999. The A beta peptide of Alzheimer's disease directly produces hydrogen peroxide through metal ion reduction. Biochemistry 38 (24), 7609–7616. Ishikawa, Y., Satoh, T., Enokido, Y., Nishio, C., Ikeuchi, T., Hatanaka, H., 1999. Generation of reactive oxygen species, release of L-glutamate and activation of caspase are required for oxygen-induced apoptosis of embryonic hippocampal neurons in culture. Brain Research 824, 71–80. Iversen, L.L., Mortishier-Smith, R.J., Pollack, S.J., Shearman, M.S., 1995. The toxicity in vitro of β-amyloid protein. The Biochemical Journal 311, 1–16. Ivins, K.J., Ivins, J.K., Sharp, J.P., Cotman, C.W., 1999. Multiple pathways of apoptosis in PC12 cells. CrmA inhibits apoptosis induced by beta-amyloid. The Journal of Biological Chemistry 274 (4), 2107–2112. Jeon, S.Y., Bae, K.W., Seong, Y.H., Song, K.S., 2003. Green tea catechins as a BACE1 (β-secretase) inhibitor. Bioogranic and Medicinal Chemistry Letters 13, 3905–3908. Koh, J.Y., Yang, L.L., Cotman, C.W., 1990. β-Amyloid protein increases the vulnerability of cultured neurons to excitotoxic damage. Brain Research 533, 315–320.

J.Y. Ban et al. / Life Sciences 79 (2006) 2251–2259 Komatsu, M., Hiramatsu, M., 2000. The efficacy of an antioxidant cocktail on lipid peroxide level and superoxide dismutase activity in aged rat brain and DNA damage in iron-induced epileptogenic foci. Toxicology 148, 143–148. Lee, Z.W., Kweon, S.M., Kim, B.C., Leem, S.H., Shin, I.C., Kim, J.H., Ha, K.S., 1998. Phosphatidic acid-induced elevation of intracellular Ca2+ is mediated by RhoA and H2O2 in rat-2 fibroblasts. The Journal of Biological Chemistry 273, 12710–12715. Lee, H., Bae, J.H., See, S.R., 2004. Protective effect of green tea polyphenol EGCG against neuronal damage and brain edema after unilateral cerebral ischemia in gerbils. Journal of Neuroscience Research 77 (6), 892–900. Lee, B.Y., Ban, J.Y., Seong, Y.H., 2005. Chronic stimulation of GABAA receptor with muscimol reduces amyloid is β protein (25–35)-induced neurotoxicity in cultured rat cortical cells. Neuroscience Research 52, 347–356. Levites, Y., Weinreb, O., Maor, G., Youdim, M.B., Mandel, S., 2001. Green tea polyphenol (−)-epigallocatechin-3-gallate prevents N-methl-4-phenyl1,2,3,6-tetrahydropyridine-induced dopaminergic neurodegeneration. Journal of Neurochemistry 78 (5), 1073–1082. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurements with Foin phenol reagent. Journal of Biological Chemistry 193, 265–275. Mandel, S., Weinreb, O., Amit, T., Youdim, M.B., 2004. Cell signaling pathways in the neuroprotective actions of the green tea polyphenol (−)epigallocatechin-3-gallate: implications for neurodegenerative diseases. Journal of Neurochemistry 88 (6), 1555–1569. Marambaud, P., Zhao, H., Davies, P., 2005. Resveratrol promotes clearance of Alzheimer's disease amyloid-β peptides. The Journal of Biological Chemistry 280 (45), 37377–37382. Miranda, S., Opazo, C., Larrondo, L.F., Munoz, F.J., Ruiz, F., Leighton, F., Inestrosa, N.C., 2000. The role of oxidative stress in the toxicity induced by amyloid β-peptide in Alzheimer's disease. Progress in Neurobiology 62, 633–648. Nicholson, D.W., Thornberry, N.A., 1997. Caspase: killer proteases. Trends in Biochemical Sciences 22, 299–306.

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Pereira, C.F., Oliveira, C.R., 2000. Oxidative glutamate toxicity involves mitochondrial dysfunction and perturbation of intracellular Ca2+ homeostasis. Neuroscience Research 37, 227–236. Pike, C.J., Ramezan-Arab, N., Cotman, C.W., 1997. Beta-amyloid neurotoxicity in vitro: evidence of oxidative stress but not protection by antioxidants. Journal of Neurochemistry 69, 1601–1611. Rezai-Zadeh, K., Shytle, D., Sun, N., Mori, T., Hou, H., Jeanniton, D., Ehrhart, J., Townsend, K., Zeng, J., Morgan, D., Hardy, J., Town, T., Tan, J., 2005. Green tea epigallocatechin-3-gallate (EGCG) modulates amyloid precursor protein cleavage and reduces cerebral amyloidosis in Alzheimer transgenic mice. Neurobiology of Disease 25 (38), 8807–8814. Su, J.H., Zhao, M., Anderson, A.J., Srinivasan, A., Cotman, C.W., 2001. Activated caspase-3 expression in Alzheimer's and aged control brain: correlation with Alzheimer pathology. Brain Research 898, 350–357. Tibor, H., Maria, S., Csaba, K., Botond, P., Paul, G.M., Csaba, N., 1999. Neuroprotective Approaches in Experimental Models of β-Amyloid Neurotoxicity: Relevance to Alzheimer's Disease. Tripetch, K., Kotomi, S., Ryoji, K., Kazuo, Y., Mohamed, S.K., Mahmoud, H.M., 2002. Stilbene and 2-arylbenzofuran glucosides from the rhizomes of Schoenocaulon officinale. Chemical and Pharmaceutical Bulletin 50, 863–865. Ueda, K., Fukui, Y., Kageyama, H., 1994. Amyloid β protein-induced neuronal cell death: neurotoxic properties of aggregated amyloid β protein. Brain Research 639, 240–244. Ueda, K., Shinohara, S., Yagami, T., Asakura, K., Kawasaki, K., 1997. Amyloid beta protein potentiates Ca2+ influx through L-type voltage-sensitive Ca2+ channel: a possible involvement of free radicals. Journal of Neurochemistry 68, 265–271. Yan, X.T., Qiao, J.T., Dou, Y., Qiao, Z.D., 1999. β-amyloid peptide fragment 31–35 induces apoptosis in cultured cortical neurons. Neuroscience 92, 177–184. Zaveri, N.T., 2006. Green tea and its polyphenolic catechins: medicinal uses in cancer and noncancer applications. Life Sciences 78, 2073–2080.