Neuroprotective biflavonoids of Chamaecyparis obtusa leaves against glutamate-induced oxidative stress in HT22 hippocampal cells

Food and Chemical Toxicology 64 (2014) 397–402

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Neuroprotective biflavonoids of Chamaecyparis obtusa leaves against glutamate-induced oxidative stress in HT22 hippocampal cells Eun Ju Jeong a, Lim Hwang b, Mina Lee b, Ki Yong Lee c, Mi-Jeong Ahn d, Sang Hyun Sung b,⇑ a

Department of Agronomy & Medicinal Plant Resources, College of Life Sciences and Natural Resources, Gyeongnam National University of Science and Technology, Jinju 660-758, Republic of Korea b College of Pharmacy and Research Institute of Pharmaceutical Science, Seoul National University, Seoul 151-742, Republic of Korea c College of Pharmacy, Korea University, Sejong 339-700, Republic of Korea d College of Pharmacy and Research Institute of Pharmaceutical Sciences, Gyeongsang National University, Jinju 660-751, Republic of Korea

a r t i c l e

i n f o

Article history: Received 7 October 2013 Accepted 3 December 2013 Available online 6 December 2013 Keywords: Chamaecyparis obtusa Biflavonoid Neuroprotection Oxidative stress HT22 ERK

a b s t r a c t Four biflavonoids (1–4), five flavonoids glycosides (5–9), two catechins (10, 11), two lignans (12–13), neolignan glycoside (14) and phenylpropanoid glycoside (15) were isolated from the leaves of Chamaecyparis obtusa (Cupressaceae). Neuroprotective effects of the isolated compounds were evaluated employing HT22 mouse hippocampal cells, a model system to study glutamate-induced oxidative stress. The glutamate injured HT22 cells were protected significantly by amentoflavone (3), ginkgetin (4) and ()-epitaxifolin 3-O-b-D-xylopyranoside (9). The reduced activities of antioxidant enzymes, superoxide dismutase (SOD), glutathione reductase (GR) in response to high concentration of glutamate were preserved by pre-treatment of 3, 4 or 9, while the activities of glutathione peroxidase (Gpx) and catalase (CAT) were little affected. The reduced content of GSH induced by glutamate was also recovered by 3, 4 or 9 in accommodation with the decrease in ROS production. In addition, the phosphorylation of ERK1/2 induced by glutamate insult was clearly prevented by 3, while little changed by 4. Taken together, amentoflavone (3), ginkgetin (4) and ()-epitaxifolin 3-O-b-D-xylopyranoside (9) derived from C. obtusa could protect HT22 neuronal cells against glutamate-induced oxidative damage through preserving antioxidant enzymes activities and/or inhibiting ERK1/2 activation. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Neuronal cell death is a major factor in the progress of neurological disorders including Alzheimer’s disease, Parkinson’s disease, Huntington’s disease or stroke. Selective neuronal cell death observed in these acute or chronic diseases is often caused by oxidative stress (Coyle and Puttfarcken, 1993). Glutamate is known to cause neuronal cell death via both glutamate receptor-mediated excitotoxicity and reactive oxygen species-mediated oxidative glutamate toxicity (Satoh et al., 2000a,b). HT22 cell, the immortalized mouse hippocampal cell line, which lack ionotropic glutamate receptor is excellent model to evaluate oxidative glutamate toxicity. Glutamate-induced neurotoxicity in HT22 cells is characterized as inhibition of glutamate/cysteine antiporter and decrease in intracellular level of cysteine and depletion of glutathione. Subsequent accumulation of reactive oxygen species (ROS) and rapid influx of Ca2+ contribute to neuronal cell death (Stanciu et al., 2000). During searching for natural products with neuroprotective effects using glutamate-injured HT22 hippocampal cells as an ⇑ Corresponding author. Tel.: +82 2 880 7859; fax: +82 2 877 7859. E-mail address: [email protected] (S.H. Sung). 0278-6915/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fct.2013.12.003

in vitro system deduced that the 80% methanolic extract of the leaves of Chamaecyparis obtusa significantly protected HT22 cells against glutamate-induced oxidative stress. C. obtusa (Sieb.et zucc.) Endl., commonly known as hinoki, is one of the most important Japanese coniferous species. C. obtusa belongs to the family of Cupressaceae, and plants of Cupressaceae have a rich source of sesquiterpenes, diterpenes, flavones and lignans (Xu et al., 2006; Barrero et al., 2004; Okasaka et al., 2006; Smith et al., 2007; Gadek and Quinn, 1987). Several studies on the chemical composition of C. obtusa revealed that it contains terpenoids, lignan, flavonoid, and some of them are considered to possess antitumor, antimalarial, and antibacterial activities (Hieda et al., 1996; Kuo and Chen, 2001; Kuo et al., 2001; Chien et al., 2007). Recently, the pharmacological effects of the constituent derived from C. obtusa, especially essential oil, on nervous system have been reported. Na et al. (1999) demonstrated that essential oil of C. obtusa affected central nervous system similar to the way a sedative does in experimental mice. In Alzheimer’s disease (AD) model induced by i.c.v. injection of b-amyloid peptides (Ab) into the hippocampus of rat brain, inhaled essential oil of C. obtusa suppressed AD-related neuronal cell death and the dysfunction of the memory system (Bae et al., 2012). Also, the essential oil showed both anxiolytic-like and stress

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mitigation effects via improving stress-related biomarkers (Kasuya et al., 2013). Nevertherless, scientific evidences on molecular actions underlying neuroprotective functionality and the involved bioactive constituents of this plant are still insufficient. In the present study, we attempted to investigate neuroprotective constituents of C. obtusa other than essential oil against glutamate-induced neurotoxicity in HT22 hippocampal cells. 2. Materials and methods 2.1. Plant material

of antioxidant enzyme activity and GSH contents. The activity of superoxide dismutase (SOD) was determined according to the method of McCord and Fridovich (1969) by the xanthine–xanthine oxidase reaction. Glutathione reductase (GR) activity was measured according to the method of Carlberg and Mannervik (1975) based on the reduction of GSSG by GSSG reductase in the presence of NADPH. The activity of glutathione peroxidase (Gpx) was determined by quantifying the rate of oxidation of GSH to GSSG by cumene hydroperoxide (Flohe and Gunzler, 1984). Values shown are the mean ± S.D. 2.6. Determination of glutathione content Total glutathione in the supernatant was determined spectrophotometrically using the enzymatic cycling method (Tietz, 1969). Values shown are the mean ± S.D.

Dried leaves of C. obtusa were provided by SK E&C (Korea) in Chungju, Korea, and identified by Dr. Jong Hee Park, a professor of the College of Pharmacy, Pusan National University. A voucher specimen (CS-222 and CS-223) has been deposited in the Herbarium of the Medicinal plant Garden, College of Pharmacy, Seoul National University.

Protein concentrations were determined using a bicinchoninic acid (BCA) kit (Sigma, MO, USA) with bovine serum albumin as a standard (Smith et al., 1985).

2.2. Isolation of compounds 1–15 from C. obtusa leaves

2.8. Western blot

The air-dried plant material (3.8 kg) was extracted three times with MeOH in an ultrasonic apparatus. Removal of the solvent in vacuo yielded a methanolic extract (673.0 g). The methanolic extract was then suspended in distilled water and partitioned successively with n-hexane, CHCl3, EtOAc, and n-BuOH. The EtOAc soluble fraction (108.0 g) which showed the protective effects on glutamate-injured HT22 cells was used to elucidate bioactive compounds. The EtOAc fraction was subjected to silica gel column using mixtures of CHCl3MeOH of increasing polarity as eluents to give 10 fractions (EI–X). Fractions EIV, EV, EVIII, EVIIII were further purified by recrystallization with MeOH to yield compounds 1 (51.1 mg), 2 (1 mg), 3 (12.8 mg), 4 (1.1 mg), respectively. EI was subjected to column chromatography on Sephadex LH-20 (MeOH) to give compounds 12 (2 mg) and 13 (152.7 mg). EX was subjected to silica gel column chromatography with a gradient elution of CHCl3MeOH–Water (100:4:1 ? MeOH) to give 13 subfractions (EX-1-13). Compound 8 (40.0 mg) was obtained from EX-10-1 by recrystallization with MeOH. Compound 15 (3.3 mg) was obtained from EX-10-7 by additional C18 RP HPLC (MeOHH2O 40:60, 2.0 mL/min, 254 nm). EX-11 was applied to C18 RP MPLC with a gradient elution of MeOH–Water to give 11 subfractions (EX-11–1–11). Compound 7 (70.8 mg) was obtained from EX-11-11. Compounds 9 (0.3 mg), 10 (2.0 mg), 11 (2.0 mg) were isolated form EX-12 by additional C18 RP HPLC (MeOHH2O 65:35, 2.0 mL/min, 254 nm). Compound 6 (15.4 mg) was isolated from EX-13 by recrystallization with MeOH. EX-13 was further subjected to silica gel column chromatography with a gradient elution of CHCl3MeOH (20:1 ? MeOH) to give 7 subfractions (EX-13–1–7). Compound 5 (155.5 mg) was obtained from EX13-6. Compound 14 (1.3 mg) was also obtained from EX-13-6 by additional C18 RP HPLC (MeOHH2O 50:50, 2.0 mL/min, 254 nm).

HT22 cells were plated overnight in 6-well plates at a density of 1  104 cells/ ml. The medium was changed to fresh one and treated with test samples for 30 min before exposure to 5 mM glutamate. After 15 min or 30 min incubation, cells were washed twice with cold PBS. The washed cell pellets were lysed in 200ul of extraction lysis buffer per well and incubated for 20 min at 4 °C. Cell lysates were centrifuged at 12,000 rpm for 15 min at 4 °C and the supernatants were collected. Protein content was determined using Bio-Rad protein assay reagent according to the manufacture’s instruction. Equal amounts of protein (40 lg) were loaded per lane onto 10% SDS–PAGE gel. Proteins were transferred to PVDF membranes and subsequently blocked in 5% skim milk for 30 min at room temperature. Anti-ERK1/2 and Anti-phospho-ERK1/2 (1:1000 dilution; Cell Signaling Technology) were employed in 1% skim milk. The membranes were incubated with the primary antibody overnight at 4 °C. After washing three times with TBST, the immunoreactive bands were visualized by using immunopure peroxidase conjugated goat antirabbit IgG (1:10000 dilution; Pierce, Rockford, IL, USA). Blots were washed three times and then developed by enhanced chemiluminescence (Bio-Rad Life Sciences).

2.3. HT22 cell culture and treatment HT22 hippocampal cells were seeded onto 48-well plates at a density of 1  104 cells/ml. Cells were grown in DMEM containing 10% heat-inactivated FBS with penicillin (100 IU/ml) and streptomycin (100 lg/ml) at 37 °C in a humidified atmosphere of 95% air–5% CO2. After 12–24 h incubation, cells were pretreated with the test samples 1 h before exposure to 5 mM L-glutamate. After 8 h, the cultures were assessed for neurotoxicity. Neuronal cell viability was quantified by MTT assay. All experiments were performed with Ethical Approval from the Seoul National University. 2.4. Measurement of cellular peroxide The relative level of free radicals, that is peroxide, in cultured cells was measured with the oxidation-sensitive compound, 2,7-dichlorofluoresein diacetate (DCF-DA) by the method of Goodman and Mattson (1994). HT22 hippocampal cells were seeded onto 24-well plates at a density of 1  104 cells/ml. Cells were treated with compound to be tested for 1 h followed by 5 mM glutamate treatment for 10 h. After washing with phosphate-buffered saline (PBS, pH 7.4), cells were loaded with 5 lM DCF-DA for 30 min followed by three times washes with PBS. The cells were extracted with 1% Triton X-100 in PBS for 10 min. DCF fluorescence was then determined by measuring light emitted at 530 nm of exciting cells with light at 485 nm. Values shown are the mean ± S.D. 2.5. Assay for activities of antioxidant enzymes Cells were seeded onto 6-well plates at a density of 1  104 cells/ml and incubated for 24 h. Cells were treated with compound to be tested for 1 h followed by 5 mM glutamate insult. After overnight, cells were washed two times with cold PBS and extracted with cell lysis buffer. The cell lysate was centrifuged for 15 min at 12,000 rpm at 4 °C and the supernatant was used for the measurements

2.7. Determination of protein concentrations

3. Results 3.1. Isolation of 1–15 from the leaves of C. obtusa The methanolic extract of C. obtusa leaves were suspended in water and successively partitioned with n-Hexane, CHCl3, EtOAc and n-BuOH. Each fraction was evaluated for its protective activity on glutamate-injured HT22 hippocampal cells. Total methanolic extract of C. obtuse and its fractions, the EtOAc and n-BuOH soluble fractions significantly protected HT22 cells against gluatamate-induced oxidative stress at the concentration ranging from 10 lg/ml to 100 lg/ml. Relative protection of total extract, EtOAc and n-BuOH fractions for HT22 hippocampal cells was 95.2 ± 3.1, 82.6 ± 2.9, 71.5 ± 2.3, respectively. The EtOAc soluble fraction which showed the most potent activity was further subjected to repeated column chromatography to elucidate neuroprotective constituents of C. obtusa. Fifteen compounds including four biflavonoids (1–4), five flavonoids glycosides (5–9), two catechins (10, 11), two lignans (12–13), neolignan glycoside (14) and phenylpropanoid glycoside (15) were obtained. The isolated compounds were identified as hinokiflavone (1) (Markham et al., 1987), isocryptomerin (2) (Shin and Kim, 1991), amentoflavone (3) (Shin and Kim, 1991), ginkgetin (4) (Gard and Mitra, 1971), quercetin 3-O-a-L-rhamnopyranoside (5) (Lu et al., 1997), hypolaetin 7-O-b-D-xylopyranoside (6) (Nakanishi et al., 2004), kaempferol 3-O-a-L-rhamnopyranoside (7) (Nakano et al., 1983), isoscutellarein 7-O-b-D-xylopyranoside (8) (Nakanishi et al., 2004), ()-epitaxifolin 3-O-b-D-xylopyranoside (9) (Nonaka et al., 1987), (+)-catechin (10) (Agrawal and Bansak, 1989), ()-epicatechin (11) (Agrawal and Bansak, 1989), ()-dihydrosesamin (12) (Takaku et al., 2001), ()-savinin (13) (Lee et al., 2010), icariside E4 (14) (Nakanishi et al., 2004), citrusin D (15) (Kishida and Akita, 2005), respectively, by comparison of spectroscopic data

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with the literature values. Compounds 3–9 and 13 were reported for the first time from this plant (Fig. 1).

using DCF-DA staining (Fig. 2). Intracellular ROS levels were two fold increased by the treatment with 5 mM glutamate compared to untreated control cells. The increased ROS level was effectively reduced by pretreatment of 3, 4 and 9 to 57.7, 40.2 and 63.6% of glutamate-only treated cells at the concentration of 50 lM, respectively.

3.2. Neuroprotective effects of 1–15 in glutamate-injured HT22 hippocampal cells Protective effects of compounds 1–15 against glutamate-induced neurotoxicity in HT22 hippocampal cells were evaluated (Table 1). The treatment of 5 mM glutamate for 8 h resulted in a decrease in cell viability of HT22 to 65.2 ± 0.2% over untreated control cells. Among the compounds isolated, the pretreatment of HT22 cells with compounds 3, 4 and 9 significantly protected cells against neurotoxicity induced by glutamate insult, preserving cellular viability to 73.8 ± 4.9, 64.6 ± 5.9 and 71.9 ± 2.7% of untreated control, respectively, at the concentration of 100 lM. All compounds tested showed no cytotoxicity in HT22 cells under the concentration of 100 lM.

3.4. Effects of 3, 4 and 9 on the activities of antioxidant enzymes and glutathione contents in glutamate-injured HT22 hippocampal cells To further elucidate antioxidant effects of 3, 4 and 9, the effects on the activities of antioxidant enzymes and on cellular glutathione level were determined. Glutamate insult resulted in a significant reduction in the activities of SOD, GR, Gpx, CAT, (Table 2) and the contents of GSH (Table 3) in HT22 hippocampal cells. The treatment of HT22 cells with compounds 3, 4 or 9 preserved the activities of SOD and GR. The activity of Gpx decreased by glutamate was little affected by the treatment of 3 or 4, while which was recovered by 9 with statistical significance. The activity of CAT decreased by glutamate was unaffected by compounds treatment. The reduced GSH contents reduced by glutamate were also significantly reversed by 3, 4 and 9. The restoration of GSH was most remarkable in the treatment of 4, in which the reduced GSH

3.3. Effects of 3, 4 and 9 on cellular peroxide in glutamate-injured HT22 hippocampal cells The intracellular ROS levels in HT22 cells were determined treated with or without 5 mM glutamate and compounds 3, 4 and 9 OH RO

OR

O

HO

O HO

OH

O

O HO

O

OH OH

O

1: R=H 2: R=CH3

O

OH

O

OH

3: R=H 4: R=CH3

O

R1 OH

OH

R 2O

O

5 6 7 8

R3 OH

O

R2 H O-β-D-xylose H O-β-D-xylose

R1 OH OH H H

OH HO O O

O

OH OH

HO

OH

OH

O

OH

OH

OH OH

HO

R3 O-α-L-rhamnose H O-α-L-rhamnose H

O

HO

O

OH

OH

OH

9

OH

10

11

O O

O

O

O

OH

O

O

HO O HO HO

OH

O

O

O

12

O

O H3 CO

13

H 3CO

14

HO H3 CO

OH O

O

15

O HO

OH OH OH

Fig. 1. The structures of compounds 1–15 isolated from C. obtusa leaves.

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Table 1 Protective effects of compounds 1–15 isolated from C. obtusa leaves against glutamate-induced neurotoxicity in HT22 cells. Relative protection (%)

1 2 3 4 5 6 7 8

Relative protection (%)

10 lM

50 lM

100 lM

2.1 ± 0.7 2.0 ± 0.2 0.3 ± 0.0 33.6 ± 7.7* 1.1 ± 0.5 0.1 ± 0.0 5.0 ± 0.3 16.1 ± 0.8*

5.3 ± 1.5 8.0 ± 0.5 42.1 ± 1.0** 59.2 ± 5.5** 2.0 ± 0.3 2.1 ± 0.1 17.6 ± 0.3* 13.0 ± 0.5*

10.4 ± 2.4* 22.0 ± 2.3* 73.8 ± 4.9** 64.6 ± 5.9** 2.3 ± 0.2 1.1 ± 0.0 10.0 ± 2.1* 16.2 ± 0.5*

9 10 11 12 13 14 15 Trolox

10 lM

50 lM

100 lM

20.0 ± 1.6* 4.0 ± 0.1 8.9 ± 0.7 2.3 ± 0.1 4.6 ± 0.9 8.0 ± 0.5 7.8 ± 0.5 39.1 ± 2.0*

57.4 ± 5.3** 12.1 ± 1.6* 27.8 ± 1.9** 1.2 ± 0.0 15.1 ± 0.8 8.0 ± 0.7 9.9 ± 2.0 95.2 ± 1.2**

71.9 ± 2.7** 41.0 ± 5.4** 40.8 ± 1.1** 13.7 ± 0.2 20.0 ± 0.1 7.5 ± 0.2 0.7 ± 0.0

HT22 cells were treated with compounds for 1 h before exposure to 5 mM glutamate for 8 h. The values shown are the mean ± S.D. of data from three independent experiments. Glutamate only-treated values differ significantly from non-treated control at a level of p < 0.001. Trolox was used as a positive control. * Results differ significantly from the glutamate only-treated, p < 0.01 ** Results differ significantly from the glutamate only-treated, p < 0.001.

1800

10 μM 50 μM

DCF fluorescence (Activity Unit)

1600 1400 1200

*

*

*

*

1000 800

*

Western blot. HT22 cells were pre-treated with 50uM of 3 or 4 for 30 min, followed by glutamate insult. Fifteen or 30 min after glutamate insult, the expression levels of p-ERK1/2 and total-ERK1/2 protein were analyzed. The result showed a remarkable increase in phosphorylation level of ERK1/2 at 30 min after glutamate treatment compared to untreated control. Glutamate-induced phophorylation of ERK1/2 was significantly attenuated by 3. The amount of p-ERK1/2 was reduced to 35.8% of the level observed in glutamate-only treated cells by the pre-treatment of 3.

600

4. Discussion

400 200 0 Control

Glutamate

3

4

9

Groups Fig. 2. The effects of 3, 4 and 9 on cellular peroxidation induced by glutamate in HT22 neuronal cells. HT22 cells were treated with compounds 3, 4 or 9 for 1 h followed by 5 mM glutamate insult and then maintained for 10 h. The content of intracellular peroxide was determined using fluorescent dye DCF-DA as described in Material and methods. The values are expressed as the means ± S.D. of three independent experiments. Results differ significantly from the value of glutamatetreated *p < 0.01.

contents was recovered to the level comparable with untreated control cells at the concentration of 10 lM. 3.5. Effect of 4 on ERK activation induced by glutamate in HT22 hippocampal cells We next investigated whether 3 and 4, potent biflavonoids from C. obtusa regulate the activation of ERK in response to glutamate by

Amentoflavone (3) and ginkgetin (4) are amentoflavone-type biflavonoids consisting of apigenin dimer linked by a C30 –C800 covalent bond, which are the most common types of biflavonoids reported so far. The diverse biological/pharmacological activities of biflavonoids have been reported on antibacterial, antifungal, antialergic, antiviral, anticancer, and recently the growing line of evidences has been accumulated on neuroprotection of naturally occurring biflavonoids. Kolaviron, a seed derived biflavonid, protected SH-SY5Y and PC12 cells against atrazine-induced neurotoxicity (Abarikwu et al., 2011a,b). Kang et al. (2005) reported that amentoflavone, ginkgetin and isoginkgetin protected SH-SY5Y neuroblastoma cells injured by cytotoxic insults of ROS or amyloid b, suggesting that amentoflavone-type biflavonoids with C30 –C800 linkage are potent in neuroprotection against oxidative stress induced cell death. Also, the administration of amentoflavone strongly protected the neonatal brain from hypoxia-ischemic injury (Shin et al., 2006). However, there have been no reports on neuroprotective activities of biflavonoids against glutamate oxidative stress in HT22 hippocampal cells. Therefore, in this study, we attempted to identify the possible actions of biflavonoids, amentoflavone (3) and ginkgetin (4) of C. obtusa in glutamate-injured HT22 cells.

Table 2 The effects of 3, 4 and 9 on the activities of glutathione peroxidase, glutathione reductase and SOD, CAT in glutamate-treated HT22 hippocampal cells. Groups Control Glutamate 3 4 9

Conc. (lM)

SOD (lmol NADPH oxidized/min/mg protein)

GR (lmol NADPH oxidized/min/mg protein)

Gpx (lmol NADPH oxidized/min/mg protein)

CAT (units/ml/mg protein)

10 50 10 50 10 50

45.22 ± 1.17 28.45 ± 3.22# 30.51 ± 2.12 38.47 ± 2.23* 34.79 ± 0.71* 41.43 ± 1.42* 28.30 ± 1.68 37.75 ± 1.11*

5.81 ± 3.22 3.46 ± 1.37# 3.25 ± 1.33 4.11 ± 0.28* 4.69 ± 1.65* 6.01 ± 1.96* 4.51 ± 1.23* 5.53 ± 0.45*

0.78 ± 0.02 0.53 ± 0.03# 0.50 ± 0.03 0.55 ± 0.06 0.50 ± 0.05 0.56 ± 0.02 0.60 ± 0.03* 0.68 ± 0.05*

0.150 ± 0.002 0.053 ± 0.004# 0.052 ± 0.003 0.055 ± 0.005 0.056 ± 0.004 0.054 ± 0.002 0.049 ± 0.002 0.053 ± 0.002

The activity of each enzyme was measured as described in materials and methods. Each value presents the mean ± S.D.. # Results significantly differ from the values of control: P < 0.01 * Glutamate-injured cultures: P < 0.01

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E.J. Jeong et al. / Food and Chemical Toxicology 64 (2014) 397–402 Table 3 The effects of 3, 4 and 9 on the contents of GSH in glutamate-treated HT22 hippocampal cells. Groups Control Glutamate 3 4 9

Conc. (lM)

Total GSH (nmol/mg protein)

Reduced GSH (nmol/mg protein)

GSSG/Total GSH ratio

10 50 10 50 10 50

20.75 ± 1.37 13.74 ± 2.23# 17.44 ± 1.62* 18.01 ± 2.20* 19.89 ± 1.32* 21.20 ± 1.51* 18.85 ± 0.83* 19.05 ± 0.54*

9.37 ± 0.54 10.11 ± 1.30# 9.15 ± 0.98 8.98 ± 1.54 8.56 ± 0.77 8.10 ± 0.89* 9.02 ± 1.20 9.00 ± 1.26

0.45 ± 0.02 0.73 ± 0.05# 0.52 ± 0.04* 0.49 ± 0.01* 0.43 ± 0.03* 0.38 ± 0.04* 0.47 ± 0.05* 0.47 ± 0.03*

The content of GSH was measured as described in materials and methods. Each value presents the mean ± S.D.. # Results significantly differ from the values of control: P < 0.01. * Glutamate-injured cultures: P < 0.01.

Fig. 3. The effect of 3 and 4 on glutamate-induced ERK activation in HT22 hippocampal cells. HT22 cells were treated with 3 or 4 for 30 min followed by 5 mM glutamate for 15 min or 30 min. The phosphorylated or total ERK1/2 in equivalent amounts of total cell lysate protein (40 lg) were visualized by Western blot analysis using an ERK-specific antibody as described in Material and methods.

A number of studies have indicated that glutamate-mediated neuronal cell death is closely associated with oxidative stress including excessive reactive oxygen species production. Neuronal damage induced by glutamate is caused through not only the excitotoxic pathway mediated by ionotropic glutamate receptors, but also through the oxidative glutamate pathway. High concentrations of glutamate insult prevent cystine uptake into the cells through the glutamate/cystine antiporter (Murphy et al., 1989; Davis, 1994). Since cystine is a precursor for the synthesis of glutathione, which results in intracellular glutathione loss and reactive oxygen species (ROS) accumulation under conditions of oxidative stress (Simonian, 1996). In these cases, reducing cystine levels in the medium can mimic the effect of glutamate, whereas increasing cystine concentrations reverse glutamate-induced toxicity (Murphy et al., 1989). This experimental paradigm serves as a good in vitro model to study the molecular events in neuronal injury. The reduction of ROS accumulation by antioxidants also prevents cell death. The treatment of HT22 cells with high dose of glutamate resulted in a significant increase in intracellular ROS. The increased production of ROS has been clearly attenuated by pre-treatment of 3, 4 and 9 (Fig. 2). The activities of antioxidant enzymes, SOD, GR, Gpx and CAT were also decreased by 5 mM glutamate insult (Table 2). It was shown that the damaged activities of SOD and GR were significantly preserved by pre-treatment of 3, 4 and 9. The most remarkable change in the activities of antioxidant enzymes by compound treatment was the increase in the activity of GR by 4 which recovered the reduced activity of GR to the level higher than untreated control. The activity of Gpx was little changed by 3 or 4 while the activity of Gpx was significantly increased by 9 with statistical significance. The reduced activity of CAT was unaffected by compounds treated. The reduced contents of GSH induced by glutamate were also recovered by the treatment of 3, 4 and 9 (Table 3). There is accumulating evidences that MEK is specifically involved in oxidative neuronal injury. ERK activation, although often involved in neuronal cell survival and/or proliferation, is also be associated with neuronal cell death that occurs in response to

specific insults suggesting the specificity of ERK activation and its functional significance depending on cell type and/or stimulus (Alessandrini et al., 1999; Murray et al., 1998). Respecting ERK inhibition in cell survival, it has been reported that a novel MAPK/ERK kinase (MEK) specific inhibitor U0126 profoundly protected HT22 cells against oxidative stress induced by glutamate, which was accompanied by an inhibition of phosphorylation of ERK1/2 (Satoh et al., 2000a,b). Also, persistent nuclear retention of activated ERK1/2 promoted cell death generated by oxidative toxicity suggesting critical role of activated ERK in eliciting proapoptotic effects in HT22 cells (Stanciu and DeFranco, 2001). ERK is one of many pharmacological targets of biflavonoids underlying its anti-cancer, anti-atherogenic, anti-inflammatory and UV-blocking effects (Lee et al., 2012; Oh et al., in press; Suh et al., 2006; Pang et al., 2009). Recently, the inhibitory effect of amentoflavone on ERK in various cell lines has been revealed. In RAW264.7 macrophages, amentoflavone was found to inhibit LPS-induced inflammation via strongly suppressing ERK’s kinase and disrupting the association between ERK and c-Fos (Oh et al., in press). In our experiment, the phosphorylated ERK1/2 in respond to glutamate stimulation in HT22 cells was effectively attenuated by 3, but little changed by 4 (Fig. 3). Though antioxidant effects of 4 on ROS production and antioxidant enzymes preservation was found to be more potent than 3, the inhibition of ERK activation was not significant in the treatment of 4. Further study on possible roles of 3 and 4 against glutamate-induced neurotoxicity in hippocampal cells will be necessary. In summary, we have demonstrated that biflavonoids, amentoflavone (3) and ginkgetin (4) derived from C. obtusa leaves scavenges ROS and exerts protective effect against oxidative damage induced by glutamate via maintaining the activities of antioxidant enzymes and/or inhibiting ERK1/2 activation. Conflict of Interest The authors declare that there are no conflicts of interest.

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Acknowledgements This work was carried out with the support of ‘‘Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ009804)’’ Rural Development Administration, Republic of Korea, and also supported by Gyeongnam National University of Science and Technology Grant. References Abarikwu, S.O., Farombi, E.O., Kashyap, M.P., Pant, A.B., 2011a. Kolaviron protects apoptotic cell death in PC12 cells exposed to atrazine. Free Radical Res. 45, 10611073. Abarikwu, S.O., Farombi, E.O., Pant, A.B., 2011b. Biflavanone-kolaviron protects human dopaminergic SH-SY5Y cells against atrazine induced toxic insult. Toxicol. in vitro 25, 848858. Agrawal, P.K., Bansak, M.C., Agrawal, P.K. (Eds.), 1989. Carbon 13 NMR of Flavonoids. Elservier, Amsterdam. Alessandrini, A., Namura, S., Moskowitz, M.A., Bonventre, J.V., 1999. MEK1 protein kinase inhibition protects against damage resulting from focal cerebral ischemia. Proc. Natl. Acad. Sci. USA 95, 12866–12869. Bae, D., Seol, H., Yoon, H.G., Na, J.R., Oh, K., Choi, C.Y., Lee, D.W., Jun, W., Youl Lee, K., Lee, J., Hwang, K., Lee, Y.H., Kim, S., 2012. Inhaled essential oil from Chamaecyparis obtuse ameliorates the impairments of cognitive function induced by injection of b-amyloid in rats. Pharm. Biol. 50, 900–910. Barrero, A.F., Quilez del Moral, J.F., Mar Herrador, M., Akssira, M., Bennamara, A., Akkad, S., Aitigri, M., 2004. Oxygenated diterpenes and other constituents from Moroccan Juniperus phoenicea and Juniperus thurifera var. africana. Phytochemistry 65, 2507–2515. Carlberg, I., Mannervik, B., 1975. Purification and characterization of the flavoenzyme glutathione reductase from rat liver. J. Biol. Chem. 25, 54755480. Chien, S.C., Chang, J.Y., Kuo, C.C., Hsieh, C.C., 2007. Cytotoxic and novel skeleton compounds from the heartwood of Chamaecyparis obtusa var. formosana. Tetrahedron Lett. 48, 1567–1569. Coyle, J.T., Puttfarcken, P., 1993. Oxidative stress, glutamate, and neurodegenerative disorders. Science 262, 689–695. Davis, J.B., Maher, P., 1994. Protein kinase C activation inhibits glutamate-induced cytotoxicity in a neuronal cell line. Brain Res. 652, 169–173. Flohe, L., Gunzler, W.A., 1984. Assays of glutathione peroxidase. Method Enzymol. 105, 114121. Gadek, P.A., Quinn, C.J., 1987. Biflavones and the affinities of Cupressus funebris. Phytochemistry 26, 2551–2552. Gard, H.S., Mitra, C.R., 1971. Putraflavone, A New biflavonoid from Putranjiva toxburghii. Phytochemistry 10, 2787–2791. Goodman, Y., Mattson, M.P., 1994. Staurosporine and K-252 compounds protect hippocampal neurons against amyloid beta-peptide toxicity and oxidative injury. Brain Res. 650, 170174. Hieda, T., Tazaki, M., Morishita, Y., Aoki, T., 1996. Sesquiterpene alcohols from Chamaecyparis obtusa leaf oil. Phytochemistry 42, 159–162. Kang, S.S., Lee, J.Y., Choi, Y.K., Song, S.S., Kim, J.S., Jeon, S.J., Han, Y.N., Son, K.H., Han, B.H., 2005. Neuroprotective effects of naturally occuring biflavonoids. Bioorg. Med. Chem. Lett. 15, 3588–3591. Kasuya, H., Hata, E., Satou, T., Yoshikawa, M., Hayashi, S., Masuo, Y., Koike, K., 2013. Effect on emotional behavior and stress by inhalation of the essential oil from Chamaecyparis obtusa. Nat. Prod. Commun. 8, 515–518. Kishida, M., Akita, H., 2005. Simple preparation of phenylpropanoid b-Dglucopyranoside congeners by Mizoroki–Heck type reaction using Organoboron reagents. Tetrahedron 61, 10559–10568. Kuo, Y.H., Chen, C.H., 2001. A novel 7(6.2)abeoabietane-type diterpene, obtusanal, from the Heartwood of Chamaecyparis obtusa var. formosana. Tetrahedron Lett. 42, 2985–2986. Kuo, Y.H., Chen, C.H., Chiang, Y.M., 2001. Three novel and one new lignan, chamaecypanones A, B, obtulignolide and isootobanone from the heartwood of Chamaecyparis obtusa var. formosana. Tetrahedron Lett. 42, 6731–6735. Lee, K.Y., Hwang, L., Jeong, E.J., Kim, S.H., Kim, Y.C., Sung, S.H., 2010. Effect of neuroprotective flavonoids of Agrimonia eupatoria on glutamate-induced oxidative injury to HT22 hippocampal cells. Biosci. Biotechnol. Biochem. 74, 1704–1706. Lee, C.W., Na, Y., Park, N.H., Kim, H.S., Ahn, S.M., Kim, J.W., Kim, H.K., Jang, Y.P., 2012. Amentoflavone inhibits UVB-induced matrix metalloproteinase-1 expression through the modulation of AP-1 components in normal human fibroblasts. Appl. Biochem. Biotechnol. 166, 1137–1147.

Lu, Y., Foo, L.Y., 1997. Identification and quantification of major polyphenols in apple pomace. Food Chem. 59, 187–194. Markham, K.R., Sheppard, C., Geiger, H., 1987. 13C NMR studies of some narurally occurring amentoflavone and hinokiflavone biflavonoids. Phytochemistry 26, 3335–3337. McCord, J.M., Fridovich, I., 1969. The utility of superoxide dismutase in studying free radical reactions. I. Radicals generated by interaction of sulfite, dimethyl sulfoxide, and oxygen. J. Biol. Chem. 25, 60566063. Murphy, T.H., Miyamoto, M., Sastre, A., Schnaar, R.L., Coyle, J.T., 1989. Glutamate toxicity in a neuronal cell line involves inhibition of cystine transport leading to oxidative stress. Neuron 2, 1547–1558. Murray, B., Alessandrini, A., Cole, A.J., Yee, A.G., Furshpanl, E.J., 1998. Inhibition of the p44/42 MAP kinase pathway protects hippocampal neurons in a cell-culture model of seizure activity. Proc. Natl. Acad. Sci. USA 95, 11975–11980. Na, K.J., Kang, H.Y., Yoon, S.K., Jeung, E.B., 1999. Biological efficacy of essential oils from softwood. Korean J. Lab. Animal Sci. 15, 7981. Nakanishi, T., Iida, N., Inatomi, Y., Murata, H., Inada, A., Murata, J., Lang, F.A., Iinuma, M., Tanaka, T., 2004. Neoliganan and Flavonoid glucosides in Juniperus communis var. depressa. Phytochemistry 65, 207–213. Nakano, K., Takatani, M., Tomimatsu, T., Nohara, T., 1983. Four Kaempferol Glycosides From Leaves of Cinnamomum Sieboldii. Phytochemistry 22, 2831– 2833. Nonaka, G., Goto, Y., Kinjo, J., Nohara, T., Nishioka, I., 1987. Tannins related compounds. Studies on the constituents of the leaves of Thujopsis dolabrata SIEB. Et ZUCC. Chem. Pharm. Bull. 35, 1105–1108. Oh, J., Rho, H.S., Yang, Y., Yoon, J.Y., Lee, J., Hong, Y.D., Kim, H.C., Choi, S.S., Kim, T.W., Shin, S.S., Cho, J.Y., in press. Extracellular signal-regulated kinase is a direct target of the anti-inflammatory compound amentoflavone derived from Torreya nucifera. Mediators Inflamm. Okasaka, M., Takaishi, Y., Kashiwada, Y., Kodzhimatov, O., Ashurmetov, O., Lin, A.J., Consentino, L.M., Lee, K.H., 2006. Terpenoids from Juniperus polycarpus var. seravschanica. Phytochemistry 67, 2635–2640. Pang, X., Yi, T., Yi, Z., Cho, S.G., Qu, W., Pinkaew, D., Fujise, K., Liu, M., 2009. Morelloflavone, a biflavonoid, inhibits tumor angiogenesis by targeting rho GTPases and extracellular signal-regulated kinase signaling pathways. Cancer Res. 69, 518–525. Satoh, T., Nakatsuka, D., Watanabe, Y., Nagata, I., 2000a. Neuroprotection by MAPK/ ERK kinase inhibition with U0126 against oxidative stress in a mouse neuronal cell line and rat primary cultured cortical neurons. Neurosci. Lett. 288, 163–166. Satoh, T., Nakatsuka, D., Watanabe, Y., Nagata, I., Kikuchi, H., Namura, S., 2000b. Neuroprotection by MAPK/ERK kinase inhibition with U0126 against oxidative stress in a mouse neuronal cell line and rat primary cultured cortical neurons. Neurosci. Lett. 288, 163166. Shin, D.I., Kim, J., 1991. Flavonoid constituents of Selaginella tamariscina. Korean J. Pharmacog. 22, 207–210. Shin, D.H., Bae, Y.C., Kim-Han, J.S., Lee, J.H., Choi, I.Y., Son, K.H., Kang, S.S., Kim, W.K., Han, B.H., 2006. Polyphenol amentoflavone affords neuroprotection against neonatal hypoxic-ischemic brain damage via multiple mechanisms. J. Neurochem. 96, 561–572. Simonian, N.A., Coyle, J.T., 1996. Annu. Rev. Pharmacol. Toxicol. 36, 83–106. Smith, P.K., Krohn, R.I., Hermanson, G.T., Mallia, A.K., Gartner, F.H., Provenzano, M.D., Fujimoto, E.K., Goeke, N.M., Olson, B.J., Klenk, D.C., 1985. Measurement of protein using bicinchoninic acid. Anal. Biochem. 150, 7685. Smith, E.C.J., Williamson, E.M., Wareham, N., Kaatz, G.W., Gibbons, S., 2007. Antibacterials and modulators of bacterial resistance from the immature cones of Chamaecyparis lawsoniana. Phytochemistry 68, 210–217. Stanciu, M., DeFranco, D.B., 2001. Prolonged nuclear retention of activated extracellular signal-regulated protein kinase promotes cell death generated by oxidative toxicity or proteasome inhibition in a neuronal cell line. J. Biol. Chem. 277, 40104017. Stanciu, M., Wang, Y., Kentor, R., Burke, N., Watkins, S., Kress, G., Reynolds, I., Klann, E., Angiolieri, M.R., Johnson, J.W., DeFranco, D.B., 2000. Persistent activation of ERK contributes to glutamate-induced oxidative toxicity in a neuronal cell line and primary cortical neuron cultures. J. Biol. Chem. 275, 12200–12206. Suh, S.J., Jin, U.H., Kim, S.H., Chang, H.W., Son, J.K., Lee, S.H., Son, K.H., Kim, C.H., 2006. Ochnaflavone inhibits TNF-alpha-induced human VSMC proliferation via regulation of cell cycle, ERK1/2, and MMP-9. J. Cell Biochem. 99, 1298–1307. Takaku, N., Choi, D.H., Mikame, K., Okunishi, T., Suzuki, S., Ohashi, H., Umezawa, T., Shimada, M., 2001. Lignans of Chamaecyparis obtuse. J. Wood Sci. 47, 476–482. Tietz, F., 1969. Enzymatic method of quantitative determination of nanogram amounts of total and oxidized glutathione. Anal. Biochem. 27, 502522. Xu, J.F., Cao, D.H., Tan, N.H., Liu, Z.L., Zhang, Y.M., Yang, Y.B.J., 2006. New lignan glycosides from Cupressus duclouxian (Cupessaceae). J. Asian Nat. Prod. Res. 8, 181–185.