Neuroprotective Benzyl Benzoate Glycosides from Disporum

Nov 18, 2013 - ABSTRACT: Bioassay-guided fractionation of the EtOAc extract from Disporum viridescens roots led to the isolation of five new benzyl be...
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Neuroprotective Benzyl Benzoate Glycosides from Disporum viridescens Roots in HT22 Hippocampal Neuronal Cells Namki Cho,† Heejung Yang,† Mina Lee,† Jungmoo Huh,† Hyeon-Woo Kim,† Hong-Pyo Kim,‡ and Sang-Hyun Sung*,† †

College of Pharmacy and Research Institute of Pharmaceutical Science, Seoul National University, Seoul 151-742, Republic of Korea College of Pharmacy, Ajou University, Suwon 443-749, Republic of Korea

‡

S Supporting Information *

ABSTRACT: Bioassay-guided fractionation of the EtOAc extract from Disporum viridescens roots led to the isolation of five new benzyl benzoate glycosides, BBGs (1−5). The neuroprotective activities of the BBGs were screened using neuronal HT22 hippocampal cells. BBG-D (4) significantly protected murine hippocampal HT22 cells against glutamateinduced neurotoxicity by maintaining the antioxidative defense systems such as superoxide dismutase, glutathione reductase, glutathione peroxidase, and the glutathione content. BBG-D, in a dose-and time-dependent manner, increased HO-1 expression through the selective activation of pERK signaling among the MAPK pathways. These results suggest that BBGD could be a promising candidate for the treatment of neurodegenerative diseases related to glutamate-induced oxidative neuronal cytotoxicity.

A

from D. viridescens roots using bioassay-guided fractionation employing neuronal HT22 hippocampal cells. Our study revealed that benzyl benzoate glycosides (BBGs) isolated from D. viridescens exert potent neuroprotective effects on glutamate-induced excitotoxicity in HT-22 cells. To elucidate the antioxidant effects of BBG in HT-22 cells, we evaluated the antioxidant defense mechanisms including superoxide dismutase (SOD), glutathione reductase (GR), glutathione peroxidase (GSH-Px), and the contents of the brain GSH in glutamate-induced HT-22 cells. In addition, we attempted to elucidate their neuroprotective mechanisms by inducing HO-1 expression, which is activated through the ERK, JNK, and p38 MAPK pathways.

number of neurodegenerative diseases including stroke, Alzheimer’s disease (AD), and Parkinson’s disease (PD) are implicated in oxidative stress in the central nervous system (CNS).1,2 The neuronal system is prone to oxidative stressinduced damage, which causes neuronal injury, cell death, and necrosis resulting in neuronal diseases.2 Glutamate is a wellknown critical contributor to pathological neuronal cell death within the CNS by inducing oxidative stress.3 Glutamateinduced oxidative injury depletes glutathione and causes pathological neuronal cell death by suppressing cysteine uptake through the cysteine/glutamate antiporter.4 Murine hippocampal HT22 cells have been used as a valuable in vitro model to study the mechanisms of glutamate-induced oxidative neuronal cytotoxicity.5 Any compound that can reduce the damage caused by glutamate might be a potential therapeutic agent for pathologies associated with oxidative stress. Therefore, we have attempted to identify an antioxidative agent that displays neuroprotective effects against excitotoxicity that causes glutamate-induced cell death.6 During a search for antioxidant agents from medicinal plants using glutamateinjured HT22 hippocampal cells, we observed that the EtOAc extract from Disporum viridescens (Liliaceae) roots exhibited a significant neuroprotective activity against glutamate-induced toxicity in HT-22 cells. D. viridescens roots are widely used to treat gastritis, asthma, and tuberculosis and are called “Bo joo cho” in Korean traditional medicine. However, a study aimed at assessing the in vitro biological effects or isolating the active components from D. viridescens has not yet been reported. Here, we describe the isolation of the chemical constituents © 2013 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION Bioassay-guided fractionation of the methanolic extract from D. viridescens roots revealed that, compared with the other fractions, the EtOAc fraction exhibited the most protective effects against glutamate injury in HT22 cells. Further fractionation and separation of the EtOAc fraction by activityguided chromatographic methods yielded five new benzyl benzoate glycosides (1−5) and henryoside (6).7 The structures of the new compounds (1−5) were elucidated by using 1D and 2D NMR and MS data. Compound 1 was obtained as a white, amorphous powder. Its molecular formula was determined to be C27H34O15 by the Received: August 19, 2013 Published: November 18, 2013 2291

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positive HRFABMS at m/z 599.1982 [M + H]+ and the 13C NMR data. The IR spectrum of 1 indicated the presence of hydroxy (3500 cm−1) and carbonyl (1659 cm−1) groups. The 1 H NMR spectrum suggested the presence of two aromatic rings, one of which is tetrasubstituted with two ortho-aromatic protons at δH 6.97 (1H, d, J = 9.0 Hz, H-4) and 6.61 (1H, d, J = 9.0 Hz, H-5) and the other is ortho-disubstituted with four aromatic protons at δH 7.15 (1H, d, J = 8.1 Hz, H-3′), 7.28 (1H, t, J = 7.1 Hz, H-4′), 7.05 (1H, t, J = 7.4 Hz, H-5′), and 7.39 (1H, d, J = 7.4 Hz, H-6′). The 13C NMR spectrum revealed signals for an ester carbonyl at δC 165.4 (C-7), an oxygenated methylene group at δC 61.2 (C-7′), and a methoxy group at δC 56.4 (OCH3) (Table 1). Analysis of the 1H and 13C NMR spectra of compound 1 indicated that it was a benzyl Table 1. 1H and 13C NMR Data of Compounds 1−5 1a position 1 2 3 4 5 6 7 1′ 2′ 3′ 4′

δH (J in Hz)

2b δC

δH (J in Hz)

3c δC

7.15, d (8.1) 7.28, t (7.1)

113.8 144.3 142.9 113.3 105.6 147.0 165.4 124.9 154.4 114.8 128.7

7.15, d (7.4) 7.29, t (7.4)

120.2 144.3 144.9 117.8 111.7 145.1 165.1 124.6 154.7 114.8 129.1

5′ 6′

7.05, t (7.4) 7.39, d (7.4)

121.8 127.4

7.05, t (7.4) 7.37, d (7.4)

121.8 128.0

7′

5.38, s

61.2

61.6

OCH3 glucose 1′ 2′ 3′ 4′ 5′ 6′ rhamnose A 1′ 2′ 3′ 4′ 5′ 6′ rhamnose B 1′ 2′ 3′ 4′ 5′ 6′ OAc 1″ 2″

3.79, s

56.4

5.35 d (13.3), 5.43, d (13.3) 3.71, s

60.5

4.87, d (7.2) 3.21−3.38, m 3.21−3.38, m 3.21−3.38, m 3.21−3.38, m 3.47, 3.71, m

100.9 73.2 76.5 71.7 77.0 60.6

4.87, d (7.0) 3.21−3.38, m 3.21−3.38, m 3.21−3.38, m 3.21−3.38, m 3.47, 3.71, m

100.7 74.2 76.5 71.7 77.0 60.6

5.24, s 3.77, m 3.76, m 3.20, m 3.50−3.55, m 1.1, d (6.1)

99.4 69.6 70.0 70.3 69.4 17.8

5.20, s 3.76, m 3.75, m 3.19, m 3.50−3.53, m 1.1, d (6.1)

99.7 69.6 70.0 70.3 69.5 17.8

6.97, d (9.0) 6.61, d (9.0)

6.87, d (9.0) 6.79, d (9.0)

δH (J in Hz)

6.87, d (9.2) 6.76, d (9.2)

7.17, d (7.8) 7.31,dt (7.8, 1.8, 1.4) 7.04, dt (7.8, 0.9) 7.4,dd (7.8, 1.8, 1.4) 5.25, d (12.8) 5.33,d (12.8) 3.68, s

4b δC 120.2 144.3 144.9 117.8 111.7 145.1 165.6 124.2 153.6 114.0 129.4

δH (J in Hz)

5c δC

δH (J in Hz)

δC

7.16, d (8.2) 7.31, t (8.2)

120.1 144.4 144.6 117.9 111.3 145.0 165.2 124.6 154.8 114.7 129.2

7.15, d (7.8) 7.28, dt (1.8, 7.3)

114.0 143.4 140.4 116.1 106.3 146.0 165.7 125.0 154.4 114.6 128.7

121.5 129.1

7.06, t (7.5) 7.36, d (7.5)

121.7 127.9

7.05, t (7.3) 7.39, d (7.8)

121.8 127.4

61.4

5.47, d (13.3), 5.35, d (13.3) 3.71, s

61.8

5.39, s

61.2

60.6

4.89, d (7.3) 3.0−3.4, m 3.0−3.4, m 3.0−3.4, m 3.0−3.4, m 3.72, 3.46, m

100.8 73.3 76.5 70.1 77.0 60.7

4.87, d (7.3) 3.21−3.38, m 3.21−3.38, m 3.21−3.38, m 3.21−3.38, m 3.49, 3.71, m

100.9 73.2 76.5 71.7 77.0 60.6

5.32, s 3.83 m, 3.62−3.72, m 4.83, t (9.8, 5.6) 3.60−3.70, m 0.9, d (6.1)

98.8 69.7 67.9 73.4 67.0 17.4

5.16, d (1.8) 3.75, m 3.55−3.65, m 3.21, m 3.50−3.60, m 1.1, d (6.4)

99.8 70.0 69.6 71.7 69.6 17.8

2.1, s

170.0 20.9

60.4

5.42, d (1.4) 3.89, m 3.71, m 3.28, m 3.45−3.55, m 1.1 d (6.1)

99.7 70.0 70.5 71.7 69.6 17.8

5.18, d (1.9) 3.89, m 3.72, m 3.28, m 3.45−3.55, m 1.1 d (6.4)

98.1 70.0 70.3 71.6 69.5 17.8

6.88, d (9.0) 6.79, d (9.0)

6.77, d (8.7) 6.49, d (8.7)

a1

H (400 MHz) and 13C (100 MHz) NMR data were obtained in DMSO-d6. b1H (500 MHz) and 13C (125 MHz) NMR data were obtained in DMSO-d6. c1H (600 MHz) and 13C (150 MHz) NMR data were obtained in DMSO-d6. 2292

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Figure 1. 1H−1H COSY, HMBC, and NOSEY correlations of compounds 1 and 4.

Figure 2. (A) Neuroprotective effects of compounds 1−6 from D. viridescens roots. The results differ significantly from the glutamate-treated cells; *p < 0.05, **p < 0.01 and ***p < 0.001. (B) Protective effects of compounds 2−5 on the glutamate-induced toxicity in HT22 cells.: (a) untreated control cells; (b) glutamate alone; (c) glutamate + 100 μM compound 2; (d) glutamate + 100 μM compound 3; (e) glutamate + 100 μM compound 4; (f) glutamate + 100 μM compound 5.

residue was assigned by the carbon−proton one-bond correlation through a 13C-decoupled 1H detected HSQC spectrum. The carbon−proton coupling constants (J = 175.6 Hz) led to the assignment for the α-configuration of the rhamnose moiety.9 Therefore, the structure of compound 1 was established as 2′-β-D-glucopyranosyloxybenzyl 6-α-L-rhamnopyranosyloxy-2-hydroxy-3-methoxybenzoate. Compound 2 was obtained as a white, amorphous powder. The molecular formula was determined to be C27H34O15, which was the same as that of compound 1, according to the [M + H]+ ion at m/z 599.1972 in the positive HRFABMS. Its 13C NMR spectra were similar to those of 1, except for the signals of the benzoyl and methoxy groups (δC 60.5) (Table 1). The methoxy group was located at C-2 based on the downfield shift of C-1 (+δC 6.4) upon comparison with that of 1 and the

benzoate glycoside, closely related to isoleiocarposide from Solidago decurrens.8 In the HMBC spectrum, the correlations of −OCH3 (δH 3.79) and H-5 with C-3, between H-4 and C-2, and from an anomeric proton (δH 5.24) and H-4 to C-6 suggested that the methoxy, hydroxy, and rhamnose groups were at C-3, C-2, and C-6, respectively (Figure 1). The NOESY experiment demonstrated correlations between 3-OCH3 and H-4, H-4 and H-5, and H-5 and the anomeric proton of the rhamnose moiety attached to C-6 (Figure 1). The sugar residues obtained from acid hydrolysis of 1 were identified as Dglucose and L-rhamnose by GC analysis of their respective trimethylsilyl L-cysteine derivatives. The coupling constant of the doublet at δH 4.87 (J = 7.2 Hz, H-1′) and anormeric resonance at δC 100.9 established the β-configuration of the glucose moiety. The anomeric configuration of the rhamnose 2293

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Table 2. Protective Effects of BBG-D on the Antioxidant Enzymatic Activities and Glutathione Levels in Glutamate-Injured HT22 Cellsa group

conc, μM (mg/mL)

control glutamate BBG-D a

10 50 100

SOD (U/mg protein) 34.65 16.10 29.78 29.05 30.71

± ± ± ± ±

GR (mU/mg protein)

0.70 0.54 0.70** 1.00** 1.89**

1.65 0.94 0.72 0.90 1.14

± ± ± ± ±

0.39 0.19 0.06 0.35 0.21

GSH-Px (mU/mg protein) 0.077 0.036 0.056 0.055 0.059

± ± ± ± ±

0.014 0.001 0.006 0.001* 0.004*

GSH/GSSG ratio 3.452 1.105 1.594 2.300 2.824

± ± ± ± ±

1.093 0.112 0.190 0.173* 0.430*

*p < 0.05. **p < 0.01.

HMBC correlation between the carbon resonance at δ 144.3 (C-2) and the proton resonance at δ 3.71. Therefore, the structure of compound 2 was established as 2′-β-D-glucopyranosyloxybenzyl 6-α-L-rhamnopyranosyloxy-3-hydroxy-2-methoxybenzoate. Compound 3 was obtained as a colorless powder, and its molecular formula was established as C27H34O14 by the positive HRFABMS at m/z 605.1851 [M + Na]+ together with the 13C NMR data. Its 1H and 13C NMR spectra were similar to those of 2, except for the signals of one of the sugar moieties. The C2′ sugar was identified as a L-rhamnose in 3 rather than Dglucose in 2 by analysis of its NMR data and GC analysis of its hydrolysate. Therefore, the structure of compound 3 was established as 2′-α-L-rhamnopyranosyloxybenzyl 6-α-L-rhamnopyranosyloxy-3-hydroxy-2-methoxybenzoate. Compound 4 was a colorless powder, and its molecular formula was established as C29H36O16 by the positive HRFABMS at m/z 663.1907 [M + Na]+ together with the 13 C NMR data. Its 1H and 13C NMR spectra were similar to those of 2, except for the deshielded H-4rha (δH 4.83) and an additional signal at δH 2.1 (3H, s, OAc-2″) that corresponded to an acetyl function. In the HMBC spectrum, the correlations between the carbon resonance at δC 170.0 (COCH3-1″) and the proton at δH 4.83 (H-4rha) and 2.1 (COCH3-2″) confirmed the presence of an acetyl group at C-4rha (Figure 1). Therefore, the structure of compound 4 was established as 2′-β-Dglucopyranosyloxybenzyl 6-α-L-(4′-O-acetyl) rhamnopyranosyloxy-3-hydroxy-2-methoxybenzoate. Compound 5 was obtained as a white, amorphous powder, and the molecular formula was determined to be C26H32O15, according to the [M + H]+ ion at m/z 585.1822 in the positive HRFABMS. The HRFABMS data revealed that the molecular mass of 5 was 14 (CH2) units smaller than 1. Its 1H and 13C NMR spectra were similar to those of 1, except for the absence of a methoxy group at C-3. Comparison of the 1H NMR spectra of 1 and 5 indicated a shielded H-4 resonance due to the occurrence of a hydroxy group at C-3 instead of a methoxy group. Therefore, the structure of compound 5 was established as 2′-β-D-glucopyranosyloxybenzyl 6-α-L-rhamnopyranosyloxy2,3-dihydroxybenzoate. Oxidative damage is closely associated with the hallmark pathologies of neurodegenerative diseases including AD.2 Many antioxidants, such as resveratrol, curcumin, and rosmarinic acid, have been suggested to reduce oxidative stress associated with AD.10,11 These polyphenolic compounds are composed of two phenolic rings at both extremities with several atom linkers. A previous report also suggested that benzyl benzoate with particular C6−linkers−C6 structure could efficiently inhibit Aβ fibrillogenesis in AD.10 Given these previous findings, we investigated the antioxidant and neuroprotective effects of BBGs isolated from the EtOAc fraction of D. viridescens roots using in vitro models. The

cytotoxicity of these compounds was measured with the MTT assay using BV2 and HT22 cells. The isolated compounds exhibit little toxicity at high concentrations (data not shown). We also observed NO inhibitory effects in LPS-stimulated BV2 microglia cells using the Griess reaction (see Supporting Information, S31). On the basis of these results, the neuroprotective effects of these compounds against glutamate-induced toxicity in HT22 hippocampal cells were measured with the MTT assay at concentrations ranging from 10 to 100 μM. Among these BBGs, compounds 1−4 exhibited significant neuroprotective activities (Figure 2A). Notably, the neuroprotective activity of compound 2 was superior to that of compound 5 despite their structural similarity (Figure 2B). The difference in the neuroprotective activity of these two compounds could be explained by the placement of the methoxy group at C-6. Furthermore, the results obtained from a comparison of compounds 2 and 4 (BBG-D) suggest that the C-4 acetylation of rhamnose slightly increases the neuroprotective activity of BBGs. However, a difference between compounds 2 and 3 was not observed, suggesting that these types of sugar moieties are not important for neuroprotection in glutamate-injured HT-22 cells. We further investigated the effect of BBG-D on GSH contents and antioxidant enzymatic activities in glutamateinjured HT22 hippocampal cells. The glutathione system is an important endogenous defense system against oxidative stress.12 Under oxidative damage, GSH is reversibly oxidized to glutathione disulfide (GSSG), which thus decreases GSH levels and changes the GSH/GSSG ratio.12 Under our experimental conditions, exposing HT22 cells to glutamate depleted the GSH content and significantly reduced the activities of SOD, GR, and GSH-Px by 53.5%, 43.0%, and 53.3%, respectively (Table 2). The activities of SOD and GSHPx reduced by glutamate insult were restored by BBG-D at concentrations ranging from 10 to 100 μM. Although the change in GR activity was not statistically significant after BBGD treatment, it increased to some extent at 100 μM. GSH-Px reduces toxic free radicals using GSH as a substrate, subsequent to the oxidation of GSH to GSSG.13 GSSG is in turn reduced to GSH by GR at the expense of NADPH, forming a redox cycle.14 On the basis of the restoration system for the activities of GSH-Px and GR, our results demonstrated that the GSH/ GSSG ratio value was restored significantly by BBG-D in a dose-dependent manner. These findings indicated that BBG-D exhibited a protective effect by reducing oxidative stress. We further evaluated whether BBG-D affected heme oxygenase (HO)-1 expression. HO-1, which is an inducible enzyme essential for heme degradation, is an important therapeutic target in neuronal oxidative damage.15 As part of our ongoing study evaluating antioxidant effects, we examined the effects of BBG-D on the protein expression of HO-1. We observed that BBG-D induced HO-1 expression in HT22 cells 2294

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Figure 3. Effects of BBG-D on heme oxygenase (HO)-1 expression and MAPK activation in HT22 cells. (A) The cells were incubated for 12 h (A) and 0.5 h (C) at the indicated periods and concentrations and with 100 μM of BBG-D (B). Each value represents the mean ± SD of three experiments. The results differ significantly from those of the glutamate-treated cells, *p < 0.05.

in a concentration-dependent manner (Figure 3A). At 100 μM BBG-D, HO-1 expression was first detected at 6 h and peaked at approximately 18 h (Figure 3B). Furthermore, the involvement of HO-1 in the neuroprotective activity of BBGD was also examined with an HO-1 inhibitor, SnPP. This specific inhibitor significantly blocked the BBG-D-mediated suppression of glutamate-induced cell death (Figure 4B). These results suggest that the neuroprotective effects of BBG-D could be due to HO-1 expression.16 MAPKs are a group of serine/ threonine kinases that include p38, JRK, and ERK, which participate in responding to extracellular signals to evoke cellular responses.17 MAPK activation has been known to modulate HO-1 protein expression.17,18 Given the numerous previous reports of MAPK activation modulating HO-1 expression, we further investigated whether BBG-D affected the MAPK families.17,18 At 100 μM, which strongly induced HO-1 and restored GSH, BBG-D induced ERK phosphorylation in HT22 cells. In contrast, phosphorylation of JNK and p38 kinases was not observed even at the high concentration of 100 μM (Figure 3C). Furthermore, to investigate the role of MAPK in HO-1 expression in HT22 cells, we examined the effects of specific p38 (SB203580), JNK (SP600125), and ERK (PD98059) inhibitors on HO-1 levels using Western blot analysis. We observed that BBG-D-induced HO-1 expression was not affected by the p38 and JNK inhibitors but was suppressed by the ERK pathway inhibitor (Figure 4A). These

findings indicate that BBG-D may induce HO-1 via the ERK pathway among the MAPK families. In conclusion, we isolated and identified five new benzyl benzoate glycosides from D. viridescens roots. Analysis of the effects of the BBGs on antioxidant enzymes demonstrated that BBG-D upregulated the protein expression of the main antioxidant enzymes SOD, GPx, GR, and HO-1. This small molecule, with two phenolic rings at both extremities linked by three atoms, increased intracellular GSH levels by maintaining antioxidative defense systems, including SOD, GR, and GSHPx. In addition, we demonstrated that the BBG-D-induced HO1 gene expression was associated with the ERK MAPK pathway. The results of these in vitro studies suggest that the BBGs isolated from D. viridescens roots may represent neuroprotective candidates and could be used to attenuate the progression of neurodegenerative diseases with glutamateinduced oxidative stress etiologies.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were recorded with a JASCO DIP-1000 digital polarimeter. UV spectra were measured on a Shimadzu UV-2101 spectrophotometer. IR spectra were recorded on a Perkin-Elmer 1710 spectrophotometer. 1H and 13C NMR,1H−1H COSY, HSQC, and HMBC spectra were recorded on a Bruker AMX 400 or 500 spectrometer in DMSO-d6. Column chromatography (CC) was performed on Kiesgel 60 silica gels (40−60 μm, 230−400 mesh, Merck, USA), YMC-GEL ODS-A 2295

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gel MPLC (MeOH−H2O = 2:8, 4:6). Compounds 2 (16 mg) and 4 (25 mg) were obtained from DE5 by ODS silica gel HPLC with MeOH−H2O (8:2, 2 mL/min). The DE3 fraction was further separated into four fractions (DE3A−3D) on Sephadex LH-20 with MeOH, and DE3C was chromatographed using ODS silica gel HPLC (MeOH−H2O = 4:6, 2 mL/min) to yield compounds 1 (7 mg), 3 (4 mg), and 5 (4 mg). 2′-β-D-Glucopyranosyloxybenzyl 6-α-L-rhamnopyranosyloxy-2-hydroxy-3-methoxybenzoate (1): white, amorphous powder; [α]25D −36 (c 0.1, MeOH); IR νmax (MeOH) 3352, 1725 cm−1; 1 H (400 MHz) and 13C (100 MHz) NMR data, see Table 1; FABMS m/z 621 [M + Na]+; HRFABMS 599.1982 [M + H]+ (calcd for C27H34O15H, 599.1976). 2′-β-D-Glucopyranosyloxybenzyl 6-α-L-rhamnopyranosyloxy-3-hydroxy-2-methoxybenzoate (2): white, amorphous powder; [α]25D −43 (c 0.1, MeOH); IR νmax (MeOH) 3348, 1727 cm−1; 1 H (500 MHz) and 13C (125 MHz) NMR data, see Table 1; FABMS m/z 599 [M + H]+; HRFABMS 599.1972 [M + H]+ (calcd for C27H34O15H, 599.1976). 2′-α-L-Rhamnopyranosyloxybenzyl 6-α-L-rhamnopyranosyloxy-3-hydroxy-2-methoxybenzoate (3): white, amorphous powder; [α]25D −50 (c 0.1, MeOH); IR νmax (MeOH) 3383, 1729 cm−1; 1 H (600 MHz) and 13C (150 MHz) NMR data, see Table 1; FABMS m/z 605 [M + Na]+; HRFABMS 605.1851 [M + Na]+ (calcd for C27H34O14 Na, 605. 1846). 2′-β-D-Glucopyranosyloxybenzyl 6-α-L-(4′-O-acetyl)-rhamnopyranosyloxy-3-hydroxy-2-methoxybenzoate (4): white, amorphous powder; [α]25D −32 (c 0.1, MeOH); IR νmax (MeOH) 3365, 1729 cm−1; 1H (500 MHz) and 13C (125 MHz) NMR data, see Table 1; FABMS m/z 663 [M + Na]+; HRFABMS 663.1907 [M + Na]+ (calcd for C29H36O16 Na, 663. 1901). 2′-β-D-Glucopyranosyloxybenzyl 6-α-L-rhamnopyranosyloxy-2,3-dihydroxybenzoate (5): white, amorphous powder; [α]25D −54 (c 0.3, MeOH); IR νmax (MeOH) 3358, 1726 cm−1; 1H (600 MHz) and 13C (150 MHz) NMR data, see Table 1; FABMS m/z 585 [M + H]+; HRFABMS 585.1822 [M + H]+ (calcd for C26H32O15 H, 585.1819). Acid Hydrolysis and Chiral Derivatization. Each compound (2 mg) was dissolved in 3 N HCl (dioxane−H2O, 1:1, 1 mL), and the solution was heated at 90 °C for 2 h. After neutralizing the acidic solution with NaHCO3, the solvent was evaporated to dryness under N2. The reaction mixture was extracted with CHCl3 and H2O, successively, and the aqueous layer was concentrated to dryness. Each residue was dissolved in dry pyridine (0.1 mL), and L-cysteine methyl ester hydrochloride in dry pyridine (0.06 M, 0.1 mL) was added. Each mixture was reacted at 60 °C for 2 h, and 0.1 mL of (trimethylsilyl)imidazole dissolved in H2O was added, followed by heating to dryness at 60 °C for 2 h. Each dried reactant was partitioned with n-hexane and H2O (0.1 mL, each). The n-hexane fraction was subjected to GC; column: DB-5, i.d. 0.25 mm, length 30 m, detector: FID, column temperature: 210 °C, injector temperature: 270 °C, detector temperature: 300 °C, carrier gas: He (2.0 kg/cm3). Under these conditions, the sugars were identified by comparison with authentic samples: tR (min) 10.45 (D-glucose), 11.04 (L-glucose), 8.92 (Drhamnose), and 8.66 (L-rhamnose). Cell Viability. The mouse hippocampal HT22 cell line was obtained from Dr. Ki-Sun Kwon at the Korea Research Institute of Bioscience & Biotechnology. HT22 cells were seeded onto 48-well plates at a density of 3 × 104 cells in 300 μL of DMEM per well. The mouse hippocampal HT22 cells were pretreated with a test compound dissolved in DMSO (final culture concentration of 0.1%) for 1 h and exposed to 5 mM L-glutamate. After an additional 12 h of incubation, the cellular viability of the cultures was assessed using the MTT (3[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. Trolox (Sigma, >97% purity) was used as a positive control in our study. Measurement of the Antioxidant Enzymatic Activities. Cells from three culture plates were pooled in 2 mL of 0.1 M phosphate buffer (pH 7.4) and homogenized. The homogenate was centrifuged for 25 min at 3000g at 4 °C, and the supernatant was collected for

Figure 4. (A) Effects of MAPK inhibitors on HO-1 expression by BGG-D in HT22 cells. (B) Effects of the HO-1 inhibitor on neuroprotection by BGG-D against glutamate-induced cytotoxicity in HT22 cells. The results differ significantly from those of the glutamatetreated cells, **p < 0.01, and from the glutamate and BBG-D-treated cells, #p < 0.05. (5−150 μm, YMC, Kyoto, Japan), and Sephadex LH-20 (25−100 μm, Pharmacia, NJ, USA), and TLC was performed on Kiesgel 60 F254 coated normal silica gels and on RP-18 F254 coated reversed-phase silica gels. The HPLC system consisted of a G-321 pump (Gilson Co. Ltd., USA), a G-151 UV detector (Gilson Co. Ltd., USA), and a YMCPack Pro C18 column (250 × 10 mm, 5 μm, YMC, Kyoto, Japan), and all chromatograms were monitored at 210 nm. Plant Material. D. viridescens were collected at the Medicinal Plant Garden, Seoul National University, Goyang, Korea, in May 2011. They were authenticated by Prof. Jong Hee Park, Pusan National University. A voucher specimen (SNU-0316) has been deposited in the Herbarium of the Medicinal Plant Garden of the College of Pharmacy, Seoul National University. Extraction and Isolation. D. viridescens roots (1.2 kg) were ground and extracted three times with 80% MeOH (4 L) in an ultrasonic apparatus at room temperature. Removal of the solvent in vacuo yielded a methanolic extract (350 g). The methanolic extract was suspended in H2O and partitioned successively with n-hexane, EtOAc, and n-BuOH (each 2 L × 3) to yield n-hexane (2 g), EtOAc (40 g), and n-BuOH (30 g) fractions. The EtOAc fraction (40 g), which displayed neuroprotective activity on HT22 cells, was subjected to silica gel CC (45 × 10 cm) eluted with mixtures of CHCl3− MeOH−H2O (25:4:1, 15:4:1, and 6:5:1) to yield six fractions (DE1− DE6). Compound 6 (10 mg) was isolated from DE1 using ODS silica 2296

dx.doi.org/10.1021/np400676b | J. Nat. Prod. 2013, 76, 2291−2297

Journal of Natural Products

Article

(7) Rosendal, S.; Nielsen, B. J.; Norn, V. Phytochemistry 1979, 18, 904. (8) Shiraiwa, K.; Yuan, S.; Fujiyama, A.; Matsuo, Y.; Tanaka, T.; Jiang, Z. H.; Kouno, I. J. Nat. Prod. 2012, 75, 88−92. (9) Agrawal, P. K. Phytochemistry 1992, 31, 3307−3330. (10) Rivière, C.; Richard, T.; Quentin, L.; Krisa, S.; Mérillon, J. M.; Monti, J. P. Bioorg. Med. Chem. 2007, 15, 1160−1167. (11) Rivière, C.; Richard, T.; Vitrac, X.; Mérillon, J. M.; Valls, J.; Monti, J. P. Bioorg. Med. Chem. Lett. 2008, 18, 828−831. (12) Pandey, K. B.; Rizvi, S. I. Oxid. Med. Cell Longev. 2010, 3, 2−12. (13) Cho, N.; Choi, J. H.; Yang, H.; Jeong, E. J.; Lee, K. Y.; Kim, Y. C.; Sung, S. H. Food Chem. Toxicol. 2012, 50, 1940−1945. (14) Jeong, E. J.; Ma, C. J.; Lee, K. Y.; Kim, S. H.; Sung, S. H.; Kim, Y. C. J. Ethnopharmacol. 2009, 121, 98−105. (15) Ryter, S. W.; Otterbein, L. E.; Morse, D.; Choi, A. M. Mol. Cell. Biochem. 2002, 234−235, 249−263. (16) Jeong, G. S.; Li, B.; Lee, D. S.; Kim, K. H.; Lee, I. K.; Lee, K. R.; Kim, Y. C. Int. Immunopharmacol. 2010, 10, 1587−1594. (17) Liu, Y.; Shepherd, E. G.; Nelin, L. D. Nat. Rev. Immunol. 2007, 7, 202−212. (18) 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. J. Biol. Chem. 2000, 275, 12200−12206.

measuring the antioxidant enzymatic activities and GSH contents. The total GSH in the supernatant was determined spectrophotometrically with the enzymatic cycling method. The activity of SOD was determined with the xanthine−xanthine oxidase reaction. The GR activity was measured based on the reduction of oxidized GSH (GSSG) by GR in the presence of NADPH. The activity of GSH-Px was determined by quantifying the rate of oxidation of GSH to GSSG using cumene hydroperoxide. The presented values are the mean ± SD of three experiments. The protein concentrations were determined using a bicinchoninic acid (BCA) kit (Sigma, St Louis, MO, USA) with a bovine serum albumin standard. Western Blot Analysis. HT22 cells were seeded in 12 wells and treated with BBG for 30 min or 12 h. The cells were washed twice with cold PBS and then lysed in an ice-cold modified RIPA buffer (50 mM Tris-HCl, pH 7.4, 1% Nonidet P-40, 0.25% Na-deoxycholate, 150 mM NaCl, 2 mM EDTA, 1 mM Na3VO4, and 1 mM NaF) containing protease inhibitors. The lysate was centrifuged for 15 min at 13000g at 4 °C, and the supernatants were collected. Equal amounts of protein (30 μg) were loaded in each lane onto a 10% SDS-PAGE gel. The proteins were separated by SDS-polyacrylamide gel electrophoresis, transferred to a PVDF membrane, and subsequently blocked in 4% bovine serum albumin (BSA)−TBST (100 mM Tris, pH 8.0, 150 mM NaCl, and 0.1% Tween 20) for 1 h at room temperature. The membranes were washed for 3 × 10 min in 0.1% Tween−20 PBS between each of following steps: 1 h block in 5% milk; overnight incubation at 4 °C with primary antibodies (HO-1, ERK or p-ERK, JNK or p-JNK, p38 or p-p38, and β-actin). The immunoreactive bands were visualized with secondary antibodies and an ECL chemiluminescence detection kit (Amersham Biosciences, USA). Statistical Analysis. The data were evaluated for statistical significance by an ANOVA test using a computerized statistical package. The data were considered to be statistically significant if the probability was ≤0.05.

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ASSOCIATED CONTENT

S Supporting Information *

The NMR spectra of compounds 1−5 and the NO inhibitory effects on the BV2 cell lines are available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: 82-2-880-7859. Fax: 82-2-877-7859. E-mail: shsung@snu. ac.kr. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (NRF-2013R1A2A2A01016296). We would like to thank S. I. Han (Medicinal Plant Garden, Seoul National University, Goyang, Korea) for kindly providing the D. viridescens.

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REFERENCES

(1) Gibson, G. E.; Zhang, H. J. Alzheimers Dis. 2001, 3, 329−338. (2) Hald, A.; Lotharius, J. Exp. Neurol. 2005, 193, 279−290. (3) Love, S. Brain Pathol. 1999, 9, 119−131. (4) Murphy, T. H.; Miyamato, M.; Sastre, A.; Schnaar, R. L.; Coyle, J. T. Neuron 1989, 2, 1547−1558. (5) Maher, P.; Schubert, D. Cell. Mol. Life Sci. 2000, 57, 1287−1305. (6) Jin, D. Q.; Lim, C. S.; Hwang, J. K.; Ha, I.; Han, J. S. Biochem. Biophys. Res. Commun. 2005, 331, 1264−1269. 2297

dx.doi.org/10.1021/np400676b | J. Nat. Prod. 2013, 76, 2291−2297