Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

1 Contents lists available at ScienceDirect 2 3 4 5 6 journal homepage: www.elsevier.com/locate/jep 7 8 9 Research Paper 10 11 12 13 14 15 Kyeong Wan Woo a, Oh Wook Kwon b, Sun Yeou Kim c,e,f, Sang Zin Choi d, Mi Won Son d, 16 a a,n Q1 Ki Hyun Kim , Kang Ro Lee 17 a 18 Natural Products Laboratory, School of Pharmacy, Sungkyunkwan University, 300 Chonchon-dong, Jangan-ku, Suwon, Gyeonggi-do 440-746, Republic of Korea 19 b Graduate School of East-West Medical Science, Kyung Hee University Global Campus, Yongin 446-701, Republic of Korea 20 c College of Pharmacy, Gachon University, Incheon 406-799, Republic of Korea 21 d Dong-A Pharm Institute, Kiheung, Yongin 449-905, Republic of Korea 22 Q3 e Gachon Institute of Pharmaceutical Science, Gachon University, Incheon 406-799, Republic of Korea f Gachon Medical Research Institute, Gil Medical Center, Inchon 405-760, Republic of Korea 23 24 25 26 art ic l e i nf o a b s t r a c t 27 Article history: Ethnopharmacological relevance: Dioscorea nipponica (Dioscoreaceae) have been used as traditional 28 Received 27 March 2014 medicines for diabetes, inflammatory and neurodegenerative diseases in Korea. The aim of the study 29 Received in revised form was to isolate the bioactive components from the rhizomes of Dioscorea nipponica and to evaluate their 30 16 June 2014 anti-neuroinfalmmatory and neuroprotective activities. 31 Accepted 17 June 2014 Material and methods: The phytochemical investigation of 50% EtOH extract of Dioscorea nipponica using 32 successive column chromatography over silica gel, Sephadex LH-20, and preparative high performance 33 Keywords: liquid chromatography (HPLC) resulted in the isolation and identification of 17 phenolic derivatives, 34 Dioscorea nipponica including four new phenolic compounds (1–4). The structural elucidation of these compounds was based 35 Dioscoreaceae on spectroscopic methods, including 1D and 2D nuclear magnetic resonance (NMR) spectroscopy Stilbene 36 techniques, mass spectrometry, and optical rotation. All isolated compounds were evaluated for their Phenanthrene 37 effects on nerve growth factor (NGF) secretion in a C6 rat glioma cell line and nitric oxide (NO) NGF induction 38 production in lipopolysaccharide (LPS)-activated BV2 cells. The neurite outgrowth of compound 16 was Chemical compounds studied in this article: further evaluated by using mouse neuroblastoma N2a cell lines. 39 G N -monomethyl-L-arginine (PubChem CID: Results: Three new stilbene derivatives, diosniponol C (1), D (2) and diosniposide A (3) and one new 40 135242) phenanthrene glycoside, diosniposide B (4), together with 13 known compounds were isolated from the 41 rhizomes of Dioscorea nipponica. Of the tested compounds (1–17), phenanthrene, 3,7-dihydroxy-2,4,642 trimethoxy-phenanthrene (16) was the most potent NGF inducer, with 162.357 16.18% stimulation, and 43 strongly reduced NO levels with an IC50 value of 19.56 μM in BV2 microglial cells. Also, it significantly 44 increased neurite outgrowth in N2a cells. 45 Conclusions: This study supports the ethnopharmacological use of Dioscorea nipponica rhizomes as 46 traditional medicine. 47 & 2014 Published by Elsevier Ireland Ltd. 48 49 50 51 1. Introduction 52 53 The onset of neurological disorders may incidentally begin with Abbreviations: NMR, nuclear magnetic resonance; NO, nitric oxide; LPS, lipopo54 lysaccharide; UV, ultraviolet; IR, infrared; HR, high resolution; FAB, fast atom the onset of chronic inflammation and abnormal metabolism such 55 bombardment; MS, mass spectrometry; COZY, correlation spectroscopy; HMQC, as diabetes etc. The most common thesis is that cerebrovascular 56 heteronuclear multiple quantum coherence; HMBC, heteronuclear multiple bond events such as diabetic polyneuropathy, peripheral neuropathies, 57 correlation; HPLC, preparative high performance liquid chromatography; RP, infections, hyperlipidemia and neurological injuries are the onset reversed-phase; LPLC, low-pressure liquid chromatography; TLC, thin-layer chro58 matography; DMEM, Dulbecco's modified Eagle medium; FBS, fetal bovine serum; causes. Specially, recent study reported that diabetic neuropathy 59 L-NMMA, NG-monomethyl-L-arginine; NOS, nitric oxide synthase; PS, penicillinmay increase the risk of neurodegenerative disease (Abrams et al., 60 streptomycin; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bro2009). Therefore, regulation of neuroinflammation and neuro61 mide; CNS, central nervous system; JNK, c-Jun N-terminal kinases trophic factor may prevent and cure neurodegenerative-related n Corresponding author. Tel.: þ 82 31 290 7710; fax: þ82 31 290 7730. 62 diseases. Particularly, induction and substitution of nerve growth E-mail address: [email protected] (K. Ro Lee). 63 64 http://dx.doi.org/10.1016/j.jep.2014.06.043 65 0378-8741/& 2014 Published by Elsevier Ireland Ltd. 66

Journal of Ethnopharmacology

Phenolic derivatives from the rhizomes of Dioscorea nipponica and their anti-neuroinflammatory and neuroprotective activities

Please cite this article as: Wan Woo, K., et al., Phenolic derivatives from the rhizomes of Dioscorea nipponica and their antineuroinflammatory and neuroprotective activities. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.06.043i

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factor may show promising results in that. So, many researchers try to find materials with NGF mimetic activity and the induction of NGF secretion. Recently, Zhao et al. (2014) group demonstrated that neurotrophic factor might prevent LPS-induced neuroinflammation in microglia by inhibition of JNK signaling. These facts suggest that neurotrophic factor may be a potential target for the treatment of neuroinflammation in the central nervous system (CNS) disorders. The neurotrophic factor inducer or mimetics with anti-neuroinflammatory activity can synergistically recover neuronal damage, furthermore, regulate neurodegenerative disease. Therefore, it is very meaningful research that focused on searching novel materials that regulate the integration of neuroinflammation and neurotrophic factor in neurological disorders. Dioscorea nipponica, known as ‘Buchema’ is a typical aura medicine which eliminates or improves giheo symptoms. Giheo symtoms means diarrhea, chronic fatigue, malnutrition, loss of appetite. The rhizomes of Dioscorea have been used for replenishing the spleen and stomach. Therefore, it can promote fluid secretion. And, it had regulated the frequency of urination or diabetes due to deficiency condition of the kidney and seminal emission, too. Traditionally, it has been known that increase of energy in the kidney may prevent neurodegenerative diseases. In recent pharmacological study, the extract of this plant showed neurotrophic activity (Kim et al., 2011; Lee et al., 2013), which has also been confirmed by our screening tests exhibiting that the Dioscorea nipponica EtOH extract had considerable NGF agonistic activity against glioma cell line in C6 rats. Based on this theory, we investigated to isolate active compound which may act on antineuroinflammation and neurotrophic factor from Dioscorea nipponica. Column chromatographic purification resulted in the isolation of three new stilbene derivatives, diosniponol C (1), D (2) and diosniposide A (3) and one new phenanthrene glycoside, diosniposide B (4), as well as 13 known compounds (5–17). Herein, we report the isolation of bioactive components from the rhizomes of Dioscorea nipponica and evaluate their antineuroinflammatory and neurotrophic activities.

2. Material and methods 2.1. Plant material The rhizomes of Dioscorea nipponica were imported from Heilongjiang, China, in January, 2009, and the plant was identified by one of the authors (K. R. Lee). A voucher specimen (SKKU NPL 0913) was deposited in the herbarium of the School of Pharmacy, Sungkyunkwan University, Suwon, Korea. 2.2. General Thin-layer chromatography (TLC) was performed using Merck precoated Silica gel F254 plates and reversed-phase (RP)-18 F254s plates. Spots were detected on TLC under ultraviolet (UV) light or by heating after spraying 10% H2SO4 in C2H5OH (v/v). Packing material of molecular sieve column chromatography was Sephadex LH-20 (Pharmacia Co. Ltd.). Low pressure liquid chromatography (LPLC) was carried out over a Merck LiChroprep Lobars-A Si 60 (240  10 mm) and LiChroprep Lobars-A RP-C18 (240  10 mm) column with an FMI QSY-0 pump (ISCO). Semi-preparative high performance liquid chromatography (HPLC) was performed using a Gilson 306 pump (Gilson, Middleton, WI) with a Shodex refractive index detector (Shodex, New York, NY) and Econosils RP-C18 10 u column (250  10 mm). Optical rotations were obtained on a JASCO P-1020 Polarimeter (Jasco, Easton, MD). Infrared (IR) spectra were recorded on a Bruker IFS-66/S FT-IR spectrometer. Nuclear magnetic resonance (NMR) spectra, including 1H–1H correlation spectroscopy (COZY),

heteronuclear multiple quantum coherence (HMQC) and hetero nuclear multiple bond correlation (HMBC), were recorded on a Varian UNITY INOVA NMR spectrometer operating at 500 MHz (1H) and 125 MHz (13C), with chemical shifts given in ppm (δ). High resolution (HR)-fast atom bombardment mass spectrometry (FABMS) and FABMS spectra were obtained on a JEOL JMS 700 mass spectrometer.

2.2.1. Extraction and isolation The rhizomes of Dioscorea nipponica (10 kg) were extracted with 50% aqueous EtOH (3  4 L every 3 days) at room temperature and filtered. The filtrate was evaporated under reduced pressure to give an EtOH extract (1 kg), which was suspended in water (800 mL) and solvent-partitioned to give n-hexane (1 g), CHCl3 (35 g), EtOAc (10 g), and BuOH (200 g) fractions. The CHCl3 fraction (9.5 g) was separated over a silica gel column with a solvent system of CHCl3/MeOH (60:1 to 1:1) to obtain 11 fractions (A–K). The B fraction (180 mg) was chromatographed on a Sephadex LH-20 column (CH2Cl2/MeOH, 1:1) to give two subfractions (B1–B2). Subfraction B1 (130 mg) was applied to LPLC on a LiChroprep Lobar-A Si column eluted with CHCl3/MeOH (90:1) to give three subfractions (B11–B13). Subfraction B13 (23 mg) and B22 (39 mg) was purified with reverse phase-C18 silica gel semi-prep. HPLC (65% MeOH and 50% MeCN) to obtain compounds 5 (6 mg), 16 (6 mg), 1 (4 mg), and 2 (6 mg). The C fraction (55 mg) was chromatographed on Sephadex LH-20 (CH2Cl2/MeOH, 1:1) to yield three subfractions (C1–C3). Subfractions C2 (15 mg) and C3 (11 mg) were purified with RP-C18 silica gel semi-prep. HPLC (50% MeCN and 70% MeOH) to obtain compounds 11 (3 mg), 15 (3 mg) and 17 (2 mg). The H fraction (310 mg) was chromatographed on a Sephadex LH-20 column (CH2Cl2/MeOH, 1:1) and separated over an RP-18 open column with a solvent system of 60% MeOH, and then purified with RP-C18 silica gel semi-prep. HPLC (50% MeOH) to yield compound 6 (5 mg). The EtOAc fraction (10.0 g) was separated over a silica gel column with a solvent system of CHCl3/MeOH (20:1 to 1:1) to obtain 10 fractions (A–J). The B fraction was chromatographed on a Sephadex LH-20 column (CH2Cl2/MeOH, 1:1) and purified with RP-C18 silica gel semi-prep. HPLC (50% MeOH) to yield compound 8 (4 mg). The C fraction (560 mg) was separated over the RP-18 open column with a solvent system of 70% MeOH to yield six subfractions (C1–C6). Subfraction C1 (330 mg) was purified with RP-C18 silica gel semi-prep. HPLC (35% and 50% MeOH) to yield compounds 7 (22 mg) and 14 (3 mg). The D fraction (650 mg) was separated over the RP-18 open column with a solvent system of 50% MeOH to yield seventeen subfractions (D1–D17). Subfraction D2 (35 mg) was purified with RP-C18 silica gel semi-prep. HPLC (50% MeOH) to yield compound 13 (4 mg). Subfraction D11 (280 mg) was chromatographed on a Sephadex LH-20 column (80% MeOH) to yield three subfractions (D111–D113). Subfractions D113 (70 mg) and D111 (100 mg) were purified with RP-C18 silica gel semi-prep. HPLC (25 and 40% MeOH) to yield compounds 9 (70 mg) and 12 (9 mg). The E fraction (1.4 g) was separated over the RP-18 open column with a solvent system of 50% MeOH to yield six subfractions (E1–E6). Subfraction E5 (50 mg) was purified with RP-C18 silica gel semi-prep. HPLC (50% MeOH) to yield compound 10 (25 mg). The F fraction (1.3 g) was separated over the RP-18 open column with a solvent system of 50% MeOH to yield 11 subfractions (F1–F11). Subfraction F2 (160 mg) was purified with RP-C18 silica gel semi-prep. HPLC (40% and 50% MeOH) to yield compounds 4 (3 mg) and 3 (2 mg). Diosniponol C (1): Colorless gum; ½α25 D þ11.0 (c 0.10 EtOH); IR (KBr) νmax 3672, 2922, 2864, 1744, 1650, 1455, 1362, 1276, 1243, 1166, 1033, 796, 700 and 617 cm  1; 1H NMR (500 MHz, CD3OD)

Please cite this article as: Wan Woo, K., et al., Phenolic derivatives from the rhizomes of Dioscorea nipponica and their antineuroinflammatory and neuroprotective activities. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.06.043i

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and 13C NMR (125 MHz, CD3OD) data, see Table 1; HR-FAB-MS 257.0813 m/z [M þH  H2O] þ (calcd for 257.0814). Diosniponol D (2): Colorless gum; ½α25 D 2.3 (c 0.15 EtOH); IR (KBr) νmax 3383, 2939, 2832, 1731, 1678, 1458, 1414, 1369, 1297, 1033 and 757 cm  1; 1H NMR (500 MHz, CD3OD) and 13C NMR (500 MHz, CD3OD) data, see Table 1; HR-FAB-MS 301.0713 m/z [Mþ H H2O] þ (calcd for 301.0712). Diosniposide A (3): Colorless gum; IR (KBr) νmax 3382, 2938, 2864, 1650, 1455, 1361, 1033 and 618 cm  1; 1H NMR (500 MHz, CD3OD) and 13C NMR (500 MHz, CD3OD) data, see Table 2; HRFAB-MS 429.1525 m/z [MþNa] þ (calcd for 429.1527). Diosniposide B (4): Colorless gum; IR (KBr) νmax 3420, 2923, 2864, 1739, 1611, 1581, 1455, 1421, 1346, 1259, 1241, 1033 and 801 cm  1; 1H NMR (500 MHz, CD3OD) and 13C NMR (500 MHz, CD3OD) data, see Table 2; HR-FAB-MS 473.1422 m/z [M þNa] þ (calcd for 473.1423). 2.3. NGF assay We used C6 glial cells to measure NGF release into the medium (Mosmann, 1983). C6 cells were purchased from the Korean Cell Line Bank (Seoul, Korea) and maintained in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin (PS) in a humidified incubator with 5% CO2. To measure NGF content in medium and cell viability, C6 cells were seeded onto 24-well plates (1  105 cells/well). After 24 h, the cells were treated with DMEM containing 2% FBS and 1% PS with 20 μM of each sample for one day. Media supernatant was used for the NGF assay using an ELISA development kit (R&D System, Minneapolis, MN, USA). Cell viability was assessed by a 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay. In this study, 6-shogaol was tested as a positive control. 2.4. Nitric oxide (NO) assay The BV-2 mouse microglial cell line has been extensively used in published studies as an in vitro culture system for the investigation of primary microglial function. In this study, BV-2 cells were maintained in DMEM supplemented with 5% FBS and 1% PS. To measure nitric oxide (NO) production, BV-2 cells were plated into a 96 well plate (3  104 cells/well) and treated with 100 ng/mL lipopolysaccharide (LPS) in the presence or absence of isolates (1–17) for 24 h. Nitrite, a soluble oxidation product of NO, was Table 1 1 H and 13C NMR data of compound 1 and 2 in CD3ODa. Position

1

1 2 3 4 5 6 7 8a 8b 9 10 11 12 13 14 COOH O–CH2–O

– 7.52 m 7.44 m 7.39 m 7.44 m 7.52 m 5.61 dd (11.5, 3.0) 3.25 m 3.11 dd (16.5, 3.5) – 6.28 s – 6.25 s – – – –

2 138.7 125.9 128.3 128.3 128.3 125.9 80.4 34.7 141.9 106.8 165.6 101.0 164.3 99.9 170.1 –

– – 6.85 d (9.0) 7.20 m 6.91 m 7.43 d (7.5) 5.91 dd (10.5, 1.5) 3.18 m 3.21 m – 6.48 s – – – – – 6.08 d (1.0)

124.6 154.2 114.8 129.1 119.2 126.2 76.4 33.3 136.6 99.9 133.0 154.1 145.6 104.1 170.2 102.4

a 1 H and 13C NMR data were recorded at 500 and 125 MHz, respectively. Coupling constants (in Hz) are given in parentheses.

3

Table 2 1 H and 13C NMR data of compounds 3 and 4 in CD3ODa. Position

3

1 2 3 4 5 6 7 8 9 10 11 12 13 14 1' 2' 3' 4' 5' 6a' 6b' OCH3 OCH3

– 7.24 (m) 7.17 (m) 7.15 (m) 7.17 (m) 7.24 (m) 2.88 (m) 2.77 (m) – 6.48 (d, 1.5 Hz) – – – 6.39 (d, 1.5 Hz) 4.79 (d, 8.0 Hz) 3.48 (m) 3.45 (m) 3.38 (m) 3.39 (m) 3.85 (dd, 12.0, 1.5 Hz) 3.68 (dd, 12.0, 10.0 Hz) 3.81 (s) –

4 143.6 129.8 130.1 127.3 130.1 129.8 39.3 39.4 139.8 109.8 152.5 137.1 151.9 112.1 103.2 75.1 78.3 71.5 78.3 62.7 62.0 –

– – – – – 6.68 (s) 2.58 (m) 2.63 (m) – – – – 7.08 (d, 8.0 Hz) 6.79 (d, 8.0 Hz) 4.84 (d, 7.5 Hz) 3.54 (m) 3.47 (m) 3.42 (m) 3.43 (m) 3.88 (dd, 12.0, 1.5 Hz) 3.72 (m) 3.90 (s) 3.73 (s)

138.8 119.2 151.1 141.3 151.9 114.0 32.4 32.1 136.5 123.0 145.1 147.9 117.7 120.7 104.4 75.1 78.4 71.5 77.8 62.7 61.7 62.8

a 1 H and 13C NMR data were recorded at 500 and 125 MHz, respectively. Coupling constants (in Hz) are given in parentheses.

measured in the culture media using the Griess reaction. The supernatant (50 μL) was harvested and mixed with an equal volume of Griess reagent (1% sulfanilamide and 0.1%N-1-napthylethylenediamine dihydrochloride in 5% phosphoric acid). After 10 min, the absorbance at 540 nm was measured using a microplate reader. Sodium nitrite was used as a standard to calculate the NO2 concentration. Cell viability was assessed by the MTT assay. In this study, NG-Mono-methyl-L-arginine (L-NMMA, SigmaAldrich), a well-known nitric oxide synthase (NOS) inhibitor, was tested as a positive control (Reif and McCreedy, 1995). 2.5. Neurite outgrowth assay Mouse neuro2a (N2a) cells, derived from a neuroblastoma, are widely established as an in vitro model for measuring the neurite outgrowth (Goshima et al., 1993; Wu et al., 2009). In this study, N2a cell lines were originally obtained from American Type Culture Collection (Manassas, VA, USA) and were maintained in DMEM (Invitrogen, Carlsbad, CA, USA) supplemented with 10% FBS (Invitrogen) plus 1% PS (Invitrogen) in an incubation chamber gassed with 5% CO2 and 95% air at 37 1C. For the neurite outgrowth experiments, N2a cells were seeded onto six-well plates at a density of 1  104 cells/well, and treated with the indicated reagent(s) for 3 days. These cells cease to proliferate and begin to differentiate, as evidenced by neurite outgrowth, in response to serum starvation, retinoic acid, or growth factors such as neurotrophins and glial cell-derived neurotrophic factor family ligands. Neurite lengths of N2a cells were measured using an incucyte imaging system (Essen Instruments, Ann Arbor, MI, USA).

3. Results and discussion 3.1. Isolation of compounds 1–17 from the rhizomes of Dioscorea nipponica The 50% ethanol extract of rhizomes of Dioscorea nipponica was successively partitioned with n-hexane, CHCl3, EtOAc, and

Please cite this article as: Wan Woo, K., et al., Phenolic derivatives from the rhizomes of Dioscorea nipponica and their antineuroinflammatory and neuroprotective activities. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.06.043i

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differences in the substituted pattern of ring A, which was identified as 1-substitued aromatic ring, and disappearance of one aromatic proton in ring B. An analysis of the 1H–1H COZY, HMQC, and HMBC correlations led to the establishment of the planar structure for 1 (Fig. 2). The optical rotation of 1 exhibited a positive value (½α25 D þ 11.0, EtOH), indicating that the absolute configuration at C-7 in 1 had to be the S form (Kim et al., 2013). Thus, the structure of compound 1 was determined as shown in Fig. 1 and named diosniponol C. Compound (2) was obtained as a colorless gum with negative optical rotation (½α25 D –2.3, EtOH). The molecular formula of 2 was determined to be C16H14O7 by positive mode HR-FAB-MS data at 301.0713 m/z [M þH H2O] þ (calcd for 301.0712). The 1H and 13C NMR spectra were similar to those of 1. The major differences were that the substitution pattern of ring A in 2, was identified as 1,2substitued aromatic ring, instead of one 1-substitued in 1, and the appearance of the methylenedioxy functional group (δH 6.08; δC 102.4, respectively). The full NMR assignments and connectives of 2 were determined by 1H–1H COZY, HMQC, and HMBC spectroscopic data analysis (Fig. 2). The absolute configuration of 2 was determined to be 7R by comparing the negative optical rotation value (Kim et al., 2013). Taken together, the structure of compound 2 was determined as shown and named diosniponol D. Compound (3) was obtained as a colorless gum. The HR-FABMS ([MþNa] þ at m/z 429.1525, calcd for 429. 1527) and 1H and 13C NMR spectra of 3 gave the molecular formula of C21H26O8. The 1H and 13C NMR data (Table 2) were very similar to that of 3,5dihydroxy-4-methoxybibenzyl, which was isolated from Dioscorea opposita (Yang et al., 2009). The only difference was the additional glucose group [δH 4.79 (1H, d, J ¼8.0 Hz, H-10 ), 3.85 (1H, dd, J¼ 12.0, 1.5 Hz, H-6a0 ), 3.68 (1H, dd, J ¼12.0, 10.0 Hz, H-6b0 ), 3.48 (1H, m, H-20 ), 3.45 (1H, m, H-30 ), 3.39 (1H, m, H-50 ), and 3.38 (1H, m, H-40 ) in the 1H NMR; δC 103.2, 78.3, 78.3, 75.1, 71.5 and 62.7 in 13C NMR]. The J values of the anomeric proton at δH 4.79 (d, J ¼8.0 Hz) indicated the presence of a β-glucopyranosyl unit (Woo et al., 2012). The position of glucose was confirmed to be at

n-BuOH. Repeated chromatographic purification of CHCl3 and EtOAc soluble fraction afforded three new stilbene derivatives, diosniponol C (1), D (2) and diosniposide A (3) and one new phenanthrene glycoside, diosniposide B (4), together with 13 known compounds (5–17) which were identified as, ( þ)-syringaresinol (5) (Rahman et al., 2007), (þ)-syringaresinol-O-β-D-glucopyranoside (6) (Shahat et al., 2004), vanillic acid (7) (Lee et al., 2010), 4-hydoxybenzaldehyde (8) (Lee et al., 2010), 4hydroxybenzoic acid (9) (Pyo et al., 2002), protocatechuic acid (10) (Lee et al., 2012), 4-(4-hydroxyphenyl)butan-2-one (11) (Back et al., 2011), N-acetyltyramine (12) (Zhao et al., 2006), tyramine (13) (Hedge et al., 1997), liquiritigenin (14) (Fu et al., 2005), 6methoxycoelonin (15) (Lu et al., 2010), 3,7-dihydroxy-2,4,6-trimethoxy-phenanthrene (16) (Lu et al., 2010), and 4,7-dihydro-2,6dimethoxy-9,10-dihydrophenanthrene (17) (Aquino et al., 1985) by comparing the 1H and 13C NMR, and MS spectra with the literatures. (Fig. 1). 3.2. Structures elucidation of new compounds Compound (1) was obtained as a colorless gum with positive optical rotation (½α25 D 711.0, EtOH). The molecular formula of 1 was determined to be C15H14O5 by positive mode HR-FAB-MS data at 257.0813 m/z [MþH H2O] þ (calcd for 257.0814). The 1H NMR spectrum of 1 showed the presence of seven aromatic protons at δH 7.52 (2H, m, H-2, 6), 7.44 (2H, m, H-3, 5), 7.39 (1H, m, H-4), 6.28 (1H, s, H-10), and 6.25 (1H, s, H-12), an oxygenated methine proton at δH 5.61 (1H, dd, J¼ 11.5, 3.0 Hz, H-7), a methylene proton at δH 3.25 (1H, m, H-8a) and 3.11 (1H, dd, J¼16.5, 3.5 Hz, H-8b). The 13C NMR spectrum (Table 1) displayed the appearance of 15 carbon signals, including a carbonyl carbon at δC 170.1, one oxygenated methine carbon at δC 80.4, one methylene carbon δC 34.7, and twelve aromatic carbons, which were classified by HMQC experiments. These NMR data were similar to those of 2-hydroxy-6-[2-hydroxy-2-(4-methoxyphenyl)ethyl]benzoic acid (Suzuki et al., 1979) but with apparent

3 4 5

1 (S) 8 6

8

7 2 9

7

OH

OH

COOH 14 OH

COOH OH

(R)

13

10

11

OH

12

OH 1

OH HO HO HO

O

O 2 OCH3 OH

H

O H3CO

OCH3

1'

OH 3

O R1

4

O

3

2 10 11

14 13 12

OCH3 OH O O

OH

1'

OH OH

HO

4

7

OH OCH3 OH

5

H

8

OH

H

H

6

Glc

9

OH

H

OH

OH

OH

H N

11

R2

R

10 OH

5

OCH3 H3CO

HO

R2 R

9

O

OCH3

O

H

RO

O

HO

R1

R

1

6

O

HO NH2

12

HO 13

OH HO

O

O 14

OH H3CO

HO H3CO

H3CO 15

HO

OH H3CO OCH3 16

OCH3

OH OH

OCH3

17

Fig. 1. Structures of compounds 1–17.

Please cite this article as: Wan Woo, K., et al., Phenolic derivatives from the rhizomes of Dioscorea nipponica and their antineuroinflammatory and neuroprotective activities. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.06.043i

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OH

COOH OH OH

COOH OH

OH OH

O

1 1

Table 3 Effects of compounds 1–17 on NGF secretion and cell viability in C6 cellsa.

O

2

1

Fig. 2. H– H COZY (bold lines) and HMBC (arrow) correlations for compounds 1 and 2.

OH HO HO HO

OCH3

O OH

O

3 1

HO H3CO

OCH3 OH O

O HO

OH OH OH

4

1

Fig. 3. H– H COZY (bold lines) and HMBC (arrow) correlations for compounds 3 and 4.

C-11 by HMBC cross-peaks of H-10 /C-11 (Fig. 3). Therefore, the structure of compound 3 was determined as shown and named diosniposide A. Compound (4) was obtained as a colorless gum. The molecular formula of 4 was determined to be C22H26O10 by positive mode HRFAB-MS data at 473.1422 m/z [MþNa] þ (calcd for 473. 1423). The 13 C NMR data of 4 showed 14 carbon signals, two aromatic rings and two methylenes, which were classified by the HMQC experiment suggesting 4 to be a phenanthrene (Kovacs et al., 2008). In addition, two methoxy signals [δH 3.90 (3H, s, 3-OCH3), and 3.73 (3H, s, 4-OCH3) in the 1H NMR; δC 62.8 and 61.7 in the 13C NMR] and glucose moiety [δH 4.84 (1H, d, J¼7.5 Hz, H-10 ), 3.88 (1H, dd, J¼12.0, 1.5 Hz, H-6a0 ), 3.72 (1H, m, H-6b0 ), 3.54 (1H, m, H-20 ), 3.47 (1H, m, H-30 ), 3.43(1H, m, H-50 ), and 3.42 (1H, m, H-40 ) in the 1H NMR; δC 104.4, 78.4, 77.8, 75.1, 71.5, and 62.7 in 13C NMR] were observed. Overall, the proton and carbon signals in 1H and 13C NMR spectra of 4 were very similar to those of icariside A2 (Miyase et al., 1988). The major differences were the position of anomeric proton and two methoxy signals. The connection of two methoxy groups was confirmed to be at C-3 and C-4, respectively by HMBC cross-peaks of 3-OCH3/C-3 and 4-OCH3/C-4, respectively. The sugar moiety is linked at C-12, which was confirmed by the HMBC correlation between C-12 (δC 147.9) and H-10 (δH 4.84). The coupling constant (J¼7.5 Hz) of the anomeric proton was indicated to be of β-form (Woo et al., 2012) (Fig. 3). From the above evidence, the structure of compound 4 was determined as shown and named diosniposide B. 3.3. Biological activities studies With our interest in searching for materials with neurotrophic activity, we measured the effects of isolates (1–17) on NGF secretion from C6 glial cells. Of the tested compounds, compound 16 was the potent stimulant of NGF release, with 162.35716.18% stimulation and compounds 4, 6, 9, and 17 also increased NGF level up to 140.8770.91%, 150.7870.01%, 142.3674.30%, and 147.93711.91%, respectiviely without cell toxicity (Table 3). To search for materials with anti-neuroinflammatory activity, we also measured the NO production in LPS-activated microglia BV-2 cell. Compound 16 significantly reduced NO level with IC50 values of 19.56 μM (Table 4). In addition, to study the effects of compound 16 on neurite outgrowth, we exposed compound 16 (5, 20 μM) and NGF (2 ng/ml) as positive control to N2a cells, finding morphological changes. Interestingly, each of compound 16 and NGF significantly stimulated

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b

Compounds

NGF secretion

Cell viability

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 6-shogaol

113.107 0.04 117.80 7 0.01 99.96 7 11.17 140.87 7 0.91 108.737 0.21 150.78 7 0.01 131.02 7 14.21 116.08 7 10.61 142.36 7 4.30 133.577 5.45 135.407 0.04 109.477 6.32 128.187 10.75 106.707 5.68 110.80 7 5.35 162.357 16.18 147.93 7 11.91 133.547 3.59

85.47 73.24 97.27 72.02 92.21 73.28 100.94 70.66 85.05 75.44 91.71 72.57 101.68 70.16 102.74 71.51 97.73 70.63 96.62 70.72 85.2474.88 96.27 70.44 96.71 71.05 102.11 70.14 97.7071.54 101.46 70.74 95.83 70.31 107.30 74.11

6-shogaol as a positive control.

a C6 cells were treated with 20 μM of compounds 1–17. After 24 h, the content of NGF secretion in C6-conditioned media was measured by ELISA, and the cell viability was determined by MTT assay. The level of secreted NGF and viable cells are expressed as percentages of the untreated control. The data shown represent the mean 7 SD of three independent experiments performed in triplicate.

Table 4 Inhibitory effect of compounds 1–17 on NO production in LPS-stimulated BV-2 cells. Compounds IC50 (μM)a

Cell viability (%)b

Compounds IC50 (μM)a

1 2 3 4 5 6 7 8 9

109.717 1.53 113.26 7 3.41 112.017 0.88 113.79 7 4.10 95.92 7 5.66 113.99 7 6.79 114.017 2.67 115.58 7 2.25 128.077 1.99

10 11 12 13 14 15 16 17 NMMAc

26.03 20.47 41.99 45.80 19.71 41.03 32.34 28.05 41.97

4500 43.18 4500 4500 19.31 19.41 19.56 63.55 16.27

Cell viability (%)b 127.747 4.31 123.377 3.30 111.86 7 3.22 113.577 0.88 117.277 1.89 115.89 7 1.18 112.137 4.08 108.577 2.00 98.2 7 2.6

a IC50 value of each compound was defined as the concentration (μM) that caused 50% inhibition of NO production in LPS-activated BV-2 cells. b Cell viability after treatment with 20 μM of each extract was expressed as percentage (%) of the LPS treatment group. The results are averages of three independent experiments, and the data are expressed as mean 7 SD. c NMMA as a positive control.

neurite outgrowth in N2a cells (Figs. 4 and 5). Compound 16 significantly induced NGF secretion in C6 cells, and decreased nitric Q4 oxide in LPS-induced activated microglia cells. It supports the fact that compound 16 had both neurotrophic effect and antineuroinflammation effect. Compound 16 may have a role in LPSinduced neuroinflammation in microglia by inhibition of c-Jun N-terminal kinases (JNK) signaling. But, this can directly or indirectly act on JNK signaling target. Additional research for that is needed. Especially, compound 16 will show a strong effect on neurological disorders which integrate neuroinflammation and neurotrophic factor. Namely, it is supposed that the interesting compound might represent a synergistic effect on neurodegenerative diseases. Excessive release of proinflammatory factors such as cytokines and nitric oxide (NO) from microglia can cause damage to neurons. (Jeohn et al., 2000). As a result, damaged neurons slowly die via apoptosis. But in this process, NGF can repair the damaged neurons back to normal condition (Apfel et al., 1994). In that respect, we want to emphasize the significance of our result.

Please cite this article as: Wan Woo, K., et al., Phenolic derivatives from the rhizomes of Dioscorea nipponica and their antineuroinflammatory and neuroprotective activities. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.06.043i

K. Wan Woo et al. / Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 Q6 Q5 55 56 57 58 59 60 61 62 63 64 65 66

Fig. 4. Effect of compound 16 on neurite morphology in N2a cells. Neurite length was measured at regular intervals over a time span of 72 h (a) and image of cells was taken at the end point (b) in N2a cells. In figure a, dot plot illustrates that the neurite length in N2a cells was treated by NGF and compound 16. Each point in the dot plot represents neurite length average value of treated N2a cells. To be measured by assaying neurite outgrowth of N2a cells, compound 16 (5 uM and 20 uM) was treated with NGF 2 ng/ml for 72 h. In the graph, each line indicated extent of neurite outgrowth of no treatment (purple), NGF 2 ng/ml (blue), compound 16 5 uM (blue-green), 20 uM (pink). In figure b, neurite length labels (red arrow) were described in the N2a cells for each group (  100) for 3 days. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

4. Conclusions This study shows that the rhizomes of Dioscorea nipponica contain the phenolic derivatives as bioactive components with neuroprotective activity and the observed effect supports the traditional use of the plant. Above all, 3,7-dihydroxy-2,4,6-trimethoxy-phenanthrene

(16) show significant neuroprotective activity through the increase of NGF secretion in C6 glioma cells and inhibition of NO production in lipopolysaccharide-activated BV-2 cells. In addition, it showed an increase of neurite outgrowth in N2a cells. Therefore, Dioscorea nipponica and its active components may be used as a potential agent for the regulation of neurodegenerative diseases.

Please cite this article as: Wan Woo, K., et al., Phenolic derivatives from the rhizomes of Dioscorea nipponica and their antineuroinflammatory and neuroprotective activities. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.06.043i

K. Wan Woo et al. / Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

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Uncited references (Shen et al., (2009)). Acknowledgments This research was supported by the Global Leading Technology Program of the Office of Strategic R&D Planning (OSP) funded by the Ministry of Knowledge Economy, Republic of Korea (10039303). We are thankful to the Korea Basic Science Institute (KBSI) for the measurements of NMR and MS spectra. References Abrams, M.B., Dominguez, C., Pernold, K., Reger, R., Wiesenfeld-Hallin, Z., Olson, L., Prockop, D., 2009. Multipotent mesenchymal stromal cells attenuate chronic inflammation and injury-induced sensitivity to mechanical stimuli in experimental spinal cord injury. Restorative Neurology and Neuroscience 27, 307–321. Apfel, S.C., Arezzo, J.C., Brownlee, M., Federoff, H., Kessler, J.A., 1994. Nerve growth factor administration protects against experimental diabetic sensory neuropathy. Brain Research 634, 7–12. Aquino, R., Behar, I., Simone, F.D., Pizza, C., 1985. Natural dihydrophenanthrene derivatives from Tamus communis. Journal of Natural Products 48, 811–813. Back, S.W., Kim, E.R., Kim, J.W., Kim, Y.C., 2011. Chemical constituents of Abies koreana leaves with inhibitory activity against nitric oxide production in BV2 microglia cells. Natural Product Sciences 17, 175–180. Fu, B., Li, H., Wang, X., Lee, F.S., Cui, S., 2005. Isolation and identification of flavonoids in licorice and a study of their inhibitory effects on tyrosinase. Journal of Agricultural and Food Chemistry 53, 7408–7414. Goshima, Y., Ohsako, S., Yamauchi, T., 1993. Overexpression of Ca2 þ/calmodulindependent protein kinase II in Neuro2a and NG108-15 neuroblastoma cell lines promotes neurite outgrowth and growth cone motility. Journal of Neuroscience 13, 559–567. Hedge, V.R., Dai, P., Ladislaw, C., Patel, M.G., Puar, M.S., Pachter, J.A., 1997. D4 dopamine receptor-selective compounds from the chines plant Phoebe chekiangensis. Bioorganic & Medicinal Chemistry Letters 7, 1207–1212. Jeohn, G.H., Kim, W.G., Hong, J.S., 2000. Time dependency of the action of nitric oxide in lipopolysaccharide-interferon-gamma-induced neuronal cell death in murine primary neuron-glia co-cultures. Brain Research 880, 173–177. Kim, N., Kim, S.H., Kim, Y.J., Kim, J.K., Nam, M.K., Rhim, H., Yoon Kim, S., Choi, S.Z., Son, M.W., Kim, S.Y., Kuh, H.J., 2011. Neutrophic activity of DA-9801, a mixture extract of Dioscorea japonica Thunb, and Dioscorea nipponica Makino, in vitro. Journal of Ethnopharmacology 137, 312–319. Kim, S., Choi, Y.K., Hong, J., Park, J., Kim, M.J., 2013. Candida antarctica lipase A and Pseudomonas stutzeri lipase as a pair of stereocomplementary enzymes for the resolution of 1,2-diarylethanols and 1,2-diarylethanamines. Tetrahedron Letters 54, 1185–1188. Kovacs, A., Vasas, A., Hohmann, J., 2008. Natural phenanthrenes and their biological activity. Phytochemistry 69, 1084–1110.

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Please cite this article as: Wan Woo, K., et al., Phenolic derivatives from the rhizomes of Dioscorea nipponica and their antineuroinflammatory and neuroprotective activities. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.06.043i

Phenolic derivatives from the rhizomes of Dioscorea nipponica and their anti-neuroinflammatory and neuroprotective activities.

Dioscorea nipponica (Dioscoreaceae) have been used as traditional medicines for diabetes, inflammatory and neurodegenerative diseases in Korea. The ai...
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