Wasabisides A E, Lignan Glycosides from the Roots of...

6
Wasabisides AE, Lignan Glycosides from the Roots of Wasabia japonica Chung Sub Kim, Lalita Subedi, ,§ Oh Wook Kwon, Hyun Bong Park, ,Sun Yeou Kim, ,§ Sang Un Choi, # and Kang Ro Lee* ,Natural Products Laboratory, School of Pharmacy, Sungkyunkwan University, Suwon 16419, Republic of Korea Gachon Institute of Pharmaceutical Science, Gachon University, Incheon 21936, Republic of Korea § College of Pharmacy, Gachon University, #191, Hambakmoero, Yeonsu-gu, Incheon 21936, Republic of Korea Natural F&P Corp., 152 Saemal-ro, Songpa-gu, Seoul 05802, Republic of Korea Department of Chemistry, Yale University, New Haven, Connecticut 06520, United States Chemical Biology Institute, Yale University, West Haven, Connecticut 06516, United States # Korea Research Institute of Chemical Technology, Daejeon 34114, Republic of Korea * S Supporting Information ABSTRACT: Five new lignan glycosides, wasabisides AE(15), and four known phenolic compounds (69), were isolated from the roots of Wasabia japonica. The chemical structures of the new compounds (15) were determined through spectroscopic analysis and chemical methods. All isolated compounds (19) were evaluated for their potential neuroprotective eects through induction of nerve growth factor in C6 glioma cells, for their eects on nitric oxide levels in lipopolysaccharide- stimulated murine microglia BV2 cells, and for their cytotoxicity against four human tumor cell lines (A549, SK-OV-3, SK-MEL- 2, and BT549). W asabi [Wasabia japonica (Miq.) Matsum., Brassicaceae] is a perennial herb cultivated in Korea and Japan. The paste of its roots has been used for a long time as a traditional Japanese pungent spice to garnish dishes such as sushi and sashimi. 1,2 The characteristic pungent avor of W. japonica is derived from several isothiocyanates, mainly allyl isothiocya- nate. Although 6-methylsulnylhexyl isothiocyanate and 6- methylthiohexyl isothiocyanate are derivatives of allyl iso- thiocyanate present in W. japonica, they are not pungent. 3 These isothiocyanate analogues are the major sulfur com- pounds in W. japonica and have various biological eects, including anticancer, anti-inammatory, antimicrobial, antiox- idant, and anti-blood-clotting activities. 4 In addition, previous investigations on W. japonica led to reports of several phenylpropanoid glycosides and avonoid glycosides with antioxidant activities. 2,5 Since most previous research on W. japonica has focused on the biological activities of these isothiocyanate compounds, an investigation into another chemical class possessing biological activity in W. japonica was pursued, namely, the lignan glycoside constituents. In a continuing search for bioactive constituents from Korean medicinal plants, ve new lignan glycosides, wasabisides AE (15), were isolated and characterized structurally, along with four known phenolic compounds (69) from the roots of W. japonica. The structures of the new compounds (15) were elucidated by NMR ( 1 H and 13 C NMR, 1 H1 H COSY, HSQC, HMBC, and NOESY), HRMS, ECD, and chemical methods. All isolated compounds (19) were evaluated for their cytotoxicity, potential neuroprotective activity, and eects on nitric oxide (NO) levels. To the best of our knowledge, this is the rst report of biologically active lignan derivatives from W. japonica. RESULTS AND DISCUSSION Wasabiside A (1) was isolated as a colorless gum. The molecular formula was determined to be C 26 H 32 O 12 from the [M + Na] + ion peak obtained by positive-ion HRFABMS. The Received: June 25, 2016 Published: October 4, 2016 Article pubs.acs.org/jnp © 2016 American Chemical Society and American Society of Pharmacognosy 2652 DOI: 10.1021/acs.jnatprod.6b00582 J. Nat. Prod. 2016, 79, 26522657

Transcript of Wasabisides A E, Lignan Glycosides from the Roots of...

Wasabisides A−E, Lignan Glycosides from the Roots of WasabiajaponicaChung Sub Kim,† Lalita Subedi,‡,§ Oh Wook Kwon,⊥ Hyun Bong Park,∥,¶ Sun Yeou Kim,‡,§

Sang Un Choi,# and Kang Ro Lee*,†

†Natural Products Laboratory, School of Pharmacy, Sungkyunkwan University, Suwon 16419, Republic of Korea‡Gachon Institute of Pharmaceutical Science, Gachon University, Incheon 21936, Republic of Korea§College of Pharmacy, Gachon University, #191, Hambakmoero, Yeonsu-gu, Incheon 21936, Republic of Korea⊥Natural F&P Corp., 152 Saemal-ro, Songpa-gu, Seoul 05802, Republic of Korea∥Department of Chemistry, Yale University, New Haven, Connecticut 06520, United States¶Chemical Biology Institute, Yale University, West Haven, Connecticut 06516, United States#Korea Research Institute of Chemical Technology, Daejeon 34114, Republic of Korea

*S Supporting Information

ABSTRACT: Five new lignan glycosides, wasabisides A−E (1−5), and four known phenolic compounds (6−9), were isolatedfrom the roots of Wasabia japonica. The chemical structures of the new compounds (1−5) were determined throughspectroscopic analysis and chemical methods. All isolated compounds (1−9) were evaluated for their potential neuroprotectiveeffects through induction of nerve growth factor in C6 glioma cells, for their effects on nitric oxide levels in lipopolysaccharide-stimulated murine microglia BV2 cells, and for their cytotoxicity against four human tumor cell lines (A549, SK-OV-3, SK-MEL-2, and BT549).

Wasabi [Wasabia japonica (Miq.) Matsum., Brassicaceae]is a perennial herb cultivated in Korea and Japan. The

paste of its roots has been used for a long time as a traditionalJapanese pungent spice to garnish dishes such as sushi andsashimi.1,2 The characteristic pungent flavor of W. japonica isderived from several isothiocyanates, mainly allyl isothiocya-nate. Although 6-methylsulfinylhexyl isothiocyanate and 6-methylthiohexyl isothiocyanate are derivatives of allyl iso-thiocyanate present in W. japonica, they are not pungent.3

These isothiocyanate analogues are the major sulfur com-pounds in W. japonica and have various biological effects,including anticancer, anti-inflammatory, antimicrobial, antiox-idant, and anti-blood-clotting activities.4 In addition, previousinvestigations on W. japonica led to reports of severalphenylpropanoid glycosides and flavonoid glycosides withantioxidant activities.2,5 Since most previous research onW. japonica has focused on the biological activities of theseisothiocyanate compounds, an investigation into anotherchemical class possessing biological activity in W. japonicawas pursued, namely, the lignan glycoside constituents.

In a continuing search for bioactive constituents from Koreanmedicinal plants, five new lignan glycosides, wasabisides A−E(1−5), were isolated and characterized structurally, along withfour known phenolic compounds (6−9) from the roots ofW. japonica. The structures of the new compounds (1−5) wereelucidated by NMR (1H and 13C NMR, 1H−1H COSY, HSQC,HMBC, and NOESY), HRMS, ECD, and chemical methods.All isolated compounds (1−9) were evaluated for theircytotoxicity, potential neuroprotective activity, and effects onnitric oxide (NO) levels. To the best of our knowledge, this isthe first report of biologically active lignan derivatives fromW. japonica.

■ RESULTS AND DISCUSSION

Wasabiside A (1) was isolated as a colorless gum. Themolecular formula was determined to be C26H32O12 from the[M + Na]+ ion peak obtained by positive-ion HRFABMS. The

Received: June 25, 2016Published: October 4, 2016

Article

pubs.acs.org/jnp

© 2016 American Chemical Society andAmerican Society of Pharmacognosy 2652 DOI: 10.1021/acs.jnatprod.6b00582

J. Nat. Prod. 2016, 79, 2652−2657

1H NMR spectrum of 1 showed the presence of two 1,3,4-trisubstituted aromatic rings [δH 6.89 (1H, brs), 6.77 (2H,overlap), 6.70 (1H, d, J = 8.0 Hz), 6.70 (1H, d, J = 1.8 Hz), and6.58 (1H, dd, J = 8.0, 1.8 Hz)], an oxygenated methine [δH 4.75(1H, d, J = 6.1 Hz)], an oxygenated methylene [δH 4.16 (1H,dd, J = 9.0, 7.3 Hz) and 3.99 (1H, t, J = 9.0 Hz)], two methines[δH 3.03 (1H, m) and 2.79 (1H, m)], two methoxy groups [δH3.85 (3H, s) and 3.82 (3H, s)], a methylene [δH 2.98 (1H, dd, J= 13.8, 5.6 Hz) and 2.91 (1H, dd, J = 13.8, 5.6 Hz)], and aglucopyranosyl unit [δH 4.37 (1H, d, J = 7.2 Hz), 3.69 (1H, dd,J = 11.8, 2.4 Hz), 3.58 (1H, dd, J = 11.8, 5.4 Hz), 3.33 (1H,overlap), 3.31 (2H, overlap), and 3.11 (1H, ddd, J = 9.1, 5.4,2.4 Hz)]. The 13C NMR spectrum of 1 displayed 26 carbonsignals including 12 aromatic carbons (from δC 111.9 to 149.0),a carbonyl carbon (δC 182.1), two oxygenated carbons (δC 83.8and 69.8), three methylene and methine carbons (δC 45.8, 44.9,and 35.8), two methoxy carbons [δC 56.5 (×2)], and a group ofglucopyranose carbons (δC 104.2, 78.6, 78.0, 76.0, 71.5, and62.8). These 1H and 13C NMR data of 1 (Table 1) were similarto those of (−)-7(S)-hydroxymatairesinol,6 except for thepresence of the glucopyranose signals (see above). The planarstructure of 1 was determined through 2D NMR analysis,including 1H−1H COSY, HSQC, and HMBC spectra. TheHMBC cross-peak of H-7/C-1″ indicated that the glucopyr-anosyl unit is located at C-7 (Figure 1), and the couplingconstant of the anomeric proton (J = 7.2 Hz) confirmed it asbeing in the β-form.7−10 Acid hydrolysis of 1 afforded D-glucopyranose, which was identified by its specific rotation{[α]D

25 +60.5 (c 0.01, H2O)} and GC/MS analysis.7−10 Therelative configuration of 1 was elucidated from NOESYcorrelations and enzymatic hydrolysis. In the NOESY spectrumof 1, strong and weak correlations of H-8/H-9b and H-9a,respectively, and a correlation of H-9a/H-8′ were observed,which confirmed that H-8 and H-8′ are in the trans form, withboth C-8 and C-8′ assigned as R* (Figure 1). A relatively smallcoupling constant value (6.6 Hz) between H-7 and H-8 of 1a,the hydrolysis product of 1, was identical to that of (−)-7(S)-hydroxymatairesinol (7S,8R,8′R; 6.6 Hz), rather than(−)-7(R)-hydroxymatairesinol (7R,8R,8′R; 7.8 Hz), whichcorroborated the relative configuration of 1 as 7S*,8R*,8′R*.6In the ECD spectrum of 1, a positive Cotton effect at 233 nmwas observed, indicating the absolute configuration of 1 to be8S,8′S.11,12 Thus, the structure of 1 was established as(7R,8S,8S′)-7-hydroxymatairesinol 7-O-β-D-glucopyranoside.Wasabiside B (2) was obtained as a colorless gum with the

same molecular formula as 1 (C26H32O12). Inspection of theNMR data of 2 indicated that this compound is structurallyquite similar to 1, with the major difference being an upfield-shifted carbon signal at C-7 (δC 74.7, 2; δC 83.8, 1), suggestingthat the glucopyranosyl unit is at a location other than C-7. The

HMBC correlation of H-1′/C-4 confirmed the location of theglucopyranosyl unit to be at C-4 (Figure 2). NMR analysis ofthe 1H−1H COSY, HSQC, and HMBC data corroborated theplanar structure of 2, and the absolute configuration of 2 wasdetermined to be the same as that of 1 through hydrolysis andthe observation of a positive Cotton effect at 233 nm in theECD spectrum. Thus, the structure of 2 was shown to be(7R,8S,8S′)-7-hydroxymatairesinol 4-O-β-D-glucopyranoside.Wasabiside C (3) gave a molecular formula of C27H34O13 as

established by HRFABMS. The 1H and 13C NMR data of 3resembled those of 2, except for the signals of a symmetric1,3,4,5-tetrasubstituted aromatic ring [δH 6.59 (2H, s); δC 154.3(×2), 140.9, 135.4, and 104.7 (×2)] instead of those of a 1,3,4-trisubstituted aromatic ring. Thus, 3 was observed to be a 5-methoxy derivative of 2, which was confirmed by the HMBCcorrelations of OCH3-5/C-5 (Figure 2). 2D NMR data analysiswas used to determine the planar structure and relativeconfiguration of 3. The absolute configuration of thiscompound was corroborated as being the same as 1 and 2 byhydrolysis and ECD spectroscopic analysis. Thus, the structureof 3 was determined as (7R,8S,8S′)-7-hydroxy-5-methoxyma-tairesinol 4-O-β-D-glucopyranoside.Wasabiside D (4) was purified as a colorless gum. The

molecular formula of this isolate was found to be the same asthat of 2 (C26H32O12) from the HRFABMS. The NMR data of4 were quite similar to those of 2, with the main differencesbeing that the signals for the aromatic protons in 4 [δH 7.02(1H, d, J = 8.2 Hz), 6.80 (1H, d, J = 1.7 Hz), 6.74 (1H, d, J =8.2 Hz), 6.71 (1H, dd, J = 8.2, 1.7 Hz), 6.66 (1H, dd, J = 8.2,1.9 Hz), and 6.65 (1H, d, J = 1.9 Hz)] were shifted slightlycompared to those of 2 [δH 7.11 (1H, d, J = 8.3 Hz), 6.84 (1H,dd, J = 8.3, 2.0 Hz), 6.83 (1H, d, J = 8.3 Hz), 6.64 (1H, d, J =8.0 Hz), 6.57 (1H, d, J = 2.0 Hz), and 6.45 (1H, dd, J = 8.0, 2.0Hz)]. This indicated the glucopyranose unit in 4 is located atC-4′. Furthermore, the HMBC cross-peak of H-1′/C-4′corroborated the location of the glucopyranose unit (Figure2). The planar structure of 4 was determined through 2D NMRdata analysis, and the relative and absolute configurations of 4were confirmed as being the same as those of 1−3 throughhydrolysis and ECD spectroscopic analysis. Thus, the structureof 4 was assigned as (7R,8S,8S′)-7-hydroxymatairesinol 4′-O-β-D-glucopyranoside.Wasabiside E (5) was obtained as a colorless gum with a

molecular formula of C26H30O12. The1H and 13C NMR spectra

of this compound resembled those of 1, but a quite downfield-shifted carbon signal at δC 198.1 was detected instead of thecarbon signal at δC 83.8 (C-7). The occurrence of a carbonylgroup at C-7 was confirmed through the HMBC cross-peaks ofH-2, H-6, H-8, H-9, and H-8′ to C-7, and the location of theglucopyranosyl unit was corroborated by the HMBCcorrelation as being between H-1″ and C-4 (Figure 3). Theβ- and D-form of the glucopyranosyl unit in compound 5 weredetermined through the same method as described for 1. Therelative configuration between H-8 and H-8′ was confirmed astrans by the NOESY cross-peaks of H-8/H-2′ and H-7′b(Figure 3). A negative Cotton effect at 233 nm was observed inthe ECD spectrum of 5, indicating that the absoluteconfiguration of this compound is 8R,8′R.11,12 Therefore, thestructure of 5 was elucidated as (8R,8′R)-7-oxomatairesinol 4-O-β-D-glucopyranoside.The four known compounds isolated (6−9) were identified

as trans-p-coumaric acid (6),13 trans-ferulic acid (7),14 benzoic

Chart 1

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DOI: 10.1021/acs.jnatprod.6b00582J. Nat. Prod. 2016, 79, 2652−2657

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Table

1.1 H

[ppm

,mult.,

(Jin

Hz)]and

13C

NMRSpectroscopicDataof

Com

poun

ds1−

5in

CD

3OD

12

34

5

positio

nδ C

δ Hδ C

δ Hδ C

δ Hδ C

δ Hδ C

δ H

1132.3

138.8

140.9

135.6

132.0

2111.9

6.89,b

rs111.4

6.83,d

(2.0)

104.7

6.59,s

110.7

6.80,d

(1.7)

112.6

7.32,d

(2.1)

3149.0

150.8

154.3

149.1

150.9

4147.6

147.4

135.4

147.1

153.0

5115.9

6.77,o

verlap

117.5

7.11,d

(8.3)

154.3

116.1

6.74,d

(8.2)

116.1

7.14,d

(8.5)

6121.1

6.77,o

verlap

119.6

6.84,d

d(8.3,2

.0)

104.7

6.59,s

119.7

6.71,d

d(8.2,1

.7)

124.3

7.29,d

d(8.5,2

.1)

783.8

4.75,d

(6.1)

74.7

4.72,d

(4.7)

74.5

4.72,d

(4.3)

75.0

4.67,d

(5.1)

198.1

845.8

2.79,m

46.7

2.65,m

46.7

2.64,m

46.9

2.62,m

48.5

4.35,q

(8.2)

9a69.8

4.16,d

d(9.0,7

.3)

71.1

4.16,o

verlap

71.2

4.23,o

verlap

70.9

4.16,d

d(9.2,6

.2)

70.3

4.56,d

d(8.7,8

.2)

9b3.99,t

(9.0)

4.11,t

(8.7)

4.16,d

d(8.7,8

.2)

1′130.7

130.7

130.6

134.2

130.4

2′114.5

6.70,d

(1.8)

114.1

6.57,d

(2.0)

114.0

6.60,d

(1.8)

114.9

6.65,d

(1.9)

113.9

6.68,d

(1.3)

3′149.0

148.9

148.9

150.7

149.1

4′146.5

146.4

146.4

146.9

146.6

5′116.2

6.70,d

(8.0)

116.2

6.64,d

(8.0)

116.2

6.62,d

(8.0)

117.6

7.02,d

(8.2)

116.4

6.59,d

(8.0)

6′123.5

6.58,d

d(8.0,1

.8)

123.2

6.45,d

d(8.0,2

.0)

123.2

6.36,d

d(8.0,1

.8)

123.4

6.66,d

d(8.2,1

.9)

123.1

6.58,d

d(8.0,1

.3)

7′a

35.8

2.98,d

d(13.8,

5.6)

36.4

2.85,d

d(13.5,

5.2)

36.3

2.83,d

d(13.8,

7.1)

36.3

2.92,d

d(13.3,

6.6)

36.3

3.16,d

d(14.0,

4.9)

7′b

2.91,d

d(13.8,

5.6)

2.76,o

verlap

2.74,d

d(13.8,

5.3)

2.81,d

d(13.3,

5.0)

2.78,d

d(14.0,

9.6)

8′44.9

3.03,m

44.2

2.90,o

verlap

43.9

2.91,o

verlap

44.3

2.94,m

47.5

3.44,o

verlap

9′182.1

182.5

182.5

182.3

180.0

OCH

3-3

56.5

3.85,s

56.7

3.82,s

57.0

3.83,s

56.5

3.82,s

56.8

3.88,s

OCH

3-5

57.0

3.83,s

OCH

3-3′

56.5

3.82,s

56.5

3.78,s

56.4

3.78,s

56.7

3.80,s

56.3

3.69,s

1″104.2

4.37,d

(7.2)

102.9

4.93,d

(7.5)

105.5

4.90,d

(7.5)

103.0

4.89,d

(7.3)

102.0

5.04,d

(7.6)

2″76.0

3.31,o

verlap

75.1

3.52,o

verlap

75.9

3.47,o

verlap

75.0

3.51,o

verlap

74.9

3.55,d

d(9.2,7

.6)

3″78.6

3.33,o

verlap

78.0

3.50,o

verlap

77.9

3.51,o

verlap

78.0

3.49,o

verlap

78.0

3.52,o

verlap

4″71.5

3.31,o

verlap

71.5

3.42,o

verlap

71.4

3.45,o

verlap

71.5

3.41,o

verlap

71.4

3.43,o

verlap

5″78.0

3.11,d

dd(9.1,5

.4,2

.4)

78.3

3.44,o

verlap

78.4

3.28,d

dd(9.3,5

.0,2

.3)

78.3

3.43,d

dd(9.7,5

.3,2

.1)

78.5

3.52,o

verlap

6″a

62.8

3.69,d

d(11.8,

2.4)

62.7

3.91,d

d(12.1,

2.0)

62.7

3.79,d

d(12.1,

2.3)

62.7

3.90,d

d(12.1,

2.1)

62.6

3.95,d

d(12.1,

2.2)

6″b

3.58,d

d(11.8,

5.4)

3.73,d

d(12.1,

5.2)

3.69,d

d(12.1,

5.0)

3.71,d

d(12.1,

5.3)

3.74,d

d(12.1,

5.8)

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acid (8),15 and syringic acid (9)16 by comparison with NMRand MS data in the literature.Many phenolic compounds were reported to be associated

with neuroprotective activity, as measured by secretion of nervegrowth factor (NGF) from C6 glioma cells in a previousstudy.7−11,17,18 Therefore, the NGF secretion effects weretested for compounds 1−9 using an enzyme-linked immuno-sorbent assay (ELISA) development kit. NGF release wasmeasured into the medium and cell viability with an MTTassay. As shown in Table 2, compound 1 induced NGFsecretion moderately by 150.7%, without producing a cytotoxiceffect at 20 μM. The other compounds evaluated showed weakactivity (99.4−129.2%).To investigate the effect of the isolated compounds (1−9)

on neuroinflammation, NO levels were measured in murinemicroglia BV2 cells stimulated with lipopolysaccharide (LPS).However, none of the compounds showed any activity (IC50 >50 μM) in this assay.The cytotoxic activities of compounds 1−9 were evaluated

against the A549 (non-small-cell lung adenocarcinoma), SK-OV-3 (ovary malignant ascites), SK-MEL-2 (skin melanoma),and BT549 (invasive ductal carcinoma) cell lines using thesulforhodamine B (SRB) bioassay. Compound 6, trans-p-coumaric acid, showed cytotoxic activity against the BT549 cellline, with an IC50 value of 10 μM. The other compounds testedwere inactive (IC50 > 10 μM) for all cancer cell lines used.

■ EXPERIMENTAL SECTIONGeneral Experimental Procedures. Optical rotations were

measured on a JASCO P-1020 polarimeter (JASCO, Easton, MD,USA). IR spectra were recorded on a Bruker IFS-66/S Fourier-transform IR spectrometer (Bruker, Karlsruhe, Germany). UV spectrawere recorded with a Shimadzu UV-1601 UV−visible spectropho-tometer (Shimadzu, Tokyo, Japan). ECD spectra were recorded with aJASCO J-810 spectropolarimeter (JASCO, Tokyo, Japan). NMRspectra were recorded on a Bruker AVANCE III 700 NMRspectrometer at 700 MHz (1H) and 175 MHz (13C). HRFABMSand HRESIMS were measured on either a Waters SYNAPT G2(Milford, MA, USA) or a JEOL JMS700 mass spectrometer (JEOL,Peabody, MA, USA). The semipreparative HPLC system used had a

Gilson 306 pump (Middleton, WI, USA) with a Shodex refractiveindex detector (New York, NY, USA). Column chromatography wasperformed with silica gel 60 (70−230 and 230−400 mesh; Merck,Darmstadt, Germany) and RP-C18 silica gel (Merck, 230−400 mesh).Merck precoated silica gel F254 plates and RP-18 F254s plates (Merck)were used for thin-layer chromatography (TLC). Spots were detectedon TLC under UV light or by heating after spraying the samples withanisaldehyde-sulfuric acid.

Plant Material. The roots ofW. japonica (3.3 kg) were collected inHanam, Republic of Korea, in October 2014. The plant was identifiedby one of the authors (K.R.L.). A voucher specimen (SKKU-NPL1409) of the plant has been deposited at the herbarium of the Schoolof Pharmacy, Sungkyunkwan University, Suwon, Republic of Korea.

Extraction and Isolation. The roots of W. japonica (3.3 kg) wereextracted with 80% aqueous MeOH at room temperature and filtered.The filtrate was evaporated under reduced pressure to obtain theMeOH extract (750 g), which was suspended in distilled H2O andsuccessively partitioned with hexane, CHCl3, EtOAc, and n-BuOH,yielding 1.3, 5.6, 5.8, and 2.7 g of each residue, respectively. TheEtOAc-soluble fraction (5.8 g) was separated by passage over DiaionHP-20 resin by elution with MeOH−H2O (0:1, 1:4, 2:3, 3:2, 4:1, and

Figure 1. 1H−1H COSY (bold), HMBC (plain arrows), and NOESY(dashed) correlations of compound 1.

Figure 2. Key HMBC (plain arrows) correlations of compounds 2−4.

Figure 3. 1H−1H COSY (bold), HMBC (plain arrow), and NOESY(dashed) correlations of compound 5.

Table 2. Effects of Compounds 1−9 on NGF Secretion in C6Cells

compound NGF secretiona (%) cell viabilityb (%)

1 150.7 100.4 ± 2.552 103.3 98.2 ± 3.473 99.4 101.6 ± 2.714 104.3 102.3 ± 6.735 122.1 100.9 ± 7.216 129.2 198.0 ± 4.837 110.7 95.0 ± 0.918 114.5 96.4 ± 1.989 125.5 95.9 ± 3.496-shogaolc 165.7 115.3 ± 6.32

aC6 cells were treated with 20 μM of each compound. After 24 h, thecontent of NGF secreted in the C6-conditioned medium wasmeasured by ELISA. The level of secreted NGF is expressed as thepercentage of the untreated control (set as 100%). bCell viability aftertreatment with 20 μM of each compound was determined by an MTTassay and is expressed as a percentage (%). Results are the means ofthree independent experiments, and the data are expressed as mean ±SD. cPositive control substance.

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1:0), to give six fractions (E1−E6). Fraction E4 (200 mg) wasseparated over a silica gel column (CHCl3−MeOH−H2O, 3:1:0.15) toyield nine subfractions (E41−E49). Subfraction E43 (25 mg) waspurified by semipreparative HPLC (2 mL/min, 25% aqueous MeCN)to give compound 6 (2 mg). Subfraction E46 (40 mg) was purified bysemipreparative HPLC (CHCl3−MeOH−H2O, 6:1:0.1) to givecompound 1 (10 mg). Fraction E5 (160 mg) was chromatographedon a silica gel column (CHCl3−MeOH−H2O, 3:1:0.15) to yield fivesubfractions (E51−E55). Compounds 7 (2 mg), 8 (2 mg), and 9 (2mg) were obtained by purifying subfraction E52 (17 mg) usingsemipreparative HPLC (45% aqueous MeOH). Subfraction E55 (13mg) was purified by semipreparative HPLC (45% aqueous MeOH) togive compound 5 (4 mg). The n-BuOH-soluble fraction (2.7 g) wasseparated over Diaion HP-20 resin with MeOH−H2O (0:1 and 1:0)and further purified over a silica gel column (CHCl3−MeOH−H2O,3:1:0.1) to yield 10 subfractions (B1−B10). Subfraction B3 (250 mg)was subjected to passage over a Lobar-A RP-C18 column with 40%aqueous MeOH and further purified by semipreparative HPLC(EtOAc−MeOH−H2O, 7:1:0.1) to give compounds 2 (6 mg) and 3(7 mg). Subfraction B4 (300 mg) was separated using a Lobar-A RP-C18 column with 20% aqueous MeOH and further purified bysemipreparative HPLC (35% aqueous MeOH) to give compound 4 (1mg).Wasabiside A (1): colorless gum; [α]D

25 +9.6 (c 0.5, MeOH); IR(KBr) νmax 3358, 2945, 2832, 1452, 1033 cm

−1; UV (MeOH) λmax (logε) 280 (1.11), 232 (2.17) nm; CD (MeOH) λmax (Δε) 282 (−2.29),233 (+6.91), 214 (−2.77) nm; 1H (700 MHz) and 13C (175 MHz)NMR data in CD3OD, see Table 1; HRFABMS (positive-ion mode)m/z 559.1789 [M + Na]+ (calcd for C26H32O12Na, 559.1791).Wasabiside B (2): colorless gum; [α]D

25 +30.4 (c 0.01, MeOH); IR(KBr) νmax 3360, 2955, 2830, 1460, 1030 cm

−1; UV (MeOH) λmax (logε) 284 (1.17), 231 (2.14) nm; CD (MeOH) λmax (Δε) 281 (−2.18),233 (+6.83), 213 (−2.75) nm; 1H (700 MHz) and 13C (175 MHz)NMR data in CD3OD, see Table 1; HRFABMS (positive-ion mode)m/z 559.1792 [M + Na]+ (calcd for C26H32O12Na, 559.1791).Wasabiside C (3): colorless gum; [α]D

25 +10.3 (c 0.3, MeOH); IR(KBr) νmax 3358, 2948, 2831, 1454, 1035 cm

−1; UV (MeOH) λmax (logε) 280 (1.15), 234 (2.20) nm; CD (MeOH) λmax (Δε) 284 (−2.25),234 (+6.61), 214 (−2.87) nm; 1H (700 MHz) and 13C (175 MHz)NMR data in CD3OD, see Table 1;

1H NMR (pyridine-d5, 700 MHz)δ 7.65 (1H, d, J = 3.3 Hz, OH-7), 7.17 (1H, d, J = 8.0 Hz, H-5′), 7.02(1H, d, J = 1.4 Hz, H-2′), 6.97 (2H, s, H-2 and H-6), 6.87 (1H, dd, J =8.0, 1.4 Hz, H-6′), 5.82 (1H, d, J = 6.7, H-1″), 5.03 (1H, t, J = 4.0 Hz,H-7), 4.41 (1H, dd, J = 12.0, 2.0 Hz, H-6″a), 4.35 (1H, overlap, H-2″),4.34 (3H, overlap, H-9a, H-4″ and H-5″), 4.33 (1H, overlap, H-6″b),4.17 (1H, t, J = 8.6 Hz, H-9b), 3.97 (1H, m, H-3″), 3.78 (6H, s,OCH3-3 and OCH3-5), 3.77 (3H, s, OCH3-3′), 3.44 (1H, m, H-8′),3.30 (1H, dd, J = 13.8, 5.5 Hz, H-7′a), 3.08 (1H, dd, J = 13.8, 5.7 Hz,H-7′b), 2.97 (1H, m, H-8); 13C NMR (pyridine-d5, 175 MHz) δ 180.4(C-9′), 154.3 (C-3 and C-5), 149.0 (C-3′), 147.4 (C-4′) 140.6 (C-1),135.7 (C-4), 129.9 (C-1′), 123.6 (C-5′), 116.8 (C-6′), 114.6 (C-2′),105.5 (C-1″), 105.2 (C-2 and C-6), 79.2 (C-3″), 78.9 (C-5″), 76.6 (C-2″), 74.3 (C-7), 72.1 (C-4″), 69.9 (C-9), 63.1 (C-6″), 57.0 (OCH3-3and OCH3-5), 56.3 (OCH3-3′), 46.4 (C-8), 43.8 (C-8′), 35.9 (C-7′);HRFABMS (positive-ion mode) m/z 589.1899 [M + Na]+ (calcd forC27H34O13Na, 589.1897).Wasabiside D (4): colorless gum; [α]D

25 +57.7 (c 0.5, MeOH); IR(KBr) νmax 3362, 2952, 2832, 1451, 1035 cm

−1; UV (MeOH) λmax (logε) 280 (1.12), 232 (2.20) nm; CD (MeOH) λmax (Δε) 282 (−2.30),233 (+6.99), 212 (−2.71) nm; 1H (700 MHz) and 13C (175 MHz)NMR data in CD3OD, see Table 1; HRFABMS (positive-ion mode)m/z 559.1790 [M + Na]+ (calcd for C26H32O12Na, 559.1791).Wasabiside E (5): colorless gum; [α]D

25 +70.0 (c 0.003, MeOH); IR(KBr) νmax 3401, 2941, 2832, 1613, 1449 cm

−1; UV (MeOH) λmax (logε) 274 (1.16), 230 (2.73) nm; CD (MeOH) λmax (Δε) 316 (+2.55),274 (−2.45), 233 (−3.87), 217 (+4.79) nm; 1H (700 MHz) and 13C(175 MHz) NMR data in CD3OD, see Table 1; HRESIMS (positive-ion mode) m/z 535.1812 [M + H]+ (calcd for C26H31O12, 535.1816).Enzymatic Hydrolysis of Compounds 1−5. A solution of each

sample (0.5−1.0 mg) in H2O (1.5 mL) was individually hydrolyzed

with cellulase (20 mg, from Aspergillus niger; ICN Biomedicals, Inc.) at37 °C for 24 h. Each reaction mixture was extracted with EtOAc toyield 0.3−0.5 mg of 1a (from 1, 2, and 4), 3a (from 3), and 5a (from5).

(−)-7(S)-Hydroxymatairesinol (1a): colorless gum; [α]D25 −15.0 (c

0.01, CHCl3);1H NMR (CDCl3, 700 MHz) δ 6.89 (1H, d, J = 8.1

Hz), 6.81 (1H, d, J = 7.7 Hz), 6.75 (1H, dd, J = 8.1, 1.7 Hz), 6.70 (1H,d, J = 1.6 Hz), 6.63 (2H, overlap), 5.67 (1H, s), 5.55 (1H, s), 4.66(1H, d, J = 6.6 Hz), 3.97 (2H, overlap), 3.88 (3H, s), 3.84 (3H, s),3.05 (1H, m), 2.95 (2H, overlap), 2.63 (1H, m); FABMS (positive-ionmode) m/z 375.1 [M + H]+.

(−)-7(S)-Hydroxy-5-methoxymatairesinol (3a): colorless gum;[α]D

25 −32.0 (c 0.01, CHCl3);1H NMR (CD3OD, 700 MHz) δ 6.61

(1H, d, J = 7.9 Hz), 6.49 (1H, d, J = 2.0 Hz), 6.48 (2H, s), 6.42 (1H,dd, J = 7.9, 2.0 Hz), 4.65 (1H, d, J = 4.7 Hz), 4.16 (1H, dd, J = 9.1, 5.8Hz), 4.12 (1H, dd, J = 9.1, 8.1 Hz) 3.78 (6H, s), 3.72 (3H, s) 2.87(1H, dt, J = 6.9, 5.6 Hz), 2.80 (1H, dd, J = 13.7, 6.9 Hz), 2.73 (1H, dd,J = 13.7, 5.5 Hz), 2.58 (1H, m); FABMS (positive-ion mode) m/z405.1 [M + H]+.

(+)-7-Oxomatairesinol (5a): colorless gum; [α]D25 +37.0 (c 0.01,

CHCl3);1H NMR (CDCl3, 700 MHz) δ 7.38 (1H, d, J = 2.1 Hz), 7.26

(1H, dd, J = 8.4, 2.2 Hz), 6.85 (1H, d, J = 8.4 Hz), 6.76 (1H, d, J = 8.0Hz), 6.65 (1H, d, J = 1.9 Hz), 6.58 (1H, dd, J = 8.0, 1.8 Hz), 5.52 (s,1H), 4.41 (1H, m), 4.13 (2H, overlap), 3.94 (3H, s), 3.78 (3H, s), 3.53(1H, m), 3.04 (2H, overlap); FABMS (positive-ion mode) m/z 373.1[M + H]+.

Acid Hydrolysis of Compounds 1−5 and Sugar Analysis.Compounds 1−5 (each 0.5−1.0 mg) were refluxed with 1 mL of 1 NHCl for 1 h at 100 °C. The hydrolysate was extracted with EtOAc, andthe aqueous layer was neutralized by passage through an AmberliteIRA-67 column (Rohm and Haas) and was repeatedly evaporated togive D-glucopyranose {[α]D

25 +60.5 (c 0.05, H2O)}, which was detectedby co-injection of the hydrolysate with standard silylated samples,giving a single peak at 9.721 min by GC-MS analysis under thefollowing conditions: capillary column, HP-5MS UI (30 m × 0.25 mm× 0.25 μm, Agilent), column temperature, 230 °C; injectiontemperature, 250 °C; carrier gas, N2. An authentic sample of D-glucopyranose (Sigma) treated in the same way showed a single peakat 9.726 min.

NGF and Cell Viability Assays. C6 glioma cells (purchased fromthe Korean Cell Line Bank, Seoul, Korea) were used to measure therelease of NGF into the culture medium. C6 cells were seeded onto24-well plates at a density of 1 × 105 cells/well, and, after 24 h, thecells were treated with serum-free DMEM and different concentrationsof compound for an additional 24 h. The medium supernatant wascollected from the culture plates, and NGF levels were measured usingan ELISA development kit. Cell viability was measured using a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay.The results are expressed as a percentage of the control group(untreated cells). 6-Shogaol was used as the positive control.

Measurement of NO Production and Cell Viability in LPS-Activated BV-2 Cells. The inhibitory effect of the test compounds onLPS-stimulated NO production was studied using BV2 cells. BV2 cellswere originally developed by Dr. V. Bocchini at the University ofPergia (Pergia, Italy). The cells were seeded on a 96-well plate (4 ×104 cells/well) and treated with or without different concentrations ofthe compounds. These cells were stimulated with LPS (100 ng/mL)and incubated for 24 h. The concentration of nitrite (NO2), a solubleoxidation product of NO, in the culture medium was measured usingGries reagent (0.1% N-1-naphthylethylenediamine dihydrochlorideand 1% sulfanilamide in 5% phosphoric acid). Fifty microliters ofsupernatant was mixed with an equal volume of Gries reagent.Absorbance was measured after 10 min using a microplate reader(Emax, Molecular Devices, Sunnyvale, CA, USA) at 570 nmwavelength. NG-Monomethyl-L-arginine, a well-known nitric oxidesynthase inhibitor, was used as a positive control (IC50 17.6 μM).Graded sodium nitrite solution was used as a standard to calculatenitrite concentrations. Cell viability was evaluated by observing theability of viable cells to reduce the yellow-colored MTT to purple-colored formazan in the MTT assay.

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Cytotoxicity Assessment. The cytotoxicity of the compoundsagainst the cultured human tumor cell lines A549, SK-OV-3, SK-MEL-2, and BT549 was evaluated by the SRB method.19 All the cells testedwere purchased from the American Type Culture Collection(Manassas, VA, USA) and maintained at the Korea Research Instituteof Chemical Technology. Cisplatin was used as the positive control.This compound exhibited IC50 values of 1.12, 1.82, 1.27, and 1.25 μMagainst the A549, SK-OV-3, SK-MEL-2, and BT549 cell lines,respectively.

■ ASSOCIATED CONTENT*S Supporting InformationThe Supporting Information is available free of charge on theACS Publications website at DOI: 10.1021/acs.jnat-prod.6b00582.

1D and 2D NMR data of 1−5; CD data of 1 and 5(PDF)

■ AUTHOR INFORMATIONCorresponding Author*Tel (K. R. Lee): 82-31-290-7710. Fax: 82-31-290-7730. E-mail: [email protected] authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThis work was supported by the Postdoctoral ResearchProgram of Sungkyunkwan University (2015). We are thankfulto the Korea Basic Science Institute (KBSI) for the measure-ments of mass spectra.

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