Bioactive Glycosides from the Twigs of Litsea cubeba - ACS Publications

Article pubs.acs.org/jnp

Bioactive Glycosides from the Twigs of Litsea cubeba Ling-Yan Wang,†,⊥ Ming-Hua Chen,‡,⊥ Jiang Wu,§ Hua Sun,† Wei Liu,† Yu-Hong Qu,† Yan-Cheng Li,† Yu-Zhuo Wu,† Rui Li,† Dan Zhang,† Su-Juan Wang,† and Sheng Lin*,† †

State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, and ‡Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, People’s Republic of China § College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, People’s Republic of China S Supporting Information *

ABSTRACT: The air-dried twigs of Litsea cubeba, a traditional Chinese medicinal tree, afforded 10 new aromatic glycosides (1− 10) and 26 known analogues. Their structures were assigned by extensive 1D and 2D NMR experiments, and the absolute configurations were resolved by chemical methods, electronic circular dichroism, specific rotation, and X-ray crystallographic analysis. Compound 4 is the first example of a naturally occurring homoneolignan glucoside. Compounds 4, 6−8, and the known neolignan glucosides (11, 12, and 14) at respective 10 μM concentrations were found to reduce acetaminophen-induced HepG2 cell injury with 30.5−46.0% inhibitions. Furthermore, compounds 12 and 15 demonstrated moderate inhibitory activities against HDAC1, with IC50 values of 3.6 and 4.6 μM, respectively. 26 known glycosides. Compound 4 is the first example of a naturally occurring homoneolignan glucoside. Reported below are the isolation and structural elucidation of the 10 new compounds, 1−10, in addition to the biological assessment of the isolates.

P

lants from the Litsea species (Lauraceae) are sources of secondary metabolites that have significant bioactivities and interesting chemical structures. Litsea cubeba is mainly grown in the east and south of China. The fruits and roots of L. cubeba, known as “bi-cheng-qie” and “dou-chi-jiang” as recorded in the Chinese Pharmacopoeia (2015 edition) and Chinese materia medica, respectively, are two important traditional Chinese medicines for treating various ailments including cerebral apoplexy, coronary disease, rheumatic arthritis, and asthma.1 The chemical constituents of the fruits and roots of L. cubeba have been extensively studied, resulting in the identification of aporphine-type alkaloids, lignans, phenolic constituents, and essential oils.2 Some of these aporphine-type alkaloids and lignans exhibit antithrombotic, anti-inflammatory, and antinociceptive properties2d,3 and thus may be considered as major active components of this plant. To date, there has been only one report that describes some amides and sterols derived from the twigs of L. cubeba.4 As part of an ongoing investigation on the biological and chemical diversity of traditional Chinese medicines, the current study was launched to search for novel bioactive secondary metabolites in the twigs of L. cubeba and led to the characterization of 10 new aromatic glycosides (1−10) and © 2017 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION The EtOH extract of the twigs of L. cubeba was processed by solvent partitioning between EtOAc and H2O, in which the aqueous phase was separated systematically by column chromatography followed by RP C18 HPLC to afford 10 new glycosides (1−10) and 26 known compounds. Compound 1 was a white, amorphous powder that showed [α]20 D −18 (c 0.5, MeOH). Infrared (IR) absorption bands at 3394, 1647, 1521, and 1468 cm−1 illustrated the presence of conjugated carbonyl, hydroxy, and aromatic ring functional groups. Its molecular formula was shown to be C23H34O11 based on the [M − H]− ion at m/z 485.2031 in the HRESIMS. Of the seven indices of hydrogen deficiency, five were assigned Received: January 3, 2017 Published: May 25, 2017 1808

DOI: 10.1021/acs.jnatprod.6b01189 J. Nat. Prod. 2017, 80, 1808−1818

Journal of Natural Products

Article

Chart 1

to a 1,2,4-substituted aromatic nucleus with a β-glucopyranosyl and an aromatic methoxy unit due to the characteristic 1H and 13 C NMR signals at δH 6.36 (1H, d, J = 3.0 Hz, H-3), 6.83 (d, J = 8.5 Hz, H-5), 6.16 (dd, J = 8.5, 3.0 Hz, H-6), 4.56 (1H, d, J = 7.0 Hz, H-1′), 3.31 (1H, dd, J = 7.0, 8.5 Hz, H-2′), 3.28 (1H, dd, J = 8.5, 9.0 Hz, H-3′), 3.25 (1H, dd, J = 9.5, 9.0 Hz, H-4′), 3.43 (1H, ddd, J = 9.5, 6.5, 1.5 Hz, H-5′), 4.29 (1H, dd, J = 12.0, 1.5 Hz, H-6a′), 4.17 (1H, dd, J = 12.0, 6.5 Hz, H-6b′), and 3.70 (3H, s, OMe); δC 141.1 (C-1), 152.6 (C-2), 102.1 (C-3), 155.3 (C-4), 107.8 (C-5), 121.1 (C-6), 104.6 (C-1′), 75.3 (C2′), 78.1 (C-3′), 72.1 (C-4′), 75.9 (C-5′), 64.2 (C-6′), and 56.8 (OMe). The sugar obtained by the acid hydrolysis of 1 with 1 N HCl was shown to be D-glucose using GC comparison of its trimethylsilyl-L-cysteine derivative with that of the D-glucose standard.5 The remaining two indices of hydrogen deficiency, combined with the NMR signals ascribed to one trisubstituted double bond [δH 5.64 (s, H-2″); δC 116.6 (C-2″) and 162.7 (C3″)], an olefinic methyl singlet [δH 2.03 (H3-10″); δC 19.5 (C10″)], two methyl singlets [δH 1.05, 1.09 (H3-8″ and H3-9″); δC 25.0, 26.3 (C-8″ and C-9″)], one oxygenated methine at δH 3.14 (d, J = 10.5 Hz, H-6″), two methylenes [δH 2.12, 2.34 (each 1H, m, H2-4″) and 1.35, 1.73 (each 1H, m, H2-5″); δC 39.5, 30.6 (C-4″ and C-5″)], one oxygenated tertiary carbon at δC 74.1, and one ester carbon at δC 168.4, suggested the

presence of a linear monoterpenoidal ester moiety in 1. Comprehensive analyses of the 2D NMR data facilitated the construction of a 6,7-dihydroxy-3,7-dimethyl-2-octenoyl moiety in 1, as indicated by 1H−1H COSY correlations between H-2″/ H3-10″ and H2-4″/H2-5″/H-6″, as well as key HMBC crosspeaks from H3-10″ to C-1″, C-2″, and C-4″, from both H3-8″ and H3-9″ to C-6″ and C-7″, from H-6″ to C-4″, and from H2″ to C-4″ and C-10″. In addition, HMBC cross-peaks of the methoxy protons to C-2, H-3 to C-1 and C-5, H-5 to C-1 and C-3, and H-6 to C-2 and C-4, indicating that 1 contained a 2methoxyhydroquinone aglycone moiety, were observed. Finally, the connectivity between the 2-methoxyhydroquinone, β-Dglucopyranosyl, and 6,7-dihydroxy-3,7-dimethyl-2-octenoyl moiety was based on the HMBC cross-peaks from H-1′ and H2-6′ to C-1 and C-1″, respectively, thereby completing the 2D structure of 1. The stereochemistry of the 6,7-dihydroxy-3,7-dimethyl-2octenoyl moiety in 1 was confirmed via chemical methods and X-ray crystallography. Alkaline hydrolysis of 1 afforded 6,7dihydroxy-3,7-dimethyl-2-octenoic acid (1a) (Figures S10− S12, Supporting Information). Suitable crystals of 1a were acquired by a vapor diffusion protocol using MeOH, and the structure of 1a, including its absolute configuration, was then readily assigned by X-ray crystallographic analyses using the 1809



Article

anomalous scattering of Cu Kα radiation (Figure 1). Therefore, 1 was defined as (−)-1-O-{6-O-[(6R,3E)-6,7-dihydroxy-3,7dimethyl-2-octenoyl]}-β-D-glucopyranosyl-2-methoxyhydroquinone.

D-glucopyranosyloxycatechin, which was also obtained in the present investigation. A Δ2‴(3‴) E-double bond was assigned according to the NOESY correlation between H3-10‴ and H2-4‴. The absolute configuration of 2 was established using specific rotation and electronic circular dichroism (ECD) data. Alkaline hydrolysis of 2 yielded (2E)-8-hydroxy-2,6-dimethyl-2-octenoic acid (HDOA, 2a) with an [α]D value of +6.8 (Figures S23 and S24, Supporting Information), which indicated the (6R) configuration by comparison of the [α]D value to those of RHDOA ([α]D +5.6) and S-HDOA ([α]D −8.3).7 The (2R,3S) absolute configuration of 2 was deduced from the ECD data, indicating negative Cotton effects (CEs) at 275 and 221 nm (Figure S14, Supporting Information), typical of (2R,3S)flavan-3-ols.8 Consequently, the structure of 2 was defined as (+)-(2R,3S)-catechin 7-{6-O-[(6R,2E)-8-hydroxy-2,6-dimethyl2-octenoyloxy]}-β-D-glucopyranoside. Compound 3 was obtained as a white powder with [α]20 D +84 (c 0.3, MeOH). It analyzed for a molecular formula of C26H38O12 via the HRESIMS ion at m/z 565.2259 [M + Na]+. Infrared absorptions at 3393, 1701, 1603, and 1520 cm−1 provided evidence for hydroxy, carbonyl, and aromatic moieties. The characteristic signals for a guaiacylglycerol and a β-glucopyranosyl moiety were observed in the 1H and 13C NMR spectra of 3 (Table 1).9 The D-glucopyranosyl unit in 3 was confirmed through hydrolyzing 3 with 1 N HCl using the same procedure as described for 1 and 2. Additionally, analysis of the NMR data of 3 revealed the presence of a 6-hydroxy-2,6dimethylocta-2,7-dienoyl moiety with resonances corresponding to one trisubstituted double bond [δH 6.75 (t, J = 6.5 Hz, H-3″; δC 128.6 (C-2″) and 144.1 (C-3″)], a terminal double bond [δH 5.81 (dd, J = 17.5, 10.5 Hz, H-7″), 5.13 (dd, J = 17.5, 1.5 Hz, H-8″a), and 4.97 (dd, J = 10.5, 1.5 Hz, H-8″b); δC 146.2 (C-7″) and 112.9 (C-8″)], an olefinic methyl singlet [δH 1.75 (H3-10″); δC 12.6 (C-10″)], a methyl singlet [δH 1.17 (H39″); δC 28.4 (C-9″)], two methylenes [δH 2.14 (m, H2-4″) and 1.51 (m, H2-5″); δC 24.8 (C-4″) and 42.0 (C-5″)], one oxygenated tertiary carbon at δC 73.6, and one ester carbonyl carbon at δC 169.8.10 This was supported by COSY correlations between H3-10″/H-3″/H2-4″/H2-5″ and H-7″/H2-8″ and HMBC couplings from H3-10″ to C-1″, C-2″, and C-3″, from H3-9″ to C-5″ and C-7″, from H-7″ to C-9″ and C-5″, and from H2-8″ to C-6″. The connectivity between the guaiacylglyceryl, β-D-glucopyranoyl, and 6-hydroxy-2,6-dimethylocta-2,7-dienoyl moieties was established by HMBC crosspeaks of the anomeric proton to C-8 and H2-6′ to C-1″. An E Δ2‴(3‴) double bond was deduced from the key NOESY correlation between H3-10″ and H2-4″. However, efforts to assign the C-6 absolute configuration of the (2E)-6-hydroxy2,6-dimethylocta-2,7-dienoyl moiety via the same approach as described for 2 were unsuccessful possibly due to its decomposition under the prevailing reaction condition. Further attempts to experimentally corroborate the configuration were not feasible due to the limited amount of material available after biological evaluations. Only 2′,7-epoxyguaiacylglycerol 8-O-β-Dglucopyranoside (ficuscarpanoside A) (3a) (Figures S35−S39, Supporting Information) was isolated from the alkaline hydrolysate of 3.11 The large coupling constant of H-7 (J = 9.6 Hz) demonstrated a trans-diaxial relationship between H-7 and H-8 in 3a. Therefore, a 7,8-threo configuration of 3 was a prerequisite. In addition, the ΔδC8−C7 value in methanol-d4 was larger than 12.0 ppm, which supported the 7,8-threo

Figure 1. ORTEP drawing for compound 1a depicting its absolute configuration.

Compound 2 was isolated as an amorphous, white solid with [α]20 D +78 (c 0.4, MeOH). The HRESIMS data showed a sodium adduct molecular ion at m/z 643.2366 [M + Na]+, which corresponded to a molecular formula of C31H40O13Na. This indicated 12 indices of hydrogen deficiency. The IR spectrum exhibited absorptions of aromatic (1604 and 1516 cm−1), carbonyl (1694 cm−1), and hydroxy (3353 cm−1) moieties. The 1H NMR spectrum of 2 displayed characteristic signals for a catechin nucleus with resonances at δH 4.52 (1H, d, J = 7.5 Hz, H-2), 3.95 (1H, ddd, J = 7.5, 8.0, 5.0 Hz, H-3), 2.52 (1H, dd, J = 16.0, 8.0 Hz, H-4β), 2.88 (1H, dd, J = 16.0, 5.0 Hz, H-4α), 6.14 (2H, brs, H-6 and H-8), 6.83 (1H, brs, H-2′), 6.76 (1H, d, J = 8.0 Hz, H-5′), and 6.61 (1H, brd, J = 8.0 Hz, H-6′).6 Also observed were an olefinic methine at δH 6.74 (t, J = 7.0 Hz, H-3‴), an olefinic methyl singlet at δH 1.71 (H3-10‴), a methyl doublet at δH 0.88 (J = 6.0 Hz, H3-9‴), one oxygenbearing methylene at δH 3.56 (m, H2-8‴), and a βglucopyranosyl moiety (Table 1). Acid hydrolysis followed by GC analysis identified the D-glucose unit in 2.5 Besides the carbon signals assigned to the catechin nucleus and β-Dglucopyranosyl unit (Table 1), the 10 additional carbon resonances in the 13C NMR spectrum were classified via HSQC data into two methyls, four methylenes (including one oxygen-bearing methylene), one methine, a trisubstituted double bond, and an ester carbonyl carbon. These data, coupled with the chemical shifts and index of hydrogen deficiency, suggested that 2 was composed of a linear monoterpenoidal acyl chain coupled via an ester linkage to a catechin glucoside. An 8-hydroxy-2,6-dimethyl-2-octenoyl moiety was suggested to be linked to the 6-CH2OH position of the β-D-glucopyranosyl unit, as implied by the 1H−1H COSY sequences between H-3‴/H2-4‴/H2-5‴/H-6‴/H2-7‴/H2-8‴ and H-6‴/H3-10‴ and HMBC correlations from H3-10‴ to C1‴, C-2‴, and C-3‴, from H3-9‴ to C-5‴ and C-7‴, from H28‴ to C-6‴, and from H2-6″ to C-1‴. The β-D-glucopyranosyl moiety was connected to OH-7 of the catechin moiety, as was exemplified by a long-range HMBC coupling from the anomeric proton to C-7. Thus, compound 2 was identified as a 6″-O-8-hydroxy-2,6-dimethyl-2-octenoyl derivative of 7-O-β1810


1 2 3 4α 4β 5 6 7 8 9a 9b 10 1′ 2′ 3′ 4′ 5′ 6′a 6′b 1″ 2″ 3″ 4″ 5″a 5″b 6″a 6″b 7″ 8″a 8″b 9″ 10″ 1‴ 2‴ 3‴ 4‴ 5‴a 5‴b 6‴a 6‴b 7‴a

no.

6.76 d (8.0) 6.61 brd (8.0) 4.79 3.32 3.45 3.40 3.63 4.51 brd (12.0) 4.14 dd (12.0, 7.5)

104.6 75.3 78.1 72.1 75.9 64.2

1811

168.4 116.6 162.7 39.5 30.6

79.1

74.1 25.0

26.3 19.5

1.09 s 2.03 s

m; 2.34 m m m d (10.5)

1.05 s

2.12 1.73 1.35 3.14

5.64 s

d (7.0) dd (7.0, 8.5) dd (8.5, 9.0) dd (9.5, 9.0) ddd (9.5, 6.5, 1.5) dd (12.0, 1.5) dd (12.0, 6.5)

t (7.0) m m m m

1.57 m

6.74 2.07 1.36 1.20 1.55

d (7.5) dd (9.0, 7.5) dd (8.5, 9.0) dd (9.0, 8.5) m

6.83 brs

6.14 brs

4.56 3.31 3.28 3.25 3.43 4.29 4.17

d (7.5) ddd (7.5, 8.0, 5.0) dd (16.0, 5.0) dd (16.0, 8.0)

6.14 brs

4.52 3.95 2.88 2.52

δH (mult, J, Hz)

107.8 121.1

141.1 152.6 102.1 155.3

δC (mult)

2

6.83 d (8.5) 6.16 dd (8.5, 3.0)

6.36 d (3.0)

δH (mult, J, Hz)

1

d (8.0) dd (8.0, 9.0) dd (8.5, 9.0) dd (9.0, 8.5) m dd (12.0, 2.0) dd (12.0, 6.5)

d (8.0) dd (8.0, 2.0) d (7.0) m dd (12.0, 4.0) dd (12.0, 3.6) d (7.8) dd (9.6, 7.8) dd (9.6, 8.4) dd (8.4, 9.6) m dd(11.4, 2.4) dd(11.4, 6.0)

d (8.4) dd (8.4, 1.8) d (9.6) m d (12.0) d (12.0) 99.8 80.8 75.1 71.9 79.8 62.6

116.1 121.9 80.2 82.7 62.1

130.1 112.4 149.0 148.0

δC (mult)

5.89 4.40 3.92 3.51 3.31 3.28

d (7.8) dd (7.8, 8.4) dd (8.4, 9.0) dd (9.0, 8.4) m

(brs) (d, 3.0) dd (6.0, 3.0) ddd (7.2,6.0, 4.8) dd (12.6, 4.8) dd (12.6, 7.2)

6.85 (d, 8.4) 7.58 (d, 8.4, 2.4)

7.50 (d, 2.4)

6.79 (d, 1.8)

6.44 (d, 1.8)

δH (mult, J, Hz)

40.9

3.64 brd (12.0) 3.51dd (12.0, 6.0)

28.4 12.6

146.2 112.9

4.58 3.14 3.58 3.39 3.46 3.90 3.78

6.77 6.85 4.43 3.80 3.42 3.38

6.98 d (1.8)

δH (mult, J, Hz)

9b

30.7

dd (17.5, 10.5) dd (17.5, 1.5) dd (10.5, 1.5) s brs

73.6

169.8 128.6 144.1 24.8 42.0

105.7 75.7 78.0 72.1 76.0 65.1

116.3 121.2 75.1 88.4 63.2

133.7 111.9 149.3 147.8

δC (mult)

3a

4.45 2.98 3.15 3.11 3.13

5.81 5.13 4.97 1.17 1.75

6.75 t (6.5) 2.14 m 1.51 m

4.37 3.25 3.33 3.29 3.45 4.45 4.12

6.71 6.76 4.64 3.69 3.47 3.26

6.94 d (2.0)

δH (mult, J, Hz)

3

170.1 128.7 145.0 27.4 37.2

65.4

102.4 75.1 78.2 72.2 75.8

104.0 132.4 115.6 146.6 146.6 116.3 120.4

157.3 96.8 159.0 97.9 157.8

83.4 69.1 29.2

δC (mult)

Table 1. NMR Data of Compounds 1−3, 3a, 9, and 10 in Methanol-d4a

61.2

102.3 73.9 77.4 70.1 77.2

106.5 89.5 76..2 86.3 60.8

106.1 121.0 116.1 145.7 149.3 116.1 122.5

161.3 100.0 162.1 94.9 156.4

158.3 133.9 178.2

δC (mult)

4.86 3.42 3.40 3.37 3.35 3.81 3.66

d (7.5) dd (7.5, 9.0) dd (8.5, 9.0) dd (8.5, 9.0) m brd (11.5) dd (11.5, 3.0)

7.62 s

6.83 s

3.52 t (6.5) 2.91 t (6.5)

δH (mult, J, Hz)

10c

136.3 103.1 75.2 78.2 71.4 78.4 62.6

112.0 154.7 147.0 117.5 122.8

49.8 28.6

167.1

δC (mult)

Journal of Natural Products Article


56.5 3.80 s

configuration of 3.9 A positive CE at 231 nm in the ECD spectrum indicated the (8S) configuration (Figure S34, Supporting Information).12 Thus, the structure of 3 was elucidated as (+)-(7S,8S)-guaiacylglycerol 8- {6-O-[(2E)-6hydroxy-2,6-dimethylocta-2,7-dienoyloxy]}-β-D-glucopyranoside. Compound 4 was an amorphous solid, which analyzed for the molecular formula C29H42O14 via the HRESIMS ion at m/z 637.2469 [M + Na]+. The presence of aromatic (1600 and 1494 cm−1) and hydroxy (3398 cm−1) moieties was indicated by the IR spectrum. The characteristic NMR data of compound 4 were comparable to those of symplocosneolignan (4,7,9,4′,9′pentahydroxy-3,5,3′,5′-tetramethoxy-8,4′-oxyneolignan-4-O-βD-glucopyranoside), which is a known neolignan glucoside that was first isolated from Symplocos cochinchinensis var. philippinensis.13 When the 1D and 2D NMR data were compared with the NMR data of symplocosneolignan, a major change in the side chain at C-1′ was revealed, where the 1-hydroxypropyl unit in symplocosneolignan was replaced by a 4-substituted 2hydroxybutyl moiety in 4. The signals of the latter unit occurred at δH 2.63 and 2.54 (each 1H, m, H2-7′), 1.66 (2H, m, H2-8′), 3.70 (1H, m, H-9′), and 1.13 (3H, d, J = 6.0 Hz, H39′a) in the 1H NMR spectrum, as well as at δC 33.8 (CH2, C7′), 42.3 (CH2, C-8′), 68.2 (CH, C-9′), and 23.9 (CH3, C-9′a) in the 13C NMR and DEPT spectra. The 1H−1H COSY sequences between H2-7′/H2-8′/H-9′/H3-9a′ and key longrange HMBC couplings of H2-7′/C-2′(C-6′), H2-8′/C-1, and H-2′(H-6′)/C-7′ confirmed the structure assigned for the side chain moiety. The β-D-glucopyranosyl moiety in 4 was confirmed by using the same protocol as earlier described. The J7,8 value of 5.0 Hz and ΔδC8−C7 value of 13.2 ppm in methanol-d4 suggested an erythro configuration for 4.9,12,14 A positive CE at 244 nm in the ECD spectrum indicated that 4 possessed the (8S) configuration (Figure S41, Supporting Information).9 Given the small amount of the material available, no attempts were made to assign the C-9′ configuration. Therefore, the structure of 4 was confirmed as (+)-(7R,8S)-4,7,9,4′,9′-pentahydroxy-3,5,3′,5′-tetramethoxy9′a-homo-8,4′-oxyneolignan-4-O-β-D-glucopyranoside. Compound 5 gave a molecular formula of C34H48O19 as indicated by HRESIMS. The IR spectrum exhibited absorption bands at 3391, 1589, and 1417 cm−1 due to the aromatic and hydroxy groups. The NMR data of 5 showed resonances similar to those of 4; however, the C-1′ side chain of 4, namely, the 4substituted 2-hydroxybutyl unit, was replaced by a transarylpropenoxy moiety [δH 6.62 (1H, d, J = 15.5 Hz, H-7′), 6.31 (1H, dt, J = 15.5, 6.0 Hz, H-8′), and 4.53 (2H, d, J = 6.0 Hz, H2-9′); δC 133.5 (C-7′), 126.6 (C-8′), and 70.7 (C-9′)]. An additional β-glucopyranoyl unit was also present (Table 2). Acid hydrolysis of 5 gave D-glucose.5 Analyses of 2D NMR COSY, HMBC, and HSQC allowed all proton and carbon signals to be assigned. Key HMBC couplings from H-1″ to C-4 and H-1‴ to C-9′ located the two β-D-glucopyranoyl units at C4 and C-9′. The erythro configuration of 5 was confirmed by the J7,8 value of 6.0 Hz and ΔδC8−C7 (12.9 ppm) in methanold4.9,12,14 The (7S,8R) configuration was deduced from its ECD spectrum, which displayed a negative CE at 236 nm (Figure S52, Supporting Information).9 On this basis, the structure of 5 was defined as (−)-(7S,8R,7′E)-4,7,9,4′,9′-pentahydroxy3,5,3′,5′-tetramethoxy-8,4′-oxyneolignan-7′-ene-4,9′-di-O-β-Dglucopyranoside. The molecular formula of compound 6 was C33H44O17 from the HRESIMS data. The NMR data of 6 displayed a close

13

56.7

Article

NMR data (δ) were measured at 600 or 500 MHz for H NMR and at 150 or 125 MHz for C NMR. Proton coupling constants (J) in Hz are given in parentheses. The assignments were based on DEPT, 1H−1H COSY, HSQC, and HMBC experiments. bData of the α-L-rhamnopyranose at C-7 of 9: δH 5.56 (1H, brs, H-1⁗), 3.84 (1H, brs, H-2⁗), 3.63 (1H, brd, J = 9.0 Hz, H-3⁗), 3.31 (1H, dd, J = 9.0, 8.4, Hz, H-4⁗), 3.43 (1H, dq, J = 8.4, 6.0, Hz, H-5⁗), 1.12 (3H, d, J = 6.0 Hz, H3-6⁗); δC 98.8 (C-1⁗), 70.3 (C-2⁗), 70.7 (C-3⁗), 72.1 (C-4⁗), 70.5 (C-5⁗), 18.4 (C-6⁗). cData of the N-Me of 10: δH 3.05 (3H, s); δC 35.6.

57.0 3.82 s

δH (mult, J, Hz) δH (mult, J, Hz) δH (mult, J, Hz)

3.81 s

1

61.3 12.9 20.1 m m d (6.5) brs 3.70 s

56.8

1.33 3.56 0.88 1.71 7‴b 8‴ 9‴ 10‴ (10″) OMe

a

δH (mult, J, Hz) δH (mult, J, Hz) δH (mult, J, Hz) no.

Table 1. continued

1

δC (mult)

2

δC (mult)

3

δC (mult)

3a

δC (mult)

9b

δC (mult)

10c

δC (mult)


1812


no.

1813

s d (5.0) m dd (12.0, 5.0) dd (12.0, 2.0)

s m m m m d (6.0) d (7.5) dd (7.5, 8.5) dd (8.5, 9.0) dd (8.5, 9.0) m dd (12.0, 2.0) dd (12.0, 5.5)

3.78 s 3.73 s

6.48 2.63 2.54 1.66 3.70 1.13 4.78 3.41 3.36 3.36 3.14 3.71 3.59

6.48 s

6.69 4.88 4.12 3.83 3.49

6.69 s

57.3 57.0

42.3 68.2 23.9 105.8 76.0 78.1 71.6 78.7 62.9

140.6 107.2 154.6 135.0 154.6 107.2 33.8

61.7

57.0 56.7

103.4 75.1 78.1 71.7 78.0 62.6

105.6 75.7 77.8 71.3 78.4 62.8

4.79 3.45 3.41 3.40 3.21 3.88 3.65 4.36 3.22 3.35 3.29 3.27 3.76 3.66 3.82 3.80

d (7.8) dd (7.8, 8.4) dd (8.4, 9.0) dd (8.4, 9.0) m dd (11.4, 2.4) dd (11.4, 5.4) d (7.8) dd (7.8, 8.4) dd (8.4, 9.0) dd (8.4, 9.0) m (11.4, 2.4) dd (11.4, 5.4) s s

126.6 70.7

135.5 105.0 154.5 134.5 154.5 105.0 133.5

61.7

139.5 106.0 153.8 136.6 153.8 106.0 74.1 87.0

δC (mult)

6.31 dt (15.5,6.0) 4.53 d (6.0)

6.73 s 6.62 d (15.5)

6.73 s

6.73 s 4.91d (6.0) 4.27 m 3.89 dd (12.0, 7.2) 3.61 dd (12.0, 3.6)

6.73 s

δH (mult, J, Hz)

139.8 106.2 154.2 135.7 154.2 106.2 74.3 87.5

δC (mult)

δH (mult, J, Hz)

s d (6.0) m dd (10.2, 2.4) dd (10.2, 6.0)

4.84 3.46 3.40 3.39 3.20 3.88 3.65 4.35 3.21 3.34 3.28 3.26 3.76 3.66 3.89 3.81

d (7.8) dd (7.8, 8.4) dd (8.4, 9.0) dd (8.4, 9.0) dd m dd (12.0, 2.4) dd (12.0.5.4) d (7.8) dd (7.8, 8.4) dd (8.4, 9.0) dd (8.4, 9.0) m dd (12.0, 2.4) dd (12.0, 5.4) s s

6.23 dt (15.6, 6.0)

6.73 brs 6.61 d (15.6)

6.96 brs

6.73 5.59 3.46 3.88 3.78

6.73 s

δH (mult, J, Hz)

6

56.8 57.0

103.2 75.2 78.2 71.7 78.0 62.6

105.3 75.7 77.8 71.4 78.4 62.8

124.5 70.9

132.6 112.3 145.6 149.3 129.9 116.7 134.1

65.0

140.0 104.5 154.5 135.8 154.5 104.5 88.9 55.5

δC (mult)

d (8.4) dd (8.4, 1.8) d (6.0) m dd (9.6, 7.8) dd (9.6, 2.4)

d (7.8) dd (7.8, 9.0) dd (8.4, 9.0) dd (8.4, 9.0) m dd (11.4, 2.4) dd (11.4, 5.4) d (1.2) dd (1.2, 1.8) (1.8, 8.4) m m (6.6) 3.82 s 3.85 s

4.33 3.21 3.33 3.28 3.39 3.96 3.62 4.73 3.82 3.64 3.34 3.66 1.22

1.81 m 3.56 t (6.6)

6.79 brs 2.62 t (7.8)

6.72 brs

6.75 6.84 5.59 3.63 4.03 3.85

6.98 d (1.8)

δH (mult)

7

56.5 56.8

102.2 72.2 72.4 74.0 69.9 18.1

104.3 75.1 78.2 71.8 76.9 68.2

35.8 62.3

137.0 114.1 145.1 147.5 129.6 118.2 32.9

72.5

134.5 110.7 149.0 147.5 116.1 119.8 89.2 53.1

δC (mult)

d (8.0) dd (8.0, 9.0) dd (8.5, 9.0) dd (8.5, 9.0) m dd (11.0, 2.0) dd (11.0, 6.0) d (1.0) dd (1.0, 1.5) dd (1.5, 8.5) m m d (6.5) 3.85s

4.94 3.44 3.35 3.25 3.29 3.81 3.49 4.55 3.54 3.47 3.26 3.41 1.09

1.12 t (7.0)

3.00 q (7.0)

7.25 s

7.25 s

δH (mult)

8

57.4

102.3 72.4 72.6 74.2 70.0 18.3

104.6 75.9 78.2 72.0 77.7 68.3

9.0

134.6 107.4 154.7 140.4 154.7 107.4 202.4 32.8

δC (mult)

a NMR data (δ) were measured at 600 or 500 MHz for 1H NMR and at 150 or 125 MHz for 13C NMR. Proton coupling constants (J) in Hz are given in parentheses. The assignments were based on DEPT, 1H−1H COSY, HSQC, and HMBC experiments.

8′ 9′ 9′a 1″ 2″ 3″ 4″ 5″ 6″a 6″b 1‴ 2‴ 3‴ 4‴ 5‴ 6‴a 6‴b OMe-3(5) OMe-3′(5′)

1′ 2′ 3′ 4′ 5′ 6′ 7′

1 2 3 4 5 6 7 8 9

5

4

Table 2. NMR Data (δ) for Compounds 4−8 in Methanol-d4a

Journal of Natural Products Article



Article

of 9. The 13C NMR shifts of the aglycone portion of 9 corresponded to the shifts for quercetin, and the only significant differences were those of C-2, C-3, and C-7. These shifts are analogous to those reported when the 3- and 7hydroxy groups are glycosylated in a flavonol glycoside.19 One anomeric proton representing a β-configured sugar moiety δH at 4.45 (1H, d, J = 7.8 Hz, H-1′″) and two anomeric protons representing α-configured sugar moieties at δH 5.89 (1H, brs, H-1″) and 5.56 (1H, brs, H-1″″) were readily identified in the 1 H NMR spectrum of 9. These anomeric protons were correlated to carbons at δC 102.3, 106.5, and 98.8, respectively. The assigned aglycone and sugar values indicated that one αrhamnopyranosyloxy group and a disaccharide chain, [βglucopyranosyl-(1→2)]-α-arabifuranosyloxy unit, were respectively attached to C-7 and C-3 of the quercetin aglycone.20 This was confirmed by the results of 2D COSY, HSQC, and HMBC experiments. In particular, HMBC correlations between H-1‴ and C-2″, between H-1″ and C-3, and between H-1″″ and C-7 clearly established the sequence of the disaccharide chain and the location of the sugar moieties. The D-glucopyranosyl, Lrhamnopyranosyl, and L-arabifuranosyl moieties in 9 were established by acid hydrolysis of 9 using the same protocol as earlier described. Therefore, the structure of 9 was defined as quercetin 3-O-β-D-glucopyranosyl(1→2)-α-L-arabifuranosyl-7O-α-L-rhamnopyranoside. Compound 10 was isolated as an amorphous, white powder with a molecular formula of C17H23NO8 as determined by HRESIMS data. Absorption peaks corresponding to hydroxy (3443 cm−1) and carbonyl (1632 cm−1) functionalities were shown by the IR spectrum. Besides the characteristic resonances for a β-glucopyranosyl moiety, the 1H NMR of 10 revealed signals for two para aromatic protons at δH 6.83 (1H, s, H-5) and 7.62 (1H, s, H-8), an N-methyl at δH3.05, a methoxy group at δH 3.82, and two mutually coupled methylenes at δH 3.52 (2H, t, J = 6.5 Hz, H2-3) and 2.91 (2H, t, J = 6.5 Hz, H2-4) based on the 1H−1H COSY spectrum. The 13C NMR and DEPT spectra of 10 showed the presence of two aromatic quaternary carbons, two oxygenated aromatic tertiary carbons, and an amide carbonyl carbon at δC 167.1. These data, in conjunction with the chemical shifts and molecular composition, disclosed that 10 was a glycosidic tetrahydroisoquinolone with a methoxy and an N-methyl group.21 The sugar obtained from the acid hydrolysis of 10 was identified as D-glucose, which was based on the same protocol as earlier described. Further analyses of 2D NMR data resulted in the assignment of all proton and carbon signals. In the HMBC spectrum, key HMBC cross-peaks from the Nmethyl to C-1 and C-3, from H-8 to C-1, from H-5 to C-4, from OMe to C-6, and from the anomeric proton to C-7 confirmed 10 as the 7-O-β-D-glucopyranosyl derivative of thalifoline. 21 The locations of the methoxy and β- D glucopyranosyl groups were also supported by an NOE enhancement of H-1′ by irradiation of H-8 in the NOE difference spectrum of 10 (Figure S109, Supporting Information). Thus, the structure of 10 was defined as 7-β-Dglucopyranosyloxythalifoline. The structures of the known compounds were defined as (7S,8R)-4,9′-di-β-D-glucopyranosyloxydehydrodiconiferyl alcohol (11),15 (7S,8R)-4-β-D-glucopyranosyloxydihydrodehydrodiconiferyl alcohol (12), (7R,8S)-4-β- D -glucopyranosyloxydihydrodehydrodiconiferyl alcohol (13),17 (7S,8R)-4-β-Dglucopyranosyloxy-5-methoxydihydrodehydrodiconiferyl alcohol (14),22 lanicepside A, 23 (+)-3a-β- D-glucopyranosyl-

resemblance to those of the co-occurring (7S,8R)-4,9′-di-β-Dglucopyranosyloxydihydrodiconiferyl alcohol (11),15 with the only difference being the replacement of the 1,2,4-trisubstituted aromatic moiety with a symmetrically tetrasubstituted benzene moiety and an additional methoxy group. These data indicated that 6 was the 5-methoxy analogue of 11, which was evident from acid hydrolysis and 2D NMR experiments of 6. NOESY correlations of H-7 with H2-9 and H-8 with H-2(6) required a trans-arrangement between H-7 and H-8 as previously reported for 4′,7-epoxy-8,3′-neolignans.16 According to the reversed helicity rule for the 1Lb ECD band of the 7-methoxy-2,3dihydrobenzo[b]furan chromophore, a positive CE at 272 nm suggested that 6 had the (7S,8R) configuration (Figure S62, Supporting Information).12,14,16 On the basis of these data, the structure of 6 was defined as (−)-(7S,8R,7′E)-4,9,9′-trihydroxy3,5,3′,5′-tetramethoxy-4′,7-epoxy-8,3′-neolignan-7′-ene-4,9′-diO-β-D-glucopyranoside. Compound 7 was obtained as an amorphous powder whose molecular formula was deduced to be C32H44O15 by HRESIMS. The NMR spectra of 7 were highly similar to those of the cooccurring (7S,8R)-4-β-D-glucopyranosyloxydehydrodiconiferyl alcohol (12), except for the presence of an α-L-rhamnopyranosyl moiety.17 This was confirmed by hydrolysis of 7 using 1 N HCl and subsequent GC analysis of the hydrolysate. Comparison of the NMR data of 7 with those of 12 showed a deshielded 13C NMR shift of 10 and 6.5 ppm for C-9 and C-6″, respectively. These data demonstrated that the β-D-glucopyranosyloxy moiety was placed at C-9 and that the α-Lrhamnopyranosyloxy unit was linked to C-6 of the β-Dglucopyranosyloxy unit. This inference was confirmed by the HMBC cross-peaks of H-1″/C-9 and H-1‴/C-6″. Analysis of the NOESY data revealed that 7 had the same relative configuration as 6. The absolute configuration at C-7 and C-8 was assessed to be S and R, respectively, based on the positive ECD CE at 243 nm and the negative CE at 221 nm (Figure S73, Supporting Information).16b,17 On the basis of these observations, the structure of 7 was assigned as (−)-(7S,8R)4,9,9′-trihydroxy-3′,5-dimethoxy-4′,7-epoxy-8,3′-neoligan-9-O[α-L-rhamnopyranosyl(1→6)]-β-D-glucopyranoside. The molecular formula of compound 8 was C23H34O13 as indicated by the HRESIMS ion at 541.1909 [M + Na]+. The NMR spectra of 8 were similar to those observed for 2,6dimethoxy-4-propionylphenyl O-β-D-glucopyranoside,18 except for a set of resonances that were characteristic for an α-Lrhamnopyranosyl moiety and the C-6′ deshielding of the resonance. These observations suggested that 8 was the 6′-O-αL-rhamnopyranosyl derivative of 2,6-dimethoxy-4-propionylphenyl O-β-D-glucopyranoside, and the structure was confirmed by the 2D NMR HSQC, COSY, and HMBC data, as well as acid hydrolysis. Therefore, the structure of 8 was defined as 2,6dimethoxy-4-propionylphenyl O-[α-L-rhamnopyranosyl(1→ 6)]-β-D-glucopyranoside. Compound 9 was purified as an amorphous, yellow powder and shown to have the molecular formula C32H38O20, as established by the HRESIMS ion at m/z 765.1847 [M + Na]+. The IR spectrum revealed absorption peaks of conjugated carbonyl (1655 cm−1), hydroxy (3389 cm−1), and aromatic (1599 and 1493 cm−1) moieties. In the 1H NMR spectrum, the signals for a 1,2,4-substituted benzene ring at δH 7.50 (1H, d, J = 2.4 Hz, H-2′), 6.85 (1H, d, J = 8.4 Hz, H-5′), and 7.58 (1H, dd, J = 8.4, 2.4 Hz, H-6′), and for two meta-coupled protons at δH 6.44 (1H, d, J = 1.8 Hz, H-6) and 6.79 (1H, d, J = 1.8 Hz, H-8) were used to characterize the quercetin aglycone moiety 1814



Article

oxylyoniresinol,24 styraxjaponoside B,25 matairesinoside,25,26 (−)-(2R)-1-O-β-D-glucopyranosyl-2-{2,6-dimethoxy-4-[1-(E)propen-3-ol]phenoxyl}propan-3-ol,27 syringin,28 psoralenoside (15), isopsoralenoside,29 scopolin,30 (+)-7-β-D-glucopyranosyloxycatechin, (+)-5-β-D-glucopyranosyloxycatechin,6a (+)-7-β-Dglucopyranosyloxy-3′-O-methylepicatechin,31 kaempferitrin,32 kaempferol 3-O-β-D-glucopyranosyl(1→2)-β-D-glucopyranoside-7-O-α-L-rhamnopyranoside,33 quercetin-3-O-α-L-rhamnopyranoside, quercetin-3-O-β-D-glucopyranoside,34 3-hydroxy4,5-dimethoxyphenyl-1-O-β-D-glucopyranoside,35 2,6-dimethoxy-4-hydroxyphenol-1-O-β-D-glucopyranoside,36 3,4,5-trimethoxyphenyl-1-O-β-D-glucopyranoside,37 prismaconnatoside,38 2-(4-hydroxyphenyl)ethyl β- D -glucopyranoside,39 2-(3,4dihydroxyphenyl)ethyl β-D-glucopyranoside,40 and canthoside A,41 by spectroscopic analysis and comparison to literature values. In the in vitro assays, compounds 4, 6, 7, 8, 11, 12, and 14 reduced acetaminophen-induced HepG2 cell injury with 38.9 ± 3.5%, 30.5 ± 2.6%, 37.4 ± 4.3%, 37.6 ± 5.1%, 46.0 ± 6.2%, 38.4 ± 4.8%, and 31.2 ± 5.2% inhibitions at 10 μM (Table 3, the

acquired on a Nicolet Impact 400 FT-IR spectrophotometer. Standard pulse sequences were used for all NMR experiments, which were run on either a Bruker spectrometer (600 MHz for 1H or 150 MHz for 13 C) or a Varian INOVA spectrometer (500 MHz for 1H or 125 MHz for 13C) equipped with an inverse detection probe. Residual solvent shifts for methanol-d4 were referenced to δH 3.31 and δC 49.15, respectively. Accurate mass measurements were obtained on a Q-Trap LC/MS/MS (Turbo ionspray source) spectrometer. Column chromatography was run using MCI gel (CHP20P), silica gel (200− 300 mesh, Qingdao Marine Chemical Inc., China), and Sephadex LH20 (Pharmacia Biotech AB, Uppsala Sweden). HPLC separation was done on Waters HPLC components comprising a Waters 600 pump, a Waters 600 controller, and a Waters 2487 dual λ absorbance, with GRACE semipreparative (250 × 10 mm) and preparative (250 × 19 mm) Rp C18 (5 μm) columns. Plant Material. The twigs of L. cubeba were collected in Zhaotong, Yunnan Province, People’s Republic of China, in May 2013, and identified by Prof. Gan-peng Li at Yunnan Minzu University. A herbarium specimen was deposited at the Herbarium of the Department of Medicinal Plants, Institute of Materia Medica, Beijing 100050, People’s Republic of China (herbarium no. 2013-05-10). Extraction and Isolation. The air-dried twigs of L. cubeba (12 kg) were ground and extracted using 30.0 L of 95% EtOH under ambient temperature for 3 × 48 h. The EtOH extract was concentrated in vacuo, and the residue was suspended in H2O, then partitioned with EtOAc, to afford EtOAc- (300 g) and H2O (380 g)-soluble extracts. The H2O-soluble extract was subjected to separation over an HP-20 macroporous adsorbent resin column, eluting successively with 10%, 30%, 70%, and 95% EtOH (3000 mL each), to yield four fractions, A (165 g), B (96 g), C (63 g), and D (28 g). Fraction B was further fractionated by MPLC over reversed-phase C18 silica gel using gradient elution (100% H2O to 50% MeOH) to give seven fractions (F1−F7) based on TLC analysis. Fraction F2 (4.8 g) was subjected to Sephadex LH-20 column chromatography eluting with MeOH to afford five subfractions (F21−B25). F23 (2.4 g) was further separated via silica gel column chromatography using CHCl3−MeOH mixtures (15:1 → 1:1), resulting in nine subfractions (F231−F239). F233 was purified by RP C18 HPLC (C18 preparative columm, 5 μm, 250 × 19 mm, 254 nm, MeOH−H2O−HOAc, 15:85:1) to give 9 (15.0 mg). F235 was chromatographed over a silica gel column eluting with CHCl3−MeOH mixtures (15:1 → 5:1) and further separated by RP C18 HPLC (C18 semipreparative, 5 μm, 250 × 10 mm, 254 nm, MeOH−H2O−HOAc, 33:67:1) to afford 5 (2.8 mg) and 6 (3.3 mg). F24 (1.5 g) was subjected to silica gel column chromatography using CHCl3−MeOH mixtures (15:1 → 1:1), followed by RP C18 HPLC (C18 preparative, 5 μm, 250 × 19 mm, 254 nm, MeOH−H2O−HOAc, 32:68:1), to give 8 (12.6 mg). F3 (3.9 g) was subjected to separation over Sephadex LH20 column chromatography eluting with MeOH to yield five subfractions (F31−F35). F32 (1.4 g) was chromatographed over a silica gel column using CHCl3−MeOH mixtures (15:1 → 1:1) and then further separated using RP C18 HPLC (C18 semipreparative, 5 μm, 250 × 10 mm, 254 nm, MeCN−H2O−HOAc, 13:87:1) to afford 7 (7.5 mg). F4 (5.2 g) was separated by Sephadex LH-20 column chromatography with MeOH as eluent to yield four subfractions (F41−F44). F43 (1.2 g) was fractionated by silica gel column chromatography using CHCl3−MeOH mixtures (15:1 → 1:1) and further separated with RP C18 HPLC (C18 semipreparative, 5 μm, 250 × 10 mm, 254 nm, MeCN−H2O−HOAc, 28:72:1), to yield 4 (2.3 mg). Fraction C was further separated by MPLC over reversed-phase C18 silica gel using gradient elution (90% H2O to 80% MeOH) to give six fractions (f1−f6) on the basis of TLC analysis. Fraction f3 was chromatographed over a Sephadex LH-20 column eluting with MeOH to yield five subfractions (f3A−f3E). Subfractions f3B and f3C were separately purified by RP C18 HPLC (C18 semipreparative, 5 μm, 250 × 10 mm, 254 nm, MeCN−H2O−HOAc, 34:66:1) to give 1 (25.6 mg), 2 (7.8 mg), and 3 (6.4 mg). Subfraction f8 was separated by RP C18 HPLC (C18 semipreparative, 5 μm, 250 × 10 mm, 254 nm, MeCN−H2O−HOAc, 32:68:1) to afford 10 (6.5 mg).

Table 3. Activities of Selected Compounds to Inhibit Acetaminophen-Induced HepG2 Damage compound

relative protection (%) (10 μM)

control acetaminophen-treated 4 6 7 8 11 12 14 Bicyclol

100.00 ± 1.9 0.0 ± 2.0*** 38.9 ± 3.5## 30.5 ± 2.6 37.4 ± 4.3# 37.6 ± 5.1# 46.0 ± 6.2### 38.4 ± 4.8## 31.2 ± 5.2 34.8 ± 3.8###

The data are expressed as mean ± SD of four independent experiments. (***p < 0.001 vs control; #p < 0.05, ##p < 0.01, ###p < 0.01 vs APAP-treated group).

a

positive control, bicyclol, exhibited 34.8 ± 3.8% inhibition), respectively. However, (7R,8S)-4-β-D -glucopyranosyloxydihydrodehydrodiconiferyl alcohol (13), which differs from active compound 12 only in the configuration at C-7 and C-8, displayed no inhibitory activity at the same concentration. This difference clearly indicates that the (7S,8R) configuration is essential to this particular activity. Compared to 11, 6 showed decreased inhibition against acetaminophen-induced HepG2 cell injury. A similar result was also observed between 12 and 14. These results suggested that OMe-5 in the 4′,7-epoxy-8,3′neolignan nucleus could reduce biological activity. In addition, compounds 12 and 15 showed moderate inhibitory activities against HDAC1, with an IC50 value of 3.6 and 4.6 μM, respectively. The isolates were also evaluated for TNF-α secretion inhibition in mouse peritoneal macrophages and cytotoxic properties toward HCT-8 colon, A2780 ovary, BGC823 stomach, Bel-7402 hepatoma, and A549 lung cell lines,42 but were inactive at 10 μM.

■

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a Rudolph Research Autopol III automatic polarimeter. UV spectra were measured on a Cary 300 spectrometer. ECD spectra were recorded on a JASCO J-815 spectrometer. IR spectra were 1815



Article

Quercetin 3-O-β-D-glucopyranosyl(1 → 2)-α-L-arabifuranosyl-7O-α-L-rhamnopyranoside (9): yellow, amorphous powder, [α]20 D −11 (c 0.5, MeOH); UV (MeOH) λmax (log ε) 203 (4.35), 265 (3.62), 358 (2.46) nm; IR (KBr) νmax 3389, 2934, 1655, 1598, 1483, 1452 cm−1; 1 H NMR (methanol-d4, 600 MHz) and 13C NMR (methanol-d4, 150 MHz) data, see Table 2; ESIMS m/z 765 [M + Na]+; HRESIMS m/z 765.1847 [M + Na]+ (calcd for C32H38O20Na, 765.1849). 7-β-D-Glucopyranosyloxythalifoline (10): white, amorphous powder, [α]20 D +39 (c 0.3, MeOH); UV (MeOH) λmax (log ε) 218 (3.65), 260 (0.93), 294 (0.46) nm; IR (KBr) νmax 3443, 2945, 1632, 1603, 1583, 1493, 1390, 1344, 1288, 1262, 1208, 1106, 1073, 1045, 1016, 897 cm−1; 1H NMR (methanol-d4, 500 MHz) and 13C NMR (methanol-d4, 125 MHz) data, see Table 1; ESIMS m/z 392 [M + Na]+; HRESIMS m/z 370.1493 [M + H]+ (calcd for C17H24NO8, 370.1502). Alkaline Hydrolysis of 1−3. Each compound (4−10 mg) was separately hydrolyzed using 0.5 N NaOH (3 mL) at 40 °C for 1 h. The reaction was terminated by addition of 2 N HCl, and the mixture was applied to a C18 solid phase extraction column and eluted with H2O (20 mL) and MeOH (20 mL) successively. The MeOH fraction was concentrated to 0.5 mL and separated by RP C18 HPLC (C18 semipreparative, 5 μm, 250 × 10 mm, 254 nm, MeOH−H2O− HOAc, 25:75:0.1, for 1 and 2; MeOH−H2O−HOAc, 35:65:0.1, for 3) to afford 1a (3.0 mg), 2a (1.1 mg), and 3a (1.6 mg), respectively. (6R,3Z)-6,7-Dihydroxy-3,7-dimethyl-2-octenoic acid (1a): color1 less plate crystals, mp 112−113 °C; [α]20 D −29 (c 0.2, MeOH); H NMR (methanol-d4, 500 MHz) δ 5.65 (1H, s, H-2), 2.37, 2.13 (each 1H, m, H2-4), 1.75, 1.39 (each 1H, m, H2-5), 3.18 (1H, dd, J = 10.5, 1.5 Hz, H-6), 1.08 (3H, s, H3-8), 1.12 (3H, s, H3-9), 2.09 (3H, s, H310); 13C NMR (methanol-d4, 125 MHz) δ 170.7 (C-1), 117.3 (C-2), 161.7 (C-3), 39.4 (C-4), 30.5 (C-5), 79.0 (C-6), 74.0 (C-7), 24.9 (C8), 26.2 (C-9), 19.2 (C-10); negative-mode ESIMS m/z 201 [M − H]− and 403 [2 M − H]−; HRESIMS m/z 201.1129 [M − H]− (calcd for C10H17O4, 201.1132). 1 (R)-HDOA (2a): colorless solid, [α]20 D +7 (c 0.1, MeOH); H NMR (methanol-d4, 600 MHz) δ 6.67 (1H, t, J = 6.5 Hz, H-3), 2.19 (2H, m, H2-4), 1.46 (1H, m, H-5a), 1.28 (1H, m, H-5b), 1.59 (H, m, H-6), 1.60 (1H, m, H-7a), 1.38 (1H, m, H-7b), 3.58 (2H, m, H2-8), 0.92 (3H, d, J = 7.0 Hz, H3-9), 1.81 (3H, s, H3-10); 13C NMR (methanold4, 150 MHz) δ 27.1 (C-4), 37.2 (C-5), 30.5 (C-6), 40.7 (C-7), 61.0 (C-8), 13.1 (C-9), 19.8 (C-10); negative-mode ESIMS m/z 185 [M − H]−. Ficuscarpanoside A (3a): white powder; [α]20 D +12 (c 0.1, MeOH); 1 H NMR (methanol-d4, 600 MHz) and 13C NMR (methanol-d4, 150 MHz) data, see Table 1; positive-mode ESIMS m/z 381 [M + Na]+. X-ray Analysis of 1a. Crystals of compound 1a were grown using slow evaporation of a methanol solution. A colorless plate crystal was fixed on fiberglass. A Bruker SMART CCD diffractometer with Cu Kα radiation (λ = 1.5418 Å) from a normal focus sealed tube was used for all measurements. A total of 3546 frames were detected with the detector set at different positions of 2θ. Each frame covered 0.20° in ω. Of the 3546 reflections accumulated with 9.16° ≤ 2θ ≤ 142.22°, 2042 were independent (Rint = 0.0248) and representing 99.91% of the unique data to the maximum value of 2θ. The SHELXTL package was used in structure determination. Anisotropic displacement parameters and atomic coordinates were refined for the non-hydrogen atoms. The full-matrix least-squares (on F2) of the 133 variables produced values of the conventional crystallographic residuals R1 = 0.0349 (wR2 = 0.0907) for the 2042 observed data points with I > 2σ(I) and R1 = 0.0371 (wR2 = 0.0931) for all data. The goodness-of-fit was 1.066. A final difference Fourier map indicated a residual density between −0.181 and 0.224 e/Å3. The absolute structure parameter with a refined value of 0.0(2) was used to assign the absolute configuration. Crystal data of 1a: C10H18O4; fw 202.24; space group P21212; orthorhombic; colorless plate; 0.55 × 0.25 × 0.06 mm; unit cell dimensions a = 19.2960(8) Å, b = 8.0943(4) Å, c = 6.9478(3) Å, V = 1085.15(9) Å3; Z = 4; dcalc = 1.238 Mg/m3. Crystallographic data of 1a were deposited in the Cambridge Crystallographic Data Centre (deposition no. CCDC 1510936). The data can be downloaded from www.ccdc.cam.ac.uk/deposit free of charge or from the Cambridge

(−)-1-O-{6-O-[(6R,3E)-6,7-Dihydroxy-3,7-dimethyl-2-octenoyl]}-βwhite, amorphous (log ε) 203 (4.10), 280 (0.36); IR (KBr) νmax 3394, 3189, 2921, 2850, 1699, 1647, 1512, 1468, 1420, 1216, 1158, 1074, 955, 805, 649 cm−1; 1H NMR (methanol-d4, 500 MHz) and 13C NMR (methanol-d4, 125 MHz) data, see Table 1; ESIMS m/z 509 [M + Na]+ and 485 [M − H]−; HRESIMS m/z 485.2031 [M − H]− (calcd for C23H33O11, 485.2028). (+)-(2R,3S)-Catechin 7-{6-O-[(6R,2E)-8-hydroxy-2,6-dimethyl-2octenoyloxy]}-β-D-glucopyranoside (2): white, amorphous solid, [α]20 D +80 (c 0.4, MeOH); UV (MeOH) λmax (log ε) 209 (4.21), 276 (0.86) nm; ECD (MeOH) 275 (Δε −0.90), 221 (Δε −5.04); IR (KBr) νmax 3353, 2926, 1694, 1604, 1516, 1444, 1278, 1171, 1076, 877, 820 cm−1; 1H NMR (methanol-d4, 500 MHz) and 13C NMR (methanol-d4, 125 MHz) data, see Table 1; ESIMS m/z 643 [M + Na]+; HRESIMS m/z 643.2366 [M + Na]+ (calcd for C31H40O13Na, 643.2367). (+)-(7S,8S)-Guaiacylglycerol 8-{6-O-[(2E)-6-hydroxy-2,6-dimethylocta-2,7-dienoyloxy]} β-D-glucopyranoside (3): white powder, [α]20 D +84 (c 0.3, MeOH); UV (MeOH) λmax (log ε) 203 (3.65), 279 (0.26) nm; ECD (MeOH) 231 (Δε +1.03), 279 (Δε +0.17); IR (KBr) νmax 3393, 2921, 2850, 1701, 1646, 1603, 1520, 1466, 1420, 1279, 1159, 1079, 923, 862, 649 cm−1; 1H NMR (methanol-d4, 500 MHz) and 13C NMR (methanol-d4, 125 MHz) data, see Table 1; ESIMS m/z 565 [M + Na]+, 541 [M − H]−, and 557 [M + Cl]−; HRESIMS m/z 565.2259 [M + Na]+ (calcd for C26H38O12Na, 565.2261). (−)-(7R,8S)-4,7,9,4′,9′-Pentahydroxy-3,5,3′,5′-tetramethoxy-9′ahomo-8,4′-oxyneolignan-4-O-β-D-glucopyranoside (4): amorphous solid, [α]20 D −10 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 207 (4.12), 275 (0.14) nm; ECD (MeOH) 244 (Δε +1.69), 219(Δε +2.00); IR (KBr) νmax 3398, 2920, 2851, 1600, 1494, 1453, 1375, 1328, 1227, 1125, 1072, 1029, 754, 698 cm−1; 1H NMR (methanol-d4, 500 MHz) and 13C NMR (methanol-d4, 125 MHz) data, see Table 2; ESIMS m/z 637 [M + Na]+ and 613 [M − H]−; HRESIMS m/z 637.2469 [M + Na]+ (calcd for C29H42O14Na, 637.2472). (−)-(7S,8R,7′E)-4,7,9,4′,9′-Pentahydroxy-3,5,3′,5′-tetramethoxy8,4′-oxyneolignan-7′-ene-4,9′-di-O-β-D-glucopyranoside (5): amorphous solid, [α]20 D −11 (c 0.3, MeOH); UV (MeOH) λmax (log ε) 239 (1.02), 273(3.36) nm; ECD (MeOH) 236 (Δε −1.43), 218 (Δε +2.94); IR (KBr) νmax 3391, 2933, 1589, 1508, 1459, 1417, 1339, 1237, 1125, 1069, 1034 cm−1; 1H NMR (methanol-d4, 600 MHz) and 13 C NMR (methanol-d4, 150 MHz) data, see Table 2; ESIMS m/z 783 [M + Na]+ and 759 [M − H]−; HRESIMS m/z 783.2698 [M + Na]+ (calcd for C34H48O19Na, 783.2687). (−)-(7S,8R,7′E)-4,9,9′-Trihydroxy-3,5,3′5′-tetramethoxy-4′,7epoxy-8,3′-neolignan-7′-ene-4,9′-di-O-β-D-glucopyranoside (6): amorphous solid, [α]20 D −9 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 240 (1.01), 277 (3.35) nm; ECD (MeOH) 272 (Δε +6.41), 238 (Δε −5.23); IR (KBr) νmax 3389, 2922, 2851, 1647, 1598, 1502, 1464, 1422, 1333, 1222, 1123, 1074, 897, 821 cm−1; 1H NMR (methanol-d4, 600 MHz) and 13C NMR (methanol-d4, 150 MHz) data, see Table 2; ESIMS m/z 735 [M + Na]+ and 747 [M + Cl]−; HRESIMS m/z 735.2486 [M + Na]+ (calcd for C33H44O17Na, 735.2476). (−)-(7S,8R)-4,9,9′-Trihydroxy-3′,5,-dimethoxy-4′,7-epoxy-8,3′neoligan-9-O-[α-L-rhamnopyranosyl(1 → 6)]-β-D-glucopyranoside (7): amorphous powder, [α]20 D −12 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 203 (4.22), 234 (sh), 282 (0.56) nm; ECD (MeOH) 288 (Δε +0.43), 243 (Δε +0.75), 221 (Δε −0.40); IR (KBr) νmax 3383, 2933, 1604, 1516, 1454, 1426, 1275, 1215, 1139, 1047, 880, 811 cm−1; 1H NMR (methanol-d4, 600 MHz) and 13C NMR (methanol-d4, 150 MHz) data, see Table 2; ESIMS m/z 691 [M + Na]+, 667 [M − H]− and 703 [M + Cl]−; HRESIMS m/z 691.2571 [M + Na]+ (calcd for C32H44O15Na, 691.2578). 2,6-Dimethoxy-4-propionylphenyl O-[α-L-rhamnopyranosyl(1 → 6)]-β-D-glucopyranoside (8): amorphous powder, [α]20 D −19 (c 0.4, MeOH); UV (MeOH) λmax (log ε) 279 (2.92) nm; IR (KBr) νmax 3432, 2969, 1682, 1590, 1502, 1457, 1415, 1353, 1311, 1118, 1070, 1041, 1018, 973, 902, 834, 799 cm−1; 1H NMR (methanol-d4, 500 MHz) and 13C NMR (methanol-d4, 125 MHz) data, see Table 1; ESIMS m/z 541 [M + Na]+ and 553 [M + Cl]−; HRESIMS m/z 541.1909 [M + Na]+ (calcd for C23H34O13Na, 541.1897). D-glucopyranosyl-2-methoxyhydroquinone (1): solid, [α]20 D −18 (c 0.5, MeOH); UV (MeOH) λmax

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Crystallographic Data Centre, 12 Union Road, Cambridge CB21EZ, UK [fax: (+44) 1223-336-033 or e-mail: [email protected]]. Sugar Analysis. See ref 5. Protcetive Effect of Acetaminophen-Induced HepG2 Cell Injury. HepG2 cells were maintained in DMEM media at a density of 1.5 × 104 cells per well in 96-well culture plates. Cultures were maintained in a humidified incubator at 37 °C in 5% CO2. All compounds, at a concentration of 10 μM and 8 mM acetaminophen, were added to the cells simultaneously after incubation for 24 h. Meanwhile, a control was prepared by adding 10 μM bicyclol and 8 mM acetaminophen to the cells. The culture supernatant was discarded, and 100 μL of the 0.5 mg/mL MTT was added to the cells and maintained for 4 h after culturing for 48 h. The optical density (OD) of the formazan solution was determined by using a microplate reader at a wavelength of 570 nm. Inhibition (%) was calculated by using the following equation:

/(OD(normal) − OD(control))] × 100 All values were expressed as the mean ± SD. The Student’s t-test for unpaired observations between the normals or controls and tested samples was used to identify statistical differences. p values of

Bioactive Glycosides from the Twigs of Litsea cubeba - ACS Publications

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