Article pubs.acs.org/jnp
Diarylheptanoids and Flavonoids from Viscum album Inhibit LPS-Stimulated Production of Pro-inflammatory Cytokines in Bone Marrow-Derived Dendritic Cells Nguyen X. Nhiem,†,‡ Phan V. Kiem,‡ Chau V. Minh,‡ Nanyoung Kim,† SeonJu Park,† Hwa Young Lee,† Eun Sil Kim,§ Young Ho Kim,⊥ Sohyun Kim,∥ Young-Sang Koh,∥ and Seung Hyun Kim*,† †
College of Pharmacy, Yonsei Institute of Pharmaceutical Sciences, Yonsei University, Incheon 406-840, Korea Institute of Marine Biochemistry, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet, Caugiay, Hanoi, Vietnam § Wildlife Genetic Resources Center, National Institute of Biological Resources, Incheon 404-708, Korea ⊥ College of Pharmacy, Chungnam National University, Daejeon 305-764, Korea ∥ School of Medicine and Brain Korea 21 Program, Jeju National University, Jeju 690-756, South Korea ‡
S Supporting Information *
ABSTRACT: Three new diarylheptanoids, (3S,5R)-3-hydroxy-5-methoxy-1,7-bis(4-hydroxyphenyl)-6E-heptene (1), (3S,5S)-3hydroxy-5-methoxy-1,7-bis(4-hydroxyphenyl)-6E-heptene (2), and (3S)-3-hydroxy-1,7-bis(4-hydroxyphenyl)-6E-hepten-5-one (3), four new flavonoid glycosides, 3,7,3′-tri-O-methylquercetin-4′-O-β-D-apiofuranosyl-(1→2)-O-β-D-glucopyranoside (4), 7,3′-di-O-methylquercetin-4′-O-β-D-glucopyranosyl-3-O-[6‴-(3-hydroxy-3-methylglutaroyl)]-α-D-glucopyranoside (5), 7,3′-di-Omethylquercetin-4′-O-β-D-glucopyranosyl-3-O-[(6⁗′→5⁗)-O-1⁗′-(sinap-4-yl)-β-D-glucopyranosyl-6‴-(3-hydroxy-3-methylglutaroyl)]α-D-glucopyranoside (6), and (2S)-5-hydroxy-7,3′-dimethoxyflavanone-4′-O-β-D-apiofuranosyl-(1→5)-O-β-D-apiofuranosyl-(1→2)-O-βD-glucopyranoside (9), and 17 known compounds were isolated from the leaves and twigs of Viscum album. Compounds 1, 4, and 19 significantly inhibited LPS-stimulated production of TNF-α, IL-6, and IL-12p40 with IC50 values ranging from 0.09 ± 0.01 to 8.96 ± 0.45 μM. (+)-Medioresinol (13) showed inhibitory effects on LPS-stimulated production of IL-12p40 with an IC50 value of 2.00 ± 0.15 μM.
N
interesting molecule, as it has both pro- and anti-inflammatory effects.5 In addition, IL-12 has been shown to have both early pro-inflammatory and late anti-inflammatory effects.6 Natural products have served as an important source of drugs since ancient times, and about half of current drugs are derived from natural sources.7 Medicinal plants still play important roles in drug discovery due to their chemical and pharmacological diversity. Viscum album L. var. coloratum OHWI (Loranthaceae) (mistletoe) is a hemiparasitic shrub that grows on the stems of other trees. It has been used as a remedy in traditional oriental
umerous molecules are involved in the induction and maintenance of inflammatory responses. Among the various inflammatory cytokines, tumor necrosis factor (TNF)α is a major factor in a number of inflammatory processes. Elevated TNF-α expression has been found during the development of diabetes, septic shock, tumorgenesis, cardiovascular diseases, rheumatoid arthritis, and inflammatory bowel disease.1 Thus, many studies have been conducted to identify TNF-α inhibitors. In practice, anti-TNF-α therapies are used to treat several autoimmune diseases, such as rheumatoid2 and Crohn’s diseases.3 Another important cytokine, interleukin (IL)-6, promotes inflammatory events by stimulating the activation and proliferation of lymphocytes, differentiation of B cells, recruitment of leukocytes, and the induction of an acute phase protein response in the liver.4 IL-6 is a particularly © XXXX American Chemical Society and American Society of Pharmacognosy
Received: July 13, 2012
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dx.doi.org/10.1021/np300490v | J. Nat. Prod. XXXX, XXX, XXX−XXX
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Table 1. NMR Spectroscopic Data for Compounds 1−3 1
a
2
3
δC,a,b type
δHa,c (J in Hz)
δC,a,b type
δHa,c (J in Hz)
δC,a,b type
δHa,c (J in Hz)
1
32.1, CH2
32.0, CH2
41.2, CH2 68.2, CH 45.0, CH2
2.56, m 2.67, m 1.74, m 4.06, m 2.74, d (4.8, 15.6) 2.83, dd (8.2, 15.6)
5 6 7 1′ 2′, 6′ 3′, 5′ 4′ 1″ 2″, 6″ 3″, 5″ 4″ 5-OMe
80.9, CH 127.6, CH 134.4, CH 133.6, C 130.3, CH 116.4, CH 156.3, C 129.6, C 128.8, CH 116.1, CH 158.5, C 56.4, CH3
2.55, m 2.63, m 1.70, m 3.64, m 1.65, m 1.83, m 3.90, m 5.79, dd (8.4, 16.0) 6.50, d (16.0)
33.0, CH2
2 3 4
2.56, m 2.67, m 1.70, m 3.77, m 1.59, m 1.75, m 3.94, dt (3.4, 8.4) 5.85, dd (8.4, 16.0) 6.46, d (16.0)
pos.
7.01, d (8.4) 6.67, d (8.4)
7.24, d (8.4) 6.73, d (8.4) 3.29, s
41.0, CH2 69.3, CH 44.1, CH2 82.5, CH 127.1, CH 134.7, CH 134.3, C 130.3, CH 116.4, CH 156.3, C 129.5, C 128.9, CH 116.1, CH 158.5, C 56.1, CH3
6.98, d (8.4) 6.65, d (8.4)
7.25, d (8.4) 6.73, d (8.4)
40.6, CH2 68.8, CH 48.7, CH2 201.9, C 124.4, CH 145.6, CH 134.1, C 131.6, CH 116.1, CH 156.4, C 127.1, C 130.3, CH 117.0, CH 162.0, C
6.63, d (16.0) 7.53, d (16.0) 7.01, d (8.4) 6.68, d (8.4)
7.46, d (8.4) 6.80, d (8.4)
3.27, s
Measured in methanol-d4. b100 MHz. c400 MHz, assignments were done by HMQC, HMBC, and COSY experiments.
[C-4 (δC 44.0) and C-5 (δC 82.2)] and (3S,5R) [C-4 (δC 44.8) and C-5 (δC 80.6)].15 Consequently, compound 1 was defined as (3S,5R)-3-hydroxy-5-methoxy-1,7-bis(4-hydroxyphenyl)-6Eheptene. Compound 2 was also obtained as a white amorphous powder. HRESIMS experiments resulted in the same molecular formula as that of 1. The results of 1H, 13C, HSQC, HMBC, and COSY analyses indicated that 2 had the same constitution as 1. The 3S absolute configuration was determined by the advanced Mosher’s method16 (Table 2), suggesting that 2 was a C-5 epimer of 1. Moreover, the 5S absolute configuration of 2 was defined by comparison of its 13C NMR data of C-4 (δC 44.1) and C-5 (δC 82.5) to those of (3S,5S)-3-hydroxy-1-(4-hydroxyphenyl)-5-methyoxy-7-phenyl-6E-heptene [C-4 (δC 44.0) and C-5 (δC 82.2)] and (3S,5R) [C-4 (δC 44.8) and C-5 (δC 80.6)].15 Thus, compound 2 was defined as (3S,5S)-3-hydroxy-5-methoxy1,7-bis(4-hydroxyphenyl)-6E-heptene. The molecular formula of 3 was determined as C19H20O4 by the HRESIMS ion at m/z 311.1290 (calcd for C19H19O4, 311.1289). The 1H and 13C NMR data were similar to those of 1, differing in the presence of a C-5 carbonyl group (Table 1). The coupling constant between H-6 and H-7, J6,7 = 16.0 Hz, defined the (E)-olefinic configuration. The HMBC correlations between H-1 (δH 2.56 and 2.67) and C-2′/C-6′ (δC 131.6) and between H-7 (δH 7.53) and C-2″/C-6″ (δC 130.3) confirmed the positions of two 4-hydroxyphenyl units at C-1 and C-7, respectively (Figure 2). The 3S absolute configuration was determined by the advanced Mosher’s method16 (Table 2). Consequently, compound 3 was defined as (3S)-3-hydroxy-1,7bis(4-hydroxyphenyl)-6E-hepten-5-one. Compound 4 was obtained as a yellow, amorphous powder, and its molecular formula was determined as C29H34O16 by the HRESIMS ion at m/z 637.1754 [M − H]− (calcd for C29H33O16, 637.1774). The 1H NMR and 13C NMR spectra exhibited a flavonol skeleton, two sugar units, and three methoxy groups (Table 3). The NMR data of 4 were similar to those of homoflavoyadorinin-B12 except for the addition of a methoxy group at C-3. The HMBC correlations between the methoxy groups at δH 3.85 (3H) and 3.87 (6H) and C-3 (δC 138.4), C-7 (δC 165.2),
medicine to treat diabetes, jaundice, indigestion, common fever, and asthma. Mistletoe exerts several biological effects such as antitumor, anticancer,8,9 and anti-inflammatory activities.10 It is well established that lectins from protein fractions of V. album contribute to its antitumor and anticancer activities.11 Flavonoids,12 lignans, phenylpropanoids,13 and triterpenes14 have also been reported from V. album. In our search for anti-inflammatory natural products from Korean medicinal plants, the aerial parts of V. album showed inhibitory activities in lipopolysaccharide (LPS)-stimulated bone marrow-derived dendritic cells (BMDCs). In the present study, three new diarylheptanoids, four new flavonoid glycosides, and 17 known compounds were isolated from this plant. These compounds were tested for inhibitory effects on the LPS-stimulated production of TNF-α, IL-6, and IL-12p40 in BMDCs.
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RESULTS AND DISCUSSION Compound 1 was obtained as a white amorphous powder, and its molecular formula was determined as C20H24O4 by the HRESIMS ion at m/z 327.1590 (calcd for C20H23O4, 327.1602). The 1H NMR spectrum showed signals for the protons of two para-substituted benzene rings at δH 6.67, 6.73, 7.01, and 7.24 (each, 2H, d, J = 8.4 Hz), two (E)-olefinic protons at δH 5.85 (dd, J = 8.4, 16.0 Hz) and 6.46 (d, J = 16.0 Hz), and methoxy protons at δH 3.29 (Table 1). The NMR data of 1 were similar to those of (3S,5R)-3-hydroxy-1-(4-hydroxyphenyl)-5-methyoxy-7-phenyl6E-heptene15 except for the addition of a hydroxy group at C-4″. The HMBC correlations between H-1 (δH 2.56 and 2.67) and C2′/C-6′ (δC 130.3) and between H-7 (δH 6.46) and C-2″/C-6″ (δC 128.8) suggested two 4-hydroxyphenyl units at C-1 and C-7, while the HMBC correlation between the methoxy protons (δH 3.29) and C-5 (δC 80.9) confirmed the position of the methoxy group at C-5 (Figure 2). The absolute configuration at C-3 in 1 was determined to be S by the advanced Mosher’s method16 (Table 2). In addition, the chemical shifts of C-4 (δC 45.0) and C-5 (δC 80.9) in 1 confirmed the 5R absolute configuration by comparison of its data with those of 3-hydroxy-1-(4hydroxyphenyl)-5-methyoxy-7-phenyl-6E-heptene: (3S,5S) B
dx.doi.org/10.1021/np300490v | J. Nat. Prod. XXXX, XXX, XXX−XXX
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Figure 1. Chemical structures of compounds 1 − 6, and 9.
Figure 2. Key HMBC correlations of compounds 1−6 and 9.
between api H-1‴ (δH 5.45) and glc C-2″ (δC 74.9), and between glc H-2″ (δH 3.60) and api C-1‴ (δC 108.3) were observed (Figure 2). These results indicated the sugar moiety of 4 to be O-β-D-apiofuranosyl-(1→2)-O-β-D-glucopyranoside and its location at C-4′. This was also supported by agreement of the 13 C NMR data reported for homoflavoyadorinin-B (8) from
and C-3′ (δC 148.5), respectively, confirmed their locations (Figure 2). The 13C NMR data showed the presence of a β-Dapiofuranose moiety and a β-D-glucopyranose moiety. Furthermore, acid hydrolysis of 4 confirmed their presence as sugar components (identified as TMS derivatives by GC). The HMBC correlations between glc H-1″ (δH 5.14) and C-4′ (δC 148.7), C
dx.doi.org/10.1021/np300490v | J. Nat. Prod. XXXX, XXX, XXX−XXX
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Table 2. 1H NMR Data for MTPA Ester Derivatives of Compounds 1−3 δHa,c (J in Hz) pos.
1b (S)
Δδ = δS − δR
1 2 3 4
2.62, m 2.01, m 5.41, m 1.84, dt (4.0, 8.0)
2.53, m 1.91, m 5.39, m 1.93, m
−0.09 −0.10 −0.02 +0.09
5 6
3.50, m 5.93, dd (8.0, 16.0) 6.31, d (16.0) 7.15, d (8.4) 7.03, d (8.4) 7.37, d (8.4) 7.07, d (8.4) 3.22, s
3.64, m 5.98, dd (8.0, 16.0) 6.42, d (16.0) 7.09, d (8.4) 7.01, d (8.4) 7.40, d (8.4) 7.09, d (8.4) 3.25, s
+0.14 +0.05
7 2′, 6′ 3′, 5′ 2″, 6″ 3″, 5″ 5-OMe a
1a (R)
δHa,c (J in Hz)
+0.11 −0.06 −0.02 +0.03 +0.02 +0.03
2a (R) 2.62, t (10.0) 2.01, m 5.19, m 1.80, m 2.11, m 3.60, m 5.89, dd (8.0, 16.0) 6.42, d (16.0) 7.14, d (8.4) 7.02, d (8.4) 7.38, d (8.4) 7.09, d (8.4) 3.19, s
δHb,c (J in Hz)
2b (S)
Δδ = δS − δR
2.49, dd (7.0, 8.6) 1.92, m 5.26, m 1.81, m 2.15, m 3.73, m 5.95, dd (8.0, 16.0) 6.47, d (16.0) 7.06, d (8.4) 6.99, d (8.4) 7.40, d (8.4) 7.09, d (8.4) 3.23, s
−0.13 −0.09 −0.07 +0.01 +0.04 +0.13 +0.06
2.75, m 2.09, m 5.68, m 3.47 3.12
6.71, d (16.0) 6.83, d (16.0)
+0.12
+0.05 −0.08 −0.03 +0.02 +0.02 +0.04
7.54, d (16.0) 7.30, d (8.4) 7.05, d (8.4) 7.68, d (8.4) 7.07, d (8.4)
+0.05 −0.09 −0.02 +0.05 +0.10
3a (R)
3b (S) 2.56, m 1.99, m 5.65, m 3.48 3.13
7.59, d (16.0) 7.21, d (8.4) 7.03, d (8.4) 7.73, d (8.4) 7.17, d (8.4)
Δδ = δS − δR −0.19 −0.10 +0.03 +0.01 +0.01
Measured in CDCl3. bMethanol-d4. c400 MHz.
V. album.12 Consequently, compound 4 was determined to be 3,7,3′-tri-O-methylquercetin-4′-O-β-D-apiofuranosyl-(1→2)-Oβ-D-glucopyranoside. The molecular formula of 5 was determined as C35H42O21 by the HRESIMS ion at m/z 797.2141 (calcd for C35H41O21, 797.2146). The 1H and 13C NMR data showed the presence of a 3-hydroxy-3-methylglutaroyl moiety17 and were similar to those of viscumneoside IV17 except for the addition of a glucosyl moiety (Table 4). Acid hydrolysis of 5 gave a D-glucose (identified as a TMS derivative by GC). The HMBC correlation between glc H-1″ (δH 5.08, d, J = 7.5 Hz) and C-4′ (δC 150.4) suggested the position of a β-D-glucopyranosyl unit at C-4′. The HMBC correlations between glc H-1‴ (δH 5.35) and C-3 (δH 135.9) and between glc H-6″′ (δH 3.75 and 3.88) and glt C-1″″ (δC 172.2) confirmed that the α-D-glucopyranosyl moiety (J1‴,2‴ = 2.0 Hz) was located at C-3 and the 3-hydroxy-3methylglutaroyl moiety was at C-6″′ of this sugar unit (Figure 2). This was confirmed by the HRESIMS/MS, which showed a pseudomolecular ion peak in the negative mode at 797.2164 [M − H]−, giving fragment ions at 635.1612 [M − (4″-glc)]− and 491.1176 [M − (3-glt + glc)]−. Thus, compound 5 was defined as 7,3′-di-O-methylquercetin-4′-O-β-D-glucopyranosyl-3-O-[6″-(3hydroxy-3-methylglutaroyl)]-α-D-glucopyranoside. The molecular formula of 6 was determined as C52H64O29 by the HRESIMS ion at m/z 1187.3223 (calcd for C52H64O29Cl, 1187.3227). The 1H and 13C NMR data were similar to those of 5 except for additional syringenin and glucose moieties at C-1″″ (Table 4). In addition, the HMBC correlations from the methoxy protons (δH 3.85) to C-3″″″/C-5″″″ (δC 154.4) and from the olefinic H-7″″″ (δH 6.54, d, J = 16.0 Hz) to C-1″″″ (δC 135.2), C-2″″″/C-6″″″ (δC 105.4), C-8″″″ (δC 130.0), and C-9″″″ (δC 63.6) confirmed the presence of a syringenin moiety in 6. The HMBC correlations between the methoxy groups at δH 3.95 and 4.02 and C-7 (δC 167.4) and C-3′ (δC 149.9), respectively, suggested substitution at C-7 and C-3′ of the flavonol moiety (Figure 2). Acid hydrolysis of 6 gave a D-glucose (identified as a TMS derivative by GC). Two O-β-D-glucopyranosyl (J1,2 = 7.5) and one O-α-D-glucopyranosyl (J1,2 = 4.0 Hz) group were deduced from the coupling constants. The location of one of the β-D-glucopyranosyl moieties at C-4′ was determined by the HMBC correlation between glc H-1″ (δH 5.14) and C-4′ (δC 150.4). The HMBC correlations between glc H-1‴ (δH 5.46) and C-3 (δH 135.9) and between glc H-1″″′ (δH 4.94) and syr C-4″″″
(δC 135.2) suggested that the remaining O-α- and O-β-Dglucopyranosyl units were located at C-3 of the flavonol and C-4″″″ of the syringenin moieties, respectively. The HMBC correlations between H-6‴ (δH 4.31 and 4.39) and C-1″″ (δC 172.1) and between H-6‴″ (δH 4.17 and 4.22) and C-5″″ (δC 172.2) revealed that these glucose units were connected via a 3-hydroxy-3-methylglutaroyl moiety (Figure 2). In addition, the structure of 6 was supported by HRESIMS/MS showing a pseudomolecular ion peak in the negative mode at 1187.3234 [M − H]− and its fragment ions at m/z 989.2854 [M − (4″-glc)]− and 491.1176 [M − (3-syr + glc + glt + glc)]−. Thus, compound 6 was defined as 7,3′-di-O-methylquercetin-4′-O-β-D-glucopyranosyl3-O-[(6⁗′→5⁗)-O-1⁗′-(sinap-4-yl)-β-D-glucopyranosyl-6‴-(3hydroxy-3-methylglutaroyl)]-α-D-glucopyranoside. The molecular formula of 9 was determined as C33H42O19 by the HRESIMS ion at m/z 741.2284 (calcd for C33H41O19, 741.2248). The 1H NMR spectrum showed signals for five aromatic protons at δH 6.04, 6.09, 7.00, 7.12, and 7.13; three protons at δH 2.77 (dd, J = 2.8, 17.2 Hz), 3.15 (m), and 5.40 (dd, J = 2.8, 12.4 Hz), suggesting the presence of a flavanone moiety;18 and three anomeric protons at δH 4.80 (br s), 5.02 (d, J = 7.6 Hz), and 5.50 (br s), suggesting the presence of three sugar units. The NMR data of 9 were similar to those of (2S)-5hydroxy-7,3′-dimethoxyflavanone-4′-O-β-D-apiofuranosyl-(1→ 2)-O-β-D-glucopyranoside (10)18 except for the addition of an apiose unit at C-5‴ (Table 3). The locations of the methoxy groups at C-7 and C-3′ were assigned on the basis of the HMBC correlations between the methoxy protons (δH 3.77 and 3.87) and C-7 (δC 169.5) and C-3′ (δC 150.9), respectively (Figure 2). Comparison of the NMR data of 9 with those of viscumneoside V (12)19 implied similar sugar linkages. Furthermore, acid hydrolysis of 9 revealed D-apiose and D-glucose units (identified as TMS derivatives by GC). HMBC correlations between glc H-1″ (δH 5.02) and C-4′ (δC 148.2); between api H-1‴ (δH 5.50) and glc C-2″ (δC 77.5); and between api H-1″″ (δH 4.80) and api C-2″′ (δC 78.6) were observed. Thus, the sugar moiety was identified as O-β-D-apiofuranosyl-(1→5)-O-β-D-apiofuranosyl(1→2)-O-β-D-glucopyranoside and located at C-4′ (Figure 2). The 2S configuration was assigned by electronic circular dichroism spectroscopy (positive Cotton effect at 331 nm and negative Cotton effect at 288 nm).12 Consequently, compound 9 was defined as (2S)-5-hydroxy-7,3′-dimethoxyflavanone-4′-O-β-D-apiofuranosyl(1→5)-O-β-D-apiofuranosyl -(1→2)-O-β-D-glucopyranoside. D
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The inhibitory effects of the extracts and the isolated compounds on pro-inflammatory cytokines were evaluated by measuring the production of TNF-α, IL-6, and IL-12p40 in LPS-stimulated BMDCs. We first used a colorimetric MTT assay (Sigma, St. Louis, MO, USA) to test cell viability, and it was confirmed that these tested samples had no or little cytotoxicity at the concentration tested (data not shown). The MeOH extract showed inhibition of TNF-α and IL-12p40 production. The CHCl3 fraction exhibited significant inhibition of production of all pro-inflammatory cytokines, while the EtOAc fraction showed potent inhibitory effects against IL-6 and IL-12p40 production (Table 5). However, the H2O fraction showed no activity against the production of any tested cytokines. Candidate compounds with inhibitory effects on pro-inflammatory cytokines over 50% at a concentration of 50 μM were further tested at concentrations of 2, 5, 10, 25, and 50 μM. SB203580, an inhibitor of cytokine suppressive binding protein/p38 kinase, was used as a positive control. SBS203580 inhibited TNF-α, IL-6, and IL-12p40 production with IC50 values of 7.53 ± 0.43, 3.45 ± 0.12, and 5.21 ± 0.32 μM, respectively (Table 6). Among the compounds tested, compounds 1, 4, and 19 showed potent inhibitory effects on production of TNF-α, IL-6, and IL-12p40 with IC50 values ranging from 0.09 ± 0.01 to 8.96 ± 0.45 μM, while compound 13 showed significant inhibitory activity only on IL-12p40, with an IC50 of 2.00 ± 0.15 μM. Compounds 2, 14, 15, and 20 showed moderate inhibitory activities on the production of TNF-α, IL-6, and IL-12p40. The remaining compounds did not show significant activity (IC50 > 50 μM). The chloroform and ethyl acetate fractions as well as compounds 1, 4, and 19 from the leaves and twigs of V. album significantly inhibited LPS-stimulated production of TNF-α, IL-6, and IL-12p40 in BMDCs. Further studies are needed to assess the mechanisms of action and the potential for their use as new anti-inflammatory agents.
Table 3. NMR Spectroscopic Data for Compounds 4 and 9 4 pos.
δC,a,c type
δHa,d (J in Hz)
9 δC,b,c type
δHb,d (J in Hz) 5.40, dd (2.4, 12.4) 2.77, dd (2.8, 17.2) 3.15, m
2
155.2, C
80.5, CH
3
138.4, C
44.2, CH2
4 5 6 7 8 9 10 1′ 2′ 3′ 4′ 5′ 6′ 3-OMe 7-OMe 3′-OMe 4′-O-Glc 1″ 2″ 3″ 4″ 5″ 6″
178.1, C 160.8, C 98.1, CH 165.2, C 92.5, CH 156.3, C 105.2, C 123.2, C 111.9, CH 148.5, C 148.7, C 114.7, CH 121.6, CH 59.8, CH3 55.8, CH3 56.1, CH3
198.0, C 165.2, C 95.0, CH 169.5, C 95.8, CH 164.5, C 104.1, C 134.5, C 111.8, CH 150.9, C 148.2, C 116.9, CH 120.0, CH
97.8, CH 74.9, CH 77.1, CH 69.9, CH 77.0, CH 60.6, CH2
2″-O-Api 1‴ 2‴ 3‴ 4‴
108.3, CH 76.1, CH 79.3, C 73.9, CH2
5‴
64.5, CH2
6.37, s 6.80, s
7.69, s
7.25, d (8.5) 7.67, d (8.5) 3.85, s 3.87, s 3.87, s
6.09* 6.04, d (2.0)
7.12*
7.13* 7.00, d (8.0)
56.4, CH3 56.5, CH3
3.77, s 3.87, s
5.14, d (8.5) 3.60* 3.55* 3.22, m 3.42, m 3.48* 3.72, dd (5.6, 10.0)
100.7, CH 77.5, CH 78.8, CH 71.4, CH 78.1, CH 62.5, CH2
5.02, d (7.6) 3.73 3.60 3.41 3.41 3.67 3.86
5.45, s 3.79, s
110.3, CH 78.6, CH 79.8, C 75.7, CH2
5.50, br s 3.94, d (0.8)
3.62, d (9.5) 4.08, d (9.5) 3.32, br s
72.0, CH2
5‴-O-Api 1″″ 2″″ 3″″ 4″″
110.5, CH 77.8, CH 80.4, C 74.9, CH2
5″″
65.5, CH2
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EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were determined on a Jasco DIP-370 digital polarimeter. Electronic circular dichroism spectra were determined on a Chirascan CD spectrometer. The IR spectra were obtained from a Tensor 37 FT-IR spectrometer (Bruker, Ettlingen, Germany). 1H NMR (400 MHz) and 13C NMR (100 MHz) spectra were recorded on an Agilent 400-MR NMR spectrometer, and TMS was used as an internal standard. Data processing was carried out with the MestReNova 6.0.2 program. HRESIMS spectra were obtained using an Agilent 6550 iFunnel Q-TOF LC/MS system. GC was recorded on a Shimadzu GC-2010 Plus. Preparative HPLC was carried out using an Agilent 1290 HPLC system. Column chromatography was performed on silica gel (Kieselgel 60, 70−230 mesh and 230−400 mesh, Merck) and YMC RP-18 resins. Plant Material. The leaves and twigs of Viscum album were collected in Inje, Gangwon Province, Korea, during December 2010, and authenticated by Dr. Jeong Eun Han at National Institute of Biological Resources, Korea. A voucher specimen (NIBRVP0000274646) was deposited at the Herbarium of Pharmacy, Yonsei Institute of Pharmaceutical Sciences, Yonsei University, Incheon, Korea, and Wildlife Genetic Resources Center, National Institute of Biological Resources, Incheon, Korea. Extraction and Isolation. The dried leaves and twigs of V. album (3.4 kg) were extracted with hot MeOH three times (3 × 10 L) under reflux for 15 h to yield 658 g of extract after evaporation of the solvent. This extract was suspended in H2O and successively partitioned with CHCl3 and EtOAc to obtain the CHCl3 (VA1, 91.0 g), EtOAc (VA2, 18.0 g), and H2O (VA3, 550.0 g) fractions after removal of the solvents in vacuo. The VA1 fraction (91.0 g) was chromatographed on a silica gel column eluting with a gradient of n-hexane−acetone (40:1 → 0:1, v/v) to obtain five subfractions, VA1A (19.0 g), VA1B (17.0 g),
3.75, d (9.6) 4.21, d (9.6) 3.41 3.71 4.80, br s 3.82 3.67 3.82 3.49, br s
a
Measured in DMSO-d6. bMethanol-d4. c100 MHz. d400 MHz. *Overlapped signals. Assignments were done by HMQC, HMBC, and COSY experiments. Glc, glucopyranosyl; Api, apiofuranosyl.
The known compounds were characterized as 3′-methoxyapiin (7),20 homoflavoyadorinin-B (8),12 (2S)-5-hydroxy-7,3′dimethoxyflavanone-4′-O-β-[apiofuranosyl-(1→2)]-glucopyranoside (10),18 (2S)-homoeriodictyol-7-O-[apiofuranosyl-(1→ 2)]-glucopyranoside (11),12 viscumneoside V (12),19 (+)-medioresinol (13),21 (+)-pinoresinol (14),22 (−)-lyoniresinol 3α-Oβ-D-glucopyranoside (15), (+)-lyoniresinol 3α-O-β-D-glucopyranoside (16),23 syringin (17),24 syringenin 4′-O-β-D-apiofuranosyl-(1→2)-β-O-D-glucopyranoside (18),25 trans-phytol (19),26 nerolidol (20),27 erythrodiol (21),28 oleanolic acid (22), β-amyrin acetate (23),29 and betulinic acid (24),30 by comparing their observed and reported physical data. E
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Table 4. NMR Spectroscopic Data for Compounds 5 and 6 5 pos.
δC,a,b type
2 3 4 5 6 7 8
158.3, C 135.9, C 179.4, C 162.7, C 99.3, CH 167.4, C 93.3, CH
9 10 1′ 2′ 3′ 4′ 5′ 6′ 7-OMe 3′-OMe 4′-O-Glc 1″ 2″ 3″ 4″ 5″ 6″
158.4, C 106.7, C 125.8, C 114.9, CH 149.9, C 150.4, C 116.6, CH 123.7, CH 56.6, CH3 57.0, CH3
6 δHa,c (J in Hz)
δC,a,b type 158.3, C 135.9, C 179.5, C 162.7, C 99.3, CH 167.4, C 93.5, CH
6.33, s 6.60, s
7.93, s
7.26, d (8.0) 7.69, d (8.0) 3.89, s 3.96, s
158.3, C 106.7, C 125.9, C 115.1, CH 150.0, C 150.4, C 116.7, CH 123.7, CH 56.6, CH3 57.2, CH3
δHa,c (J in Hz)
6.39, s 6.66, s
8.01, s
7.33, d (8.0) 7.76, d (8.0) 3.95, s 4.02, s
102.1, CH 74.8, CH 77.8, CH 71.3, CH 78.3, CH 62.5, CH2
5.08, d (7.5) 3.57 3.55 3.46 3.53, m 3.75, dd (5.0, 12.0) 3.88*
102.1, CH 74.8, CH 77.9, CH 71.3, CH 78.3, CH 62.5, CH2
5.14, d (7.5) 3.63 3.58 3.51 3.56 3.80, dd (5.0, 12.0) 3.94, dd (7.0, 12.0)
3-O-Glc 1″′ 2″′ 3″′ 4″′ 5″′ 6″′
103.7, CH 75.8, CH 77.8, CH 71.6, CH 75.8, CH 64.5, CH2
5.35, d (2.0) 3.47* 3.48* 3.30* 3.49, m 4.11, dd (6.0, 12.0) 4.23, d (12.0)
103.6, CH 75.8, CH 77.7, CH 71.7, CH 75.7, CH 64.5, CH2
5.46, d (4.0) 3.54* 3.56* 3.43* 3.44* 4.31, d (12.0) 4.39, d (12.0)
6″-Glt 1″″ 2″″
172.2, C 46.2, CH2
2.47, d (7.5)
172.1, C 46.1, CH2
3″″ 4″″
70.6, C 46.0, CH2
2.44, d (6.0)
70.6, C 46.1, CH2
5″″ 6″″
175.3, C 27.6, CH3
1.17, s
172.2, C 27.7, CH3
pos.
δC,a,b type
δHa,c (J in Hz)
5″″-O-Glc 1″″′ 2″″′ 3″″′ 4″″′ 5″″′ 6″″′
105.0, CH 75.8, CH 78.3, CH 71.8, CH 77.9, CH 64.6, CH2
4.94, d (7.5) 3.57* 3.57* 3.33* 3.51* 4.17, dd (7.0, 12.0) 4.22, dd (5.0, 12.0)
4″″′-O-Syr 1″″″ 2″″″ 3″″″ 4″″″ 5″″″ 6″″″ 7″″″ 8″″″ 9″″″ 3″″″, 5″″″-OMe
135.2, C 105.4, CH 154.4, C 135.2, C 154.4, C 105.4, CH 131.3, CH 130.0, CH 63.6, CH2 57.0, CH3
6.70, s
6.70, s 6.54, d (16.0) 6.33, dt (5.0, 16.0) 4.27, d (5.0) 3.85, s
2.39, d (15.0) 2.47, d (15.0) 2.43, d (15.0) 2.49, d (15.0) 1.17, s
Measured in methanol-d4. 100 MHz. 400 MHz. *Overlapped signals. Assignments were done by HMQC, HMBC, and COSY experiments. Glc, glucopyranosyl; Glt, 3-hydroxy-3-methylglutaroyl; Syr, syringenin. a
b
c
(8:1, v/v) to give four fractions, VA1D1 (3.2 g), VA1D2 (2.4 g), VA1D3 (2.8 g), and VA1D4 (3.0 g). The VA1D2 fraction was chromatographed on HPLC using a J’sphere ODS H-80 250 mm × 20 mm column, with 70% MeCN(aq) and a flow rate of 5 mL/min, to yield 13 (100.0 mg) and 14 (45.0 mg). The VA1D3 fraction was chromatographed on HPLC using a J’sphere ODS H-80 250 mm × 20 mm column, with 70% MeCN(aq) and a flow rate of 5 mL/min, to yield 1 (9.0 mg), 2 (9.0 mg), and 3 (10.0 mg). The VA2 fraction was chromatographed on a silica gel column eluting with a gradient of CHCl3−MeOH (10:1 → 2:1, v/v) to give four fractions, VA2A (4.0 g), VA2B (6.3 g), VA2C (3.8 g), and VA2D (2.6 g). The VA2C fraction was chromatographed on a Sephadex LH-20 column eluting with MeOH to give 4 (28.0 mg), 7 (4.1 mg), and 8 (2.0 g).
VA1C (10.0 g), VA1D (13.0 g), and VA1E (10.0 g). The VA1B fraction was chromatographed on a silica gel column eluting with n-hexane− EtOAc (10:1, v/v) to give four smaller fractions, VA1B1 (4.0 g), VA1B2 (3.0 g), VA1B3 (5.8 g), and VA1B4 (2.8 g). The VA1B1 fraction was chromatographed on a YMC RP-18 column eluting with acetone−H2O (4:1, v/v) to yield 19 (130.0 mg) and 23 (200.0 mg). The VA1B3 fraction was chromatographed on a YMC RP-18 column eluting with acetone−H2O (4:1, v/v) to yield 20 (7.0 mg) and 24 (25.0 mg). The VA1C fraction was chromatographed on a silica gel column eluting with n-hexane−EtOAc (5:1, v/v) to give three fractions, VA1C1 (3.0 g), VA1C2 (2.4 g), and VA1C3 (3.6 g). The VA1C2 fraction was chromatographed on a YMC RP-18 column eluting with acetone−H2O (3.5: 1, v/v) to yield 21 (18.0 mg) and 22 (30.0 mg). The VA1D fraction was chromatographed on a silica gel column eluting CHCl3−MeOH F
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a silica gel column eluting with a gradient of CHCl3−MeOH (10:1 → 2:1, v/v) to give five fractions, VA3D1 (3.5 g), VA3D2 (7.0 g), VA3D3 (8.2 g), VA3D4 (4.2 g), and VA3D5 (4.4 g). The VA3D2 fraction was chromatographed on a YMC RP-18 column eluting with MeOH−H2O (1:1, v/v) to yield 10 (9.0 mg) and 12 (250.0 mg). The VA3D3 fraction was chromatographed on a YMC RP-18 column eluting with MeOH− H2O (1:1, v/v) to yield 9 (18.0 mg) and 11 (400.0 mg). (3S,5R)-3-Hydroxy-5-methoxy-1,7-bis(4-hydroxyphenyl)-6E-heptene (1): white, amorphous powder; [α]20D −10 (c 0.1, MeOH); IR (film) νmax 3350, 1602, 1460, 1090 cm−1; 1H and 13C NMR, see Table 1; HRESIMS m/z 327.1590 [M − H]− (calcd for C20H23O4, 327.1602), 363.1345 [M + Cl]− (calcd for C20H24O4Cl, 363.1369). (3S,5S)-3-Hydroxy-5-methoxy-1,7-bis(4-hydroxyphenyl)-6E-heptene (2): white, amorphous powder; [α]20D −8 (c 0.1, MeOH); IR (film) νmax 3349, 1600, 1460, 1090 cm−1; 1H and 13C NMR, see Table 1; HRESIMS m/z 327.1597 [M − H]− (calcd for C20H23O4, 327.1602), 363.1344 [M + Cl]− (calcd for C20H24O4Cl, 363.1369). (3S)-3-Hydroxy-1,7-bis(4-hydroxyphenyl)-6E-hepten-5-one (3): white, amorphous powder; [α]20D −18 (c 0.1, MeOH); IR (film) νmax 3244, 1717, 1590, 1420 cm−1; 1H and 13C NMR, see Table 1; HRESIMS m/z 311.1290 [M − H]− (calcd for C19H19O4, 311.1289), 347.1060 [M + Cl]− (calcd for C19H20O4Cl, 347.1056). 3,7,3′-Tri-O-methylquercetin-4′-O-β-D-apiofuranosyl-(1→2)-O-β20 D-glucopyranoside (4): yellow, amorphous powder; [α] D −8 (c 0.1, MeOH); IR (film) νmax 3325, 1654, 1600, 1500, 1070 cm−1; 1H and 13 C NMR, see Table 3; HRESIMS m/z 637.1754 [M − H]− (calcd for C29H33O16, 637.1774), 673.1541 [M + Cl]− (calcd for C29H34O16Cl, 637.1774). 7,3′-Di-O-methylquercetin-4′-O-β-D-glucopyranosyl-3-O-[6‴-(3hydroxy-3-methylglutaroyl)]-α-D-glucopyranoside (5): yellow, amorphous powder; [α]20D −75 (c 0.1, MeOH); IR (film) νmax 3142, 1668, 1606, 1510, 1068 cm−1; 1H and 13C NMR, see Table 4; HRESIMS m/z 797.2141 [M − H]− (calcd for C35H41O21, 797.2146). 7,3′-Di-O-methylquercetin-4′-O-β-D-glucopyranosyl-3-O-[(6⁗′→ 5⁗)-O-1⁗′-(sinap-4-yl)-β-D-glucopyranosyl-6‴-(3-hydroxy-3-methylglutaroyl)]-α-D-glucopyranoside (6): yellow, amorphous powder; [α]20D −21 (c 0.1, MeOH); IR (film) νmax 3142, 1660, 1602, 1505, 1069 cm−1; 1H and 13C NMR, see Table 4; HRESIMS m/z 1187.3223 [M + Cl]− (calcd for C52H64O29Cl, 1187.3227). (2S)-5-Hydroxy-7,3′-dimethoxyflavanone-4′-O-β-D-apiofuranosyl-(1→5)-O-β-D-apiofuranosyl-(1→2)-O-β-D-glucopyranoside (9): colorless, amorphous powder; [α]20D −90 (c 0.1, MeOH); ECD (c 6.0 × 10−4 M, MeOH) [θ]25 (nm) +6696 (331) (positive maximum), −24 658 (288) (negative maximum); IR (film) νmax 3411, 1620, 1590, 1070, 1022 cm−1; 1H and 13C NMR, see Table 3; HRESIMS m/z 741.2284 [M − H]− (calcd for C33H41O19, 741.2248), 777.2051 [M + Cl]− (calcd for C33H42O19Cl, 777.2014). Preparation of MTPA Esters. A solution of each compound (1, 2, and 3: 2.0 mg) in 300 μL of anhydrous CH2Cl2 was reacted with (R)-MTPA chloride (8.0 μL) in the presence of 4-DMAP (3.0 mg); the mixture was stirred occasionally at room temperature for 1 h. After adding 300 μL of CH2Cl2, the solution was washed with H2O (300 μL) and 5% HCl (300 μL), successively. The organic layer was evaporated, and the residue was purified by preparative TLC silica gel (0.25 mm thickness), developed with n-hexane−EtOAc (4:1, v/v), and eluted with MeOH to afford the ester, 1a, 2a, and 3a (each 2.5 mg), while those with (S)-MTPA chloride were subjected to normal-phase preparative TLC with n-hexane−EtOAc (4:1, v/v) and then eluted with MeOH to afford the esters 1b, 2b, and 3b (each 2.5 mg). For 1H NMR data of 1a, 1b, 2a, 2b, 3a, and 3b, see Table 2. Acid Hydrolysis. Each compound (4, 5, 6, and 9: 0.5 mg) was separately dissolved in 1 N HCl (dioxane−H2O, 1:1, 1 mL) and heated to 80 °C in a water bath for 3 h. The acidic solutions were neutralized with Ag2CO3, and the solvent was evaporated under N2 gas overnight. After extraction with CHCl3, the aqueous layers were concentrated to dryness using N2 gas. The residues were dissolved in 0.1 mL of dry pyridine, and then L-cysteine methyl ester hydrochloride in pyridine (0.06 M, 0.1 mL) was added to the solutions. The mixtures were heated at 60 °C for 2 h, and 0.1 mL of TMS was added, followed by heating at 60 °C for 1.5 h. The dried products were partitioned with n-hexane and
Table 5. Anti-inflammatory Effects of Extract and Fractions on LPS-Stimulated BMDCs IC50 (μg/mL)b extract
TNF-α
IL-6
IL-12p40
MeOH extract CHCl3 fraction EtOAc fraction H2O fraction SB203580a
10.18 ± 0.56 1.14 ± 0.04 20.40 ± 1.54 >50 3.65 ± 0.20
>50 1.86 ± 0.06 0.97 ± 0.04 >50 1.67 ± 0.05
4.75 ± 0.14 0.23 ± 0.01 0.12 ± 0.00 >50 2.52 ± 0.15
SB203580 was used as a positive control. bIC50 values < 50 μg/mL are considered to be active. a
Table 6. Anti-inflammatory Effects of Compounds 1−24 on LPS-Stimulated BMDCs IC50 (μM)b compound
TNF-α
IL-6
IL-12p40
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 SB203580a
0.32 ± 0.01 >50 >50 1.01 ± 0.06 >50 >50 >50 >50 >50 >50 >50 >50 22.29 ± 1.37 >50 >50 >50 >50 >50 1.22 ± 0.04 2.02 ± 0.07 >50 >50 >50 19.45 ± 1.56 7.53 ± 0.43
0.68 ± 0.02 >50 >50 8.96 ± 0.45 >50 >50 >50 >50 >50 >50 >50 >50 20.87 ± 1.65 >50 >50 >50 >50 >50 2.02 ± 0.09 20.19 ± 0.98 >50 >50 >50 21.27 ± 1.02 3.45 ± 0.12
0.09 ± 0.01 11.84 ± 0.34 >50 3.86 ± 0.12 >50 >50 76.79 ± 2.35 >50 >50 >50 >50 >50 2.00 ± 0.15 28.58 ± 1.25 14.55 ± 0.55 >50 >50 >50 3.62 ± 0.12 7.57 ± 0.25 13.51 ± 0.78 >50 >50 3.61 ± 0.12 5.21 ± 0.32
a SB203580 was used as a positive control. bIC50 values < 50 μM are considered to be active.
The water-soluble fraction (VA3, 550 g) was chromatographed on a Diaion HP-20P column eluting with H2O containing increasing concentrations of MeOH (0, 25, 50, 75, and 100%) to obtain five subfractions, VA3A (438.0 g), VA3B (25.0 g), VA3C (27.0 g), VA3D (30.0 g), and VA3E (30.0 g). The VA3B fraction was chromatographed on a silica gel column eluting with CHCl3−MeOH−H2O (5:1:0.1, v/v/v) to give 17 (80.0 mg) and 18 (120.0 mg). The VA3C fraction was chromatographed on a silica gel column eluting with a gradient of CHCl3−MeOH (10:1 → 2:1, v/v) to give four fractions, VA3C1 (6.5 g), VA3C2 (5.0 g), VA3C3 (7.2 g), and VA3C4 (5.6 g). The VA3C2 fraction was chromatographed on a Sephadex LH-20 column eluting with MeOH to give four fractions, VA3C2A (1.5 g), VA3C2B (0.9 g), VA3C2C (1.2 g), and VA3C2D (1.0 g). The fraction VA3C2A was chromatographed on HPLC using a J’sphere ODS H-80 250 mm × 20 mm column, with 20% MeCN(aq) and a flow rate of 5 mL/min, to yield 15 (46.0 mg) and 16 (100.0 mg). The VA3C2D fraction was chromatographed on HPLC using a J’sphere ODS H-80 250 mm × 20 mm, with 20% MeCN(aq) and a flow rate of 5 mL/min, to yield 5 (10.0 mg) and 6 (9.0 mg). The VA3D fraction was chromatographed on G
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H2O (0.1 mL each), and the organic layer was analyzed by GC: column SPB-1 (0.25 mm × 30 m), detector FID, column temp 210 °C, injector temp 270 °C, detector temp 300 °C, carrier gas He (2 mL/min). A peak was found at the retention time of 14.11 min for compounds 5 and 6. Two peaks were found at the retention time of 6.70 min for compounds 4 and 9. When standard solutions were prepared by the same reaction from the standard glucoses and apiose, the retention times of persilylated D-glucose, L-glucose, D-apiose, and L-apiose were 14.11, 14.26, 6.70, and 6.95 min, respectively. By comparing of retention times, sugars in compounds 4 and 9 were identified as D-glucose and D-apiose, and the sugar in compounds 5 and 6 was determined to be D-glucose. Cell Culture and Measurement of Cytokine Production. BMDCs were grown from wild-type C57BL/6 mice (Taconic Farm, NY, USA). Briefly, the mouse tibia and femur were obtained by flushing with Dulbecco’s modified Eagle’s medium to yield bone marrow cells. The cells were cultured in RPMI 1640 medium containing 10% heatinactivated fetal bovine serum (FBS) (Gibco, NY, USA), 50 μM β-mercaptoethanol, and 2 mM glutamine supplemented with a 3% J558L hybridoma cell culture supernatant containing granulocytemacrophage colony-stimulating factor. The culture medium was replaced with fresh medium every second day. At day 6 of culture, nonadherent cells and loosely adherent DC aggregates were harvested, washed, and resuspended in RPMI 1640 supplemented with 5% FBS. The DCs were incubated in 48-well plates in 0.5 mL containing 1 × 105 cells per well treated with the extracts in DMSO (1, 2, 5, and 10 μg/mL) and compounds in DMSO (2, 10, 25, and 50 μM) for 1 h before stimulation with 10 ng/mL LPS from Salmonella minnesota (Alexis, NY, USA). Supernatants were harvested 16 h after stimulation. Concentrations of murine TNF-α, IL-6, and IL-12p40 in the culture supernatant fraction were determined by ELISA (Pharmingen, CA, USA) according to the manufacturer’s instructions. The data are presented as means ± standard deviation of at least three independent experiments performed in triplicate.
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ASSOCIATED CONTENT
S Supporting Information *
1D and 2D NMR and mass spectra are included for compounds 1−6 and 9 along with the CD spectrum of compound 9. This material is available free of charge via the Internet at http://pubs. acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Tel: 82-32-749-4514. Fax: 82-32-749-4105. E-mail: kimsh11@ yonsei.ac.kr. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2011-0025129). This work was supported in part by the Yonsei University Research Fund of 2012.
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REFERENCES
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