Neolignanamides, Lignanamides, and Other Phenolic Compounds

Article pubs.acs.org/jnp

Neolignanamides, Lignanamides, and Other Phenolic Compounds from the Root Bark of Lycium chinense Jing-Xian Zhang,†,‡ Shu-Hong Guan,*,† Rui-Hong Feng,† Yang Wang,†,‡ Zhi-Yuan Wu,† Yi-Bei Zhang,† Xiao-Hui Chen,‡ Kai-Shun Bi,‡ and De-An Guo*,† †

Shanghai Research Center for Modernization of Traditional Chinese Medicine, National Engineering Laboratory for TCM Standardization Technology, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 501 Haike Road, Shanghai 201203, People’s Republic of China ‡ Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang 110016, People’s Republic of China S Supporting Information *

ABSTRACT: Seven new neolignanamides (1−7), including two pairs of cis- and trans-isomers, and a new lignanamide (8) were isolated from the EtOAc-soluble fraction of an EtOH extract of the root bark of Lycium chinense, together with 22 known phenolic compounds (9−30), four of which were obtained from the genus Lycium for the first time. Compounds 5, 6, and 7 are unusual dimers having a rare connection mode between the two cinnamic acid amide units, while compounds 6, 7, and 8 are the first naturally occurring dimers derived from two dissimilar cinnamic acid amides. The cinnamic acid amides, neolignanamides, and lignanamides possess moderate radical-scavenging activity against the DPPH (2,2-diphenyl-1-picrylhydrazyl) and superoxide radicals.

T

structure of 1 was similar to that of thoreliamide B10 by comparison of their NMR data (Table 1). The 1H and 13C NMR spectra of 1 indicated an ABX spin system at δH 7.01 (1H, d, J = 1.8 Hz, H-2′), 6.82 (1H, d, J = 8.1 Hz, H-5′), and 6.89 (1H, dd, J = 8.1, 1.8 Hz, H-6′) in 1 replacing a 1,3,4,5tetrasubstituted aromatic moiety in thoreliamide B. The methoxy group at δH 3.87 was placed at C-3′ by its HMBC correlation with C-3′. In addition, the ESIMSn experiment of compound 1 afforded the protonated molecule [M + H]+ at m/ z 478 in the positive ion mode. Its characteristic product ion peaks m/z 341 and 298 were formed, respectively, by the fission of the amide bond to eliminate a p-tyramine (137 Da) moiety and by a retro-Diels−Alder cleavage of the 1,4-dioxane ring to eliminate a coniferyl alcohol (180 Da) moiety. The proposed fragmentation pathway of 1 is shown in Figure 1. On the basis of spectroscopic data, compound 1 was assigned as (E)-3-{(2,3trans)-2-(4-hydroxy-3-methoxyphenyl)-3-hydroxymethyl-2,3dihydrobenzo[b][1,4]dioxin-6-yl}-N-(4-hydroxyphenethyl)acrylamide. The trans orientation of H−C(7′) and H−C(8′) was determined by the large coupling constant of 7.9 Hz (measured in DMSO-d6).11 Compound 2 was obtained as an optically inactive, white powder. On the basis of HREIMS analysis (m/z 477.1787; calcd 477.1788), its molecular formula was established as C27H27NO7. The IR and 1H and 13C NMR spectra of 2 were similar to those of 1 except that one cis-substituted double bond

he genus Lycium (family Solanaceae) includes about 80 species worldwide, mainly distributed in South America, South Africa, and temperate Europe and Asia. In China, there are seven species and three varieties, mostly distributed in the northwest and northern parts of China. Cortex Lycii, the root bark of L. chinense or L. barbarum, has been widely used in traditional Chinese medicine for its various biological activities, usually used as an antipyretic and for the treatment of pneumonia, night-sweats, cough, hematemesis, inflammation, and diabetes mellitus.1,2 Modern pharmacological studies showed that Cortex Lycii exhibited activities of lowering blood pressure, serum glucose, and lipid levels.3,4 Several types of compounds have been isolated from the plant, including alkaloids,5,6 peptides,7,8 flavonoids,9 terpenoids,8 organic acids, and their derivatives.9 In order to further investigate the active components of the herb, a systematic chemical study was carried out on the EtOAc-soluble fraction of an EtOH extract of the root bark of L. chinense. As a result, 30 phenolic compounds including seven new neolignanamides and one new lignanamide were isolated. Herein, we describe the isolation and structure elucidation of new compounds 1−8, as well as the antioxidant activities of the cinnamic acid amides, neolignanamides, and lignanamides.

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RESULTS AND DISCUSSION Compound 1 was obtained as an optically inactive, white powder. Its molecular formula was tentatively assigned as C27H27NO7 on the basis of HREIMS analysis at m/z 477.1791 (calcd 477.1788), suggesting 15 degrees of unsaturation. The © XXXX American Chemical Society and American Society of Pharmacognosy

Received: September 23, 2012

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dx.doi.org/10.1021/np300655y | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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Chart 1

Table 1. 1H and 13C NMR Data of Compounds 1−4 in Methanol-d4 1 position

a

δCa

1 2 3 4 5 6 7 8 9 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′

129.8 116.9 145.6 146.7 118.4 122.9 141.4 119.9 168.9 129.2 112.0 149.3 148.4 116.3 121.7 77.7 80.2 62.0

1″ 2″ 3″ 4″ 5″ 6″ 7″ 8″ 1‴ 2‴ 3‴ 4‴ 5‴ 6‴ 7‴ 8‴ OCH3

131.3 130.7 116.3 156.9 116.3 130.7 35.8 42.6

56.5

2

δHb (J in Hz) 7.14, d (2.0)

6.97, 7.08, 7.42, 6.42,

d (8.4) dd (8.4, 2.0) d (15.7) d (15.7)

7.01, d (1.8)

6.82, 6.89, 4.90, 4.09, 3.69, 3.46,

d (8.1) dd (8.1, 1.8) d (7.9)c m dd (12.3, 2.5) m

7.04, d (8.5) 6.70, d (8.5) 6.70, 7.04, 2.74, 3.45,

d (8.5) d (8.5) t (7.4) m

3.87, s

δ Ca 130.0 118.9 144.8 145.5 117.7 124.3 137.2 123.1 170.3 129.3 112.0 149.2 148.3 116.3 121.7 77.6 80.2 62.0 131.2 130.7 116.3 156.9 116.3 130.7 35.4 42.3

56.4

3

δHb (J in Hz) 7.18, d (2.0)

6.89, 6.97, 6.62, 5.87,

d (8.6) dd (8.6, 2.0) d (12.6) d (12.6)

7.01, d (1.8)

6.82, 6.89, 4.91, 4.05, 3.69, 3.48,

d (8.1) dd (8.1, 1.8) d (8.0)c m dd (12.3, 2.5) dd (12.3, 2.5)

130.4 117.2 143.9 146.4 118.8 123.2 141.1 120.4 168.8 127.9 121.8 148.6 116.3 149.2 112.0 78.2 79.4 168.9 131.3 130.8 116.3 156.9 116.3 130.8 35.5 42.6 130.9 130.7 116.3 156.9 116.3 130.7 35.8 42.3 56.4

7.00, d (8.5) 6.69, d (8.5) 6.69, 7.00, 2.68, 3.45,

δ Ca

d (8.5) d (8.5) t (7.4) t (7.4)

3.80, s

4

δHb (J in Hz) 7.20, d (1.8)

6.96, d (8.5) 7.13 dd (8.5, 1.8) 7.43, d (15.7) 6.45, d (15.7)

6.94, s 6.81, s 6.80, s 5.06, d (7.0) 4.52, d (7.0)

6.81, d (8.5) 6.71, d (8.5) 6.71, 6.81, 2.75, 3.46,

d (8.5) d (8.5) t (7.3) t (7.3)

7.05, d (8.5) 6.65, d (8.5) 6.65, 7.05, 2.45, 3.17, 3.84,

d (8.5) d (8.5) m m s

δ Ca 130.5 119.0 143.2 145.2 118.0 124.9 137.2 123.2 170.0 128.1 121.7 148.5 116.2 149.1 112.0 78.0 79.3 169.2 131.2 130.7 116.2 156.9 116.2 130.7 35.5 42.3 130.9 130.7 116.2 156.9 116.2 130.7 35.5 42.3 56.4

δHb (J in Hz) 7.23 d (1.8)

6.87 d (8.4) 6.99 dd (8.4, 1.8) 6.62, d (12.6) 5.88, d (12.6)

6.93, s 6.81, s 6.81, s 5.06, d (6.8) 4.52, d (6.8)

6.98, d (8.5) 6.64, d (8.5) 6.64, d (8.5) 6.98, d (8.5) 2.69 t (7.3) 3.38 t (7.3) 6.82, d (8.5) 6.66, d (8.5) 6.66, 6.82, 2.47, 3.17, 3.83,

d (8.5) d (8.5) m m s

Recorded at 100 MHz. bRecorded at 400 MHz. cCoupling constant was measured in DMSO-d6 at 400 MHz.

B



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Figure 1. Proposed fragmentation of compounds 1, 3, 5, 6, 7, and 8.

Figure 2. Key HMBC correlations (H→C) of compounds 3, 5, 6, and 8.

at δH 6.62 (d, J = 12.6 Hz, H-7) and 5.87 (d, J = 12.6 Hz, H-8) (Table 1) in 2 replaced the trans-substituted double bond in 1. Thus, the structure of 2 was determined as (Z)-3-{(2,3-trans)2-(4-hydroxy-3-methoxyphenyl)-3-hydroxymethyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl}-N-(4-hydroxyphenethyl)acrylamide. A literature survey shows that the trans-isomers of this type of compounds are widespread in some genera within Solanaceae

as the more stable compounds, and they will be transformed into the cis-isomers by photolysis.12,13 In our experiment, the cis-isomer was detected in trace amounts in the crude drug by HPLC-DAD-MS/MS. A relatively larger amount of 2 was formed from 1 by photolysis during the isolation and purification procedures, and then the pair of isomers was separated using semipreparative HPLC (MeOH−H2O, 50:50) C



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hydroxyphenethyl)amino]-3-oxoprop-1-en-1-yl}-2,3-dihydrobenzo[b][1,4]dioxine-2-carboxamide. Compound 5 was isolated as a yellow gum. Its positive ion HRESIMS spectrum provided an ion at 665.2483 [M + Na]+, corresponding to a molecular formula of C36H38N2O9. Fragmentation in the positive ion ESIMSn of the quasimolecular ion at m/z 643 [M + H]+ resulted in the neutral loss of 137 Da (m/z 506), consistent with the presence of a tyramine moiety. This was followed by the loss of 163 Da (m/z 343) from the precursor ion [M − 137 + H]+, implying the presence of a second tyramine fragment (Figure 1). In the lowfield region of the 1H NMR spectrum, five aromatic and/or olefinic protons as singlets were observed at δH 6.34 (2H, s, H2,6), 6.81 (1H, s, H-2′), 6.54 (1H, s, H-5′), and 7.56 (1H, s, H7). Analysis of the HMBC correlations from H-2(6) to C-4 and from the O-methyl protons at δH 3.57 (6H, s) to C-3(5) (Figure 2) permitted assignment of a symmetrical 1,3,4,5tetrasubstituted aromatic moiety. The H-7 singlet showed HMBC correlations to C-2(6) and C-9, suggesting the connection of C-7 to C-1 and C-8 to C-9. The result was further supported by the cross-peak between H-7 and H-2(6) in the NOESY spectrum. The HMBC correlation from H-8″ to C-9 indicated the linkage of C-9 to a tyramine moiety. The H2′ and H-5′ singlets were assigned to a 1,2,4,5-tetrasubstituted aromatic system by the HMBC correlations of H-2′/C-4′, C-6′ and H-5′/C-1′, C-3′ (Figure 2). In the high-field region of the 1 H NMR spectrum, there were two methylene protons at δH 2.47 (2H, m, H-7′) and 2.14 (2H, m, H-8′). The C-7′−C-8′ fragment was deduced to be linked at C-1′ and C-9′ according to the HMBC correlations from H-7′ to C-1′, C-2′, C-6′, and C-9′ and from H-8′ to C-1′ and C-9′ (Figure 2). The similar HMBC correlation from H-8‴ to C-9′ was used to confirm the connectivity of C-9′ with the second tyramine moiety. The connection of the two cinnamic acid amide units was defined by the HMBC correlations from H-7 to C-6′ and H-5′ to C-8. The E relation at the C-7−C-8 double bond was determined by the correlation between H-2 (or 6) and H-5′ in the ROESY spectrum. Furthermore, chemical shifts of H-7 and C-7 were observed similar to those of (E,E)-N,N-dityramine-4,4′dihydroxy-3,5′-dimethoxy-β,3′-bicinnamamide.16 Thus, compound 5 was characterized as (E)-2-(4,5-dihydroxy-2-{3-[(4hydroxyphenethyl)amino]-3-oxopropyl}phenyl)-3-(4-hydroxy3,5-dimethoxyphenyl)-N-(4-hydroxyphenethyl)acrylamide. Compound 6 was isolated as a yellow gum and displayed a molecular formula of C34H41N3O9, as determined by HRESIMS at m/z [M + H]+ 636.2934 (calcd for C34H42N3O9, 636.2921). The ESIMSn data in the positive ion mode showed the same daughter ion peaks at m/z 506 and 343 as those in 5 deduced from the loss of a 130 Da unit and then loss of a 163 Da unit. In the 1H NMR spectrum, there were nine aromatic and/or olefinic proton signals at δH 7.57 (1H, s, H-7), 6.93 (2H, d, J = 8.5 Hz, H-2″,6″), 6.83 (1H, s, H-2′), 6.67 (2H, d, J = 8.5 Hz, H-3″,5″), 6.58 (1H, s, H-5′), and 6.34 (2H, s, H-2,6) in the low-field region with a similar distribution to that for 5 except for the absence of two pairs of ortho-coupled protons, due to a p-tyramine moiety attached to C-9. Detailed analysis of the 1H, 13 C, and 2D NMR spectroscopic data of 6 disclosed that 6 possessed an N-(4-aminobutyl)acetamide moiety with a molecular weight of 130 Da (Figure 1). The important HMBC correlations from H-4‴ to C-2‴ and C-3‴, from H2‴ and H-3‴ to C-1‴ and C-4‴, and from H-1‴ and the methyl protons at δH 1.91 to the carbonyl carbon at δC 173.3 suggested the presence of an N-(4-acetamidobutyl) moiety.

due to their different retention times on the reverse-phase C18 column. In addition, different UV spectra were also observed using a photodiode array detector. The cis-isomer showed maximum absorption at 277 and 305 nm, which was blueshifted compared to those at 285 and 319 nm for the transisomer. However, the isomers showed identical fragmentation pathways in the ESIMSn spectra. Compound 3 was isolated as an optically inactive, white powder. The molecular formula was tentatively determined as C35H34N2O8 by HRESIMS (m/z 633.2223 [M + Na]+, calcd 633.2213), suggesting 20 degrees of unsaturation. The 1H, 13C, and DEPT NMR spectroscopic data of 3 (Table 1) displayed signals assignable to one methoxy group, two pairs of aliphatic fragments (C-7″−C-8″, C-7‴−C-8‴), two oxymethines (C-7′, C-8′), 16 aromatic and/or olefinic methines, 10 aromatic quaternary carbons, and two carbonyls (C-9, C-9′). The 1H, HSQC, and HMBC NMR experiments suggested the presence of two 1,4-disubstituted aromatic moieties [(C-2″(6″)−C3″(5″), C-2‴(6‴)−C-3‴(5‴)], an ABX spin system [6.96 (d, J = 8.5 Hz), 7.20 (d, J = 1.8 Hz), and 7.13 (dd, J = 8.5, 1.8 Hz)], and a 1,3,5-trisubstituted aromatic moiety [δH 6.80 (s), 6.81 (s), and 6.94 (s)]. The 1H NMR data showed two vicinal olefinic protons signals at δH 7.43 (1H, d, J = 15.7 Hz, H-7) and 6.45 (1H, d, J = 15.7 Hz, H-8) due to one trans-substituted double bond. The HMBC correlations of H-8/C-1 and H-7/C2, C-6, C-9 suggested the presence of a caffeoyl-like unit (Figure 2). Two pairs of vicinal methylenes (H-7″−H-8″, H7‴−H-8‴) suggested the presence of two NHCH2 CH 2 segments. Additional HMBC correlations of H-8″/C-1″, H7″/C-2″(6″), H-8‴/C-1‴, and H-7‴/C-2‴(6‴) confirmed the linkage of C-7″ to C-1″ and C-7‴ to C-1‴(Figure 2), and two p-tyramine moieties were present in 3. Furthermore, two vicinal aliphatic oxymethine signals at δ 5.06 (1H, d, J = 7.0 Hz, H-7′) and 4.52 (1H, d, J = 7.0 Hz, H-8′) were linked to C-1′ and C9′, respectively, by the HMBC correlations from H-7′ to C-1′, C-2′, C-6′, and C-9′ and from H-8′ to C-1′ and C-9′ (Figure 2). The linkages of C-9 and C-9′ to the p-tyramine moieties were determined by the HMBC correlations of H-8″/C-9 and H-8‴/C-9′. Additional HMBC correlation from H-7′ to C-4 connected C-7′ with C-4 via oxygen. A similar correlation from H-8′ to C-3 was used to connect C-8′ with C-3. Thus, the structure of 3 was assigned to be composed of two cinnamic acid amide units. A large coupling constant (J 7′,8′ = 7.0 Hz) indicated that the two protons were trans-oriented.11 Compound 3 was confirmed as (2,3-trans)-3-(3-hydroxy-5methoxyphenyl)-N-(4-hydroxyphenethyl)-7-{(E)-3-[(4hydroxyphenethyl)amino]-3-oxoprop-1-en-1-yl}-2,3-dihydrobenzo[b][1,4]dioxine-2-carboxamide. In addition, the ESIMSn experiment confirmed the proposed structure of 3. The protonated molecule [M + H]+ at m/z 611 and its predominant product ion peaks at m/z 474 and 311 were observed in the mass spectrum. The ion at m/z 474 was formed by the loss of a p-tyramine (137 Da) moiety. The significant ion peak at m/z 311 was generated by the fission of C-8′−C-9′ to eliminate a 4-(2-isocyanatoethyl)phenol (163 Da) fragment from m/z 474 (Figure 2). Compound 4 was obtained as an optically inactive, white powder. Its molecular formula was tentatively assigned as C35H34N2O8 on the basis of HRESIMS [M + Na]+ at m/z 633.2195 (calcd 633.2213). Analysis of its 1D and 2D NMR spectra led to the definition of 4 as the cis-isomer of 3. Thus, compound 4 was assigned as (2,3-trans)-3-(3-hydroxy-5methoxyphenyl)-N-(4-hydroxyphenethyl)-7-{(Z)-3-[(4D



Article

The HMBC correlation of H-4‴/C-9 confirmed the connectivity of C-4‴ and C-9 via an amide linkage. The Econfiguration was determined as for 5. Therefore, compound 6 was confirmed as (E)-2-(4,5-dihydroxy-2-{3-[(4-hydroxyphenethyl)amino]-3-oxopropyl}phenyl)-3-(4-hydroxy-3,5dimethoxyphenyl)-N-(4-acetamidobutyl)acrylamide. Compound 6 represents the first naturally occurring dimer derived from two dissimilar cinnamic acid amides. Compound 7 was obtained as a yellow gum. Its molecular formula was deduced to be C33H39N3O8 by HRESIMS [M + Na]+ m/z 628.2631 (calcd for C33H39N3O8Na, 628.2635). The UV, IR, and NMR spectra of 7 were similar to those of 6. The molecular weight of 7 was 30 Da less than that of 6, which was tentatively attributed to lack of one methoxy group in 7. The above deduction was proved by the absence of one methoxy signal (linking to C-6) in the 1H and 13C NMR spectra of 7. The correlation of H-6 and H-5′ in the NOESY spectrum defined the E relation of the C-7−C-8 double bond. Thus, compound 7 was determined as (E)-2-(4,5-dihydroxy-2-{3-[(4hydroxyphenethyl)amino]-3-oxopropyl}phenyl)-3-(4-hydroxy3-methoxyphenyl)-N-(4-acetamidobutyl)acrylamide. Compound 8 was isolated as an optically inactive, yellow gum. Its HRESIMS spectrum displayed a molecular ion peak at m/z [M + H]+ 634.2766 (calcd for C34H40N3O9, 634.2765) corresponding to the molecular formula C34H39N3O9. The presence of an N-(4-acetamidobutyl) moiety and a p-tyramine moiety was deduced from the 1H and 13C NMR data as described before, together with the fragments [M + H − 130]+ and [M + H − 130 − 165] + in the mass spectrum (Figure 1). The 1H NMR data showed an ABX spin system with signals at δH 6.44 (1H, d, J = 1.8 Hz, H-2′), 6.57 (1H, d, J = 8.1 Hz, H5′), and 6.38 (1H, dd, J = 8.1, 1.8 Hz, H-6′), two aromatic and/ or olefinic protons as singlets at δH 6.78 (1H, s, H-6) and 7.31 (1H, s, H-7), two aliphatic protons at δH 4.79 (1H, s, H-7′) and 3.66 (1H, s, H-8′), and two methoxy groups at δH 3.54 (3H, s) and 3.91 (3H, s). Detailed analysis of the HMBC correlations (Figure 2) of H-7/C-1, C-2, C-6, C-8′, C-9, H-7′/C-1, C-3, C8, C-9′, and H-8′/C-7, C-9, C-1′ revealed the presence of a dihydronaphthalene core structure that resembled the structure of 7-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-N2,N3-bis(4hydroxyphenethyl)-6-methoxy-1,2-dihydronaphthalene-2,3-dicarboxamide.14 The difference between the two compounds was that the p-tyramine moiety linking to C-9 in the known compound was replaced by an N-(4-aminobutyl)acetamide moiety in 8. Thus, the planar structure of 8 was established. The relative trans configuration was suggested by the small coupling constant between H-7′ and H-8′ (proton signals as two singlets in the 1H NMR spectrum).14,15 The J (7′,8′) value was close to zero, indicating that the corresponding dihedral angle should be ca. 90°. The minimized structure obtained by molecular mechanics (MM2) calculation was used to generate dihedral angles, and an angle of 87.5° was measured, which was compatible with a trans configuration (Figure 3).16 Thus the structure of 8 was elucidated as (1,2-trans)-N3-(4-acetamidobutyl)-1-(3,4-dihydroxyphenyl)-7-hydroxy-N 2 -(4-hydroxyphenethyl)-6,8-dimethoxy-1,2-dihydronaphthalene-2,3-dicarboxamide. The known compounds were identified as trans-Nhydroxycinnamoyltyramine (9),17 trans-N-isoferuloyltyramine (10),18 trans-N-caffeoyltyramine (11),5 dihydro-N-caffeoyltyramine (12),5 trans-N-feruloyloctopamine (13),18 cis-N-feruloyloctopamine (14),18 thoreliamide B (15),10 7-hydroxy-1-(3,4dihydroxy)-N2,N3-bis(4-hydroxyphenethyl)-6,8-dimethoxy-1,2-

Figure 3. Minimized structure of compound 8.

dihydronaphthalene-2,3-dicarboxamide (16),14 gentisic acid (17),19 vanillic acid (18),20 p-coumaric acid (19),21 caffeic acid (20), 22 ferulic acid (21), 22 sinapic acid (22), 22 dihydrocaffeic acid (23),23 isoscopoletin (24),20 fraxidin (25),20 aquillochin (26),24 scopolin (27),25 kaempferide (28),26 apigenin (29),26 and luteolin (30)26 by comparison of their experimental and reported spectroscopic data. Compounds 13−16 were isolated from the genus for the first time. Recently, much attention has been paid to antioxidant activity because many pathological conditions are associated with oxidative stress, and antioxidant capacity is widely used as a parameter to characterize food, medicinal plants, and their bioactive components.27 Therefore, all the cinnamic acid amides, neolignanamides, and lignanamides were evaluated for their antioxidant activity by the DPPH and NBT superoxide-scavenging assays, and the results are shown in Table 3. All the compounds exhibited moderate antioxidant activity as compared with the positive control. Compounds 6, 7, and 8, with the common N-(4-acetamidobutyl) moiety, showed relatively weak activity compared to 5 and 16, which possess a tyramine moiety in the corresponding position. The antioxidant activity of the three pairs of cis- and trans-isomers was similar.

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EXPERIMENTAL SECTION

General Experimental Procedures. UV spectra were determined using a Shimadzu UV-240 spectrophotometer. IR spectra were recorded on a Perkin-Elmer 577 spectrometer. Optical rotations were measured on a Perkin-Elmer 341 polarimeter. NMR spectra were recorded on a Bruker AM-400 spectrometer at 400 MHz for 1H NMR and 100 MHz for 13C NMR. HRMS data were recorded on a Finnigan/MAT-95 instrument or a Agilent QTOF 6520, and ESIMSn was carried out on a Bruker HCT ion trap mass spectrometer. Semipreparative HPLC was performed on an Agilent 1100 VWD spectrophotometer. Silica gel (200−300 mesh, Qingdao Haiyang Chemical Co., Ltd.) and Sephadex LH-20 gel (Amersham Biosciences) were also used for column chromatography. TLC analysis was run on GF254 precoated silica gel plates (Merck), and spots were visualized E



Article

Table 2. 1H and 13C NMR Data of Compounds 5−8 in Methanol-d4 5 position

δ Ca

1 2 3 4 5 6 7 8 9 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 1″ 2″ 3″ 4″ 5″ 6″ 7″ 8″ 1‴ 2‴ 3‴ 4‴ 5‴ 6‴ 7‴ 8‴ N-COCH3

127.1 109.0 148.7 138.0 148.7 109.0 138.4 132.1 170.3 132.5 118.2 147.3 146.2 118.2 127.0 29.8 37.7 175.0 130.9 130.7 116.2 156.9 116.2 130.7 35.4 42.7 131.2 130.7 116.4 156.8 116.4 130.7 35.6 42.3

3′-OCH3 5′-OCH3 a

56.3 56.3

6

δHb(J in Hz) 6.34, s

6.34, s 7.56, s

6.81, s

6.54, s 2.47, m 2.14, m

6.92, d (8.5) 6.66, d (8.5) 6.66, 6.92, 2.67, 3.41,

d (8.5) d (8.5) m m

6.94, d (8.5) 6.67, d (8.5) 6.67, 6.94, 2.56, 3.21,

δ Ca

7

δHb (J in Hz)

127.1 109.0 148.7 138.0 148.7 109.0 138.5 132.3 170.6 132.3 118.2 147.3 146.2 118.3 127.1 29.8 37.7 175.0 131.2 130.7 116.2 156.8 116.2 130.7 35.6 42.3 40.1 27.7 28.0 40.6

6.67, 6.93, 2.58, 3.21, 3.14, 1.47, 1.47, 3.25,

173.3 22.6 56.3 56.3

1.91, s 3.57, s 3.57, s

6.34, s

6.34, s 7.57, s

6.83, s

6.58, s 2.49, m 2.23, m

6.93, d (8.5) 6.67, d (8.5) d (8.5) d (8.5) t (7.4) m m brs brs m

δ Ca

8

δHb (J in Hz)

128.3 113.0 148.5 149.0 130.7 127.0 138.4 131.7 170.8 132.6 118.2 147.3 146.2 118.3 127.2 29.9 37.7 175.0 131.3 130.7 116.2 156.8 116.2 130.7 35.6 42.3 40.2 27.7 28.0 40.6

6.67, 6.94, 2.57, 3.19, 3.13, 1.47, 1.47, 3.23,

173.3 22.6 55.8

1.91, s 3.34, s

6.38, d (1.5)

6.65, d (8.3) 6.73, dd (8.3, 1.5) 7.58, s

6.83, s

6.57, s 2.54, m 2.22, m

6.94, d (8.4) 6.67, d (8.4) d (8.4) d (8.4) m m m brs brs m

δ Ca

δHb (J in Hz)

126.9 125.5 146.9 143.1 149.1 109.2 135.2 131.1 170.2 136.1 115.9 145.9 144.8 116.1 119.9 41.0 50.4 174.1 124.3 130.7 116.2 156.8 116.2 130.7 35.5 42.4 40.1 27.7 27.7 40.4

6.63, 6.81, 2.52, 3.22, 3.14, 1.48, 1.48, 3.20,

173.3 22.6 60.8 56.8

1.90, s 3.54, s 3.91, s

6.78, s 7.31, s

6.44, d (1.8)

6.57, 6.38, 4.79, 3.66,

d (8.1) dd (8.1,1.8) s s

6.81, d (8.2) 6.63, d (8.2) d (8.2) d (8.2) t (6.1) m m brs brs m

d (8.5) d (8.5) t (7.4) m

3.57, s 3.57, s

Recorded at 100 MHz. bRecorded at 400 MHz.

by heating after spraying with 10% H2SO4−EtOH. All solvents used for isolation were of analytical grade. Plant Material. The root bark of L. chinense was collected from Gansu Province, People’s Republic of China, in April 2010. It was identified by one of the authors (D.-A.G.). A voucher specimen (No. 20104002) was deposited at Shanghai Research Center for Modernization of Traditional Chinese Medicine, Shanghai Institute of Materia Medica. Extraction and Isolation. The crude drug (20 kg) was extracted three times with 30 L of EtOH−H2O (95:5 v/v). The solvent was evaporated, and the residue (1.8 kg) suspended in H2O and then successively extracted with petroleum ether and EtOAc. The EtOAc fraction (90 g) was subjected to column chromatography (silica gel; CHCl3−MeOH, 100:1 to 2:1) in a gradient to obtain eight fractions. Fraction 2 (5 g) was subjected to a silica gel column eluting with petroleum ether−EtOAc (20:1 to 1:1) to afford three major fractions, F2a−F2c. F2c was purified by semipreparative HPLC (MeOH−H2O, 72:28) to afford 24 (35 mg) and 25 (12 mg). Fraction 3 (19 g) was subjected to a silica gel column eluting with petroleum ether−EtOAc (10:1 to 0:1) to obtain four major fractions, F3a−F3d. F3a was subjected to a Sephadex LH-20 column (petroleum ether−CHCl3−

MeOH, 2:1:1) to obtain 18 (40 mg), 28 (17 mg), and 27 (19 mg). F3c was subjected to a Sephadex LH-20 column (petroleum ether− CHCl3−MeOH, 2:1:1) to afford three major fractions, which were further purified by semipreparative HPLC [MeOH−H2O (containing 0.03% TFA), 30:70] to afford 21 (32 mg), 19 (11 mg), and 22 (7 mg). Fraction 4 (20 g) was chromatographed on a silica gel column (petroleum ether−acetone, 20:1 to 0:1) to obtain four major fractions, F4a−F4e. F4b was further purified on a Sephadex LH-20 column (CHCl3−MeOH, 1:1) to obtain 17 (8 mg), 29 (11 mg), and 26 (8 mg). F4c was further purified by semipreparative HPLC [MeOH− H2O (containing 0.03% TFA), 40:60] to obtain 23 (15 mg), 20 (12 mg), and 30 (16 mg). Fraction 5 (8 g) was separated on a silica gel column eluting with CHCl3−acetone (20:1 to 0:1) to afford three major fractions, F5a−F5e. F5a was separated on a Sephadex LH-20 column (CHCl3−MeOH, 1:1) to obtain two major fractions, which were further purified by semipreparative HPLC (MeOH−H2O, 42:58) to obtain 9 (25 mg), 10 (22 mg), 13 (8 mg), and 14 (5 mg). F5b and F5c were subjected to semipreparative HPLC (MeOH−H2O, 45:55) to afford 11 (28 mg) and 12 (22 mg), respectively. F5e was subjected to column chromatography over silica gel and Sephadex LH-20 (CHCl3−MeOH, 1:1) to obtain five major fractions, which were F



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acetamidobutyl)acrylamide (6): yellow gum; UV (MeOH) λmax (log ε) 318 (4.18) nm; IR (KBr) νmax 3369, 2937, 1643, 1610, 1500, 1452, 1367, 1257, 1224, 1114, 833 cm−1; for 1H and 13C NMR spectroscopic data, see Table 2; HRESIMS [M + H]+ 636.2934 (calcd for C34H42N3O9, 636.2921). (E)-2-(4,5-Dihydroxy-2-{3-[(4-hydroxyphenethyl)amino]-3oxopropyl}phenyl)-3-(4-hydroxy-3-methoxyphenyl)-N-(4acetamidobutyl)acrylamide (7): yellow gum; UV (MeOH) λmax (log ε) 291 (4.04), 321 (4.10) nm; IR (KBr) νmax 3357, 2935, 1641, 1595, 1514, 1450, 1369, 1280, 1128, 1029, 821 cm−1; for 1H and 13C NMR spectroscopic data see Table 2; HRESIMS [M + Na]+ 628.2631 (calcd for C33H39N3O8Na, 628.2635). (1,2-trans)-N3-(4-Acetamidobutyl)-1-(3,4-dihydroxyphenyl)-7-hydroxy-N 2 -(4-hydroxyphenethyl)-6,8-dimethoxy-1,2-dihydronaphthalene-2,3-dicarboxamide (8): yellow gum; [α]20D 0 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 250 (3.48), 303 (4.42) nm; IR (KBr) νmax 3405, 2937, 1643, 1614, 1515, 1456, 1367, 1284, 1113, 557 cm−1; for 1H and 13C NMR spectroscopic data, see Table 2; HRESIMS [M + H]+ 634.2766 (calcd for C34H40N3O9, 634.2765). 2,2-Diphenyl-1-picrylhydrazyl (DPPH) Free Radical Scavenging Activity. The DPPH radical scavenging activity was analyzed by the following procedure.28 Briefly, in a 96-well microplate, an aliquot of each test compound (120 μL, with a concentration of 5−100 μg/ mL) was mixed with 30 μL of 0.75 mM DPPH dissolved in MeOH, each test in triplicate. The mixture was shaken vigorously in the dark at room temperature for 30 min, and then the absorbance was measured at 517 nm using an ELISA reader. The DPPH radical scavenging activity was calculated as follows: % = (1 − As /A0) × 100, where As is the absorbance of sample with DPPH solution and A0 is the absorbance of the initial DPPH solution. Trolox (5−100 μM) was used as a positive control. The final result was reported as the IC50 concentration, which is the concentration of sample required to cause 50% inhibition against the DPPH radical in reaction solution. NBT Superoxide Scavenging Assay. Superoxide radicals were generated by the xanthine/xantine oxidase system following a described procedure.27 Xanthine, xanthine oxidase, and nitroblue tetrazolium (NBT) were respectively dissolved in 1 μM NaOH solution, 0.1 mM Na2EDTA solution, and 20 mM phosphate buffer (pH 7.4). The reaction mixtures in a 96-well microplate consisted of xanthine (40 μM), xanthine oxidase (0.05 unit/mL), NBT (50 μM), and different concentrations of each sample solution with a final volume of 220 μL. After the addition of xanthine oxidase, the reaction was incubated at 37 °C for 20 min. The absorbance was measured at 560 nm against blank samples, and all determinations are the average of triplicate analyses. Trolox was used as positive control.

Table 3. Free Radical Scavenging Activity of Compounds 1− 16 IC50 ± SD (μM) compound 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Trolox

DPPH 55.2 54.1 49.8 48.2 57.1 64.7 65.2 67.0 63.5 67.7 47.6 61.2 62.4 61.2 57.3 46.5 33.5

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.2 1.6 2.1 1.4 2.0 1.5 1.9 2.2 1.9 1.1 1.8 2.3 2.0 2.1 1.8 1.7 0.8

NBT 44.4 49.9 46.4 44.1 49.9 52.7 56.5 58.0 52.6 48.8 44.1 30.2 24.6 22.2 59.5 50.1 27.2

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

2.3 1.9 1.2 1.6 0.8 1.5 2.0 2.2 2.9 3.1 1.8 1.8 2.9 3.2 1.1 0.8 0.5

further purified by semipreparative HPLC (MeOH−H2O, 50:50) to get 15 (6 mg), 1 (6 mg), 2 (3 mg), 3 (5 mg), and 4 (4 mg). In the same manner, fraction 7 (20 g) was separated on a silica gel column eluting with CHCl3−MeOH (20:1 to 1:1) to afford five major fractions, which were further purified by Sephadex LH-20 (CHCl3− MeOH, 1:1) and semipreparative HPLC (MeOH−H2O, 38:62) to obtain 5 (25 mg), 16 (28 mg), 6 (21 mg), 7 (21 mg), and 8 (19 mg). (E)-3-{(2,3-trans)-2-(4-Hydroxy-3-methoxyphenyl)-3-hydroxymethyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl}-N-(4hydroxyphenethyl)acrylamide (1): white powder; [α]20D 0 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 285 (4.07), 319 (3.97) nm; IR (KBr) νmax 3396, 2929, 1657, 1609, 1519, 1471, 1268, 605 cm−1; for 1 H and 13C NMR spectroscopic data, see Table 1; HREIMS 477.1791 (calcd for C27H27NO7, 477.1788). (Z)-3-{(2,3-trans)-2-(4-Hydroxy-3-methoxyphenyl)-3-hydroxymethyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl}-N-(4-hydroxyphenethyl)acrylamide (2): white powder; [α]20D 0 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 277 (3.69), 305 (3.54) nm; IR (KBr) νmax 3388, 2932, 1648, 1620, 1521, 1471, 1259, 1121, 1062, 835 cm−1; for 1 H and 13C NMR spectroscopic data, see Table 1; HREIMS 477.1787 (calcd for C27H27NO7, 477.1788). (2,3-trans)-3-(3-Hydroxy-5-methoxyphenyl)-N-(4-hydroxyphenethyl)-7-{(E)-3-[(4-hydroxyphenethyl)amino]-3-oxoprop-1-en-1yl}-2,3-dihydrobenzo[b][1,4]dioxine-2-carboxamide (3): white powder; [α]20D 0 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 285 (3.86), 318 (3.73) nm; IR (KBr) νmax 3421, 2922, 1657, 1614, 1516, 1438, 1271, 1209, 559 cm−1; for 1H and 13C NMR spectroscopic data, see Table 1; HRESIMS [M + Na]+ 633.2223 (calcd for C35H34N2O8Na, 633.2213). (2,3-trans)-3-(3-Hydroxy-5-methoxyphenyl)-N-(4-hydroxyphenethyl)-7-{(Z)-3-[(4-hydroxyphenethyl)amino]-3-oxoprop-1-en1-yl}-2,3-dihydrobenzo[b][1,4]dioxine-2-carboxamide (4): white powder; [α]20D 0 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 278 (3.85), 306 (3.67) nm; IR (KBr) νmax 3411, 2927, 1676, 1614, 1516, 1437, 1271, 1207, 1126, 823, 723, 561 cm−1; for 1H and 13C NMR spectroscopic data, see Table 1; HRESIMS [M + Na]+ 633.2195 (calcd for C35H34N2O8Na, 633.2213). (E)-2-(4,5-Dihydroxy-2-{3-[(4-hydroxyphenethyl)amino]-3oxopropyl}phenyl)-3-(4-hydroxy-3,5-dimethoxyphenyl)-N-(4hydroxyphenethyl)acrylamide (5): yellow gum; UV (MeOH) λmax (log ε) 320 (4.22) nm; IR (KBr) νmax 3384, 2937, 1643, 1610, 1500, 1452, 132 cm−1; for 1H and 13C NMR spectroscopic data, see Table 2; HRESIMS [M + Na]+ 665.2483 (calcd for C36H38N2O9Na, 665.2475). (E)-2-(4,5-Dihydroxy-2-{3-[(4-hydroxyphenethyl)amino]-3oxopropyl}phenyl)-3-(4-hydroxy-3,5-dimethoxyphenyl)-N-(4-

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ASSOCIATED CONTENT

* Supporting Information S

The 1H and 13C NMR, HSQC, and HMBC spectra of compounds 1−8 are available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel/Fax: +86 21 50272223. E-mail: [email protected]. cn (S.-H.G.). Tel/Fax: +86 21 50271516. E-mail: daguo@mail. shcnc.ac.cn (D.-A.G.) Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Prof. Cheng Luo and Dr. Kong-Kai Zhu for helping to define the configuration of compound 8. This study was financially supported by the 12th Five-Year National Science & Technology Support Program (No. 2012BAI29B06) and Major Projects of Knowledge Innovation Program of the Chinese Academy of Sciences (No. KSCX2-YW-R-166). This work was G



Article

also partially supported by Global Research Network for Medicinal Plants (GRNMP) and King Saud University.

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REFERENCES

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