Article pubs.acs.org/jnp
Trichinenlides A−T, Mexicanolide-Type Limonoids from Trichilia sinensis Jin-Biao Xu,† Yuan Lin,‡ Shi-Hui Dong,† Fei Wang,‡ and Jian-Min Yue*,† †
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zu Chong Zhi Road, Shanghai 201203, People’s Republic of China ‡ Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, People’s Republic of China S Supporting Information *
ABSTRACT: Twenty new mexicanolide-type limonoids, namely, trichinenlides A−T (1−20), and 11 known analogues were isolated from the leaves and twigs of Trichilia sinensis. Trichinenlides B (2) and C (3) and heytrijunolide D exhibited inhibition against lipopolysaccharide-induced nitric oxide production in RAW 264.7 macrophages with IC50 values of 2.85, 1.88, and 3.33 μM, respectively.
imonoids, a class of tetranortriterpenoids with a βsubstituted furan ring, are the major secondary metabolites of plants of the Meliaceae family1 and were demonstrated to exhibit a wide spectrum of biological activities, such as anticancer,2 antisickling,3 antifungal,4 antimalarial,5 insect antifeedant,6 and anti-inflammatory activities.7 Previous investigations of the genus Trichilia (Meliaceae) resulted in the isolation of a number of triterpenoids, limonoids, and steroids.8 Trichilia sinensis Bentv, a shrub, is native to the south of China and Vietnam.9 The whole plants have been used as a Chinese folk medicine for the treatment of abdominal pain caused by ascaris lumbricoides, chronic osteomyelitis, scabies, and eczema.10 Nitric oxide (NO) is a signaling molecule that is implicated in a variety of inflammatory conditions including rheumatoid arthritis, sepsis, Crohn’s disease, and asthma.11 Its release is one of the most characteristic inflammatory responses of cells that are exposed to outer bacterial membrane toxins such as lipopolysaccharide (LPS).12 Therefore, agents that block bacterial toxin-induced NO production might be beneficial for the treatment of inflammatory responses. To date, no chemical investigation on T. sinensis has been reported. In the current study, 20 new mexicanolide-type limonoids, trichinenlides A−T, and 11 known analogues were isolated from the leaves and twigs of T. sinensis. Herein we present the isolation, structural elucidation, and the antiinflammatory activities of these limonoids.
L
(calcd 592.2547), requiring 16 indices of hydrogen deficiency. The IR absorptions at 3437 and 1724 cm−1 showed the presence of hydroxy and carbonyl groups, respectively. The 13C NMR data (Table 4) displayed 33 carbon resonances, which were further classified by the DEPT experiment as five methyl (one O-methyl), three methylene, 15 methine (eight olefinic and three oxygenated), and 10 quaternary (three olefinic and four carbonyl) carbons. A typical β-substituted furan ring (δH 6.36, br s, 1H; 7.45, br s, 1H, and 7.55, br s, 1H; δC 109.2, 121.0, 140.6, and 143.1), an m-substituted pyridine ring (δH 7.35, dd, J = 7.9, 4.7 Hz; 8.22, d, J = 7.9 Hz; 8.75, d, J = 4.7 Hz; 9.14, s; δC 123.6, 125.1, 137.1, 150.6, and 153.8), one ketocarbonyl (δC 215.9), and three ester carbonyls (δC 164.4, 168.2, and 175.7) were evident from the NMR data (Tables 1 and 4). These functionalities accounted for 12 indices of hydrogen deficiency, thus requiring four additional rings in the structure of 1. The aforementioned data suggested that 1 was likely a mexicanolide-type limonoid. The NMR data showed high similarity to those of swietenin F,13 with the major differences involving the C-3 substituent. A proton resonance (δH 2.96, s) that did not show correlation with any carbon in the HSQC spectrum was assigned to an OH group at C-6 (δC 72.8) by the HMBC correlations from 6-OH to C-5, C-6, and C-7 (Figure 1A). The carbon resonance at δC 215.9 was assigned to C-1 by the HMBC correlations from H-2, H3-19, and H-30 to C-1. The multiple HMBC correlations of H-3/C1′, H-7′/C-1′, and H-3′/C-1′ indicated that the nicotinate moiety was attached to C-3. The HMBC correlations of
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RESULTS AND DISCUSSION Trichinenlide A (1) had the molecular formula C33H37NO9, as determined by HRESIMS based on m/z 592.2553 [M + H]+ © XXXX American Chemical Society and American Society of Pharmacognosy
Received: May 24, 2013
A
dx.doi.org/10.1021/np400408s | J. Nat. Prod. XXXX, XXX, XXX−XXX
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165.1). The coupling constant between H-2′ and H-3′ (J = 15.4 Hz) indicated an E-geometry of the 2-butenoate group. The relative configuration in the limonoid core of 2 was assigned to be identical to that of quivisianolide B by analysis of the ROESY data (Figure 2B). Trichinenlide C (3) gave a sodiated molecular ion [M + Na]+ at m/z 607.2155 in the HRESI(+)MS corresponding to the molecular formula C31H36O11. Its 1H and 13C NMR data (Tables 1 and 4) showed similarity to those of 2, except for the presence of an additional hydroxy group at C-6 (δC 69.3) of 3, which was confirmed by the HMBC correlations of 6-OH/C-5, C-6, and C-7 (Supporting Information Figure S17). As a result, the carbon signals of C-5 and C-6 were shifted downfield by ΔδC 3.3 and 38.2, respectively, due largely to the inductive effect of the 6-OH as compared with the proton in 2. The 1D NMR data of trichinenlides D (4) and E (5) (Tables 1 and 4) closely resembled those of 2 and 3, respectively, except for the presence of a tigloyloxy group at C-3 of 4 and 5 instead of the (E)-2-butenoate. These conclusions were verified by the HMBC correlations from H-3′ (δH 7.02 in 4, δH 6.98 in 5) and H-3 (δH 5.06 in 4, δH 4.91 in 5) to C-1′ (δC 166.5 in 4, δC 166.1 in 5). This was further supported by the molecular weights of 4 and 5, showing 14 mass units more than those of 2 and 3, respectively. The NMR data suggested that compounds 4 and 5 possessed the same relative configuration as those of 2 and 3, respectively. Trichinenlide F (6) possessed the elemental composition C33H37NO10 as established by the protonated molecular ion at m/z 608.2511 [M + H]+ in the HRESI(+)MS. The NMR data indicated that 6 had a structure similar to 3-angeloyl-3detigloylruageanin B.15 The differences involved the substituents at C-3, where 6 bore a nicotinate. The structural assignment for 6 was further confirmed by HMBC and ROESY data (Supporting Information Figures S33 and S34). The 1H and 13C NMR data of trichinenlides G (7) and H (8) (Tables 1, 2, and 4) resembled those of 6. Their NMR data revealed that 7 had a 6-OAc group, and 8 possessed a 6-OH group. Consequently, the H-6 resonances of 7 and 8 were shifted downfield to δH 5.56 and 4.44 in the 1H NMR spectra, respectively. The C-6 resonances at δC 71.9 and 72.1 for 7 and 8, respectively, were also in favor of the assignments. The structural assignments of 7 and 8 were confirmed by HMBC spectra (Supporting Information Figures S39 and S44). The key correlation of H-6/Ac-6 was observed for 7, while correlations of H-6/C-5 (δC 46.0) and C-7 (δC 175.4) were evident for 8 in the HMBC spectra. The spectroscopic data of 9 and 10 (Tables 2 and 4) revealed that they were also mexicanolide-type limonoids, and their structures were closely related to that of quivisianolide A.15 A tigloyloxy and an (E)-2-butenoate group were attached to C-3 in 9 and 10, respectively, replacing the angeloyloxy moiety at C-3 of quivisianolide A. The above assignments were confirmed by 2D NMR data, especially HMBC, where the key correlation of H-3/C-1′ was observed for both compounds (Supporting Information Figures S49 and S55). The relative configurations of 9 and 10 were shown to be identical with that of quivisianolide A in the limonoid core by NMR analysis. Trichinenlide K (11) possessed a molecular formula of C32H38O12 as determined by HRESI(+)MS at m/z 637.2277 [M + Na]+ (calcd 637.2261), which is 16 mass units more than that of 9. The NMR data of 11 were similar to those of 9 except for the changes around C-6. H-6 (δH 4.29, s) and C-6 (δC 70.6) of 11 were markedly shifted downfield, indicating that a
CH3O/C-7 (δC 175.7) and H-6/C-5 and C-7 placed the methoxycarbonyl group at C-6. The Δ8(30) double bond was fixed by the HMBC correlations of H-30/C-1, C-3, C-9, and C14. The β-furan ring was attached to C-17 based on the HMBC correlations from H-17 to C-20, C-21, and C-22. The relative configuration of 1 was assigned by interpretation of the ROESY data (Figure 1B). The ROESY correlations of H5/H3-29, H-5/H-17, and H-12β/H-17 suggested that they were cofacial and were arbitrarily assigned a β-orientation. Consequently, the ROESY cross-peaks of H-2/H-3, H-2/H30, H-2/H-9, H-9/H3-19, H-9/H-11α, H-11α/H3-18, and H14/H3-18 indicated that H-2, H-3, Me-18, Me-19, and H-14 were α-oriented. The relative configuration of C-6 could not be assigned via the available NMR data.14 Trichinenlide B (2) was obtained as a white, amorphous powder. HRESI(+)MS analysis displayed a quasi molecular ion at m/z 591.2224 [M + Na]+ (calcd 591.2206) that gave a molecular formula of C31H36O10. The IR absorptions at 3438 and 1728 cm−1 indicated the presence of hydroxy and carbonyl groups, respectively. Analysis of the 1H and 13C NMR data (Tables 1 and 4) showed a typical feature of a mexicanolidetype limonoid possessing the functionalities of four tertiary methyls, one methoxy, one trisubstituted double bond, one trisubstituted oxirane, one ketocarbonyl, three ester carbonyls, and one β-substituted furan ring. The aforementioned data showed that the structure of 2 was similar to that of quivisianolide B,13 with the only difference being the presence of a 2-butenoate instead of an angeloyloxy group at C-3 (δC 84.8) of 2. This deduction was validated by the HMBC correlations (Figure 2A) from H-3, H-2′, and H-3′ to C-1′ (δC B
dx.doi.org/10.1021/np400408s | J. Nat. Prod. XXXX, XXX, XXX−XXX
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Table 1. 1H NMR Spectroscopic Data of Compounds 1−7a proton position 2 3 5 6 9 11α
a
1
2
3
4
5
6
7
(mult., J in Hz)
(mult., J in Hz)
(mult., J in Hz)
(mult., J in Hz)
(mult., J in Hz)
(mult., J in Hz)
(mult., J in Hz)
11β 12α
3.60, m 4.88, d (9.4) 3.61, s 4.60, s 2.34, dd (12.5, 5.0) 2.09, ddd (16.8, 12.8, 5.0) 1.81, m 1.46, m
12β
1.73, m
14
2.21, br d (5.3)
15α
2.74, dd (8.5, 5.3)
15β
2.65, d (8.5)
17 18 19 21 22 23 28 29 30 HO-2 HO-6 MeO-7 Ac-6 2′
5.54, 0.93, 1.48, 7.55, 6.36, 7.45, 1.19, 0.97, 5.39,
s s s br br br s s br
s s s
d (7.2)
2.96, s 3.82, s
3′
9.14, s
4′ 5′ 6′ 7′
8.75, d (4.7) 7.35, dd (7.9, 4.7) 8.22, br d (7.9)
5.09, s 2.97, t (6.7) 2.32, d (6.7)
4.94, s 2.89, s 4.28, s
5.06, s 3.00, t (6.7) 2.31, d (6.7)
4.91, s 2.91, s 4.27, s
5.37, 3.34, 2.39, 1.93, 1.87,
s dd (8.4, 3.1) d (8.4) m m
5.24, 3.59, 5.56, 1.98, 1.96,
s s s m m
5.82, dd (4.4, 3.3)
6.03, dd (4.8, 2.9)
5.82, dd (4.4, 3.1)
6.01, dd (4.7, 2.5)
2.12, dd (18.5, 4.4) 2.56, dd (18.5, 3.3) 1.82, dd (12.3, 6.9) 2.76, dd (18.4, 6.9) 3.07, dd (18.4, 12.3) 5.15, s 1.02, s 1.28, s 7.35, br s 6.27, br s 7.42, t (1.7) 0.82, s 0.78, s 3.43, s 4.04, br s
2.11, dd (18.7, 4.4) 2.55, dd (18.7, 3.1) 1.78, dd (12.2, 7.0) 2.69, dd (18.4, 7.0) 3.01, dd (18.4, 12.2) 5.11, s 1.00, s 1.25, s 7.32, br s 6.23, br s 7.39, t (1.7) 0.79, s 0.77, s 3.40, s 4.03, br s
1.31, m
2.00, m
2.10, m
1.60, dd (13.4, 5.5) 2.80, dd (16.3, 5.5) 3.59, dd (16.3, 13.4) 5.18, s 0.99, s 1.20, s 7.47, br s 6.41, br s 7.42, t (1.7) 0.87, s 0.86, s 3.54, s 4.10, s
1.56, dd (12.6, 6.5) 2.72, dd (16.8, 6.5) 3.38, dd (16.8, 12.6) 5.13, s 1.13, s 1.21, s 7.44, s 6.36, br s 7.41, t (1.6) 1.02, s 0.99, s 3.42, s 4.11, s
3.67, s
2.18, dd (19.2, 4.7) 2.61, dd (19.2, 2.5) 1.76, dd (11.8, 7.5) 2.67, dd (18.8, 7.5) 2.79, dd (18.8, 11.8) 5.10, s 1.01, s 1.45, s 7.31, br s 6.21, br s 7.40, t (1.7) 1.03, s 0.79, s 3.35, s 4.14, br s 3.15, br s 3.87, s
1.25, m
3.69, s
2.21, dd (18.9, 4.8) 2.64, dd (18.9, 2.9) 1.82, dd (11.8, 7.4) 2.72, dd (18.8, 7.4) 2.85, dd (18.8, 11.8) 5.14, s 1.05, s 1.49, s 7.35, br s 6.26, br s 7.43, t (1.7) 1.07, s 0.80, s 3.39, s 4.07, br s 3.00, s 3.92, s
3.75, s
3.87, s 2.20, s
5.99, dq (15.5, 1.7) 7.18, dq (15.5, 7.0) 2.01, dd (7.0, 1.7)
5.92, dq (15.5, 1.7) 7.16, dq (15.5, 7.0) 2.03, dd (7.0, 1.7)
7.02, q (7.2)
6.98, q (7.1)
9.33, d (1.3)
9.32, d (1.2)
1.91, d (7.2) 1.94, br s
1.91, d (7.1) 1.92, s
8.87, dd (4.8, 1.3) 7.56, dd (8.1, 4.8) 8.39, dt (8.1, 2.0)
8.89, dd (4.7, 1.2) 7.56, dd (7.9, 4.7) 8.37, dt (7.9, 1.9)
Data were measured in CDCl3 at 400 MHz.
hydroxy group was present at C-6. The structure of 11 was further confirmed by the HMBC spectrum (Supporting Information Figure S60). Trichinenlide L (12) was assigned a molecular formula of C36H44O13 by HRESI(+)MS at m/z 707.2684 [M + Na]+ (calcd 707.2680). Its 1H and 13C NMR data (Tables 2 and 5) suggested a mexicanolide-type limonoid possessing a tetrasubstituted double bond, a tigloyloxy group, and two O-acetyl groups. The Δ8(14) tetrasubstituted double bond was localized by the HMBC correlations from H-30 to C-8 and C-14, and H15 to C-8 (Supporting Information Figure S65). The key HMBC correlation from H-3 to C-1′ placed the tigloyloxy group at C-3. Two O-acetyl groups were located at C-15 and C30 by the key HMBC correlations from H-15 and H-30 to each of acetyl groups (Supporting Information Figure S65), respectively. The structures of compounds 13 and 14 were closely related to that of 12 on the basis of their NMR data. Analysis of the 1D NMR and MS data (Tables 2, 3, and 5) revealed that
compounds 13 and 14 were acetoxy and/or de-O-acetyl derivatives of 12. Compounds 13 and 14 were further identified as 6-acetoxy-15-O-deacetyl and 15-O-deacetyl derivatives of 12, respectively, on the basis of comprehensive analysis of 1D and 2D NMR spectra (Supporting Information Figures S71 and S77). Trichinenlides O (15) and P (16) had molecular formulas of C37H44O15 and C38H48O15 as determined by HRESI(+)MS data at m/z 751.2587 [M + Na]+ (calcd for C37H44O15Na 751.2578) and 767.2873 [M + Na]+ (calcd for C38H48O15Na 767.2891), respectively. The NMR data (Tables 3 and 5) of 15 and 16 indicated that they possessed similar structures to that of heytrijunolide C,16 the differences being the presence of (E)-2butenoate and 2-methylbutanoate groups at C-3 of 15 and 16 instead of the tigloyloxy group in heytrijunolide C. The absolute configuration of the 2-methylbutanoate moiety in 16 was not assigned. These assignments were confirmed by HMBC data (Supporting Information Figures S83 and S88). C
dx.doi.org/10.1021/np400408s | J. Nat. Prod. XXXX, XXX, XXX−XXX
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Table 2. 1H NMR Spectroscopic Data of Compounds 8−13a proton position
a
8
9
10
11
12
13
(mult., J in Hz)
(mult., J in Hz)
(mult., J in Hz)
(mult., J in Hz)
(mult., J in Hz)
(mult., J in Hz)
5.13, s 3.27, dd (7.6, 5.7) a 2.47, dd (16.5, 5.7) b 2.36, dd (16.5, 7.6)
5.16, s 3.24, dd (7.6, 5.4) a 2.46, dd (16.5, 5.4) b 2.36, dd (16.5, 7.6)
4.93, s 3.29, s 4.29, s
3.25, s
3.25, s
3.66, s
1.99, 2.39, 1.63, 2.75, 3.59, 4.99, 1.07, 0.90, 7.29, 6.16, 7.39, 0.88, 0.79, 3.42, 4.07,
2.00, 2.42, 1.68, 2.79, 3.63, 5.01, 1.10, 0.91, 7.33, 6.21, 7.41, 0.88 0.80 3.45, 4.03,
2.10, 2.25, 1.61, 2.75, 3.05, 4.99, 1.09, 1.15, 7.28, 6.14, 7.39, 1.07, 0.85, 3.32, 4.16,
3 5 6
4.95, s 3.34, s 4.44, s
9 11α 11β 12α 12β 14 15α 15β 17 18 19 21 22 23 28 29 30 HO-2 HO-15 HO-6 MeO-7 Ac-6 Ac-15 Ac-30 2′ 3′ 4′ 5′
1.95, 1.80, 1.91, 1.38, 2.09, 1.59, 2.85, 3.18, 5.11, 1.07, 1.43, 7.42, 6.36, 7.44, 1.03, 0.86, 3.22, 4.05,
m m m m m dd (11.8,7.5) dd (17.6, 7.5) dd (17.6, 11.8) s s s br s m t (1.7) s s s br s
3.93, s
5.98, dq (15.5, 1.7) 7.17, dq (15.5, 7.0) 2.01, dd (7.0, 1.7)
d (15.3) d (15.3) dd (12.5, 7.0) dd (18.6, 7.0) dd (18.6, 12.5) s s s s s t (1.6) s s s br s
3.73, s
7.06, q (7.1) 1.93, t (7.1) 1.96, s
d (15.5) d (15.5) dd (12.5, 6.8) dd (18.6, 6.8) dd (18.6, 12.5) s s s s m t (1.7)
s s
d (16.0) d (16.0) dd (12.4, 7.2) dd (18.9, 7.2) dd (18.9, 12.4) s s s s br s t (1.6) s s s s
3.25, s 3.96, s
3.74, s
6.01, dq (15.5, 1.7) 7.22, dq (15.5, 7.0) 2.02, dd (7.0, 1.7)
7.02, m 1.95, m 1.96, s
5.13, s 3.39, dd (10.2, 2.8) 2.41, m
5.04, s 3.56, s 5.50, s
2.42, 1.82, 1.89, 1.05, 1.92,
2.41, 1.83, 2.00, 1.18, 1.86,
m m m m m
m m m m m
6.60, d (2.1)
5.04, m
5.67, 1.11, 1.29, 7.66, 6.52, 7.43, 0.78, 0.74, 5.72, 4.10,
5.53, 1.09, 1.31, 7.59, 6.49, 7.45, 1.09, 0.86, 6.08, 4.17, 3.87,
s s s s m t (1.7) s s s br s
3.74, s
s s s s m t (1.6) s s s s d (2.9)
3.76, s 2.19, s
2.05, s 1.99, s
2.02, s
6.83, dq (7.0, 1.5) 1.80, dd (7.0, 1.1) 2.03, m
6.80, dq (7.0, 1.2) 1.79, dd (7.0, 0.7) 1.90, br s
Data were measured in CDCl3 at 400 MHz.
hydroxybutenolide motif at C-17. This deduction was supported by the HMBC cross-peaks of H-17/C-20 and C-22. The relative configuration of the limonoid core in compounds 12−20 was identical on the basis of NMR data, and that of 18 was defined by a ROESY experiment (Figure 3). In the ROESY spectrum of 18, the correlations of H3-18/H11α, H3-18/H-22, H3-19/H-9, and H-9/11α indicated that they were cofacial and were assigned arbitrarily in an α-orientation. Thus, the ROESY correlations of H-17/H-15, H-17/H-5, H15/H-30, and H-5/H3-29 revealed that they were β-oriented. The C-23 configuration in 20 was not assigned by the available data. Similarly, the relative configuration of C-6 in compounds 3, 5, 7, 8, 11, 13, 15, 16, 18, and 20 could not be established.14 Eleven known limonoids, heytrijunolides A−D,16 swietemahonin G,13 methyl-3β-tigloyloxy-2,6-dihydroxy-1-oxomeliac8(30)-enate,17 humilinolide E,18 methyl-2-hydroxy-3-β-tigloyloxy-1-oxomeliac-8(30)-enate,19 E-volkendousin,20 proceranolide,21 and (6R)-hydroxymexicanolide,22 were also isolated. Their structures were identified on the basis of spectroscopic analysis and comparison with reported data. Compounds 2−9, 13, 14, 16−18, 20, heytrijunolide D, and swietemahonin G were examined for anti-inflammatory activities by monitoring the inhibition of LPS-induced NO production in RAW 264.7 macrophages. Trichinenlides B (2) and C (3) and heytrijunolide D exhibited significant anti-
HRESIMS analysis revealed that trichinenlides Q−S (17− 19) had the molecular formulas C36H46O12, C41H50O15, and C39H48O13 by HRESIMS, respectively. The 1H and 13C NMR spectra of 17−19 (Tables 3 and 5) showed similarities to those of 12−14, with the differences being the substitution patterns at C-6, C-15, and C-30. A comparison of NMR data of 17 with those of 14 indicated that an isobutyryloxy group was located at C-30 in 17 instead of an acetoxy group in 14, which was confirmed by HMBC data (Supporting Information Figure S93). The 1H and 13C NMR data of 18 showed the presence of one more tigloyl group as compared to those of 13. The extra tigloyl group was assigned at C-15 by the key HMBC correlation of H-15/C-(15-tig-1″) (Supporting Information Figure S99). Compound 19 was assigned as the 6-deacetoxy derivative of 18 by comparing their NMR data (Supporting Information Figures S96−S99 and S103−S105). Trichinenlide T (20) was obtained as a white, amorphous powder, having a molecular formula of C38H46O17 as established on the basis of HRESI(+)MS at m/z 797.2632 [M + Na]+ (calcd 797.2633). Its 1H and 13C NMR data (Tables 3 and 5) were similar to those of heytrijunolide C,16 except for the presence of a γ-hydroxybutenolide (δH 6.39, br s; 7.37, s; δC 99.2, 133.6, 149.9, and 169.3) group in place of the typical C-17 β-furan ring in heytrijunolide C. This suggested that 20 was an oxygenated product of heytrijunolide C and had a γD
dx.doi.org/10.1021/np400408s | J. Nat. Prod. XXXX, XXX, XXX−XXX
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Table 3. 1H NMR Spectroscopic Data of Compounds 14−20a 14 proton position
a
(mult., J in Hz)
15
16
(mult., J in Hz)
(mult., J in Hz)
17
18
(mult., J in Hz)
(mult., J in Hz)
3 5 6
5.13, s 3.34, br d (10.0) 2.41, br d (10.0)
5.06, s 3.54, s 5.51, s
5.19, s 3.44, s 5.47, s
5.12, s 3.34, dd (9.4, 3.4) 2.42, m
5.05, s 3.64, s 5.58, s
9 11α 11β 12α 12β 15 17 18 19 21 22 23 28 29 30 HO-2 HO-15 MeO-7 Ac-6 Ac-15 Ac-30 2′ 3′
2.37, 1.75, 1.90, 1.13, 1.77, 5.11, 5.61, 1.10, 1.29, 7.63, 6.50, 7.42, 0.79, 0.72, 6.10, 4.21, 3.98, 3.72,
2.44, 1.85, 2.03, 1.20, 1.91, 6.57, 5.66, 1.11, 1.30, 7.64, 6.51, 7.45, 1.04, 0.85, 5.73, 4.09,
d (7.3) m m m m d (2.0) s s s s br s t (1.6) s s s s
2.45, 1.91, 2.02, 1.24, 1.85, 6.67, 5.71, 1.13, 1.30, 7.58, 6.48, 7.43, 1.04, 0.82, 5.88, 4.13,
2.47, 1.93, 2.06, 1.26, 1.91, 6.60, 5.73, 1.10, 1.29, 7.71, 6.57, 7.57, 1.06, 0.87, 5.72,
3.78, 2.07, 2.19, 1.91, 5.98, 7.07,
s s s s dq (15.6, 1.6) dq (15.6, 6.9)
4′ 5′ 15-Tig-3″ 15-Tig-4″ 15-Tig-5″
1.80, d (7.0) 1.90, s
3.73, s 2.10, s 2.19, s 1.99, s 2.93, m a 1.90, m b 1.47, m 0.99, t (7.5) 1.20, d (7.4)
2.36, 1.77, 1.89, 1.20, 1.81, 5.10, 5.61, 1.09, 1.29, 7.61, 6.50, 7.42, 0.79, 0.72, 6.08, 4.14, 4.01, 3.73,
m m m m m m s s s s s br s s s s s d (2.7) s
2.02, s 6.82, q (7.0)
1.91, dd (6.9, 1.6)
m m m m m d (1.8) s s s s br s t (1.7) s s s s
19
20
(mult., J in Hz)
(mult., J in Hz)
5.13, s 3.43, d (11.5) a 2.67, d (17.1) b 2.51, dd (17.1, 11.5) 2.41, m 1.87, m 2.03, m 1.18, m 1.90, m 6.67, d (1.9) 5.79, s 1.11, s 1.29, s 7.73, s 6.59, br s 7.56, t (1.7) 0.80, s 0.73, s 5.75, s
5.07, s 3.51, s 5.61, s
3.80, s 2.18, s
3.76, s
1.84, s 6.82, dq (7.0, 1.4) 1.80, d (7.0) 1.90, s
m m m m m s s s s s br s t (1.7) s s s br s s s
m m m m m s s s s s s br s s s s
2.51, 1.88, 1.93, 1.36, 1.91, 6.54, 5.65, 1.08, 1.32,
m m m m m s s s s
7.37, 6.39, 1.03, 0.86, 5.71, 4.16,
s br s s s s br s
1.84, s
3.74, 2.05, 2.17, 1.97,
s s s s
6.84, q (6.9)
6.89, q (7.2)
6.73, q (6.9)
1.78, 2.00, 6.69, 1.78, 1.81,
1.79, 2.01, 6.71, 1.79, 1.81,
1.78, d (6.9) 1.98, s
d (6.9) s q (7.0) d (7.0) s
d (7.2) s q (7.0) d (7.0) s
Data for compounds 14−17 and 20 were measured in CDCl3, and compounds 18 and 19 in methanol-d4 at 400 MHz. were used for column chromatography, and precoated silica gel GF254 plates (Qingdao Marine Chemical Plant, Qingdao, People’s Republic of China) were used for TLC. All solvents used were of analytical grade (Shanghai Chemical Reagents Company, Ltd.), and solvents used for HPLC were of HPLC grade (J & K Scientific Ltd.). Plant Material. The leaves and twigs of T. sinensis Bentv. were collected in April 2008 from Sanya of Hainan Province, People’s Republic of China, and authenticated by Professor S. M. Huang of the Department of Biology, Hainan University. A voucher specimen (accession number TSB-2008-01y) has been deposited at Shanghai Institute of Materia Medica. Extraction and Isolation. The air-dried powders of the leaves and twigs of T. sinensis (5 kg) were extracted three times with 95% EtOH (each 10 L, each 3 days) at room temperature to give an EtOH extract (300 g), which was partitioned between EtOAc and H2O to obtain the EtOAc-soluble fraction (200 g). The EtOAc-soluble fraction was fractionated over a column of D101-macroporous absorption resin (EtOH/H2O, 30/70, 50/50, 80/20, and 100/0), and the major fraction (EtOH/H2O = 80/20, 150 g) was subjected to passage over a column of MCI gel (MeOH/H2O, 50/50 to 90/10), to produce six fractions, A−F. Fraction C (16 g) was chromatographed over a silica gel column, eluted with petroleum ether/acetone (20/1 to 1/1), to afford 10 fractions, C1−C10. Fraction C8 (7.7 g) was subjected to a column of reversed-phase silica gel eluted with a MeOH/H2O (50/50 to 100/0) gradient to yield four fractions, C8a−C8d. Fraction C8b (45 mg) was purified by semipreparative HPLC with 70% MeOH in H2O
inflammatory activities, with IC50 values of 2.85, 1.88, and 3.33 μM, respectively. Curcumin, with an IC50 value of 5 μM, was used as a positive control. In summary, 20 new and 11 known mexicanolide limonoids were isolated from the leaves and twigs of T. sinensis for the first time. Compounds 2, 3, and heytrijunolide D exhibited noteworthy anti-inflammatory activities, with IC50 values of 2.85, 1.88, and 3.33 μM, respectively.
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EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were measured on a Perkin-Elmer 341 polarimeter at room temperature. UV spectra were recorded on a Shimadzu UV-2550 UV−visible spectrophotometer. IR spectra were recorded on a Perkin-Elmer 577 IR spectrometer with KBr disks. NMR spectra were acquired on a Bruker AM-400 NMR spectrometer with TMS as internal standard. ESIMS was measured with a Bruker Daltonics Esquire 3000plus instrument. HRESIMS was carried out on an LCT Premier XE (Waters) mass spectrometer. Semipreparative HPLC was run on a Waters 1525 pump with a Waters 2489 detector (254 and 210 nm) and a YMC-Pack ODS-A column (250 × 10 mm, S-5 μm, 12 nm). Silica gel (300−400 mesh), C18 reversed-phase silica gel (150−200 mesh, Merck), Sephadex LH-20 (Amersham Biosciences), and MCI gel (CHP20P, 75−150 μM, Mitsubishi Chemical Industries, Ltd.) E
dx.doi.org/10.1021/np400408s | J. Nat. Prod. XXXX, XXX, XXX−XXX
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Table 4. 13C NMR Spectroscopic Data of Compounds 1−10a carbon
1
2
3
4
5
6
7
8
9
10
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 28 29 30 MeO-7 Ac-6
215.9 48.7 79.4 39.1 45.4 72.8 175.7 139.1 57.4 50.4 21.2 34.5 36.6 45.1 29.6 168.2 76.7 21.3 16.4 121.0 140.6 109.2 143.1 22.8 22.9 123.0 53.5 170.3 22.7 164.4 125.1 150.6
210.1 78.7 84.8 39.2 47.4 31.1 173.1 60.8 137.1 51.2 126.9 31.7 35.2 39.2 29.2 169.6 81.9 23.2 14.5 121.8 140.6 109.4 143.6 20.0 23.7 64.2 52.3
209.4 78.8 85.8 40.2 50.7 69.3 175.8 60.5 137.3 52.1 126.2 31.6 34.7 38.4 28.9 169.2 82.5 23.1 15.1 121.8 140.5 109.2 143.8 21.9 22.2 64.2 53.5
210.0 79.0 85.0 39.5 47.3 31.0 173.0 60.9 136.9 51.3 126.8 31.7 35.1 39.1 29.2 169.2 81.9 23.6 14.6 121.8 140.5 109.3 143.5 20.1 23.2 64.2 52.2
209.4 79.0 85.9 40.2 50.4 69.1 175.8 60.5 136.9 52.0 126.4 31.3 34.5 38.1 28.8 169.0 82.3 22.9 15.0 121.7 140.4 109.1 143.6 21.8 22.1 64.0 53.2
212.7 78.2 85.8 40.1 44.9 33.0 173.9 63.3 54.9 49.1 19.5 32.9 36.1 42.3 33.5 170.8 79.2 26.3 16.1 120.2 140.9 110.1 143.2 20.6 22.0 67.1 52.6
211.6 78.2 86.8 40.7 45.2 71.9 170.9 63.0 54.8 49.3 20.1 32.7 35.8 44.0 33.0 169.5 79.9 26.6 16.1 120.4 140.8 109.9 143.4 20.9 22.3 67.0 53.7
212.6 77.9 86.0 40.4 46.0 72.1 175.4 62.5 54.6 49.2 20.3 32.3 35.6 43.5 32.5 170.8 80.7 26.9 18.3 120.7 140.6 109.7 143.5 22.2 22.2 67.3 53.6
208.7 78.3 84.9 39.3 42.9 31.8 173.4 61.6 62.1 50.4 56.2 27.4 34.0 38.4 28.8 168.8 82.6 23.2 10.7 121.8 140.6 109.2 143.6 23.8 20.8 64.5 52.6
208.8 78.0 84.5 39.0 42.9 31.7 173.5 61.6 62.3 50.3 56.4 27.5 34.1 38.4 28.8 169.1 82.7 23.3 10.9 120.9 140.7 109.3 143.7 23.7 20.7 64.6 52.7
165.1 121.1 148.0 18.3
164.7 121.2 147.5 18.2
166.5 127.8 139.4 14.3 12.5
166.1 128.0 138.7 14.5 12.3
164.4 123.9 151.3
164.3 124.8 151.2
164.9 121.1 148.0 17.1
165.0 121.8 148.5 18.4
154.3 124.9 136.8
154.4 123.9 136.8
166.5 127.8 139.6 14.6 12.6
1′ 2′ 3′ 4′ 5′ 6′ 7′ a
153.8 123.6 137.1
Data were measured in CDCl3 at 100 MHz. (20 mg), 10 (3 mg), and 11 (45 mg), respectively. By using the same purification procedures, C9c (1.5 g) yielded compounds 1 (5 mg), 7 (7 mg), 13 (7 mg), 14 (6 mg), heytrijunolide A (5 mg), 20 (4 mg), and methyl-3β-tigloyloxy-2,6-dihydroxy-1-oxomeliac-8(30)-enate (8 mg). Fraction D was subjected to a column of Sephadex LH-20 gel eluted with MeOH to obtain five major subfractions, each of which was then purified by semipreparative HPLC with the mobile phase MeOH/H2O (70/30) to produce compounds 16 (5 mg), 17 (8 mg), 18 (10 mg), 19 (4 mg), and proceranolide (4 mg), respectively. Trichinenlide A (1): white, amorphous powder; [α]22D −392 (c 0.08, MeOH); UV (MeOH) λmax (log ε) 212 (4.63) nm; IR (KBr) νmax 3437, 2949, 1724, 1593, 1435, 1358, 1281, 1221, 1134, 1026 cm−1; 1H NMR, Table 1; 13C NMR, Table 4; HRESIMS m/z 592.2553 [M + H]+ (calcd for C33H38NO9 592.2547). Trichinenlide B (2): white, amorphous powder; [α]21D −59 (c 0.12, MeOH); UV (MeOH) λmax (log ε) 210 (4.46) nm; IR (KBr) νmax 3438, 2995, 1728, 1655, 1504, 1458, 1439, 1360, 1271, 1174, 1018, 970, 877, 602 cm−1; 1H NMR, Table 1; 13C NMR, Table 4; HRESIMS m/z 591.2224 [M + Na]+ (calcd for C31H36O10Na 591.2206). Trichinenlide C (3): white, amorphous powder; [α]21D −151 (c 0.08, MeOH); UV (MeOH) λmax (log ε) 213 (4.54) nm; IR (KBr) νmax 3454, 2952, 1736, 1651, 1504, 1441, 1387, 1252, 1169, 1109, 1072, 1016, 973 cm−1; 1H NMR, Table 1; 13C NMR, Table 4;
as the mobile phase to yield compound 2 (5 mg) and swietemahonin G (8 mg). C8c (300 mg) and C8d (2.2 g) were applied to silica gel eluted with DCM/MeOH (100/1 to 10/1) to yield two fractions (C8c1 and C8c2) and four fractions (C8d1 to C8d4), respectively. Fraction C8c1 (85 mg) was purified by semipreparative HPLC with 70% MeOH in H2O as the mobile phase to yield compounds 4 (30 mg), heytrijunolide D (8 mg), and (6R)-hydroxymexicanolide (4 mg), and in a similar procedure, fraction C8d2 (150 mg) yielded heytrijunolide C (60 mg), 12 (3 mg), and E-volkendousin (3 mg). Fraction C8d1 (250 mg) was separated on a column of Sephadex LH20 gel to obtain two fractions (C8d1A and C8d1B). C8d1B (120 mg) was purified by semipreparative HPLC with 80% MeOH in H2O as the mobile phase to yield heytrijunolide B (3 mg), humilinolide E (50 mg), and methyl-2-hydroxy-3-β-tigloyloxy-1-oxomeliac-8(30)-enate (10 mg). Fraction C8d3 (40 mg) was subjected to a column of Sephadex LH-20 gel and then purified by semipreparative HPLC to afford compounds 15 (4 mg) and 6 (10 mg). Fraction C9 (15.5 g) was separated on silica gel eluted with petroleum ether/EtOAc (10/1−1/ 1) to obtain six fractions (C9a to C9f). Fraction C9b (2.5 g) was subjected to a silica gel column eluted with CHCl3/MeOH (200/1− 10/1) to afford four fractions (C9b1 to C9b4). Fraction C9b2 (350 mg) was chromatographed over a column of Sephadex LH-20 gel eluted with MeOH to give seven major components, and each of them was purified by semipreparative HPLC with the mobile phase MeOH/ H2O (70/30) to afford compounds 3 (5 mg), 5 (30 mg), 8 (5 mg), 9 F
dx.doi.org/10.1021/np400408s | J. Nat. Prod. XXXX, XXX, XXX−XXX
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Table 1; 13C NMR, Table 4; HRESIMS m/z 608.2511 [M + H]+ (calcd for C33H38NO10 608.2496). Trichinenlide G (7): white, amorphous powder; [α]21D −118 (c 0.19, MeOH); UV (MeOH) λmax (log ε) 212 (4.74) nm; IR (KBr) νmax 3444, 2954, 1736, 1593, 1458, 1375, 1271, 1219, 1103, 1026 cm−1; 1H NMR, Table 1; 13C NMR, Table 4; HRESIMS m/z 666.2546 [M + H]+ (calcd for C35H40NO12 666.2551). Trichinenlide H (8): white, amorphous powder; [α]21D −132 (c 0.17, MeOH); UV (MeOH) λmax (log ε) 214 (4.43) nm; IR (KBr) νmax 3469, 2954, 1731, 1651, 1441, 1371,1259, 1165, 1103, 1024, 970 cm−1; 1H NMR, Table 2; 13C NMR, Table 4; HRESIMS m/z 609.2295 [M + Na]+ (calcd for C31H38O11Na 609.2312). Trichinenlide I (9): white, amorphous powder; [α]21D −96 (c 0.12, MeOH); UV (MeOH) λmax (log ε) 214 (4.32) nm; IR (KBr) νmax 3392, 2970, 1734, 1716, 1651, 1439, 1360, 1257, 1122, 1062, 1038 cm−1; 1H NMR, Table 2; 13C NMR, Table 4; HRESIMS m/z 621.2319 [M + Na]+ (calcd for C32H38O11Na 621.2312). Trichinenlide J (10): white, amorphous powder; [α]22D −168 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 209 (4.69) nm; IR (KBr) νmax 3446, 2952, 2920, 1736, 1651, 1439, 1375, 1259, 1205, 1169, 1072, 1024 cm−1; 1H NMR, Table 2; 13C NMR, Table 4; HRESIMS m/z 607.2156 [M + Na]+ (calcd for C31H36O11Na 607.2155). Trichinenlide K (11): white, amorphous powder; [α]21D −78 (c 0.16, MeOH); UV (MeOH) λmax (log ε) 217 (4.52) nm; IR (KBr) νmax 3479, 2956, 1738, 1649, 1439, 1367, 1257, 1223, 1113, 1070, 1028 cm−1; 1H NMR, Table 2; 13C NMR, Table 5; HRESIMS m/z 637.2277 [M + Na]+ (calcd for C32H38O12Na 637.2261). Trichinenlide L (12): white, amorphous powder; [α]22D −37 (c 0.06, MeOH); UV (MeOH) λmax (log ε) 211 (4.40) nm; IR (KBr) νmax 3460, 2972, 2951, 1753, 1724, 1649, 1371, 1223, 1199, 1043, 1024 cm−1; 1H NMR, Table 2; 13C NMR, Table 5; HRESIMS m/z 707.2684 [M + Na]+ (calcd for C36H44O13Na 707.2680). Trichinenlide M (13): white, amorphous powder; [α]22D −30 (c 0.12, MeOH); UV (MeOH) λmax (log ε) 212 (4.39) nm; IR (KBr) νmax 3467, 2954, 1751, 1728, 1649, 1373, 1220, 1124, 1024 cm−1; 1H NMR, Table 2; 13C NMR, Table 5; HRESIMS m/z 723.2619 [M + Na]+ (calcd for C36H44O14Na 723.2629). Trichinenlide N (14): white, amorphous powder; [α]22D −58 (c 0.09, MeOH); UV (MeOH) λmax (log ε) 211 (4.44) nm; IR (KBr) νmax 3462, 2952, 1724, 1647, 1373, 1259, 1225, 1201, 1124, 1024 cm−1; 1H NMR, Table 3; 13C NMR, Table 5; HRESIMS m/z 665.2587 [M + Na]+ (calcd for C34H42O12Na 665.2574). Trichinenlide O (15): white, amorphous powder; [α]22D −8 (c 0.07, MeOH); UV (MeOH) λmax (log ε) 210 (4.50) nm; IR (KBr) νmax 3458, 2979, 1755, 1732, 1655, 1371, 1284, 1219, 1157, 1090, 1009 cm−1; 1H NMR, Table 3; 13C NMR, Table 5; HRESIMS m/z 751.2587 [M + Na]+ (calcd for C37H44O15Na 751.2578). Trichinenlide P (16): white, amorphous powder; [α]22D −7 (c 0.11, MeOH); UV (MeOH) λmax (log ε) 213 (4.10) nm; IR (KBr) νmax 3458, 2978, 1753, 1367, 1281, 1225, 1086, 1022 cm−1; 1H NMR, Table 3; 13C NMR, Table 5; HRESIMS m/z 767.2873 [M + Na]+ (calcd for C38H48O15Na 767.2891). Trichinenlide Q (17): white, amorphous powder; [α]22D −62 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 214 (4.64) nm; IR (KBr) νmax 3458, 2974, 1724, 1649, 1385, 1257, 1120, 1149, 1024 cm−1; 1H NMR, Table 3; 13C NMR, Table 5; HRESIMS m/z 693.2887 [M + Na]+ (calcd for C36H46O12Na 693.2887). Trichinenlide R (18): white, amorphous powder; [α]22D −21 (c 0.15, MeOH); UV (MeOH) λmax (log ε) 214 (4.51) nm; IR (KBr) νmax 3462, 2954, 1763, 1728, 1651, 1371, 1282, 1217, 1117, 1086, 1024 cm−1; 1H NMR, Table 3; 13C NMR, Table 5; HRESIMS m/z 805.3058 [M + Na]+ (calcd for C41H50O15Na 805.3047). Trichinenlide S (19): white, amorphous powder; [α]22D −47 (c 0.12, MeOH); UV (MeOH) λmax (log ε) 214 (4.52) nm; IR (KBr) νmax 3473, 2972, 1761, 1724, 1649, 1373, 1227, 1142, 1119, 1066, 1026 cm−1; 1H NMR, Table 3; 13C NMR, Table 5; HRESIMS m/z 747.2971 [M + Na]+ (calcd for C39H48O13Na 747.2993). Trichinenlide T (20): white, amorphous powder; [α]22D +244 (c 0.08, MeOH); UV (MeOH) λmax (log ε) 212.8(4.50) nm; IR (KBr) νmax 3606, 3454, 1763, 1736, 1649, 1373, 1228, 1197, 1122, 1026
Figure 1. Selected HMBC (H→C) and ROESY (H↔H) correlations of 1.
Figure 2. Selected HMBC (H→C) and ROESY (H↔H) correlations of 2.
HRESIMS m/z 607.2161 [M + Na]+ (calcd for C31H36O11Na 607.2155). Trichinenlide D (4): white, amorphous powder; [α]21D −81 (c 0.12, MeOH); UV (MeOH) λmax (log ε) 213 (4.47) nm; IR (KBr) νmax 3411, 2968, 1734, 1712, 1651, 1504, 1439, 1358, 1263, 1225, 1124, 1032 cm−1; 1H NMR, Table 1; 13C NMR, Table 4; HRESIMS m/z 605.2363 [M + Na]+ (calcd for C32H38O10Na 605.2363). Trichinenlide E (5): white, amorphous powder; [α]21D −102 (c 0.17, MeOH); UV (MeOH) λmax (log ε) 206 (4.44) nm; IR (KBr) νmax 3477, 2993, 1738, 1649, 1441, 1360, 1255, 1219, 1136, 1070, 1030 cm−1; 1H NMR, Table 1; 13C NMR, Table 4; HRESIMS m/z 621.2327 [M + Na]+ (calcd for C32H38O11Na 621.2312). Trichinenlide F (6): white, amorphous powder; [α]21D −114 (c 0.17, MeOH); UV (MeOH) λmax (log ε) 214 (4.28) nm; IR (KBr) νmax 3413, 2966, 1730, 1593, 1269, 1232, 1103, 1022 cm−1; 1H NMR, G
dx.doi.org/10.1021/np400408s | J. Nat. Prod. XXXX, XXX, XXX−XXX
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Table 5. 13C NMR Spectroscopic Data of Compounds 11−20a carbon 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 28 29 30 MeO-7 Ac-6
11
12
13
14
15
16
17
18
19
20
208.2 78.2 86.0 40.5 46.6 70.6 175.4 61.2 62.1 50.8 54.9 27.8 33.9 38.1 29.0 168.5 82.5 23.2 10.9 121.4 140.6 109.1 143.8 22.6 22.1 64.5 53.9
212.9 79.1 85.6 39.8 41.1 33.0 174.2 132.7 47.4 51.9 18.4 28.3 39.2 139.4 64.5 167.8 80.2 17.2 17.1 120.5 142.2 109.9 143.0 21.2 23.6 74.1 52.3
212.4 78.3 86.3 40.2 44.8 72.6 171.4 131.3 48.6 52.2 18.3 28.8 86.3 143.4 64.6 171.7 80.7 17.4 17.0 120.3 141.8 109.8 143.2 22.2 23.1 74.6 53.4 169.7 20.9
213.3 78.4 85.7 39.7 41.1 33.0 174.1 131.4 47.8 51.8 18.5 28.6 38.7 143.0 64.6 171.8 80.7 17.4 17.0 120.5 142.0 109.9 143.0 19.3 23.4 74.7 52.3
211.3 79.0 86.3 40.2 44.4 72.5 171.7 134.1 48.7 52.1 17.8 28.7 38.9 139.3 64.3 168.3 80.7 16.3 16.2 120.3 141.9 109.4 143.3 21.1 22.3 74.4 52.6 169.8 19.3
212.7 79.1 85.7 39.8 40.7 32.1 175.0 134.0 48.0 51.8 17.8 28.4 38.9 138.8 64.4 168.5 80.8 16.1 16.0 120.4 142.0 109.4 143.1 18.1 22.5 74.4 51.3
166.3 128.0 139.1 14.6 12.5
170.0 21.2 166.7 129.3 138.8 14.4 12.6
170.1 21.2 166.9 129.3 138.8 14.5 12.6
212.3 78.9 85.3 40.1 44.9 72.4 171.1 133.0 48.4 51.9 18.2 28.6 39.3 139.8 64.6 167.3 80.3 17.3 17.0 120.2 141.9 109.8 143.1 22.5 22.6 74.0 53.3 169.6 21.3 169.6 20.8 168.0 17.1 175.2 40.3 25.5 11.2 17.1
213.3 78.4 85.6 39.6 41.1 33.0 174.0 131.3 48.0 51.7 18.5 28.8 38.5 142.9 64.6 171.6 80.6 17.6 17.0 120.4 141.9 109.9 142.9 19.3 23.3 74.4 52.2
169.5 20.9 168.2 19.3 167.1 131.0 137.1 14.4 12.8
211.9 79.0 85.9 39.9 44.9 72.6 171.4 132.7 48.2 52.2 18.2 28.6 39.3 139.9 64.7 167.6 80.5 17.2 17.2 120.3 142.1 109.9 143.1 22.3 23.1 74.1 53.5 169.6 21.2 169.7 21.0 168.1 20.9 165.0 121.8 147.6 18.3
168.7 19.4 166.8 130.8 136.5 12.9 10.8 165.9 128.8 136.9 12.9 11.5
168.8 19.4 167.0 130.8 136.5 12.9 10.7 165.9 128.8 136.8 12.9 11.5
212.2 78.4 86.0 40.3 44.5 72.6 170.4 136.4 50.5 51.3 19.0 30.3 39.1 138.2 64.1 167.6 79.1 19.1 16.8 133.6 169.3 149.9 99.2 22.1 23.1 75.2 53.3 169.2 21.1 169.4 20.9 168.1 20.9 166.7 130.9 137.1 14.5 12.7
Ac-15 Ac-30 1′ 2′ 3′ 4′ 5′ 15-Tig-1″ 15-Tig-2″ 15-Tig-3″ 15-Tig-4″ 15-Tig-5″ a
166.9 129.2 138.8 14.4 12.6
Data for compounds 14−17 and 20 were measured in CDCl3, and compounds 18 and 19 in methanol-d4 at 100 MHz. Anti-inflammatory Bioassay. The RAW 264.7 macrophages (obtained from Shanghai Institutes for Biological Science, Shanghai, China) were maintained in DEMEM/high-glucose medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% (v/v) newborn calf serum and antibiotics (100 U/mL penicillin and 0.1 g/L streptomycin) at 37 °C in the presence of 5% CO2. The RAW 264.7 macrophages were seeded in 96-well plates with 1 × 104 cells/ well and allowed to adhere for 6 h at 37 °C in a humidified atmosphere containing 5% CO2. After that the RAW 264.7 macrophages were pretreated with compounds for 2 h, followed by 1 mg/L LPS for 24 h. Curcumin (5 μM) was used as a positive control. NO production in the cell culture medium was determined by using a commercially available kit (Beyotime, Haimen, China). Nitrite production was measured at OD 550. Percent inhibition was calculated using the following equation: % inhibition = (A − B)/(A − C) × 100, where A = LPS (+), sample (−); B = LPS (+), sample (+); and C = LPS (−), sample (−).
Figure 3. Key ROESY (H↔H) correlations of 18. cm−1; 1H NMR, Table 3; 13C NMR, Table 5; HRESIMS m/z 797.2632 [M + Na]+ (calcd for C38H46O17Na 797.2633). H
dx.doi.org/10.1021/np400408s | J. Nat. Prod. XXXX, XXX, XXX−XXX
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Article
(19) Okorie, D. A.; Taylor, D. A. H. Phytochemistry 1971, 10, 469− 470. (20) Rogers, L. L.; Zeng, L.; McLaughlin, J. L. J. Org. Chem. 1998, 63, 3781−3785. (21) Sondengam, B. L.; Kamga, C. S.; Connolly, J. D. Phytochemistry 1980, 19, 2488. (22) Connolly, J. D.; McCrindle, R.; Overton, K. H.; Warnock, W. D. C. Tetrahedron 1968, 24, 1507−1515.
ASSOCIATED CONTENT
S Supporting Information *
IR, HRESIMS, and 1D and 2D NMR spectra of compounds 1− 20. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Tel: +86-21-50806718. Fax: +86-21-50806718. E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS Financial support from the National Natural Science Foundation (Grant Nos. 81273398, 20932007) of the People’s Republic of China is gratefully acknowledged. We thank Prof. S.-M. Huang of Hainan University for the identification of the plant material.
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