Article pubs.acs.org/jnp
Cafestol-Type Diterpenoids from the Twigs of Tricalysia f ruticosa with Potential Anti-inflammatory Activity Chuan-Pu Shen, Jian-Guang Luo, Ming-Hua Yang, and Ling-Yi Kong* State Key Laboratory of Natural Medicines, Department of Natural Medicinal Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, People’s Republic of China S Supporting Information *
ABSTRACT: Eight new cafestol-type diterpenoids, tricalysins A−H (1−8), along with five known analogues (9−13), were isolated from the twigs of Tricalysia fruticosa. The structures of 1−8 were elucidated by the application of spectroscopic methods. Inhibitory effects of the isolates on nitric oxide (NO) production in lipopolysaccaride-activated RAW 264.7 macrophages were evaluated, and compound 8 exhibited the most potent bioactivity, with an IC50 value of 6.6 ± 0.4 μM. It was shown further that compound 8 inhibits inflammatory responses via suppression of the expression of iNOS and reduction of the production of the pro-inflammatory cytokines IL-6 and TNF-α, resulting from activation of nuclear factor-kappaB (NF-κB) and phosphorylation of MAPKs (ERK, JNK, and p38).
D
iterpenoids occur in many terrestrial plants and have been investigated for their potential anti-inflammatory activity.1−4 Cafestol derivatives are a rare class of compounds rearranged from an ent-kauranoid framework and occur only in the genera Tricalysia and Caf fea in the family Rubiaceae.5−7 Significantly, cafestol derivatives from unroasted coffee beans have been reported to exhibit good anti-inflammatory activity due to their suppressive effects on the expression of COX-2 and the activation of NF-κB.8 Tricalysia f ruticosa (Hemsl.) K. Schum. ex E. Pritz. (Rubiaceae), a small shrub or tree, is distributed widely in the southern part of mainland China.9 The species, together with Tricalysia dubia, has been used as a potential therapeutic agent for the treatment of skin ulcers and contusions. Previously, phytochemical research on T. dubia has led to the isolation of about 20 cafestol derivatives.5−7 However, there are few comprehensive and detailed reports on the antiinflammatory activity of T. f ruticosa and its constituents. In the course of ongoing investigations on anti-inflammatory natural products,10−12 eight new cafestol-type diterpenoids (1− 8), as well as five known compounds, were isolated from the twigs of T. f ruticosa. Herein, the isolation and identification of these compounds are described as well as the inhibitory activity of LPS-induced NO production in RAW 264.7 macrophage cells. Additionally, the mechanism of action of the most potent bioactive compound 8 is reported, focusing on the suppressive effects of the expression of iNOS and pro-inflammatory cytokines IL-6 and TNF-α, activation of NF-κB, and phosphorylation of MAPKs (ERK, JNK, and p38).
ether and EtOAc. The EtOAc-soluble fraction was subjected to column chromatography over silica gel, MCI gel, Sephadex LH20, and ODS and then purified by preparative HPLC to afford 13 cafestol-type diterpenoids (1−13). Tricalysin A (1) was obtained as a colorless gum. The molecular formula was determined as C20H28O6 by HRESIMS
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RESULTS AND DISCUSSION The ethanol extract from the twigs of T. f ruticosa was suspended in water and partitioned successively by petroleum © XXXX American Chemical Society and American Society of Pharmacognosy
Received: February 18, 2015
A
DOI: 10.1021/acs.jnatprod.5b00165 J. Nat. Prod. XXXX, XXX, XXX−XXX
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from the [M + H]+ peak at m/z 365.1960. The IR spectrum indicated that 1 possesses hydroxy (3427 cm−1) and α,βunsaturated-γ-lactone (1739 cm−1) groups.4,6 The 1H NMR spectrum (Table 1) exhibited a characteristic olefinic signal (δH
tricalysiolide B except for the presence of an additional hydroxy group.6 The HMBC correlations of OH-15 (δH 4.72) with C-15 (δC 81.0) and H-15 (δH 3.27) with C-17 (δC 64.7) and C-8 (δC 46.7) demonstrated the additional hydroxy group to be located at C-15. The relative configuration of compound 1 was determined from the ROESY spectrum, as shown (Figure 1b). The ROESY correlations from CH3-20 to H-14 revealed H-14 to be α-oriented. Also, the ROESY correlations of OH-3 to H5, H-5 to H-9, H-9 to H-15, and H-15 to H-17 suggested that OH-3, H-5, H-9, H-15, and H-17 are all β-oriented. The absolute configuration at C-3 was established from the ECD spectrum (a π−π* transition in the α,β-unsaturated-γ-lactone moiety). The observed negative Cotton effect at ca. 225 nm revealed the 3R configuration (Figure S25, Supporting Information).13−17 As supported by the ROESY correlations, the absolute configuration of tricalysin A was determined as shown in structure 1. Tricalysin B (2), a colorless gum, was found to possess a molecular formula of C20H28O6 based on the HRESIMS ion at m/z 382.2227 [M + NH4]+. The 1H and 13C NMR spectroscopic data (Tables 1 and 2) closely resembled those of the known compound tricalysiolide B6, except for an additional oxygenated carbon signal (δC 79.2). When taking the spectroscopic data into consideration, it was deduced that compound 2 is 13-hydroxytricalysiolide B, which was confirmed by the HMBC correlations from H-17 to C-13 and H-14 to C13. Its relative configuration was determined to be the same as that of tricalysiolide B, and the OH-13 group was assigned as βoriented as indicated by the ROESY spectrum. Tricalysin C (3) was assigned the molecular formula C22H30O7, as established from the [M + H]+ peak at m/z 407.2063. Its 1H and 13C NMR data (Tables 1 and 2) were very similar to those of compound 1 except for the presence of signals due to an acetyl group (δC 170.5, 18.6 and δH 2.02). The deshielded H-17 resonance indicated the acetyl group to be located at C-17, which was confirmed by the HMBC correlation between H-17 (δH 4.21, d, J = 11.0 Hz; 4.04, d, J = 11.0 Hz) and the acetyl carbonyl carbon (δC 170.5). Its relative configuration was determined to be the same as that of compound 1 from the ROESY spectrum. Tricalysin D (4) was obtained as a colorless gum and displayed a [M + Na] + ion peak at m/z 349.2013, corresponding to a molecular formula of C20H28O5. Analysis of its NMR spectra revealed that the structure of compound 4 differs from that of compound 1 primarily in the region around C-3, since the hemiketal carbon in 1 (δC 104.4) was replaced by an oxygenated methine carbon signal (δC 75.0) in 4. In addition, apart from two hydroxy groups at C-16 and C-17, a third hydroxy group could be assigned at C-9 by the HMBC correlations from H-20 (δH 0.86, s) and H-15 to C-9 (δC 75.0). The relative configuration of compound 4 was established using the ROESY spectrum, in which correlations of H-3 to H-5 and H-5 to OH-9 confirmed the latter group to be β-oriented. Therefore, the structure of compound 4 was elucidated as shown. For support of the absolute configurations assigned for compounds 2−4, the ECD spectra were measured (Figure S25, Supporting Information). The ECD spectra obtained were in close agreement with that of 1 at ca. 225 nm, thus indicating that the absolute configuration of C-3 in 2−4 is R, the same as that of 1. Tricalysin E (5), a colorless gum, showed a molecular formula of C20H26O5 according to the HRESIMS data at m/z
Table 1. 1H NMR Spectroscopic Data for 1−4 (δ in ppm, 500 MHz)
position
2b
δH (J in Hz)
δH (J in Hz)
1a 1b 2a
1.68 m 1.52 m 2.12 m
2b 3 5
1.42 m
6a 6b 7a 7b 9
1.54 m
11a
1.54 overlapped
11b 12a
2.19 m
1.68, m 1.52, m 1.26 d (6.5)
12b 13
1.56 overlapped 1.37 m 1.98 m
14a
1.17 m
14b
1.77 m
15a 15b 17a
3.27 d (7.0)
17b 18 20 CH3CO-17 OH-3 OH-9 OH-15 OH-16 OH-17 a
1a
3.51 dd (11.5, 6.5) 3.45 dd (11.5, 6.5) 5.73 d (2.0) 0.75 s 7.23 s
1.74 m 1.51 m 2.52 d (13.5) 1.96 m
3a
4a
δH (J in Hz)
δH (J in Hz)
1.71 m 1.53 m 2.12 m
1.98 m 1.50 m 2.28 m
1.44 m
1.88 m 4.80 brt (7.5) 2.71 brd (12.0) 1.61 m 1.52 m 1.92 m 1.18 m
2.60 d (10.0) 1.52 m 1.37 m
2.19 d (7.0)
1.52 m 1.25 d (8.5) 1.87 m
1.53 m 1.26 d (6.5) 1.53 m
1.65 m
1.73 m 2.14 m
1.46 m 1.46 m
1.37 m 1.44 m
2.04 overlapped 1.72 m
1.88 m
1.18 m
4.21 d (11.0)
1.70 d 3.5) 2.22 d 0.94 d 3.50 d
4.04 d (11.0)
3.38 d (11.0)
5.73 d (2.0)
5.70 brs
0.77 s 2.02 s 7.23 s
0.86 s
1.42 m 1.47 m
1.97 m
2.22 d (11.0) 2.10 d (11.0) 1.96 m 1.84 m 4.20 d (11.0) 4.10 d (11.0) 5.79 d (1.5) 0.82 s
3.38 d (6.5)
2.05 d (11.0) (11.0, (12.0) (12.0) (11.0)
4.20 s 4.72 d (7.0) 4.27 s 4.34 t (6.5)
5.10 d (6.5) 4.38 s
3.89 s 4.32 brs
Measured in DMSO-d6. bMeasured in pyridine-d5.
5.73, d, J = 2.0 Hz) and a methyl group signal (δH 0.75, s). The 13 C NMR spectrum (Table 2), when combined with the HSQC experiment, revealed 20 carbons, including a carbonyl carbon (δC 170.5), a pair of olefinic carbons (δC 111.8 and 172.6), a hemiketal carbon (δC 104.4), and three oxygenated carbons (δC 81.0, 80.1, and 64.7). The presence of an α,β-unsaturated-γlactone moiety was supported by HMBC correlations (recorded in DMSO-d6, Figure 1a) from H-18 (δH 5.73) to C-3 (δC 172.6), C-4 (δC 104.4), and C-19 (δC 170.5) and from OH-3 (δH 7.23) to C-2 (δC 33.7), C-3, and C-4. The aforementioned NMR data suggested that compound 1 possesses a comparable structure to the known compound B
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Table 2. 13C NMR Spectroscopic Data for 1−8 (δ in ppm, 125 MHz) 1a
a
2b
3a
4a
5a
6a
7a
8b
position
δC
δC
δC
δC
δC
δC
δC
δC
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 1′ 2′ CH3CO-17 CH3CO-17
35.2 33.7 104.4 172.6 46.6 19.0 35.5 46.7 51.8 43.0 20.5 25.1 42.1 35.1 81.0 80.1 64.7 111.8 170.5 14.1
36.7 35.7 106.2 174.3 47.9 22.6 41.1 42.2 53.4 44.3 21.5 34.7 79.2 45.6 53.0 81.0 66.5 113.2 172.1 15.0
33.5 33.6 104.2 172.4 46.6 25.1 20.7 46.6 51.6 46.4 18.6 25.1 42.9 35.4 80.5 78.3 67.1 112.1 170.7 14.1
28.6 29.5 80.5 177.0 41.4 21.9 34.8 49.2 75.0 46.6 26.7 29.6 45.9 38.1 47.0 80.5 65.1 110.7 173.0 16.0
38.8 107.7 148.8 159.7 42.2 20.4 33.3 41.3 50.6 44.8 18.1 25.0 44.8 35.2 80.8 80.0 64.6 109.7 169.8 15.0
49.6 186.7 146.6 141.9 38.0 21.9 35.0 48.6 43.5 48.8 26.3 28.7 80.6 38.1 46.9 75.0 65.1 110.5 148.6 17.1
161.0 125.8 198.8 40.3 45.0 25.8 19.6 40.9 47.2 41.0 40.5 32.8 78.9 44.3 51.1 77.4 64.0
162.2 126.7 189.1 146.6 52.6 22.8 40.5 42.4 48.1 44.5 21.1 34.2 80.7 45.2 52.3 79.1 66.2 124.5 168.5 19.3 61.4 14.7
16.8
170.5 20.5
Measured in DMSO-d6. bMeasured in pyridine-d5.
Figure 1. Key HMBC (a) and ROESY (b) correlations of compound 1.
347.1854 [M + H]+, which was less than that of 1 by two hydrogen atoms. The 1H and 13C NMR spectra (Tables 2 and 3) were generally similar to those of 1, except for the C-2 and C-3 signals. Through the analysis of the HSQC and HMBC spectra, a double bond between C-2 and C-3 was suggested from the correlations from H-2 (δH 5.77) to C-1 (δC 38.8), C-3 (δC 148.8), and C-4 (δC 159.7) and from H-18 (δH 6.04) to C3 (δC 148.8), C-4 (δC 159.7), and C-19 (δC 169.8). Thus, the structure of 5 (tricalysin E) was assigned as shown. Tricalysin F (6) was isolated as a colorless gum and designated with an elemental formula of C20H26O5 by HRESIMS at m/z 369.1669 from the [M + Na]+ ion. The 1 H NMR spectrum demonstrated a pair of olefinic protons [δH 7.93 (d, J = 1.5 Hz) and 6.66 (d, J = 1.5 Hz)]. The 13C NMR spectrum with the aid of the HSQC and HMBC spectra revealed that compound 6 is also a cafestol-type diterpenoid with a furan ring attached to the A ring and an additional conjugated carbonyl carbon (δC 186.7). In the HMBC
spectrum, the correlation from H-1 to C-2 (δC 186.7) revealed the conjugated carbonyl to be located at C-2. The relative configuration of 6 was determined from the ROESY spectrum, in which the correlations between H-5/H-9, H-9/H-15a, and H-15a/H2-17 demonstrated H-5, H-9, H-15a, and CH2-17 to be β-oriented, while the correlations between H-20/H-14a indicated that CH3-20, H-14, and OH-13 are α-oriented. Thus, compound 6 was elucidated as 13β-hydroxy-2-oxocafestol. Tricalysin G (7) was purified as a colorless gum, and its molecular formula was established as C18H26O4 from its 13C NMR spectroscopic data and the HRESIMS that showed a pseudomolecular [M + H]+ ion at m/z 329.1720. The 1H NMR spectrum of 7 exhibited resonances for the presence of a pair of cis-olefinic protons [δH 7.20 (d, J = 10.0 Hz) and 5.69 (d, J = 10.0 Hz)] and a characteristic singlet methyl group (δH 1.15, s). The 13C NMR spectrum, with the support of the HSQC experiment, gave 18 carbons including an α,β-unsaturatedketone carbon (δC 198.8, 161.0, and 126.8) and three C
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dubia and was named tricalysione A.5 The only difference between 7 and tricalysione A was found to be an additional hydroxy group located at C-13 in 7, which was deduced by HMBC correlations from H-14 (δH 1.64), H-15 (δH 1.40 and 1.55), and H-17 (δH 3.45 and 3.43) to C-13 (δC 78.9). The ROESY spectrum showed a correlation from OH-13 to H-17 and suggested the β-orientation of OH-13. Tricalysin H (8) was obtained as a colorless gum, and its molecular formula was defined as C22H30O6, based on its 13C NMR and HRESIMS data (m/z 413.1937 [M + H]+), indicating seven degrees of unsaturation. The 1H NMR data (Table 3) showed the presence of three olefinic proton signals [δH 7.14 (d, J = 10.0 Hz), 6.06 (d, J = 10.0 Hz), and 6.02 (d, J = 2.0 Hz)], one characteristic singlet methyl (δH 1.14, s), and an ethoxy group [δH 4.36 (q, J = 7.5 Hz) and 1.26 (t, J = 7.5 Hz)]. The 13C NMR data (Table 3), combined with the HSQC spectrum, revealed the presence of 22 carbons, including two carbonyl carbons (δC 189.1 and 168.5) and four olefinic carbons (δC 80.7, 79.1, 66.2, and 61.4). The aforementioned data suggested that compound 8 is a lactone seco-cafestol-type diterpenoid, as confirmed by the HMBC correlations (Figure 3a) from H-1 (δH 7.14) to C-3 (δC 189.1), C-9 (δC 48.1), and C-10 (δC 44.5), from H-2 (δH 6.06) to C-4 (δC 146.6) and C10, and from H-18 (δH 6.02) to C-3, C-4, and C-19 (δC 168.5). The linkage between the ethoxy group and the 19-carbonyl was confirmed by the HMBC correlation of H-1′ (δH 4.36) to C-19. The relative configurations of the B, C, and D rings in compound 8 were shown to be the same as compound 7 using the ROESY spectrum (Figure 3b). Additionally, the ROESY correlation between H-18 and H-6 suggested the geometry of the double bond of C-3 and C-18 is Z. Thus, the structure of 8 was elucidated as shown. Compound 8 is the first representative of a lactone seco cafestol-type diterpenoid. The ethyl group is considered to be generated from the extraction procedure used (Supporting Information). By comparing spectroscopic data (MS, 1H NMR, and 13C NMR) with literature values, five known compounds also isolated were identified as tricalysiolide A (9),6 tricalysiolide B (10),6 tricalysiolide E (11),6 tricalysiolide F (12),6 and tricalysiolide H (13),18 respectively. Lipopolysaccharide (LPS)-induced macrophages lead to the activation of several inflammatory signaling pathways and may be used for the evaluation of the anti-inflammatory effects. Nitric oxide (NO) is one of the most important inflammatory modulators produced by LPS-induced macrophages.19 Therefore, all isolated compounds were evaluated for their inhibitory effects on NO production of LPS-activated RAW 264.7 macrophages. Cell viability was determined initially by the MTT method to determine if the inhibition of NO production was due to the cytotoxicity of the compounds tested. As a result, no obvious cytotoxic effects (over 90% cell survival) of
Table 3. 1H NMR Spectroscopic Data for 5−8 (δ in ppm, 500 MHz) 5a position
δH (J in Hz)
1a 1b 2
1.98 m
6a δH (J in Hz)
δH (J in Hz)
δH (J in Hz)
7.20 d (10.0)
7.14 d (10.0)
6.69 d (10.0)
6.06 d (10.0)
4a 4b 5
2.00 m
3.36 m
6a 6b 7a 7b 9 11a 11b 12a 12b 13 14a 14b 15a 15b 17a
1.52 1.46 1.91 1.45 1.39 1.53
m m m m m m
1.58 1.40 2.46 1.66 1.52 3.27
m m m m m m
1.93 1.83 2.03 1.26 1.88 1.67 1.40 1.86 1.29
17b
3.45 d (11.0)
18 19 20 1′
6.04 s
2′ OH-13 OH-15 OH-16 OH-17 a
0.88 s
4.66 br s 4.33 s 4.29 s
8b
2.92 d (17.0) 2.34 d (17.0)
5.77 dt (6.5, 2.0)
3.52 d (11.0)
7a
m m m m m m m m m
1.96 m 1.65 m 2.19 d (17.0) 0.97 d (17.0) 3.51 dd (11.0, 3.5) 3.39 dd (11.0, 3.5) 6.66 d (1.5) 7.93 d (1.5) 0.96 s
2.35 dd (17.5, 14.0) 2.11 dd (17.5, 3.5) 1.79 m 1.45 1.31 1.90 1.79 1.11 1.44
m m m m d (7.0) m
1.65 m 1.88 m 1.64 s 1.40 m 1.55 m 3.45 dd (12.5, 4.5) 3.43 dd (12.5, 4.5)
2.43 dt (10.0, 2.0) 1.43 m 1.60 1.46 1.21 1.94 1.84 2.19 1.95
m m d (7.5) m m m m
2.24 1.95 1.96 1.81 4.20
d d d d d
(11.0) (11.0) (13.5) (13.5) (11.0)
4.15 d (11.0) 6.02 d (2.0)
1.15 s
4.25 s
4.20 s
3.90 s 4.34 brs
3.88 s 4.26 brs
1.14 s 4.36 dq (13.5, 7.5) 1.26 t (7.5)
Measured in DMSO-d6. bMeasured in pyridine-d5.
oxygenated carbons (δ C 78.9, 77.4 and 64.0). These spectroscopic data suggested compound 7 is a bisnorditerpenoid.5 The C-4 of compound 7 was shown to be a methylene carbon but not an olefinic carbon, on analysis of the HSQC and HMBC spectra (Figure 2a), consistent with 7 being a bisnorkauranoid due to the degradation of C-18 and C-19. This type of bisnor-diterpenoid has been isolated previously only from T.
Figure 2. Key HMBC (a) and ROESY (b) correlations of compound 7. D
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Figure 3. Key HMBC (a) and ROESY (b) correlations of compound 8.
all isolates at concentrations up to 100 μM on RAW 264.7 cells were observed. For all isolates (1−13), only 7 and 8 inhibited NO release, with IC50 values of 27.1 ± 1.2 and 6.6 ± 0.4 μM, respectively, while the other compounds were considered to be inactive (IC50 > 100 μM). The compound N-monomethyl-Larginine was used as the positive control (IC50 = 40.5 μM). The results indicated that the presence of the α,β-unsaturatedketone moiety between C-1 and C-3 of ring A plays an important role in mediating NO inhibitory activity. Since compound 8 inhibited LPS-induced NO release with the most potent inhibitory activity, it was selected for further investigation. iNOS are the key enzymes for the production of NO,20 so the expression levels of iNOS protein were examined in LPS-stimulated RAW 264.7 cells by Western blots. Compound 8 strongly suppressed the expression of iNOS protein in a concentration-dependent manner (Figure 4). Also,
Figure 5. Effects of 8 on the production of TNF-α (A) and IL-6 (B) in LPS-stimulated RAW 264.7 cells. Data are expressed as means ± SD (p < 0.05 indicates statistically significant differences from the LPS group; *p < 0.05 or **p < 0.01 versus the LPS group, ##p < 0.01 versus the control group).
The activation of NF-κB is a key step for the expression of the production of pro-inflammatory mediators (e.g., TNF-α and IL-6) and iNOS in LPS-stimulated macrophages.22−24 NFκB occurs as an inactive p50/p65 heterodimer associated with inhibitory κB (IκB) proteins in the cytoplasma of the resting macrophages.25 The phosphorylation of IκB and its subsequent degradation is known to be a critical step in NF-κB activation by LPS.26 It was deduced from Figure 6A that 8 has an inhibitory effect on the activation of NF-κBp65 in LPS-induced macrophages. To determine whether the NF-κBp65 activation is related to IκBα phosphorylation, the effects of 8 on LPSinduced p-IκBα were evaluated by Western blots, which showed that the phosphorylation of IκBα in RAW264.7 cells increased after LPS administration but was significantly inhibited by 8 in a dose-dependent manner (Figure 6B and C). Besides the activation of NF-κB, the phosphorylation of MAPKs also regulates the expression of inflammatory enzymes and the production of pro-inflammatory cytokines.27 To determine whether the inhibitory response of 8 on the inflammatory procedure was mediated through the MAPKs signaling pathway, the LPS-induced phosphorylation of ERK, JNK, and p38 was analyzed by Western blots in RAW264.7 macrophage cells. As shown in Figure 7, LPS stimulation
Figure 4. Effect of 8 on the expression of iNOS protein in LPSstimulated RAW 264.7 cells. iNOS protein expression was tested by Western blot analysis (**p < 0.01 versus LPS group, ##p < 0.01 versus the control group).
pro-inflammatory cytokines (e.g., IL-6 and TNF-α) play an important role in modulating the inflammatory reponse.21 The effects of 8 on the expression of IL-6 and TNF-α in LPSstimulated RAW 264.7 macrophage cells were further evaluated. As shown in Figure 5, in the response of LPS, the expression of IL-6 and TNF-α were upregulated when compared to the control group. Then, after treatment with compound 8, the expression of IL-6 and TNF-α was attenuated in a dose-dependent manner. The underlying molecular mechanism for these effects in LPS-stimulated macrophage cells was then probed further in the following manner. E
DOI: 10.1021/acs.jnatprod.5b00165 J. Nat. Prod. XXXX, XXX, XXX−XXX
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Figure 6. Effects of 8 on the activation of NF-κB protein (A) and phosphorylation of IκB (B and C) in LPS-stimulated RAW 264.7 cells by Western blot (*p < 0.05 or **p < 0.01 versus LPS group, ##p < 0.01 versus the control group). chromatography. Semipreparative HPLC was carried out using a Shimadzu SCL-10A series instrument with a Shimpak RP-C18 column and an SPD-6A variable-wavelength detector. All solvents were of analytical grade. Plant Material. The twigs of T. f ruticosa were collected at Xishuangbanna, Yunnan Province, People’s Republic of China, in November 2012 and identified by Prof. Mian Zhang of the Department of Medicinal Plants, China Pharmaceutical University. A voucher specimen was deposited at the Department of Natural Medicinal Chemistry, China Pharmaceutical University. Extraction and Isolation. The twigs of T. f ruticosa (15.0 kg) were cut into small pieces and extracted with 95% ethanol (3 × 4 h) to yield a dried extract (600.0 g), which was suspended in water (1.0 L) and partitioned successively by petroleum ether (1.0 L × 4) and EtOAc (1.0 L × 4). The EtOAc-soluble fraction was subjected to silica gel chromatography eluted by petroleum ether/EtOAc mixtures (10:1−5:1−1:1) to give five fractions (E1−E5). Fraction E4 (40.0 g) was subjected to passage over a silica gel column eluted by CH2Cl2/MeOH gradient mixtures (100:1−50:1−25:1−10:1), to give nine subfractions (E41− E49). Fr.E44 was subjected to MCI gel column chromatography (MeOH/H2O, 30:70−50:50−70:30−90:10−100:0) to give seven additional subfractions (Fr.E44a−E44g). Fr.E44d was purified by Sephadex LH-20 and further purified by semipreparative HPLC using MeOH/H2O (60:40) to give 8 (10.0 mg, 19.1 min) and 10 (8.2 mg, 22.3 min). Fr.E44c was subjected to separation over Sephadex LH-20 to give nine subfractions (Fr.E44ca−E44ci). E443ci was purified by semipreparative HPLC using CH3CN/H2O (30:70) to afford 2 (3.3 mg, 34.0 min) and 1 (5.3 mg, 38.0 min). E443ch was purified by semipreparative HPLC with CH3CN/H2O (30:70) to obtain 9 (4.3 mg, 24.2 min), 13 (6.3 mg, 28.0 min), and 12 (6.3 mg, 31.0 min). E443 cd was purified by semipreparative HPLC using CH3CN/H2O
significantly increased the phosphorylation of ERK, JNK, and p38, while 8 decreased the levels of phosphor-ERK, phosphorJNK, and phosphor-p38 in LPS-induced macrophage cells. The levels of nonphosphorylated MAPK isoforms did not vary significantly among groups. In conclusion, the current study describes the isolation and elucidation of eight new cafestol-type diterpenoids (1−8), of which compound 8 was proven to be the most potent inhibitor of NO release in LPS-stimulated RAW264.7 macrophage cells. Further biological evaluation in vitro demonstrated that 8 reduces the production of TNF-α and IL-6 and suppresses the expression of inflammatory enzymes of iNOS by the activation of NF-κB and phosphorylation of MAPKs (ERK, JNK, and p38) in LPS-stimulated macrophages. These results show that 8 exhibits therapeutic potential for ameliorating inflammation.
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EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were measured with a JASCO P-1020 polarimeter. UV spectra were recorded on a Shimadzu UV-2450 spectrophotometer. ECD spectra were obtained on a JASCO 810 spectropolarimeter (JASCO, Tokyo, Japan). IR spectra were recorded on KBr disks on a Bruker Tensor 27 IR spectrometer. NMR spectra were recorded on a Bruker Avance III500 instrument (1H: 500 MHz, 13C: 125 MHz), with TMS as internal standard. Mass spectra were acquired using an MS Agilent 1100 series LC/MSD ion-trap mass spectrometer (ESIMS) and an Agilent UPLCQ-TOF (6520B) spectrometer (HRESIMS), respectively. Silica gel (100−200 and 200−300 mesh, Qingdao Haiyang, Qingdao, People’s Republic of China), Sephadex LH-20 (Pharmacia, Uppsala, Sweden), and RP-C18 (40−63 μm, Fuji, Tokyo, Japan) were used for column F
DOI: 10.1021/acs.jnatprod.5b00165 J. Nat. Prod. XXXX, XXX, XXX−XXX
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Figure 7. Effects of 8 on LPS-induced phosphorylation of ERK, JNK, and p38 in RAW 264.7 cells by Western blot. The phosphorylation of ERK (A), JNK (B), and p38 (C) MAPKs stimulated by LPS was dramatically suppressed by 8. Data are expressed as means ± SD (*p < 0.05 or **p < 0.01 versus LPS, ##p < 0.01 versus the control group). 3; ESIMS m/z 347.3 [M + H]+ and 381.2 [M + Cl]−; HRESIMS m/z 347.1854 [M + H]+ (calcd for C20H27O5 347.1853). Tricalysin F (6): colorless gum; [α]25D −57.7 (c 0.19, MeOH); UV (MeOH) λmax (log ε) 249 (4.25) nm; IR (KBr) νmax 3426, 2930, 1658, 1433, 1049 cm−1; 1H and 13C NMR data, see Tables 2 and 3; ESIMS m/z 347.1 [M + H]+ and 381.2 [M + Cl]−; HRESIMS m/z 369.1669 [M + Na]+ (calcd for C20H26O5Na 369.1672). Tricalysin G (7): colorless gum; [α]25D −55.6 (c 0.23, MeOH); UV (MeOH) λmax (log ε) 217 (4.16), 250 (3.23) nm; IR (KBr) νmax 3432, 2928, 1735, 1441, 1298 cm−1; 1H and 13C NMR data, see Tables 2 and 3; ESIMS m/z 307.1 [M + H]+; HRESIMS m/z 329.1720 [M + Na]+ (calcd for C18H26O4Na 329.1723). Tricalysin H (8): colorless gum; [α]25D −34.2 (c 0.21, MeOH); UV (MeOH) λmax (log ε) 230 (4.21) nm; IR (KBr) νmax 3412, 2927, 1745, 1451, 1198 cm−1; 1H and 13C NMR data, see Tables 2 and 3; ESIMS m/z 391.3 [M + H]+ and 424.3 [M + Cl]−; HRESIMS m/z 413.1937 [M + Na]+ (calcd for C22H30O6Na 413.1935). Cell Culture. The RAW 264.7 mouse macrophage cell line was purchased from the Cell Bank of Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, People’s Republic of China), and was cultured in Dulbecco’s modified Eagle medium (DMEM, Gibco Invitrogen Corp., Carlsbad, CA, USA). The cells were supplemented with 3.0 mM glutamine, antibiotics (100 U/ mL penicillin and 100 U/mL streptomycin), and 10% heat-inactivated fetal bovine serum at 37 °C under a humidified atmosphere of 5% CO2. In all experiments, macrophages were incubated in the presence or absence of various concentration of 8 (2.5, 5.0, 10.0 μM), which was solubilized with DMSO and added 1 h before LPS (1.0 μg/mL) stimulation. Measurement of Nitrite Concentration. Raw 264.7 macrophage cells were cultured onto 96-well plates at a density of 2 × 104 cells/mL and then incubated with or without LPS (1.0 g/mL) in the absence or presence of various concentrations of 8 (2.5, 5.0, 10.0 μM) for up to
(25:75) to give 11 (7.1 mg, 44.1 min). Fr.E45 were subjected to purification over an MCI gel column (MeOH/H2O, 30:70−50:50− 70:30−90:10−100:0) to give seven subfractions (Fr.E45a−Fr.E45g). E45c was purified further using Sephadex LH-20 and semipreparative HPLC with CH3CN/H2O (20:80) to yield 7 (2.1 mg, 20.2 min) and 4 (2.5 mg, 25.5 min). E45d was subjected to separation over LH-20 and then semipreparative HPLC with CH3CN/H2O (25:75) to afford 5 (2.2 mg, 15.4 min) and 6 (2.5 mg, 17.6 min). Finally, E45f was subjected to Sephadex LH-20 separation and semipreparative HPLC using MeOH/H2O (55:45) to obtain 3 (110 mg, 15.0 min). Tricalysin A (1): colorless gum; [α]25D −36.4 (c 0.19, MeOH); UV (MeOH) λmax (log ε) 217 (4.23) nm; IR (KBr) νmax 3378, 2932, 1776, 1640, 1044, 1025 cm−1; 1H and 13C NMR data, see Tables 1 and 2; ESIMS m/z 365.2 [M + H]+ and 399.2 [M + Cl]−; HRESIMS m/z 365.1960 [M + H]+ (calcd for C20H29O6, 365.1959). Tricalysin B (2): colorless gum; [α]25D −25.3 (c 0.19, MeOH); UV (MeOH) λmax (log ε) 218 (4.56) nm; IR (KBr) νmax 3444, 2928, 1738, 1385, 1222 cm−1; 1H and 13C NMR data, see Tables 1 and 2; ESIMS m/z 365.2 [M + H]+ and 399.2 [M + Cl]−; HRESIMS m/z 382.2227 [M + NH4]+ (calcd for C20H32NO6, 382.2224). Tricalysin C (3): colorless gum; [α]25D −25.3 (c 0.19, MeOH); UV (MeOH) λmax (log ε) 217 (4.35) nm; IR (KBr) νmax 3410, 2926, 1736, 1346, 1233 cm−1; 1H and 13C NMR data, see Tables 1 and 2; ESIMS m/z 406.1 [M + H]+; HRESIMS m/z 407.2063 [M + H]+ (calcd for C22H31O7 407.2064). Tricalysin D (4): colorless gum; [α]25D −25.3 (c 0.19, MeOH); UV (MeOH) λmax (log ε) 217 (4.44) nm; IR (KBr) νmax 3398, 2933, 1738, 1335, 1241 cm−1; 1H and 13C NMR data, see Tables 1 and 2; ESIMS m/z 435.2 [M + H]+; HRESIMS m/z 349.2013 [M + H]+ (calcd for C20H29O5 349.2010). Tricalysin E (5): colorless gum; [α]25D −67.3 (c 0.19, MeOH); UV (MeOH) λmax (log ε) 278 (3.81) nm; IR (KBr) νmax 3434, 2923, 1736, 1604, 1462, 1380, 1258 cm−1; 1H and 13C NMR data, see Tables 2 and G
DOI: 10.1021/acs.jnatprod.5b00165 J. Nat. Prod. XXXX, XXX, XXX−XXX
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24 h. Dexamethasone (1.0 μM) was used as a positive control. The nitrite accumulation in the supernatant was assessed by the Griess reaction. Each 50 μL of culture supernatant was mixed with an equal volume of Griess reagent (0.1% naphthylethylene diamine, 1.0% sulfanilamide in 2.5% phophoric acid solution) and incubated at room temperature for 10 min. The absorbance at 550 nm was measured in an automated microplate reader (TECAN, Grödig, Austria). The concentrations of NO were determined by a sodium nitrite standard curve. Western Blotting Analysis. RAW 264.7 macrophage cells were plated onto six-well plates (1 × 106 cells/well) and treated with 1 μg/ mL of LPS in the presence or absence of compound 8 (2.5, 5.0, or 10.0 μM). Dexamethasone (1.0 μM) was used as a positive control. Cells were homogenized in ice-cold RIPA buffer containing 0.1% phenylmethylsulfonyl fluoride. The dissolved proteins were collected from the supernatant after centrifugation at 12000g for 20 min. Protein extracts were separated by an SDS-polyacrylamide gel electrophoresis and then transferred onto a PVDF membrane. The membrane was blocked with 5% skim milk in Tris buffer saline and then incubated at 4 °C overnight with the respective primary antibodies. After being washed with tris-buffered saline-Tween 20 (TBST), the membranes were incubated with a horseradish peroxidase conjugated secondary antibody (1:12 000) for 1.5 h at room temperature. The antibodyreactive bands were visualized using enhanced chemiluminescence detection reagents and a gel imaging system (Tanon Science & Technology Co., Ltd., Shanghai, People’s Republic of China). Pro-inflammatory Cytokine (TNF-α and IL-6) Assay. Compound 8 solubilized with DMSO was diluted with DMEM before treatment. RAW 264.7 cells were plated onto 24-well plates (4 × 105 cells/well) and were incubated in the presence of either 1.0 mg/mL LPS alone or LPS plus various concentrations of 8 (2.5, 5.0, or 10.0 μM) for 18 h. Cell-free supernatants in the pro-inflammatory cytokine assays were subsequently employed using a mouse ELISA kit (R&D Systems, Minneapolis, MN, USA), following the manufacturer’s protocol (BioLegend). Statistical Analysis. All values were presented as means ± SD. The Student’s t-test was employed to analyze the differences between sets of data, with a p value of
