2014 Volume 62 Issue 2 Pages 203-208
Nine cembranoid diterpenes 1–9, including four new compounds, crassumols D–G (1–4), were isolated from the methanol extract of the Vietnamese soft coral Lobophytum crassum. Spectroscopic methods were used to elucidate the structures of these compounds. Compound 5 exhibited a potent inhibitory effect on tumor necrosis factor-alpha (TNFα)-induced nuclear factor-kappa B (NF-κB) transcriptional activation in HepG2 cells and significantly inhibited the mRNA expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) in a dose-dependent manner.
Soft corals, which occur widely in the coral reefs throughout the world, are marine invertebrates of the order Alcyonacea, subclass Octocorallia, class Anthozoa, and phylum Cnidaria. These marine invertebrates are a rich source of secondary metabolites, especially diterpenoids and hydroxylated sterols. Cnidarian-algal symbiotic associations are common in marine environments and are of great ecological importance, and many of the secondary metabolites of cembranoid diterpenes from the soft corals may be involved in ecological interactions.1) Soft corals of the genera Cespitularia, Clavularia, Gersemia, Lobophytum, Nephthea, Sarcophyton, and Sinluaria are the most prolific.2) Among Lobophytum soft corals, the species L. crassum is abundantly found in Vietnam. Published investigations indicated that cembranoid diterpenes and sterols are the main constituents of this species. The cembranoid diterpenes and sterols of L. crassum exhibit various biological activities, including cytotoxicity,3–8) anti-infammatory,8–10) immunomodulation,11) inhibition of 5-reductase,12) nitric oxide (NO) production,13) and the release of elastase by human neutrophils.14)
Previously, we reported four sterols from L. crassum and evaluation of their anti-inflammatory activity.15) In this study, which constitutes a continuation of our investigations of this soft coral, we report the isolation and structure elucidation of nine cembranoid diterpenes including four new compounds, crassumols D–G (1–4). The anti-inflammatory effects of the isolated compounds were also evaluated.
A methanol extract (75.0 g) of L. crassum was suspended in water and successively extracted with n-hexane and CH2Cl2. After combined column chromatography, the n-hexane residue (36.2 g) afforded nine cembranoid diterpenes 1–9 (Fig. 1). The known compounds were elucidated as (1R,4R,2E,7E,11E)-cembra-2,7,11-trien-4-ol (5),16) 7S,8S-epoxy-1,3,11-cembratriene-16-oic methyl ester (6),17) 2,16:7S,8S-diepoxy-1,3,11,15-cembratetraene (7),18) (2S,7S,8S)-sarcophytoxide (8),18) and (2S,7S,8S)-sarcophine (9)19) by detailed analyses of their spectroscopic data (one dimennsional (1D)-, 2D-NMR and MS) and comparison with previously reported values.
Crassumol D (1) was isolated as a colorless oil. The molecular formula C20H30O4 was determined using Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) which demonstrated six degrees of unsaturation. The NMR features indicated a diterpene, a main constituent of soft corals. The presence of hydroxyl group was suggested by a broad absorption band at 3441 cm−1 in the IR spectrum. The 1H-NMR spectrum of 1 displayed signals corresponding to an oxymethine [δH 4.11 (1H, t, J=8.0 Hz, H-7)], an oxymethylene [δH 4.64 (1H, dd, J=12.0, 1.0 Hz, Ha-16) and 4.47 (1H, dd, J=12.0, 3.5 Hz, Hb-16)], and four tertiary methyl [δH 1.64 (3H, d, J=1.0 Hz, H-17), 1.37 (3H, s, H-18), 1.05 (3H, s, H-19), and 1.23 (3H, s, H-20)] groups. The 13C-NMR spectrum revealed 20 carbon signals, of which the presence of one oxymethine, one oxymethylene, and four methyl carbons was demonstrated at δC 85.2 (CH, C-7), 78.8 (CH2, C-16), 9.4 (CH3, C-17), 28.9 (CH3, C-18), 26.5 (CH3, C-19), and 25.6 (CH3, C-20), respectively. Typical carbon signals for a dioxygenated quaternary carbon [δC 115.8 (C-2)], three oxygenated quaternary carbons [δC 86.9 (C-4), 74.2 (C-8), and 79.9 (C-12)], and one fully substituted double bond [δC 132.5 (C-1)/123.9 (C-15)] were also observed. Thus, with five remaining degrees of unsaturation (six total minus one double bond), 1 was determined to be a pentacyclic diterpene. The 1H–1H correlation spectroscopy (COSY) led to assignment of the following connectivities: H-5/H-6/H-7, H-9/H-10/H-11/H-3, and H-13/H-14. The COSY evidence and the cross-peaks of H-3 (δH 2.06) with C-1 (δC 132.5), C-2 (δC 115.8), C-4 (δC 86.9), C-5 (δC 34.6), and C-11 (δC 54.1) in heteronuclear multiple-bond correlation (HMBC) spectrum confirmed the cyclization between C-3 and C-11. The HMBC correlations of H-19 (δH 1.05) with C-7 (δC 85.2), C-8 (δC 74.2), and C-9 (δC 40.6) indicated the location of the hydroxyl group at C-8. Detailed analysis of other HMBC correlations (Fig. 2) clearly elucidated the planar structure of 1, a novel 3,11-cyclocembranoid diterpene. The relative configuration of 1 was assigned using nuclear Overhauser effect spectroscopy (NOESY). The spatial proximities of H-18 (δH 1.37) with H-3 (δH 2.06)/Hb-5 (δH 1.92) and H-7 (δH 4.11) with Hb-5 (δH 1.92)/H-19 (δH 1.05) suggested the β-orientation of H-3, Hb-5, H-7, Me-18, and Me-19. A large coupling constant between H-3 and H-11 (J=11.0 Hz) indicated a trans relationship of these two protons. Moreover, H-11 (δH 1.74) exhibited a NOE correlation with H-20 (δH 1.23) suggesting that H-11 and Me-20 are both in the α-orientation (Fig. 3).
The molecular formula of crassumol E (2) was also identified as C20H30O4 by FT-ICR-MS. The 1H- and 13C-NMR data (in CDCl3) were characteristic of a cembranoid diterpene containing two oxygenated quaternary carbons [δC 80.8 (C-1) and 83.2 (C-8)], three oxymethine groups [δH 4.57 (1H, d, J=8.0 Hz, H-2), 3.40 (1H, br d, J=6.0 Hz, H-7), and 4.21 (1H, br d, J=8.0 Hz, H-11)/δC 84.9 (C-2), 77.1 (C-7), and 77.7 (C-11)], and one oxymethylene group [δH 4.47 and 4.50, each 1H, dt, J=13.5, 2.5 Hz, H-16/δC 69.8 (C-16)]. The presence of hydroxyl groups was also supported by a broad absorption band at 3410 cm−1 in the IR spectrum. Carbon signals at δC 118.3 (CH, C-3)/144.3 (C, C-4), 146.6 (C, C-12)/111.2 (CH2, C-20), and 154.0 (C, C-15)/105.4 (CH2, C-17) indicated the presence of one trisubstituted double bond and two 1,1-disubstituted double bonds. Moreover, two tertiary methyl groups were identified at δH 1.80 (3H, br s, H-18) and 1.18 (3H, s, H-19)/δC 19.5 (C-18) and 20.6 (C-19). The 1H- and 13C-NMR data of 2 were similar to those of sinumaximol D,20) apart from significantly different data for C-7, C-8, and C-11. The carbon signals at C-8 and C-11 of 2 were strongly shifted downfield relative to those of sinumaximol D suggesting for the presence of an epoxy group at C-8/C-11, which was further confirmed by the HMBC cross-peak between H-11 (δH 4.21) and C-8 (δC 83.2). Detailed analysis of other HMBC correlations clearly elucidated the planar structure of 2 (Fig. 2). The relative configurations of 2 at C-1 and C-2 were found to be identical with those of sinumaximol D20) by agreement of the 1H- and 13C-NMR data for these two compounds and by the spatial proximities of H-2 (δH 4.41) and H-18 (δH 1.67)/1-OH (δH 4.95) in NOESY of 2 in DMSO-d6. Proton Ha-5 (δH 2.14) exhibited NOESY correlations with H-3 (δH 5.61) and H-19 (δH 1.04), suggesting that H-3, Ha-5, and Me-19 are all in the α-orientation. Moreover, H-7 (δH 3.14) exhibited a strong correlation with H-11 (δH 4.11) but no correlations with H-19 (δH 1.04), thus confirming the β-orientation for both H-7 and H-11 (Fig. 3).
The molecular formula of crassumol F (3) was identical to that of 1 and 2 as determined by FT-ICR-MS. The 1H- and 13C-NMR data of 3 were similar to those of 2 except for big difference in the data for C-7, C-8, and C-11. The carbon signal at C-7 of 3 was strongly shifted downfield relative to that of 2, suggesting for the presence of an epoxy group at C-7/C-11. The presence of a C-7/C-11 epoxy was further confirmed by the HMBC cross-peak between H-11 (δH 3.72) and C-7 (δC 87.7). The planar structure of 3 was unambiguously elucidated using other HMBC correlations (Fig. 2). The 13C-NMR data for C-1 and C-2 of 3 were essentially identical to those of 2 suggesting for the same configurations at C-2 and C-3 in these two compounds. In the NOESY experiment, correlation of H-2 (δH 4.75) with H-18 (δH 1.78) as well as Hb-5 (δH 2.40) with H-18 (δH 1.78) and H-19 (δH 1.17) suggested that H-2, Hb-5, Me-18, and Me-19 are all in the β-orientation. Moreover, spatial proximities between H-7 (δH 3.08) and H-3 (δH 5.05)/H-11 (δH 3.72) were observed, thereby supporting α-orientation of H-3, H-7, and H-11.
Crassumol G (4) was also isolated as a colorless oil and exhibited spectroscopic features typical for a cembranoid diterpene. Its molecular formula, C20H28O4, was determined by FT-ICR-MS. The 1H- and 13C-NMR data of 4 were similar to those of 2. The differences between them are the appearance of a fully substituted double bond [δC 162.1 (C-1) and 123.3 (C-15)], a carbonyl carbon [δC 174.9 (C-16)], and a tertiary methyl group [δC 8.8 (C-17)/δH 1.87 (H-17)] in 4 instead of a 1,1-disubstituted double bond, a quaternary oxygenated carbon, and an oxymethylene group in 2. The absorption bands at 3420 and 1755 cm−1 in the IR spectrum suggested the presence of hydroxyl and carbonyl groups, respectively. Consideration of the structure of the structure of 2 suggested the placement of the fully substituted double bond C-1/C-15, carbonyl carbon C-16, and tertiary methyl C-17 in 4, which was further confirmed by HMBC correlation of H-2 (δH 5.48) with C-1 (δC 162.1) and H-17 (δH 1.87) with C-1 (δC 162.1)/C-15 (δC 123.3)/C-16 (δC 174.9). NOESY spatial proximities were observed between H-18 (δH 1.84) and H-2 (δH 5.48)/Hb-6 (δH 1.48) and between H-19 (δH 1.19) and Hb-6 (δH 1.48)/H-11 (δH 4.55), suggesting that H-2, Hb-6, H-11, H-18, and H-19 are all in the β-orientation. Moreover, H-3 (δH 4.97) exhibited a NOESY correlation with H-7 (δH 3.36), which suggested an α-orientation for these two protons.
The anti-inflammatory activity of the isolated compounds was evaluated by the inhibitory effect on tumor necrosis factor-alpha (TNFα)-induced nuclear factor-kappa B (NF-κB) transcriptional activation in HepG2 cells.21) Potent inhibition of NF-κB activation with IC50 value of 1.65±0.2 µM was observed for compound 5. Compound 2 (IC50=9.23±1.66 µM) exhibited significant inhibitory effect, whereas the other compounds were inactive. NF-κB is highly activated at sites of inflammation in various diseases and can induce expression of matrix metalloproteinases (MMPs), cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS).22) Thus, the influence of compounds 2 and 5 on the expression of COX-2 and iNOS in TNFα-stimulated Hep-G2 cells was evaluated using reverse transcription polymerase chain reaction (RT-PCR) analysis. In accordance with the inhibitory effect on NF-κB activation, compound 5 significantly inhibited the mRNA expression of COX-2 and iNOS in a dose-dependent manner (Fig. 4.), indicating that this compound reduced transcription of these genes. Moreover, the housekeeping protein β-actin was unaltered by the presence of 2 and 5 at all tested concentrations.
Sulfasalazine 1 µM (Suf) was used as the positive control.
The specimens of Lobophytum crassum were collected in Conco, Quang Tri, Vietnam, during May 2013 and deep frozen until used. The sample was identified by Professor Do Cong Thung (Institute of Marine Environment and Resources). A voucher of specimen (No. LC0513) was deposited at the Institute of Marine Biochemistry and the Institute of Marine Resources and Environment, VAST, Vietnam.
Extraction and IsolationFreeze-dried bodies of the soft coral L. crassum (1.0 kg) were well chopped and extracted three times with hot MeOH (at 50°C for 5 h each time). The obtained solutions were filtered, combined, and concentrated under reduced pressure to yield a dark brown viscous residue (75.0 g). This residue was suspended in water (1 L) and partitioned with n-hexane and CH2Cl2 (3×3 L). The combined n-hexane soluble portions were evaporated under reduced pressure to afford an n-hexane fraction (36.2 g). This fraction was crudely separated on silica gel column chromatography (CC) with gradient concentrations of ethyl acetate in n-hexane from 0 to 100% as eluent to yield six fractions, H-1 to H-6. Fraction H-2 (1.5 g) was further separated on silica gel CC using n-hexane–EtOAc (25 : 1) as eluent, to give three sub-fractions, H-2.1 to H-2.3. Sub-fraction H-2.1 (0.3 g) was then separated on silica gel CC with eluent of n-hexane–acetone (30 : 1) and further purified by YMC RP-18 CC eluting with MeOH–acetone–H2O (9 : 1 : 1) to afford 7 (6.8 mg). Compound 8 (8.2 mg) was purified from sub-fraction H-2.3 (0.8 g) using silica gel CC with n-hexane–EtOAc (20 : 1) as eluent. Fraction H-4 (2.5 g) was separated on YMC RP-18 CC with eluent of MeOH–acetone–H2O (95 : 2 : 3) to yield four subfractions, H-4.1 to H-4.4. Subfraction H-4.1 (0.8 g) was further separated on YMC RP-18 CC, eluting with MeOH–H2O (8 : 1) to yield 9 (9.8 mg). Sub-fraction H-4.2 (0.5 g) was separated into four smaller fractions, H-4.2a to H-4.2d, using YMC RP-18 CC with acetone–water (85 : 15) as eluent. Subfraction H-4.2a (0.17 g) afforded compounds 5 (9.8 mg) and 6 (10.4 mg), after subjecting it to silica gel CC with eluent of dichloromethane–acetone (35 : 1). Then, sub-fraction H-4.2b (0.16 g) was separated on CC over silica gel eluting with CH2Cl2–acetone (28 : 1), followed by YMC RP-18 CC with MeOH–H2O (5.5 : 1), to give 4 (5.5 mg). Sub-fraction H-4.2c (0.1 g) was chromatographed on silica gel CC with eluent of CH2Cl2–EtOAc (35 : 1) and further separated by YMC RP-18 CC with acetone–H2O (5.5 : 1) to obtain compounds 1 (5.6 mg), 2 (4.8 mg), and 3 (6.3 mg).
Crassumol D (1): Colorless oil; [α]D25 +5.8° (c=0.10, CHCl3); FT-ICR-MS m/z 335.22219 [M+H]+ (Calcd for C20H31O4, 335.22224); UV λmax (MeOH) nm (log ε): 222 (3.68); IR (KBr) νmax 3441, 1660, and 1055 cm−1; 1H- and 13C-NMR data, see Tables 1 and 2.
| Position | 1 | 2 | 2a) | 3 | 4 |
|---|---|---|---|---|---|
| 1 | 132.5 | 80.8 | 79.6 | 81.0 | 162.1 |
| 2 | 115.8 | 84.9 | 83.8 | 84.8 | 80.6 |
| 3 | 61.8 | 118.3 | 118.6 | 119.0 | 120.8 |
| 4 | 86.9 | 144.3 | 139.2 | 141.4 | 143.4 |
| 5 | 34.6 | 34.5 | 34.0 | 40.8 | 36.7 |
| 6 | 30.2 | 30.5 | 29.3 | 24.9 | 29.9 |
| 7 | 85.2 | 77.1 | 75.0 | 87.7 | 75.2 |
| 8 | 74.2 | 83.2 | 83.1 | 69.7 | 85.3 |
| 9 | 40.6 | 36.4 | 35.8 | 40.2 | 36.0 |
| 10 | 23.5 | 29.1 | 28.6 | 27.0 | 30.3 |
| 11 | 54.1 | 77.7 | 76.5 | 75.8 | 84.4 |
| 12 | 79.9 | 146.6 | 147.3 | 148.9 | 147.9 |
| 13 | 30.2 | 30.5 | 30.1 | 29.3 | 26.8 |
| 14 | 19.3 | 36.6 | 36.8 | 32.3 | 26.5 |
| 15 | 123.9 | 154.0 | 154.7 | 153.1 | 123.3 |
| 16 | 78.8 | 69.8 | 69.2 | 68.9 | 174.9 |
| 17 | 9.4 | 105.4 | 104.3 | 105.9 | 8.8 |
| 18 | 28.9 | 19.5 | 19.1 | 15.6 | 17.1 |
| 19 | 26.5 | 20.6 | 20.7 | 20.3 | 20.0 |
| 20 | 25.6 | 111.2 | 110.1 | 110.3 | 112.7 |
a) Recorded in DMSO-d6; all assignments were done by HSQC, HMBC, 1H–1H COSY, and NOESY experiments.
| Position | 1 | 2 | 2a) | 3 | 4 |
|---|---|---|---|---|---|
| 1 | — | — | — | — | — |
| 2 | — | 4.57, d (8.0) | 4.41, d (9.0) | 4.75, d (11.5) | 5.48, d (9.5) |
| 3 | 2.06, d (11.0) | 5.72, dd (8.0, 1.0) | 5.61, d (9.0) | 5.05, d (11.5) | 4.97, dd (9.5, 1.5) |
| 4 | — | — | — | — | — |
| 5 | 2.36, m | 2.35, m | 2.14, m | 2.40, m | 2.27, m |
| 1.92, m | 2.20, m | 2.06, dt (2.5, 13.5) | 2.22, m | ||
| 6 | 2.22, m | 2.40, m | 2.07, m | 1.82, m | 1.89, m |
| 1.90, m | 1.47, m | 1.37, m | 1.59, m | 1.48, m | |
| 7 | 4.11, t (8.0) | 3.40, br d (6.0) | 3.14, t (7.0) | 3.08, d (10.0) | 3.36, dd (8.0, 6.0) |
| 8 | — | — | — | — | — |
| 9 | 1.80, m | 2.05, m | 2.08, m | 1.91, m | 2.26, m |
| 1.72, m | 1.80, m | 1.57, m | 1.63, m | 1.72, m | |
| 10 | 1.71, m | 1.88, m | 1.79, m | 1.87, m | 1.97, m |
| 1.45, m | 1.82, m | 1.63, m | 1.61, m | 1.89, m | |
| 11 | 1.74, m | 4.21, br d (8.0) | 4.11, d (9.0) | 3.72, br d (8.5) | 4.55, dd (6.5, 9.0) |
| 12 | — | — | — | — | — |
| 13 | 1.77, m | 2.70, m | 2.48, m | 2.33, m | 2.31, m |
| 1.47, m | 1.95, m | 1.81, m | 2.22, m | 2.12, m | |
| 14 | 2.43, dd (15.5, 8.0) | 2.10, m | 1.94, m | 1.71, m | 2.73, dt (13.5, 4.5) |
| 2.27, m | 1.55, m | 1.29, m | 1.67, m | 2.20, m | |
| 15 | — | — | — | — | — |
| 16 | 4.64, dd (12.0, 1.0) | 4.50, dt (13.5, 2.5) | 4.32, m | 4.66, dt (13.5, 2.0) | — |
| 4.47, dd (12.0, 3.5) | 4.47, dt (13.5, 2.5) | 4.44, br d (13.5) | |||
| 17 | 1.64, d (1.0) | 5.21, t (2.5) | 5.08, br s | 5.19, t (2.0) | 1.87, s |
| 5.11, t (2.5) | 5.02, br s | 5.07, t (2.0) | |||
| 18 | 1.37, s | 1.80, br s | 1.67, s | 1.78, s | 1.84, d (1.5) |
| 19 | 1.05, s | 1.18, s | 1.04, s | 1.17, s | 1.19, s |
| 20 | 1.23, s | 4.90, br s | 4.77, s | 4.94, s | 5.10, s |
| 4.89, br s | 4.77, s | 4.93, s | |||
| 1-OH | 4.95, s | ||||
| 7-OH | 4.76, d (7.0) |
a) Recorded in DMSO-d6; all assignments were done by HSQC, HMBC, 1H–1H COSY, and NOESY experiments.
Crassumol E (2): Colorless oil; [α]D25 −4.6° (c=0.10, CHCl3); FT-ICR-MS m/z 335.22241 [M+H]+ (Calcd for C20H31O4, 335.22224); UV λmax (MeOH) nm (log ε): 220 (3.76); IR (KBr) νmax 3410, 1665, and 1062 cm−1; 1H- and 13C-NMR data, see Tables 1 and 2.
Crassumol F (3): Colorless oil; [α]D25 +10.4° (c=0.10, CHCl3); FT-ICR-MS m/z 335.22242 [M+H]+ (Calcd for C20H31O4, 335.22224); IR (KBr) νmax 3395, 1660, and 1065 cm−1; 1H- and 13C-NMR data, see Tables 1 and 2.
Crassumol G (4): Colorless oil; [α]D25 +7.9° (c=0.10, CHCl3); FT-ICR-MS m/z 333.20611 [M+H]+ (Calcd for C20H29O4, 333.20659); UV λmax (MeOH) nm (log ε): 201 (3.43); IR (KBr) νmax 3420, 1755, 1660, and 1060 cm−1; 1H- and 13C-NMR data, see Tables 1 and 2.
This study was supported by Vietnam National Foundation for Science & Technology Development (Project No: 104.01-2012.37), the framework of international cooperation program managed by National Research Foundation of Korea (2012K2A1A2032970), and Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology (20120006681), Republic of Korea. The authors are grateful to Institute of Chemistry, VAST and KBSI for the provision of the spectroscopic instrument.
