Bioactive Diterpenes, Norditerpenes, and Sesquiterpenes from a Formosan Soft Coral Cespitularia sp.

Chemical investigation of the soft coral Cespitularia sp. led to the discovery of twelve new verticillane-type diterpenes and norditerpenes: cespitulins H–O (1–8), one cyclic diterpenoidal amide cespitulactam L (9), norditerpenes cespitulin P (10), cespitulins Q and R (11 and 12), four new sesquiterpenes: cespilins A–C (13–15) and cespitulolide (16), along with twelve known metabolites. The structures of these metabolites were established by extensive spectroscopic analyses, including 2D NMR experiments. Anti-inflammatory effects of the isolated compounds were studied by evaluating the suppression of pro-inflammatory protein tumor necrosis factor-α (TNF-α) and nitric oxide (NO) overproduction, and the inhibition of the gene expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), in lipopolysaccharide-induced dendritic cells. A number of these metabolites were found to exhibit promising anti-inflammatory activities.


Introduction
In the inflammatory stimuli, the inflammatory mediators such as tumor necrosis factor-α (TNF-α), prostaglandin E2 (PGE2), and nitric oxide (NO) are known to be secreted through lipopolysaccharide (LPS)-induced activation of macrophages [1][2][3] and dendritic cells [4][5][6][7]. Furthermore, the overexpression of two inducible proteins, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) produced the excess amount of NO and PGE2 in the inflammatory process. It has been well known that natural products have a great potential in drug discovery, thus the anti-inflammatory activity screening by evaluating the suppression of TNF-α and NO overproduction, and the inhibition of iNOS and COX-2 protein and gene expression, in LPS-induced macrophages or dendritic cells (DCs) is one of the important methods for searching for anti-inflammatory agents from natural compounds [8][9][10][11][12][13][14].
Additionally, in order to discover bioactive substances for future medicinal application, the anti-inflammatory activities of the inhibition of TNF-α and NO, and the suppression of iNOS and COX-2 gene expression in LPS-induced DCs of the isolated compounds 1- 28 were also evaluated and are reported herein.
Cespitulin H (1) was isolated as a white amorphous powder and its molecular formula was established as C 23 H 30 O 6 by HRESIMS (m/z 425.1932 [M + Na] + ), accounting for nine degrees of unsaturation. The IR spectrum of 1 exhibited the absorption peaks of hydroxy (3480 cm -1 ) and carbonyl (1741 cm -1 ) groups. Assignment of two germinal methyls (δ C 26.8 and 24.9,both CH 3 ; δ H 1. 35 and 0.72, both s), a methyl (δ C 19.2,CH 3 ; δ H 2.11, s), a vinyl group (δ C 132.6, CH 2 and 127. 6,CH;δ H 6.15,br d,J = 17.5 Hz,5.16,dd,J = 10.5,1.0 Hz and 5.77,dd,J = 17.5,10.5 Hz), a 1,1-disubstituted double bond (δ C 144.1, C and 116.5, CH 2 ; δ H 5.09 and 4.77, both s), a trisubstituted double bond (δ C 148. 5,C and 129.4,CH;δ H 6.13,s), an acetal (δ C 101. 8,CH;δ H 5.96,s), three other sp 3 oxygenated carbons (δ C 94. 6, 80.0, and 72.8, C), a conjugated ester carbonyl (δ C 164.9, C), and a conjugated ketone (δ C 197.7, C) of verticillane-type diterpene were supported by analysis of the 13 C and 1 H NMR signals along with heteronuclear single quantum coherence (HSQC) spectrum (Tables 1 and 2). Table 1. 13 C NMR spectroscopic data of compounds 1-6.   4 -7 /12 -15 29.8, 29.7, 29.5, 29.3    The planar structure of 1 was further determined by analysis of correlations spectroscopy (COSY) and heteronuclear multiple bond correlation (HMBC) correlations ( Figure 3). The HMBC correlations of H 2 -9 (δ H 3.00 and 2.21, both d, J = 16.0 Hz) to C-10 (δ C 94.6, C) and C-11 (δ C 72.8, C), assigned a possible 10,11-tetrasubstituted epoxide moiety. Additionally, HMBC correlations of a hydroxy proton (δ H 2.25, br s) to both C-12 (δ C 80.0, C) and C-13 (δ C 26.8, CH 2 ), as well as an acetal proton H-20 (δ H 5.96, s) to both C-12 and ester carbonyl carbon (δ C 164.9, C, C-21), positioned a hydroxy group at C-12 and an acrylate group at C-20. The above findings and the remaining one degree of unsaturation were used to establish a polyoxygenated epoxytetrahydrofuran ring, as shown in formula of 1. The relative stereochemistry of 1 was determined by the analysis of nuclear Overhauser effect spectroscopy (NOESY) correlations and molecular modeling from energyminimized (MM2) force field calculation. Assuming the β-orientation of H-1 (δ H 1. 19 46, m) and H 3 -17; thus the above methylene protons were characterized as H-14β and H-13β, while the rest protons were assigned as H-14α (δ H 1.11, ddd, J = 14.0, 6.0, 3.5 Hz) and H-13α (δ H 1.57, td, J = 14.0, 3.5 Hz). Subsequently, H-20 (δ H 5.96, s) exhibited NOE interactions with both H-13α and 12-OH (δ H 2.25, br s), revealing that H-20 and the 12-hydroxy group were positioned on the α face. Moreover, H-7 (δ H 6.13, br s) exhibited an NOE response with one proton of H-9 (δ H 2.21, d, J = 16.0 Hz), while H 3 -19 (δ H 2.11, s) showed an NOE interaction with the other proton of H-9 (δ H 3.00, d, J = 16.0 Hz) but not with H-7, confirming the E geometry of trisubstituted double bond at C-7/C-8. The above NOE results were shown to be well matched with a molecular model of minimized energy generated from MM2 calculation in Figure 4. Additionally, conformational searching of compound 1 by molecular mechanics model with MMFF force field calculation in the Spartan'14 program [35] was further performed. In a relative energy window of 0−3 Kcal/mol, the results of the calculation displayed nine lowest energy conformers for 1 (Table S2 and Figure S113) which were shown to fit from the observed NOE correlations. From the above findings, the relative configuration of 1 was elucidated as that for formula 1. Cespitulin I (2) appeared as a white amorphous powder with the molecular formula C 23 H 32 O 6 as indicated by the HRESIMS (m/z 427.2089 [M + Na] + ) spectrum, suggesting the presence of eight degrees of unsaturation. The IR spectrum showed the absorptions of hydroxy (3440 cm -1 ) and carbonyl (1740 cm -1 ) groups. The NMR data of 2 (Tables 1 and 2) revealed this compound to be a tricyclic verticillane-type diterpene and should be very similar to cespihypotin H [28] except for the position of a tetrasubstituted epoxide and the hydroxy group in the tetrahydrosubstituted furan ring. Similar to 1, this tetrasubstituted epoxide was located between C-10 (δ C 94.7, C) and C-11 (δ C 72.4, C), and the hydroxy group was found at C-12 (δ C 79.8, C) on the basis of the assistance of HMBC correlations. The position of the acrylate group at C-20 was also confirmed by the HMBC correlations from H-20 (δ H 5.70, s) and H-22 (δ H 6.14, dd, J = 17.2, 10.4 Hz) to C-21 (δ C 164.8, C). These observations, together with analysis of other COSY and HMBC correlations, enabled the gross structure of 2 to be established reasonably ( Figure 3).
Cespitulin J (3) was isolated as a colorless oil. The HRESIMS (m/z 611.4282 [M + Na] + ) and NMR data (Tables 1 and 2) of 3 exhibited a molecular formula of C 36 H 60 O 6 , acquiring seven degrees of unsaturation. The IR spectrum suggested the presence of hydroxy (3446 cm -1 ) and ester carbonyl (1758 cm -1 ) groups. Comparison of the NMR spectroscopic data of 3 and 2 indicated that the structure of 3 was highly similar to that of 2, with the exception of an acrylate ester group in 2 being replaced by a long-chain ester moiety in 3. Furthermore, it is reasonable to elucidate the hexadecanoyl ester group at C-20 (δ C 100.8, CH) by HRESIMS and 2D NMR spectroscopic data, including HMBC and COSY correlations. Thus, the structural framework of 3 was established to be a verticillane-type diterpene, including a polyoxygenated epoxytetrahydrofuran ring, too ( Figure 3). The analysis of the NOESY spectrum revealed that 3 possessed the same relative configurations at C-1, C-6, C-10, C-11, and C-12 as those of compound 2. A difference in the stereochemistry of H-20 between 2 and 3 was demonstrated with the assistance of the NOESY experiment which revealed that H-20 (δ H 5.63, s) had an NOESY correlation with 12-OH (δ H 2.52, br s), indicating that H-20 of 3 should be α-oriented and accomplished the elucidation of the relative configuration of 3.
Cespitulin K (4) was obtained as a colorless oi1 that gave a sodiated adduct ion peak at m/z 637.4440 [M + Na] + in the HRESIMS spectrum, suggesting the molecular formula C 38 H 62 O 6 with eight degrees of unsaturation. IR absorptions at 3420 and 1748 cm -1 showed the presence of hydroxy and ester carbonyl functionalities, too. The 13 C and 1 H NMR spectroscopic data (Tables 1 and 2) of 4 were found to be very similar to those of 3, with the exception that the hexadecanoyl ester at C-20 in 3 was converted to the octadecenoyl ester group in 4 by the HRESIMS data and 2D NMR (HMBC and COSY) correlations ( Figure 3) of 3. The remaining one degree of unsaturation has arisen from the cis C-9 /C-10 double bond of the octadecenoyl ester group in 4 by comparison of 13 C NMR spectroscopic data of this ester side chain at C-20 with those reported previously [36,37]. Finally, the Z geometry of the 9 , 10 -double bond was also deduced from a 10.5 Hz coupling constant between H-9 and H-10 in the 1 H NMR spectrum.
The relative configuration of 4 was also determined by a NOESY experiment. The Cespitulin L (5) was isolated as a white amorphous powder. Its molecular formula, C 21 H 32 O 5 , was established by HRESIMS (m/z 365.2315 [M + H] + ), implying six degrees of unsaturation. The IR spectrum showed the presence of the hydroxy moiety (3445 cm -1 ). The 13 C and 1 H NMR spectroscopic data revealed that 5 was found to possess a 10,20-ether linkage tetrahydrosufuran ring (δ C 104.6, CH, C-20 and 94.3, C, C-10; δ H 4.36, s, H-20) and a 10,11-tetrasubstituted epoxide (δ C 72.8, C, C-11), as well as the same verticillane core skeleton of compounds 2-4 (Tables 1 and 2). The presence of a methoxy group at C-20 of 5 was further established by an HMBC correlation from H 3 -21 (δ H 3.47, s) to C-20 ( Figure 3). These results suggested that the relative configuration of 5 was nearly the same as those of 2-4. Further, the 20-acetal proton (δ H 4.36, s) was found to show an NOE interaction with H-13β (δ H 1.70, br d, J = 14.0 Hz), while the 12-OH (δ H 3.29, s) exhibited interactions with both H-13α (δ H 1.58, m) and H 3 -21, indicating the β-orientation of H-20 and the α-orientation of the 21-methoxy group ( Figure 4).
Cespitulin M (6) was found to possess the same molecular formula, C 21 H 32 O 5 , as that of 5 from the HRESIMS data (387.2142 [M + Na] + ). Analysis of the 1D NMR spectroscopic data (Tables 1 and 3) and the 2D NMR (HSQC, COSY, and HMBC) correlations enabled the planar structure of 6 to be established the same as 5 ( Figure 3). The 13 C NMR spectroscopic data of 6 were nearly similar to those of 5, with the exception of downfield shifts observed at C-12 (∆δ C +1.6) and C-20 (∆δ C +4.5) relative to 5, revealing that 6 should be the C-12 or C-20 isomer of 5. Further analysis of NOE correlations revealed that 6 possessed the identical relative configurations at C-1, C-6, C-10, C-11, and C-12 as those of 5. A difference in relative configuration for C-20 of the tetrahydrofuran ring between 5 and 6 was characterized by a comparison of their key NOE correlations ( Figure 4). Cespitulin N (7) had the molecular formula C 22 H 32 O 5 as determined by HRESIMS (m/z 399.2142 [M + Na] + ). The IR spectrum of 7 showed the presence of hydroxy (3446 cm -1 ) and ester carbonyl (1733 cm -1 ) groups. All the proton and carbon signals of 7 were assigned from the 13 C and 1 H NMR spectroscopic data (Tables 3 and 4), along with HSQC spectrum, which established the structure of 7 as a tetracyclic verticillane-type diterpene with an acetoxy group (δ C 170. 2,C and 21.3,CH 3 ; δ H 2.02, s). In addition, the NMR data of 7 were found to resemble those of cespitulin G [27]. Detailed analysis of 2D NMR spectra (COSY, HMBC, and NOESY), revealed that 7 possesses a 10,20-ether linkage trihydrosubstituted furan ring (δ C 95.9, C, C-10 and 75.5, CH 2 , C-20; δ H 3.59 and 3.43, both d, J = 9.0 Hz), a 10,11-epoxide (δ C 74.0, C, C-11), and a hydroxy group at C-12 (δ C 78.7, C; δ H 1.96, br d, J = 2.0 Hz, 12-OH). Furthermore, key NOE correlations of the 12-OH to H-13α (δ H 1.76, m) and H-20α (δ H 3.43, d, J = 9.0 Hz) indicated that the 12-hydroxy group should be positioned on the α face.  (8) at m/z 393.2267 in HRES-IMS indicated a molecular formula C 22 H 32 O 6 . The IR absorptions showed the presence of the hydroxy (3419 cm -1 ) and an ester carbonyl (1733 cm -1 ) groups. The 13 C and 1 H spectroscopic data (Tables 3 and 4) of 8 were very similar to those of cespihypotin I [28] and had the same molecular formula. However, the gross structure of 8 was established as a 10,11-epoxy-10,11,12,20-tetrahydrosubstituted-furanyl diterpene containing an acetoxy group at C-20 by the results of 2D NMR experiments (including COSY and HMBC correlations, Figure 5). Further analysis of the NOE correlations revealed that H-20 (δ H 5.76, s) showed NOE interaction with H-13β (δ H 1.81, m), while 12-OH (δ H 2.68, br s) with H-13α (δ H 1.62, m), confirming the β-orientation of the acetoxy group at C-20 ( Figure 6).
The assignment of the relative stereochemistry of the nonprotonated carbons C-10 and C-11 of the epoxy ring in compounds 1-8 was also based on the observed NOE correlations and molecular model calculation. For example, the relative configurations of C-10 and C-11 of compound 5 as shown in Figure 7 were assigned on the basis that the distance between H 3 -16 and one proton of H 2 -9 is 2.22 Å and that between H 3 -17 and this proton of H 2 -9 is 4.01 Å in the molecular model generated from MM2 calculation, which well match the NOE correlation observed between H 3 -16 and this proton of H 2 -9, and not fit the correlations between H 3 -17 and the same proton at C-9. Moreover, for the conformer of the isomeric 10,11-epoxide (Figure 8), the NOE correlations for H 3 -17/H-20, and between both H 3 -16 and H 3 -17 with this H-9 should be found as the distances of H 3 -17/H-20, H 3 -16, and H 3 -17 with this H-9 proton were calculated to be 3.11, 2.13, and 3.16 Å, respectively. However, only the NOE correlation between H 3 -16 and this specific H-9 was detected, suggesting the relative configuration of 5 and the other related compounds should be the same as those described in Figure 1.    The HRESIMS data (m/z 368.2195 [M + Na] + ) of cespitulactam L (9) established the molecular formula C 21 H 31 O 3 N, consistent with seven degrees of unsaturation. The IR spectrum suggested the presence of hydroxy and/or amide (3245 cm -1 ) and conjugated carbonyl (1698 cm -1 ) groups. Compound 9 and cespitulactam F [22] were found to have the same α,β-unsaturated lactam ring by comparison of their 1D and 2D NMR spectroscopic data. Likewise, the 1 H and 13 C NMR data of 9 (Tables 3 and 4) were highly similar with those of cespitulactam F, with the difference that the presence of a methoxy group (δ C 50.4, CH 3 ; δ H 3.13, s) at C-10 (δ C 93.9, C) in 9 was found, instead of a hydroxy group in cespitulactam F. Cespitulactam L (9) is the 10-methoxy derivative of cespitulactam F. The relative stereochemistry of 9 was deduced from the analysis of the observed NOE correlations. The known β-oriented H 3 -17 (δ H 1.24, s) exhibited NOE interactions with the methoxy protons (δ H 3.13, s), indicating the β-orientation of 10-methoxy group. By the biogenetic consideration and other detailed NOE correlations (Figure 9), cespitulactam L (9) was found to possess the same relative configuration as that of cespitulactam F. The presence of the hydroxy, ester carbonyl, and ketone groups was observed by IR ab-sorptions at 3445, 1732, and 1715 cm -1 , respectively. The 13 C and 1 H NMR data (Table 5) of 10 revealed that three degrees of unsaturation were contributed from a 1,1-disubstituted double bond (δ C 147.1, C and 113.2, CH 2 ; δ H 4.87, s), a trisubstituted double bond (δ C 131. 7,CH and 122.2,C;δ H 5.32,d,J = 8.5 Hz), and an ester carbonyl (δ C 171.4, C). The remaining two degrees of unsaturation were arisen from a 2-hydroxy-6,6-dimethylcyclohexan-1-one moiety by inspection of 2D NMR correlations ( Figure 5). The NMR spectroscopic data of 10 resemble those of known norditerpenoid cespitularin Q (17) [18,26], except for the presence of a methoxy group (δ C 51.8, CH 3 ; δ H 3.69, s) at C-15 (δ C 171.4, C) and a hydroxy group at C-3 (δ C 71.4, CH) in 10, and also the absence of a 14-membered lactone ring linkage between C-10 (δ C 169.7, C) and C-12 (δ C 72.2, CH) which is present in 17, indicating that a linear terpenoidal ester 10 might be arisen from cespitularin Q by hydrolysis and further esterification. It was also found that the molecular skeleton of 10 is nearly the same as that of retinoids with missing of the methyl group at C-5, while normal retinoids are originated from the oxidative cleavage of β-carotene [38]. In the NOESY spectrum, a strong interaction between H-6β (δ H 1.79, m) and H 3 -17 (δ H 1.32, s) showed the β-orientation of H [3][4][5][6][7][8][9][10][11][12][13][14][15][16][17]m) showed NOE correlations with both H 3 -17 and one proton of H-4 (δ H 2.31, m), as did the 3-OH (δ H 3.67, br d, J = 3.5 Hz) with the other proton of H-4 (δ H 1.58, m), reflecting that H-3 should be β-oriented while the hydroxy group at C-3 was assigned as α-oriented ( Figure 9). By the analysis of the above NOE correlations and the biosynthetic relation of 10 and 17, the relative configuration of 10 was elucidated and named cespitulin P. The new norditerpene cespitulin Q (11) was obtained as a colorless oil, which showed the pseudomolecular ion peak [M + H] + at m/z 305.2108 in HRESIMS, appropriate for the molecular formula of C 19 H 28 O 3 and six degrees of unsaturation. The IR absorptions at 3418 and 1699 cm -1 indicated the presence of the hydroxy and carbonyl groups, respectively. The carbon NMR signals (Table 4) at δ C 202.6 (C), 150.3 (C), and 134.6 (CH), as well as the proton NMR signal (Table 3) at δ H 6.10 (d, J = 3.5 Hz), were characteristic resonances for an α,β-unsaturated ketone unit in 11.
The analyses of COSY and HMBC correlations were used to establish the planar structure of 11 ( Figure 5). Moreover, the NMR spectroscopic data of 11 were found to close to those of known metabolite cespitularin E (18) [17], with the exception of the carbon signal of C-13 resonating at δ C 23.9 (CH 2 ) in 18 was downfield shifted to δ C 65.9 (CH) in 11, suggesting that 11 is the C-13 oxidation derivative of cespitularin E (18). From the NOE correlations ( Figure 10) of 11, one of the methylene protons at C-3 (δ H 2.59, dd, J = 15.0, 11.0 Hz) displayed an NOE correlation with the β-oriented H 3 -16 (δ H 1.09, s), which correlated with the known β-oriented H-1 (δ H 1.81, m), and thus was characterized as H-3β, while the other (δ H 1.86, m) was assigned as H-3α. The NOE correlation between H-13 (δ H 4.50, m) and H-3α determined the β-orientation of the hydroxy group at C-13. From the all NOE correlations observed, the relative configuration of 11 was thus established. The molecular formula of cespitulin R (12) was found to be C 19 H 26 O 3 , as deduced by HRESIMS (m/z 325.1777 [M + Na] + ). IR absorptions at 3420 and 1748 cm -1 of the corresponding hydroxy and carbonyl moieties were also confirmed. Comparison of the 1 H and 13 C NMR spectroscopic data (Tables 3 and 4) of 11 and 12 suggested that both compounds are the same bicyclic verticillane-type norditerpenes, except that a hydroxy group at C-13 (δ C 65.9, CH; δ H 4.50, m) in 11 was replaced by a ketone (δ C 199.0, C) in 12. The planar structure of 12 was further determined from analysis of the HMBC and COSY correlations, as shown in Figure 5. From the above results and on the basis of the analysis of NOE correlations (Figure 10), the relative configuration of cespitulin R (12) was established.
Cespilin C (15) has the molecular formula C 15 H 22 O 3 as shown by HRESIMS spectrum (m/z 273.1464 [M + Na] + ). The IR spectrum of 15 showed the absorption of an α,βunsaturated ketone (1683 cm -1 ) which was further characterized from the corresponding 13 C NMR signals (Table 6) of δ C 197.7 (C), 162.7 (C), and 128.6 (CH). The NMR signals at δ C 84.0 (C) and δ H 7.42 (1H, br s) revealed the presence of a hydroperoxy group at the sp 3 nonprotonated carbon. Analysis of the COSY spectrum of 15 identified one proton sequence from H-4 to H 2 -8 via H-10, which assembled the major part of the planar structure of 15 with the crucial HMBC correlations as shown in Figure 5.
However, the connection of C-8/C-9 and C-9/C-10 could not be observed by COSY and HMBC correlations; instead, the two single bonds were established to fulfill the cadinane skeleton of 15 by the molecular formula and the tetrahedron nature of sp 3 carbons. The relative structure of 15 was elucidated by the analysis of NOE correlations ( Figure 11). Assuming the β-orientation of H-4 (δ H 3.26, d, J = 13.5 Hz), NOE correlations of H-4 with H-10 (δ H 2.90, br d, J = 13.5 Hz) and H-10 with H-6 (δ H 1.61, m) implied the β-orientation of both H-6 and H-10, while the α-orientation of the isopropenyl group at C-4 and the methyl group (δ H 0.90, d, J = 6.5 Hz) at C-6. Subsequently, one proton of H 2 -7 (δ H 1.54, m) displayed NOE correlations with both the hydroperoxy proton (δ H 7.42, s) and H-6, while the other proton of H 2 -7 (δ H 1.22, m) correlated with H 3 -15, reflecting that the 9-hydroperoxy group should be situated on the β-face. Finally, the relative stereochemistry of 15 was thus established as 4S*, 6R*, 9R*, and 10S*.
Compound 16 was isolated as a white amorphous powder. The HRESIMS of 16 exhibited a sodiated pseudomolecular ion peak at m/z 287.1255 [M + Na] + and revealed a molecular formula of C 15 H 20 O 4 , implying six degrees of unsaturation. The IR absorptions displayed the presence of the hydroxy (3418 cm -1 ) and carbonyl (1732 cm -1 ) groups. A comparison of the NMR data (Tables 6 and 7) of 16 to those of a known metabolite atractylenolide III (25) [30][31][32][33], could well describe the molecular framework of 16 as eudesmane-type sesquiterpenoid. A difference was found that the methylene (H 2 -3) of 25 was substituted with a hydroxy group in 16 (Figures 1 and 5).

Anti-Inflammatory Activities of the EtOAc Extract and the Isolated Compounds 1-28
The anti-inflammatory activities of the EtOAc extract were screened in terms of the suppression of TNF-α production and NO release, as well as the inhibition of upregulation of pro-inflammatory iNOS and COX-2 gene, in LPS-induced DCs. The results of a preliminary study at a concentration of 100 µg/mL showed that the relative activities of this extract in inhibiting the production of TNF-α and NO were 84.1 ± 4.2 and 76.1 ± 1.4%, respectively, and it could reduce the levels of iNOS and COX-2 gene to 15.9 ± 0.4, and 28.6 ± 4.1%, respectively, too. For the discovery of bioactive compounds with anti-inflammatory abilities by inhibition of TNF-α and NO overproduction, 1-28 isolated from this extract were further assayed (Table 8). At a concentration of 100 µM, 1-3 could potently inhibit 95.0 ± 0.2, 95.7 ± 0.4, and 95.8 ± 0.1% TNF-α production, respectively, relative to the control cells treated with LPS only. The respective IC 50 values of 1-3, 47.2, 48.6, and 41.1 µM, were further measured. Compounds 2, 12, 19, and 21 showed significant activities to inhibit NO releasing at 63.3 ± 1.6, 61.1 ± 0.5, 63.7 ± 0.8, and 61.7 ± 1.0%, respectively, at the same concentration. The IC 50 values of 49.7, 51.9, and 57.4 µM, respectively, of 2, 19, and 21 in inhibiting the NO production were also measured. On the other hand, the anti-inflammatory potentials of compounds 1-28 in inhibition toward the accumulation of pro-inflammatory iNOS and COX-2 gene expression in the same LPS-induced DCs model were also evaluated (Figures 12 and 13, and Table 9). At a concentration of 25 µM, 2 was found to effectively reduce the gene expression of iNOS and COX-2 to 0.3 ± 0.1 and 2.9 ± 0.6%, respectively, relative to the control cells stimulated with LPS only. Meanwhile, 1, 13-15, 20, and 28 were found to conspicuously reduce the gene expression of iNOS to 3.6 ± 1.8, 7.4 ± 2.9, 1.5 ± 0.8, 4.6 ± 2.9, 0.2 ± 0.1, and 1.2 ± 0.5%, respectively, while 1, 13, 18, and 22 could strongly reduce the COX-2 gene level to 4.2 ± 0.1, 4.4 ± 3.5, 4.5 ± 0.5, and 2.1 ± 0.4%, respectively, at a concentration of 100 µM. On the contrary, 6 significantly enhanced the gene expression of iNOS to 281.2 ± 15.4%, and 16 exhibited obvious activity of enhancing 196.9 ± 55.1% COX-2 gene expression, at the same concentration of 100 µM. Table 8. Inhibitory effects of compounds 1-28 on TNF-α expression and NO production in LPSinduced dendritic cells.

Discussion
The pro-inflammatory cytokine TNF-α, and reactive nitrogen species (RNS), such as NO, are shown to involve in the physiological regulation of immune responses [39][40][41]. The inducible enzymes iNOS and COX-2 are also critical regulators of inflammation [40,42]. iNOS and COX-2 are also known to express together in inflamed responses and overproduction of NO can enhance the expression of COX-2 protein [43]. Generally, the inappropriate production of TNF-α and NO, as well as high iNOS and COX-2 protein and gene expression were found to be related to the pathogenesis of many inflammatory related diseases [44][45][46][47][48][49][50][51] such as AIDS, Alzheimer's, arthritis, cancer, diabetes, stroke, multiple sclerosis, obesity, and Parkinson's disease. Therefore, substances with inhibitory ability toward the overproduction of these inflammatory mediators are candidates for the development of new pharmaceutics in the treatment of chronic inflammation and autoimmune diseases [52,53].
The anti-inflammatory potential of all isolated compounds revealed that compounds 1-3 and 12-15 might represent promising anti-inflammatory agents, in particular, 1 and 2 not only could significantly inhibit the production of TNF-α and NO but also displayed potent suppression to the expression of iNOS and COX-2 gene. Compound 13 might also be regarded as a promising inducible enzyme inhibitor as it can potently inhibit the expression of both iNOS and COX-2 genes.
From the structure−activity relationship (SAR), the tetracyclic verticillane-type diterpenes 1 and 2 were showing significant activities for each biological study relative to 3-8, owing to the presence of an acrylate group at C-20. Furthermore, 2 exhibited stronger anti-inflammatory abilities at lower concentrations (25 µM) than 1 at higher concentrations (100 µM). Thus, the acrylate group at C-20 and the hydroxy group at C-6 in epoxyfuranyl verticillane-type metabolites could effectively enhance the anti-inflammatory activity. The bicyclic verticillane-type norditerpene 12 displayed more effective anti-inflammatory activities than 11, suggesting that the presence of the α,β-conjugated ketone at C-13 as in 12 could strengthen activities from the allylic hydroxy group at C-13 as in 11. On the other hand, the cadinane-type squiterpenes 13-15 exhibited significant inhibition toward iNOS gene expression, however, the presence of a hydroperoxy group and/or conjugated enone group as shown in 15 might promote the COX-2 gene expression.

General Experimental Procedures
Values of specific optical rotation were determined on a JASCO P-1020 digital polarimeter. UV spectra were recorded on a JASCO V-650 spectrophotometer. IR spectra were measured on a JASCO FT-IR-4100 and Nicolet iS5 FT-IR infrared spectrophotometers. ESIMS and HRESIMS data were obtained with a Bruker APEX II mass spectrometer. NMR spectra were recorded on a JEOL ECZ600R FT-NMR (or a Varian Unity INOVA 500 FT-NMR, or a Varian MR 400 FT-NMR) instrument at 600 MHz (or 500 MHz, or 400 MHz) for 1 H and 150 MHz (or 125 MHz, or 100 MHz) for 13 C, respectively. All NMR experiments were measured using CDCl 3 or benzene-d 6 as the solvent. Silica gel (Merck, and  were used for column chromatography. High-performance liquid chromatography (HPLC) was performed on a HiTachi L-7100 HPLC system apparatus with a Supelco C18 (250 mm × 21.2 mm, 5 µm) or Hibar 250-10 C18 (250 mm × 21.2 mm, 5 µm) column.

Animal Material
The soft coral Cespitularia sp. was collected by hand using SCUBA at Green Island, which is located off the southeastern coast of Taiwan, in June 2007, at a depth of 10-15 m, and was stored in a freezer until extraction. A voucher specimen was deposited in the Department of Marine Biotechnology, National Sun Yat-sen University, Kaohsiung, Taiwan.

Measurement of Nitric Oxide (NO) Production by DCs
DC cells were seeded in 24-well plates at a density of 1 × 10 6 /mL. DCs were treated with each compound for 1 h and then stimulated with 100 ng/mL LPS for 24 h. The nitrite concentration in the medium was measured as an indicator of NO production through the Griess reaction. Briefly, 100 µL of cell culture supernatant was reacted with 100 µL of Griess reagent (1:1 mixture of 2% sulfanilamide and 0.2% N-(1-naphthyl-)ethylenediamine dihydrochloride in water) in 96-well plate at room temperature for 10 min, and absorbance at 540 nm was recorded using sandwich ELISA assays [6,7].

Measurement of Pro-Inflammatory Inducible NO Synthase (iNOS) and Cyclooxygenase-2 (COX-2) Gene Expression by DCs
The suppression activities of compounds were measured by the examining suppression of LPS-induced upregulation of pro-inflammatory iNOS and COX-2 gene expression in DCs using real-time polymerase chain reaction (PCR) [14]. Briefly, DCs (1 × 10 6 /mL) were incubated in 6-well plates and treated with each compound for 1 h, and then were added the LPS (100 ng/mL), stimulating for 24 h. Subsequently, cells were harvested and isolated total RNA using Trizol reagent. A total of 2 µg RNA was reverse-transcribed using M-MLV Reverse Transcriptase to synthesize cDNA (Applied Biosystems). Gene expression levels of iNOS and COX-2 were analyzed using SYBR-Green PCR Master Mix with StepOne PCR System (Applied Biosystems; Thermo Fisher Scientific). Relative gene expression levels were calculated using the 2 −∆∆Ct method and normalized to GAPDH; all the primers which were used are listed in Table 10 [54]. Table 10. Primers of quantitative RT-PCR.

Statistical Analysis
The results are expressed as the mean ± SEM, and comparisons were made using one-way ANOVA by Tukey's post hoc test (Graphpad Prism 5.0, GraphPad Software, San Diego, CA, USA). A probability value of 0.05 or less was considered significant. The software Sigma Plot was used for the statistical analysis.

Conclusions
In conclusion, our chemical investigation demonstrated that the soft coral Cespitularia sp. could be a good source of bioactive substances. Eight new tricyclic verticillane-type diterpenes 1-9, one novel norditerpene 10, two new dicyclic verticillane-type norditerpenes 11 and 12, three cadinane-type sesquiterpenes 13-15, and one eudesmane-type sesquiterpenoid 16, along with twelve known metabolites 17-28, were isolated from this investigation. The structural framework of verticillane-type derivatives was found to be close to the tricyclic taxane skeleton [55] and obtained from marine organisms only in the soft coral genus Cespitularia [15]. Furthermore, the cadinane-type sesquiterpenes 13-15 were isolated from the soft coral genus Cespitularia for the first time. From the results of the evaluated biological activities, it appears that compounds 1, 2, and 13 might be promising compounds for further marine anti-inflammatory drug development.