New Anti-Inflammatory 9,11-Secosterols with a Rare Tricyclo[5,2,1,1]decane Ring from a Formosan Gorgonian Pinnigorgia sp.

Pinnigorgiols D (1) and E (2), two new 9,11-secosterols with a rearranged carbon skeleton, were isolated from a Taiwan gorgonian Pinnigorgia sp. The structures of these two compounds were elucidated on the basis of spectroscopic methods and were proven to possess a tricyclo[5,2,1,1]decane ring. The new secosterols 1 and 2 displayed significant inhibitory effects on the generation of superoxide anions and the release of elastase by human neutrophils.


Introduction
Gorgonian corals belonging to the genus Pinnigorgia (Family Gorgoniidae), have proven to be rich sources of bioactive sterols [1][2][3][4]. Previous bioassay results of these sterol analogues have demonstrated cytotoxic and anti-inflammatory activities. Following the above investigations, with the aim of discovering bioactive substances for new drug development in the future, we continue here to carry out an investigation on a Taiwan gorgonian Pinnigorgia sp., and two novel 9,11-secosterols, pinnigorgiols D (1) and E (2), with a rare carbon skeleton arrangement, were discovered ( Figure 1). The ability of these two compounds to inhibit the generation of superoxide anions and the release of elastase in N-formyl methionyl leucylphenylalanine/cytochalasin B (fMLP/CB)-induced neutrophils were also evaluated.

Results and Discussion
Pinnigorgiol D (1) was obtained as an oil and had the molecular formula C 30 H 46 O 6 as determined by high-resolution electrospray ionization mass spectrum (HRESIMS) at m/z 525.31883 (calcd. for C 30 H 46 O 6 + Na, 525.31866), requiring eight degrees of unsaturation. The IR absorptions of 1 showed the presence of hydroxy (ν max 3446 cm −1 ), ester (ν max 1739 cm −1 ), and ketonic carbonyl (ν max 1717 cm −1 ) groups. The 13 C NMR and distortionless enhancement of polarization transfer (DEPT) data of 1 (Table 1) indicated the presence of 30 carbons, including seven methyls, seven sp 3 methylenes, eight sp 3 methines, a disubstituted double bond, and six quaternary carbons. The 1 H NMR spectrum (Table 1)  The signal at δ H 4.54 (1H, ddd, J = 11.6, 11.6, 4.4 Hz) and 3.86 (1H, ddd, J = 11.6, 11.6, 6.0 Hz) were assumed to be an oxymethylene group. It was found that the NMR signals of 1 were similar to those of a known 9,11-secosterol analogue, pinnigorgiol A (3) (Figure 1) [1], except that the signals corresponding to the 11-hydroxy group in 3 were replaced by signals for an acetoxy group in 1 ( Table 2). The correlations from a nuclear Overhauser effect spectroscopy (NOESY) experiment of 1 also revealed that the stereochemistry of this metabolite was identical to that of 3. Thus, pinnigorgiol D (1) was found to be the 11-O-acetyl derivative of 3.  Pinnigorgiol E (2) was obtained as an oil and had the molecular formula C 30 H 48 O 6 as determined by HRESIMS at m/z 527.33444 (calcd. for C 30 H 48 O 6 + Na, 527.33431) and by analysis of NMR spectral data, requiring seven degrees of unsaturation. Initial analyses of the 1 H and 13 C NMR spectral data of 2 illustrated features very similar to those of 1 (Table 1) and a known secosterol, pinnigorgiol B (4) (Figure 1) [1], except that the signals corresponding to the 11-hydroxy group in 4 were replaced by signals for an acetoxy group in 2 (Table 3). Based on the above observations, pinnigorgiol E (2) was assigned as the 11-O-acetyl derivative of pinnigorgiol B (4). The CD spectra of pinnigoriols D (1), E (2), A (3), and B (4) in methanol displayed positive Cotton effects at 216 nm (∆ε = +4.0), 214 nm (∆ε = +4.6), 216 nm (∆ε = +4.6), and 218 nm (∆ε = +2.7), respectively, [1] (Figure 2). These highlights confirmed that secosterols 1-4 possess the same configurations. The CD spectra of pinnigoriols D (1), E (2), A (3), and B (4) in methanol displayed positive Cotton effects at 216 nm (Δε = +4.0), 214 nm (Δε = +4.6), 216 nm (Δε = +4.6), and 218 nm (Δε = +2.7), respectively, [1] (Figure 2). These highlights confirmed that secosterols 1-4 possess the same configurations.  The hepatic stellate cell is the major cell type involved in liver fibrosis, which is the formation of scar tissue in response to liver damage. Secosterols 1 and 2 were tested against the HSC-T6 rat hepatic stellate cell line. Compounds 1 and 2 decreased the viability of HSC-T6 cells to 31.9% and 51.7%, respectively, at a concentration of 10 µM (Figure 3). The hepatic stellate cell is the major cell type involved in liver fibrosis, which is the formation of scar tissue in response to liver damage. Secosterols 1 and 2 were tested against the HSC-T6 rat hepatic stellate cell line. Compounds 1 and 2 decreased the viability of HSC-T6 cells to 31.9% and 51.7%, respectively, at a concentration of 10 μM (Figure 3). In a previous study, pinnigorgiols A (3) and B (4) were reported to significantly decrease the cell viability of HSC-T6 cells to 13.0% and 20.8%, respectively, at a concentration of 10 μM (Table 4) [1]. It seemed that the C-11 hydroxy group and the double bond between C-22/23 are critical for the cytotoxic activity of secosterols 1-4. The in vitro anti-inflammatory effects of secosterols 1 and 2 were tested. Pinnigorgiols D (1) and In a previous study, pinnigorgiols A (3) and B (4) were reported to significantly decrease the cell viability of HSC-T6 cells to 13.0% and 20.8%, respectively, at a concentration of 10 µM (Table 4) [1]. It seemed that the C-11 hydroxy group and the double bond between C-22/23 are critical for the cytotoxic activity of secosterols 1-4. The in vitro anti-inflammatory effects of secosterols 1 and 2 were tested. Pinnigorgiols D (1) and E (2) were found to exhibit inhibitory effects on the generation of superoxide anions (IC 50 = 3.5 and 3.9 µM, respectively) and the release of elastase (IC 50 = 2.1 and 1.6 µM, respectively) by human neutrophils (Table 5). Pinnigorgiols A (3) and B (4) were also found to display inhibitory effects on the generation of superoxide anions (IC 50 = 4.0 and 2.5 µM, respectively) and the release of elastase (IC 50 = 5.3 and 3.1 µM, respectively) [1]. Secosterol 2 show stronger activity in the inhibitory effect on the release of elastase, which indicated that an acetoxy substituent at C-11 and the absence of C-22/23 double bond would enhance the activity by comparison with the structure and anti-inflammatory data of 2 with those of 1, 3, and 4. Table 5. Inhibitory effects of secosterols 1-4 on the generation of superoxide anions and the release of elastase by human neutrophils in response to fMet-Leu-Phe/cytochalastin B (fMLP/CB).

Animal Material
Specimens of the gorgonian corals Pinnigorgia sp. were collected by hand via scuba off the coast of Green Island, Taiwan, in August 2012 and stored in a freezer until extraction. A voucher specimen (NMMBA-TW-GC-2012-130) was deposited in the National Museum of Marine Biology & Aquarium, Taiwan. This organism was identified by a comparison with previous descriptions [5].

Anti-Hepatofibric Assay
The anti-hepatofibric effects of tested secosterols 1 and 2 were assayed using a WST-1 assay method. Anti-hepatofibric assays were carried out according to procedures described previously [6].

Generation of Superoxide Anions and Release of Elastase by Human Neutrophils
Human neutrophils were obtained by means of dextran sedimentation and Ficoll centrifugation. Measurements of superoxide anion generation and elastase release were carried out according to previously described procedures [7,8]. Briefly, superoxide anion production was assayed by monitoring the superoxide dismutase-inhibitable reduction of ferricytochrome c. Elastase release experiments were performed using MeO-Suc-Ala-Ala-Pro-Valp-nitroanilide as the elastase substrate.

Conclusions
Pinnigorgiols D (1) and E (2) are rare sterols containing a tricyclo[5,2,1,1]decane ring in their structures. Prior to this study, only four compounds of this type had been isolated from sea hare Aplysia kurodai [9] and gorgonian coral Pinnigorgia sp. [1]. In an anti-inflammatory activity test, secosterol 2 displayed significantly inhibitory effects on the release of elastase by human neutrophils and may become lead compounds in future marine anti-inflammatory drug development [10,11]. The gorgonian coral Pinnigorgia sp. will be transplanted to culturing tanks located in the National Museum of Marine Biology & Aquarium, Taiwan, for the extraction of additional natural products to establish a stable supply of bioactive material.