Rumphellaoic Acid A, a Novel Sesquiterpenoid from the Formosan Gorgonian Coral Rumphella antipathies

A novel sesquiterpenoid, rumphellaoic acid A (1), was isolated from the gorgonian coral Rumphella antipathies, and was found to possess a carbon skeleton that was obtained for the first time from a natural sources. The structure of 1 was elucidated by spectroscopic methods and this compound and was found to exert a moderate inhibitory effect on the release of elastase by human neutrophils.

The relative configuration of 1 was established from the interactions observed in nuclear Overhauser effect spectroscopy (NOESY) spectra (Figure 1). In the NOESY spectra of 1, the correlation of H-5 with one proton of the C-11 methylene (δH 1.94), but not with H-2, indicated that these protons were situated on the same face, and these were assigned as β protons, since H-2 is α-substituted at C-2. It was found that one of the methylene protons at C-3 (δH 1.56) exhibited a correlation with H-2, and therefore it was assigned as H-3α, and the other C-3 proton (δH 1.35) as H-3β. The C-13 methyl showed a correlation with H-3β, but not with H-3α, demonstrating that the C-1 chiral carbon possesses an S*-configuration. Furthermore, the carboxyl group at C-8 was proven to possess an S*-configuration by modeling analysis. Based on the above findings, the structure of 1 was elucidated and the chiral carbons for 1 were assigned as 1S*, 2S*, 5R* and 8S*.  It is worth noting that a sesquiterpenoid analog possessing the carbon skeleton descried for 1 was obtained from a natural source for the first time in this study. The in vitro anti-inflammatory effect of 1 was tested and this compound was found to display a modest inhibitory effect on the release of elastase (inhibition rate = 29.2%) by human neutrophils at a concentration of 10 μg/mL.

Animal Material
Specimens of the gorgonian coral Rumphella antipathies (Nutting) were collected by hand using scuba equipment off the coast of Pingtung, Southern Taiwan. This organism was identified by comparison with previous descriptions [17]. A voucher specimen (Specimen No. NMMBA-TWGC-010) was deposited in the National Museum of Marine Biology and Aquarium, Taiwan.

Extraction and Isolation
Sliced bodies of the gorgonian R. antipathies (wet weight 402 g, dry weight 144 g) were extracted with a mixture of methanol (MeOH) and dichloromethane (CH2Cl2) (1:1) at room temperature. The extract was partitioned with ethyl acetate (EtOAc) and H2O. The EtOAc layer was separated by silica gel and eluted using n-hexane/EtOAc (stepwise, 25:1-pure EtOAc) to yield 29 fractions. Every fraction was checked using the 1 H NMR spectra. Fraction 15 was re-purified by normal-phase HPLC (NP-HPLC) using a mixture of n-hexane and EtOAc as the mobile phase to afford 1 (1.6 mg, 5:1).

Human Neutrophil Elastase Release
Human neutrophils were obtained by means of dextran sedimentation and Ficoll centrifugation. Briefly, elastase release experiments were performed using MeO-Suc-Ala-Ala-Pro-Val-p-nitroanilide as the elastase substrate [18,19]. Elastatinal was used as a reference compound in the anti-inflammatory test of the inhibitory effects on the release of elastase (IC50 = 60.0 μM) by human neutrophils in response to fMet-Leu-Phe/Cytochalastin B (fMLP/CB). In the in vitro anti-inflammatory bioassay, the inhibitory effects on the release of elastase by activated neutrophils were used as indicators. At a concentration of 10 μg/mL, for the significant activity of pure compounds, an inhibition rate ≥50% is required (inhibition rate ≤ 10%, not active; 20% ≥ inhibition rate ≥ 10%, weakly anti-inflammatory; 50% ≥ inhibition rate ≥ 20%, modestly anti-inflammatory).

Conclusions
In continuing studies of new substances from marine invertebrates collected off the waters of Taiwan, a new sesquiterpenoid, rumphellaoic acid A (1), was isolated from R. antipathies. The structure of sesquiterpenoid 1 was elucidated on the basis of spectroscopic methods, and this compound was found to display an inhibitory effect on the release of elastase by human neutrophils. The sesquiterpenoid analogues prepared by chemical methods and biotransformation by Collado's group [20][21][22] possessed the same carbon skeleton as that of 1. However, to the best of our knowledge, this is the first time that compound 1 has been obtained from a natural source.