*3.2. Animal Materials*

The marine organism *S. cherbonnieri* was collected and preserved as described previously [24].

### *3.3. Extraction and Isolation*

By using the procedure reported previously, 1.2 kg (wet weight) of organism *S. cherbonnieri* was dehydrated, minced, extracted, and concentrated to afford 10.2 g of residue. The residue was fractionated by chromatography to yield 19 fractions [24]. Fraction 10, eluting with *n*-hexane–acetone (4:1), was further purified over silica gel using *n*-hexane–acetone (6:1) to afford seven subfractions (A1–A7). Subfraction A3 was further separated by reverse-phase HPLC using acetonitrile–H2O (1:1.1) to afford **2** (1.4 mg). Subfraction A4 was purified by reverse-phase HPLC using acetonitrile–H2O (1:1.2) to afford **4** (8.8 mg), and subfraction A6 was purified by reverse-phase HPLC acetonitrile–H2O (2:1) to afford **5** (3.1 mg). Fractions 11 and 12, obtained by eluting with *n*-hexane–acetone 3:1 and 2:1, respectively, were combined and further eluted with acetone by a Sephadex LH-20 column to afford six subfractions (B1–B6). The purification of subfractions B4 and B5 using reverse-phase HPLC by elution of acetonitrile–H2O (1:1.3) and MeOH–H2O (3:2) afforded **6** (12.4 mg) and **7** (33.1 mg), respectively. Fraction 13, eluting with *n*-hexane–acetone (1:1), was purified by eluting with acetone on Sephadex LH-20 to yield five subfractions (C1–C5). Subfraction C2 was further separated by reverse-phase HPLC using acetonitrile–H2O (1:1.4) to afford **1** (3.3 mg) and **3** (10.8 mg).

Cherbonolide F (**1**): colorless oil; [α]D25 +177 (*c* 0.50, CHCl3); IR (neat) νmax 3460, 2967, 2928, 2864, 1748, 1677, 1452, 1385, 1096, 984, and 755 cm–1; for 13C- and 1H-NMR data (400 MHz; C6D6), see Tables 1 and 2; ESI-MS *m*/*z* 355 [M + Na]<sup>+</sup>; HR-ESI-MS *m*/*z* 355.1879 [M + Na]+ (calculated for C20H28O4Na, 355.1880).

Cherbonolide G (**2**): colorless oil; [α]D25 +25 (*c* 0.33, CHCl3); IR (neat) νmax 3419, 2925, 2855, 1748, 1678, 1454, 1387, 1096, 987, and 755 cm–1; for 13C- and 1H-NMR data (400 MHz; C6D6), see Tables 1 and 2; ESI-MS *m*/*z* 371 [M + Na]<sup>+</sup>; HR-ESI-MS *m*/*z* 371.1830 [M + Na]+ (calculated for C20H28O5Na, 371.1829).

Cherbonolide H (**3**): colorless oil; [α]D25 +41 (*c* 1.00, CHCl3); IR (neat) νmax 3445, 2928, 2864, 1747, 1679, 1455, 1387, 1094, 996, and 755 cm–1; for 13C- and 1H-NMR data (400 MHz; C6D6), see Tables 1 and 2; ESI-MS *m*/*z* 355 [M + Na]<sup>+</sup>; HR-ESI-MS *m*/*z* 355.1878 [M + Na]+ (calculated for C20H28O4Na, 355.1880).

Cherbonolide I (**4**): colorless oil; [α]D25 +13 (*c* 1.00, CHCl3); IR (neat) νmax 3420, 2925, 2855, 1747, 1541, 1390, 992, and 756 cm–1; for 13C- and 1H-NMR data (500 MHz; C6D6), see Tables 1 and 2; ESI-MS *m*/*z* 371 [M + Na]<sup>+</sup>; HR-ESI-MS *m*/*z* 371.1828 [M + Na]+ (calculated for C20H28O5Na, 371.1829).

Cherbonolide J (**5**): white powder; [α]D25 −6 (*c* 0.50, CHCl3); IR (neat) νmax 3443, 2937, 2860, 1755, 1675, 1381, 1076, 990, and 755 cm–1; CD (1.2 × 10−<sup>4</sup> M, MeOH) λmax (Δε ) 247 (−5.2), and 228 (+26.5) nm; for 13C- and 1H-NMR data (400 MHz; C6D6), see Tables 1 and 3; ESI-MS *m*/*z* 373 [M + Na]<sup>+</sup>; HR-ESI-MS *m*/*z* 373.1984 [M + Na]+ (calculated for C20H30O5Na, 373.1986).

Cherbonolide K (**6**): yellow oil; [α]D25 +12 (*c* 1.00, CHCl3); IR (neat) νmax 3444, 2927, 1763, 1435, 1386, 1241, 1083, 931, and 756 cm–1; for 13C- and 1H-NMR data (400 MHz; CDCl3), see Tables 1 and 3; ESI-MS *m*/*z* 355 [M + Na]<sup>+</sup>; HR-ESI-MS *m*/*z* 355.1880 [M + Na]+ (calculated for C20H28O4Na, 355.1880).

Cherbonolide L (**7**): yellow oil; [α]D25 +33 (*c* 1.00, CHCl3) ; IR (neat) νmax 3445, 2929, 2872, 1752, 1665, 1455, 1384, 1050, 927, and 756 cm–1; for 13C- and 1H-NMR data (400 MHz; CDCl3), see Tables 1 and 3; ESI-MS *m*/*z* 355 [M + Na]<sup>+</sup>; HR-ESI-MS *m*/*z* 355.1877 [M + Na]+ (calculated for C20H28O4Na, 355.1880).

### *3.4. Reduction of Cherbonolide I (***4***)*

The solution of compound **4** (1.4 mg) in diethyl ether (5.0 mL) was added to an excess amount of triphenylphosphine (1.3 mg), and the mixture was stirred at room temperature for 4 h. The solvent of the solution was evaporated under reduced pressure to afford a residue, which was purified by silica gel column chromatography using *n*-hexane–acetone (3:1) as an eluent to yield **3** (1.0 mg, 75%).

### *3.5. In Vitro Anti-Inflammatory Assay*

### 3.5.1. Primary Human Neutrophils

Blood was obtained from the elbow vein of healthy adult volunteers (with ages 20–30). Neutrophils were enriched by means of dextran sedimentation, Ficoll–Hypaque centrifugation, and hypotonic lysis. Neutrophils were incubated in an ice-cold Ca2+-free Hank's Balanced Salt Solution (HBSS buffer, pH 7.4) [31]. The research protocol was granted approval by the institutional review board of Chang Gung Memorial Hospital (IRB No: 201601307A3, 20161124-20191123; 201902217A3, 20200501-20240630). All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki.

### 3.5.2. Superoxide Anion Generation

Neutrophils (6 × 10<sup>5</sup> cells·mL−1) incubated in HBSS with ferricytochrome *c* (0.5 mg·mL−1) and Ca2+ (1 mM) at 37 ◦C were treated with dimethyl sulfoxide (DMSO), as control, or with the tested compound for 5 min. Neutrophils were primed by cytochalasin B (CB, 1 <sup>μ</sup>g·mL−1) for 3 min before activating fMLF (100 nM) for 10 min (fMLF/CB). The change in superoxide anion generation was spectrophotometrically measured at 550 nm (U-3010, Hitachi, Tokyo, Japan) [32,33].
