Isolation of Two New Meroterpenoids from Sargassum siliquastrum

Experimental

General Experimental Procedures. NMR spectra were recorded in CD3OD on a Varian Mercury 300 spectrometer. 1H and 13C NMR spectra were measured using standard Varian pulse sequence programs at 300 MHz and 75 MHz, respectively. Optical rotations were taken on a Perkin-Elmer polarimeter 341 using a 5 cm cell. All chemical shifts were recorded with respect to residual CD3OD peaks. Mass spectra were obtained at the Korean Basic Science Institute, Seoul, Korea. HPLC was performed using a Dionex P580 isocratic pump equipped with a Shodex RI detector. All solvents used were spectral grade or were distilled from glass prior to use.

Table 2.Measured in CDCl3 at 300 and 75 MHz, respectively. Assignments were aided by 1H gDQCOSY, TOCSY, DEPT, gHMQC, and gHMBC experiments.

Plant Material. The brown alga Sargassum siliquastrum was collected by hand in February 2007, along the shore of Cheju Island, Korea and identified by Dr. Jee Hee Kim. A voucher specimen (No J07-11) was deposited at the Division of Marine Environment ＆ Bioscience, Korea Maritime University, Korea.

Extraction and Isolation. Shade-dried samples of S. siliquastrum were ground to powder and extracted successively for 24 h with a mixture (1:1) of acetone-CH2Cl2 (2 L × 2) and MeOH (2 L × 2). The combined crude extract (59.6 g) was evaporated under reduced pressure and then the residue was partitioned between CH2Cl2 and water. The organic layer was further partitioned between 85% aq. MeOH and n-hexane, and the aqueous layer was fractionated with n-BuOH and H2O. The resulting four fractions were evaporated to dryness in vacuo, to yield n-hexane (7.8 g), 85% aq. MeOH (8.7 g), n-BuOH (3.3 g), and water (41.9 g) fractions, respectively. The 85% aq. MeOH fraction was separated into six subfractions by C18 (YMC-GEL ODS-A, 12 nm, S-75 mm) reversed-phase vacuum flash chromatography eluting with stepwise gradient mixtures of MeOH and water (50%, 60%, 70%, 80%, and 90% aq. MeOH, and 100% MeOH). Fraction 6 was separated by reversed-phase HPLC (ODS-A, 73% aq. MeOH) to give 6 subfractions (6-1~4), in order of elution. Subfraction 6-1 was further separated by reversedphase HPLC (ODS-A, 75% aq. CH3CN) to give 3 (5.5 mg). Subfraction 6-2 was also further separated by reversed-phase HPLC with 78% aq. CH3CN to yield 1 (3.5 mg) and 2 (3.3 mg).

Sargachromanol J (1): A colorless gum; : +27.27° (c 0.13, MeOH); IR (NaCl): νmax = 3350, 1711 cm−1; 1H and 13C NMR, see Tables 7 and Tables 8; HREIMS m/z 430.3080 [M]+ (calcd for C27H42O4, 430.3083).

Sargachromanol Q (2): A colorless gum; : +42.86° (c 0.73, MeOH); IR (NaCl): νmax = 3350, 1621 cm−1; UV (MeOH) λmax(log ε) 224 (3.86) nm; 1H and 13C NMR, see Tables 7 and Tables 8; HREIMS m/z 428.2915 [M]+ (calcd for C27H40O4, 428.2927).

Sargachromanol R (3): A colorless gum; : +12.67° (c 0.12, MeOH); IR (NaCl): νmax = 3350, 1682, 1620 cm−1; UV (MeOH) λmax (log ε) 237 (3.77) nm; 1H and 13C NMR, see Table 6; HREIMS m/z 424.2609 [M]+ (calcd for C27H38O3, 424.2614).

Cell Cytotoxicity: Cytotoxic levels of the isolated compounds on cultured cells were measured using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay [6b], which is based on the conversion of MTT to MTT-formazan by mitochondrial enzyme. The cells were grown in 96-well plates at a density of 5 × 103 cells/well. After 24 h, cells were washed with fresh medium and treated with different concentrations of samples. After incubation for 48 h, the cells were rewashed and incubated with 100 μL of MTT (1 mg/mL) for 4 h. Finally, 150 μL of DMSO was added to solubilize the formed formazan crystals. The amount of formazan was determined by measuring the absorbance at 540 nm using a multidetection microplate fluorescence spectrophotometer synergy HT (Bio-Tek instruments Inc., Winooski, VT). Relative cell viability was determined by the amount of MTT converted into formazan. Viability of cells was quantified as a percentage compared with the control, and dose response curves were developed.