Leathesia nana is a
brown alga of Leathesiaceae family and widely distributed in gulf of the Yellow Sea, China. The ethanolic
extract of
Leathesia nana showed selective cytotoxicity in our in vitro biological screening against several human cancer cell lines, including KB and HT-29 cell lines with LD_~50 12.65 and 40.60 μg/ml, respectively. It showed indistinct cytotoxicity to normal cell NIH-3T3 with LD_~50 >50 μg/ml. Thin layer chromatography indicated the presence of
compounds positive to ferric chloride spray reagent. In order to searching for new bioactive lead compounds, we investigated the chemical constituents of the brown alga Leathesia nana. Leathesia nana was collected in Weihai of Shandong Province, China in May 2002. The specimen was identified by Dr. Kui-Shuang Shao (Institute of Oceanology, Chinese Academy of Sciences, China). A voucher specimen (No.200216) is deposited at the Herbarium of Institute of Oceanology, Chinese Academy of Sciences, Qingdao, China. Brown alga Leathesia nana (8.25 kg, dry weight) was extracted with 95% EtOH at room temperature for 3×72 h. After the solvent was removed under reduced pressure at <40°C, a dark residue (710 g) was obtained. The residue was suspended in water and then partitioned with EtOAc. The EtOAc soluble fraction (125g) was chromatographed over silica gel eluting with a gradual increasing Me_2CO (0%—100%) in petroleum ether. The fraction eluted by 33% Me_2CO in petroleum ether was decolored by column chromatography over Bio-Beads SX3 using CHCl_3-EtOAc (1∶2) and re-chromatographed over Sephadex LH-20 using petroleum ether-CHCl_3-MeOH (5∶5∶1) to afford three subfractions. Separation of the subfractions yielded compounds 1—7 by reverse phase preparative HPLC using MeOH-H_2O-AcOH (75∶25∶0.1) as mobile phase. Column chromatography was performed with silica gel (160-200 mesh, Qingdao Marine Chemical Inc. China) and Sephadex LH-20 (Pharmacia Biotech AB, Uppsala Sweden). HPLC separation was performed on a chromatograph consisting of Waters 600 Controller, Waters~TM 600 Pump and Waters 2487 Dual λ Absorbance Detector with an Alltima 250 cm×2.2 cm preparative column packed with C_~18 (10μm). TLC was carried out with glass precoated silica gel GF254 plates. Spots were visualized under UV light and by spraying with 2% FeCl_3 in 95% EtOH. All solvents used were either analytical grade or spectral grade, or were distilled prior to use. Melting points (uncorrected) were determined on an XT-4 micro melting point apparatus. IR spectra were recorded as KBr disks on a Nicolet Impact 400 FT-IR Spectrophotometer. NMR spectra were recorded on a Varian ~Inova 500 MHz spectrometer at 500.103 MHz for ~ 1H and 125.762 MHz for ~ ~13 C in acetone-d_6 with TMS as internal standard. EIMS and HREIMS data were measured with Micromass Autospec-Ultima ETOF spectrometer. By spectroscopic methods including IR, EIMS, 1D and 2D NMR experiments, their structures were identified as 3-(hydroxyacetyl)-1H-indole(1), 4-hydroxybenzoic acid (2), 2,3-dibromo-4,5-dihydroxybenzylaldehyde (3), 3-bromo-4,5-dihydroxybenzaldehyde (4), 2,3-dibromo-4,5-dihydroxybenzyl ethyl ether (5), bis(2,3-dibromo-4,5-dihydroxybenzyl) ether (6) and 3-bromo-4-(2,3-dibromo-4,5-dihydroxybenzyl)-5-methoxymethylpyrocatechol (7). This paper deals with the structural elucidation of 1—7. A variety of bromophenols have been isolated from marine red algae, a green alga, sponges and ascidians. Many of these compounds are antioxidant, feeding deterrent, α-glucosidase and enzymatic inhibitory and were reported to have effects of antimicrobe and anticancer. However, to the best of our knowledge, this is the first known brown alga containing bromophenols. Compounds 1—7 were isolated from this species for the first time.