70 230 mesh

All chemical reagents and solvents were obtained from Sigma Aldrich, Merck, Alfa Aesar, Fluka or TCI and were used without further purification. Microwave-assisted reactions were performed using the Biotage Initiator microwave synthesizer. TLC were performed on precoated silica plated (Merck silica gel 60, F254) and visualized with UV or stained with phosphomolybdic acid, alkaline potassium permanganate stain. Flash chromatography was performed on silica (Merck, 70-230 mesh). ¹H, ¹³C and ³¹P NMR spectra were measured on a Bruker AMX 300, 500 or 400 spectrometer (Supporting Information). Chemical shifts were reported in parts per million (δ) relative to the tetramethylsilane standard. Mass spectra were performed on a Finnigan/MAT LCQ mass spectrometer under either electron spray ionization (ESI) or electron impact (EI) techniques.

Reagents were purchased
from Aldrich and TCI Chemicals and were of analytical grade. Reactions
were monitored by thin-layer chromatography (TLC) carried out on silica
gel plates (Macherey Nagel SIL G UV254) and visualized in UV light.
Purification of the compounds was performed by filtration or by column
chromatography. Column chromatography was carried out on silica gel
(Merck, 70–230 mesh) using the appropriate solvents. PE refers
to petroleum ether 40–60 °C. ¹H and ¹³C NMR spectra were recorded on a Bruker 400 MHz Avance spectrometer.
13C NMR spectra are fully proton-decoupled. Electrospray ionization
(ESI) mass spectral analyses were performed on a mass spectrometer
MSQ Surveyor, Finnigan, using direct sample injection in the positive
or negative mode. High-resolution mass spectrometry (HRMS) spectra
were registered using a 4800 MALDI-TOF mass spectrometer (Applied
Biosystems, Foster City) in the positive reflection mode in the m/z range of 100–700. All synthesized
compounds gave satisfactory NMR spectra. All tested compounds possessed
purity > 95% based on HRMS. Data were processed using Compass Data
Analysis software and MestReNova v.14.1.1.

All chemical reagents were purchased from Merck, Fluke, or Sigma-Aldrich and used without previous purification. All solvents were distilled and stored over proper desiccants. Melting points were measured on a BUCHI M-565 equipment (BÜCHI Labortechnik GmbH, Essen, Germany) being EtOAc the last solvent used in all cases. NMR spectra were recorded at 298 K on a Varian Mercury 400 NMR spectrometer (Varian, Palo Alto, CA, USA) at 400 MHz and 101 MHz for ¹H and ¹³C respectively. All spectra were referenced using the TMS signal or the residual peak of the solvent. Chemical shifts (δ) are reported in ppm and coupling constants (J) are given in Hz. A TripleToF 6600-1 mass spectrometer (Sciex, MA, USA) was used for high-resolution mass spectrometry (HRMS). Silica gel (Merck 70–230 mesh) was used for column chromatography (CC) and silica gel plates HF254 for thin-layer chromatography (TLC). TLC spots were detected by heating after staining with cerium molybdate in H₂SO₄.

Lipids were extracted from three S. alveolata whole individuals following Folch et al.51 (link). The lipids were placed at the top of a silica gel microcolumn (30 × 5 mm internal diameter; Kieselgel; 70–230 mesh [Merck, Lyon, France]; previously heated to 450 °C and deactivated with 5% water). Neutral lipids were eluted with 10 mL of chloroform/methanol (98:2, v/v), and polar lipids were eluted with 15 mL of methanol52 . Tricosanoic acid (2.3 μg) was added as internal standard. Polar lipids were transesterified at 100 °C for 10 min with 1 mL of boron trifluoride (12% Me–OH)53 . This transesterification produces fatty acid methyl esters (FAME) from the fatty acid esterified at the sn-1 and sn-2 position of diacylphospholipids, and the sn-2 position of plasmalogen PL. It also produces dimethyl acetals (DMA) from the alkenyl chains at the sn-1 position of plasmalogens54 (link). FAME and DMA were analysed in a HP6890 GC system (Hewlett-Packard) equipped with a DB-Wax capillary column (30 m × 0.25 mm; 0.25 μm film thickness; Agilent technologies). Peaks were analysed by comparison of their retention time with those of a standard 37 component fatty acid methyl ether (FAME) mix and other standard mixes from marine bivalves. Fatty acid contents were expressed as the mole percentage of the total fatty acid content. Total DMA was used as an indicator of the plasmalogen level.

C. lepifera fronds (1.5 kg dry weight) were extracted with 15 L of 70% (v/v) aqueous methanol and 15 L of methanol as described above, and concentrated at 40 °C in vacuo to produce an aqueous residue. The aqueous residue was then adjusted to pH 7.0 with 1 M phosphate buffer, partitioned three times against an equal volume of ethyl acetate, and separated into ethyl acetate and aqueous fractions. The biological activity of the two fractions was determined using a garden cress bioassay as described above.
The ethyl acetate fraction was then evaporated to dryness and separated on a column of silica gel (100 g, silica gel 60, 70–230 mesh; Merck), and eluted with 20%, 30%, 40%, 50%, 60%, 70%, and 80% ethyl acetate in n-hexane (v/v; 100 mL per step), ethyl acetate (100 mL), and methanol (200 mL). The biological activity of all separated fractions was determined using a garden cress bioassay. Two growth inhibitory active fractions were obtained by elution with 70% (fraction 6) and 80% (fraction 7) ethyl acetate in n-hexane.

Column chromatography was performed on silica gel-60 (70–230 Mesh, by Merck). Thin-layer chromatography (TLC) was done on precoated Kieselgel-60 GF₂₅₄ aluminum (Al) sheets made by Merck. The ceric sulphate (CeSO₄) solution was sprayed for the visualization of spots and then subjected to heating, while a high-performance liquid chromatography (HPLC) device (Shimadzu SHP-600), coupled to a photodiode array detector 20-A, was employed for confirming the sample's purity of all the isolated compounds. The HPLC analysis was carried out under isocratic conditions using a C8 column (4.6mm × 150mm) packed with 5 μm diameter particles; the mobile phase was acetonitrile : water (95 : 5, v/v) containing 1.0% acetic acid with the flow rate = 1 ml/min and the run time was set for 50 minutes. Detections were made at 365 nm [8 (link)].

Solvent evaporation was carried out using a Buchi Rotavapor (R215, Buchi, Flawil, Switzerland). Column chromatography (CC) was performed via glass column using silica gel (70-230 mesh, Merck, Darmstadt, Germany), and thin layer chromatography (TLC) was performed using silica gel pre-coated plates F-254 Merck (20 × 20 cm). Compounds were visualized under UV light (254 and 365 nm), sprayed with dilute sulfuric acid and heated. The melting points of the compounds were recorded using Buchi M-560 (Buchi, Flawil, Switzerland) melting point apparatus equipped with a Buchi M-569 sample loader. The ¹H NMR (500 MHz) and ¹³C NMR (125 MHz) data were recorded via a Bruker Avance AV-500 (Bruker, Ettlingen, Germany) spectrometer in deuterated solvents, with trimethylsilane (TMS) used as the reference. Chemical shifts were given in ppm (δ), and coupling constants (J) in Hz. ESI-TOF-MS spectra were registered on a QTOF Spectrometer (Bruker, Ettlingen, Germany). A Multiskan Go microplate reader (Thermo Fischer Scientific, Waltham, MA, USA) was used to measure absorbances in the bioassays.