*3.2. Metabolic Profiling*

Metabolic profiling was performed using the liquid chromatography coupled with high-resolution electron spray ionization mass spectrometry technique (LC-HR-ESI-MS) as previously mentioned [20]. The obtained metabolites were described by comparison with several databases (e.g., DNP and METLIN).

#### *3.3. Extraction and Isolation of Pure Compounds*

The red algae *Hypnea musciformis* (430 g fresh weight) was freeze-dried, ground, and soaked in methanol (3 × 2 L) at room temperature. Then, the combined methanol extracts were evaporated under a vacuum, resulting in 32 g of green residue. The total methanol extract was then fractionated by partitioning with different solvents in increasing order of polarity several times to afford an *n*-hexane fraction (3.67 g), a chloroform fraction (2.33 g), an ethyl acetate fraction (7.43 g), an *n*-butanol fraction (3.07 g), and finally an aqueous fraction. The ethyl acetate fraction (Hp-EA, 7.43 g) was chromatographed with SiO2 column chromatography using hexane:EtOAc:methanol (80:20:0 up to 0:75:25) gradient elution, giving seven subfractions, Hp-EA-(1-7). Subfraction Hp-EA-4 (437 mg) was rechromatographed on silica gel using hexane:EtOAc:methanol (50:50:0 up to 0:90:10) gradient elution, giving five subfractions, Hp-EA-4-(1-5). One of the resultant subfractions, Hp-EA-4-2, was applied to a sephadex LH-20 column eluted with CHCl3-MeOH (1:1), giving 3 subfractions, Hp-EA-4-2-(1-3). Among them, ceramide **A** (**1**) (13 mg, white powder) was afforded from subfraction Hp-EA-4-2-2. Ceramide **A** (**1**) was finally purified on an open ODS column using MeOH/H2O (8:2). Another subfraction, Hp-EA-4-2-3, was subjected to silica gel column chromatography using EtOAc:methanol (95:5) to afford ceramide **B** (**2**) (15 mg, white powder) and ceramide **C** (**3**) (6 mg, white powder). Regarding the chloroform fraction (Hp-C, 2.33 g), it was chromatographed with SiO2 column chromatography using hexane:EtOAc (100:0 up to 25:75) gradient elution, giving 3 subfractions, Hp-C-(1-3). Subfraction Hp-C-1 (223 mg) was rechromatographed on silica gel using hexane:EtOAc (90:10 up to 70:30) gradient elution, giving two subfractions, Hp-C-1-(1-2). The first one,

Hp-C-1-1, afforded compound **4**, white powder 10 mg in weight, while the second subfraction (Hp-C-1-2) was applied to a sephadex LH-20 column eluted with CHCl3-MeOH (1:1), giving compound **5** (12 mg, white powder) and compound **6** (13 mg, white powder).

#### *3.4. In Vitro Cytotoxic Activity of Ceramides* **A** (**1**)*,* **B** (**2**)*, and* **C** (**3**) 3.4.1. In Vitro Cell Culture

The human breast adenocarcinoma (MCF-7) cell line was purchased from the American Type Culture Collection (ATCC HTB-22), Minnesota, USA). The tumor cell lines were maintained at the National Cancer Institute in Cairo, Egypt by serial subculturing. The cells were subcultured on RPMI 1640 medium supplemented by 1% penicillin/streptomycin and 10% fetal bovine serum [20].
