Extraction of crude lipids

30 g fresh edible portions (aliquoted in falcon tubes and stored at −80 °C) were transferred to 250 mL conical flask

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Samples were homogenized with 150 mL isopropyl alcohol/cyclohexane (10:12, v/v) containing 0.075% butylated hydroxytoluene (BHT: w/v; antioxidant)

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Bath sonication (JAC-2010; 300 w, 60 Hz, for 10 min) for the efficient disintegration and complete extraction

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Centrifuged at 8000× g (10 min at 4 °C), the supernatant was collected, and pellets were extracted again with 100 mL of cyclohexane

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Supernatants from both extractions were pooled (total volume of ~200 mL) and partitioned with 100 mL of 1 M sodium chloride (NaCl)

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The upper cyclohexane phase containing crude lipids were collected, filtered over anhydrous sodium sulfate, transferred to a preweighted 250 mL round bottom flask, and vacuum-dried in a rotary evaporator at 35 °C

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The contents of crude lipids were determined gravimetrically and recovered in 8 mL cyclohexane/dichloromethane (DCM) (3:1, v/v) containing 0.1% BHT and utilized as follows:

(a)One mL of the crude lipids sample was filtered through a 0.45 μm PTFE syringe filter and transferred to a 1.5 mL autosampler vial for tocols analysis by utilizing HPLC-DAD. (b)500 µL of the sample was converted to FAMEs and analyzed by GC- FID and GC-MS. (c)200 µL of the sample was hydrolyzed, silylated, and utilized to quantify the sterols using GC-MS. (d)Sample containing 20 mg of crude lipids were transferred to a 5 mL glass, vacuum-dried in a rotary evaporator at 35 °C, lipids recovered in 1 mL dimethyl sulfoxide DMSO, and utilized for cytotoxicity studies.