4.4. Metabolites Extraction
The IC-E and IC-P were freeze-dried. Then 20 mg aliquot of individual samples were preciselt weighed and were transferred to an Eppendorf tube, after the addition of 300 μL water (containing internal standard). After 30 s vortex, the samples were homogenized at 40 Hz for 4 min and sonicated for 5 min in an ice-water bath. The homogenization and sonication cycle were repeated 3 times. Then the samples were extracted over night at 4 °C on a shaker, then centrifuged at 12,000 rpm (RCF = 13,800 (×g), R = 8.6 cm) for 15 min at 4 °C. The supernatant was carefully filtered through a 0.22 μm microporous membrane, then the resulting supernatants were diluted 50 times with methanol/ water mixture (