Fatty acids were in situ methylated according to Trigueros and Sendra (2015) with some modifications. Briefly, 0.50 g dried mulberry fruit were transferred into a test tube. The internal standard used was C17:0; the standard was freshly prepared as 20 mg/mL solution in HPLC grade n-hexane. 30 μL of C17:0 in n-hexane solution were added to each tube containing the dried sample. Then, 100 μL of methylene chloride and 1 mL of 0.5 N NaOH in methanol were added and the tubes were heated in a water bath at 90 °C for 10 min. 1 mL of BF3 in methanol was added and the mixture, and samples were left at room temperature (25 °C) for 30 min to prevent intraisomerization of long chain fatty acids isomers (Werner, Luedecke, & Shultz, 1992). 1 mL of distilled water and 600 μL hexane were added, and then FAMEs were extracted by vigorous shaking for about 1 min. Following centrifugation, the aliquots were dried with anhydrous sodium sulfate and the top layer was transferred into a vial, which was flushed with nitrogen, and vials were stored at −20 °C until analyzed by gas chromatography.