3.3.1. Sample Extraction

First, 2 g of each sample was weighed and added to 100 mL of 80% methanol solution (*v/v*). Sample extraction was performed in a shaking incubator for 24 h at 30 ◦C. After extraction, the solution was centrifuged (05PR-22 centrifuge, Hitachi, Tokyo, Japan) at 500× *g* for 10 min at room temperature. The supernatants were gathered after centrifugation and filtered through Whatman No. 42 filter paper (Whatman Inc., Clifton, NJ, USA). The filtrate was concentrated using a vacuum rotary evaporator (Eyela Co., Tokyo, Japan) at 40 ◦C. The samples were freeze-dried in a vacuum freeze-dryer (Christ Alpaha 1–4, Germany). Finally, the samples were stored in a refrigerator at −20 ◦C for subsequent experiments.
