**4. Materials and Methods**

#### *4.1. Preparation of Adlay Extracts*

The adlay utilized in this experiment was purchased from a local farmer in Taichung, and the variety of adlay was Taichung Shuenyu no. 4 (TCS 4) of *Coix lachryma-jobi* L. var. *ma-yuen Stapf*. Sample preparation and extraction used previously reported methods from our laboratory [22]. Briefly, adlay seeds were air-dried at 40 ◦C and ground to obtain AH, and further ground to separate dehulled adlay and AT by an electric fan. After these procedures, the whole grain contained 7.2 ± 0.4% water, while that in AH and AT contained 5.2 ± 0.1% and 7.2 ± 0.1%, respectively. A total of 12 kg of AT was extracted with 120 L of ethanol 3 times at room temperature for 24 h, and filtered through #1 filter paper (Cytiva, Washington, D.C., USA). The filtrate was then concentrated to dryness to yield approximately 538 g of dried ATE (Figure 1). The dried extracts were stored at −20 ◦C until use. ATE was then suspended in a 10% methanol solution and partitioned with *n*-hexane to obtain the lipid-rich ATE-Hex fraction, yielding approximately 144 g. The defatted ATE was further partitioned with Ea to obtain about 160 g of ATE-Ea. The remaining ATE was partitioned with butanol to obtain about 50 g of ATE-BuOH. Finally, the residual extract was the ATE-H2O fraction. ATE-Ea was dissolved in Ea and subjected to column chromatography on a silica gel to yield ATE-Ea subfractions a–h. The gradient used for elution was a hexane/Ea gradient from 0 to 100% EA (every 10%), and the sub-fractions with similar chemical compositions on thin-layer chromatography (TLC) were combined and concentrated to dryness under a vacuum at 60 ◦C. Subfractions were stored at −20 ◦C until use. On the other hand, ATE-BuOH was dissolved in methanol and subjected to column chromatography on a Diaion HP-20 resin to yield ATE-BuOH subfractions a–f. The gradient used for elution was an H2O/MeOH gradient from 0 to 100% MeOH (every 25%) and was washed with 100% EA. Sub-fractions with similar chemical compositions on TLC were combined and concentrated to dryness in a vacuum at 60 ◦C and stored at −20 ◦C until use.
