2.1.4. Heart and Kidney Analysis

Heart and kidney samples were homogenized in a buffer solution (50 mM Tris-HCl, 150 mM NaCl, 0.1% SDS, 1% NP-40, pH 7.5), and suspensions were centrifuged at 1000× *g* at 4 ◦C for 10 min. ACE activity levels of the hearts and kidneys were determined using the method described by Yang et al. [16].

#### *2.2. Human Study*

Participants were recruited through announcement posters at the I-Shou University (Kaohsiung, Taiwan). People with diagnoses of diabetes, cardiovascular diseases, eating disorders, or liver, kidney, or other digestive diseases; medications and supplement users; pregnant or lactating individuals; and people with an adlay allergy were excluded. We recruited 25 volunteers, and among them, 23 participants with a body mass index (BMI, kg/m2) within the range of 25 to 35 (inclusive) or waist circumference within an indicated sex-specific range (>90 cm for men and >80 cm for women) were included in the experiment as shown in Figure 1. We explained the purpose and design and of the study, as well as the risks involved to all participants, and obtained their written informed consent.

**Figure 1.** Flowchart illustrating the number of volunteers who were screened, included, and completed the study.

#### 2.2.1. Experimental Design

We implemented this study following the protocol approved by the Institutional Review Board (IRB) of E-Da Hospital according to the guidelines in the Declaration of Helsinki (IRB No.: EMRP41104N). During the 6-week experimental period, 23 participants were asked to consume two packages (30 g/package) of dehulled adlay powder per day to replace three corresponding portions of cereal, according to the Food Exchange List of Taiwan and the dosage consumed daily in a previous study [13]. Those participants came to our center weekly to receive dietary consultation, bring back the used container, and obtain their adlay powder packages for the coming week. During the experimental period, the consumption of other supplements and foods not specified in the study was prohibited, and all participants were asked to maintain their normal dietary habits and physical activities.

#### 2.2.2. Blood Pressure Measurement

Participants visited our research center at 07:30 a.m. at the baseline and the end of the study after a fasting period of at least 8 h. Blood pressure was measured using an automatic blood-pressure monitor on their right arm (Microlife, Taipei, Taiwan) after participants sat in a chair for at least 10 min. SBP and DBP were calculated as the average of three separate measurements.
