*2.1. Animal Study*

We purchased dehulled adlay powder (Taichung No. 3) from the Nantou County Tsao-Tun Production Association (Nantou County, Taiwan). Eight-week-old Wistar Kyoto (WKY) rats and spontaneously hypertensive rats (SHRs) were purchased from the National Laboratory Animal Breeding and Research Center. Rats were housed in the Experimental Animal Center as per guidelines reviewed by the Institutional Animal Care and Use Committee of I-Shou University (Approval ID: AUP-105-43-01). Rats were maintained in an environment with a constant temperature (23 ± 2 ◦C) and humidity (55 ± 10%) and were exposed to a 12 h light–dark cycle in accordance with the Animal Protection Act and the regulations of the Animal Care and Use Committee of the Council of Agriculture, Executive Yuan. Rats were fed a standard rat chow diet for 1 week for acclimatization. Then, SHRs were randomly assigned to three groups: an SHR group fed a standard AIN-93M rodent diet (*n* = 10), an SHR + LA group fed AIN-93M with a low dose of 12% adlay powder (*w*/*w*), and an SHR + HA group fed AIN-93M with a high dose of 24% adlay powder (*w*/*w*), WKY rats were used as the normotensive control and also received a standard AIN-93M rodent diet (*n* = 10). We used adlay powder substituted for part of the components from the standard AIN-93M diet to ensure equal nutrient composition among the diets, as shown in Table 1. During the 12-week experimental period, food and water were provided ad libitum. Rats' food intake was recorded daily and their body weights recorded weekly. At the end of the study, we collected the previous 24 h of urine of rats using metabolic cages, and thereafter sacrificed the rats to obtain blood, heart, and kidney samples for analysis.

#### 2.1.1. Measurement of Blood Pressure

SBP and DBP were measured using a noninvasive tail-cuff system (MK-2000ST, Muromachi Kikai, Tokyo, Japan) every 4 weeks. Rats were placed in restrainers, and we recorded at least five readings to calculate the mean of blood pressure over the course of the measurement.


**Table 1.** Dietary compositions (g/kg) of groups in the murine trial.

Corn starch, dextrin, casein, soy oil, cellulose (non-nutritive bulk), AIN-93M vitamin and mineral mixture were obtained from ICN Biochemicals (Aurora, OH, USA). Choline bitartrate and cystine were obtained from Sigma (St. Louis, MO, USA). Dehulled adlay (Taichung No. 3) powder was purchased from the Nantou County Tsao-Tun Production Association (Nantou County, Taiwan). SHR + LA, low-dose (12%, *w*/*w*) dehulled adlay powder in diet; SHR + HA, high-dose (24%, *w*/*w*) dehulled adlay powder in diet.

#### 2.1.2. Blood Analysis

Blood samples were collected and centrifuged at 1500× *g* and 4 ◦C for 15 min for plasma separation. Plasma samples were collected to analyze the aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatinine (Cr), uric acid, and phosphorus concentrations by using a Roche Modular P800 Autoanalyzer (Diagnostics Roche, Basel, Switzerland). ACE activity was analyzed according to the method previously described by Vermeirssen et al. [15]. C-reactive protein (CRP; Invitrogen, CA, USA), plasminogen activator inhibitor-1 (PAI-1; HYPHEN BioMed, Neuville-sur-Oise, France), and endothelin-1 (ET-1; Enzo Life Sciences, New York, USA) were analyzed using commercial kits as per the manufacturer's instructions.

### 2.1.3. Urine Analysis

The 24 h of urine samples were collected using metabolic cages. Urinary protein excretion, urine urea nitrogen (UUN), and Cr levels were analyzed using a Roche Modular P800 Autoanalyzer (Diagnostics Roche, Basel, Switzerland). All urine values were corrected in accordance with the Cr level.
