*3.1. Effects of Dehulled Adlay Intake on Body Weight in SHRs*

The results of the animal experiment demonstrated that using a diet containing dehulled adlay did not affect food and energy intake among groups. In addition, we also discovered no significant differences in body weight among the groups over the experiment period (Figure 2a).

**Figure 2.** Food intake (**a**), SBP (**b**), and plasma and tissue ACE activities (**c**) of rats in different groups. Values are presented as mean <sup>±</sup> SD (*<sup>n</sup>* = 10). abc Different superscript letters indicate a significant difference (*<sup>p</sup>* < 0.05). SBP, systolic blood pressure; ACE, angiotensin-converting enzyme. WKY, Wistar Kyoto) rats fed an AIN-93M diet; SHR, spontaneously hypertensive rats fed an AIN-93M diet; SHR + LA, SHRs fed an AIN-93M diet containing 12% dehulled adlay powder; SHR + HA, SHRs fed an AIN-93M diet containing 24% dehulled adlay powder.

#### *3.2. Effects of Dehulled Adlay Intake on Blood Pressure and ACE Activity in SHRs*

The SHR, SHR + LA, and SHR + HA groups all had higher SBP levels than the WKY group throughout the 12-week experimental period. Both the SHR + LA and SHR + HA groups had lower SBP than the SHR group after the fourth week, and this difference was maintained until the end of the study (Figure 2b). Although the SHR group had higher renal ACE activity than the WKY group (WKY vs. SHR, *p* = 0.0004), we found no significant difference in plasma, heart, and kidney ACE activity among the SHR, SHR + LA, and SHR + HA groups (Figure 2c). The results indicated that daily dehulled adlay intake in replacement of part of the diet composition could prevent blood-pressure increases among SHRs, but these effects may not be explained by the inhibition of ACE activity alone.

#### *3.3. Effects of Dehulled Adlay Intake on AST, ALT, and Renal Functions in SHRs*

At the end of the study, we found that the SHR group had higher plasma AST (WKY vs. SHR, *p* < 0.0001; SHR + LA vs. SHR, *p* = 0.0011; SHR + HA vs. SHR, *p* = 0.0076) and ALT (WKY vs. SHR, *p* < 0.0001) activities than the WKY group did, and that both the SHR + LA and SHR + HA groups had lower AST activities than the SHR group did (Figure 3a). In

addition, plasma Cr concentrations (WKY vs. SHR, *p* = 0.0289) and the total protein-to-Cr ratios in the rat urine were significantly higher in the SHR groups than in WKY (WKY vs. SHR, *p* = 0.0042). Both dehulled adlay groups had lower plasma Cr concentrations than the SHR group did (SHR + LA vs. SHR, *p* = 0.0020; SHR + HA vs. SHR, *p* < 0.0001), but no significant differences were observed in total protein/Cr, UUN/Cr, and plasma phosphorus concentrations in urine among the three SHR groups. Additionally, plasma uric acid levels were significantly higher in the SHR group and lower in the HA group (WKY vs. SHR, *p* = 0.0022; SHR + HA vs. SHR, *p* = 0.0002) (Figure 3b,c). The results also indicated that elevation of blood pressure may increase the risk of renal tissue injuries, and that dehulled adlay consumption may ameliorate these risks.

**Figure 3.** Plasma hepatic (**a**), renal function parameters (**b**), and urine analysis (**c**) of rats in different groups. Values are presented as mean <sup>±</sup> SD (*<sup>n</sup>* = 10). abc Different superscript letters indicate a significant difference (*<sup>p</sup>* < 0.05). AST, aspartate aminotransferase; ALT, alanine aminotransferase; UUN, urine urea nitrogen. WKYWistar Kyoto rats fed an AIN-93M diet; SHR, spontaneously hypertensive rats fed an AIN-93M diet; SHR + LA, SHRs fed an AIN-93M diet containing 12% dehulled adlay powder; SHR + HA, SHRs fed an AIN-93M diet containing 24% dehulled adlay powder.

#### *3.4. Effects of Dehulled Adlay Intake on Indicators of Endothelial Function in SHRs*

Over the course of the experiment, plasma CRP and PAI-1 levels tended to decrease in both the SHR + LA and SHR + HA groups when compared with the SHR group. Both dehulled-adlay-consuming groups also had lower plasma ET-1 levels than did the SHR group (SHR + LA vs. SHR, *p* = 0.0164; SHR + HA vs. SHE, *p* = 0.0345), and no significant difference in ET-1 level was observed compared with the WKY group (Figure 4). The results showed that adlay may retard the elevation of blood pressure through improving endothelial function.

**Figure 4.** Plasma CRP, PAI-1, and ET-1 levels of rats in different groups. Values are presented as the mean ± SD (*n* = 10). ab Different superscript letters indicate a significant difference (*p* < 0.05). CRP, C-reactive protein; PAI-1, plasminogen activator inhibitor-1; ET-1, endothelin-1. WKYWistar Kyoto rats fed an AIN-93M diet; SHR, spontaneously hypertensive rats fed an AIN-93M diet; SHR + LA, SHRs fed an AIN-93M diet containing 12% dehulled adlay powder; SHR + HA, SHRs fed an AIN-93M diet containing 24% dehulled adlay powder.

## *3.5. Effects of Daily Dehulled Adlay Intake on Blood Pressure and Endothelial Function in Participants*

Furthermore, we performed an interventional human study to observe the effects of a dehulled adlay-rich dietary pattern on blood-pressure regulation in participants. During the experimental period, participants were asked to maintain their normal dietary and physical activity, but to replace 60 g of refined grain products with dehulled adlay powder either in their beverage or meal under the guidance of a dietitian; subjects brought back the used and empty containers to our center every week, and no subjective adverse effects were reported. We learned that the SBP of our participants decreased over the 6-week experimental period (6-week vs. 0-week, *p* = 0.006) (Figure 5a). Additionally, we categorized participants into subgroups according to their baseline SBP (<120 mmHg, *n* = 5; 120–130 mmHg, *n* = 11; >130 mmHg, *n* = 7), and discovered that the effects of dehulled adlay consumption on blood-pressure change were more obvious in the participants with higher baseline SBP (ΔSBP: >130 vs. <120, *p* = 0.0243) (Figure 5b). We also noted a trend of decreasing plasma ET-1 levels in participants (*p* = 0.07) (Figure 5c). These results indicated a beneficial effect of dehulled adlay on blood-pressure modulation, and that these effects may be related to baseline SBP.

**Figure 5.** Baseline and end SBP (**a**) and ET-1 (**b**) levels, and changes in SBP and DBP (**c**) of participants subgrouping by baseline SBP after the 6-week dehulled adlay intervention. Values are presented as mean <sup>±</sup> SD (*<sup>n</sup>* = 23). ab Different superscript letters indicate a significant difference (*p* < 0.05). SBP, systolic blood pressure; DBP, diastolic blood pressure; ET-1, endothelin-1.

#### **4. Discussion**

In this study, we found that dehulled adlay consumption retarded the elevation of blood pressure in SHRs, and may be beneficial in blood pressure modulation in overweight and obese adults. Dietary intake of whole-grain foods may lower cardiovascular risks. A randomized controlled trial concluded that daily consumption of whole grains (50 g/1000 kcal) resulted in greater improvements in blood pressure than a refined grain diet did in adults with overweight and obesity [17]. Results from the Furukawa Nutrition and Health Study also indicated that higher intake of whole-grain foods may reduce hypertension risk [7]. Adlay is a common grain in Asian diets, and is used in traditional Chinese medicines to treat cardiovascular diseases. Studies have also reported that adlay has various beneficial effects. For example, adlay bran was discovered to have anti-inflammatory [18] and anti-tumor effects [19], and dehulled adlay also had a gastroprotective effect in vitro [20]. Although one previous study reported that adlay-derived peptides may have antihypertensive effects [14], studies focused on adlay's influence on blood-pressure management remains scarce. To our knowledge, this is the first study to explore the potential effects of daily consumption of dehulled adlay in reducing blood pressure in hypertensive rats and in overweight and obese participants.

Increased daily whole-grain consumption has positive effects on blood-pressure control. The 2020 International Society of Hypertension's Global Hypertension Practice Guidelines also include a suggestion to eat a healthy diet rich in whole grains to treat hypertension [21]. Dehulled adlay is one of the ingredients recommended to replace polished rice in some Asian diets. According to the Nutrient Composition Database of the Food

and Drug Administration of the Ministry of Health and Welfare, every 100 g of adlay seed contains 199 mg of magnesium, which is approximately 10 times of the level in white rice. Magnesium has been shown to regulate blood pressure through directly stimulating prostacyclin and nitric oxide production [22]. These blood-pressure-reducing effects may be caused by endothelium-dependent and endothelium-independent vasodilation [23,24]. Furthermore, magnesium may also prevent vascular injury due to its antioxidant and anti-inflammatory effects [25]. In the present study, we discovered that in hypertensive rats, partial dietary replacement with dehulled adlay could limit the progression of hypertension without affecting food intake or body weight in hypertensive rats. We also found that daily intake of 60 g dehulled adlay could lower blood pressure in human participants with high baseline SBP. These results suggested that dehulled adlay has potential use for the treatment or prevention of hypertension.

Blood pressure is regulated by numerous mechanisms in vivo, and the renin-angiotensin system is one of such major regulatory mechanism. Increased ACE activity reveals the formation of angiotensin II, which leads to vessel constriction and elevated blood pressure. In 2017, Li et al. [14] observed potent anti-hypertensive peptides in Coix glutelin. However, we found no significant effect of dehulled adlay on plasma, kidney, and heart ACE activities in SHRs; these rats did, however, have significantly lower SBP at the end of the 12-week experimental period. Therefore, the lowered blood pressure associated with consuming dehulled adlay cannot be explained by its ACE inhibitory activity. Conversely, we found that both dehulled adlay intervention groups had significant lower plasma ET-1 and Cr concentrations than the non-treated SHR group did. Studies have indicated that ET-1 secretion raises blood pressure and accelerates the progression of nephropathy by stimulating vasoconstriction and the retention of water and sodium [26]. Therefore, our results indicated that dehulled adlay may not only retard the elevation of blood pressure in hypertensive rats, but also reduce the risk of kidney injury. In addition, we discovered, through human trials, that replacing part of daily staple food intake with dehulled adlay intake for up to six weeks produced positive effects of lower blood pressure. However, no significant change in ET-1 was found six 6 weeks. On the basis of these results, future studies should extend the experimental period or increase the sample size to further clarify the mechanisms underlying such outcomes.

Recent studies have found that uric acid is strongly linked to high blood pressure. A cross-sectional study reported that each 1 mg/dL increase in plasma uric acid increases the risk of hypertension by 20% [27]. Uric acid may directly cause endothelial dysfunction. When uric acid crystals are deposited in blood vessels, vascular inflammation and endothelial damage arise [28]. Moreover, uric acid could also affect vascular function through crystalline-independent pathways. Otani et al. [29] revealed that uric acid has the potential to reduce the phosphorylation of endothelial nitric oxide synthase and to damage endothelial function. Furthermore, hyperuricemia leads to increased ET-1 expression and renal injury [30]. In a hyperuricemic rat model, dehulled adlay extract effectively decreased serum uric acid levels by inhibiting xanthine oxidase [31]. In this study, we observed that rats fed high-dose dehulled adlay exhibited lower plasma uric acid levels than the SHR group, but the level was not significantly different from those of the WKY group. In addition, we ascertained that levels of the inflammatory-response indicators CRP and PAI-1 tended to decline as a result of consumption of an adlay-rich diet. These results indicated that the blood-pressure reduction associated with dehulled adlay may be related to moderating effects on uric acid and ET-1 levels.

This is the first study to apply dehulled adlay intake in the daily diet of participants with a high risk of hypertension, and we observed that replacing 60 g of staple food in daily diet with dehulled adlay helped moderate high blood pressure. In the animal study, we also discovered that dehulled adlay intake curbed blood-pressure elevation and lessened uric acid and ET-1 levels. However, some limitations were present in our study and may be rectified by further studies. First, we used a non-invasive tail-cuff method with a four-week interval to investigate the change of blood pressure in this study. A telemetry system may be more ideal, and could be used to record more hemodynamic information in future long-lasting experiments. Second, the number of human participants in this study was limited and lacked a control group. The single-arm study design followed by a pre-post evaluation can only offer preliminary information, and may not completely exclude the placebo effects of the intervention [32]. Although there currently are not enough previous references available about the effects of adlay on blood pressure, the results of this study can be used as a basis for further research. Future studies may increase the number of participants, extend the duration of the intervention period, use a control group consuming refined cereals, and measure dietary intake throughout the intervention to better observe more influential results and to clarify the related underlying mechanisms. Third, we used participants with high risks of cardiovascular diseases, such as overweight and obesity, in this study, and discovered that the effects of dehulled adlay intake on blood-pressure reduction was more evident in participants with high basal blood pressure. Therefore, future studies also could focus on patients diagnosed as having hypertension to further explore whether combining drug treatment with daily dehulled adlay consumption would have synergistic effects. The pathways related to uric acid and ET-1 also could be emphasized in future investigations.

#### **5. Conclusions**

In conclusion, our results suggested that daily intake of 60 g dehulled adlay had beneficial effects on blood-pressure management. Future studies could further clarify the possible underlying mechanisms for the consuming of dehulled adlay as a beneficial dietary approach for people at risk of hypertension.

**Author Contributions:** W.-J.Y. and H.-Y.Y. designed the study; W.-J.Y., J.K., W.-Y.C., and H.-Y.Y. conducted the experiments; and W.-J.Y., J.K., and H.-Y.Y. wrote the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by E-Da Hospital (Kaohsiung, Taiwan), grant number ISU 104-IUC-03. The APC received no external funding.

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institutional Review Board of E-Da Hospital (EMRP41104N 2016/02/04).

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.

**Conflicts of Interest:** The authors declare no conflict of interest, and all authors adhered to the Committee on Publication Ethics' guidelines on research and publication ethics.

#### **References**

