Effects of Roasting and Extrusion Puffing on the Antihypertensive Activity of Blended Grains in Spontaneously Hypertensive Rats

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Introduction
Hypertension is a severe disease with a high mortality rate worldwide that can lead to complications such as heart failure, arteriosclerosis, and stroke [1,2].Because essential hypertension accounts for over 80% of all hypertensive cases and occurs without any clear cause, research has been conducted to improve essential hypertension [3,4].Drugs such as captopril and ramipril are used to treat hypertension; however, they have been reported having side efects, including edema and dizziness [5].Consequently, there is an increasing interest in exploring natural foods for the treatment of hypertension [6].
Grains, one of the most consumed foods worldwide, may help alleviate cardiovascular disease and metabolic syndrome owing to their high phytochemical content, including polyphenol compounds, favonoids, and tannins [7,8].Sorghum (Sorghum bicolor) is abundant in various phenolic compounds, particularly 3-deoxyanthocyanidins and tannins, which can potentially reduce the risk of cardiovascular diseases [9,10].Adzuki bean (Vigna angularis) has been reported to be associated with the improvement of vascular oxidative stress and infammation owing to its rich content of phytochemicals, particularly catechins, procyanidins, and saponins [11,12].Finger millet (Eleusine coracana), a type of millet, is efective in maintaining blood pressure due to its elevated levels of polyphenols such as ferulic acid and proanthocyanidins, as well as its higher calcium content than other millet varieties [13,14].Accordingly, the antihypertensive efects of various grains, including sorghum, adzuki bean, and fnger millet, have been extensively investigated.
Grains are usually processed to enhance digestibility and eliminate antinutritional factors [15][16][17].Processing methods such as roasting and extrusion pufng are widely used in grain-based food production.Roasting involves a high-heat process for a short period of time, resulting in a crispy texture and enhanced favor [18].Extrusion pufng, however, utilizes gas pressure and thermal phase changes, which thereby increases volume and improves digestibility [19,20].As such, processing improves various aspects of grains; however, several studies have reported that thermal processing can result in a reduction in the bioactive compound content of the grains, thereby losing their potential health benefts [21,22].Tus, research on processing methods that can minimize the reduction in the antihypertensive activity of grains is necessary.
Nevertheless, research on processing methods without losing their biological activity of grains is limited, and comparative studies on the antihypertensive efects of roasting and extrusion pufng have not been conducted.Terefore, this study aimed to investigate the antihypertensive efects of blended grains to identify a processing method that preserves their antihypertensive activity.

Sample Preparation. Sorghum (Sorghum bicolor,
Sodamchal), adzuki bean (Vigna angularis, Arari), and fnger millet (Eleusine coracana, Finger1ho) were grown in 2021 and provided by the National Institute of Crop Science (Rural Development Administration, Suwon, Korea).After being fully ground, sorghum, adzuki bean, and fnger millet were blended in a ratio of 35 : 35 : 30.Te blending ratio was determined based on preliminary experiments evaluating the antihypertensive efects at various mixing ratios.Roasting was conducted at 220 °C for 5 min, and extrusion pufng was performed using a Welly snack machine S3 model (Jain Inc., Bucheon, Korea) at 120 °C and 120 rpm.

Determination of TPC and TFC in Blended Grain Extracts.
Te total phenolic content (TPC) and total favonoid content (TFC) of each sample were measured as follows: samples were extracted with 80% ethanol at a ratio of 1 : 10 (w/v) and concentrated using a rotary evaporator (PuSHB-3S, Daihan Scientifc, Wonju, Korea).TPC was determined using a method described by Xiang et al. [23], with some modifcations.In brief, 200 μL of the sample was mixed with 500 μL of 0.2 N Folin-Ciocalteu reagent and allowed to react for 5 min.Ten, 500 μL of 7.5% (w/v) Na 2 CO 3 was added, and the mixture was allowed to react for another at 50 °C 10 min.Subsequently, the absorbance at 760 nm was determined with a microplate reader (Bio Tek, Winooski, VT, USA).Gallic acid (Sigma-Aldrich, St. Louis, MO, USA) was used as a standard to quantify TPC.TFC was determined using a method described by Xiang et al. [23], with some modifcations.In brief, 300 μL of the sample was mixed with 300 μL of a 2% (w/v) aluminum chloride solution and allowed to react at room temperature for 10 min.Ten, the absorbance at 415 nm was determined with a microplate reader (Bio Tek).Quercetin (Sigma-Aldrich) was used as the standard to quantify TFC.

Analysis of the ACE Inhibitory Activity in Blended Grain
Extracts.An angiotensin-converting enzyme (ACE) inhibition assay was performed by slightly modifying the method described by Cushman and Cheung [24].Te amount of hippuric acid generated by the reaction of the substrate, hippuryl-His-Leu, with ACE was measured.Te ACE solution was extracted from lung acetone powder from rabbit (Sigma-Aldrich).Te substrate solution was prepared by dissolving hippuryl-His-Leu acetate salt (Sigma-Aldrich) in 0.1 M sodium borate bufer.Te substrate solution (100 μL) was mixed with the sample (50 μL) and reacted at 37 °C for 10 min.ACE solution (50 μL) was added and the reaction was conducted at 37 °C for 60 min.Te reaction was stopped by adding 250 μL of 1 N HCl.Te mixture was then partitioned with 1 mL of ethyl acetate (Samchun Chemicals, Seoul, Korea).Te supernatant (800 μL) was collected and dried.Next, the dried residue was dissolved in 1 mL of 0.1 M sodium borate bufer, and the absorbance thereof was measured at 228 nm using a spectrophotometer (GEN10S UV-Vis, Termo Fisher Scientifc, Waltham, MA, USA).Te ACE inhibitory activity was calculated using the following formula: ACE inhibitory activity (%) �  1.During the experimental period, body weight was measured once per week and feed intake was measured twice a week.After the experiment was completed, the animals were fasted for 12 h and then anesthetized via an intraperitoneal injection of xylazine (15 mg/kg BW) and ketamine (100 mg/kg BW).Blood was collected via cardiac puncture, and the kidneys and aortas were recovered via laparotomy.Te blood and organs were stored at −80 °C until analysis.

Growth Performance Analysis.
During the experimental period, body weight was measured once a week and feed intake was measured twice a week for 8 weeks to investigate the efects of blended grain intake on body weight, feed intake, and feed efciency ratio (FER).Te FER was calculated by dividing the weight gain during the experiment by the amount of feed intake during the same period.
2.6.Blood Pressure Measurement.During the experimental period, systolic blood pressure (SBP) and diastolic blood pressure (DBP) were measured once a week for 8 weeks using the tail-cuf method.Te rats were placed in a BP-2000 holder (Visitech Systems, Apex, NC, USA) and allowed to acclimate for 5 min in a heating chamber at 36 °C before the tail-cuf was attached to measure blood pressure.To minimize measurement errors, the blood pressure of each rat was measured at least three times.

Blood Biochemical Analysis.
Blood was collected via cardiac puncture, placed in tubes containing ethylenediaminetetraacetic acid (EDTA), and centrifuged to separate the plasma.Serum levels of total cholesterol (TC), triglycerides (TG), aspartate aminotransferase (AST), and alanine transaminase (ALT) were measured using commercial kits (Asan Pharmaceutical, Seoul, Korea).Serum ACE activity was measured using the method described by Schwager et al. [25], with some modifcations.In brief, 3 μL of serum was mixed with 30 μL of 5.7 mM hippuryl-His-Leu (Sigma-Aldrich) and incubated at 37 °C for 25 min.Ten, 117 μL of 0.28 M NaOH (Sigma-Aldrich) and 45 μL of 20 mg/mL o-phthaldialdehyde (Sigma-Aldrich) were added, and the mixture was incubated at room temperature for 10 min.Te reaction was stopped by adding 75 μL of 3 M HCl (Sigma-Aldrich), and the upper layer was extracted after centrifugation.Te fuorescence intensity was measured at excitation and emission wavelengths of 360 and 460 nm, respectively.Serum angiotensin II levels were measured using an angiotensin II ELISA kit (RayBiotech, Inc., Norcross, GA, USA).
2.9.Histological Analysis of the Aorta.On the day of autopsy, the recovered aortas were fxed in 10% neutral bufered formalin (Sigma-Aldrich), dehydrated, and embedded in parafn to prepare tissue sections.Te sections were stained with hematoxylin and eosin (H&E) to measure the intimamedia thickness (IMT) and lumen diameter (LD) of the blood vessel.Te IMT and LD of the aorta were measured using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Statistical Analysis.
All statistical analyses were performed using the GraphPad Prism 9 software (GraphPad Software, San Diego, CA, USA).Results are expressed as the mean ± standard error of the mean (SEM).One-way ANOVA was performed for comparisons among multiple groups, followed by Tukey's post hoc test.When the p value was less than 0.05 (p < 0.05), the results were considered statistically signifcant.

Results and Discussion
3.1.TPC, TFC, and ACE Inhibitory Activities.Te TPC, TFC, and ACE inhibitory activities of the blended grains are shown in Figure 1.TPC and TFC were signifcantly lower in both RBG and EPBG than in NPBG (p < 0.05).Among the processed blended grain groups, RBG showed signifcantly higher TPC and TFC than EPBG (p < 0.05).Tese fndings are consistent with those of a previous study that reported a decrease in phenolic compounds in processed grains [26].
Te reduction in TPC and TFC can be attributed to the high temperature during heating, which causes the oxidative breakdown of phenolic compounds in grains, such as sorghum or adzuki bean [27,28].Similarly, heat processes, such as roasting and boiling, can diminish the antioxidant activity of fnger millet via oxidation and decomposition reactions [29].Tese results suggested that thermal processing, including roasting and extrusion pufng, can negatively afect the TPC and TFC of grains.However, it is worth noting that roasting may retain higher TPC and TFC than extrusion pufng.A previous study reported that roasting could preserve the antioxidant activity by generating Maillard reaction products [30].In addition, roasting can damage the cellular structure of cereal grains, leading to the release of bound phenolic compounds [31].Furthermore, the thermal decomposition of protein-lipid complexes that occurs during roasting may be associated with an increase in carotenoid content [32].Based on these studies, it can be inferred that roasting might more greatly mitigate the decrease of TPC and TFC than extrusion pufng.
ACE inhibitory activity was signifcantly lower in RBG and EPBG than in NPBG (p < 0.05).In addition, EPBG signifcantly decreased ACE inhibitory activity compared to RBG (p < 0.05).Te decrease in the ACE inhibitory activity can be attributed to a reduction in TPC and TFC during processing.Phenolic compounds can suppress ACE by competing with its active site [33].Similarly, a previous study showed that the ACE inhibitory activity of whole rice was signifcantly reduced by processing because of the decrease in phenolic compounds [34].In conclusion, roasting and extrusion pufng decreased the content of bioactive compounds and ACE inhibitory activity in blended grains; however, roasting may better retain bioactive compounds and ACE inhibitory activity than extrusion pufng.

Growth Performance.
Te growth performances of the experimental animals are shown in Table 2. Body weights were signifcantly lower in all SHR experimental groups (MC, NPBG, RBG, and EPBG) than in the NC group (p < 0.05).Tese results are consistent with those of previous studies showing that SHRs have a lower weight gain rate than WKY due to genetic diferences [35,36].Notably, the fnal body weight of the NPBG rats was signifcantly higher than that of the MC, RBG, and EPBG rats (p < 0.05).
Tese fndings suggest that NPBG may help promote growth by overcoming the weight gain resistance attributed to hypertension-related genes.In addition, FER was signifcantly higher in the NC group than in the MC, NPBG, RBG, and EPBG groups (p < 0.05).However, there were no signifcant diferences in feed intake and FER among the MC, NPBG, RBG, and EPBG groups.In summary, the efects of weight gain were observed only in the NPBG group, whereas the consumption of blended grains did not afect feed intake and FER in SHR.

Systolic Blood Pressure and Diastolic Blood Pressure.
Changes in SBP and DBP in the experimental animals are shown in Figure 2. Initially, all SHRs exhibited elevated SBP and DBP, confrming the successful establishment of essential hypertension.Troughout the experimental period, the NC maintained a normal range of blood pressure, whereas the MC showed a progressive increase in blood pressure.Starting from the third week of the experiment, SBP in the NPBG, RBG, and EPBG groups was signifcantly lower than that in the MC group (p < 0.05).In addition, SBP in the fnal week was signifcantly decreased in all blended grain groups compared to MC (p < 0.05); NPBG exhibited the greatest reduction, followed by RBG, and then EPBG.As for DBP in the fnal week, only the NPBG and RBG showed a signifcant decrease compared with the MC (p < 0.05), whereas the reduction observed in the EPBG was not statistically signifcant.
Te blood pressure-lowering efects of blended grains, including adzuki bean, fnger millet, and sorghum, are supported by the results of previous studies.Adzuki bean and fnger millet extract alleviated blood pressure by regulating the renin-angiotensin system or inhibiting infammatory cytokines [37][38][39].In addition, sorghum consumption reduces blood pressure and protects against oxidative stress-induced vascular damage [40].More importantly, this study revealed that roasting had superior blood pressure-lowering efects compared to extrusion pufng.Our in vitro investigations found that the TPC and TFC of the blended grains decreased after processing, which may correlate with the blood pressure results.A previous study has shown that polyphenols, which are abundant in the bran of grains, can reduce blood pressure in spontaneously hypertensive rats via nitric oxide-mediated vasodilation [41].In addition, dietary favonoids exert blood pressure-lowering efects in spontaneously hypertensive rats by improving endothelial function [42].
Tus, roasting, which exhibits a lesser reduction in bioactive compounds than extrusion pufng, can be an efective processing method for maintaining antihypertensive activity.Considering that most grains undergo processing before consumption, it is essential to highlight the potential of roasting as a promising processing for preserving the antihypertensive properties of blended grains.

Blood Biochemical Analysis.
Te AST, ALT, TG, and TC levels in the experimental animals are presented in Table 3. AST and ALT are liver enzymes that increase with liver 4 Journal of Food Biochemistry damage and are indicators of hepatic toxicity [43,44].Serum AST levels were not signifcantly diferent among all experimental groups; however, serum ALT levels were signifcantly increased in MC compared with NC (p < 0.05), and there were no signifcant diferences in NPBG, RBG, and EPBG compared with MC.Tese results are consistent with a previous study that reported that higher levels of ALT in hypertension are associated with the infammatory response in hypertension [45].Importantly, the ASTand ALT levels of all experimental groups were within the normal range (<40 IU/L), suggesting that there was no hepatotoxicity from consuming the blended grains.Serum TG levels were not signifcantly diferent among all experimental groups; however, serum TC levels were signifcantly increased in the NC group compared to all SHR experimental groups (p < 0.05).However, there was no signifcant diference in TC levels among the SHR experimental groups.In summary, serum TG and TC levels did not exhibit signifcant diferences among all SHR experimental groups, indicating that the consumption of blended grains did not afect serum lipid profles.

ACE Activity and the Angiotensin II Level in Serum and
mRNA Expression of Renin in the Kidney.Te reninangiotensin system plays a crucial role in regulating blood pressure.ACE converts angiotensin I to angiotensin II, resulting in vasoconstriction and an increased blood pressure [46,47].A previous study showed that decreased ACE activity is associated with reduced systolic blood pressure in SHR [48].Angiotensin II is generated by ACE via the removal of two C-terminal residues (His-Leu) from angiotensin I [25].Renin is a hormone secreted by the kidneys that participates in aldosterone synthesis and angiotensin activation.Decreased renin mRNA expression is associated with improved hypertension [49].
In this study, serum ACE activity, angiotensin II levels, and renal mRNA expression of renin in rats were analyzed (Figure 3).MC exhibited higher ACE activity, angiotensin II levels, and renin mRNA expression in the kidney than NC (p < 0.05).Tese results are consistent with those of a previous study showing that SHRs have enhanced activity within the renin-angiotensin system associated with hypertension [50].Notably, NPBG, RBG, and EPBG signifcantly reduced the serum ACE activity compared to that in the MC group (p < 0.05).Tis can be attributed to the ACE inhibitory potentials of grains enriched in polyphenols, which bind to the ACE active site [51,52].In addition, all blended grain groups showed signifcantly decreased angiotensin II levels compared with the MC group (p < 0.05).Specifcally, angiotensin II levels were signifcantly lower in the NPBG and RBG than in the EPBG (p < 0.05).Furthermore, the renin mRNA expression was signifcantly decreased in the NPBG and RBG compared with that in the MC (p < 0.05).
Interestingly, NPBG and RBG showed a higher ability to modulate the renin-angiotensin system than EPBG, which can be attributed to the impact of processing on polyphenol content and bioavailability.A previous study demonstrated the potential loss of bioactive constituents during extrusion pufng [53,54].In addition, the low efectiveness of EPBG in regulating the renin-angiotensin system was supported by in vitro experiments that demonstrated low ACE inhibitory activity of EPBG.Overall, all the blended grains regulated the renin-angiotensin system, which is the main mechanism of hypertension, with NPBG and RBG exhibiting greater modulatory efects than EPBG.3.6.Histological Analysis of the Aorta.Histological changes in the aorta are illustrated in Figure 4. Te IMTand ratio of IMT to LD of the aorta signifcantly increased in the MC group compared to those in the NC group (p < 0.05), indicating elevated vascular resistance.Te diference in vascular thickness between WKY and SHR is consistent with a previous study that SHR are stimulated by angiotensin, leading to thicker vascular walls [55].Notably, NPBG signifcantly decreased IMT and the ratio of IMT to LD of the aorta compared to MC (p < 0.05), whereas RBG and EPBG showed no signifcant diferences compared to MC.However, there was no signifcant diference in the LD of the blood vessels among the experimental groups.While previous studies have demonstrated the positive efect of grains on vascular remodeling, this is the frst study to investigate the efects of diferently processed grains on the aorta.A previous study demonstrated that the ACE inhibitory activity plays a signifcant role in ameliorating vascular remodeling by suppressing vascular constriction [56].In line with these fndings, NPBG showed a remarkable improvement in vascular remodeling of the aorta, which may be attributed to its ACE inhibitory activity.However, RBG and EPBG, which demonstrated ACE inhibitory efects, did not improve vascular remodeling in the aorta.Terefore, the NPBG has the greatest potential to mitigate vascular remodeling compared with the RBG and EPBG.Further research is required to explore processing methods beyond roasting and extrusion pufng, which may have a positive impact on vascular remodeling.

Conclusions
In this study, the intake of all blended grains dramatically reduced SBP in SHR, and the efects of reduction are as follows: NPBG > RBG > EPBG.In addition, NPBG and RBG signifcantly lowered DBP.Furthermore, all blended grains resulted in a considerable reduction in serum ACE activity and angiotensin II levels, with NPBG and RBG showing the most signifcant reduction in angiotensin II levels.NPBG and RBG also signifcantly decreased renin mRNA expression in SHR, and NPBG remarkably improved aortic vascular remodeling.Tese fndings indicated that the antihypertensive efects of the blended grains aligned with the trend observed in vitro.Considering that most grains undergo processing before consumption, roasting could be an efective method to minimize the reduction in the antihypertensive properties of blended grains.Terefore, roasting can be used to develop processed products from blended grains.

Figure 2 :Figure 3 :
Figure 2: (a) Weekly systolic blood pressure, (b) systolic blood pressure at week 0 and week 8, (c) weekly diastolic blood pressure, and (d) diastolic blood pressure at week 0 and week 8 in the rats supplemented with diferently processed blended grains for 8 weeks.NC, normal control; MC, model control; NPBG, nonprocessed blended grains; RBG, roasted blended grains; EPBG, extrusion pufed blended grains.Data are expressed as the means ± standard error of the means.* Signifcant diferences from MC at p < 0.05. (A-B) Te diference in systolic blood pressure at week 0 indicates signifcant diferences at p < 0.05. (a-d) Te diference in systolic blood pressure at week 8 indicates signifcant diferences at p < 0.05.

Table 2 :
Efects of diferently processed blended grains on growth performance in rats for 8 weeks.NC, normal control; MC, model control; NPBG, nonprocessed blended grains; RBG, roasted blended grains; EPBG, extrusion pufed blended grains; FER, feed efciency ratio.Data are expressed as the means ± standard error of the means.(a-c)Valueswith diferent letters within the same row are signifcantly diferent at p < 0.05.

Table 3 :
Efects of diferently processed blended grains on blood biochemical parameters in the serum of the rats for 8 weeks.RBG, roasted blended grains; EPBG, extrusion pufed blended grains.Data are expressed as the means ± standard error of the means.(a-c)Valueswith diferent letters within the same row are signifcantly diferent at p < 0.05.