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Antihypertensive and Immunomodulatory Effects of Defatted Corn Germ Hydrolysates: An in vivo Study

This study investigated the antihypertensive and immunomodulatory effects of defatted corn germ hydrolysates (DCGHs) in vivo and their potential regulatory mechanisms. The systolic blood pressure (SBP) of spontaneously hypertensive rats (SHRs) was significantly reduced (10.30%) by the long-term intragastric administration of DCGHs (high doses). Also, there was drastic inhibition of angiotensin-I-converting enzyme (ACE) activity in the lung, kidney, and heart tissues by 24.53, 22.28, and 12.93%, respectively. It could regulate the blood pressure by adjusting the balance between endothelium-derived vasoconstrictor factors and endothelium-derived relaxing factors. Meanwhile, DCGHs enhanced the phagocytosis of mononuclear macrophages, cellular immunity, and humoral immunity of ICR mice by increasing the phagocytic index of mononuclear macrophages (23.71%), ear swelling degree (44.82%), and antibody levels (52.32%). Moreover, it stimulated the release of immunoactive substances (e.g., lysozyme, interferon-γ, immunoglobulin G, and complement 3). Consequently, DCGHs could suitably be used in the formulation of novel functional foods with antihypertensive and immunomodulatory properties.


INTRODUCTION
Defatted corn germ (DCG), a by-product of corn germ oil production, is often used as feed, resulting in a tremendous waste of nutritional value (1). The proteins in DCG are rich and balanced in most of the essential amino acids and mainly consist of soluble alkali proteins, albumins, and globulins, with a protein efficiency ratio similar to that of soy protein, and thus, DCG can be used as a high-quality protein source (2,3). DCG can produce a significant quantity of active peptides via protease hydrolysis. Studies have shown that polypeptides have antihypertensive and immunomodulatory activities, which play an essential role in metabolic regulation and can serve as functional food ingredients, nutraceuticals, and pharmaceutical additives to improve human health and prevent diseases (4)(5)(6).
Hypertension is a chronic disease in which the blood pressure is higher than usual [systolic blood pressure (SBP) ≥140 mmHg, diastolic blood pressure ≥90 mmHg] (7). It has the characteristics of high incidence and gradual rejuvenation and can cause various diseases, such as cardiovascular disease, nephropathy, and myocardial infarction (8). The control of blood pressure in the body is dependent on the regulation of the renin-angiotensin system (RAS), and the angiotensin-converting enzyme (ACE) plays a vital role in the RAS (9). In RAS, renin acts on angiotensinogen to convert it into angiotensin I (Ang I), and under the action of ACE, Ang I is converted into angiotensin II (Ang II), causing an increase in blood pressure (10). At present, the drugs used to treat hypertension mainly include calcium channel antagonists, ACE inhibitors, angiotensin receptor blockers, and β-receptor blockers. However, these drugs have many side effects, such as skin rashes, disturbance of taste, dry cough, and allergic reactions (11,12). Maes et al. (13) reported that milk protein-derived peptides could lower blood pressure in vivo by inhibiting the release of endothelin-1 (ET-1) from the endothelial cells of spontaneously hypertensive rats (SHRs). Alashi et al. (14) indicated that rapeseed peptides induced antihypertensive effects on SHRs. Therefore, bioactive substances derived from natural sources, which exert non-toxic side effects and hypotensive effects, have gradually become the focus of research.
Immunity protects organisms against harmful foreign bacteria and viruses (15). The immune system of the body is regulated by the interactions among macrophages, lymphocytes, and their immunoactive substances, which is manifested in the promotion of the growth of the immune organs, proliferation of immune cells, and release of immunoactive substances (16). Mao et al. reported that casein peptides obtained via alkaline protease hydrolysis could mediate immunoregulation by stimulating the release of ConA-induced lymphocyte proliferation and interleukin (IL)-2 in mice (5). Sun et al. reported that the polypeptides from Pleurotus eryngii mycelium could exert immunomodulatory effects by promoting the proliferation of macrophages and stimulating the release of tumor necrosis factor-α (TNF-α), IL-6, nitric oxide (NO), and H 2 O 2 (17). Hence, the study of food-derived substances with immunomodulatory properties is gradually receiving widespread research attention.
Therefore, the purpose of this study was to evaluate the antihypertensive and immunomodulatory effects of DCGHs through animal experiments to study the more effective utilization of corn germ oil by-products. The antihypertensive effect of DCGHs in vivo was evaluated in terms of body weight (BW), blood pressure, tissue ACE activity, and serum biochemical indices of SHRs after acute oral and long-term intragastric administration. Meanwhile, the immunoregulatory effect of DCGHs was studied by testing the immune organ indices, phagocytic index of mononuclear macrophages, ear swelling degree, antibody levels, and serum immunoactive substance levels in ICR mice.

Preparation of DCGHs
DCGHs was prepared from DCG using the ultrasonicassisted enzymatic hydrolysis method, as described in a previous study (19). Briefly, energy-concentrated dualfrequency ultrasonic equipment was utilized in pretreating the substrate before enzymatic hydrolysis. Ultrasonic treatment was achieved by employing the optimal ultrasonic conditions from our preliminary study, that is, ultrasonic power density of 120 W/L, 15 min sonication time, and treatment temperature of 30 • C. After the pretreatment, the substrate was subjected to enzymatic hydrolysis to obtain DCGHs under the hydrolysis conditions with a temperature of 50 • C, a pH of 9.0, an enzyme dosage of 3,000 U/g, and reaction time of 30 min. From the above conditions, a total protein conversion degree of 63.34% was obtained. Figure 1 displays the preparation process and the in vivo study of DCGHs.

Animals and Experimental Protocol
All the experiments were approved by the Institutional Animal Care and Use Committee of Jiangsu University and met the guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Thirty 13-week-old malespecific pathogen-free SHRs were purchased from Vital River Experimental Animal Technical Co. Ltd. (Beijing, China) (No. 11400700249349). They were raised in the barrier system of the Laboratory Animal Research Center of Jiangsu University. The conditions of the breeding room were temperature of 25 ± 2 • C, humidity of 50 ± 5%, and daily light-dark cycle of 12 h. Before the administration test, the SHRs were subjected to a 1-week adaptation period. After a week of acclimatization, 30 SHRs were randomly divided into five groups (n = 6): control (administered a sterile saline solution), captopril (administered captopril at a dose of 30 mg/kg BW), lowdose (administered DCGHs at a dose of 500 mg/kg BW), middle-dose (administered DCGHs at a dose of 1,000 mg/kg BW), and high-dose (administered DCGHs at a dose of 1,500 mg/kg BW). At the beginning of the administration of these substances, the SBP of SHRs was measured and recorded every 3 from 0 to 12 h to perform the acute oral test. The SHRs were then intragastrically administered the aforementioned substances (depending on the group) daily for 5 weeks to perform a long-term administration test. The SBP of SHRs was measured using the tail-cuff method with an ALC-NIBP noninvasive blood pressure system once a week (Allcott Biotech Co. Ltd. Shanghai, China). Each measurement was repeated three times and the average was recorded. The same person performed all measurements at the same time of day and within the same environment to minimize blood pressure measurement errors.

Determination of Body Weight and Organ Index
In the long-term intragastric administration experiment, the BW of the SHRs was measured once a week. After 5 weeks, 1.5 mL of chloral hydrate solution (10%) was injected into the abdominal cavity of SHRs to induce anesthesia, following which the rats were euthanized and dissected. The celiac artery blood of the SHRs was collected using blood collection tubes not containing anticoagulants and stored at 25 ± 2 • C for subsequent use. The heart, liver, spleen, lung, and kidney of SHRs were excised and weighed, and the organ index was calculated according to the following Equation (1):

Determination of Tissue ACE Activity
The extraction of ACE and determination of its activity in the lung, kidney, heart, and artery were performed using the method described by Guo et al. (20).

Biochemical Index Analysis of Serum
The celiac artery blood of the SHRs collected as described in section Determination of Body Weight and Organ Index was stored for 1 h to coagulate spontaneously and then centrifuged at 3,000 rpm for 10 min. The supernatant (serum) was aliquoted into different centrifuge tubes and stored at 4 • C for further use. The levels of renin, Ang II, ET-1, NO, PGI 2 , TNF-α, GST, and CK-MB in the serum of the SHRs were determined using the experimental kits described in section Materials. The operation method referred to the kit instructions (20).

Animals and Experimental Protocol
One hundred and sixty 6-week-old female-specific pathogenfree ICR mice were obtained from the Laboratory Animal  I, II, III, and IV) consisted of 40 ICR mice, which were randomly divided into four groups (n = 10): control (administered a sterile saline solution), lowdose (DCGHs administered at a dose of 100 mg/kg BW), middledose (DCGHs administered at a dose of 500 mg/kg BW), and high-dose groups (DCGHs administered at a dose of 1,000 mg/kg BW). These substances were administered intragastrically daily for 5 weeks. After the fifth week, the BW of ICR mice in the four tests were recorded and the body weight gain before and after the long-term administration experiment was calculated. Simultaneously, the spleen, thymus, heart, lung, liver, and kidney of ICR mice subjected to the test IV were excised and weighed to study the effect of DCGHs on the organ index of ICR mice. The organ index was calculated by Equation (1) in section Determination of Body Weight and Organ Index. Then, ICR mice subjected to tests I, II, III, and IV underwent carbon clearance, DTH, serum hemolysin, and serum immunoactive substance tests, respectively, to analyze the immunomodulatory effect of DCGHs on the body after long-term intragastric administration.

Determination of Macrophage Phagocytic Index, Ear Swelling Degree, and Antibody Levels
The macrophage phagocytic index, ear swelling degree, and antibody levels of ICR mice were measured using the carbon clearance (I), DTH (II), and serum hemolysin (III) tests, respectively. The carbon clearance test (I) referred to the method described by Suzuki et al. (21) with some modifications. After long-term intragastric administration, the tail vein of ICR mice was injected with diluted Indian ink (100 mL/kg BW). Blood samples (20 µL) were collected from the retro-orbital venous plexus of ICR mice at the second and tenth minute after the injection and then immediately added to 2 mL of 0.1% Na 2 CO 3 solution. The optical density (OD) at 600 nm was measured using a Multiskan spectrum microplate spectrophotometer (Infinite 200 PRO, Tecan Co., Ltd., Germany), and Na 2 CO 3 solution was used as the control. The phagocytosis index was calculated using the following equation: where OD 1 and OD 2 are the ODs at times t 1 (second minute) and t 2 (tenth minute), respectively.
The DTH test (II) was performed as described by Sakai et al. (22) with some modifications. The abdominal skin of each mouse was wiped with barium sulfide, shaved, and then 50 µL of 1% DNFB in acetone-seed oil (1:1) was evenly applied to the area to sensitize the skin of the mice. After 5 days, the right ears were challenged with 10 µL of 1% DNFB in acetone-seed oil (1:1), and the left ears were untreated. After 24 h, the ears with a diameter of 8 mm were removed using a hole punch and weighed. Ear swelling was expressed as the weight difference between the left and right ears.
The serum hemolysin test (III) was performed as described by Tort et al. (23). After the long-term administration experiment, the ICR mice in test III were intraperitoneally injected with 0.2 mL 2% SRBC. After 5 days, the retro-orbital venous plexus blood of ICR mice was collected in blood collection tubes not containing anticoagulants and stored at room temperature for 1 h for spontaneous blood coagulation. Next, the blood samples were centrifuged at 3,000 rpm for 10 min, and the serum was collected and diluted different multiples by the double dilution method. Then, 100 µL serum of different concentrations and 100 µL 0.5% (v/v) SRBC suspension were added to the micro hemagglutination test plate in turn. After mixing evenly, they were placed in a moist flat plate and incubated in an incubator at 37 • C for 3 h. The degree of hemagglutination was observed and the antibody level was calculated by the following equation: where 1, 2, 3. . . , n, represent the index of double dilution, S represents the level of agglutination.

Determination of Serum Immunoactive Substances
The process of immune regulation is often accompanied by changes in the content of immunoactive substances. The ICR mice subjected to test IV underwent a serum immunoactive substances test, and the determination method referred to the article by Huang et al. (16). The serum of ICR mice was collected by the aforementioned method in the serum hemolysin test (III). After centrifugation, the serum was aliquoted into different centrifuge tubes and stored at 4 • C for use. The levels of lysozyme, IFN-γ, IgA, IgG, IgM, and C3 in the serum of ICR mice were determined using the experimental kits described in section Materials. The operation method referred to the kit instructions (17).

Statistical Analysis
The experimental data were expressed as the mean ± standard deviation (SD). The differences between groups were evaluated using the one-way analysis of variance (ANOVA). The statistical tests were analyzed using SPSS 19.0 (SPSS Inc., USA) at the significance levels of p < 0.05 and p < 0.01.

Molecular Weight Distribution
The molecular weight composition of peptides is an important index to evaluate the nutritional value of enzymatic hydrolysate, which is closely related to the absorption rate and physiological activity of enzymatic hydrolysate (24,25). The molecular weight distributions of the DCGHs prepared using ultrasonic-assisted enzymatic hydrolysis are presented in Table 1. The fraction with a molecular weight of more than 2,000 Da accounted for the largest proportion (36.56%), followed by the range of 200-1,000 Da (35.97%), 1,000-2,000 Da (17.15%), and <200 Da (10.32%). Studies have shown that the polypeptide fractions with larger relative molecular weight prepared by hydrolysis could effectively utilize the protease in the gastrointestinal tract and have high biological activity after gastrointestinal digestion, even reaching or exceeding the functional activity level of traditional polypeptides (26). The result showed that DCGHs with molecular weight >2,000 and 200-1,000 Da accounted for more than 80%, which is similar to the results of the molecular weight distribution of walnut proteolysis products studied by Liu et al. (27), indicating that DCGHs may have good biological activity for application.

BW and Organ Indices of the SHRs
The BW gain and organ indices are shown in Table 2. In the whole experiment, the BW gain of the SHRs in the tested groups was not significantly different from that of the control group (p > 0.05). In contrast, the BW gain of the SHRs in the captopril group was significantly lower than that in the control group (p < 0.01). Although captopril (an ACE inhibitor) has been prescribed for the treatment of cardiovascular disease, long-term use could cause some side effects such as vascular edema, rash, and dyspepsia (28). Hence, the weight of SHRs in the captopril group was significantly lower than that of the control group. Also, the organ indices of the SHRs in the tested groups and captopril group were not significantly different from those in the control group (p > 0.05), indicating that DCGHs and captopril did not affect the growth of organs in SHRs.

Blood Pressure of SHRs
In the acute oral intragastric administration experiment, variations in SBP in different groups of SHRs are shown in Figure 2A.   group exerted a more rapid blood pressure-lowering effect than the tested groups initially, there was no difference between the tested groups and the captopril group after 6 h. Cui et al. (29) studied the antihypertensive effect of milk macromolecular peptides in the acute oral intragastric administration experiment. They found that the blood pressure of SHRs in the tested groups also reached the maximum decline at 6 h. The results initially showed that DCGHs had an antihypertensive effect in vivo in a short time, which was consistent with the result of milk macromolecular peptides.
The SBP of the control, tested, and captopril groups during the long-term intragastric administration test is shown in Figure 2B. The SBP of the captopril group was significantly lower than that of the control group from the first week to the fifth week and reached the maximum decrease of 29.25 mmHg at the fifth week (p < 0.01), revealing that the antihypertensive effect of captopril was rapid and long-lasting. All the tested groups presented a significant antihypertensive effect on SHRs from the second week (p < 0.05). The SBP of the high-dose group decreased more rapidly than that of the control group in the second week and maintained a downward trend until the fifth week, which attained the maximum decrease of 18.94 mmHg. In comparison with the control group, the SBP of the low-dose and middle-dose groups tended to be stable from the fourth week to the last week and decreased by 8.97 mmHg (p < 0.05) and 13.93 mmHg (p < 0.01), respectively, in the last week. The results showed that different doses of DCGHs also had a long-term and stable antihypertensive effect on SHRs, especially in the high-dose group. Wu et al. (30) found that different doses of ACE inhibitory peptides prepared from soy protein could significantly reduce the blood pressure of SHRs compared with the control group, which was consistent with our results. Therefore, DCGHs has good antihypertensive effect in vivo.

ACE Activity in the Tissues of SHRs
The ACE activity values are presented in Table 3. The ACE activity of the lung, kidney, heart, and aorta in the tested groups and captopril group decreased to different degrees than those in the control group. In comparison with the control group, ACE activity in the lung tissues of the middle-dose, high-dose, and captopril groups exhibited a significant reduction of 17.10, 24.53, and 30.83% (p < 0.01), while the low-dose group showed no significant changes (p > 0.05). Compared to the control group, ACE activity in the kidney and heart tissues of the high-dose group showed a significant reduction of 22.28% (p < 0.01) and 12.93% (p < 0.05), respectively, and the captopril group exhibited a marked decrease of 34.11% (p < 0.01) and 25.70% (p < 0.01), respectively, while the other tested groups showed minimal changes (p > 0.05). There were limited changes in ACE activity in the aorta among the control, tested, and captopril groups. Previous studies have shown that ACE exists widely in epithelial cells, neuroepithelial cells, and vascular endothelial cells in many organs (31). As shown in Table 3, the low-dose group showed limited effects on ACE activity in the tissues, the middle-dose group showed significantly reduced ACE activity in the lung tissues, and the high-dose and captopril groups showed significantly reduced ACE activity in the lung, kidney, and heart tissues. Moreover, DCGHs showed a doseresponse relationship with ACE inhibitory activity. Therefore, DCGHs could exert an antihypertensive effect by inhibiting ACE activity in the lung, kidney, and heart tissues. Guo et al. (32) found that the corn germ peptides, prepared by an enzymatic membrane reactor with a new gradient diafiltration feeding working-mode (processing duration of 660 min), could significantly increase the tissue ACE activity of SHRs in a long-term intragastric administration. DCGHs can therefore be compared to corn germ peptides since they have similar ACE inhibitory activity in the body, and its preparation process (ultrasonic-assisted) makes full use of the protein resources of DCG, which dramatically shortens the enzymatic hydrolysis time and reduces energy consumption.

Serum Biochemical Indices of SHRs
The endothelium-derived vasoconstrictor factors (EDCFs) and endothelium-derived relaxing factors (EDRFs) secreted by  vascular endothelial cells can regulate blood pressure and endothelial function (20). To further explore the mechanism underlying the antihypertensive effects of DCGHs in vivo, the serum biochemical indicators of SHRs were analyzed. As shown in Table 4, compared with the control group, the content of renin in the middle-dose and high-dose groups showed a marked decrease of 14.01 and 14.62% (p < 0.05), while the content of renin in the captopril group significantly increased by 12.57% (p < 0.05). In addition to the low-dose group, the content of Ang II in the middle-dose, high-dose, and captopril groups was significantly different from that in the control group (p < 0.01), representing a decrease of 10.60, 10.80, and 13.30%, respectively. Renin (an EDCF) initiates the RAS chain reaction and causes an increase in blood pressure. The expression of RAS promotes the production of Ang II (an EDCF), which regulates vasoconstriction and stimulates the vascular endothelial cells to secrete ET-1 (an EDCF) (33). The results showed that DCGHs could regulate blood pressure by promoting the production of renin and Ang II, while captopril had a rapid antihypertensive effect, resulting in a fast decrease in Ang II levels. This led to an increase in renin content to maintain the balance of the RAS. As shown in Table 4, the content of ET-1 (an EDCF) in the high-dose and captopril groups decreased significantly by 23.62% (p < 0.01) and 28.17% (p < 0.01), respectively, compared with the control group. The content of NO (an EDRF) in the tested and captopril groups increased significantly compared with the control group (p < 0.05); particularly, in the high-dose group, NO content increased by 36.53% (p < 0.01). ET-1 (an EDCF), a subtype of endothelin, has the effect of regulating vasoconstriction (13). NO (an EDRF) has the effect of protecting vascular smooth muscle tissue, regulating angiogenesis, and preventing harmful blood vessel contraction (34). The results indicated that DCGHs could exert a hypotensive effect by inhibiting the release of ET-1 and promoting the release of NO. PGI 2 (an EDRF) can relax smooth muscle cells of different blood vessel beds to regulate systemic vascular tone (35). The results in Table 4 show that PGI 2 content in the tested groups showed less change compared with the control group (p > 0.05), while that of the captopril group increased significantly by 21.58% compared with the control group (p < 0.01). Therefore, DCGHs had little effect on blood pressure regulation through PGI 2 . The increase in TNF-α (an EDCF) content in the serum can cause hypertrophy and proliferation of vascular smooth muscle cells, leading to an increase in blood pressure (36). As described in Table 4, the content of TNF-α in the tested and captopril groups was significantly higher than that in the control group (p < 0.05), indicating that DCGHs can regulate blood pressure through TNF-α. GST has antioxidative and detoxifying effects, and its concentration changes in serum reflect the degree of damage to the liver (37). The concentration of CK-MB in the serum can increase to a certain extent due to myocardial injury or necrosis (38). As observed in Table 4, in comparison with the control group, the content of GST and CK-MB in the tested groups showed less change (p > 0.05), while the content of GST and CK-MB in the captopril group increased by 15.81% (p < 0.01) and 6.63% (p < 0.05), respectively. Captopril possibly caused damage to the myocardium and liver of SHRs, and DCGHs were safe for the body of SHRs. Thus, the assessment of the serum biochemical indicators showed that the mechanism underlying the antihypertensive effects of DCGHs involved the regulation of the balance between EDCFs and EDRFs in the serum (39).  In vivo Immunomodulation Experiment

BW and Organ Indices of ICR Mice
The results of BW gain and organ indices of ICR mice in the long-term intragastric administration experiment are shown in , the weights of the spleen, thymus, heart, lung, liver, and kidney of ICR mice between the control group and tested groups were not significantly different (p > 0.05), which confirmed the non-toxic side effects and safety use of DCGHs. Therefore, DCGHs can be preliminarily used as a food-borne substance that is harmless to the body.

Macrophage Phagocytic Index, Ear Swelling Degree, and Antibody Levels of ICR Mice
The results of the carbon clearance, DTH, and serum hemolysin tests are shown in Table 6, which reflect the phagocytic function of mononuclear macrophages, cellular immune function, and humoral immune function, respectively (40). The phagocytic index of mononuclear macrophages in the middle-dose and high-dose groups was significantly higher than that in the control group, increasing by 17.18% (p < 0.01) and 23.71% (p < 0.01), respectively. The low-dose group showed less difference compared with the control group (p > 0.05). The phagocytic index reflects the phagocytic ability of mononuclear macrophages. The higher the phagocytic index, the stronger the phagocytic capacity. The results showed that DCGHs could improve the phagocytic capacity of mononuclear macrophages in ICR mice. DTH reflects the level of cellular immune function, and the magnitude of it is expressed by ear swelling degree. As shown in Table 6, the ear swelling degree of ICR mice in the low-dose group was not significantly different from that of the control group, while the ear swelling degree of ICR mice in the middle-dose and high-dose groups was 21.95% (p < 0.05) and 44.82% (p < 0.01) higher than that of the mice in the control group, indicating that DCGHs could improve the  (41) found that detoxified rapeseed peptides had a significant effect on the phagocytic ability and ear swelling degree of ICR mice, which presented a good regulatory effect on immunity. Therefore, DCGHs also have a good immunomodulatory effect in vivo compared with the detoxified rapeseed peptides.

Immunoactive Substances in the Serum of ICR Mice
To further explore the immunomodulatory effects of DCGHs in vivo, the immunoactive substances in the serum of ICR mice were determined. As shown in Table 7, lysozyme content in the tested groups was significantly higher than that in the control group (p < 0.05). Particularly, in the high-dose group, lysozyme content increased by 34.97% (p < 0.01). Lysozyme can kill pathogenic bacteria and alleviate inflammation, reflecting the degree of nonspecific immune regulation (42). Therefore, DCGHs promoted the release of lysozyme in ICR mice to regulate immune function. IFN-γ, a cytokine, can activate immune cells, improve the ability to kill pathogens and tumor cells, and promote phagocytosis of macrophages (43,44). Table 7 shows that the content of IFN-γ in the high-dose group increased by 18.53% (p < 0.05) compared with the control group, but there was no significant change in the other tested groups (p > 0.05). Studies have shown that low levels of IFN-γ can cause immune system dysfunction in mice, resulting in a decline in immunity (45). Therefore, the results showed that DCGHs could stimulate the release of IFN-γ to enhance the immune function of the body. Immunoglobulin is a soluble globulin with humoral immune function. As shown in Table 7, IgA and IgM content in the tested groups was not significantly different from that in the control group (p > 0.05), while the content of IgG in the middle-dose and highdose groups was significantly higher than that in the control group (p < 0.01), showing an increase of 38.42 and 21.72%, respectively. IgG levels were the highest in the serum, and this immunoglobulin has various immunomodulatory activities, such as antibacterial, antiviral, and antitoxin effects. It can also promote the connection between tumor cells and effector cells, causing cytotoxicity to kill tumor cells and other target cells (46). The results showed that DCGHs could act as immunomodulators by increasing the IgG content in the serum. C3, the most abundant protein in the complement system, can be cleaved into biologically active fragments after activation, which assists phagocytes and antibodies in killing pathogenic bacteria and strengthening immune function (47). As indicated in Table 7, C3 content in the middle-dose and high-dose groups was significantly higher than that in the control group (p < 0.01), increasing by 6.13 and 6.68%, respectively. The results showed that DCGHs could increase C3 levels in the serum of ICR mice to exert immunoregulatory effects. Ratajczak et al. (48) studied the good immune regulation function of corn oil in vivo. Lee et al. (49) found that corn byproduct poly-saccharides isolated from corn steep liquor could exert an immunomodulatory effect by stimulating the release of Immunoactive substances. Compared with the immunomodulatory effects of corn oil and corn byproduct poly-saccharides, DCGHs also have a good immune regulation effect, which provides a new guide for the comprehensive development and utilization of corn byproducts.

CONCLUSIONS
In this study, we investigated the antihypertensive and immunomodulatory effects of DCGHs in vivo. Analysis of tissue ACE activity demonstrated that DCGHs could exert an antihypertensive effect mainly by inhibiting ACE activity in the kidney, lung, and heart tissues. Moreover, the serum biochemical factor test results indicated that the DCGHs could adjust the balance between EDCFs (e.g., renin, Ang II, TNF-α, and ET-1) and EDRFs (e.g., NO) to regulate blood pressure. Furthermore, the results of analyses of the immunomodulatory effects showed that DCGHs could enhance the phagocytosis of mononuclear macrophages, cellular immunity, and humoral immunity by increasing the phagocytic index of mononuclear macrophages, ear swelling degree, and antibody levels in ICR mice, in addition to stimulating the release of immunoactive substances (e.g., lysozyme, IFN-γ, IgG, and C3). The results herein, may add to existing knowledge on the bioactive properties of DCGHs and guide the development and utilization of cereal-based by-products.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.

ETHICS STATEMENT
The animal study was reviewed and approved by Institutional Animal Care and Use Committee of the Jiangsu University.