Jingjing Dong, Shen Wng, Xioyo Yin, Min Fng, Zhiyong Gong,*, Yongning Wu

a Key Laboratory for Deep Processing of Major Grain and Oil of Ministry of Education, College of Food Science and Engineering,Wuhan Polytechnic University, Wuhan 430023, China.

a HC Key Laboratory of Food Safety Risk Assessment, Food Safety Research Unit (2019RU014) of Chinese Academy of Medical Science,China National Center for Food Safety Risk Assessment, Beijing 100021, China

ABSTRACT

In this paper, the antihypertension effect of rice peptide (RP) was studied. With spontaneously hypertensive rats (SHR) and Wistar Kyoto (WKY) as the research objects, RP disposable gastric and long-term gastric irrigation experiments were carried out and systolic blood pressure (SBP) was measured. At the end of the long-term gastric irrigation experiment, the content of nitric oxide (NO), angiotensin-converting enzyme (ACE), angiotensin II (Ang II) and renin in the plasma and the activity of ACE were determined.The results showed that RP could reduce systolic pressure of SHR and had time-dose dependence while high-dose RP significantly reduced systolic pressure by 24.6 and 17.2 mmHg, respectively after a single and long-term gastric irrigation test. RP also could inhibit the activity of ACE and increase the release of NO.These results suggested that the decompression mechanism of RP is likely to be related to the regulation of the renin-angiotensin system (RAS) and NO.

Keywords:

Rice peptide

Angiotensin-converting enzyme (ACE)

Antihypertensive effect

1. Introduction

Hypertension is the most common and serious chronic health disease in the world today, and it is a high risk factor associated with cardiovascular disease, such as atherosclerosis and myocardial infarction [1-3]. The mortality rate from cardiovascular diseases has remained high in recent years [4], with higher mortality rates than other diseases. Human blood pressure is regulated mainly by the renin-angiotensin system (RAS) [5]. The angiotensin in the plasma is hydrolysed by renin to produce angiotensin I (Ang I), and then Ang I is hydrolysed by angiotensin-converting enzyme (ACE) in the endothelial cells of the blood vessels to form Ang II, which stimulates the release of aldosterone in the adrenal cortical globular band [6].Aldosterone can significantly enhance the kidney’s reabsorption of sodium, but also indirectly promote the reabsorption of water, so that the blood volume increases, and thus raise blood pressure. Therefore,inhibiting ACE activity is an effective way to treat hypertension, some ACE inhibitors have also made valid progress in hypertension-related diseases [7]. In addition, some studies [3,8] also have shown that the release of NO causes blood vessels to dilate, which also can play a certain role in reducing pressure. However, traditional ACE inhibitors can cause adverse reactions during dehypertension [9,10],like coughing. Therefore, ACE inhibition products which are high-safety, and non-toxic side effects are required. In recent years,peptides produced by food protein partially enzymatic products have become more and more popular with food workers. These peptides are inactive in the sequence of the parent protein, but can be released during enzyme digestion or food processing [7]. At the same time, the proportion of amino acid composition of cereal protein in rice and rice bran is closer to the United Nations Food Agriculture Organization/World Health Organization (FAO/WHO) recommended model and belongs to high-quality plant protein, which has a nutritional value comparable to egg, fish, shrimp and beef protein. And compared with soy protein and milk protein, the protein in rice and rice bran has the advantages of hypoallergenic. The protein content of rice varies between 6.0% and 15.7% in China. In the processing of rice,only starch is effectively utilized, and a large amount of high-quality rice protein in the by-products such as broken rice, rice bran and rice husk cannot be effectively exploited and utilized, resulting in a great waste of protein resources. Meanwhile, several million tons of rice polluted by heavy metals in China every year, and they could be used to develop rice protein and rice starch products. Therefore,the development of rice protein and rice peptides has a very broad application prospects and space. Therefore, this study mainly discusses the antihypertension effect of rice peptide (RP) through the spontaneously hypertensive rats (SHR) model and explore its anti-blood pressure system. The experiment is divided into the following parts: 1) extracting the rice protein by alkaline,and RP was extracted by alkaline proteinase-trypsin method;2) determining the molecular mass and amino acid composition of RP using high performance liquid chromatography; 3) studying the anti-pressure effect of RP and traditional bucking substances as positive control groups in SHR model. It is hoped that the results of this paper can be of reference value to other researchers who are engaged in the antihypertensive mechanism of rice bioactive peptide or related product development.

2. Materials and methods

2.1 Materials and reagents

The rice used in this experiment is “Fuwa” brand rice, purchased from Hubei Fuwa Company (China); alkaline protease (2.4 U/g),trypsin (1250 USP), purchased by Novo Nordisk, Denmark;captopril was purchased by Sigma Corporation of the United States;SHRs (10 weeks old, (240 ± 20) g) male, SBP 180 mmHg, Wistar Kyoto (WKY) (10 weeks old, (242 ± 10) g) male, SBP 150 mmHg,are purchased in Beijing Vitolihua Experimental Animal Technology Company (China); Ang II, renin, NO, ACE content and its kit purchased Nanjing Jiancheng Bioengineering Institute (China).

2.2 Preparation of RP

The extraction craft of RP were as follows: the extractant was 0.1% sodium hydroxide, solid-liquid ratio was 1:6, magnetic force stirred 60 min under 50 °C. And the slurry was centrifuged 20 min at 4 000 r/min. After the acid deposition of the supernatant, it was centrifuged again. The sediment was stored at -40 °C for vacuum freeze-drying to obtain rice protein. The rice protein was treated with alkaline protease and trypsin respectively, separated with DA201-C large hole adsorption resin adsorption resin, and the RP was collected after vacuum freeze-drying.

2.3 Determination of molecular weight, amino acid composition, content of Val-Asn-Pro (VNP) and Val-Trp-Pro(VWP) of the purified peptides

Multi-angle laser light scattering combined with gel permeation chromatography (MALLS/GPC) was used to determine the molecular weight of RP. Amino acid analysis of RP was determined following the method of Adeyeye [11] with some modifications. About 60 mg purified RP was weighed and placed into glass ampoules. 5 mL of 6 mol/L HCl with 1% phenol was added and oxygen was expelled by pumping into nitrogen. The glass ampoules were sealed with Bunsen flame and put into the oven at 105 °C for 24 h. The ampoules were allowed to cool and thefiltrate was added to 250 mL to the constant volume. Amino acid analysis was performed using RP-HPLC with Cat Ex resin column (0.4 cm × 25 cm) which was eluted with nacitrate-naborate (1:1) at a flow rate of 0.4 mL/min. VARIAN 363 fluorescence detection (Palo Alto, USA) was used with an excitation wavelength of 338 nm and an emission wavelength of 425 nm for quantitation.

Sedex 90 evaporative light scattering detector (ELSD) (Alfortville,France) combined with HPLC (Palo Alto, USA) was used to analyze the content of VNP, VWP in RP. The mobile phase used for these experiments was a binary mixture of water (A)-methanol (B) (90:10,V/V) with 0.1% TFA at a flow rate of 0.3 mL/min. The gradient of 70%–50% A in 9 min, and another 50%–70% A in 1 min.

2.4 Animal breeding and grouping

SHR and WKY were kept in the SPF-class small animal laboratory at Tongji Medical College and received humane care in compliance with the Ethics Committee of Huazhong University of Science and Technology (No. 0290308). The feeding ambient temperature maintained at (25 ± 1) °C and the daily lighting were 12 h of light-dark alternate, with lighting hours from 8:00 to 20:00. Before the start of the experiment, SHR and WKY had a one-week period of environmental adaptation, SHR was randomly divided into 5 groups of 6 in each group, including: positive control group (captopril, dose of 10 mg/kg), RP high, medium, low dose (100, 50 and 20 mg/kg),and negative control group (distilled water). WKY was randomly divided into 3 groups of 6 in each group, including: positive control group (captopril), high dose group of RP and negative control group (distilled water).

2.5 Measurement of blood pressure

The arterial blood pressure of SHR and WKY rats was determined by an instrument for the BP-100A (Softron, Tokyo, Japan) according to the tail cuff method.

2.6 A reduction in systolic blood pressure after a single intragastric administration

SHR and WKY rats were given a gastric infusion of stomach in group doses of the experimental animals, after which their systolic blood pressure was measured at 0, 2, 4, 6, 8, 10 and 24 h, respectively.

2.7 A reduction in systolic blood pressure after long-term intragastric administration

SHR and WKY were given gavage once a day for 6 weeks, their weight and blood pressure were recorded at afixed time each day.

2.8 Determination of plasma renin, ACE, Ang II, NO content and ACE activity

After the long-term gastric irrigation experiment, the serotonin was injected into the abdomen of SHR rats at a dose of 50 mg/kg, and when SHR and WKY was unconscious, collected the blood samples,centrifuged at 1 200 ×gfor 10 min after 30 min. The supernatants collected were combined, stored at -20 °C for analysis within one week.

2.9 Data analysis

All data were expressed as mean ± SEM. Analysis of one-way analysis of variance (ANOVA) was carried out using the SPSS 19.0 package software. Differences were considered significant atP< 0.05.

3. Results

3.1 Molecular weight, amino acid composition, the content of VNP, VWP and IC50 value of the purified peptides

Table 1 displayed the purified ACE-inhibitory peptides from rice had a high quantity (69.0%) of oligopeptide between 549 and 576, and a low quantity (21.6%) between 1 113 and 1 158. As was shown in Table 2, high level of essential amino acids (35.87%) and hydrophobic amino acids (32.07%) were observed in rice peptide.Proline (Pro), alanine, valine, isoleucine, leucine, tyrosine and phenylalanine were hydrophobic amino acids. Meanwhile, it was found that the content of VNP, VWP in rice peptides was 18.51%,1.29%, respectively.

Table 1Molecular weight of rice peptide (mean ± SEM, n = 3).

Table 2Amino acid composition of rice peptide (g/100 g, mean ± SEM, n = 3).

3.2 Effects on blood pressure after one gastric buck

Table 3 indicated the change of SBP within 24 h after SHR were given different samples. All dose groups of RP in SHR decreased within 2 h after being gavaged, and the decrease range of blood pressure in RP dose group increased with the increase of RP dose,reaching the maximum at 100 mg/kg, while the positive control group had the largest reduction of blood pressure. Compared to the negative control group, SBP decreased significantly (P< 0.05) after oral RP (100 mg/kg) and captopril, with the largest decrease at 6 h at 24.6 and 38.4 mmHg, respectively. In addition, the SBP decreased significantly (P< 0.05) after WKY rats took captopril (10 mg/kg)orally. RP (100 mg/kg) has no buck effect on WKY rats. The buck effect on SHR and WKY from once infusion of stomach was relatively short, and after 24 h it restored to its initial level.

3.3 Effects of long-term intragastric administration of RP on SBP

Table 4 revealed that SBP of SHR was significantly reduced (P< 0.05) after oral administration of RP (100 mg/kg) and captopril (10 mg/kg) for 6 weeks compared to the negative control group.Compared to the initial SBP value, the positive control group of SHR decreased the most, at 18.8 mmHg, followed by the RP (100 mg/kg)dose group at 17.1 mmHg and the RP (50 mg/kg) dose group at 8.2 mmHg. The negative control group of SHR rose by 14.3 mmHg after 6 weeks, in line with the reference value of the SHR model mouse age-blood pressure value relationship provided by Beijing-based Vitong Lihua [12]. Blood pressure increased by 1.3 mmHg after 6 weeks in the RP (20 mg/kg) dose group, but decreased compared to the negative control group. In addition, after 6 weeks of oral captopril (10 mg/kg) in WKY rats, SBP decreased significantly (P< 0.05), while RP (100 mg/kg) had no buck effect after 6 weeks of oral administration. Many studies have reported the antihypertensive effect of food extracts on SHR [13,14].

Table 3SBP values of SHR rats after a single oral administration (mmHg, mean ± SEM, n = 6).

Table 4Long-term effects on SBP values of SHR and WKY rats (mmHg, mean ± SEM, n = 6).

3.4 Measurement of plasma renin, ACE, Ang II, NO content and ACE activity

Fig. 1 showed that there was no significant difference in renin content between the SHR and WKY groups (P> 0.05). Compared to the negative control group, the content of ACE in the plasma decreased significantly (P< 0.01) after 6 weeks of SHR-injected captopril (10 mg/kg) and RP (100, 50 mg/kg) also decreased significantly(P< 0.05). At the same time, the content of Ang II decreased significantly (P< 0.05) in rat plasma in the SHR-positive control group(captopril), and in the RP group decreased gradually with the increase of the dose, but the difference was not significant. After long-term intragastric administration of captopril (10 mg/kg) and RP (100,50 mg/kg), the content of NO in SHR plasma increased significantly(P< 0.01) and ACE activity decreased significantly (P< 0.01).WKY were given captopril (10 mg/kg) for 6 weeks, the increase of NO content in the plasma was very significant (P< 0.01), the activity of Ang II and ACE decreased significantly (P< 0.05), and the difference in ACE content was not significant.

Fig. 1 Effects of RP on plasma levels of renin, ACE, Ang II, NO and ACE activity in (A) SHR and (B) WKY by intragastric administration. Rats were treated with RP (100, 50 and 20 mg/kg) and captopril (10 mg/kg) every day for 6 weeks. Data are expressed as mean ± SEM (n = 6).

4. Discussion

The hydrolysates from enzymatic hydrolysis reaction displayed excellent physicochemical properties. The present study showed that relative molecular mass of many active antihypertensive peptides derived from a variety of proteins mainly distributed below 2 000 Da. Ourfinding indicated that the molecular weight of RP mainly distributed from 549 Da to 1 158 Da. The composition proportion of amino acids was abundant, and the content of hydrophobic amino acids was 45.2 g/100 g, which was closely associated with ACE inhibitory activity [15]. Meanwhile, Liu et al. [16] reported that ACE inhibitory peptides obtained from many marine organisms such as rotifers, sea cucumbers, sea shrimp and jellyfish were rich in glycine, glutamic,proline, alanine, aspartic acid, which were also the main component of RP with the content of 40.12%.

The ACE inhibitory activity and antihypertensive activities of RP were investigated in the research. It was found that a single administration (100, 50 mg/kg) of RP and captopril (10 mg/kg)showed a strong suppressive effect on SBP of SHRs while RP (20 mg/kg) made little effect on SBP of SHRs. Besides, RP had no effect on SBP of WKYs. The results of long-term oral administration experiments showed that the SBP of SHR treated with RP (100 mg/kg)and captopril was lowered significantly (P< 0.05) after 6 weeks.Meanwhile, the SBP of WKY treated with captopril was lowered significantly (P< 0.05) while the SBP of WKY treated with RP (100 mg/kg) had not changed almost. This result seemed to be consistent with previous studies, which showed whey protein hydrolysate and peptide fractions extracted from Spanish dry-cured ham had antihypertensive effect on SBP of SHRs [17]. Judging from the results of single and long-term gavage, the drop of blood pressure in a single gavage is greater. This may be because the single action time was short, and the blood pressure was measured during the relatively dense time when the foreign substance entered the body.This may be the time when the effect of exogenous substances is the most obvious, but as the body’s metabolism and drug power decrease,blood pressure will slowly return to its initial value, and according to our single gavage data, this is indeed the case. Long-term gavage tends to be an overall result obtained after long-term exposure to foreign substances. Although the blood pressure drop is small, it does not return to the initial value, and there is a dose-dependent relationship between the RP group, which shows long-term gavage of RP can indeed help SHR blood pressure to a certain extent.

The body secretes a protein hydrolysis enzyme called renin from the kidneys as a defense against lower blood pressure [18].The secreted renin catalyzes angiotensin (continuously secreted by the liver) into an inactive ten peptide called angiotensin I.ACEs found in plasma convert inactive angiotensin I into active octapeptide angiotensin II [19,20]. Angiotensin is 40 times stronger than norepinephrine and is considered as one of the strongest vassals.In addition to blocking in the synthesis of angiotensin, ACE inhibitors also cause decrease in vasodilation and blood pressure by inhibiting the destruction of kinase 2 enzymes and reducing the destruction of retardants [21]. Studies had also showed that the mechanism sedation of these peptides is stimulated by peripheral opioid receptors firstly, followed by the release of NO causing blood vesseld dilation [19,22,23]. In this study, the ACE content and ACE activity decreased significantly (P < 0.05) in the long-term intragastric administration of RP (100 mg/kg) and RP (50 mg/kg) groups, and the NO content increased significantly. The results of this study are consistent with the above mechanism. Therefore, the RP decompression mechanism is likely to be related to the regulation of RAS system and NO.

5. Conclusion

This study revealed that RP can effectively inhibit the activity of ACE and thus lower blood pressure. These results indicated that RP could be a source of bioactive peptides for functional foods or nutraceuticals.

Declaration of interests

All authors declare that they have no known competingfinancial interests or personal relationships that could have appeared to in fluence the work reported in this paper.

Acknowledgements

National Science and Technology Project for Grain Industry of China (201513006).