CN117646053A

CN117646053A - Preparation method and application of rice peptide with antihypertensive activity

Info

Publication number: CN117646053A
Application number: CN202311727727.5A
Authority: CN
Inventors: 张钊; 魏玮; 张会; 李会允; 王芳; 王惠娟
Original assignee: ZHONGSHI DUQING (SHANDONG) BIOTECH CO Ltd
Current assignee: ZHONGSHI DUQING (SHANDONG) BIOTECH CO Ltd
Priority date: 2023-12-15
Filing date: 2023-12-15
Publication date: 2024-03-05

Abstract

The patent provides a preparation method of rice peptide with antihypertensive activity, which comprises the following steps: s1, adding 12-square deionized water into an enzymolysis tank, adding 1kg of alkaline protease, and fully stirring to dissolve the protease; s2, preparing 1000kg of rice protein, adding the rice protein into an enzymolysis tank of S1, fully stirring and dissolving, continuously adding 5kg of alkaline protease, continuously adding 2kg of neutral protease, performing enzymolysis for 3 hours, and heating to perform enzyme deactivation treatment; s3, filtering the enzymolysis liquid in the S2 by a ceramic membrane with the aperture of 0.2 mu m, and controlling the treatment pressure and the flow rate of the enzymolysis liquid; s4, treating the enzymatic hydrolysate filtered by the ceramic membrane by using an organic filter membrane with the aperture of 10nm, controlling the treatment pressure and the flow rate of the enzymatic hydrolysate, and performing spray drying treatment to finally obtain the rice protein polypeptide. Use of rice peptide with blood pressure lowering activity for maintaining blood pressure health level is provided. The advantages are that: the rice peptide is simple to prepare and low in cost, and the obtained rice peptide has a good effect of reducing hypertension.

Description

Preparation method and application of rice peptide with antihypertensive activity

Technical Field

The invention relates to the technical field of deep processing application of rice, in particular to a preparation method and application of rice peptide with antihypertensive activity.

Background

Rice has a long history of eating and is one of the most important grains in the world today. The world grain and agriculture organization statistics in 2019 show that the total yield of the rice in the world reaches 7.13 hundred million tons, and the yield of the rice in China accounts for about 30 percent. Rice is an indispensable important component in the diet of residents in China, and the consumption of the rice is up to more than 80%. The main nutritional components in the whole grain rice comprise 80% of starch and 6% -8% of protein. Meanwhile, the amino acid ratio of the rice protein is more similar to the FAO/WHO recommended mode, the rice protein belongs to high-quality protein, and the protein nutrition of the rice protein is comparable to animal proteins such as eggs.

The rice protein is hydrolyzed by protease to prepare bioactive peptide, and compared with rice protein, the small molecular polypeptide can be absorbed more rapidly and effectively, and has the characteristic of low sensitization. According to the reports of various documents at home and abroad, the rice protein polypeptide has various biological activities, such as immunity enhancing, blood fat reducing and other functions. According to the invention, through optimizing the preparation process of the rice protein peptide and continuously separating and purifying, the rice protein peptide is found to have the activity of obviously inhibiting ACE, and further, animal experiments prove that the rice protein peptide has obvious blood pressure reducing activity.

Hypertension, which is the most common chronic health condition in the world today, is a high risk factor associated with cardiovascular diseases such as arteriosclerosis, stroke and myocardial infarction. The mortality rate of cardiovascular and cerebrovascular diseases has been high in recent years, and is higher than that of other diseases. The main types of drugs for treating hypertension which are common at present include: diuretics, calcium antagonists, angiotensin converting enzyme inhibitors, etc., with angiotensin converting enzyme inhibitors being the most common. The drugs mainly have competitive inhibition with Angiotensin Converting Enzyme (ACE), so as to interfere synthesis of angiotensin and play a role in reducing blood pressure. These drugs have several side effects, mainly including renal toxicity, nausea, vomiting, etc. The rice protein polypeptide serving as an ACE inhibitor can effectively reduce hypertension through a renin-angiotensin system, has the characteristics of high safety and no toxic or side effect, and provides an important basis for the product development of natural antihypertensive polypeptides.

Disclosure of Invention

Based on the above, it is necessary to provide a method for preparing rice peptide with antihypertensive activity and application thereof.

The invention adopts the following technical scheme to solve the technical problems:

a preparation method of rice peptide with antihypertensive activity comprises the following steps:

s1, adding 12-square deionized water into an enzymolysis tank, regulating the pH to be only 8.8-9.0 by adopting 1M sodium hydroxide, heating to 53-55 ℃, adding 1kg of alkaline protease, and fully stirring to dissolve the protease;

s2, preparing 1000kg of rice protein, adding the rice protein into an enzymolysis tank of S1, fully stirring and dissolving, measuring the pH, adopting 1M sodium hydroxide to adjust the pH to 8.8-9.0, continuously adding 5kg of alkaline protease, fully stirring and carrying out enzymolysis for 3 hours, continuously adding 2kg of neutral protease, carrying out enzymolysis for 3 hours, and then heating to carry out enzyme deactivation treatment;

s3, filtering the enzymolysis liquid in the S2 by a ceramic membrane with the aperture of 0.2 mu m, controlling the processing pressure and the flow rate of the enzymolysis liquid, and fully filtering out fat macromolecular proteins and other impurities by the ceramic membrane so as to greatly improve the quality of products;

s4, treating the enzymatic hydrolysate filtered by the ceramic membrane by using an organic filter membrane with the aperture of 10nm, controlling the treatment pressure and the flow rate of the enzymatic hydrolysate, increasing the concentration of the enzymatic hydrolysate concentrated by using the organic membrane to 32% -36%, and then performing spray drying treatment under the condition that the air inlet temperature is 135 ℃ and the air outlet temperature is 75 ℃, so as to finally obtain the rice protein polypeptide;

s5, separating and purifying the rice peptide by adopting a preparative high performance liquid chromatograph.

Further, in the step S1, the rotation speed of the stirring mechanism is 60 rpm when the protease is stirred and dissolved.

Further, in the step S2, the temperature after the temperature rise is 100 ℃.

Further, when the enzymolysis liquid in the step S3 is subjected to ceramic membrane filtration treatment, the treatment pressure is 1.2MPa, and the flow rate of the enzymolysis liquid is stabilized to 2000L/h.

Further, when the enzymolysis liquid in the step S4 is treated by the organic filtering membrane, the treatment pressure is 2.3MPa, and the flow rate is stable at 2800L/h.

Further, the step of separating and purifying the rice peptide in the step S5 specifically comprises the following steps:

s51, balancing 6 column volumes at a speed of 5ml/min by adopting a mobile phase A until a baseline is stable;

s52, dissolving the rice peptide hydrolyzed by pepsin and trypsin by using a mobile phase A, preparing a sample with the concentration of 10mg/mL, centrifuging for 5min at 10000r/min, filtering the supernatant by using a filter membrane with the thickness of 0.22 mu m, and loading the sample with the sample size of 15mL;

s53, performing gradient elution by adopting a mixed solution of a mobile phase A and a mobile phase B;

s54, sample collection is carried out, and ACE enzyme inhibition activity detection is carried out after freeze drying;

s55, further separating and purifying to continuously improve the purity of the single component, wherein the purity and structure of the single component after purification are identified by adopting a nano-upgrading liquid chromatography-Q EXACTIVE mass spectrometry system;

s56, purifying the rice peptide component by using nano-upgrading liquid chromatography-Q EXACTIVE mass spectrometry to obtain 1 polypeptide, identifying the single-chain polypeptide with the structure of RIPAGTTY, and synthesizing the single-chain polypeptide.

Further, mobile phase A in S51 adopts 0.1% trifluoroacetic acid+99.9% ultrapure water, mobile phase B adopts 0.1% trifluoroacetic acid+99.9% acetonitrile, the detection wavelength is 214nm, the detection time is 36min, and the column temperature is 21-25 ℃.

Further, the elution gradient of S53 is specifically as follows: the mobile phase B concentration is gradually increased from 0% to 5% from 0 to 5 min; 5 to 30min, the mobile phase B concentration is increased from 5% gradient to 80%; the elution is kept with mobile phase B with concentration of 80% for 30 to 40min all the time; the mobile phase B concentration was reduced from 80% gradient to 5% for 40 to 45 min.

The invention also provides application of the rice peptide with the antihypertensive activity in maintaining blood pressure health level.

The invention has the advantages that:

1. the rice peptide has wide sources of raw materials, low cost of various raw materials involved in preparation, simple and easy operation of the preparation method, and provides the preparation method of the rice peptide with low cost and simple preparation process;

2. the rice peptide prepared by the method can effectively reduce hypertension and has no side effect.

Drawings

FIG. 1 is a rice peptide separation and purification chromatogram;

FIG. 2 is a chromatogram of rice peptide fraction 3 after purification;

FIG. 3 is a secondary mass spectrum of rice peptide D1;

FIG. 4 is a graph of data relating to the effect of rice peptide on body weight of rats in each group;

FIG. 5 is a graph of data showing the effect of rice peptide on systolic blood pressure in groups of rats;

FIG. 6 is a graph of data relating to the effect of rice peptide on diastolic blood pressure in each group of rats;

FIG. 7 is a graph of data showing the effect of rice peptide on heart rate in rats of each group;

FIG. 8 is a graph of data relating to the effect of rice peptide on ultrasound in the heart and carotid arteries of rats in each group;

FIG. 9 is a representative image of cardiac ultrasound for each group of rats;

FIG. 10 is a graph of data showing the effect of rice peptide on blood glucose and oxidative stress in groups of rats;

FIG. 11 is a graph of data showing the effect of rice peptide on creatinine and urea in various groups of rats;

FIG. 12 is a graph of data showing the effect of rice peptide on blood lipid in rats of each group.

Detailed Description

The following will clearly and fully describe the technical solutions of the embodiments of the present patent, and it is apparent that the described embodiments are only some of the embodiments of the present patent, not all of them. All other embodiments, based on the embodiments in this patent, which would be within the purview of one of ordinary skill in the art without the creative effort, are contemplated to be within the scope of protection of this patent.

Example 1

S1, adding 12-square deionized water into an enzymolysis tank, adopting 1M sodium hydroxide to adjust the pH to be only 8.8-9.0, heating to 53-55 ℃, adding 1kg of alkaline protease, fully stirring to dissolve the protease, and controlling the rotating speed to be 60 revolutions per minute;

s2, preparing 1000kg of rice protein (tin-free gold biotechnology Co., ltd.) and adding into an enzymolysis tank of S1, fully stirring and dissolving, regulating the pH to be only 8.8-9.0 by adopting 1M sodium hydroxide after measuring the pH, continuously adding 5kg of alkaline protease (NoveXin biotechnology Co., ltd.) and fully stirring and carrying out enzymolysis for 3 hours, continuously adding 2kg of neutral protease (NoveXin biotechnology Co., ltd.) and carrying out enzymolysis for 3 hours, and then heating to 100 ℃ for enzyme deactivation treatment;

s3, filtering the enzymolysis liquid obtained in the step 2 by using a ceramic membrane with the aperture of 0.2 mu m, wherein the treatment pressure is 1.2MPa, the flow rate is stabilized to 2000L/h, and the ceramic membrane can sufficiently filter out fat macromolecular proteins and other impurities so as to greatly improve the quality of products;

s4, treating the enzymatic hydrolysate filtered by the ceramic membrane by using an organic filter membrane with the aperture of 10nm, wherein the treatment pressure is 2.3MPa, the flow rate is stable at 2800L/h, the concentration of the hydrolysate concentrated by using the organic membrane is increased to 32-36%, and then spray drying treatment is carried out, wherein the spray drying condition is that the air inlet temperature is 135 ℃, and the air outlet temperature is 75 ℃, so as to finally obtain the rice protein polypeptide.

Example two

The ACE inhibitory activity of the isolated and purified rice peptide component is determined by using an Angiotensin Converting Enzyme (ACE) inhibitor activity detection kit. The test was performed in 2mL centrifuge tubes, with a total volume of 0.2mL for each test. Ma Niaoxian group aminoacyl leucine (HHL) was dissolved in 0.1M borate buffer (ph=8.3) containing 0.3M sodium chloride to prepare a 5mM HHL solution for use. 80. Mu.L of HHT, 100. Mu.L of deionized water and 10. Mu.L of hydrolysate are sequentially added into a centrifuge tube, the mixture is placed into a constant temperature water bath at 37 ℃ for 3min, and then 10. Mu.LACE enzyme solution is added to start the reaction. Maintaining at constant temperature for 30min, adding 0.2mL of 1M HCl to stop reaction, adding 1.2mL of cold (-20deg.C) ethyl acetate, uniformly mixing for about 15s to extract hippuric acid released by ACE, centrifuging (5500 r/min) for 5min, taking out 0.8m L ester layer, transferring into another test tube, oven drying at 120deg.C for about 1 hr, redissolving in 0.8m L deionized water, and measuring absorbance at 228 nm. Blank control the same procedure was followed except that 0.2mL of 1 mol.L-1 HCl was added to terminate the reaction prior to the reaction. Wherein, the inhibition rate is calculated as follows:

inhibition (%) = (OD _A -OD _B )÷(OD _A -OD _C ))

ODA is the absorbance value in the absence of inhibitor (deionized water instead of hydrolysate); ODB is the absorbance in the presence of inhibitor and enzyme; ODC is the absorbance value in the absence of both inhibitor and enzyme (deionized water was used instead of the hydrolysate and the reaction was stopped by adding hydrochloric acid prior to the reaction).

Example III

Preparing artificial gastric juice: taking 240mLHCl solution (pH=2), 1.8g of pepsin (3000-3500U/g), shaking uniformly, adding water to dilute to 300mL, and placing in a refrigerator at 4 ℃ for standby. Preparation of artificial intestinal juice: taking 2g of monopotassium phosphate, adding 150mL of water to dissolve the monopotassium phosphate, adjusting the pH value to 6.8 by using 0.1mol/LNaOH solution, taking 3g of pancreatin, adding a proper amount of water to dissolve the pancreatin, mixing the prepared solutions, adding water to dilute the solution to 300mL, and placing the solution in a refrigerator at the temperature of 4 ℃ for standby.

The artificial gastric juice and intestinal juice are taken as solvents, and the concentration of the added sample is controlled to be 4mg/mL. Taking 1mg of sample, adding 20mL of artificial gastric juice, shaking at 37 ℃ for 2 hours at 100r/min, adjusting the pH to 6.8, transferring into 20mL of artificial intestinal juice, shaking uniformly at 37 ℃ for 3 hours at 100r/min, inactivating enzyme at 85 ℃ for 10 minutes, cooling to room temperature, performing ultrafiltration and centrifugation, retaining the filtrate, and determining the ACE enzyme inhibition activity of the digested rice peptide according to the method of the second embodiment.

Sample concentration (ug/ml)	0.032	0.16	0.8	4	20	100	500
								Pre-digestion inhibition (%)	2.74	4.85	5.77	9.04	14.42	39.41	81.33
Post-digestion inhibition (%)	3.01	4.92	6.01	8.86	15.96	42.11	83.01

List one

Example IV

According to previous literature studies, the biological activity and functionality of peptides depends on the amino acid composition, sequence and molecular weight of the polypeptides. According to the invention, rice protein is taken as a raw material and subjected to enzymolysis treatment by using protease to generate a plurality of polypeptide components, so that the preparation type liquid phase is continuously adopted to separate and purify rice peptide, the activity of different separated components is evaluated by adopting in-vitro ACE inhibitory activity, and the amino acid sequence identification is further carried out on the components with ACE inhibitory activity by adopting Madli-TOF MS.

Analytical liquid chromatography conditions:

adopting a preparative high performance liquid chromatograph to separate and purify the rice peptide:

mobile phase a:0.1% trifluoroacetic acid +99.9% ultrapure water; mobile phase B:0.1% trifluoroacetic acid +99.9% acetonitrile;

detection wavelength: 214nm; the detection time is 36min; column temperature: 21-25 ℃.

(1) The 6 column volumes were equilibrated with mobile phase a at a rate of 5ml/min until the baseline stabilized.

(2) Dissolving pepsin and trypsin hydrolyzed rice peptide with mobile phase A, preparing sample with concentration of 10mg/mL, centrifuging at 10000r/min for 5min, filtering supernatant with 0.22 μm filter membrane, and loading sample with 15mL.

(3) And (3) carrying out gradient elution by adopting a mixed solution of the mobile phase A and the mobile phase B, wherein the elution gradient is as follows:

the mobile phase B concentration is gradually increased from 0% to 5% from 0 to 5 min; 5 to 30min, the mobile phase B concentration is increased from 5% gradient to 80%; the elution is kept with mobile phase B with concentration of 80% for 30 to 40min all the time; the concentration of mobile phase B is reduced from 80% gradient to 5% gradient from 40 to 45 min;

(4) Samples were collected according to Table II, and after lyophilization, ACE enzyme inhibition activity was examined.

Peak time	Sample name	Inhibition ratio (%)	Peak time	Sample name	Inhibition ratio (%)
						5min～7min	Component 1	25.6％	15min～17min	Component 6	12.6％
7min～9min	Component 2	59.1％	17min～19min	Component 7	8.3％
						9min～11min	Component 3	81.5％	19min～21min	Component 8	15.6％
11min～13min	Component 4	38.1％	21min～23min	Component 9	21.2％
						13min～15min	Component 5	51.2％	23min～25min	Component 10	3.6％

Watch II

The rice peptide component 3 is selected for further separation and purification, so that the purity of the single component is continuously improved. The single purified component adopts a nano-upgrading liquid chromatography-Q EXACTIVE mass spectrometry system to carry out purity and structure identification.

1. Chromatographic conditions:

(1) Mobile phase A is 100% pure water and 0.1% formic acid; phase B, 100% acetonitrile +0.1% formic acid;

(2) The flow rate of the mobile phase is 300nl/min;

(3) Sample injection amount is 1 mu L of supernatant;

(4) The mobile phase gradient procedure is shown in table three below.

Time (minutes)	0	2.0	36.0	38.0	41.0	42.0	45.0
								A(％)	97	97	63	10	10	97	97
B(％)	3	3	37	90	90	3	3

Watch III

3 polypeptides are purified from soybean peptide D4 by using nano-upgrading liquid chromatography-Q EXACTIVE mass spectrometry, single-chain polypeptide with the structure of RIPAGTTY is identified, and the mass percentage of the single-chain polypeptide in soybean peptide (prepared in example I) component 3 is 3.61% by using a liquid chromatography-mass spectrometry technology. Therefore, the single-chain polypeptide was designated as rice peptide D1, and was synthesized by Shanghai shou Biotechnology Co., ltd.

The rice peptide of example four helps to maintain verification of blood pressure health level function:

1. principle of experiment

The animal model of male spontaneous hypertension rat (Spontaneously hypertensive rats, SHR) is given with different doses of the tested sample (rice peptide), the influence of the tested sample (rice peptide) on the indexes such as blood pressure, heart rate and the like of the animal model of hypertension is observed, and the antihypertensive effect of the tested sample (rice peptide) is evaluated.

2. Materials and methods

2.1 sample rice peptide, supplied by Shandong Biotechnology Co. The sample was pale yellow powder. Preserving in a refrigerator at-20 ℃.

2.2 laboratory animals

10 SPF-class 9-week-old male Wistar-Kyoto (WKY) rats and 50 SHRs (weight 200+ -20 g) were purchased from Beijing Veitz laboratory animal technology Co., ltd, and the laboratory animal production license number was SCXK (Beijing) 2021-0006. The experimental unit uses license number SYXK 20190005. Animal feeding is carried out in an animal house with constant temperature (22+/-2) DEG C, constant humidity (55+/-5) percent and alternating light and shade period of 12 hours/12 hours, and common feed is purchased from Jinan Pengyue laboratory animal breeding Limited company and is free to ingest and drink water.

2.3 grouping and administration methods for animals

After one week of adaptive feeding, 10 WKY rats served as a normal control group (gavage administration of distilled water, a.wky control group). Male 50 SHR rats were selected and randomly divided into 5 groups according to blood pressure and body weight: hypertension model group (gavage administration of distilled water, b.shr model group), low-dose rice peptide group (gavage administration of 20mg/kg/d, c.low-rice peptide group), medium-dose rice peptide group (gavage administration of 100mg/kg/d, d.medium-rice peptide group), high-dose rice peptide group (gavage administration of 500mg/kg/d, e.high-rice peptide group), captopril positive control group (gavage administration of 30mg/kg/d, k.captopril group). Each group of 10 animals, single cage and single animal, and is fed with common feed, and is administered by intragastric administration 1 time a day for 10 weeks. Body weight, food intake and blood pressure were collected weekly during the experiment.

2.4 major instrumentation and reagents

An electronic balance of Shanghai smart sky JT1001N, an Shanghai barrier solid JA5003A analytical balance, a Beckmann ALLEGRA X-15R low-temperature centrifuge, a Beckmann AU480 full-automatic biochemical analyzer, a Thermo paraffin microtome, a Thermo full-closed coloring agent, a Thermo automatic dehydrator, a Thermo embedding machine, an OLYMPUS ZX70-112 straight microscope, a TECAN Infinitem200PRO enzyme-labeled instrument, a Beijing soft-tipped rat noninvasive tail artery blood pressure instrument, and a Vevo ultra-high resolution small animal color Doppler ultrasound imaging system. The low density lipoprotein cholesterol and malondialdehyde detection kit is provided by the institute of biological engineering, built by south Beijing, and other biochemical kits are provided by Ningbo health biotechnology Co.

2.5 determination of blood pressure and heart rate

The tail artery systolic pressure (Systolic blood pressure, SBP), diastolic pressure (Diastolic blood pressure, DBP) and heart rate of each group of rats in a quiet and awake state were measured using a rat noninvasive tail artery blood pressure meter (Softron BP-2010A), the effective values were continuously measured 3 to 5 times, and the average value was calculated 1 time/week.

2.6 determination of heart and carotid ultrasound:

at week 10 of intervention, isoflurane was used for anesthesia and ultrasound indicators were detected using a special purpose ultrasound instrument for small animals.

2.7 determination of blood glucose, blood lipid, oxidative stress, renal function and liver function indicators

After the end of the experiment, the rats of each group were fasted overnight, blood was taken from the abdominal aorta after anesthesia, serum was separated, and rapidly stored in a-80 ℃ refrigerator. Rapidly taking out relevant tissue, placing in pre-cooled physiological saline at 4deg.C, cleaning blood, sucking with filter paper, packaging tissue, and storing at-80deg.C. Serum Glucose (GLU), high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C), total cholesterol (T-CH), triglyceride (TG), malondialdehyde (MDA), creatinine (CR), urea (Urea), aspartate Aminotransferase (AST), and alanine Aminotransferase (ALT) were each assayed by reference to the protocol.

2.8 collection of cardiac, vascular and renal tissue and pathological morphological observations

After the experiment is finished, tissues such as heart, blood vessels, kidneys and the like of each group of rats are taken and placed in 4% paraformaldehyde for fixation, paraffin embedding is carried out, hematoxylin and Eosin (HE) staining is carried out, and histopathological morphology such as heart, blood vessels, kidneys and the like is observed by using a light microscope and photographed for analysis.

2.9 data processing and result determination

The results were expressed as mean ± standard error. Statistical analysis was performed using SPSS data analysis software. The comparison between groups adopts analysis of variance, and the comparison between groups is tested by LSD. P <0.05 indicates that the difference is statistically significant.

3. Results

3.1 general Condition observations

The animals in each group have normal growth and development and activity, good mental state and no poisoning and death.

3.2 Effect of rice peptide on body weight of rats in each group

Male WKY rats were subjected to gavage with distilled water (A.WKY control group), male spontaneous hypertension rats were subjected to gavage with distilled water (B.SHR model group), low dose of rice peptide (20 mg/kg/d, C.Low rice peptide group), medium dose of rice peptide (100 mg/kg/d, D.medium rice peptide group), high dose of rice peptide (500 mg/kg/d, E.high rice peptide group), positive control group of captopril (30 mg/kg/d, K.captopril group), and the weights of rats of the different groups were observed for 10 consecutive weeks. At week 10 of intervention, the body weight of SHR model, low and medium dose rice peptide rats was significantly reduced (P < 0.05) compared to the WKY control group, and the body weight of high dose rice peptide rats was not significantly different (P > 0.05). There was no significant difference in body weight (P > 0.05) between the rats in each dose group of the test sample compared to the SHR model group. See table four and fig. 4.

Table IV Effect of rice peptides on body weight (BW, g) of rats in each group

3.3 Effect of rice peptides on the Systolic Blood Pressure (SBP) of rats in each group

The tail artery systolic pressure (SBP), diastolic pressure (DBP) and Heart Rate (HR) indices of each group of rats were observed using a rat noninvasive tail artery blood pressure meter. As shown in table five and fig. 5, at week 10 of intervention, the systolic blood pressure was significantly increased (P < 0.05) in SHR model group and each dose of rice peptide group rats compared to WKY control group. The systolic blood pressure of rats was significantly reduced (P < 0.05) for each dose of rice peptide group compared to the SHR model group.

Time (week)

WKY control group

SHR model group

C. Low rice peptide group

D. Medium rice peptide group

E. High rice peptide group

K. Captopril group

0

133.53±3.87

187.96±5.20 ^***

189.88±4.11 ^***

192.80±3.02 ^***

190.52±5.44 ^***

174.57±6.55 ^***

1

135.64±3.53

196.49±4.15 ^***

194.76±3.32 ^***

191.81±5.85 ^***

194.91±4.56 ^***

178.66±4.82 ^***#

2

136.84±4.46

189.48±4.69 ^***

195.51±5.00 ^***

191.64±3.84 ^***

196.78±2.67 ^***

179.27±5.39 ^***

3

138.24±3.15

203.06±7.00 ^***

199.70±3.53 ^***

188.84±5.95 ^***

194.24±6.99 ^***

168.56±4.58 ^***###

4

137.34±2.82

204.32±3.82 ^***

195.88±4.55 ^***

192.98±4.82 ^***

187.49±3.87 ^***##

156.79±6.02 ^**###

5

133.90±4.43

199.60±1.56 ^***

194.68±3.41 ^***

197.47±3.56 ^***

196.52±2.97 ^***

160.61±9.13 ^***###

6

139.26±4.86

200.60±1.57 ^***

183.26±5.73 ^***#

188.38±5.24 ^***

183.60±4.01 ^***#

162.69±10.08 ^**###

7

139.42±3.59

195.23±5.65 ^***

198.18±4.72 ^***

186.96±6.38 ^***

187.06±5.79 ^***

164.81±6.27 ^**###

8

139.66±3.40

192.76±3.87 ^***

182.48±7.90 ^***

189.64±3.68 ^***

180.34±4.91 ^***

163.31±9.07 ^**#*

9

131.88±3.30

203.03±3.49 ^***

186.36±4.74 ^***#＃

191.97±3.71 ^***

180.99±4.46 ^***###

10

122.47±5.83

198.99±3.98 ^***

178.28±6.22 ^***##

182.64±4.37 ^***#

182.70±3.79 ^***#

Table five Effect of rice peptides on the systolic blood pressure (SBP, mmHg) of rats in each group

3.4 Effect of rice peptide on Diastolic Blood Pressure (DBP) in rats of each group

The tail artery systolic pressure (SBP), diastolic pressure (DBP) and Heart Rate (HR) indices of each group of rats were observed using a rat noninvasive tail artery blood pressure meter. As shown in table 3 and fig. 3, at week 10 of intervention, the diastolic blood pressure was significantly increased (P < 0.05) in SHR model group and each dose of rice peptide group rats compared to WKY control group. The diastolic blood pressure of rats was significantly reduced (P < 0.05) for each dose of rice peptide group compared to SHR model group.

Time (week)

WKY control group

SHR model group

C. Low rice peptide group

D. Medium rice peptide group

E. High rice peptide group

K. Captopril group

0

101.90±4.38

156.16±5.17 ^***

156.72±435 ^***

158.02±4.13 ^***

159.03±7.26 ^***

150.67±640 ^***

1

100.60±3.11

161.93±5.54 ^***

159.34±4.14 ^***

159.65±6.11 ^***

165.04±4.17 ^***

150.96±6.03 ^***

2

107.48±3.20

157.98±4.90 ^***

166.29±4.65 ^***

161.61±2.33 ^***

167.74±3.44 ^***

152.00±6.98 ^***

3

105.88±2.69

172.47±7.48 ^***

168.64±334 ^***

158.70±5.94 ^***

164.79±6.06 ^***

117.50±6.76 ^###

4

104.27±2.25

176.82±5.47 ^***

164.77±3.42 ^***

160.76±5.21 ^***#

161.64±568 ^***#

113.41±5.86 ^###

5

88.95±1.67

143.99±4.15 ^***

140.76±3.82 ^***

144.08±3.40 ^***

145.20±3.16 ^***

120.70±7.85 ^***###

6

90.81±3.56

154.01±2.96 ^***

134.50±5.15 ^***##

135.39±4.66 ^***##

137.63±3.09 ^***#

118.84±9.02 ^***###

7

101.39±2.85

145.22±2.62 ^***

155.89±5.61 ^***

144.69±4.22 ^***

143.79±343 ^***

126.94±5.79 ^***##

8

92.89±4.19

145.26±4.51 ^***

136.62±8.56 ^***

146.13±5.25 ^***

138.16±3.48 ^***

126.41±7.89 ^***#

9

95.40±3.95

152.18±3.48 ^***

144.68±2.57 ^***

147.37±2.58 ^***

135.69±3.49 ^***##

10

83.21±3.25

154.99±3.18 ^***

136.21±6.85 ^***##

140.77±3.93 ^***#

139.07±2.26 ^***#

Table five Effect of Rice peptides on diastolic blood pressure (DBP, mmHg) in rats of each group

3.5 Effect of rice peptide on Heart Rate (HR) in rats of each group

The tail artery systolic pressure (SBP), diastolic pressure (DBP) and Heart Rate (HR) indices of each group of rats were observed using a rat noninvasive tail artery blood pressure meter. As shown in table six and fig. 6, the heart rate was significantly increased (P < 0.05) in SHR model group and each dose of rice peptide group rats compared to WKY control group at week 10 of intervention. The heart rate of rats was significantly reduced (P < 0.05) for each dose of the rice peptide group compared to the SHR model group.

Influence of the Table Liu Rice peptide on the heart Rate (HR, bpm) of rats in each group

3.6 Effect of rice peptide on ultrasound of rat heart and carotid artery of groups

Before the experiment is finished, the ultrasonic indexes of the heart and carotid artery of each group of rats are detected by using a special ultrasonic instrument for small animals.

As shown in fig. 7, 8, 9 and table seven, left Ventricular Mass (LVM) and carotid intima-media thickness (cIMT) were significantly increased (P < 0.05) in SHR model rats compared to WKY control group. The left ventricular mass and carotid intima-media thickness were significantly reduced (P < 0.05) in rats of each dose of rice peptide group compared to SHR model group.

Influence of Sevoflurane on ultrasound of heart and carotid arteries of rats in each group

3.7 Effect of rice peptide on blood sugar and oxidative stress in rats of each group

Different doses of rice peptide were administered to spontaneously hypertensive rats by gavage, and after 10 weeks of continuous intervention, biochemical indicators of blood glucose and oxidative stress were observed for different groups of rats. As shown in table nine and fig. 10, at week 10 of intervention, the blood glucose was significantly elevated (P < 0.05) in SHR model rats compared to the WKY control group, with no significant change in malondialdehyde (P > 0.05). Compared with SHR model group, the blood sugar of the rats of the low-dose rice peptide group is obviously reduced (P < 0.05), the malondialdehyde of the rats of the high-dose rice peptide group is obviously reduced (P < 0.05), and the blood sugar and malondialdehyde of the rats of other groups are not obviously different (P > 0.05).

Group of	Glucose (mmol/L)	Malondialdehyde (mu mol/L)
			WKY control group	3.73±0.26	17.10±3.02
SHR model group	5.46±0.50 ^**	23.92±5.56
			C. Low rice peptide group	3.97±0.37 ^##	22.94±4.45
D. Medium rice peptide group	4.83±0.38 ^*	26.36±6.11
			E. High rice peptide group	4.71±0.38	6.41±1.18 ^##
K. Captopril group	5.80±0.27 ^*	14.81±4.51

Influence of Table eight Rice peptides on blood glucose and oxidative stress in groups of rats

3.8 Effect of rice peptide on creatinine and Urea in groups of rats

The different doses of rice peptide are administrated to spontaneous hypertension rats through gastric lavage, and after continuous intervention for 10 weeks, biochemical indexes of kidney functions of different groups of rats are observed. As shown in table nine and fig. 11, at week 10 of intervention, the creatinine was significantly increased (P < 0.05) in the SHR model group rats, significantly decreased (P < 0.05) in the rice peptide group rats at each dose, and significantly increased (P < 0.05) in the urea in the SHR model group and each dose of rice peptide group rats, as compared to the WKY control group. Compared with SHR model group, creatinine of rats of each dose of rice peptide group is significantly reduced (P < 0.05), urea of rats of high dose of rice peptide group and captopril group is significantly reduced (P < 0.05), while creatinine and urea of rats of other dose group are not significantly different (P > 0.05).

Group of	Creatinine (mu mol/L)	Urea (mmol/L)
			WKY control group	43.00±1.51	7.30±0.35
SHR model group	49.14±2.55 ^*	10.15±0.40 ^***
			C. Low rice peptide group	36.56±1.11 ^**###	9.28±0.24 ^***
D. Medium rice peptide group	32.89±1.81 ^***###	9.16±0.21 ^***
			E. High rice peptide group	35.25±0.77 ^**###	8.53±0.55 ^*##
K. Captopril group	45.57±2.64	7.87±0.52 ^＃＃＃

Table nine influence of Rice peptide on creatinine and Urea in groups of rats

3.9 Effect of Rice peptide on blood lipid in rats of each group

The rice peptide with different doses is administrated to spontaneous hypertension rats through gastric lavage, and biochemical indexes of blood fat of rats with different groups are observed after continuous intervention for 10 weeks. As shown in table ten and fig. 12, at week 10 of intervention, the SHR model, the test sample doses and captopril significantly reduced high density lipoprotein cholesterol, total cholesterol and triglyceride (P < 0.05) in rats without significantly affecting low density lipoprotein cholesterol (P > 0.05) compared to the WKY control. Compared with the SHR model group, the low and medium doses of the rice peptide and the captopril significantly raise high-density lipoprotein cholesterol (P < 0.05) of the rat, the rice peptide and the captopril significantly lower the low-density lipoprotein cholesterol (P > 0.05) of the rat, the captopril significantly raise the total cholesterol of the rat, the rice peptide and the captopril do not have significant influence on the total cholesterol of the rat (P > 0.05), and the rice peptide and the captopril do not have significant influence on the triglyceride of the rat (P > 0.05).

Influence of Ten rice peptides on blood lipid of rats of each group

This patent and its embodiments have been described in a non-limiting sense, and the actual structure is not limited to this, but is shown in the drawings and is merely one of the embodiments of this patent. In summary, if one of ordinary skill in the art is informed by this disclosure, structural modes and embodiments similar to the technical scheme are not creatively designed without departing from the gist of the patent and are all intended to be within the protection scope of the patent.

Claims

1. The preparation method of the rice peptide with the antihypertensive activity is characterized by comprising the following steps of:

2. The method for preparing rice peptide with antihypertensive activity according to claim 1, wherein: when the protease is stirred and dissolved in the step S1, the rotating speed of the stirring mechanism is 60 revolutions per minute.

3. The method for preparing rice peptide with antihypertensive activity according to claim 1, wherein: in the step S2, the temperature after the temperature rise is 100 ℃.

4. The method for preparing rice peptide with antihypertensive activity according to claim 1, wherein: and when the enzymolysis liquid in the step S3 is subjected to ceramic membrane filtration treatment, the treatment pressure is 1.2MPa, and the flow rate of the enzymolysis liquid is stabilized to 2000L/h.

5. The method for preparing rice peptide with antihypertensive activity according to claim 1, wherein: and when the enzymolysis liquid in the step S4 is treated by the organic filtering membrane, the treatment pressure is 2.3MPa, and the flow rate is stable at 2800L/h.

6. The method for preparing rice peptide with antihypertensive activity according to claim 1, wherein: the step S5 of separating and purifying the rice peptide specifically comprises the following steps:

7. The method for preparing rice peptide with antihypertensive activity according to claim 6, wherein: the mobile phase A in the S51 adopts 0.1% trifluoroacetic acid and 99.9% ultrapure water, the mobile phase B adopts 0.1% trifluoroacetic acid and 99.9% acetonitrile, the detection wavelength is 214nm, the detection time is 36min, and the column temperature is 21-25 ℃.

8. The method for preparing rice peptide with antihypertensive activity according to claim 6, wherein: the elution gradient of S53 is specifically as follows: the mobile phase B concentration is gradually increased from 0% to 5% from 0 to 5 min; 5 to 30min, the mobile phase B concentration is increased from 5% gradient to 80%; the elution is kept with mobile phase B with concentration of 80% for 30 to 40min all the time; the mobile phase B concentration was reduced from 80% gradient to 5% for 40 to 45 min.

9. Use of a rice peptide with antihypertensive activity according to any one of claims 1-8 for maintaining blood pressure health levels.