Coix seed biological peptide with blood pressure and lipid lowering activity

Abstract

The invention discloses an amino acid sequence of coix seed glutelin source polypeptide with angiotensin converting enzyme (ACE) inhibitory activity and blood pressure and lipid lowering activity and application of the coix seed glutelin source polypeptide. The amino acid sequence of the polypeptide is shown as a sequence 1 in a sequence table. The polypeptide disclosed by the invention has an obvious inhibiting effect on angiotensin converting enzyme, and has obvious blood pressure reducing and lipid reducing effects. The polypeptide can be used for preparing an angiotensin converting enzyme inhibitor and can be used for preparing blood pressure and lipid lowering medicines.

Description

Technical Field

[0001] The invention relates to an amino acid sequence of a coix seed glutenin-derived polypeptide with blood pressure and lipid lowering activities and application thereof. Background Art

[0002] Cardiovascular disease (CVD) is the world's largest health killer, accounting for about one-third of the global mortality rate. Hypertension (HTN) and hyperlipidemia (HLP) are the main risk factors for CVD. Hypertension combined with hyperlipidemia (HTN-HLP) greatly increases the risk of CVD. In the United States, hypertension combined with hyperlipidemia is the most common clinical chronic disease combination; in China, the incidence of hypertension is 23.2%, and the incidence of hyperlipidemia is 41.9%; the proportion of patients with hypertension and hyperlipidemia is 2.1 times that of patients with normal blood pressure and hyperlipidemia. HTN-HLP can cause endothelial dysfunction and affect endothelial-dependent vasodilation, while weakening the self-regulation ability of blood flow in the central nervous system and optic nerve head. Therefore, it is of great clinical value to control blood pressure and blood lipid abnormalities at the same time. However, it is difficult for commonly used clinical drugs to achieve multiple effects with one drug, and there are even serious side effects, such as gastrointestinal reactions, skeletal muscle pain, respiratory infections, headaches, etc. Some antihypertensive drugs may also aggravate dyslipidemia. Therefore, the development of drugs with dual lipid-lowering and blood pressure-lowering effects has important clinical application value.

[0003] Bioactive peptides are peptide compounds that play a variety of physiological functions in the human body. They are composed of 2 to 20 amino acids and have a molecular weight generally less than 6 kDa. As a functional protein fragment, they can be obtained through enzymatic hydrolysis, microbial fermentation, and chemical hydrolysis. Biopeptides have the effects of lowering blood pressure, lowering blood lipids, lowering blood sugar, inhibiting bacteria, and anti-oxidation. At the same time, small molecule biopeptides have the characteristics of high bioavailability, no toxic side effects, and multifunctionality. The development and utilization of natural biopeptides have broad prospects in the fields of medicine and food. Coix lachryma-Jobi L. var. ma-yuen (Roman.) Stapf, a plant of the Gramineae family, is a commonly used diuretic and dampness-infiltrating drug in the clinical practice of traditional Chinese medicine in my country. It is a medicine and food of the same origin. It tastes sweet and light, is cool in nature, and enters the spleen, stomach, and lung meridians. It has the effects of strengthening the spleen and removing dampness, lightening the body and invigorating qi, relaxing muscles and removing numbness, etc. It can be used to treat yin deficiency, thirst, dizziness, edema, and oliguria. Coix seed is listed as a top-grade product in "Shennong Bencao Jing" and is known as the "king of the world's grasses" and "the grass of life and health". Modern clinical research has shown that coix seed has good blood pressure and lipid-lowering effects. Summary of the invention

[0004] The object of the present invention is to provide a polypeptide, the amino acid sequence IALNRF of the polypeptide is as shown in sequence 1 in the sequence list.

[0005] The polypeptide of the present invention has an inhibitory effect on angiotensin converting enzyme.

[0006] The polypeptide of the present invention has a blood pressure lowering effect.

[0007] The polypeptide of the present invention has the effects of increasing serum HDLC level, reducing liver CHO and TG levels, alleviating liver fatty degeneration and improving liver fat metabolism.

[0008] The polypeptide of the present invention has the effect of improving blood rheology.

[0009] The polypeptide of the present invention can be used to prepare angiotensin converting enzyme inhibitors.

[0010] The polypeptide of the present invention can be used to prepare blood pressure and lipid lowering drugs.

[0011] Another object of the present invention is to provide an angiotensin converting enzyme inhibitor, the active ingredient of which is the polypeptide.

[0012] Another object of the present invention is to provide a blood pressure and lipid lowering drug, the active ingredient of which is the polypeptide.

[0013] The polypeptide of the present invention has a significant inhibitory effect on angiotensin converting enzyme and has a significant blood pressure and lipid lowering effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is the secondary mass spectrum of the hexapeptide IALNRF.

[0015] Figure 2 The HPLC chromatograms of the in vitro ACEI activity of each sample are shown in Table 1.

[0016] Among them, a is a blank control, b is a captopril positive control, and c is the hexapeptide IALNRF.

[0017] Figure 3 This is the ACE inhibition rate curve of the hexapeptide IALNRF at different concentrations.

[0018] Figure 4 This is a graph showing changes in blood pressure in HTN-HLP rats after long-term administration of the hexapeptide IALNRF.

[0019] Figure 5 This is a graph showing the changes in liver lipids in HTN-HLP rats after long-term administration of the hexapeptide IALNRF.

[0020] Figure 6This is the blood rheology of HTN-HLP rats after long-term administration of the hexapeptide IALNRF.

[0021] Figure 7 This is the liver tissue pathology of HTN-HLP rats after long-term administration of the hexapeptide IALNRF. DETAILED DESCRIPTION

[0022] The inventors of the present invention discovered the hexapeptide IALNRF with angiotensin converting enzyme inhibitory (ACEI) activity through the following studies:

[0023] Mass spectrometric identification of the hexapeptide IALNRF

[0024] The protein was separated and purified from the enzymatic hydrolysate of coix seed protein, and the amino acid sequence of the peptide fragment was analyzed and identified by liquid chromatography-tandem mass spectrometry (ESI-MS / MS), and the hexapeptide IALNRF (Ile-Ala-Leu-Asn-Arg-Phe) was obtained. The secondary mass spectrum is shown in Figure 1 shown.

[0025] Example 1. Hexapeptide IALNRF

[0026] (I) Artificially synthesized hexapeptide IALNRF

[0027] The hexapeptide IALNRF (molar mass 733.65; purity 99.86%) was artificially synthesized at Beijing Zhongke Yaguang Biotechnology Co., Ltd.

[0028] (II) In vitro ACE inhibitory activity assay

[0029] Unless otherwise specified, the materials and instruments used in this example can be obtained from commercial sources, such as:

[0030] Angiotensin converting enzyme (ACE) Sigma, USA

[0031] Captopril Reference Material China National Institute for the Control of Pharmaceutical and Biological Products

[0032] Analytical HPLC Waters

[0033] The in vitro activity of ACEI was determined by high performance liquid chromatography.

[0034] The hexapeptide IALNRF was dissolved in boric acid buffer (0.1M boric acid, 0.3M NaCl, pH 8.3) to prepare a sample solution. The reaction system was: 10μL sample solution, 20μL ACE and 20μL HHL. After the sample solution was mixed with ACE, it was incubated at 37°C for 10min, and HHL was added to continue incubation for 80min. Finally, 100μL acetonitrile was added to terminate the reaction. Boric acid buffer was used as a blank control and captopril was used as a positive control. The analysis was performed on a C18 column (250×4.6mm, 5um, TIANHE), with mobile phase A being 100% acetonitrile and B being water (containing 0.05% trifluoroacetic acid, V / V), elution conditions A:B=25:75, flow rate 1mL / min, column temperature 30°C, sample volume 10μL, detection at 228nm, and ACE inhibition rate calculated based on HA peak area:

[0035] ACE inhibition rate (%) = (ab) / a×100

[0036] Wherein, a is the HA peak area produced by the blank control, and b is the HA peak area produced under the conditions where the sample participates in the reaction. The results of the in vitro ACEI activity of the hexapeptide IALNRF determined by reverse phase-high performance liquid chromatography (RP-HPLC) are as follows Figure 2 shown. Figure 2 a is the blank control group, which has no ACE inhibitory activity and thus produces a larger amount of HA; Figure 2 b is the positive control group (final concentration is 2×10 -8 M) ACE inhibition rate was 96.47%; Figure 2 c is the hexapeptide IALNRF, which has a significant ACE inhibitory effect. The inhibition rate at each final concentration of IALNRF was determined as follows Figure 3 As shown, the IC is calculated using SAS 50 =116.47±22.13μM.

[0037] (III) Long-term pharmacodynamic evaluation of the hexapeptide IALNRF

[0038] Unless otherwise specified, the materials and instruments used in this example can be obtained from commercial sources, such as:

[0039] Captopril Shanghai Shuangji Pharmaceutical Co., Ltd.

[0040] Wistar rats and essential hypertensive rats (SHR) Beijing Weitonglihua Experimental Animal Technology Co., Ltd.

[0041] BP2000 Non-Invasive Blood Pressure Monitor Visitech Systems, USA

[0042] Wistar rats: 3 weeks old, male, SPF grade. SHR rats: 3 weeks old, male, SPF grade. Wistar rats and SHR rats were kept in the animal room of Beijing University of Chinese Medicine, with a temperature of 23±2℃, humidity of 55±5%, and a light-dark interval of 12h, and free food and water during the period. After 1 week of adaptive feeding, SHR rats were randomly divided into SHR control group (10 rats) and model group (39 rats), and Wistar rats were used as normal control group (10 rats); the control group was fed with ordinary feed, and the model group was fed with high-fat feed for 6 consecutive weeks to establish the HTN-HLP rat model. After 6 weeks of modeling, the tail systolic blood pressure of the SHR control group and the model group was basically above 180mmHg, and compared with the SHR control group, the HDLC level of the model group was significantly reduced, and the LDLC level was significantly increased, indicating that the HTN-HLP model was successfully established.

[0043] After the modeling was completed, the model group rats that met the modeling criteria were randomly divided into 3 groups, each with 9 rats, namely (1) model control group; (2) positive drug group; (3) peptide drug group; and (4) SHR control group; (5) Wistar normal control group. Hexapeptide IALNRF and captopril tablets were dissolved in double distilled water and administered orally with a volume of 5 mL / kg. The feeding dose is shown in Table 1.

[0044] Table 1. Dosage table for long-term administration of hexapeptide IALNRF

[0045]

[0046] 1. Evaluation of antihypertensive pharmacodynamics

[0047] Blood pressure measurement of rats: put rats into the insulation bag of BP2000 non-invasive blood pressure meter according to their numbers, preheat at 38℃ for 10 minutes, and after a stable pulse waveform of rats appears on the computer monitor, pressurize, hold the air for 10 seconds, then release the air, and record the systolic blood pressure (SBP) value in the waveform. Repeat this process several times, and select the three closest blood pressure values ​​for statistical analysis.

[0048] The rats were given intragastrically according to the dosage designed in Table 1 for 6 consecutive weeks, once every morning at 8:00. The blood pressure of the rats was measured at a fixed time every week, and the changes in the systolic blood pressure of the rats in each experimental group were observed and recorded.

[0049] The trend of blood pressure changes in rats in each group after long-term administration is as follows Figure 4As shown in the figure, during the drug administration period, the blood pressure of rats in the Wistar control group was maintained at about 140 mmHg, while the systolic blood pressure of the SHR control group and the HTN-HLP model group showed an upward trend. The systolic blood pressure change trends of the positive drug group and the polypeptide drug group were basically the same. During the drug administration period, both were significantly lower than those of the SHR control group and the model group (P < 0.05). Compared with before intervention, the blood pressure of the positive drug group and the polypeptide drug group decreased by 35.69 mmHg and 34.74 mmHg, respectively, and there was no significant difference between the two groups (P > 0.05). The results of systolic blood pressure (SBP) measurement of each group after long-term drug administration are shown in Table 2.

[0050] Table 2. Blood pressure measurements of rats in the long-term administration of hexapeptide IALNRF (mmHg, )

[0051]

[0052]

[0053] Note: Different letters indicate significant differences between groups (P < 0.05)

[0054] 2. Lipid-lowering pharmacodynamics evaluation

[0055] Determination of rat body weight: The body weight of rats in each group was tested every week. The test results are shown in Table 3.

[0056] At the third week of HTN-HLP administration, the body weight of the hexapeptide IALNRF administration group was significantly lower than that of the model group (P < 0.05), while there was no significant difference in body weight between the positive drug group and the model group (P > 0.05).

[0057] Table 3 Changes in body weight of rats during long-term administration of hexapeptide IALNRF (g, )

[0058]

[0059] Note: Different letters indicate significant differences between groups (P < 0.05)

[0060] Determination of blood lipid content in rats: After continuous administration for 6 weeks, blood was collected from the orbits of rats in each group, and the serum was separated. The contents of CHO, HDLC, LDLC and TG in the serum were measured using an automatic biochemical analyzer. The test results are shown in Table 4.

[0061] Compared with the SHR control group, the HDLC level in the model group was significantly decreased (P < 0.05), and the LDLC level was significantly increased (P < 0.05), which was consistent with the clinical characteristics of hyperlipidemia. Compared with the model group, the HDLC in the peptide drug group increased by 25% (P < 0.05.

[0062] Table 4 Effects of hexapeptide IALNRF on blood lipids in HTN-HLP rats (μM, )

[0063]

[0064] Note: Different letters indicate significant differences between groups (P < 0.05)

[0065] Lipid content detection in rat liver: After 6 weeks of continuous administration, liver tissues of rats in each group were isolated and homogenized for CHO and TG content determination. The results were as follows: Figure 5 As shown, Figure 5 A is the CHO content, Figure 5 B is the TG content.

[0066] The CHO and TG levels in the model group were significantly higher than those in the SHR control group (P < 0.05), and decreased significantly after administration (P < 0.05). The CHO and TG contents in the liver decreased by 13% and 38%, respectively, indicating that the hexapeptide IALNRF has a clear effect on reducing the lipid content in the liver.

[0067] Blood rheology test in rats: After continuous administration for 6 weeks, blood was collected from the abdominal aorta of rats in each group into negative pressure blood collection tubes treated with anticoagulants, and blood rheology indicators were measured using a fully automatic blood rheometer within 4 hours. Figure 6 As shown, Figure 6 A is the viscosity of whole blood, Figure 6 B is the reduced viscosity of whole blood.

[0068] Compared with the SHR control group, the medium and high shear whole blood viscosity and whole blood reduced viscosity in the HTN-HLP model group were significantly increased (P < 0.05). Compared with the model group, there were no significant differences in the various blood rheological indicators in the captopril administration group (P > 0.05); while the low shear, medium shear, and high shear whole blood viscosity in the polypeptide drug group were significantly reduced (P < 0.05), by 25.7%, 31.3%, and 32.4%, respectively, and the medium and high shear whole blood reduced viscosity were also significantly reduced (P < 0.05), by 43.0% and 56.8%, respectively.

[0069] Rat liver histopathological study: After 6 weeks of continuous administration, liver tissues of rats in each group were isolated and sliced ​​for H&E staining. Figure 7 As shown, A is the Wistar control group, B is the SHR control group, C is the model group, D is the positive drug group, and E is the polypeptide drug group.

[0070] The results of H&E staining of liver tissue showed that: the SHR control group had obvious inflammatory cell aggregation; the liver tissue cells in the model group were arranged chaotically, with blurred boundaries, large areas of severe fatty degeneration and obvious inflammatory cells; the positive drug group had large areas of fatty degeneration, a large number of fat vacuoles in the cytoplasm, and a relatively disordered arrangement of cell nuclei, but no obvious inflammatory infiltration was observed; the fatty degeneration of the liver in the polypeptide drug group was significantly reduced, and no obvious inflammatory infiltration was observed, indicating that liver fat metabolism was significantly improved after long-term administration of the hexapeptide IALNRF.

[0071] Example 1 shows that the hexapeptide IALNRF has significant in vitro ACE inhibitory activity and in vivo antihypertensive and lipid-lowering effects. The in vitro ACE inhibitory activity IC50 is 116.47±22.13μM. In vivo efficacy experiments show that the hexapeptide IALNRF has similar antihypertensive effects as the positive drug captopril, and the hexapeptide IALNRF can significantly improve the hemorheological indexes of HTN-HLP rats, reverse liver fatty degeneration, and improve liver fat metabolism. Based on the good antihypertensive and lipid-lowering effects of long-term administration of the hexapeptide IALNRF, it can be used to prepare antihypertensive and lipid-lowering drugs.

Claims

1. A polypeptide, the amino acid sequence of which is shown in Sequence 1 in the sequence listing.

2. The polypeptide according to claim 1, characterized in that The polypeptide has an inhibitory effect on angiotensin converting enzyme.

3. The polypeptide according to claim 1, characterized in that The polypeptide has the effects of lowering blood pressure and reducing blood lipids.

4. Use of the polypeptide according to claim 1 in the preparation of angiotensin converting enzyme inhibitors.

5. Use of the polypeptide according to claim 1 in the preparation of blood pressure and lipid lowering drugs.

6. An angiotensin converting enzyme inhibitor, the active ingredient of which is the polypeptide according to claim 1.

7. A blood pressure and lipid lowering drug, wherein the active ingredient is the polypeptide according to claim 1.

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