Polypeptides with selective inhibitory action on ace domains and use in reducing blood pressure

Abstract

The application discloses a polypeptide with ACE domain selective inhibition and application in blood pressure reduction, the polypeptide has high selective inhibition to the C domain of ACE and weak inhibition to the N domain, can effectively control blood pressure while avoiding bradykinin accumulation, thus having low side effects, easy absorption and other characteristics, can be developed into a functional medicine with blood pressure reduction effect, and food or health care product for helping to maintain a healthy blood pressure level, not only solves the problems of poor selectivity and large side effects of existing ACE inhibitors, but also provides a new technical approach for developing safe and effective blood pressure reduction functional products.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedicine, in particular to polypeptides with ACE domain selective inhibitory effect and application in reducing blood pressure. BACKGROUND

[0002] Blood pressure is defined as systolic and diastolic blood pressure (SBP / DBP) higher than 140 / 90 mmHg, which is a factor of cardiovascular disease, obesity, diabetes and hyperlipidemia, and is an important public health problem that can lead to myocardial infarction, stroke, etc.

[0003] At present, drug therapy is a common means of treating hypertension in clinical practice, which mainly regulates key targets such as renin-angiotensin-aldosterone system, vascular endothelial system and sympathetic nervous system, so that the body blood pressure is maintained at a normal level. Common antihypertensive drugs in clinical practice include ACE inhibitors, Ang II receptor antagonists, calcium channel blockers and other drugs. However, these drugs are generally chemical synthetics. Although these drugs can effectively reduce blood pressure in the short term, the drug efficacy time is short, and patients need to take them for a long time, often accompanied by various side effects. Long-term use can cause serious side effects such as cough, hyperkalemia, rash, and some drugs even have the risk of carcinogenesis.

[0004] Angiotensin-converting enzyme (ACE) plays a key role in blood pressure regulation, and inhibiting the activity of ACE in the body can achieve the purpose of preventing and treating hypertension. At present, most of the antihypertensive drugs used in clinical practice are chemically synthesized, such as captopril and ramipril. These drugs have strong antihypertensive effect, but the drug efficacy time is short, patients need to take them for a long time, and the side effects are large. Food-derived ACE inhibitory peptides are polypeptides isolated from food proteins that have ACE inhibitory activity. Their advantages are abundant source, low cost, and no toxic side effects, making them a research hotspot in the functional food, pharmaceutical and other industries.

[0005] At present, ACE inhibitory peptides from various sources have been found to have significant antihypertensive activity. Some have been proven to effectively reduce the blood pressure of spontaneous hypertensive rats (SHR). Two tripeptides (IQP and VEP) isolated and purified from spirulina protein hydrolysate have been shown to have significant antihypertensive effect on spontaneous hypertensive rats. IPP, VPP and LPP (commonly known as milk-derived tripeptides) derived from cheese protein have been proven to effectively reduce blood pressure in hypertensive patients through clinical trials, and have no effect on blood pressure in normal blood pressure individuals. However, existing research mainly focuses on the evaluation of ACE overall inhibitory activity, and there is a relative lack of research on the selective inhibition of ACE domains by food-derived polypeptides.

[0006] ACE is a zinc-containing dipeptidyl carboxypeptidase, which consists of two highly homologous (about 60% sequence homology) but functionally significantly different domains (N domain and C domain). Both domains contain HEXXH zinc-binding sites and can independently exert catalytic function, but their substrate specificity and physiological functions are significantly different: the C domain is mainly responsible for the conversion of angiotensin I to angiotensin II, which causes vasoconstriction by activating the AT1 receptor, and is a key site for blood pressure regulation; at the same time, the C domain is also involved in the metabolism of atrial natriuretic peptide (ANP). The N domain mainly mediates the degradation of bradykinin, which is closely related to inflammation and side effects such as dry cough.

[0007] Most of the ACE inhibitors currently used in clinical practice are non-selective inhibitors that simultaneously inhibit the activity of both domains, leading to the accumulation of bradykinin in the body, which is an important cause of adverse reactions such as dry cough and severe angioedema. Most ACE inhibitor drugs such as captopril, enalapril, and benazepril are chemically synthesized drugs, and hypertensive patients need to take these drugs long-term to control blood pressure, which can easily cause side effects.

[0008] Hu et al. compared the risk of cough induced by different ACEIs through systematic review and network meta-analysis, and compared it with placebo, ARB and CCB. The results showed that all ACEIs have a similar risk of inducing cough. The risk of moexipril-induced cough is the highest (SUCRA 80.4%), followed by enalapril and lisinopril, while the risk of cilazapril and captopril is the lowest. Compared with placebo, the risk of cough induced by ACEIs is significantly increased (RR 2.21, 95% CI: 2.05-2.39), and is higher than that of ARB (RR 3.2, 95% CI: 2.91-3.51) and CCB (RR 6.5, 95% CI: 5.07-8.34). Therefore, for patients at risk of cough, ACEIs should be avoided, and ARB or CCB should be considered as an alternative according to the patient's comorbidities.

[0009] Professor Laurent Azouay's team published an article in BMJ, which showed that ACEIs increase the risk of lung cancer through the accumulation of bradykinin and substance P. The study found that people who took ACEIs long-term had a 31% higher risk of developing cancer, and compared with patients who used angiotensin receptor blockers (ARBs), the overall risk of lung cancer in patients who used ACEIs increased by 14%. The risk of lung cancer also increased with the length of time ACEIs were used. Compared with patients who used them for less than five years, the risk increased by 22% for those who used them for five to ten years, and by 31% for those who used them for more than ten years.

[0010] Therefore, developing an active polypeptide with selective inhibition on the C domain can not only maintain good antihypertensive effect, but also reduce adverse reactions by reducing inhibition on the N domain, which has important clinical application value. SUMMARY

[0011] The present application provides a polypeptide with selective inhibition on ACE domains, which has high selective inhibition on the C domain of ACE and weak inhibition on the N domain, can effectively control blood pressure while avoiding bradykinin accumulation, and can significantly reduce side effects.

[0012] Therefore, the present application provides the following solutions:

[0013] The first aspect of the present application is to provide a polypeptide with selective inhibition on ACE domains, wherein the amino acid sequence of the polypeptide is Leu-Arg-Asn-Ile-Ser-Pro.

[0014] The second aspect of the present application is to provide the use of the polypeptide with selective inhibition on ACE domains in the preparation of antihypertensive drugs.

[0015] Further, the drug comprises a pharmaceutically acceptable carrier, and / or an excipient, and / or a diluent.

[0016] The third aspect of the present application is to provide the use of the polypeptide with selective inhibition on ACE domains in the preparation of health products for maintaining a healthy level of blood pressure.

[0017] The fourth aspect of the present application is to provide a preparation method of the polypeptide with selective inhibition on ACE domains, comprising the following steps:

[0018] S1. Extracting a mixed polypeptide from a moringa seed protein powder by taking moringa seed protein, using alkaline protease for enzymatic hydrolysis of the moringa protein, centrifuging the enzymatic hydrolysis solution to obtain the supernatant, and drying to obtain the mixed polypeptide;

[0019] S2. Separating and purifying the mixed polypeptide, and analyzing the separation effect by using thin layer chromatography;

[0020] S3. Screening high-inhibitory-activity components by detecting the in vitro ACE inhibitory activity of the high-inhibitory-activity components;

[0021] S4. Analyzing the high-inhibitory-activity components by LC-MS / MS to determine the main polypeptide composition, and respectively performing molecular docking with ACE to select several polypeptides with low binding;

[0022] S5. Selecting the polypeptides with high inhibitory activity on the ACE C domain and low inhibitory effect on the N domain from the polypeptides obtained in step S4 as target polypeptides.

[0023] The target polypeptide includes a polypeptide with an amino acid sequence of Leu-Arg-Asn-Ile-Ser-Pro.

[0024] Further, in step S1, the enzymolysis process has a pH value of 8-11, an enzymolysis temperature of 35-70 DEG C, and an enzymolysis time of 3-6 h.

[0025] Further, in step S2, the separation and purification process uses an ultrafiltration membrane separation or a silica gel column chromatography.

[0026] Further, the target polypeptide has an in-vitro ACE inhibition activity of 80% or more.

[0027] Further, the target polypeptide has an in-vitro ACE inhibition activity IC 50 of 0.05 μmol / mL.

[0028] Compared with the prior art, the present application has the following beneficial effects:

[0029] The polypeptide disclosed by the present application is derived from a moringa oleifera protein, has a high ACE inhibition activity, has an amino acid sequence different from a reported blood pressure lowering peptide sequence derived from a moringa oleifera, and belongs to a newly discovered blood pressure lowering peptide.

[0030] The polypeptide disclosed by the present application has a high selective inhibition effect on the C domain of ACE and a weak inhibition effect on the N domain, effectively controls blood pressure while avoiding the accumulation of bradykinin, and thus has low side effects, easy absorption, and the like, can be developed as a functional drug with a blood pressure lowering effect, and a food or health care product that helps maintain a healthy blood pressure level, not only solves the problems of poor selectivity and large side effects of existing ACE inhibitors, but also provides a new technical approach for developing safe and effective blood pressure lowering functional products. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 FIG. 1 is a thin layer chromatography result of different components of a moringa oleifera polypeptide after separation and purification in Example 1 of the present application.

[0032] Figure 2 FIG. 3 is an ACE inhibition rate result of different moringa oleifera polypeptide components in Example 1 of the present application.

[0033] Figure 3 FIG. 4 is an ACE inhibition rate result of a polypeptide with a low ACE binding ability in a 12 BV moringa oleifera polypeptide component after ultrafiltration in Example 1 of the present application.

[0034] Figure 4 FIG. 5 is an in-vitro ACE C domain and N domain inhibition activity test result of different polypeptides in Example 1 of the present application.

[0035] Figure 5 Toxicity of polypeptide LRNISP to cells in Example 2 of the present application.

[0036] Figure 6 Effects of polypeptide LRNISP of different doses on systolic and diastolic blood pressure of SHR rats in Example 3 of the present application. DETAILED DESCRIPTION

[0037] The technical solutions of the present application will be described below in connection with preferred embodiments, obviously, the described embodiments are only a part of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the protection scope of the present application.

[0038] In one embodiment, a moringa angiotensin converting enzyme (ACE) domain selective inhibition polypeptide is provided, the amino acid sequence of the moringa angiotensin converting enzyme inhibition peptide from N-terminal to C-terminal is: Leu-Arg-Asn-Ile-Ser-Pro, abbreviated as LRNISP, the sequence is shown as SEQ ID NO: 1. The active peptide can effectively and selectively inhibit different domains of ACE, and has high inhibition effect on ACE C domain, but poor inhibition effect on N domain. It is of great significance for the development of drugs with lipid-lowering and blood pressure-lowering functions, and for the development of functional nutrition products and health foods that help maintain healthy blood pressure.

[0039] In the above embodiment, the molecular weight of the polypeptide LRNISP is 788.88, the in vitro ACE inhibition activity IC 50 is 0.05 μmol / mL, and is a white powder, easily soluble in water.

[0040] In another embodiment, by injecting different doses of polypeptide LRNISP into the tail vein of rats every other day, it is found that in the case of low dose and high dose, blood pressure can be reduced, and the effect of blood pressure reduction is more significant in the case of high dose. Therefore, the recommended dose for use as a blood pressure-lowering drug is 0.1-2 mg / kg.

[0041] Example 1

[0042] 1) Preparation of Moringa oleifera peptide mixture, 10 times the volume of petroleum ether and ethanol (petroleum ether: ethanol = 4: 1) was added to the Moringa oleifera powder, and ultrasonic was performed for 30 min. After centrifugation, the precipitate was allowed to stand to fully evaporate the organic solvent, so as to remove the oil. 10 times the volume of PBS was added to the Moringa oleifera powder, and ultrasonic was performed for 1 h to dissolve the Moringa oleifera protein. Ethanol was slowly added to the Moringa oleifera extract to make the concentration of ethanol 70%, and then it was stirred uniformly and allowed to stand at 4°C for 1 h to allow the Moringa oleifera protein to be eluted. The solution was centrifuged at 5000g at 4°C for 10 min, and the precipitate was collected. The ethanol precipitated Moringa oleifera protein was vacuum freeze-dried to obtain Moringa oleifera protein powder.

[0043] 2) A certain amount of Moringa oleifera protein was taken, 10 times the volume of PBS was added, and the precipitate was removed by centrifugation. The Moringa oleifera protein was prepared into a 50 mg / mL solution, 30000 U / g of alkaline protease was added, the pH of the enzymolysis solution was controlled at 9, the enzymolysis temperature was 60°C, and the enzymolysis time was 6 h. After enzymolysis, boiling water bath was performed for 10 min. After standing to room temperature, centrifugation was performed at 5000g for 10 min, and the supernatant was vacuum dried to obtain a Moringa oleifera mixed peptide sample.

[0044] 3) A certain concentration of Moringa oleifera peptide was prepared, and ultrafiltration centrifuge tubes with molecular weight cut off (Molecular Weight Cut Off, MWCO) of 3 kDa and 1 kDa were used for fractionation, respectively. The component with molecular weight <1 kDa was collected and freeze-dried. The dried Moringa oleifera peptide component was subjected to silica gel column chromatography, and 150 mL of eluent and pure water were used for elution at a ratio of dichloroethane:methanol = 1:0, 9:1, 8:2, 6:4, 4:6, 3:7, 2:8, 15:85, 1:9, 5:95, and 0:1, respectively. Thin layer chromatography was used for rapid qualitative analysis of the Moringa oleifera mixed peptide sample, and appropriate Moringa oleifera peptide components were selected. The thin layer chromatography analysis results of different components of the Moringa oleifera peptide after separation and purification are shown in Figure 1

[0045] 4) The separated components were detected by RP-HPLC method for ACE in vitro inhibition activity

[0046] The ACE inhibition rate of each component was determined by the in vitro ACE inhibition activity determination method, and the ACE inhibition rates of different Moringa oleifera peptide components at a concentration of 0.01 mg / mL are shown in Figure 2 It can be seen that the ACE inhibition rate of 12BV is 100%, which is the strongest part of ACE inhibition activity. The ACE inhibition rate of 1 kDa ultrafiltration 12BV Moringa oleifera peptide component after ultrafiltration is shown in Figure 3 It can be seen that the active peptide is mainly distributed in the part with molecular weight less than 1 kDa. Therefore, the part is further separated.

[0047] ​5) Using LC-MS / MS method to identify the amino acid sequence and molecular weight of the components with a molecular weight of <1 kDa, a total of 48 peptides were obtained.

[0048] 6) The bioactive peptides from Moringa oleifera screened in step 5) were docked with the receptor protein (ACE). According to the results of molecular docking, 5 Moringa oleifera polypeptide fragments with lower binding energy were selected for chemical synthesis, with a purity of more than 95%, and the inhibitory activity of the synthesized polypeptides was determined using the method of step 4). The results are shown in Table 1.

[0049] 7) Two polypeptides with good ACE enzyme inhibitory effect in vitro and good binding effect on ACE C domain and poor binding effect on ACE N domain were selected, and the results are shown in Table 2. Figure 4 Table 2: Inhibition rate of different polypeptides on ACE C domain and N domain

[0050] Table 1: ACE inhibition rate of 100 μg / mL synthetic polypeptides

[0051]

[0052] The polypeptide with the best selective inhibitory effect on different domains of ACE in vitro was finally determined to be the synthetic polypeptide No. 76, with the sequence Leu-Arg-Asn-Ile-Ser-Pro.

[0053] Example 2

[0054] The effect of different polypeptide concentrations on the activity of RAW264.7 cells was determined by CCK-8 experiment, and the results are shown in Table 3. Figure 5 The results showed that the Moringa oleifera polypeptides had no cytotoxicity at the experimental concentration and had strong safety.

[0055] Example 3

[0056] The antihypertensive effect of the polypeptide was evaluated by administering it to the tail vein of SHR rats. Physiological saline (CON group) and test groups with doses of 1 mg / kg (LOW group), 5 mg / kg (MID group), and 10 mg / kg (HIG group) were set up, and the injection was performed every other day for four weeks. The effects of the control group and different dose groups on the systolic blood pressure (SBP) and diastolic blood pressure (DBP) of SHR rats were tested. The results are shown in Table 4. Figure 6The results show that the SBP of the SHR rats is lowered in the three dose groups of the polypeptide LRNISP, and the DBP is also lowered to different degrees. The HIG group has the most significant effect on lowering the SBP of the SHR rats, indicating that the polypeptide LRNISP has a significant blood pressure lowering effect, and the converted dose for human consumption is about 1.59 mg / kg.

[0057] While the embodiments of the application have been illustrated and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the spirit and scope of the application, which is defined by the appended claims and their equivalents.

Claims

1. A polypeptide having ACE domain-selective inhibitory action, characterized in that, The amino acid sequence of the polypeptide is Leu-Arg-Asn-Ile-Ser-Pro.

2. Use of the polypeptide with ACE domain selective inhibitory effect according to claim 1 in the preparation of a blood pressure lowering drug.

3. Use according to claim 2, characterized in that, The drug comprises a pharmaceutically acceptable excipient and / or diluent.

4. Use of the polypeptide with ACE domain selective inhibitory effect according to claim 1 in the preparation of a health product for helping to maintain a healthy level of blood pressure.

5. The method of producing a polypeptide having ACE domain-selective inhibitory action according to claim 1, wherein the step of Comprise: S1. Extracting moringa oleifera protein from moringa oleifera seed protein powder, using alkaline protease for enzymolysis of the moringa oleifera protein, centrifuging the enzymolysis solution to obtain supernatant, and drying to obtain mixed moringa oleifera polypeptide; the pH value of the enzymolysis process is 8-11, the enzymolysis temperature is 35-70 ℃, and the enzymolysis time is 3-6 h; S2. Separating and purifying the mixed polypeptide, and analyzing the separation effect by thin layer chromatography; the separation and purification process uses ultrafiltration membrane separation or silica gel column chromatography; S3. Screening high inhibitory activity components by detecting ACE in vitro inhibitory activity of high activity components; S4. LC-MS / MS analysis of high inhibitory activity components to determine the main polypeptide composition, and molecular docking with ACE respectively, and selecting several polypeptides with low binding; S5. Selecting polypeptides with high inhibitory activity to ACE C domain and low inhibitory effect to N domain from the polypeptides obtained in step S4 as target polypeptides; The target polypeptide comprises a polypeptide with an amino acid sequence of Leu-Arg-Asn-Ile-Ser-Pro.

6. The preparation method according to claim 5, characterized in that, The in vitro inhibitory activity of the target polypeptide to ACE is more than 80%.

7. The preparation method according to claim 5, characterized in that, The in vitro ACE inhibitory activity IC of the target polypeptide 50 was 0.05 μmol / mL.

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