Polypeptide with ACE structural domain selective inhibition effect and application of polypeptide in blood pressure reduction

Abstract

The invention discloses a polypeptide with an ACE structural domain selective inhibition effect and application in blood pressure lowering, the polypeptide has a high-selectivity inhibition effect on a C structural domain of ACE and a weak inhibition effect on an N structural domain of ACE, and can effectively control blood pressure and avoid accumulation of bradykinin, so that the polypeptide has the characteristics of low side effect, easiness in absorption and the like; the ACE inhibitor disclosed by the invention can be developed into a functional medicine with a blood pressure reducing effect and a food or a health-care product beneficial to maintaining the blood pressure health level, so that not only are the problems of poor selectivity and large side effect generally existing in the conventional ACE inhibitor solved, but also a new technical approach is provided for developing a safe and effective blood pressure reducing functional product.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and specifically to polypeptides with selective inhibitory effects on the ACE domain and their applications in blood pressure reduction. Background Art

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

[0003] Currently, drug treatment is a common means of treating hypertension clinically. It mainly regulates key targets such as the renin-angiotensin-aldosterone system, vascular endothelial system, and sympathetic nervous system to maintain the body's blood pressure at a normal level. Commonly used clinical antihypertensive drugs include ACE inhibitors, Ang II receptor antagonists, calcium channel blockers, etc. However, these drugs are generally chemically synthesized. Although these drugs can effectively reduce blood pressure in the short term, their 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, and rash, and some drugs even have a carcinogenic risk.

[0004] Angiotensin-converting enzyme (ACE) plays a key role in blood pressure regulation. By inhibiting the activity of ACE in the body, the purpose of preventing and treating hypertension can be achieved. Currently, most of the antihypertensive drugs used clinically are chemically synthesized, such as captopril, ramipril, etc. These drugs have a strong antihypertensive effect, but their efficacy time is short, patients need to take them for a long time, and the side effects are relatively large. Food-derived ACE inhibitory peptides refer to polypeptides isolated from food proteins with ACE inhibitory activity. Their advantages are rich sources, low costs, and no toxic side effects, and they are a research hotspot in industries such as functional foods and medicine.

[0005] Currently, ACE inhibitory peptides from various sources have been found to have significant antihypertensive activities. Some have been proven to be able to effectively reduce the blood pressure of spontaneous hypertensive rats (SHR). Two tripeptides (IQP and VEP) have been isolated and purified from spirulina protein hydrolysates and have an obvious blood pressure-lowering effect on spontaneous hypertensive rats. IPP, VPP, and LPP (commonly known as milk-derived tripeptides) from milk casein have passed clinical trials and have been proven to be able to effectively reduce the blood pressure of hypertensive patients and have no effect on the blood pressure of those with normal blood pressure. However, existing research mainly focuses on the evaluation of the overall inhibitory activity of ACE, and there is relatively little research on the selective inhibition of different domains of ACE by food-derived polypeptides.

[0006] ACE is a zinc-containing dipeptidyl carboxypeptidase, composed of two highly homologous domains (N domain and C domain, with approximately 60% sequence homology) but with significantly different functions. Both domains contain the HEXXH zinc-binding site and can independently perform catalytic functions, but their substrate specificities and physiological functions are significantly different: the C domain is mainly responsible for converting angiotensin I to angiotensin II, causing vasoconstriction by activating the AT1 receptor, which 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 and is closely related to side effects such as inflammatory reactions and dry cough.

[0007] Most of the ACE inhibitors currently used clinically are non-selective inhibitors, which simultaneously inhibit the activities of both domains, resulting in the abnormal degradation of bradykinin and its accumulation 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, benazepril, etc. are chemically synthesized drugs, and hypertensive patients need to take such drugs for a long time to control blood pressure, and side effects are likely to occur.

[0008] Hu et al. compared the risks of cough induction by different ACEIs through systematic reviews and network meta-analyses and compared them with placebo, ARB, and CCB. The results showed that all ACEIs have a risk of inducing cough, and the risks are similar. The risk of cough induction by moexipril is the highest (SUCRA 80.4%), followed by enalapril and lisinopril, while the risks of cilazapril and captopril are the lowest. Compared with placebo, the risk of cough induction by ACEIs is significantly increased (RR 2.21, 95% CI: 2.05 - 2.39), and the risks are higher than those 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 alternative options according to the patient's comorbidities.

[0009] Professor Laurent Azouay's team published an article in the *BMJ*. The research results showed that ACEIs increase the risk of lung cancer through the accumulation of bradykinin and substance P. The follow-up study found that the cancer risk of people taking ACEI antihypertensive drugs for a long time is as high as 31%. Compared with patients using angiotensin receptor blockers (ARBs), the overall lung cancer risk of patients using ACEIs increased by 14%. As the use time of ACEIs extends, the lung cancer risk also increases. Compared with patients using the drugs for less than five years, the risk of patients using the drugs for five to ten years increased by 22%, while the risk of patients using the drugs for more than ten years increased by 31%.

[0010] Therefore, the development of active polypeptides with selective inhibitory effects on the C domain can not only maintain good antihypertensive effects, but also reduce adverse reactions by reducing the inhibition of the N domain, which has important clinical application value. Summary of the Invention

[0011] The present invention provides a polypeptide with selective inhibitory effects on the ACE domain. This polypeptide has a high selective inhibitory effect on the C domain of ACE, while having a weak inhibitory effect on the N domain. It can avoid the accumulation of bradykinin while effectively controlling blood pressure, and can significantly reduce side effects.

[0012] In view of this, the solution of the present invention is as follows:

[0013] The first aspect of the present invention is to provide a polypeptide with selective inhibitory effects on the ACE domain, and the amino acid sequence of the polypeptide is Leu-Arg-Asn-Ile-Ser-Pro.

[0014] The second aspect of the present invention is to provide the application of the polypeptide with selective inhibitory effects on the ACE domain described in the first aspect in the preparation of antihypertensive drugs.

[0015] Furthermore, the drug includes a pharmaceutically acceptable carrier, and / or excipient, and / or diluent.

[0016] The third aspect of the present invention is to provide the application of the polypeptide with selective inhibitory effects on the ACE domain described in the first aspect in the preparation of health products that help maintain a healthy blood pressure level.

[0017] The fourth aspect of the present invention is to provide a preparation method of the polypeptide with selective inhibitory effects on the ACE domain, and the steps include:

[0018] S1. Extract moringa protein from moringa seed protein powder, enzymatically hydrolyze the moringa protein with alkaline protease, centrifuge the enzymatic hydrolysate to obtain the supernatant, and dry it to obtain moringa mixed polypeptides;

[0019] S2. Separate and purify the mixed polypeptides, and analyze the separation effect using thin layer chromatography;

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

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

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

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

[0024] Further, in step S1, the pH value of the enzymatic hydrolysis process is 8-11, the enzymatic hydrolysis temperature is 35-70 °C, and the enzymatic hydrolysis is carried out for 3-6 h.

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

[0026] Further, the in vitro inhibitory activity of the target polypeptide against ACE is more than 80%.

[0027] Further, the in vitro ACE inhibitory activity IC 50 is 0.05 μmol / mL.

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

[0029] The polypeptide of the present invention is derived from moringa protein and has a high inhibitory activity against ACE. Its amino acid sequence is different from the reported blood pressure-lowering peptide sequences derived from moringa, and it belongs to a newly discovered blood pressure-lowering peptide.

[0030] The polypeptide of the present invention has a high selective inhibitory effect on the C domain of ACE, while having a weak inhibitory effect on the N domain. While effectively controlling blood pressure, it avoids the accumulation of bradykinin. Therefore, it has the characteristics of low side effects and easy absorption, and can be developed into a functional drug with blood pressure-lowering effects, as well as foods or health products that help maintain a healthy blood pressure level. It not only solves the problems of poor selectivity and large side effects commonly existing in existing ACE inhibitors, but also provides a new technical approach for the development of safe and effective blood pressure-lowering functional products. Description of the Drawings

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

[0032] Figure 2 It is the ACE inhibition rate result of different moringa polypeptide components in Example 1 of the present invention.

[0033] Figure 3 It is the ACE inhibition rate result of the polypeptide with a relatively low ACE-binding energy in the 12 BV moringa polypeptide component after ultrafiltration in Example 1 of the present invention.

[0034] Figure 4 It is the test result of the in vitro inhibitory activities of different polypeptides against the C domain and N domain of ACE in Example 1 of the present invention.

[0035] Figure 5 This is the experimental result of the cytotoxicity of polypeptide LRNISP to cells in Example 2 of the present invention.

[0036] Figure 6 This is the result of the effects of different doses of polypeptide LRNISP on the systolic and diastolic blood pressures of SHR rats in Example 3 of the present invention. Detailed implementation manners

[0037] The technical solutions of the present invention will be clearly and completely described below in conjunction with the preferred embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the scope of protection of the present invention.

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

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

[0040] In another embodiment, different doses of polypeptide LRNISP were injected into the tail vein of rats every other day, and it was found that blood pressure could be reduced in both low-dose and high-dose cases, and the antihypertensive effect was more significant at high doses. Therefore, the recommended dosage as an antihypertensive drug is 0.1-2 mg / kg.

[0041] Example 1

[0042] 1) Prepare a moringa polypeptide mixture. Add petroleum ether and ethanol (petroleum ether:ethanol = 4:1) with a volume 10 times that of the dry weight of the material to the moringa powder, sonicate for 30 min, centrifuge, and let the precipitate stand to fully volatilize the organic solvent to achieve the purpose of degreasing. Add PBS with a volume 10 times that of the dry weight of the material and sonicate for 1 h to dissolve the moringa protein, and sonicate for 1 h. Slowly add ethanol to the moringa extract to make the ethanol concentration 70%, stir evenly, and let it stand at 4 °C for 1 h to fully precipitate the moringa protein. Centrifuge the solution at 5000 g for 10 min at 4 °C, and collect the precipitate part. The moringa protein precipitated by ethanol is vacuum freeze-dried to obtain moringa protein powder.

[0043] 2) Take a certain mass of moringa protein, add PBS with a volume 10 times that of the protein, centrifuge to remove the undissolved precipitate, prepare the moringa protein into a 50 mg / mL solution, add alkaline protease at 30000 U / g, control the pH of the enzymatic hydrolysis solution to 9, the enzymatic hydrolysis temperature to 60 °C, and enzymatically hydrolyze for 6 h. After enzymatic hydrolysis, boil in a water bath for 10 min. Let it stand to room temperature, centrifuge at 5000 g for 10 min, and vacuum-dry the supernatant to obtain a moringa mixed polypeptide sample.

[0044] 3) Prepare a moringa polypeptide with a certain concentration, and fractionate it using ultrafiltration centrifugal tubes with a molecular weight cut-off (MWCO) of 3 kDa and 1 kDa respectively. Collect the fraction with a molecular weight < 1 kDa for freeze-drying. Subject the dried moringa polypeptide fraction to silica gel column chromatography, and use eluents with ratios of dichloroethane:methanol of 1:0, 9:1, 8:2, 6:4, 4:6, 3:7, 2:8, 15:85, 1:9, 5:95, 0:1 and pure water, each with a volume of 150 mL, for elution, and collect samples with a volume of 8 column volumes. Rapidly qualitatively analyze the moringa mixed polypeptide sample by thin-layer chromatography and select the appropriate moringa polypeptide fraction. The thin-layer chromatography analysis results of different fractions after separation and purification of moringa polypeptides are as Figure 1 shown.

[0045] 4) Detect the in vitro ACE inhibitory activity of the separated fractions by RP-HPLC

[0046] Use the in vitro ACE inhibitory activity assay method to measure the ACE inhibition rate of each fraction. The ACE inhibition rates of different moringa polypeptide fractions at a concentration of 0.01 mg / mL are as Figure 2 shown. It can be seen that the ACE inhibition rate of 12BV is 100%, which is the part with the strongest ACE inhibitory activity. After ultrafiltration, the ACE inhibition rate of the 12BV moringa polypeptide fraction after 1 kDa ultrafiltration is as Figure 3 shown. It can be seen that the active polypeptides are mainly distributed in the part with a molecular weight less than 1 kDa. Therefore, further separate this part.

[0047] 5) The amino acid sequences and molecular weights of the components with a molecular weight cut-off of < 1 kDa retained by 12BV were identified using LC-MS / MS, and a total of 48 peptides were obtained.

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

[0049] 7) Two polypeptides with good inhibitory effects on ACE in vitro and showing good binding effects on the C domain of ACE but poor binding effects on the N domain of ACE as shown by the molecular docking results were selected. As shown, the polypeptide numbered 76 (LRNISP) had a high inhibitory rate on the C domain of ACE at a concentration of 250 μg / mL and a low inhibitory rate on the N domain. The inhibitory effects of the polypeptide on different domains of ACE in vitro were determined using the method in step 4). Figure 4 The synthetic polypeptide numbered 76 with the sequence Leu-Arg-Asn-Ile-Ser-Pro was finally determined to have the best selective inhibitory effect on different domains of ACE in vitro.

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

[0051]

[0052] Example 2

[0053] The cytotoxicity experiment was used to determine the effects of different polypeptide concentrations on the activity of RAW264.7 cells using the CCK-8 assay. The results are shown as follows. The results showed that Moringa oleifera polypeptides had no cytotoxicity at the experimental concentrations and had strong safety.

[0054] The effects of different polypeptide concentrations on the activity of RAW264.7 cells were determined using the CCK-8 assay. The results are shown as follows. The results showed that Moringa oleifera polypeptides had no cytotoxicity at the experimental concentrations and had strong safety. Figure 5 The results showed that Moringa oleifera polypeptides had no cytotoxicity at the experimental concentrations and had strong safety.

[0055] Example 3

[0056] The antihypertensive effects of the polypeptides were evaluated by tail vein injection in SHR rats. A physiological saline group (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. Injection was carried out every other day for four weeks, and the effects on the systolic blood pressure (SBP) and diastolic blood pressure (DBP) of SHR rats in the control group and different dose groups were measured respectively. The results are as follows. Figure 6As shown. The results showed that the SBP of SHR rats could be down-regulated in all three dosing groups of the 76 (LRNISP) polypeptide, and the DBP also decreased to varying degrees. Among them, the HIG group had the most significant effect on down-regulating the SBP of SHR rats, indicating that the polypeptide LRNISP has a significant blood pressure-lowering effect. After conversion, the dosage for human use is approximately 1.59 mg / kg.

[0057] Although the embodiments of the present invention have been shown and described, those of ordinary skill in the art can understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

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

2. Use of the polypeptide having ACE domain selective inhibitory effect according to claim 1 in the preparation of antihypertensive drugs.

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

4. Use of the polypeptide having ACE domain selective inhibitory effect according to claim 1 in the preparation of health products that help maintain healthy blood pressure levels.

5. The method for preparing the polypeptide having selective ACE domain inhibition according to claim 1, characterized in that the steps include: S1. Extract Moringa protein from Moringa seed protein powder, enzymolyze the Moringa protein using alkaline protease, centrifuge the enzymolysis solution to obtain the supernatant, and dry the mixture to obtain Moringa mixed polypeptides; S2. Separate and purify the mixed polypeptides and analyze the separation effect using thin layer chromatography; S3. Screening components with high inhibitory activity by detecting the ACE inhibitory activity in vitro of the components with high activity; S4. LC-MS / MS analysis was performed on the components with high inhibitory activity to determine the main peptide composition, and molecular docking was performed with ACE respectively to select several peptides with low binding; S5. Selecting the polypeptide obtained in step S4 that has high inhibitory activity against ACE C domain and low inhibitory effect against N domain as the target polypeptide; The target polypeptide includes 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: In step S1, the pH value of the enzymatic hydrolysis process is 8-11, the enzymatic hydrolysis temperature is 35-70° C., and the enzymatic hydrolysis is carried out for 3-6 hours.

7. The preparation method according to claim 5, characterized in that: In step S2, the separation and purification process uses ultrafiltration membrane separation or silica gel column chromatography.

8. The preparation method according to claim 5, characterized in that: The target polypeptide has an in vitro inhibitory activity on ACE of more than 80%.

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

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