Polypeptide with blood pressure lowering function and preparation method thereof

Abstract

The present application relates to the field of functional food and medicine, and particularly relates to a polypeptide with blood pressure lowering function and a preparation method thereof. The amino acid sequence of the polypeptide is as shown in any one of the following: 1) Leu-Ser-Leu; 2) Ile-Ser-Leu; 3) Leu-Ser-Ile; or 4) Ile-Ser-Ile. The polypeptide has excellent ACE inhibitory activity, good digestive stability, and can be obtained from natural edible materials, thus further ensuring safety. Therefore, the functional polypeptide can be used in health products for patients with hypertension and as a lead drug for blood pressure lowering drugs.

Description

Technical Field

[0001] This invention relates to the field of functional foods, specifically to a polypeptide with blood pressure-lowering function and its preparation method. Background Technology

[0002] Angiotensin I-converting enzyme (ACE, EC 3.4.15.1) is widely distributed in various tissues of mammals and can regulate blood pressure through the renin-angiotensin system and the kallikrein-kinin system. To effectively control blood pressure, many antihypertensive drugs based on the principle of ACE activity inhibitors have been successfully developed and used, such as captopril, fosinopril, and lisinopril.

[0003] ACE inhibitors, commonly used in first-line treatment, can cause significant side effects in clinical applications and have limited efficacy, often requiring combination with other medications. To obtain new, natural, highly effective, stable, and safe antihypertensive drugs, many food-derived peptide molecules with ACE-inhibiting activity have been identified. However, enzymatic digestion in the body often destroys their structures, and poor digestive stability and bioavailability limit the final use of many bioactive peptides.

[0004] In the prior art, CN 107964034A provides an ultrasound-assisted simulated digestion method for casein bioactive peptides and their application in health foods. This method first pre-treats casein with ultrasound, then uses protease to enzymatically hydrolyze it to prepare casein ACE-inhibiting bioactive peptides. Next, it tracks the ACE-inhibiting activity of the casein bioactive peptides by simulating gastrointestinal digestion. After absorption by Caco-2 cells simulating small intestinal epithelial cells, it characterizes casein functional peptides with high ACE-inhibiting activity after gastrointestinal digestion and absorption by Caco-2 cells simulating the small intestinal wall. However, the amino acid sequence in CN 107964034A clearly contains Pro, especially at the C-terminus. Peptides with C-terminal Pro have a highly similar core structure to captopril (methimazole proline). This core structure, because it can bind to the ACE enzyme active pocket, has long been recognized as one of the important pharmacophore structures of ACE inhibitors. Many researchers have long been conducting screening studies for ACE-inhibiting peptides based on C-terminal Pro as a structural feature; this is a well-known empirical finding in the relevant academic field. However, the IC50 of LSLP (molecular weight 428.52304 g / mol) in CN 107964034A is... 50The concentration was 21.09 μg / ml, equivalent to approximately 49215.56 nM, indicating relatively moderate in vitro inhibitory activity. Furthermore, the cells used in CN 107964034A to simulate small intestinal epithelial cell absorption were Caco-2 cells, which are human colorectal adenocarcinoma cells and do not truly reflect the actual digestive and absorptive processes in the human body. Summary of the Invention

[0005] Among known inhibitors with tripeptide structures, a structure-activity relationship of "hydrophobic amino acid + positively charged amino acid + aromatic amino acid / cyclic amino acid" has been found. However, in our research, we unexpectedly discovered that when LSL is used as a tripeptide, although it does not conform to the above structure-activity relationship, it exhibits superior in vitro inhibitory activity (IC50) compared to LSLP. 50 The ACE inhibitor has a molecular weight of 107.17 ± 2.05 nM, with hydrophobic leucine residues at both ends and a polar serine residue with a hydroxyl group in the middle. This unique structural feature implies a different inhibitory mechanism. The smaller molecular volume suggests less steric hindrance in inhibitory binding, and the appropriate ratio of hydrophilic to hydrophobic groups helps the inhibitor achieve better blood-brain barrier penetration to exert its antihypertensive effect. Furthermore, since leucine and isoleucine have extremely similar structures, it is presumed that replacing one or both leucine residues in the above-mentioned peptide with isoleucine may also result in a similar efficacy. Therefore, the above findings of this invention provide a new design reference for ACE inhibitors.

[0006] Based on the above findings, the first objective of this invention is to provide a functional polypeptide.

[0007] The second objective of this invention is to provide an oyster protein peptide and a method for preparing the same.

[0008] The aforementioned functional polypeptides and oyster protein peptides have the function of lowering blood pressure and have good digestive stability.

[0009] A third objective of this invention is to provide the application of the aforementioned functional polypeptides and oyster protein peptides in pharmaceuticals.

[0010] To achieve the above objectives, the present invention first provides a functional polypeptide with an amino acid sequence of any of the following: 1) Leu-Ser-Leu; 2) Ile-Ser-Leu; 3) Leu-Ser-Ile; or 4) Ile-Ser-Ile. All of the above polypeptides exhibit superior antihypertensive function and good digestive stability.

[0011] Those skilled in the art can obtain the above-mentioned functional peptides through artificial synthesis or by preparing them from natural proteins.

[0012] Here, the present invention specifically provides a method for preparing oyster protein peptides, which includes: using oyster protein as raw material, and obtaining an enzymatic hydrolysis product through two enzymatic hydrolysis steps;

[0013] In the two-step enzymatic hydrolysis, the enzyme used in the first step is pepsin, and the enzyme used in the second step is trypsin.

[0014] Using the methods described above, functional polypeptides with a high content of the amino acid sequence Leu-Ser-Leu can be obtained from natural edible materials, further ensuring their safety.

[0015] Preferably, in the first step of enzymatic hydrolysis, the amount of pepsin used is 1000~2000U / 1g of raw material protein, and the enzymatic hydrolysis conditions are: enzymatic hydrolysis at pH 2.0~1.2 and 36.8~37.4℃ for 2.5~3.5h.

[0016] Preferably, in the second enzymatic hydrolysis step, the amount of trypsin used is 200~400U / 1g of raw material protein, and the enzymatic hydrolysis conditions are: enzymatic hydrolysis for 2.5~3.5h under ultrasonic treatment at pH 7.0~7.5, 36.8~37.4℃, and 25~40kH.

[0017] After enzymatic hydrolysis using the above method, the content of functional polypeptides with the amino acid sequence Leu-Ser-Leu in the hydrolysate is higher, which is more conducive to enhancing its inhibitory effect on ACE.

[0018] Preferably, the preparation method further includes: separating a protein peptide solution with a molecular weight of less than 1000 Daltons from the enzymatic hydrolysate, and then separating the target peptide;

[0019] The separation of the target peptide includes:

[0020] Protein peptides with a molecular weight less than 1000 Daltons were separated by reversed-phase high-performance liquid chromatography (RP-HPLC) using a Sephadex G-25 gel column elution solution of deionized water. The corresponding elution peaks of functional peptides with the amino acid sequence Leu-Ser-Leu were collected.

[0021] The above methods can further increase the content of functional peptides in the product.

[0022] More preferably, the method for separating protein peptides with a molecular weight of less than 1000 Daltons from the enzymatic hydrolysate includes:

[0023] First, filter the enzymatic hydrolysate using diatomaceous earth and collect the filtrate;

[0024] The filtrate was then subjected to ceramic membrane ultrafiltration at 3000 Daltons and 1000 Daltons in sequence to obtain a protein peptide solution with a molecular weight of less than 1000 Daltons.

[0025] In specific implementation, before performing the two-step enzymatic hydrolysis, the method further includes: dissolving oyster protein in water to prepare an aqueous solution. Specifically, this can be achieved by stirring and homogenizing the oyster protein in the aqueous solution to prepare an oyster protein aqueous solution.

[0026] In practice, after collecting the corresponding elution peaks of the functional peptide with the amino acid sequence Leu-Ser-Leu, those skilled in the art can further concentrate and freeze-dry it, without further limitations.

[0027] Those skilled in the art can combine the above solutions to obtain preferred embodiments of the method for preparing oyster protein peptides of the present invention.

[0028] As a preferred embodiment, the method for preparing the oyster protein peptide includes the following steps:

[0029] (1) After the oyster protein is crushed and homogenized in an aqueous solution, the pH is adjusted to 2.0~1.2, and then pepsin is added for 3 hours of enzymatic hydrolysis with an enzyme activity concentration of 1000~2000 U / 1g raw protein. After 3 hours of enzymatic hydrolysis, the pH is adjusted to 7.0~7.5, and trypsin is added for 3 hours of enzymatic hydrolysis with an enzyme activity concentration of 200~400 U / 1g raw protein. The enzymatic hydrolysis temperature is 36.8~37.4℃, and the enzymatic hydrolysis process is simultaneously subjected to ultrasonic treatment at 25~40kH.

[0030] (2) After the enzymatic hydrolysis is completed, the solution is filtered through diatomaceous earth and the filtrate is collected.

[0031] (3) The collected separation liquid will be sent to a two-step ultrafiltration process, using ceramic membranes with pore sizes of 3000 Daltons and 1000 Daltons respectively, to finally obtain protein peptides with a molecular weight of less than 1000 Daltons.

[0032] (4) Peptide powder containing LSL was obtained by reversed-phase high performance liquid chromatography: the filtrate was eluted and separated by Sephadex G-25 gel column with deionized water as the elution solution. After collecting the corresponding elution peaks of functional peptides with the amino acid sequence Leu-Ser-Leu, the powder was obtained by concentration and freeze drying.

[0033] Furthermore, the present invention also provides an oyster protein peptide, which is prepared by the method described above, wherein the oyster protein peptide contains a functional peptide with the amino acid sequence Leu-Ser-Leu.

[0034] Furthermore, the present invention also provides the use of the aforementioned functional polypeptide or the aforementioned oyster protein peptide in the preparation of pharmaceuticals.

[0035] Preferably, the medicine is used in at least one of the following aspects:

[0036] (1) To prevent, alleviate or treat hypertension;

[0037] (2) Inhibits angiotensin-converting enzyme activity.

[0038] Furthermore, the present invention also provides a medicine containing the aforementioned functional polypeptide or the aforementioned oyster protein peptide.

[0039] Based on the above technical solution, the beneficial effects of the present invention are as follows:

[0040] The functional peptides of this invention possess excellent ACE inhibitory activity and good digestive stability. Furthermore, they can be obtained from natural food materials, which further ensures safety. Therefore, the functional peptides of this invention can be used in medicines for patients with hypertension. Attached Figure Description

[0041] Figure 1 This is the mass spectrum of the peptide product measured in Example 2 of the present invention. Detailed Implementation

[0042] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention.

[0043] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0044] Example 1: Preparation of LSL from natural food ingredients

[0045] This embodiment provides an oyster protein peptide, which is prepared by the following method:

[0046] (1) Oyster protein was crushed and homogenized in aqueous solution, and the pH was adjusted to 1.8. Then, pepsin was added for 3 hours of enzymatic hydrolysis, with an enzyme activity concentration of 2000 U / 1g of raw protein. After 3 hours of enzymatic hydrolysis, the pH was adjusted to 7.9, and trypsin was added for 3 hours of enzymatic hydrolysis, with an enzyme activity concentration of 200 U / 1g. The enzymatic hydrolysis temperature was 37.0℃, and the enzymatic hydrolysis process was simultaneously subjected to 30kH ultrasonic treatment.

[0047] (2) After the enzymatic hydrolysis is completed, the solution is filtered through diatomaceous earth and the filtrate is collected.

[0048] (3) The collected separation liquid will be sent to a two-step ultrafiltration process, using ceramic membranes with pore sizes of 3000 Daltons and 1000 Daltons respectively, to finally obtain protein peptides with a molecular weight of less than 1000 Daltons.

[0049] (4) Peptide powder containing LSL was obtained by reversed-phase high performance liquid chromatography: the filtrate was eluted and separated by Sephadex G-25 gel column with deionized water as the elution solution. After collecting the corresponding elution peaks of functional peptides with the amino acid sequence Leu-Ser-Leu, the powder was obtained by concentration and freeze drying.

[0050] Example 2: Small Intestinal Endothelial Absorption Simulation Experiment

[0051] NCM460 cells were loaded at a rate of 1 × 10⁻⁶. 5 Cells were seeded at a density of 1 / mL on the filter membrane of a 12-well Transwell plate. The culture medium was changed every other day. The complete culture medium consisted of DMEM supplemented with fetal bovine serotonin (FPS), penicillin, and streptomycin (double antibody), with a solution ratio of DMEM:serum:double antibody = 9:1:0.1. Cells were cultured at 37°C, 5% CO2, and constant humidity. The culture was continued until the epithelial cell resistivity reached 400 Ω / cm. 2 Before administration, the sample was washed with HBSS buffer. Oyster protein peptides prepared in Example 1 were added at a concentration of 0.1 mg / mL to the AP side (apical, intestinal lumen side). After incubation for two hours, the product solution after absorption, transport, and enzymatic digestion by NCM460 cells was collected from the BL side (basolateral, intestinal wall side). This solution was rapidly frozen and stored at -80°C, and the peptide products in the solution were subsequently determined using nano-HPLC-MS. Results (see...) Figure 1 The study found that the LSL peptide (m / z of 332.2186) was still well preserved, indicating that LSL can not only be effectively transported and absorbed by normal human colonic epithelial cells NCM460, but also maintains its structural integrity well.

[0052] Example 3 Assay of ACE inhibitory activity of Leu-Ser-Leu

[0053] Determination principle: Histidine-histylleucine (HHL) can be hydrolyzed into hippuric acid (Hip) and histidine-leucine (HL) under the action of angiotensin-converting enzyme (ACE). The content of histidine-leucine produced per unit time can be measured by the phthalaldehyde method, and then the reaction rate and inhibition rate can be calculated.

[0054] Assay Method: The tripeptide LSL used in the experiment was synthesized by Shanghai Nuoyou Biotechnology Co., Ltd., and the ACE enzyme was isolated and extracted from porcine lung. The buffer was a 0.1M borate-borax buffer containing 0.3 mol / L sodium chloride and pH 8.3. The tripeptide LSL was prepared into solutions of 4 μg / mL, 20 μg / mL, and 100 μg / mL, with captopril as a positive control. 15 μL of sample solution was taken, and 30 μL of 4.7 mM HCl solution and 30 μL of ACE enzyme solution were added sequentially. The enzymatic digestion reaction was carried out at 37℃ for 45 min. After digestion, 150 μL of 1M sodium hydroxide solution was added to terminate the digestion. Then, 20 μL of OPA solution was added, mixed, and reacted at 37℃ for 20 min. Finally, 90 μL of 2M hydrochloric acid was added to terminate the derivatization reaction. After appropriate dilution, the fluorescence values ​​were detected promptly under the conditions of "excitation wavelength 340 nm, emission wavelength 455 nm".

[0055] The measured values ​​when both inhibitor and ACE enzyme are present are recorded as A; the measured values ​​when both inhibitor and ACE enzyme are replaced with blank buffer solution are recorded as D; the measured values ​​when ACE enzyme is present but inhibitor is absent are recorded as B; and the measured values ​​when inhibitor is present but ACE enzyme is absent are recorded as C. The inhibition rate is then calculated as 1 - (AC) / (BD) × 100%. The inhibition rate measurement results are shown in Table 1. After measuring the inhibition rate at different concentrations, the IC50 can be calculated through nonlinear fitting. 50 .

[0056] Table 1. Inhibition rate of ACE by different concentrations of Leu-Ser-Leu

[0057]

[0058] Based on the data in Table 1, the half-maximal inhibitory concentration (IC50) of Leu-Ser-Leu against ACE was calculated to be 107.17 ± 2.05 nM; meanwhile, the half-maximal inhibitory concentration (IC50) of captopril was measured to be 10.22 ± 0.45 nM, demonstrating that this natural polypeptide has good potential for application as a lead compound.

[0059] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. The application of functional peptides in the preparation of pharmaceuticals, characterized in that, The amino acid sequence of the functional polypeptide is Leu-Ser-Leu; The drug is used in at least one of the following aspects: (1) To prevent, alleviate or treat hypertension; (2) Inhibits angiotensin-converting enzyme activity.

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