A sea squirt oligopeptide for lowering blood pressure, and a preparation method and application thereof

Abstract

The application discloses a sea squirt siphon blood pressure reducing oligopeptide with angiotensin converting enzyme (ACE) inhibitory activity and a preparation method and application thereof, the amino acid sequence of the blood pressure reducing oligopeptide is Phe-Cys-Arg-Leu-His-Pro (FCRLHP), and the ESI / MS detection molecular weight is 771.93 Da. The sea squirt siphon is crushed, ultrasonically pretreated, enzymatically hydrolyzed, ultrafiltered, gel chromatographically purified, and RP-HPLC purified to obtain the blood pressure reducing oligopeptide Phe-Cys-Arg-Leu-His-Pro (FCRLHP). The active peptide Phe-Cys-Arg-Leu-His-Pro (FCRLHP) prepared by the application has significant angiotensin converting enzyme (ACE) inhibitory activity and human umbilical vein endothelial cell (HUVEC) protection effect, and can be developed as a hypertension treatment drug and an auxiliary treatment product.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a cuttlebone-based oligopeptide for lowering blood pressure, its preparation method, and its application. Background Technology

[0002] Hypertension is a clinical syndrome characterized primarily by elevated systemic arterial blood pressure (systolic and / or diastolic blood pressure) (systolic blood pressure ≥140 mmHg, diastolic blood pressure ≥90 mmHg), which may be accompanied by functional or organic damage to organs such as the heart, brain, and kidneys. Hypertension is the most common chronic disease and a major risk factor for cardiovascular and cerebrovascular diseases.

[0003] Angiotensin-converting enzyme (ACE), also known as kininase II, is a membrane-bound enzyme of vascular endothelial cells and is widely distributed in various tissues of the human body. ACE has two main functions: catalyzing the conversion of angiotensin I to angiotensin II, thereby activating bradykinin. Because of these two functions, angiotensin-converting enzyme has become an effective target for treating diseases such as hypertension, heart failure, and type 2 diabetes. Angiotensin-converting enzyme inhibitors (ACEIs) have been extensively studied for the prevention and control of hypertension; however, synthetically produced ACEIs have various adverse side effects. Summary of the Invention

[0004] This invention uses cuttlebone as raw material and employs enzyme engineering and chromatography techniques to prepare a cuttlebone-based hypotensive oligopeptide. This cuttlebone-based hypotensive oligopeptide exhibits significant hypotensive effects and vascular endothelial cell protection, and can be used to prepare drugs or adjuvant therapy products for the prevention / treatment of hypertension.

[0005] On one hand, the present invention provides a cuttlebone-based blood pressure-lowering oligopeptide, wherein the amino acid sequence of the cuttlebone-based blood pressure-lowering oligopeptide is Phe-Cys-Arg-Leu-His-Pro(FCRLHP), and its molecular weight is determined to be 771.93 Da by ESI-MS.

[0006] The cuttlebone-based blood pressure-lowering oligopeptide of this invention can significantly inhibit angiotensin-converting enzyme (ACE) and has a significant protective effect against oxidative damage to human umbilical vein endothelial cells (HUVECs).

[0007] On the other hand, the present invention provides a method for preparing the above-mentioned cuttlebone antihypertensive oligopeptide, which includes the following steps: cuttlebone pretreatment and crude protein extraction, enzymatic hydrolysis with a combination of flavor protease and neutral protease to obtain enzymatic hydrolysate, and preparation of cuttlebone antihypertensive oligopeptide by ultrafiltration, ion exchange chromatography, gel filtration chromatography and high performance liquid chromatography.

[0008] Furthermore, the preparation method includes the following steps:

[0009] 1) Pretreatment and crude protein extraction of cuttlebone: Wash, dry, and pulverize the cuttlebone sample, then sieve it. Add ethyl acetate, sonicate at room temperature, centrifuge to remove the supernatant, and obtain cuttlebone powder. Add the cuttlebone powder to SDTris-HCl buffer, adjust the pH of the mixture to 6.5-7.5, sonicate at room temperature, centrifuge, and collect the supernatant. Add acetone to the supernatant until the acetone concentration reaches 80%, place in a -4℃ refrigerator for 8-12 hours, centrifuge, wash the precipitate twice with 80% acetone, and air dry to obtain crude cuttlebone protein.

[0010] 2) Preparation of Cuttlebone Enzymatic Hydrolysate: The crude cuttlebone protein was added to phosphate buffer (0.2 mol / L) at a ratio of 1 g: 10-15 mL. The pH of the mixture was adjusted to 6.5-7.5, and the temperature was adjusted to 45-55℃. Flavored protease was added, and the mixture was enzymatically hydrolyzed for 2-4 hours. The hydrolysate was then placed in boiling water for 5-10 minutes to inactivate the enzyme. The temperature of the mixture was adjusted to 50-60℃, and neutral protease was added. The mixture was then enzymatically hydrolyzed for 2-4 hours. The hydrolysate was then placed in boiling water for 5-10 minutes to inactivate the enzyme, thus obtaining the cuttlebone enzymatic hydrolysate.

[0011] 3) Ultrafiltration fractionation of cuttlebone enzymatic hydrolysate: The cuttlebone enzymatic hydrolysate was ultrafiltered using 1.0 kDa and 3.5 kDa ultrafiltration membranes. Fractions with molecular weights less than 1 kDa, 1-3.5 kDa, and greater than 3.5 kDa were collected, respectively. The angiotensin-converting enzyme (ACE) inhibitory activity of each fraction was measured. The fraction with the highest ACE inhibitory activity was the ultrafiltration enzymatic hydrolysate, which was then lyophilized to obtain the ultrafiltration enzymatic hydrolysate.

[0012] 4) Preparation of cuttlebone hypotensive peptide: The ultrafiltration enzymatic hydrolysate was purified sequentially by hydroxypropyl dextran gel (Sephadex LH-20) column chromatography and reversed-phase high-performance liquid chromatography (RP-HPLC) to obtain cuttlebone hypotensive peptide.

[0013] Preferably, the weight-to-volume ratio of cuttlebone to ethyl acetate in step 1) is 1g:5-8mL.

[0014] Preferably, the concentration of the SDS Tris-HCl buffer in step 1) is 0.05 mol / L, and the weight-to-volume ratio of the cuttlebone powder to the SDS Tris-HCl buffer is 1 g: 10-15 mL.

[0015] Preferably, the concentration of the phosphate buffer solution in step 2) is 0.2 mol / L.

[0016] Preferably, in step 2), the mass ratio of flavor protease to cuttlebone powder is 1.0–2.0%.

[0017] Preferably, in step 2), the mass ratio of neutral protease to cuttlebone powder is 1.0–2.0%.

[0018] Preferably, the hydroxypropyl dextran gel (Sephadex LH-20) chromatographic purification and RP-HPLC purification in step 4) are as follows:

[0019] Hydroxypropyl dextran gel chromatography (Sephadex LH-20): The ultrafiltration hydrolysate was prepared into a 45-55 μg / mL solution and separated by hydroxypropyl dextran gel chromatography (Sephadex LH-20). The solution was eluted with double-distilled water, and the eluted fractions were collected according to the absorbance curve at 220 nm. The peak with the highest ACE inhibitory activity was the gel chromatography hydrolysate, which was then lyophilized.

[0020] RP-HPLC purification: The above cuttlebone gel chromatography enzymatic hydrolysate was prepared into a solution of 50-60 μg / mL with double-distilled water and purified by RP-HPLC. Based on the ACE inhibitory activity of the prepared oligopeptide, a highly active oligopeptide, Phe-Cys-Arg-Leu-His-Pro (FCRLHP), was obtained. The molecular weight was determined to be 771.93 Da by ESI-MS.

[0021] More preferably, the RP-HPLC conditions are as follows: injection volume 15-20 μL; column Zorbax, SB C-18 (4.6 × 250 mm, 5 μm); mobile phase: gradient elution, acetonitrile concentration is increased uniformly from 0 to 40% within 30 min; elution rate 0.8-1.2 mL / min; UV detection wavelength 220 nm.

[0022] In another aspect, the present invention provides a pharmaceutical composition containing a therapeutically effective amount of the cuttlebone-based blood pressure-lowering oligopeptide Phe-Cys-Arg-Leu-His-Pro (FCRLHP).

[0023] In addition, the present invention provides the use of the cuttlebone hypotensive oligopeptide or a pharmaceutical composition containing it in the preparation of a drug.

[0024] Furthermore, the preparation of the drug is for use in the preparation of drugs or adjunctive treatment products for treating and / or preventing hypertension.

[0025] The cuttlebone antihypertensive peptide Phe-Cys-Arg-Leu-His-Pro (FCRLHP) of this invention has a significant inhibitory effect on ACE, with a half-maximal inhibitory rate (IC50) of 100%. 50The concentration was 4.36 μM; it had no significant toxicity to human umbilical vein endothelial cells (HUVECs), and could promote the release of endogenous relaxing factor nitric oxide (NO) and inhibit the production of endogenous contractile factor endothelin-1 (ET-1) in HUVECs. It also had a significant protective effect against H2O2 oxidative damage to human umbilical vein endothelial cells (HUVECs), and could be used as a drug or adjunctive therapy for the treatment and / or prevention of hypertension.

[0026] This invention employs a controllable and environmentally friendly enzymatic method. By monitoring the enzymatic hydrolysis process, the antihypertensive peptides from cuttlebone are released to the maximum extent, improving the utilization rate of raw materials. The preparation process is scientific and reasonable, simple to operate, and has strong industrial feasibility. The prepared antihypertensive peptides from cuttlebone are obtained by enzymatic hydrolysis of cuttlebone, are safe and have no toxic side effects, have significant ACE inhibitory activity, and have a hypotensive effect. Attached Figure Description

[0027] Figure 1 The inhibition rate (%) of angiotensin-converting enzyme (ACE) of cuttlebone enzymatic hydrolysate (SH) and its ultrafiltration fractions (SH-I to SH-III) at a concentration of 3.0 mg / mL.

[0028] Figure 2 This is a chromatogram obtained by separating and purifying SH-I using a hydroxypropyl dextran gel (Sephadex LH-20) column (2.6cm × 120cm) in an embodiment of the present invention.

[0029] Figure 3 The inhibition rate (%) of angiotensin-converting enzyme (ACE) of cuttlebone ultrafiltration hydrolysate (SH-I) and its gel chromatography-separated fractions (SH-Ia~SH-Id) at a concentration of 3.0 mg / mL.

[0030] Figure 4 This is a chromatogram obtained when SH-Ic is separated and purified using a Zorbax, SB C-18 (4.6×250mm, 5μm) column in an embodiment of the present invention.

[0031] Figure 5 This is the inhibition rate (%) of ACE by the components (SP1~SP13) prepared by RP-HPLC in the embodiments of the present invention at a concentration of 1.5 mg / mL.

[0032] Figure 6 This is the structural diagram of the cuttlebone-derived blood pressure-lowering oligopeptide Phe-Cys-Arg-Leu-His-Pro (FCRLHP).

[0033] Figure 7This is the mass spectrum of Phe-Cys-Arg-Leu-His-Pro (FCRLHP), an oligopeptide from cuttlebone that lowers blood pressure.

[0034] Figure 8 The effect of cuttlebone-derived blood pressure-lowering oligopeptide Phe-Cys-Arg-Leu-His-Pro (FCRLHP) on the activity of human umbilical vein endothelial cells (HUVEC).

[0035] Figure 9 The study investigated the effect of the cuttlebone-derived blood pressure-lowering oligopeptide Phe-Cys-Arg-Leu-His-Pro (FCRLHP) on the nitric oxide (NO) content in human umbilical vein endothelial cells (HUVECs).

[0036] Figure 10 The effect of cuttlebone-derived blood pressure-lowering oligopeptide Phe-Cys-Arg-Leu-His-Pro (FCRLHP) on the endothelin-1 (ET-1) content in human umbilical vein endothelial cells (HUVEC).

[0037] Figure 11 This study investigated the protective effect of cuttlebone-derived blood pressure-lowering oligopeptide Phe-Cys-Arg-Leu-His-Pro (FCRLHP) against H2O2-induced oxidative damage to human umbilical vein endothelial cells (HUVECs). Detailed Implementation

[0038] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0039] Example

[0040] A method for preparing a cuttlebone-based antihypertensive oligopeptide, the preparation process is as follows: cuttlebone → tissue disruption → ultrasonic pretreatment → enzymatic hydrolysis → ultrafiltration → gel chromatography purification → RP-HPLC purification → cuttlebone-based antihypertensive oligopeptide → activity evaluation.

[0041] 1) Pretreatment and crude protein extraction of cuttlebone: The cuttlebone sample was washed, dried, pulverized, and passed through a 200-mesh sieve. Ethyl acetate was added at a ratio of 1g:8mL, and the mixture was sonicated at 42kHz and 300W for 30min at room temperature. The supernatant was removed by centrifugation to obtain cuttlebone powder. The cuttlebone powder was added to 4% SDS-Tris-HCl buffer (0.05mol / L) at a ratio of 1g:10mL, and the pH of the mixture was adjusted to 7.0. The mixture was sonicated at 42kHz and 300W for 60min at room temperature, centrifuged at 6000rpm for 15min, and the supernatant was collected. Acetone was added to the supernatant until the acetone concentration reached 80%, and the mixture was placed in a -4℃ refrigerator for 12 hours. The mixture was centrifuged at 12000rpm for 25min, and the precipitate was washed twice with 80% acetone and air-dried to obtain crude cuttlebone protein.

[0042] 2) Preparation of Cuttlebone Enzyme Hydrolysate: The crude cuttlebone protein was added to phosphate buffer (0.2 mol / L) at a ratio of 1 g: 10 mL. The pH of the mixture was adjusted to 7.0, and the temperature was adjusted to 45–55 °C. Flavor protease was added at 1.5% of the weight of the cuttlebone powder, and the mixture was enzymatically hydrolyzed for 2.5 h. The hydrolysate was then placed in boiling water for 10 min to inactivate the enzyme. The temperature of the mixture was adjusted to 55 °C, and neutral protease was added at 1.5% of the weight of the cuttlebone powder, and the mixture was enzymatically hydrolyzed for 2.5 h. The hydrolysate was then placed in boiling water for 10 min to inactivate the enzyme, yielding cuttlebone enzymatic hydrolysate (SH).

[0043] 3) Ultrafiltration fractionation of cuttlebone enzymatic hydrolysate: The cuttlebone enzymatic hydrolysate was ultrafiltered using 1.0 kDa and 3.5 kDa ultrafiltration membranes. Fractions with molecular weights less than 1 kDa (SH-I), 1-3.5 kDa (SH-II), and greater than 3.5 kDa (SH-III) were collected. The angiotensin-converting enzyme (ACE) inhibitory activity of fractions SH-I, SH-II, and SH-III was measured (see...). Figure 1 The component with the highest ACE inhibitory activity is the ultrafiltration hydrolysate, which is lyophilized to obtain the ultrafiltration hydrolysate (SH-I).

[0044] 4) Preparation of cuttlebone hypotensive peptide: The ultrafiltration enzymatic hydrolysate (SH-I) was purified sequentially by hydroxypropyl dextran gel (Sephadex LH-20) column chromatography and reversed-phase high-performance liquid chromatography (RP-HPLC) to obtain cuttlebone hypotensive peptide.

[0045] ① Hydroxypropyl dextran gel (Sephadex LH-20) chromatography purification: The ultrafiltration enzymatic hydrolysate (SH-I) was prepared into a 45-55 μg / mL solution, separated by hydroxypropyl dextran gel (Sephadex LH-20) column chromatography, eluted with double-distilled water, and the eluent fractions (SH-Ia, SH-Ib, SH-Ic, and SH-Id) were collected according to the absorbance curve at 220 nm (see...). Figure 2 The angiotensin-converting enzyme (ACE) inhibitory activities of components SH-Ia, SH-Ib, SH-Ic, and SH-Id were determined (see [reference needed]). Figure 3 Among them, the peak with the highest ACE inhibitory activity was the gel chromatography enzymatic hydrolysate (SH-Ic), which was lyophilized.

[0046] ②RP-HPLC purification: The above-mentioned cuttlebone gel chromatography enzymatic hydrolysate (SH-Ic) was dissolved in double-distilled water to prepare a solution of 50-60 μg / mL. Purification was performed using RP-HPLC (injection volume 15-20 μL; column Zorbax, SB C-18 (4.6 × 250 mm, 5 μm); mobile phase: gradient elution, acetonitrile concentration increased uniformly from 0 to 40% over 30 min; elution rate 0.8-1.2 mL / min; UV detection wavelength 220 nm) (see [link to RP-HPLC purification process]). Figure 4 The angiotensin-converting enzyme (ACE) inhibition rate of each isolated fraction (SP1–SP13) was determined (see [reference]). Figure 5 SP7 has the lowest angiotensin-converting enzyme (ACE) inhibition rate, and its amino acid sequence and molecular weight were determined.

[0047] ③ Structural analysis: The fraction with the highest ACE inhibitory activity (SP7) was collected, and its amino acid sequence was determined using a protein / peptide sequencer to be Phe-Cys-Arg-Leu-His-Pro (FCRLHP) (see [link to protein / peptide sequencer]). Figure 6 The molecular weight, as determined by ESI / MS, was 771.93 Da (see...). Figure 7 ).

[0048] ④ Functional Evaluation: The ACE inhibitory activity of the cuttlebone antihypertensive peptide Phe-Cys-Arg-Leu-His-Pro (FCRLHP) prepared above was tested. The experimental results showed that the half-maximal inhibitory concentration (IC50) of this peptide was [missing information]. 50 The value is 4.36 μM.

[0049] The effects of Phe-Cys-Arg-Leu-His-Pro (FCRLHP) on human umbilical vein endothelial cells (HUVECs) and related indicators were evaluated according to the literature [Shi-Kun Suo, Yu-Qin Zhao, Yu-Mei Wang, Xiao-Yang Pan, Chang-FengChi, Bin Wang. Seventeen novel angiotensin converting enzyme (ACE) inhibitory peptides from protein hydrolysate of Mytilus edulis: Isolation, identification, molecular docking study, and protective function on HUVECs. Food & Function, 2022, 13, 7831-7846]. The results demonstrated that Phe-Cys-Arg-Leu-His-Pro (FCRLHP) had no significant toxicity to HUVECs (see [link to article]). Figure 8 It can also promote the release of the endogenous relaxing factor nitric oxide (NO) in HUVEC cells (see...). Figure 9 ) and inhibit the production of the endogenous contractile factor endothelin-1 (ET-1) (see Figure 10 It can also alleviate oxidative damage caused by H2O2 to human umbilical vein endothelial cells (HUVECs) (see...). Figure 11 Therefore, Phe-Cys-Arg-Leu-His-Pro (FCRLHP) has certain hypotensive and protective functions on HUVEC cells.

[0050] Finally, it should be noted that the above-described embodiments are merely one specific example of the present invention. Obviously, the present invention is not limited to the above embodiments and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the disclosure of the present invention should be considered within the scope of protection of the present invention.

Claims

1. A sea squirt oligopeptide for lowering blood pressure, characterized in that, The amino acid sequence of the sea squirt siphonaxin antihypertensive oligopeptide is Phe-Cys-Arg-Leu-His-Pro (FCRLHP), and the ESI-MS determination of its molecular weight is 771.93 Da.

2. A pharmaceutical composition, characterized by, The pharmaceutical composition contains a therapeutically effective amount of the sea squirt siphonaxin antihypertensive oligopeptide Phe-Cys-Arg-Leu-His-Pro (FCRLHP) as claimed in claim 1.

3. Use of the sea squirt siphonaxin antihypertensive oligopeptide as claimed in claim 1 or the pharmaceutical composition containing the same as claimed in claim 2 in the preparation of a drug for treating and / or preventing hypertension.

Images