Hypotensive hexapeptide from eucheuma and preparation method and application thereof

Abstract

This invention discloses a blood pressure-lowering hexapeptide from *Euphorbia milii* with angiotensin-converting enzyme (ACE) inhibitory activity, its preparation method, and its application. Using *Euphorbia milii* as raw material, the invention employs ultrasonic extraction of crude protein, enzymatic hydrolysis, ultrafiltration, gel chromatography purification, and reversed-phase high-performance liquid chromatography (RP-HPLC) purification to obtain the blood pressure-lowering hexapeptide. The blood pressure-lowering hexapeptide prepared by this invention exhibits significant angiotensin-converting enzyme (ACE) inhibitory activity, promotes the release of endogenous diastolic factor (NO) in HUVEC cells, inhibits the production of endothelin (ET-1), and has a significant protective effect on human umbilical vein endothelial cells (HUVECs).

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of bioengineering technology, and particularly relates to a hypotensive six-peptide of Eucheuma, a preparation method and application thereof. BACKGROUND

[0002] Hypertension refers to a clinical syndrome that arterial systolic pressure is greater than 140mmHg and / or diastolic pressure is increased by 90mmHg in a resting state, and is often accompanied by functional or organic damage of organs such as heart, brain and kidney. Hypertension is the most common chronic disease and the most important risk factor of cardiovascular and cerebrovascular diseases. Scientific research shows that the disability rate and mortality rate of severe complications of hypertension such as stroke, coronary heart disease, heart failure and kidney disease are high, which has become a heavy burden of families and society in China, and effective prevention and control of hypertension has become one of the core strategies to curb the epidemic of cardiovascular and cerebrovascular diseases in China.

[0003] Angiotensin-I-Converting Enzyme (ACE) is also known as kallikrein II or peptidyl-carboxyl peptidase, and belongs to a membrane-bound enzyme of vascular endothelial cells. ACE is widely distributed in various tissues of the human body, and the content in epididymis, testis and lung is relatively rich, and the activity of lung capillary endothelial cell ACE is the highest. It is attached to the surface of endothelial cells and can be decomposed and released into the blood circulation. ACE is a key enzyme in the angiotensin system, which can catalyze angiotensin I to angiotensin II with strong vasoconstriction effect, and inactivate bradykinin with blood pressure lowering effect. Therefore, ACE inhibitors have been applied to the prevention and control of hypertension. However, the chemical synthesis of ACE inhibitors has adverse side effects such as persistent cough, skin rash and taste disorder, and the use is limited. SUMMARY

[0004] Therefore, based on the above, the present application uses food red alga Eucheuma muricatum as raw material and uses enzyme engineering technology to prepare a hypotensive six-peptide Phe-His-Pro-Ser-Tyr-Met (FHPSYM). The oligopeptide shows significant blood pressure lowering effect and can be used as a drug or an auxiliary drug for the treatment of hypertension.

[0005] The first technical problem to be solved by the present application is to provide a hypotensive six-peptide of Eucheuma according to the above technical status. The amino acid sequence of the hypotensive six-peptide is Phe-His-Pro-Ser-Tyr-Met (FHPSYM), and the ESI-MS measured molecular weight is 766.9Da. The six-peptide has significant inhibitory effect on angiotensin converting enzyme (ACE) and significant protective effect on human umbilical vein endothelial cells (HUVEC).

[0006] The second technical problem to be solved by the present application is to provide a preparation method of a hypotensive Eucheuma peptide with ACE inhibitory effect.

[0007] The preparation method of the hypotensive Eucheuma peptide comprises the following steps: taking Eucheuma as raw material, extracting crude protein through ultrasonic extraction, and then preparing the hypotensive Eucheuma peptide through enzymolysis, ultrafiltration, gel chromatography purification and RP-HPLC purification.

[0008] The preparation method of the hypotensive Eucheuma peptide comprises the following steps:

[0009] 1) Preparation of Eucheuma protein: Eucheuma powder is taken, phosphate buffer solution (0.2M, pH 7.2) is added, and then soaked for 9-12h; ultrasonic extraction is carried out at a frequency of 53kHz and a power of 200W and at 4℃ for 45-60min; the extraction liquid is filtered through gauze to remove residues, and the filtrate is centrifuged at 8000r / min for 10-15min; the supernatant is taken and ammonium sulfate solid is added to reach 100% saturation; the precipitate is centrifuged at 8000r / min for 15-20min; the precipitate is added with appropriate amount of distilled water, and then transferred to a dialysis bag with a molecular weight cut-off of 8-14kDa; the dialysis bag is dialyzed against distilled water to remove salt; the dialysate is freeze-dried to obtain Eucheuma crude protein;

[0010] 2) Preparation of Eucheuma enzymolysis liquid: the Eucheuma crude protein in step 1) is mixed with glycine-NaOH buffer solution (0.05M), the pH value of the mixed solution is adjusted to 9.0-10.0, the temperature is adjusted to 40-45℃, and alkaline protease is added for enzymolysis for 3-5h; the enzymolysis liquid is placed in boiling water for 5-10min to inactivate the enzyme; the pH value of the solution is adjusted to 8.0-8.5, the temperature is adjusted to 35-40℃, and trypsin is added for enzymolysis for 3-5h; the enzymolysis liquid is placed in boiling water for 5-10min to inactivate the enzyme, and Eucheuma protein enzymolysis liquid is obtained;

[0011] 3) Ultrafiltration fractionation of Eucheuma enzymolysis liquid: the Eucheuma protein enzymolysis liquid is subjected to ultrafiltration treatment using 1kDa, 3kDa and 5kDa ultrafiltration membranes, and components with molecular weights less than 1kDa, 1-3kDa, 3-5kDa and greater than 5kDa are collected respectively; the angiotensin converting enzyme (ACE) inhibitory activity of the four ultrafiltration components is determined, and the component with the highest ACE inhibitory activity is the ultrafiltration enzymolysis liquid, which is freeze-dried to obtain an ultrafiltration enzymolysis product;

[0012] 4) Preparation of hypotensive Eucheuma peptide: the ultrafiltration enzymolysis product is sequentially subjected to Sephadex LH-20 column chromatography and reverse phase high performance liquid chromatography (RP-HPLC) purification to obtain the hypotensive Eucheuma peptide.

[0013] As preferred, the ratio of the powder of Eucheuma to the phosphate buffer solution in step 1) is 1g:10-15mL.

[0014] As preferred, the ratio of the crude protein of Eucheuma to the glycine-NaOH buffer (0.05M) in step 2) is 1g:6-8mL.

[0015] As preferred, the amount of the added alkaline protease in step 2) is 1.0-2.0% of the mass of the powder of Eucheuma; and the amount of the added trypsin is 1.0-1.5% of the mass of the powder of Eucheuma.

[0016] As preferred, the steps of the Sephadex LH-20 chromatography and RP-HPLC purification in step 4) are as follows:

[0017] Sephadex LH-20 chromatography: the ultrafiltration enzymatic hydrolysate of Eucheuma protein is prepared into a solution of 45-55μg / mL, and is subjected to chromatography separation through a Sephadex LH-20 column, eluted with a phosphate buffer solution (0.2M, pH 7.2), and the elution components are collected according to the absorbance curve at 218nm, wherein the peak with the highest ACE inhibitory activity is the gel chromatography enzymatic hydrolysate, which is freeze-dried.

[0018] RP-HPLC purification: the gel chromatography enzymatic hydrolysate of Eucheuma protein is prepared into a solution of 60-75μg / mL, and is subjected to purification through RP-HPLC, and one high-activity oligopeptide Phe-His-Pro-Ser-Tyr-Met (FHPSYM) is obtained according to the ACE inhibitory activity of the prepared polypeptide, and the ESI-MS measurement of the molecular weight is 766.9Da.

[0019] Further preferably, the RP-HPLC conditions are as follows: injection amount 180-200μL; chromatography column Hypersil 300AC18 (250mm×10.0mm, 10μm); mobile phase: 40% acetonitrile; elution speed 2.0-2.5mL / min; ultraviolet detection wavelength 218nm.

[0020] The third technical problem to be solved by the present application is to provide an application of the Eucheuma blood pressure-lowering hexapeptide with ACE inhibitory activity in the preparation of a hypertension treatment drug.

[0021] The Eucheuma blood pressure-lowering peptide Phe-His-Pro-Ser-Tyr-Met (FHPSYM) provided by the present application has a significant inhibitory effect on ACE, and the half-inhibitory rate (IC 50) is 19.61±2.03 μM; has no obvious toxicity to human umbilical vein endothelial cells (HUVEC), and can promote the release of endogenous vasodilator nitric oxide (NO) and inhibit the production of endogenous vasoconstrictor endothelin-1 (ET-1) in HUVEC cells; the Eucheuma hypotensive peptide Phe-His-Pro-Ser-Tyr-Met (FHPSYM) has certain hypotensive and regulating functions on HUVEC cells, and can be applied to the preparation of drugs or health foods for treating hypertension.

[0022] The application adopts a controllable and environment-friendly biological enzyme method, so that the Eucheuma hypotensive hexapeptide can be released to the maximum extent through monitoring of the enzyme hydrolysis process, and the utilization rate of raw materials is improved. The hypotensive hexapeptide prepared by the application is obtained by enzyme hydrolysis of Eucheuma, is safe and has no toxic side effects, has significant ACE inhibitory activity, and has a hypotensive effect on patients with hypertension. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 The table shows the ACE inhibition rates (%) of the Eucheuma enzymatic hydrolysate (QPH) and the ultrafiltration fractionated components (QPH-1 to STH-4) in the application at a concentration of 5 mg / mL.

[0024] Figure 2 The figure shows the chromatogram obtained when the application is subjected to separation and purification by using a Sephadex LH-20 column (2.6 cm x 100 cm).

[0025] Figure 3 The table shows the ACE inhibition rates (%) of the Eucheuma ultrafiltration enzymatic hydrolysate (QPH-1) and the gel chromatography separation components (QPH-1A to QPH-1D) in the application at a concentration of 5 mg / mL.

[0026] Figure 4 The figure shows the chromatogram obtained when the application is subjected to chromatography by using a Hypersil 300A C18 (250 mm x 10.0 mm, 10 μm) column.

[0027] Figure 5 The table shows the half-inhibition rates (IC 50 ) of the components prepared by using RP-HPLC on ACE in the application.

[0028] Figure 6 The figure shows the structure of the Eucheuma hypotensive peptide Phe-His-Pro-Ser-Tyr-Met (FHPSYM) in the application.

[0029] Figure 7Mass spectrum of Phe-His-Pro-Ser-Tyr-Met (FHPSYM) as a blood pressure lowering peptide of Eucheuma cottonii according to an embodiment of the present application.

[0030] Figure 8 Effect of Phe-His-Pro-Ser-Tyr-Met (FHPSYM) as a blood pressure lowering peptide of Eucheuma cottonii according to an embodiment of the present application on the viability of human umbilical vein endothelial cells (HUVECs).

[0031] Figure 9 Effect of Phe-His-Pro-Ser-Tyr-Met (FHPSYM) as a blood pressure lowering peptide of Eucheuma cottonii according to an embodiment of the present application on the content of nitric oxide (NO) in human umbilical vein endothelial cells (HUVECs).

[0032] Figure 10 Effect of Phe-His-Pro-Ser-Tyr-Met (FHPSYM) as a blood pressure lowering peptide of Eucheuma cottonii according to an embodiment of the present application on the content of endothelin-1 (ET-1) in human umbilical vein endothelial cells (HUVECs). DETAILED DESCRIPTION

[0033] The present application will be further described in conjunction with the following examples, which are not intended to limit or restrict the scope of the present application.

[0034] The solvent used in the present application is not particularly limited, and a commercially available conventional solvent can be used.

[0035] Preparation of glycine-NaOH buffer solution (0.05 M): 50 mL of glycine (0.2 mol / L) and 16.8 mL of NaOH solution (0.2 mol / L) were mixed, and the volume was adjusted to 200 mL.

[0036] Preparation of phosphate buffer solution (0.2 M, pH 7.2): 72 mL of disodium hydrogen phosphate (0.2 mol / L) and 28 mL of sodium dihydrogen phosphate buffer solution (0.3 mol / L) were mixed.

[0037] EXAMPLE

[0038] 1) Preparation of Eucheuma protein: Take Eucheuma powder, add phosphate buffer solution (0.2 M, pH 7.2) according to the solid-liquid ratio of 1 g: 12 mL, soak for 12 h, then ultrasonic at 4 ℃ for 50 min at a frequency of 53 kHz and a power of 200 W, filter the residue of the extract with gauze, centrifuge the filtrate at 8000 r / min for 15 min, take the supernatant, add ammonium sulfate solid to reach 100% saturation, precipitate for 60 min, then centrifuge at 8000 r / min for 20 min, transfer the precipitate to a dialysis bag with a molecular weight cutoff of 8-14 kDa, dialyze against distilled water to remove salt, freeze-dry the dialysate, and obtain Eucheuma crude protein;

[0039] 2) Preparation of Eucheuma enzymatic hydrolysate: Mix the above Eucheuma crude protein with glycine-NaOH buffer (0.05 M) according to the weight-volume ratio of 1 g: 8 mL, adjust the pH of the mixture to 10.0, adjust the temperature to 45 ℃, add alkaline protease according to 1.2% of the mass of Eucheuma powder, and hydrolyze for 4 h; after the enzyme hydrolysate is placed in boiling water for 10 min, the enzyme activity is inactivated; adjust the pH of the solution to 8.5, adjust the temperature to 37 ℃, add trypsin according to 1.2% of the mass of Eucheuma powder, and hydrolyze for 4 h; after the enzyme hydrolysate is placed in boiling water for 10 min, the enzyme activity is inactivated, and Eucheuma protein enzymatic hydrolysate (QPH) is obtained;

[0040] 3) Ultrafiltration fractionation of Eucheuma enzymatic hydrolysate: Eucheuma protein enzymatic hydrolysate QPH is subjected to ultrafiltration treatment using 1 kDa, 3 kDa and 5 kDa ultrafiltration membranes to obtain four components: QPH-1 (MW < 1 kDa), QPH-2 (1 kDa < MW < 3 kDa), QPH-3 (3 kDa < MW < 5 kDa) and QPH-4 (MW > 5 kDa), and the angiotensin converting enzyme (ACE) inhibitory activity of the four ultrafiltration components is determined according to the literature [Yu H, Cao D Q, He Y L, Xu N J. Optimization of ACE-inhibitory peptides prepared from Gracilaria lemaneiformis by enzymatic hydrolysis [J]. Journal of Food and Biotechnology, 2019, 38(2): 133-139] (results shown in Figure 1 ), and the QPH-1 component has the highest ACE inhibitory activity, which is freeze-dried to obtain an ultrafiltration hydrolysate;

[0041] 4) Preparation of Eucheuma antihypertensive peptides: The ultrafiltration hydrolysate QPH-1 is sequentially subjected to Sephadex LH-20 column chromatography and reverse phase high performance liquid chromatography (RP-HPLC) purification to obtain Eucheuma antihypertensive peptides;

[0042] The purification steps using hydroxypropyl dextran gel chromatography (Sephadex LH-20) are as follows: The ultrafiltration hydrolysate QPH-1 was prepared into a 25-30 μg / mL solution, separated by hydroxypropyl dextran gel chromatography (Sephadex LH-20) column (2.6 × 100 cm), eluted with phosphate buffer solution (0.2 M, pH 7.2), and the eluted fractions (QPH-1A~QPH-1D) were collected based on the absorbance curve at 218 nm (see results). Figure 2 The angiotensin-converting enzyme (ACE) inhibitory activity of components QPH-1A to QPH-1D was measured (results are shown in Figure 3). Among them, QPH-1D had the highest ACE inhibitory activity, which is the gel chromatography enzymatic hydrolysate.

[0043] Purification steps by RP-HPLC: The gel chromatography enzymatic hydrolysate (QPH-1D) was prepared into a 70 μg / mL solution and purified using RP-HPLC (conditions: injection volume 190 μL; Hypersil 300A C18 column (250 mm × 10.0 mm, 10 μm); mobile phase: 40% acetonitrile; elution rate 2.5 mL / min; UV detection wavelength 218 nm) (results are shown in [link to results]). Figure 4 The fractions QPP1 to QPP9 were collected and separated, and their angiotensin-converting enzyme (ACE) half-maximal inhibitory rate (IC50) was measured. 50 (Results are shown in) Figure 5 QPP7 has the lowest ACE half-repression rate (IC50). 50 ), and determine its amino acid sequence and molecular weight.

[0044] Structural analysis: Collection of ACE half-inhibition rate (IC50) 50 The lowest fraction (QPP7) was sequenced using a protein / peptide sequencer, and its amino acid sequence was determined to be Phe-His-Pro-Ser-Tyr-Met (FHPSYM) (see results). Figure 6 The molecular weight, as determined by ESI / MS, was 766.9 Da (see results below). Figure 7 ).

[0045] The ACE inhibitory activity of the prepared Euphorbia lactea antihypertensive peptide Phe-His-Pro-Ser-Tyr-Met (FHPSYM) was tested. The results showed that the half-maximal inhibitory concentration (IC50) of this peptide was significantly higher than that of other peptides. 50 The value was 19.61 ± 2.03 μM.

[0046] Reference [Shuo-Lei Zheng, Qian-Bin Luo, Shi-Kun Suo, Yu-Qin Zhao, Chang-Feng Chi, Bin Wang. Preparation, identification, molecular docking study and protective function on HUVECs of novel ACE inhibitory peptides from protein hydrolysate of skipjack tuna muscle [J]. Mar. Drugs 2022, 20, 176] evaluates the effect of Phe-His-Pro-Ser-Tyr-Met (FHPSYM) on related indicators of human umbilical vein endothelial cells (HUVEC), and the results prove that Phe-His-Pro-Ser-Tyr-Met (FHPSYM) has no significant effect on the cell viability of HUVEC, i.e. no significant cytotoxic activity (results are shown in Figure 8 ), and can promote the release of endogenous vasodilator nitric oxide (NO) and inhibit the production of endogenous vasoconstrictor endothelin-1 (ET-1) in HUVEC cells (results are shown in Figure 9 and Figure 10 ), indicating that Phe-His-Pro-Ser-Tyr-Met (FHPSYM) has certain protective function on HUVEC cells.

Claims

1. A blood pressure-lowering hexapeptide from Euphorbia milii, characterized in that... The amino acid sequence of the blood pressure reducing peptide is Phe-His-Pro-Ser-Tyr-Met, and the ESI-MS measured molecular weight is 766.9 Da.

2. The use of the blood pressure reducing hexapeptide of Eucheuma in the preparation of a drug for treating hypertension.

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