CN102643889B - Antihypertensive active rapeseed peptide and preparation method and application thereof - Google Patents

Abstract

The invention provides a kind of antihypertensive rapeseed active peptide and its preparation method and application. The method for preparing the blood pressure-lowering rapeseed active peptide is to hydrolyze the rapeseed protein liquid with pepsin and trypsin to obtain the crude extract of the blood-pressure-lowering rapeseed active peptide, and obtain the blood-pressure-lowering rapeseed active peptide after refining. The invention also provides a blood pressure-lowering rapeseed active peptide prepared by the method and its application. The rapeseed meal for preparing the blood pressure-lowering rapeseed active peptide has wide sources, simple production process, low cost and environmental protection, and is a method suitable for industrialization. The blood pressure-lowering rapeseed active peptide has little gastrointestinal reaction, can resist the hydrolysis of human digestive tract enzymes, acts on target organs in its active form, and has a good blood pressure-lowering effect. The antihypertensive rapeseed active peptide can not only inhibit ACE, but also inhibit renin activity, can avoid the bypass pathway of RAS activated by long-term administration of antihypertensive drugs ACE inhibitors, and has a good application prospect in reducing blood pressure.

Description

A kind of antihypertensive rapeseed active peptide and its preparation method and application

technical field

The invention relates to the field of biotechnology, in particular to the antihypertensive rapeseed active peptide, its preparation method and application.

Background technique

my country is the country with the largest rapeseed production in the world, with an annual output of nearly 12 million tons. Rapeseed meal is the main by-product of rapeseed oil processing, accounting for 65% of the total rapeseed, which contains 35% to 42% of protein. The amino acid composition of rapeseed protein is reasonable, and the content of lysine, cystine and methionine is relatively high, so it is an excellent vegetable protein. The development and utilization of rapeseed protein resources has great social significance and good economic benefits for the effective use of our country's plant protein resources and the improvement of the nutritional level of our people.

At present, there are about 200 million hypertensive patients in my country, and there are still 10 million new people added every year. The existing antihypertensive drugs have the following problems: On the one hand, long-term use of existing antihypertensive drugs will cause many side effects to the body, especially It has great damage to the digestive tract system; on the other hand, the target of most antihypertensive peptide research is angiotensin transferase (ACE), but long-term use of antihypertensive drugs ACE inhibitors will activate angiotensin system (RAS) bypass pathway, thereby affecting the clinical treatment of hypertension. However, renin inhibitors can block the RAS system from the source, and have more potential benefits, fewer adverse reactions, and better tolerance than ACE inhibitors, and have been shown to be effective in large-scale studies of intermediate end points. Target organ protection. However, there are fewer blood pressure-lowering drugs based on renin inhibitors.

Contents of the invention

The purpose of the present invention is to provide a method for preparing antihypertensive rapeseed active peptide by enzymatic method, which has a wide range of sources of raw materials, simple production process, low cost, and turning waste into treasure, which is beneficial to environmental protection. It is a low-cost, suitable A method for industrially mass-preparing rapeseed for lowering blood pressure.

Another object of the present invention is to provide antihypertensive rapeseed active peptide, which has a small gastrointestinal reaction, can resist the hydrolysis of human digestive tract enzymes, acts on target organs in its active form, and has a good blood pressure lowering effect .

Another object of the present invention is to provide the application of antihypertensive rapeseed active peptide in the preparation of antihypertensive drugs.

The invention provides a method for preparing antihypertensive rapeseed active peptide, comprising the following steps:

(1) Use crushed rapeseed meal as raw material, mix the raw material with water, adjust the pH value to 1.5-2.5, temperature 36.5-37.5 ℃, stir and disperse for 0.5-2 h, and obtain rapeseed protein liquid;

(2) Add pepsin to the rapeseed protein liquid described in step (1), and hydrolyze it at 36.5°C to 37.5°C for 1 to 3 hours to obtain pepsin hydrolyzate of rapeseed protein; add pepsin hydrolyzate of rapeseed protein to Adjust the pH value to 6.5.~8.0, add trypsin, hydrolyze at 36.5°C~37.5°C for 3-5 hours, inactivate the pepsin and trypsin in it, centrifuge to take the supernatant, and get the antihypertensive rapeseed active peptide crude extract;

(3) The blood pressure lowering rapeseed active peptide is obtained by refining the crude extract of the blood pressure lowering rapeseed active peptide in step (2).

The source of the rapeseed meal in step (1) is common rapeseed or double-low rapeseed, and the particle size of the crushed rapeseed meal is 10-200 μm.

The raw material of the rapeseed protein liquid in step (1) may also be protein separated from rapeseed meal.

The refining method described in step (3) is as follows: the crude extract of the antihypertensive rapeseed active peptide is ultrafiltered through an ultrafiltration membrane with a molecular weight cut-off of 3 kDa, the permeate is taken for dialysis and desalination, and freeze-dried to obtain the antihypertensive rapeseed active peptide.

The refining method described in step (3) is: the crude extract of the antihypertensive rapeseed active peptide is separated by reverse-phase high-performance liquid chromatography, and two parts with an organic solvent volume concentration of 30%~35% and 45%~50% are respectively taken. The eluent, the two parts of the eluent were freeze-dried respectively to obtain the antihypertensive rapeseed active peptide; the conditions for the reverse-phase high-performance liquid chromatography separation were: the chromatographic column was a carbon-12 or carbon-18 silica gel column; the mobile phase It is water and an organic solvent, the organic solvent is methanol or acetonitrile, and both the water and the organic solvent contain trifluoroacetic acid with a mass concentration of 0.05-0.10%, and the elution conditions are: linear elution, the organic solvent in the mobile phase Volume concentrations vary from 5% to 80%.

The mass ratio of the pepsin added in the step (2) to the protein in the rapeseed protein liquid is 1-10:100, and the mass ratio of the added pancreatin to the protein in the rapeseed protein liquid is 1-10:100. Preferably, the mass ratio of the pepsin added to the protein in the rapeseed protein liquid is 5:100, and the mass ratio of the added pancreatin to the protein in the rapeseed protein liquid is 5:100.

The present invention also provides a blood pressure-lowering rapeseed active peptide prepared by the method.

The present invention also provides the application of the antihypertensive rapeseed active peptide in the preparation of antihypertensive drugs.

Beneficial effects of the present invention:

(1) The rapeseed meal used in the method for preparing the blood pressure-lowering rapeseed active peptide of the present invention has a wide range of sources, the production process is simple, the cost is low, and it turns waste into treasure, which is beneficial to environmental protection. It is a low-cost, suitable for large-scale industrialization Method for preparing rapeseed for lowering blood pressure.

(2) The present invention directly uses rapeseed meal as raw material, simulates gastrointestinal digestion, uses pepsin and pancreatin in combination to hydrolyze rapeseed meal to prepare antihypertensive rapeseed active peptide, has little gastrointestinal reaction, and can resist human body digestion The hydrolysis of Dao enzyme acts on the target organ in its active form, and has a good blood pressure lowering effect. The reason is that: pepsin, trypsin and chymotrypsin and other gastrointestinal proteases can hydrolyze food protein to release blood pressure-lowering peptides that can resist digestive tract enzyme hydrolysis; pancreatin (pancreatin), also known as pancreatin, is a The mixture of protease, pancreatic amylase and pancreatic lipase has strong protein hydrolysis ability and can be used to prepare rapeseed blood pressure lowering peptide.

(3) In addition, the antihypertensive rapeseed active peptide obtained by two refining methods can not only inhibit angiotensin transferase (ACE), but also inhibit renin, which can avoid the blood pressure activated by long-term use of antihypertensive drugs ACE inhibitors. The bypass pathway of the angiotensin system (RAS), thus greatly improving the antihypertensive effect. the

Description of drawings

Figure 1 is a chromatogram of the crude extract of antihypertensive rapeseed active peptide separated by reversed-phase high performance liquid chromatography.

Detailed ways

The pepsin (Pepsin) used in the present invention is purchased from Sigma Company, article number is 232-629-3, product number P7000; Pancreatin (Pancreatin) is purchased from Sigma Company, article number is 232-468-9, product number P1750.

Example 1 Simulated gastrointestinal tract digestion and hydrolysis of rapeseed meal to prepare crude extract of antihypertensive rapeseed active peptide

method one)

(1) Crush the rapeseed meal, pass through a 100-mesh sieve, use the crushed rapeseed meal as the raw material, and mix the raw material with water at a solid-to-liquid ratio of 1:20, adjust the pH value to 2.0, and the temperature is 36.5°C to 37.5°C ℃, stir and disperse for 1 h to obtain rapeseed protein liquid.

The source of the rapeseed meal is common rapeseed or double-low rapeseed, and the particle size of the crushed rapeseed meal is 10-200 μm.

It is also possible to use protein isolated from rapeseed meal instead of pulverized rapeseed meal to prepare rapeseed protein liquid.

(2) According to the mass ratio of the pepsin added to the protein in the rapeseed protein liquid of 1 to 10:100, add pepsin to the rapeseed protein liquid, control the temperature and pH value, and hydrolyze for 2 h to obtain rapeseed Protein pepsin hydrolyzate; adjust the pH value of the rapeseed protein pepsin hydrolyzate to 7.0~7.5, according to the mass ratio of the added trypsin to the protein in the rapeseed protein solution is 1~10:100, add trypsin, at 36.5 Continue hydrolysis for 4 h at ℃～37.5 ℃. After the reaction, heat the mixture to 90-100°C, keep it for 10 minutes, inactivate protease and trypsin, cool to room temperature, centrifuge at 10,000 g for 1 hour, and take the supernatant to obtain the crude blood pressure-lowering rapeseed active peptide. Extraction.

Method Two)

(1) Crush the rapeseed meal, pass through a 100-mesh sieve, use the crushed rapeseed meal as raw material, and mix the raw material with water at a solid-to-liquid ratio of 1:20, adjust the pH value to 2.5, and the temperature is 36.5°C-37.5 ℃, stirring and dispersing for 2 h to obtain rapeseed protein liquid.

The source of the rapeseed meal is common rapeseed or double-low rapeseed, and the particle size of the crushed rapeseed meal is 10-200 μm.

It is also possible to use protein isolated from rapeseed meal instead of pulverized rapeseed meal to prepare rapeseed protein liquid.

(2) According to the mass ratio of the added pepsin to the protein in the rapeseed protein solution of 5:100, add pepsin to the rapeseed protein solution, control the temperature and pH value, and hydrolyze for 2 hours to obtain the rapeseed protein stomach Protease hydrolyzate; adjust the pH value of the rapeseed protein pepsin hydrolyzate to 7.0~7.5, add trypsin according to the mass ratio of the added trypsin to the protein in the rapeseed protein solution is 5:100, and continue hydrolysis at 37 °C for 4 h. After the reaction, the mixture was heated to 95°C and kept for 10 minutes to inactivate the protease and trypsin, cooled to room temperature, and centrifuged at 10,000 g for 1 hour to obtain the supernatant to obtain the crude extract of the antihypertensive rapeseed active peptide .

Example 2 Preparation of Rapeseed Active Peptide for Blood Pressure by Refining the Crude Extract of Rapeseed Active Peptide for Blood Pressure

The blood pressure lowering rapeseed active peptide is refined to obtain the blood pressure lowering rapeseed active peptide.

Refining method 1: The crude extract of the antihypertensive rapeseed active peptide is ultrafiltered with an ultrafiltration membrane with a molecular weight cut-off of 3kDa, the permeate (all polypeptides with a molecular weight less than 3KDa) is desalted by dialysis, and freeze-dried to obtain the antihypertensive rapeseed active peptide , the antihypertensive rapeseed active peptide has both renin inhibitory activity and ACE inhibitory activity.

The method for detecting ACE inhibitory activity is as follows: add 1 mL of 0.5 mM FAPGG (full name N-[3-(2-Furyl)acryloyl]-L-phenylalanyl-glycyl-glycine) to a cuvette (dissolved in 50 mM Tris-HCl buffer solution, containing 0.3 M NaCl, pH 7.5), 20 μL ACE (1U/mL, final activity 20 mU), 200 μL antihypertensive rapeseed active peptide solution (protein concentration is 1mg/mL; solvent is 50 mM Tris-HCl buffer solution, containing 0.3 M NaCl, pH 7.5), mixed at room temperature, and measured the decrease rate of absorbance at 345 nm within 2 minutes. The blank was replaced by Tris–HCl buffer. The ACE inhibitory activity was calculated according to the following formula:

ACE inhibitory activity (%)=1-[△A.min ^-1 _(sample) / △A.min ^-1 _(blank) ]×100%,

Wherein, ΔA.min ^-1 _(sample) and ΔA.min ^-1 _(blank) represent the ACE activity inhibition rates of the sample and the blank, respectively.

The detection method of renin inhibitory activity is as follows: In vitro assay of antihypertensive rapeseed active peptide inhibitory activity of angiotensin According to the method provided by the renin activity kit (purchased from Cayman Chemical, No. 10006270), it is measured according to the following steps: 96-well plate (1) blank: 20 μL substrate, 160 μL test buffer, 10 μL pure water; (2) blank control: 20 μL substrate, 150 μL test buffer, and 10 μL pure water; ( 3) Sample: 20 μL substrate, 150 μL assay buffer, and 10 μL sample. Afterwards, 10 μL of renin was added to each well (except the blank) to start the reaction. Shake the 96-well plate at room temperature for 10 s, preheat at 37 ^° C for 15 min, measure the fluorescence intensity of the sample within 10 min, the excitation wavelength is 340nm, the emission wavelength is 490nm, and the renin inhibitory activity of the sample is calculated according to the following formula:

, wherein, 100% inhibition rate (blank control) and inhibition rate (sample) represent the renin activity inhibition rate of the blank control and the sample within 10 min, respectively. Among them, the preparation of test buffer: (1) Preparation of buffer: 50 mM Tris-HCl buffer, containing 0.1 M NaCl, pH 8.0; (2) Dissolving antihypertensive rapeseed active peptide in the buffer to prepare The solution with a protein concentration of 1mg/mL is the test buffer.

The antihypertensive rapeseed active peptide prepared by the above method has ACE inhibitory activity of 50.84 % and renin inhibitory activity of 75.31 %, which has a good application prospect in lowering blood pressure.

Refining method 2: separate the crude extract of antihypertensive rapeseed active peptide by reversed-phase high-performance liquid chromatography, and take two parts of eluents with volume concentrations of organic solvents of 30%~35% and 45%~50%, respectively. The eluents of the two parts were freeze-dried respectively to obtain the antihypertensive rapeseed active peptide; the conditions for the reversed-phase high-performance liquid chromatography separation were: the chromatographic column was a carbon-12 or carbon-18 silica gel column; the mobile phase was water and an organic solvent, The organic solvent is methanol or acetonitrile, both the water and the organic solvent contain trifluoroacetic acid with a mass concentration of 0.05-0.10%, and the elution conditions are: linear elution, and the volume concentration of the organic solvent in the mobile phase changes from 5% to 80%.

When separated by reversed-phase high-performance liquid chromatography (RP-HPLC), a UV detector (detection wavelength 215 nm) was used. Among them, component 4 (elution concentration of organic solvent is 45%~50%) ACE inhibitory activity reaches 85.91%, renin inhibitory activity reaches 51.28%; component 6 (organic solvent elution concentration is 45%~50%) ACE The inhibitory activity reached 89.93%, and the renin inhibitory activity reached 61.28%. It can be seen that the components 4 and 5 separated by this method have very high ACE inhibitory activity and renin inhibitory activity, and have a great effect in lowering blood pressure. Good application prospects.

Claims (5)

1. A method for preparing hypotensive rapeseed active peptide, comprising the steps of: (1) Use crushed rapeseed meal as raw material, mix the raw material with water, adjust the pH value to 1.5-2.5, temperature 36.5-37.5 ℃, stir and disperse for 0.5-2 h, and obtain rapeseed protein liquid; (2) Add pepsin to the rapeseed protein solution described in step (1), and hydrolyze it at 36.5°C to 37.5°C for 1 to 3 hours to obtain a pepsin hydrolyzate of rapeseed protein; Adjust the pH value to 6.5~8.0, add trypsin, hydrolyze at 36.5°C~37.5°C for 3-5 hours, inactivate the pepsin and trypsin in it, centrifuge to take the supernatant, and get the crude antihypertensive rapeseed active peptide extract; (3) The blood pressure-lowering rapeseed active peptide crude extract was refined in step (2) to obtain the blood-pressure-lowering rapeseed active peptide. Phase chromatography separation, taking an eluent with an organic solvent volume concentration of 45%~50%, freeze-drying the eluent to obtain the antihypertensive rapeseed active peptide; the conditions for the reversed-phase high-performance liquid chromatography separation are: The chromatographic column is a carbon-12 or carbon-18 silica gel column; the mobile phase is water and an organic solvent, and the organic solvent is methanol or acetonitrile, and both the water and the organic solvent contain trifluoroacetic acid with a mass concentration of 0.05-0.10%. The conditions are: linear elution, the volume concentration of the organic solvent in the mobile phase is changed from 5% to 80%; The antihypertensive rapeseed active peptide has ACE and renin inhibitory activity; The mass ratio of the pepsin added in step (2) to the protein in the rapeseed protein liquid is 5:100, and the mass ratio of the added pancreatin to the protein in the rapeseed protein liquid is 5:100. 2. The method for preparing the antihypertensive rapeseed active peptide according to claim 1, characterized in that the source of the rapeseed meal in step (1) is common rapeseed or double-low rapeseed, and the crushed rapeseed meal is The diameter is 10-200 μm. 3. The method for preparing antihypertensive rapeseed active peptide according to claim 2, characterized in that the raw material of the rapeseed protein liquid in step (1) is protein separated from rapeseed meal. 4. A blood pressure-lowering rapeseed active peptide with ACE and renin inhibitory activity prepared by the method according to any one of claims 1-3. 5. The application of the antihypertensive rapeseed active peptide described in claim 4 in the preparation of antihypertensive drugs.