A blood pressure lowering peptide derived from blackberry and a screening method thereof

Abstract

The application provides a blood pressure reducing peptide derived from blackberry and a screening method thereof. The amino acid sequence of the blood pressure reducing peptide is RVSPEIWAK, the blood pressure reducing peptide has a significant inhibitory effect on angiotensin converting enzyme (ACE) and can exert a good anti-hypertensive effect, and has a good application prospect in the preparation of drugs related to hypertension treatment or health care foods that are helpful to maintaining a healthy level of blood pressure.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a blood pressure-lowering peptide derived from blackberries and its screening method. Background Technology

[0002] The World Health Organization (WHO) lists hypertension as the leading risk factor for public health, a major cause of rising mortality worldwide, affecting approximately 15%–20% of adults. In 2010, 1.38 billion people globally (31.1% of the global adult population) had hypertension. The global prevalence of hypertension is rising due to population aging and increased exposure to lifestyle risk factors such as unhealthy diets (high sodium and low potassium intake) and lack of physical activity. There are many types of antihypertensive drugs, including diuretics, angiotensin-converting enzyme (ACE) inhibitors, angiotensin II receptor antagonists, adrenergic receptor blockers, calcium channel blockers, vasodilators, ganglion and postganglionic sympathetic nerve inhibitors, and serotonin receptor antagonists.

[0003] Angiotensin-converting enzyme (ACE) belongs to the zinc protease class, which requires zinc and chloride ions to function. ACE plays an important physiological role in regulating blood pressure. Many synthetic ACE inhibitors, including captopril, enalapril, and lisinopril, are available for clinical use. Most patients tolerate ACE inhibitors well, but some adverse side effects may occur, such as cough, loss of taste, kidney damage, and angioedema.

[0004] It is clear that there is an urgent need for a new antihypertensive drug. Summary of the Invention

[0005] This invention provides a blood pressure-lowering peptide derived from blackberries and a screening method thereof, in order to at least partially solve the problems existing in the prior art.

[0006] The first aspect of this invention provides a blood pressure-lowering peptide derived from blackberries, wherein the amino acid sequence of the blood pressure-lowering peptide is RVSPEIWAK, as shown in SEQ ID NO.1.

[0007] The second aspect of this invention provides the use of the blackberry-derived antihypertensive peptide described in the first aspect in the preparation of drugs for the prevention and treatment of hypertension.

[0008] A third aspect of the present invention provides a drug for preventing and treating hypertension, the drug comprising the blood pressure-lowering peptide derived from blackberries as described in the first aspect.

[0009] Optionally, the antihypertensive drug may also include pharmaceutically acceptable excipients.

[0010] Optionally, the pharmaceutically acceptable excipients are selected from at least one of the following pharmaceutically acceptable solvents, solubilizers, emulsifiers, colorants, binders, disintegrants, fillers, wetting agents, osmotic pressure regulators, stabilizers, flavoring agents, preservatives, suspending agents, coating materials, anti-adhesives, binding agents, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, thickeners, inclusion agents, humectants, flocculants and anti-flocculation agents, filter aids, release inhibitors, and polymeric framework materials.

[0011] Optionally, the antihypertensive drug is in the form of tablets, capsules, powders, granules, oral liquids, or syrups.

[0012] A fourth aspect of this invention provides a method for screening to obtain the blood pressure-lowering peptides derived from blackberries as described in the first aspect, the method comprising the following steps: Step 1, Extraction of water-soluble protein from blackberries: Freeze-dried blackberries are crushed and sieved, defatted, and then extracted with deionized water. After filtration, the extract is concentrated and centrifuged. The supernatant is collected to precipitate with ammonium sulfate. The precipitate is reconstituted and dialyzed. The extract is then freeze-dried to obtain water-soluble protein extract from blackberries. Step 2, Preparation of blackberry water-soluble protein hydrolysate: Dissolve blackberry water-soluble protein extract in PBS buffer, add alkaline protease for enzymatic hydrolysis, inactivate the enzyme, then add trypsin for enzymatic hydrolysis to obtain blackberry water-soluble protein hydrolysate. Step 3, Isolation of blackberry blood pressure lowering peptides: Ultrafiltration of blackberry water-soluble protein hydrolysate with ultrafiltration membranes of different molecular weights was used to obtain hydrolysate components of different molecular weights. The component with the strongest ACE inhibitory activity was selected for RP-HPLC reversed-phase high-performance liquid chromatography separation. The chromatographic peak component with the strongest ACE inhibitory activity was collected and selected for mass spectrometry identification, and the sequences of the top 5 abundant peptides were determined. Step 4, Activity identification of blackberry antihypertensive peptide: The peptide obtained from sequencing in step 3 was artificially synthesized, and its ACE inhibitory activity was measured to obtain an antihypertensive peptide with the amino acid sequence RVSPEIWAK.

[0013] Optionally, the defatting in step 1 is petroleum ether soxhlet defatting; the extraction is ultrasonic-assisted extraction in a 60°C water bath; and the ammonium sulfate precipitation is performed by adding ammonium sulfate to the supernatant until the ammonium sulfate concentration reaches 60%, followed by centrifugation after the precipitate is fully separated.

[0014] Optionally, the alkaline protease hydrolysis conditions in step 2 are: pH=8-9, the amount of alkaline protease added is 1-5% of the water-soluble protein in blackberries, the temperature is 40-50℃, and the hydrolysis time is 1-5 hours; the trypsin hydrolysis conditions are: pH=6-8, the amount of trypsin added is 1-5% of the water-soluble protein in blackberries, the temperature is 40-50℃, and the hydrolysis time is 1-5 hours.

[0015] Optionally, the molecular weights of the ultrafiltration membranes with different molecular weights in step 3 are 10kD, 5kD, and 3kD, respectively; the RP-HPLC reversed-phase high-performance liquid chromatography separation conditions are: detection wavelength of 214nm, chromatographic column: C18 reversed-phase column, mobile phase A: 0.1% (v / v) trifluoroacetic acid-water, mobile phase B: acetonitrile, flow rate: 2mL / min; and the mass spectrometry identification is LC-MS / MS.

[0016] Optionally, the artificial synthesis described in step 4 is a solid-phase synthesis.

[0017] The present invention has achieved the following beneficial effects: This invention obtains a blood pressure-lowering peptide RVSPEIWAK derived from blackberry by enzymatic hydrolysis of blackberry protein extract, followed by ultrafiltration, RP-HPLC reversed-phase high-performance liquid chromatography separation, and mass spectrometry identification. This blood pressure-lowering peptide exhibits significant angiotensin-converting enzyme (ACE) inhibitory activity (IC50 = 16.89 μM) and can be used to prepare drugs related to hypertension treatment or health foods that help maintain healthy blood pressure levels.

[0018] The blood pressure-lowering peptide derived from blackberries provided in this invention offers significant comprehensive advantages as a novel blood pressure-lowering drug. Compared to traditional chemical antihypertensive drugs, its core advantages are primarily reflected in its extremely high safety and good tolerability. This antihypertensive peptide is naturally derived and has no toxic side effects, avoiding common adverse reactions of chemical antihypertensive drugs such as dry cough, dizziness, electrolyte imbalance, and liver and kidney damage. Long-term use places no additional burden on the body, making it more suitable for the long-term management needs of hypertensive patients. Secondly, its antihypertensive effect is gentle and stable, exerting its effect by inhibiting the activity of angiotensin-converting enzyme (ACE). It avoids the risks of hypotension or excessive blood pressure fluctuations caused by rapid and potent blood pressure reduction, making it particularly suitable for people with prehypertension, mild hypertension, and elderly patients with poor tolerance to antihypertensive drugs.

[0019] The blood pressure-lowering peptide derived from blackberries provided in this invention is a small molecule peptide that can be rapidly absorbed by the human body directly or after simple hydrolysis, exhibiting high bioavailability. It not only lowers blood pressure but also provides the body with high-quality nutrition, making it suitable for hypertensive individuals with weak digestive function, frail constitution, or metabolic diseases, thus broadening its applicability. Furthermore, its cost is controllable; relying on mature extraction and preparation processes, it enables the resource utilization of agricultural by-products. Compared to chemically synthesized antihypertensive drugs, it effectively reduces production and raw material costs, demonstrating promising industrialization prospects. Attached Figure Description

[0020] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 The ACE inhibition activity of each component obtained by ultrafiltration in Example 3 is shown.

[0022] Figure 2 This is a chromatogram obtained during RP-HPLC reversed-phase high-performance liquid chromatography separation in Example 3.

[0023] Figure 3 The ACE inhibitory activity of each component obtained by chromatographic separation in Example 3 is shown. Detailed Implementation

[0024] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0025] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents and other instruments whose manufacturers are not specified are all commercially available products.

[0026] Peptides derived from food proteins are considered milder and safer than synthetic drugs; in addition, these peptides are often multifunctional and easily absorbed.

[0027] Blackberries, also known as raspberries, are recorded in the Compendium of Materia Medica as having anti-aging properties, boosting immunity, promoting brain metabolism, and being used to lower blood pressure, reduce blood lipids, and combat arrhythmia, thus possessing certain medicinal value. Therefore, blackberries may also contain antihypertensive active ingredients, but research on the identification and isolation of these active ingredients in blackberries has not yet been reported.

[0028] Therefore, in this embodiment of the invention, the antihypertensive active ingredients in blackberries were identified and isolated, as detailed below: Example 1: Extraction of water-soluble protein from blackberries Take 20g of freeze-dried blackberries, crush and sieve them, defatt them with petroleum ether soxhlet, add deionized water at a solid-liquid ratio of 1:10, and extract with ultrasonic assistance in a 60℃ water bath for 1 hour. Filter to remove solid residue, concentrate the filtrate under vacuum to a certain volume, centrifuge, take the supernatant and add ammonium sulfate until the ammonium sulfate concentration reaches 60%, precipitate is fully separated and centrifuged to collect the precipitate, dissolve the precipitate in sodium hydrogen phosphate buffer, dialyze to change the buffer to remove residual ammonium sulfate, and freeze dry to obtain blackberry water-soluble protein extract.

[0029] Example 2: Preparation of water-soluble protease hydrolysate from blackberries Take the blackberry water-soluble protein extract from Example 1, dissolve it in PBS buffer at a solid-liquid ratio of 1:80, adjust the pH to 8.5, add alkaline protease at 3% of the blackberry water-soluble protein mass, adjust the temperature to 45°C, hydrolyze for 3 hours, and inactivate the enzyme in a 95°C water bath; then adjust the pH to 7, add trypsin at 2% of the blackberry water-soluble protein mass, hydrolyze for 3 hours, and inactivate the enzyme in a 95°C water bath; thus obtaining the blackberry water-soluble protease hydrolysate.

[0030] Example 3: Isolation of blackberry antihypertensive peptides.

[0031] Blackberry water-soluble protein hydrolysate was ultrafiltered using 10kD, 5kD, and 3kD ultrafiltration membranes to obtain component A (molecular weight >10kD), component B (molecular weight 5-10kD), component C (molecular weight 3-5kD), and component D (molecular weight <3kD), respectively. The ACE inhibitory activity of these four components was determined spectrophotometrically. Figure 1 As shown, component D exhibits the strongest ACE-inhibiting activity.

[0032] Component D was separated by RP-HPLC (reversed-phase high-performance liquid chromatography) at a detection wavelength of 214 nm. The chromatographic column was a C18 reversed-phase column. Mobile phase A was 0.1% (v / v) trifluoroacetic acid-water, and mobile phase B was acetonitrile. The flow rate was 2 mL / min. Different chromatographic peak components were collected, such as... Figure 2 As shown. The ACE inhibitory activity of each component was measured. (See figure.) Figure 3 As shown, component D3 exhibited the strongest ACE inhibitory activity at 10-12 min.

[0033] The D3 fraction was identified by LC-MS / MS, and the top 5 abundant peptide sequences were determined to be RVSPEIWAK, ALFGDGVK, HIHDCKP, SSGDYLRLW, and LERHKGW.

[0034] Example 3: Activity identification of blackberry antihypertensive peptides.

[0035] The peptides identified in Example 2 were synthesized by solid-phase synthesis and subjected to ACE inhibition activity experiments. The results are shown in Table 1. Among them, the RVSPEIWAK peptide showed the strongest ACE inhibition activity, with IC50 = 16.89 μM.

[0036] Table 1. Peptide activity

[0037] Based on the above exploratory research, this invention proposes that a polypeptide with the amino acid sequence RVSPEIWAK has angiotensin-converting enzyme (ACE) inhibitory activity and can be used as an antihypertensive peptide to prepare drugs for the prevention and treatment of hypertension.

[0038] Based on this, this invention proposes a blood pressure-lowering peptide derived from blackberries, wherein the amino acid sequence of the blood pressure-lowering peptide is RVSPEIWAK, as shown in SEQ ID NO.1.

[0039] This invention also proposes the use of the aforementioned blackberry-derived antihypertensive peptide in the preparation of drugs for the prevention and treatment of hypertension.

[0040] This invention also proposes a drug for preventing and treating hypertension, the drug comprising the aforementioned blood pressure-lowering peptide derived from blackberries.

[0041] Specifically, the antihypertensive drugs also include pharmaceutically acceptable excipients.

[0042] Specifically, the pharmaceutically acceptable excipients are selected from at least one of the following pharmaceutically acceptable solvents, solubilizers, emulsifiers, colorants, binders, disintegrants, fillers, wetting agents, osmotic pressure regulators, stabilizers, flavoring agents, preservatives, suspending agents, coating materials, anti-adhesives, binding agents, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, thickeners, inclusion agents, humectants, flocculants and anti-flocculation agents, filter aids, release inhibitors, and polymeric framework materials.

[0043] In this embodiment of the invention, appropriate excipients for therapeutic agents can be selected based on clinical needs, safety, functionality, and compatibility.

[0044] Specifically, the antihypertensive drugs are tablets, capsules, powders, granules, oral liquids, or syrups.

[0045] In this embodiment of the invention, a method for screening and obtaining the aforementioned blood pressure-lowering peptides derived from blackberries is also proposed, characterized in that the method includes the following steps: Step 1, Extraction of water-soluble protein from blackberries: Freeze-dried blackberries are crushed and sieved, defatted, and then extracted with deionized water. After filtration, the extract is concentrated and centrifuged. The supernatant is collected to precipitate with ammonium sulfate. The precipitate is reconstituted and dialyzed, and then freeze-dried to obtain the water-soluble protein extract from blackberries.

[0046] Step 2, Preparation of blackberry water-soluble protein hydrolysate: Dissolve blackberry water-soluble protein extract in PBS buffer, add alkaline protease for enzymatic hydrolysis, inactivate the enzyme, then add trypsin for enzymatic hydrolysis to obtain blackberry water-soluble protein hydrolysate.

[0047] Step 3, Isolation of blackberry blood pressure lowering peptides: Ultrafiltration of blackberry water-soluble protein hydrolysate with ultrafiltration membranes of different molecular weights was used to obtain hydrolysate components of different molecular weights. The component with the strongest ACE inhibitory activity was selected for RP-HPLC reversed-phase high-performance liquid chromatography separation. The chromatographic peak component with the strongest ACE inhibitory activity was collected and selected for mass spectrometry identification, and the sequences of the top 5 abundant peptides were determined.

[0048] Step 4, Activity identification of blackberry antihypertensive peptide: The peptide obtained from sequencing in step 3 was artificially synthesized, and its ACE inhibitory activity was measured to obtain an antihypertensive peptide with the amino acid sequence RVSPEIWAK.

[0049] Specifically, the defatting in step 1 is petroleum ether soxhlet defatting; the extraction is ultrasonic-assisted extraction in a 60°C water bath; and the ammonium sulfate precipitation is achieved by adding ammonium sulfate to the supernatant until the ammonium sulfate concentration reaches 60%, followed by centrifugation after the precipitate is fully separated.

[0050] Specifically, the alkaline protease hydrolysis conditions in step 2 are: pH=8-9, the amount of alkaline protease added is 1-5% of the water-soluble protein in blackberries, the temperature is 40-50℃, and the hydrolysis time is 1-5 hours; the trypsin hydrolysis conditions are: pH=6-8, the amount of trypsin added is 1-5% of the water-soluble protein in blackberries, the temperature is 40-50℃, and the hydrolysis time is 1-5 hours. Specifically, the molecular weights of the ultrafiltration membranes with different molecular weights mentioned in step 3 are 10kD, 5kD, and 3kD, respectively; the RP-HPLC reversed-phase high-performance liquid chromatography separation conditions are: detection wavelength of 214nm, chromatographic column: C18 reversed-phase column, mobile phase A: 0.1% (v / v) trifluoroacetic acid-water, mobile phase B: acetonitrile, flow rate: 2mL / min; and the mass spectrometry identification is LC-MS / MS.

[0051] Although preferred embodiments of the present invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of the embodiments of the present invention.

[0052] The above provides a detailed description of a blood pressure-lowering peptide derived from blackberries and its screening method provided by the present invention. Specific examples have been used to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only for the purpose of helping to understand the method and core ideas of the present invention. At the same time, for those skilled in the art, there will be changes in the specific implementation methods and application scope based on the ideas of the present invention. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. A blood pressure-lowering peptide derived from blackberries, characterized in that, The amino acid sequence of the blood pressure-lowering peptide is RVSPEIWAK, as shown in SEQ ID NO.

1.

2. The use of the blackberry-derived antihypertensive peptide according to claim 1 in the preparation of drugs for the prevention and treatment of hypertension.

3. A drug for preventing and treating hypertension, characterized in that, The drug includes the blood pressure-lowering peptide derived from blackberries as described in claim 1.

4. The antihypertensive drug according to claim 3, characterized in that, The drugs for the prevention and treatment of hypertension also include pharmaceutically acceptable excipients.

5. The antihypertensive drug according to claim 4, characterized in that, The pharmaceutically acceptable excipients are selected from at least one of the following pharmaceutically acceptable solvents, solubilizers, emulsifiers, colorants, binders, disintegrants, fillers, wetting agents, osmotic pressure regulators, stabilizers, flavoring agents, preservatives, suspending agents, coating materials, anti-adhesives, binding agents, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, thickeners, inclusion agents, humectants, flocculants and anti-flocculators, filter aids, release inhibitors, and polymeric framework materials.

6. The antihypertensive drug according to claim 3, characterized in that, The medications for preventing and treating hypertension are in the form of tablets, capsules, powders, granules, oral liquids, or syrups.

7. A method for screening to obtain the blood pressure-lowering peptide derived from blackberries as described in claim 1, characterized in that, The method includes the following steps: Step 1, Extraction of water-soluble protein from blackberries: Freeze-dried blackberries are crushed and sieved, defatted, and then extracted with deionized water. After filtration, the extract is concentrated and centrifuged. The supernatant is collected to precipitate with ammonium sulfate. The precipitate is reconstituted and dialyzed. The extract is then freeze-dried to obtain water-soluble protein extract from blackberries. Step 2, Preparation of blackberry water-soluble protein hydrolysate: Dissolve blackberry water-soluble protein extract in PBS buffer, add alkaline protease for enzymatic hydrolysis, inactivate the enzyme, then add trypsin for enzymatic hydrolysis to obtain blackberry water-soluble protein hydrolysate. Step 3, Isolation of blackberry blood pressure lowering peptides: Ultrafiltration of blackberry water-soluble protein hydrolysate with ultrafiltration membranes of different molecular weights was used to obtain hydrolysate components of different molecular weights. The component with the strongest ACE inhibitory activity was selected for RP-HPLC reversed-phase high-performance liquid chromatography separation. The chromatographic peak component with the strongest ACE inhibitory activity was collected and selected for mass spectrometry identification, and the sequences of the top 5 abundant peptides were determined. Step 4, Activity identification of blackberry antihypertensive peptide: The peptide obtained from sequencing in step 3 was artificially synthesized, and its ACE inhibitory activity was measured to obtain an antihypertensive peptide with the amino acid sequence RVSPEIWAK.

8. The method according to claim 7, characterized in that, The defatting in step 1 is petroleum ether soxhlet defatting; the extraction is ultrasonic-assisted extraction in a 60°C water bath; the ammonium sulfate precipitation is achieved by adding ammonium sulfate to the supernatant until the ammonium sulfate concentration reaches 60%, and centrifuging after the precipitate is fully separated.

9. The method according to claim 7, characterized in that, The alkaline protease hydrolysis conditions in step 2 are: pH=8-9, the amount of alkaline protease added is 1-5% of the water-soluble protein in blackberries, the temperature is 40-50℃, and the hydrolysis time is 1-5 hours; the trypsin hydrolysis conditions are: pH=6-8, the amount of trypsin added is 1-5% of the water-soluble protein in blackberries, the temperature is 40-50℃, and the hydrolysis time is 1-5 hours.

10. The method according to claim 3, characterized in that, The molecular weights of the ultrafiltration membranes with different molecular weights mentioned in step 3 are 10kD, 5kD, and 3kD, respectively; the RP-HPLC reversed-phase high-performance liquid chromatography separation conditions are: detection wavelength of 214nm, chromatographic column: C18 reversed-phase column, mobile phase A: 0.1% (v / v) trifluoroacetic acid-water, mobile phase B: acetonitrile, flow rate: 2mL / min; the mass spectrometry identification is LC-MS / MS.

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