An active peptide with ACE inhibitory activity and screening method and application thereof

Abstract

This invention discloses an ACE-inhibiting peptide, its screening method, and its applications, relating to the field of food and beverage technology. The active peptide with a specific amino acid sequence exhibits high ACE-inhibiting activity, with an IC50 value of [missing information]. 50 The concentration ranges from 0.31 to 1.93 mg / mL. This bioactive peptide exhibits no toxic side effects at the cellular level and is characterized by its small molecular weight, safety, stability, and easy absorption. It can be used to prepare ACE inhibitory drugs for the prevention or treatment of hypertension, or as a functional food additive to prepare supplementary blood pressure-lowering health foods for long-term health maintenance by hypertensive patients.

Description

Technical Field

[0001] This invention relates to the field of food and beverage technology, and more specifically, to an active peptide with ACE inhibitory activity, its screening method, and its application. Background Technology

[0002] Hypertension is a physiological state characterized by excessively high arterial blood pressure, with a systolic blood pressure ≥140 mmHg and a diastolic blood pressure ≥90 mmHg. It is the most prevalent chronic cardiovascular disease worldwide, seriously endangering human health. The renin-angiotensin system is an important endocrine system in the human body, composed of a series of hormones and corresponding enzymes, playing a crucial role in regulating water and electrolyte balance, blood volume, vascular tone, and blood pressure. Among them, angiotensin-converting enzyme (ACE) is a key enzyme in the renin-angiotensin system, catalyzing the conversion of angiotensin I to angiotensin II and inactivating bradykinin, thus raising blood pressure. Therefore, ACE inhibition is considered an important target for preventing hypertension and improving blood pressure.

[0003] Orange juice is a popular beverage with benefits such as improving blood pressure and lipid metabolism, reducing inflammation, regulating blood sugar and satiety, enhancing cognition, controlling uric acid and kidney stones, and improving gut microbiota. In particular, published in vitro and in vivo studies have shown that orange juice has good activity in preventing and improving hypertension. Increasing research indicates that bioactive peptides from food have positive effects on human health. The physiological functions of bioactive peptides are mainly manifested in antibacterial, antiviral, antioxidant, antitumor, blood sugar lowering, blood pressure lowering, cholesterol lowering, and immune regulation. Plant-derived bioactive peptides are widely used due to their natural properties and high nutritional value, such as soybean bioactive peptides, peanut bioactive peptides, and rapeseed bioactive peptides. However, research on bioactive peptides in orange juice is still limited.

[0004] Developing bioactive peptides with ACE inhibitory activity and independent intellectual property rights has significant academic and practical application value.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The purpose of this invention is to provide an active peptide with ACE inhibitory activity, its preparation method, and its application to solve the above-mentioned technical problems.

[0007] This invention is implemented as follows:

[0008] In a first aspect, the present invention provides an active peptide having ACE inhibitory activity, the active peptide having an amino acid sequence as shown in any one of SEQ ID NO. 1-6.

[0009] The amino acid sequence of the bioactive peptide is as follows:

[0010]

[0011] The inventors discovered that the active peptide with the above-mentioned amino acid sequence has high ACE inhibitory activity, and its IC50 value is [missing information]. 50 The concentration ranges from 0.31 to 1.93 mg / mL. This bioactive peptide is characterized by its small molecular weight, safety, stability, and easy absorption. It can be used to prepare ACE inhibitory drugs for the prevention or treatment of hypertension, or as a functional food additive to prepare supplementary blood pressure-lowering health foods for long-term health maintenance by hypertensive patients.

[0012] This invention effectively utilizes the abundant and inexpensive orange juice resources to prepare bioactive peptides with high ACE inhibitory activity. It explores a practical and efficient method for separating and purifying these bioactive peptides. Through a combination of solid-phase extraction and liquid chromatography-quadrupole time-of-flight mass spectrometry, bioactive peptides with high inhibitory activity are screened, and their amino acid composition and sequence are determined to obtain their primary structure. The purified bioactive peptides can be added to pharmaceuticals or food products to produce drugs or functional health foods with antihypertensive effects. This invention maximizes the antihypertensive function of orange juice bioactive peptides, increasing their added value and creating good economic benefits and market value. Furthermore, it helps alleviate and control the incidence of hypertension. This invention also provides a theoretical basis and experimental evidence for studying the antihypertensive mechanism of orange juice bioactive peptides.

[0013] Define efficacy as IC 50 The value is the concentration of active peptide that inhibits enzyme activity by 50%.

[0014] In a preferred embodiment of the present invention, the above-mentioned active peptide is one of chemical solid-phase synthesis, animal origin, plant origin, or microbial origin.

[0015] Chemical solid-phase synthesis methods include, but are not limited to: artificial synthesis using Fmoc-protected amino acid methods or Tboc-protected amino acid methods, or chemical solid-phase synthesis using an automated peptide synthesizer.

[0016] Microbial sources include, for example, Escherichia coli, yeast, and other microorganisms.

[0017] In a preferred embodiment of the present invention, the above-mentioned active peptide is derived from orange juice.

[0018] In a preferred embodiment of the present invention, the orange juice described above is not-from-concentrate orange juice. Not-from-concentrate (NFC) juice is 100% juice made directly from fruit using mechanical methods.

[0019] Secondly, the present invention also provides the application of active peptides with ACE inhibitory activity in the preparation of ACE inhibitory drugs.

[0020] In a preferred embodiment of the present invention, the dosage form of the drug includes, but is not limited to, tablets, capsules, suspensions, solutions, emulsions, powders, granules, injections, lyophilized powder injections, liniments, ointments, films, creams, lotions, suppositories, aerosols, sprays, powder mists, ointments, plasters, poultices, or patches.

[0021] In one alternative implementation, the drug is in the form of an injection.

[0022] In a preferred embodiment of the present invention, the drug further includes pharmaceutically acceptable additives or excipients.

[0023] In one alternative embodiment, the pharmaceutically acceptable additive or excipient is selected from one or more of the following: solvents, buffers, emulsifiers, suspending agents, disintegrants, disintegrants, dispersants, binders, excipients, stabilizers, chelating agents, diluents, gelling agents, preservatives, wetting agents, lubricants, absorption delay agents, flavorings, sweeteners, colorings, and liposomes.

[0024] In a preferred embodiment of the present invention, the drug includes a carrier, which is a polymer, liposome, ethanol, or polyol.

[0025] When the carrier is a polymer, the polymer can be biodegradable or non-biodegradable. On one hand, polymers include carbohydrates such as starch, cellulose, and dextran. On the other hand, polymers include proteins such as collagen, gelatin, fibrinogen, and albumin. On the other hand, polymers include polyesters (e.g., poly(D,L-lactide)), poly(D,L-lactide-co-glycolic acid), or polyglycolic acid. On the other hand, polymers include poly(ε-caprolactone), polyhydroxybutyrate, polyalkyl carbonate, polyanhydride, or polyorthoester. On the other hand, polymers include ethylene-vinyl acetate copolymer (EVA), silicone rubber, polyurethane, or acrylic polymers or copolymers. On the other hand, the polymerization carrier includes polyethylene glycol. On the other hand, the polymerization carrier includes 4-arm thiol PEG and 4-arm NHS PEG and may optionally contain collagen or collagen derivatives, such as methylated collagen.

[0026] In one alternative embodiment, the polymer is selected from starch, cellulose, dextran, polylactic acid-glycolic acid copolymer, chlorinated chitosan, collagen, gelatin, fibrinogen, albumin, polyester, and polyethylene glycol.

[0027] In an optional embodiment, the medicament further includes a combination drug for the prevention and / or treatment of hypertension, the combination drug being selected from PDE5 inhibitors and / or angiotensin II receptor antagonists. Angiotensin II receptor antagonists are selected from captopril, candesartan, eprosartan, irbesartan, losartan, olmesartan, medoxomil, salazine, telmisartan, and valsartan, and pharmaceutically acceptable salts thereof. PDE5 inhibitors are selected from: 5-[2-ethoxy-5-(4-methyl-1-piperazinylsulfonyl)phenyl]-1-methyl-3-n-propyl-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one (sildenafil); (6R,12aR)-2,3,6,7,12,12a-hexahydro-2-methyl-6-(3,4-methylenedioxyphenyl)-pyrazolo[2′,1′:6,1]pyrido[3,4-b]indole-1,4-dione (tadalafil); 2-[2-ethoxy-5-(4-ethyl-piperazin-1-yl-1-sulfonyl)-phenyl]-5-methyl-7-propyl-3H-imidazo[5,1-f][1,2,4]triazine-4 -one (vardenafil); 3-(1-methyl-7-oxo-3-propyl-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-5-yl)-N-(2-(1-methylpyrrolidone-2-yl)ethyl)-4-propoxybenzenesulfonamide; 5-[2-ethoxy-5-(4-ethylpiperazin-1-ylsulfonyl)pyridin-3-yl]-3-ethyl-2-[2-methoxyethyl]-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one; and 5-(5-acetyl-2-butoxy-3-pyridinyl)-3-ethyl-2-(1-ethyl-3-azacyclobutyl)-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one; and pharmaceutically acceptable salts thereof.

[0028] The term "treatment" as used herein includes relieving, treating, and preventing hypertension. The term "hypertension" includes all conditions with abnormally high blood pressure, such as primary hypertension, pulmonary hypertension, secondary hypertension, isolated systolic hypertension, diabetes-related hypertension, atherosclerotic hypertension, and renovascular hypertension; and further extends to conditions where elevated blood pressure is a known risk factor. Therefore, the term "treatment of hypertension" includes the treatment or prevention of complications and other related comorbidities resulting from hypertension, including congestive heart failure, angina, stroke, glaucoma, and impaired kidney function, including renal failure. Metabolic diseases specifically include: metabolic syndrome (also known as syndrome X); diabetes; and impaired glucose tolerance, including its complications such as diabetic retinopathy and diabetic neuropathy.

[0029] Thirdly, this invention also provides the application of bioactive peptides with ACE inhibitory activity in the preparation of health foods that help lower blood pressure. The types of these foods include, but are not limited to: breakfast powder, luncheon meat, biscuits, milk tablets, noodles, steamed buns, mantou (steamed bread), soy milk, etc.

[0030] Fourthly, the present invention also provides a method for screening active peptides with ACE inhibitory activity, which includes the following steps: solid-phase extraction of the components after filtering orange juice through a membrane, freeze-drying the extracted product, and then injecting the freeze-dried product into a liquid chromatograph-quadrupole time-of-flight mass spectrometer (LC-QTOF) to perform compositional analysis of the peptide components, selecting the peptide components with the dominant content for ACE inhibitory activity determination, and thus screening to obtain active peptides with ACE inhibitory activity;

[0031] The solid-phase extraction used a Sep-pak C18 column; the eluent consisted of acetonitrile and a 0.2% formic acid aqueous solution, with a volume ratio of acetonitrile to 0.2% formic acid aqueous solution of 10:90 to 20:80.

[0032] In one alternative embodiment, the volume ratio of acetonitrile to 0.2% formic acid aqueous solution is 15:85.

[0033] Dominant content refers to ranking multiple peptide components according to their content; the peptide components with the highest ranking are considered to have dominant content. For example, the top 10, 20, or 14 peptide components with dominant content can be selected.

[0034] In a preferred embodiment of the present invention, when performing compositional analysis of peptide components using a liquid chromatograph-quadrupole time-of-flight mass spectrometer, the chromatographic conditions used are as follows:

[0035] Gradient elution was performed using mobile phase A (0.2% formic acid aqueous solution) and mobile phase B (acetonitrile). The elution gradient was as follows: 0–11.50 min, 5%–30% mobile phase B; 11.50–11.51 min, 30–100% mobile phase B; 11.51–15.00 min, 100% mobile phase B; 15.00–15.01 min, 100%–5% mobile phase B; 15.01–18.00 min, 5% mobile phase B.

[0036] In a preferred embodiment of the present invention, the flow rate is set to 0.300 mL / min for liquid chromatography; the chromatographic column is an ACQUITY UPLC HSS T3 with dimensions of 1.8 μm and 2.1 × 100 mm.

[0037] Mass spectrometry analysis was performed using a QTOF MS instrument. The TOF MS scan parameters were set as follows: curtain gas: 25 (any unit); nebulizer pressure 1: 50 (any unit); auxiliary gas pressure 2: 50 (any unit); temperature: 500℃; ion spray voltage: 5.5kV; declustering voltage: 60V; collision energy: 10eV; IDA cycle time: 545ms, consisting of TOF MS scan (accumulation time 50ms; CE, 10eV) and scan of 15 fragment ions (accumulation time 30ms per fragment ion; CES, 35eV).

[0038] Following compositional analysis, the process also includes absolute quantification of the dominant peptide components based on liquid chromatography-triple quadrupole mass spectrometry, followed by ACE inhibitory activity determination based on the absolute quantification results. Absolute quantification provides the accurate content of the dominant peptide components. In one embodiment, only qualitative screening of peptide components is required, without needing to know their absolute content.

[0039] The liquid chromatography conditions were as follows: gradient elution was performed using mobile phase A and mobile phase B, where mobile phase A was 0.2% formic acid aqueous solution and mobile phase B was acetonitrile; the elution gradient was: 0–11.50 min, 5%–30% mobile phase B; 11.50–11.51 min, 30–100% mobile phase B; 11.51–15.00 min, 100% mobile phase B; 15.00–15.01 min, 100%–5% mobile phase B; 15.01–18.00 min, 5% mobile phase B; the chromatographic column used was an ACQUITY UPLC HSS T3 (1.8 μm, 2.1 × 100 mm); the sample loading volume was 2 μL, and the flow rate was 0.300 mL / min.

[0040] The mass spectrometry conditions used for absolute quantification were as follows: MRM mode, curtain gas: 25 (any unit); collision gas: 9 (any unit); ion spray voltage: 5.5 kV; temperature: 500 °C; nebulizer pressure 1: 50 (any unit); auxiliary gas pressure 2: 50 (any unit); enhancement voltage: 10.0; collision cell outlet potential: 9.0; duration: 18.007 min; number of cycles: 427 cycles; cycle time: 2.5302 s.

[0041] The present invention has the following beneficial effects:

[0042] The inventors discovered that active peptides with specific amino acid sequences possess high ACE inhibitory activity, with an IC50 value of [missing information]. 50The concentration ranges from 0.31 to 1.93 mg / mL. This bioactive peptide is characterized by its small molecular weight, safety, stability, and easy absorption. It can be used to prepare ACE inhibitory drugs for the prevention or treatment of hypertension, or as a functional food additive to prepare supplementary blood pressure-lowering health foods for long-term health maintenance by hypertensive patients.

[0043] This invention effectively utilizes the abundant and inexpensive orange juice resources to prepare bioactive peptides with high ACE inhibitory activity. It explores a practical and efficient method for separating and purifying these bioactive peptides. Through a combination of solid-phase extraction and liquid chromatography-quadrupole time-of-flight mass spectrometry, bioactive peptides with high inhibitory activity are screened, and their amino acid composition and sequence are determined to obtain their primary structure. The purified bioactive peptides can be added to pharmaceuticals or food products to produce drugs or functional health foods with antihypertensive effects. This invention maximizes the antihypertensive function of orange juice bioactive peptides, increasing their added value and creating good economic benefits and market value. Furthermore, it helps alleviate and control the incidence of hypertension. This invention also provides a theoretical basis and experimental evidence for studying the antihypertensive mechanism of orange juice bioactive peptides. Detailed Implementation

[0044] Reference will now be made to detailed embodiments of the present invention, one or more of which are described below. Each example is provided for explanation and not for limitation of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to the invention without departing from its scope or spirit. For example, features described or illustrated as part of one embodiment may be used in another embodiment to produce further embodiments.

[0045] Unless otherwise specified, the practice of this invention will employ conventional techniques of cell biology, molecular biology (including recombinant technologies), microbiology, biochemistry, and immunology, which are within the capabilities of those skilled in the art. This technique is well explained in the literature, such as *Molecular Cloning: A Laboratory Manual*, 2nd edition (Sambrook et al., 1989); *Oligonucleotide Synthesis* (edited by M.J. Gait, 1984); *Animal Cell Culture* (edited by R.R. Freshney, 1987); *Methods in Enzymology* (Academic Press, Inc.); *Handbook of Experimental Immunology* (edited by D.M. Weir and C.C. Blackwell); *Gene Transfer Vectors for Mammalian Cells* (edited by J.M. Miller and M.P. Calos, 1987); *Current Protocols in Molecular Biology* (edited by F.M. Mausubel et al., 1987); and *PCR: The Polymerase Chain Reaction*. The references cited in the references are: "Reaction" (Mullis et al., ed., 1994); and "Current Protocols in Immunology" (JEColigan et al., ed., 1991), each of which is explicitly incorporated herein by reference.

[0046] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0047] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0048] Example 1

[0049] This embodiment provides a method for preparing an active peptide with ACE inhibitory activity.

[0050] (1) Separation of peptide components in orange juice based on solid phase extraction.

[0051] Orange juice was collected from non-concentrated orange juice from a trusted facility. The orange juice was centrifuged at 10,000 g for 5 min, and the supernatant was passed through a 0.22 μm aqueous membrane. The resulting fraction was then subjected to solid-phase extraction.

[0052] The solid-phase extraction (SPE) column was a Sep-pak C18 (50 mg, 1 mL). The SPE column was pre-equilibrated and activated using 2.5 mL of acetonitrile and 2.5 mL of formic acid solution. 2.5 mL of centrifuged orange juice sample was added to the SPE column, and the crude extract was obtained by elution with 2.5 mL of acetonitrile and 0.2% formic acid solution (v:v = 15:85).

[0053] The crude extract was flash-frozen in liquid nitrogen, lyophilized, and reconstituted with water to 2.5 mL to obtain the peptide components of orange juice.

[0054] (2) Compositional analysis of peptide components based on liquid chromatography-quadrupole time-of-flight mass spectrometry

[0055] The chromatographic column used was an ACQUITY UPLC HSS T3 (1.8 μm, 2.1 × 100 mm). The autosampler temperatures were 40 °C and 4 °C, respectively. The injection volume and flow rate were 2 μL and 0.300 mL / min, respectively. Mobile phase A and mobile phase B were 0.2% formic acid aqueous solution and acetonitrile, respectively.

[0056] The solvent gradient was as follows: 0–11.50 min, 5%–30% mobile phase B; 11.50–11.51 min, 30%–100% mobile phase B; 11.51–15.00 min, 100% mobile phase B; 15.00–15.01 min, 100%–5% mobile phase B; 15.01–18.00 min, 5% mobile phase B.

[0057] Mass spectrometry analysis was performed using QTOF MS (TripleTOF 6600, SCIEX, USA). IDA was employed, with TOF MS scanning of peptide components and product ion scanning performed in positive mode. The TOF MS scanning parameters were set as follows: curtain gas: 25 (any unit); nebulizer pressure 1: 50 (any unit); auxiliary gas pressure 2: 50 (any unit); temperature: 500℃; ion spray voltage: 5.5kV; declustering voltage: 60V; collision energy: 10eV; IDA cycle time: 545ms, consisting of TOF MS scanning (accumulation time 50ms; CE, 10eV) and scanning of 15 fragment ions (accumulation time 30ms per fragment ion; CES, 35eV).

[0058] Based on Proteinpilot software, peptide components were analyzed and identified, and 14 peptides with high content were selected for solid-phase synthesis.

[0059] (3) Absolute quantification of orange juice peptide components based on liquid chromatography-triple quadrupole mass spectrometry

[0060] The chromatographic column used was an ACQUITY UPLC HSS T3 (1.8 μm, 2.1 × 100 mm). The autosampler temperatures were 40 °C and 4 °C. The injection volume and flow rate were 2 μL and 0.300 mL / min, respectively. Mobile phases A and B were 0.2% formic acid aqueous solution and acetonitrile, respectively. The solvent gradient was as follows: 0–11.50 min, 5%–30% B; 11.50–11.51 min, 30–100% B; 11.51–15.00 min, 100% B; 15.00–15.01 min, 100%–5% B; 15.01–18.00 min, 5% B.

[0061] The mass spectrometry conditions used for absolute quantification were as follows: MRM mode, curtain gas: 25 (any unit); collision gas: 9 (any unit); ion spray voltage: 5.5 kV; temperature: 500 °C; nebulizer pressure 1: 50 (any unit); auxiliary gas pressure 2: 50 (any unit); enhancement voltage: 10.0; collision cell outlet potential: 9.0; duration: 18.007 min; number of cycles: 427 cycles; cycle time: 2.5302 s.

[0062]

[0063]

[0064] In the positive mode, absolute quantification was performed on 14 peptides with high content, and the results are shown in Table 1.

[0065] Table 1. Sequences and contents of 14 peptides

[0066]

[0067] Experimental Example 1

[0068] The 14 peptides isolated in Example 1 were synthesized in a solid phase, and the ACE inhibitory activity of the synthesized peptides was determined.

[0069] A PBS buffer-based ACE enzyme simulation system was used to determine ACE inhibitory activity via the hippuric acid method. The specific method is as follows:

[0070] (1) In a 1.5 mL centrifuge tube, add 150 μL of hippuryl-histyl-leucine phosphate buffer (1 mM), 30 μL of pure water or positive control (10 ppm captopril) or peptides, and 30 μL of ACE enzyme (0.25 U / mL). React at 37 °C for 150 min.

[0071] (2) After the reaction was completed, 50 μL of HA-d5 (10 ppm hippuric acid isotope internal standard) and 740 μL of 0.1% formic acid aqueous solution were added to the system. After shaking and mixing, the reaction was terminated and the reaction product hippuric acid (HA) was extracted using an OASIS-HLB (1 mL, 30 mg) solid phase extraction column.

[0072] The solid-phase extraction column was activated and equilibrated with 1.0 mL of methanol and 1.0 mL of 0.1% formic acid aqueous solution. The sample after reaction was added, and the column was washed with 1.0 mL of 0.1% formic acid aqueous solution.

[0073] (3) Add methanol (1 mL) at a low flow rate, elute and collect the hippuric acid retained therein.

[0074] The specific instrument and parameters for determining the content of substance HA using liquid chromatography-mass spectrometry (LC-MS) are as follows:

[0075] The chromatographic column used was a Kinetex F5 (2.6 μm, 2.1 × 100 mm). The autosampler temperatures were 40 °C and 4 °C, respectively. The injection volume and flow rate were 2 μL and 0.300 mL / min, respectively. Mobile phases A and B were 0.1% formic acid aqueous solution and 0.1% formic acid acetonitrile solution, respectively.

[0076] The solvent gradient was as follows: 0–1.00 min, 10% B; 1.00–2.00 min, 10%–25% B; 2.00–6.00 min, 25% B; 6.00–6.10 min, 25%–95% B; 6.10–9.00 min, 95% B; 9.00–9.10 min, 95%–10% B.

[0077] Mass spectrometry analysis was performed in MRM mode. In positive mode, HA and HA-d5 were absolutely quantified, and the ACE inhibitory activity of peptide components was calculated. The IC50 of peptide components with higher ACE inhibitory activity was also calculated. 50 The values ​​are as follows:

[0078] GAVEPAK 0.31mg / mL, KVLPALN 0.70mg / mL, TTVPVEK 1.15mg / mL, VTVPK 1.22mg / mL, AASPFK 1.28mg / mL, AAPVQ 1.93mg / mL.

[0079] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An active peptide with ACE inhibitory activity, characterized in that, The amino acid sequence of the active peptide is shown in SEQ ID NO.

2.

2. The application of the bioactive peptide with ACE inhibitory activity as described in claim 1 in the preparation of health foods that help lower blood pressure.

3. The use of the active peptide with ACE inhibitory activity as described in claim 1 in the preparation of antihypertensive drugs.

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