CN117462649B - Royal jelly active peptide composition with ACE inhibition effect, and preparation method and application thereof - Google Patents
Royal jelly active peptide composition with ACE inhibition effect, and preparation method and application thereof Download PDFInfo
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- CN117462649B CN117462649B CN202311508063.3A CN202311508063A CN117462649B CN 117462649 B CN117462649 B CN 117462649B CN 202311508063 A CN202311508063 A CN 202311508063A CN 117462649 B CN117462649 B CN 117462649B
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- royal jelly
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- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 77
- 229940109850 royal jelly Drugs 0.000 title claims abstract description 55
- 239000000203 mixture Substances 0.000 title claims abstract description 29
- 230000005764 inhibitory process Effects 0.000 title claims abstract description 28
- 230000000694 effects Effects 0.000 title claims abstract description 26
- 238000002360 preparation method Methods 0.000 title claims abstract description 11
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 22
- 101000984728 Chiropsoides quadrigatus Angiotensin-converting enzyme inhibitory peptide Proteins 0.000 claims abstract description 18
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- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 2
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- UUUHXMGGBIUAPW-UHFFFAOYSA-N 1-[1-[2-[[5-amino-2-[[1-[5-(diaminomethylideneamino)-2-[[1-[3-(1h-indol-3-yl)-2-[(5-oxopyrrolidine-2-carbonyl)amino]propanoyl]pyrrolidine-2-carbonyl]amino]pentanoyl]pyrrolidine-2-carbonyl]amino]-5-oxopentanoyl]amino]-3-methylpentanoyl]pyrrolidine-2-carbon Chemical compound C1CCC(C(=O)N2C(CCC2)C(O)=O)N1C(=O)C(C(C)CC)NC(=O)C(CCC(N)=O)NC(=O)C1CCCN1C(=O)C(CCCN=C(N)N)NC(=O)C1CCCN1C(=O)C(CC=1C2=CC=CC=C2NC=1)NC(=O)C1CCC(=O)N1 UUUHXMGGBIUAPW-UHFFFAOYSA-N 0.000 abstract description 47
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- ZDLZKMDMBBMJLI-FDMDGMSGSA-N 2-[[2-[[(2s)-2-[[(e)-3-(furan-2-yl)prop-2-enoyl]amino]-3-phenylpropanoyl]amino]acetyl]amino]acetic acid Chemical compound C([C@@H](C(=O)NCC(=O)NCC(=O)O)NC(=O)\C=C\C=1OC=CC=1)C1=CC=CC=C1 ZDLZKMDMBBMJLI-FDMDGMSGSA-N 0.000 description 3
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- CZGUSIXMZVURDU-JZXHSEFVSA-N Ile(5)-angiotensin II Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC=1C=CC=CC=1)C([O-])=O)NC(=O)[C@@H](NC(=O)[C@H](CCCNC(N)=[NH2+])NC(=O)[C@@H]([NH3+])CC([O-])=O)C(C)C)C1=CC=C(O)C=C1 CZGUSIXMZVURDU-JZXHSEFVSA-N 0.000 description 2
- 102000035195 Peptidases Human genes 0.000 description 2
- ORWYRWWVDCYOMK-HBZPZAIKSA-N angiotensin I Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CC(C)C)C(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@@H](N)CC(O)=O)C(C)C)C1=CC=C(O)C=C1 ORWYRWWVDCYOMK-HBZPZAIKSA-N 0.000 description 2
- 239000002220 antihypertensive agent Substances 0.000 description 2
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- ZDLZKMDMBBMJLI-INIZCTEOSA-N 2-[[2-[[(2s)-2-[3-(furan-2-yl)prop-2-enoylamino]-3-phenylpropanoyl]amino]acetyl]amino]acetic acid Chemical compound C([C@@H](C(=O)NCC(=O)NCC(=O)O)NC(=O)C=CC=1OC=CC=1)C1=CC=CC=C1 ZDLZKMDMBBMJLI-INIZCTEOSA-N 0.000 description 1
- 102400000344 Angiotensin-1 Human genes 0.000 description 1
- 101800000734 Angiotensin-1 Proteins 0.000 description 1
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- 101800004538 Bradykinin Proteins 0.000 description 1
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- QXZGBUJJYSLZLT-UHFFFAOYSA-N H-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-OH Natural products NC(N)=NCCCC(N)C(=O)N1CCCC1C(=O)N1C(C(=O)NCC(=O)NC(CC=2C=CC=CC=2)C(=O)NC(CO)C(=O)N2C(CCC2)C(=O)NC(CC=2C=CC=CC=2)C(=O)NC(CCCN=C(N)N)C(O)=O)CCC1 QXZGBUJJYSLZLT-UHFFFAOYSA-N 0.000 description 1
- MMFKFJORZBJVNF-UWVGGRQHSA-N His-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H](N)CC1=CN=CN1 MMFKFJORZBJVNF-UWVGGRQHSA-N 0.000 description 1
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- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- QXZGBUJJYSLZLT-FDISYFBBSA-N bradykinin Chemical compound NC(=N)NCCC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N1[C@H](C(=O)NCC(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CO)C(=O)N2[C@@H](CCC2)C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)CCC1 QXZGBUJJYSLZLT-FDISYFBBSA-N 0.000 description 1
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- 108010083141 dipeptidyl carboxypeptidase Proteins 0.000 description 1
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- DBLXOVFQHHSKRC-UHFFFAOYSA-N ethanesulfonic acid;2-piperazin-1-ylethanol Chemical compound CCS(O)(=O)=O.OCCN1CCNCC1 DBLXOVFQHHSKRC-UHFFFAOYSA-N 0.000 description 1
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- 210000003254 palate Anatomy 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- A61K38/08—Peptides having 5 to 11 amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/12—Antihypertensives
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Genetics & Genomics (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Immunology (AREA)
- Cardiology (AREA)
- General Engineering & Computer Science (AREA)
- Epidemiology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Gastroenterology & Hepatology (AREA)
- Heart & Thoracic Surgery (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Mycology (AREA)
- Nutrition Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The application relates to the field of food and medicine, and in particular provides a royal jelly active peptide composition with an ACE (angiotensin converting enzyme) inhibition effect, and a preparation method and application thereof, wherein the royal jelly active peptide composition comprises at least one of 5 small molecule peptides; the amino acid sequences of the 5 small molecule peptides are respectively shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO. 5. The ACE inhibitory peptide composition prepared by the application has high activity, high yield and strong operability, can be used for industrial production, and provides experimental basis for preparing the ACE inhibitory peptide with high activity from the royal jelly.
Description
Technical Field
The application relates to the field of food and medicine, in particular to a royal jelly active peptide composition with an angiotensin converting enzyme (Angiotensin converting enzyme, ACE) inhibition effect, a preparation method and application thereof.
Background
Hypertension is a clinical syndrome characterized by an increase in systemic arterial blood pressure (systolic and/or diastolic), and can cause myocardial infarction, stroke, coronary arteriosclerosis, and other diseases. Global hypertension patients are increasing year by year, and hypertension has become an important world public health problem. ACE is a dipeptidyl carboxypeptidase containing Zn 2+ prosthetic group that is widely present in the lung, testes and kidneys. ACE is part of the renin-angiotensin system (Renin angiotensin system, RAS) and can cleave His-Leu from the C-terminus to angiotensin I (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu), converting it to angiotensin II (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe), which can constrict arterioles and accelerate heart beats leading to elevated blood pressure. On the other hand, ACE is also an important component of the kallikrein system, which catalyzes bradykinin as an inactive fragment, thereby accelerating the increase in blood pressure in the body. Thus, inhibiting ACE activity may lower blood pressure. The general antihypertensive drugs used clinically achieve the aim of reducing blood pressure by inhibiting ACE activity, however, long-term administration of the chemical synthetic antihypertensive drugs can generate a series of side effects on human bodies, so that development of ACE inhibitory peptides of food-source proteins has become a hotspot for research of domestic and foreign students.
At present, a great deal of reports on the preparation of ACE inhibitory peptides from food-source proteolysis are available. These food materials are generally protein-rich, low-cost, have special processing requirements, or have excellent physiological activity in peptide products. Royal Jelly (Royal Jelly) is a special substance secreted by glands such as developed hypopharynx gland and palate gland of young adult bees of 6-18 d age, is milky or yellowish, semitransparent, slightly viscous, has special fragrance, is sour, astringent, spicy and slightly sweet, and has various physiological activities such as resisting aging, inhibiting bacteria, resisting fatigue, diminishing inflammation, resisting oxidation, regulating blood pressure, reducing blood sugar, promoting cell growth and the like. The protein content in the royal jelly is 9-18%, the price is 160-240 yuan/kg, and compared with other raw materials for preparing active peptide, the protein content and the cost are not advantageous. However, the fresh royal jelly has poor stability, needs full cold chain storage and transportation, and has extremely high logistics storage cost; poor water solubility, and has a bad taste of sourness and pungency; in addition, the sensitive population may be allergic to royal jelly, and it is studied that allergens are major proteins 1 and 2 in royal jelly. These drawbacks greatly affect the commercial value of royal jelly, and limit the application of royal jelly in various fields. The output of the royal jelly in China accounts for more than 90% of the total output of the world, but is mostly used as a raw material outlet, and the profit is low. Therefore, the royal jelly is deeply processed, and the development of active peptide products with strong functionality, good stability, good taste, removal of sensitization factors and high added value is also necessary.
The research on the ACE inhibitory peptide of the royal jelly protein source is still in the starting stage, few research reports are presented at present, most of the research reports stay in simple hydrolysis conditions, and no products with blood pressure reducing effect are commercially developed. There are only few reports on the isolation and identification of ACE inhibitory peptide amino acid sequences from royal jelly proteins, such as: toshiro Matsui et al hydrolyzed royal jelly protein by pepsin, trypsin and chymotrypsin, and then obtained 8 ACE inhibitory peptides with IC50 value less than 10 μm by complicated chromatographic separation method.
The traditional enzymolysis method comprises enzyme type selection, single-enzyme or multi-enzyme compound action research, enzymolysis condition optimization and the like; if the pure peptide is to be separated, a complex purification process is needed, particularly, in the last step of purification, an analytical chromatographic column is needed to achieve enough resolution, and a plurality of times of purification are needed to obtain the trace amount of the pure peptide, so that the preparation cost is extremely high, the method is not suitable for industrial production, and the method is only suitable for scientific research; although the polypeptide can be synthesized in vitro according to the obtained pure peptide sequence, the synthesis cost is still high at present, and the method is not suitable for industrial production.
Therefore, there is a need for improving the conventional production methods of active peptides of royal jelly. The rapid development of bioinformatics technology in recent years can provide methods and tools for analyzing biological big data, and is gradually applied to functional research of bioactive peptides. The bioinformatics method can analyze the structure and functional relationship of the active peptide; predicting the biological activity of the peptide; simulating proteolysis; searching for potential precursor proteins of the bioactive peptide; protein and active peptide molecular docking, and the like. At present, the bioinformatics method has been applied to the preparation of some active peptides, but the application of the method to the preparation of the royal jelly active peptides has not been reported yet.
Disclosure of Invention
In order to overcome the defects in the prior art, the application provides a royal jelly active peptide composition with an ACE inhibition effect, a preparation method and application thereof, which takes a royal jelly water-soluble protein as a raw material, adopts bioinformatics virtual enzymolysis, screens enzymes or enzyme combinations suitable for producing ACE inhibition peptides of a royal jelly protein source, adopts experiments to verify the effect of virtual enzyme digestion, and adopts gel chromatography, anion exchange chromatography and reversed-phase C18 semi-preparation chromatography to purify and produce the royal jelly active peptide composition with the ACE inhibition effect.
In order to achieve the above object, the present application provides the following technical solutions:
in one aspect, the present application provides a royal jelly active peptide composition having ACE inhibitory effect, the royal jelly active peptide composition comprising at least one of 5 small molecule peptides;
The amino acid sequences of the 5 small molecule peptides are respectively shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO. 5.
In a second aspect, the present application provides a method for preparing the active peptide composition of royal jelly with ACE inhibition effect, comprising the following steps:
(1) Performing virtual enzymolysis on the royal jelly protein by using a biological information method to prepare an ACE inhibitory peptide zymolyte;
(2) Verifying the effect of virtual enzymolysis, and preparing ACE inhibition peptide zymolyte;
(3) And (3) separating and purifying the ACE inhibitory peptide zymolyte obtained in the step (2) to prepare the royal jelly active peptide composition with the ACE inhibitory effect.
Optionally, the royal jelly protein in step (1) comprises MRJP1, MRJP2 and MRJP3; wherein the protein number of MRJP1 in NCBI database is ACS66836.1; the protein number of MRJP2 in NCBI database is ACS66837.1, and the protein number of MRJP3 in NCBI database is ADC55524.1.
Optionally, the bioinformatics method in step (1) includes: biological activity prediction was performed on MRJP1, MRJP2, MRJP3 using "PROTEINS", "ANALYSIS", "CALCULATIONS", "PROFILES OF POTENTIAL BIOLOGICAL ACTIVITY" activities in BIOPEP databases; and (3) performing single-ENZYME or combined-ENZYME virtual cleavage on MRJP1, MRJP2 and MRJP3 by using functions of 'PROTEINS', 'ANALYSIS', 'ENZYME (S) ACTION' in BIOPEP databases, and obtaining target ENZYMEs or ENZYME combinations according to the frequency of ACE active peptides released by proteins after ENZYME cleavage.
Optionally, the single enzyme comprises any one of pepsin EC 3.4.23.1, trypsin EC3.4.21.4, chymotrypsin A EC3.4.21.1, bromelain EC 3.4.22.32, papain EC3.4.22.2, alkaline protease EC 3.4.21.62, protease KEC3.4.21.64 in the digestive tract of a human body; the combination enzyme comprises any two of chymotrypsin A EC3.4.21.1, bromelain EC 3.4.22.32, papain EC3.4.22.2, alkaline protease EC 3.4.21.62 and protease KEC3.4.21.64.
Optionally, the condition of the verifying in step (2) is:
1) Dissolving the royal jelly water-soluble protein dry powder into deionized water according to the mass ratio of 8-12%, adding enzyme according to the amount of 7000-9000 mu/g protein, and carrying out enzymolysis for 3-6 h under the condition of optimal enzyme so as to obtain enzymolysis liquid;
2) Inactivating enzyme of the enzymolysis liquid at 90 ℃ for 15min, cooling to room temperature, centrifuging at 5-20 ℃ for 5-10 min at 5000-10000 g, collecting supernatant, freeze-drying to determine ACE inhibitory activity, and obtaining freeze-dried product which is the enzymolysis product with ACE inhibitory activity;
the conditions for preparing the ACE inhibition peptide zymolyte in the step (2) are as follows: and after the verification is finished, the enzyme or enzyme combination required by the enzymolysis product with the maximum ACE inhibition rate and the enzymolysis condition thereof can be obtained.
Optionally, if the enzyme in the step 1) is combined enzymolysis, adding a first enzyme according to 50% of the total enzyme adding amount, adjusting to the optimal reaction condition of the first enzyme for enzymolysis for 2 hours, adding a second enzyme according to 50% of the total enzyme adding amount, and adjusting to the optimal condition of the second enzyme for continuous enzymolysis for 2 hours to obtain an enzymolysis liquid.
Optionally, the separating and purifying operation in the step (3) is to separate the ACE inhibitory peptide zymolyte obtained in the step (2) through Sephadex G-25 column chromatography, elute with deionized water, flow rate is 0.5mL/min, detect absorbance at 280nm to collect eluted components, and then further purify the components with the highest ACE inhibitory activity by adopting HITRAP DEAE-FF anion exchange;
The conditions for the HITRAP DEAE-FF anion exchange purification are as follows: the anion exchange chromatography mobile phase A solution is 0.01mol/L pH 7.0 phosphate buffer solution, the mobile phase B solution is 0.01mol/L pH 7.0 phosphate buffer solution containing 1.0mol/L NaCl, the elution condition is 100% A elution for 20min, then the elution is gradually changed to 100% B in 40min, finally the elution is carried out for 20min by 100% B, the elution flow rate is 1mL/min, and the absorbance is detected at 280nm to collect the eluted component.
Optionally, step (3) further comprises purifying the component having the highest ACE inhibition rate by C18 reverse phase high performance liquid chromatography after the anion exchange purification;
The conditions for purifying the C18 reversed-phase high performance liquid chromatography are as follows: mobile phase a was ultrapure water containing 0.1% tfa, mobile phase B was acetonitrile containing 0.1% tfa, flow rate was 3mL/min, and gradient elution procedure was: ①0-5min：100％B,②5-25min：50％A,50％B,③ 25-32min:100% A; detecting absorbance at 280nm, collecting the eluted components, and freeze-drying and concentrating the collected components to obtain the active peptide composition of the royal jelly with ACE inhibition effect.
In a third aspect, the application provides an application of the active peptide composition of royal jelly with ACE inhibition effect in preparing a medicament for assisting in reducing blood pressure.
Compared with the prior art, the application has the following beneficial effects:
1. The bioinformatics is used as a tool for predicting the potential activity of the royal jelly water-soluble protein, whether the protein raw material is suitable for developing peptides with specific functions or not can be rapidly found, and the most suitable enzymes or enzyme combinations for producing the peptides with the specific functions can be rapidly screened by virtual enzyme digestion.
2. The final step of separation and purification is completed by adopting a semi-preparative chromatographic column, and the pure peptide is not obtained, but the yield is higher, the operability is strong, and the method is suitable for industrial production. The amino acid sequence composition of the main active peptide in the obtained ACE inhibitory peptide composition is HNSDDSFHRL, SFHRL, KNYPF, DVNFR and VEIPH respectively.
3. The royal jelly protein source ACE inhibitory peptide composition provided by the application is safe, free of toxic and side effects, scientific and reasonable in process, and can be developed as a drug additive, a drug synergist and the like.
Drawings
In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only embodiments of the present application, and that other drawings can be obtained according to the provided drawings without inventive effort for a person skilled in the art.
FIG. 1 shows ACE inhibition rate after different enzymes or enzyme combinations hydrolyze water-soluble proteins of Lac Regis Apis;
FIG. 2 is a Sephadex G-25 gel chromatographic profile of the enzyme substrate with maximum ACE inhibition;
FIG. 3 shows ACE inhibition of the components of Sephadex G-25 gel chromatography;
FIG. 4 is an anion exchange chromatogram of the F3 component;
FIG. 5 shows the ACE inhibition of the components after anion exchange chromatography of the F3 component;
FIG. 6 is a reversed phase HPLC purification chromatogram of the F3-4 fraction;
FIG. 7 shows ACE inhibition ratios of the F3-4 components after reversed-phase high performance liquid chromatography separation;
FIG. 8 is a total ion flow chromatogram of the F3-4-1 component;
FIG. 9 is a one-dimensional mass spectrum analysis of the first peptide content in the F3-4-1 component;
FIG. 10 is a two-dimensional mass spectrum analysis of the first peptide content in the F3-4-1 component;
FIG. 11 is a one-dimensional mass spectrum analysis of the second peptide content in the F3-4-1 component;
FIG. 12 is a two-dimensional mass spectrum analysis of the second peptide content in the F3-4-1 component;
FIG. 13 is a one-dimensional mass spectrum analysis of the peptide of the third content in the F3-4-1 component;
FIG. 14 is a two-dimensional mass spectrum analysis of the peptide of the third content in the F3-4-1 component;
FIG. 15 is a one-dimensional mass spectrum analysis of the fourth peptide in the F3-4-1 component;
FIG. 16 is a two-dimensional mass spectrum analysis of the fourth peptide in the F3-4-1 component;
FIG. 17 is a one-dimensional mass spectrum analysis of the peptide having the fifth content in the F3-4-1 component;
FIG. 18 is a two-dimensional mass spectrum analysis of the peptide having the fifth content in the F3-4-1 fraction.
Detailed Description
The technical solutions provided by the present application are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present application.
Unless otherwise indicated, all starting materials in the examples of the present application were purchased commercially and used without any particular treatment.
Unless otherwise indicated, the analytical methods in the examples all employed conventional arrangements of instruments or equipment and conventional analytical methods.
Example 1
Prediction of potential biological activity of main protein MRJP1-3 of royal jelly
(1) The amino acid sequence of MRJP1 (ACS 66836.1), MRJP2 (ACS 66837.1), MRJP3 (ADC 55524.1) was downloaded from NCBI database (https:// www.ncbi.nlm.nih.gov).
(2) The possible activities of the royal jelly proteins after enzymolysis were investigated by means of the biological activity prediction in the BIOPEP database (http:// biochem. Edu. Pl/biopep-uwm /) "PROTEINS", "ANALYSIS", "CALCULATIONS", "PROFILES OF POTENTIAL BIOLOGICAL ACTIVITY" functions of MRJP1-3 and the results are shown in Table 1. From Table 1, it is shown that ACE inhibitory activity is inferior to DPP-IV inhibitory activity among three main proteins of royal jelly, and it is fully possible to perform enzymolysis of main protein MRJP1-3 of royal jelly to produce ACE inhibitory peptide, and specific results are shown in Table 1.
TABLE 1
Note that: a=a/N, wherein a represents the frequency of occurrence of a particular active peptide; a represents the amount of a specific active peptide in the protein; n represents the number of amino acids in the protein
Example 2
Enzymolysis of water-soluble protein of royal jelly
(1) Pepsin (pepsin) EC 3.4.23.1, trypsin (trypsin) EC3.4.21.4, chymotrypsin A (chymotrypsin A) EC3.4.21.1 in the human digestive tract were compared using the "PROTEINS", "ANALYSIS", "ENZYME (S) ACTION" functions in BIOPEP database (http:// biochem. Edu. Pl/biopep-uwm /); bromelain (Stem bromelain) EC 3.4.22.32, papain (papain) EC3.4.22.2, alkaline protease (subtilisin) EC 3.4.21.62, protease K (Proteinase K) EC3.4.21.64. The results of the single or combination enzyme virtual cleavage performed in MRJP1-3 are shown in Table 2. As can be seen from table 2, proteinase K and papain combined use released the theoretically most abundant ACE inhibiting peptides.
TABLE 2 occurrence frequency of ACE inhibitory peptides after theoretical cleavage of MRJP1, MRJP2, MRJP3
Note that: a E＝d/N,W＝AE/A
Wherein a E represents the frequency of a specific active peptide released by a specific enzymatic hydrolysis protein; d represents the amount of the specific active peptide released by the specific enzymatic hydrolysis protein; n represents the number of amino acids in the protein; w represents the ratio of the frequency of a specific active peptide released by a specific enzymatic hydrolysis protein to the frequency of occurrence of the specific active peptide
(2) Enzyme verification of water-soluble protein of royal jelly
In order to verify the effect of theoretical digestion, the study also carries out the experimental verification. Dissolving the royal jelly water-soluble protein dry powder in deionized water according to the mass ratio of 10%, adding enzyme according to the amount of 8000 mu/g protein, and carrying out enzymolysis for 4 hours under the condition of optimal enzyme so as to obtain an enzymolysis liquid. If the combined enzymolysis is the combined enzymolysis, adding a first enzyme according to 50% of the total enzyme adding amount, and regulating to the optimal reaction condition of the first enzyme for enzymolysis for 2 hours; then, adding a second enzyme according to 50% of the total enzyme adding amount, and regulating to the optimal condition of the second enzyme to continue enzymolysis for 2 hours to obtain an enzymolysis liquid. After the enzymolysis is finished, the enzymolysis liquid is put at 90 ℃ to inactivate enzyme for 15min, cooled to room temperature, centrifuged for 5-10 min at 5-20 ℃ and 5000-10000 g, the supernatant is collected, the ACE inhibitory activity is determined by freeze-drying, and the freeze-dried product is the enzymolysis product with the ACE inhibitory activity. The results are shown in FIG. 1.
Determination of ACE inhibitory Activity
70Mg/mL of a sample aqueous solution, 0.08mol/L of 4-hydroxyethylpiperazine ethanesulfonic acid buffer (HEPES), 0.005mg/mL of (N- [3- (2-furyl) acryloyl ] -L-phenylalanyl-glycyl-glycine solution) (FAPGG), and 0.1. Mu.L of an Angiotensin Converting Enzyme (ACE) solution were prepared. ACE 10. Mu.L, FAPGG. Mu.L, HEPES 40. Mu.L was added as blank wells. ACE 10. Mu.L, FAPGG. Mu.L, 40. Mu.L of sample solution was added to the sample wells. Absorbance at 340nm before reaction was measured using a microplate reader. After the mixture on the ELISA plate was allowed to react at 37℃for 30 minutes, the absorbance value of the mixture was again measured.
And (3) calculating a formula: [ (A1-A2) - (B1-B2) ]/(A1-A2) x100%
A1: initial absorbance of blank wells; a2: absorbance for 30min in blank wells; b1: initial absorbance of the sample wells; b2: absorbance after 30min of reaction in the sample wells.
The result proves that the virtual enzyme digestion is basically consistent with the experimental result, the optimal enzyme digestion process is BE combination, namely proteinase K and papain are combined for enzymolysis, and the workload of the experiment can BE greatly reduced by the virtual enzyme digestion.
Example 3
Separation and purification of royal jelly active peptide composition with ACE inhibition effect
The freeze-dried powder of the enzymolysis product prepared by the optimal enzyme digestion process selected in the example 2 is used as a raw material to purify ACE inhibitory peptide, and the method comprises the following steps:
(1) Gel chromatographic column separation
Dissolving the freeze-dried powder with pure water to prepare a solution with the concentration of 2g/mL, and filtering the solution with a filter membrane with the concentration of 0.22 mu m for standby. The method comprises the steps of selecting Sephadex G-25 gel chromatographic columns, wherein the specification phi of the chromatographic columns is 1.5 multiplied by 60cm, the loading amount is 0.5mL, water is taken as a mobile phase, the flow rate is 0.5mL/min, effluent is detected at 280nm, a chromatographic peak F1, a chromatographic peak F2, a chromatographic peak F3 and a chromatographic peak F4 are known from a spectrogram (see figure 2), corresponding fractions of each chromatographic peak are collected, ACE inhibitory activity (see figure 3) of each fraction is detected, the inhibitory activity of the corresponding fraction of the chromatographic peak F3 is optimal, the steps are repeated continuously, the corresponding fraction of the chromatographic peak F3 is collected, and the concentration and freeze drying are carried out.
(2) Ion exchange chromatography column separation
Separating the corresponding fractions of the chromatographic peak F3 by using HITRAP DEAE-FF anion exchange chromatographic column (25 mm multiplied by 150 mm), taking pH 7.0,0.01mol/L phosphate buffer solution as mobile phase A, taking pH 7.0 containing 1mol/L NaCl, 0.1mol/L phosphate buffer solution as mobile phase B, the flow rate being 1mL/min, eluting for 20min at 100% A, gradually changing to 80% A within 40min, eluting for 20min at 100% B, detecting at 280nm, obtaining the chromatographic peak F3-1, the chromatographic peak F3-2, the chromatographic peak F3-3, the chromatographic peak F3-4, the chromatographic peak F3-5 and the chromatographic peak F3-6 from a spectrogram (see figure 4), collecting the corresponding fractions of each chromatographic peak, detecting ACE inhibition activity of the corresponding fractions of the chromatographic peak F3-4, continuously repeating the steps, collecting the corresponding fractions of the chromatographic peak F3-4, and concentrating and freezing the concentrated fractions.
(3) Semi-preparative reversed phase column chromatographic separation
The fractions corresponding to the chromatographic peak F3-4 were further separated using a semi-preparative Shim-pack prep-ods C18 column (20X 250mm,15 μm) at a loading of 100. Mu.L with ultrapure water (0.1% trifluoroacetic acid) as mobile phase A and acetonitrile (0.1% trifluoroacetic acid) as mobile phase B at a flow rate of 3mL/min, and the gradient elution procedure was: ①0-5min：100％B,②5-25min：50％A,50％B,③ 25-32min:100% A. Detecting at 280nm, and obtaining chromatographic peaks F3-4-1, F3-4-2 and F3-4-3 from the spectrogram (see figure 6), collecting corresponding fractions of each chromatographic peak, detecting ACE inhibitory activity of each fraction (see figure 7), and repeating the steps to obtain corresponding fractions of the chromatographic peak F3-4-1, concentrating, and freeze drying.
Example 4
Mass spectrum identification of royal jelly active peptide composition with ACE inhibition effect
(1) Sample pretreatment
The powder samples were dissolved with 50mM NH 4HCO3, after which the final concentration was 10mmol/L by adding DTT solution and reduced in a water bath at 56℃for 1h. IAM solution was added to give a final concentration of 55mmol/L and reacted in the dark for 40min. Desalting was performed using a self-packed desalting column, and the solvent was evaporated in a vacuum centrifugal concentrator at 45 ℃.
(2) Capillary liquid chromatography conditions
Pre-column: 300 μm i.d. times 5mm,Packed with Acclaim PepMap RPLC C18,5 μm,10nm. Analytical column: 150 μm i.d.× 150mm,Packed with Acclaim PepMap RPLC C18,1.9 μm,10nm. Mobile phase a:0.1% formic acid; mobile phase B:0.1% formic acid, 80% ACN; flow rate: 600nL/min; liquid chromatography gradient: 66min.
(3) Mass spectrometry conditions
Primary mass spectrometry parameters: resolution:70000; AGCTARGET:3e6; maximumIT:100ms; SCANRANGE:100-1500m/z. Secondary mass spectrometry parameters: resolution:75000; AGCTARGET:1e5; maximumIT:50ms; topN:20, a step of; NCE/STEPPEDNCE:28.
Collecting raw data for mass spectrometry results can generate a total ion flow chromatogram (fig. 8). Mass spectrometry results showed that the most abundant peptides were: HNSDDSFHRL, SFHRL, KNYPF, DVNFR and VEIPH, it can be seen that the major components of ACE inhibiting peptide compositions are small peptides of less than 2000Da, the five peptides being more than 50% of the total peptides. The results of primary and secondary mass spectrometry experiments on these small peptides are shown in FIGS. 9-18.
Table 3 mass spectrum data for five small peptides
The foregoing is merely a preferred embodiment of the present application and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present application, which are intended to be comprehended within the scope of the present application.
Claims (4)
1. A preparation method of a royal jelly active peptide composition with an ACE inhibition effect is characterized by comprising the following steps:
(1) Performing virtual enzymolysis on the royal jelly protein by using a biological information method to prepare an ACE inhibitory peptide zymolyte;
(2) Verifying the effect of virtual enzymolysis, and preparing ACE inhibition peptide zymolyte;
(3) Separating and purifying the ACE inhibitory peptide zymolyte obtained in the step (2) to prepare the royal jelly active peptide composition with the ACE inhibitory effect;
The royal jelly active peptide composition comprises 5 small molecule peptides;
the amino acid sequences of the 5 small molecule peptides are respectively shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO. 5;
in the step (1), the royal jelly proteins are MRJP1, MRJP2 and MRJP3; wherein the protein number of MRJP1 in NCBI database is ACS66836.1; the protein number of MRJP2 in NCBI database is ACS66837.1, and the protein number of MRJP3 in NCBI database is ADC55524.1;
In step (1), the enzymatic hydrolysis is performed using a combination of protease KEC3.4.21.64 and papain EC3.4.22.2.
2. The method for preparing a royal jelly active peptide composition having ACE inhibitory effect according to claim 1, wherein the condition of the verification in the step (2) is as follows:
1) Dissolving the royal jelly water-soluble protein dry powder into deionized water according to the mass ratio of 8-12%, adding enzyme according to the amount of 7000-9000 mu/g protein, and carrying out enzymolysis for 3-6 h under the condition of optimal enzyme so as to obtain enzymolysis liquid;
2) Inactivating enzyme of the enzymolysis liquid at 90 ℃ for 15min, cooling to room temperature, centrifuging at 5-20 ℃ for 5-10 min at 5000-10000 g, collecting supernatant, freeze-drying to determine ACE inhibitory activity, and obtaining freeze-dried product which is the enzymolysis product with ACE inhibitory activity;
the conditions for preparing the ACE inhibition peptide zymolyte in the step (2) are as follows: and after the verification is finished, the enzyme or enzyme combination required by the enzymolysis product with the maximum ACE inhibition rate and the enzymolysis condition thereof can be obtained.
3. The method for preparing a royal jelly active peptide composition with an ACE inhibitory effect as claimed in claim 2, wherein the enzyme in the step 1) is combined enzymolysis, wherein a first enzyme is added according to 50% of the total enzyme addition, the enzymolysis is performed under the optimal reaction condition of the first enzyme, the enzymolysis is performed for 2 hours, and then a second enzyme is added according to 50% of the total enzyme addition, the enzymolysis is performed for 2 hours under the optimal condition of the second enzyme, so that an enzymolysis solution is obtained.
4. The method for preparing a royal jelly active peptide composition with an ACE inhibitory effect as claimed in claim 1, wherein the separation and purification operation in the step (3) is to separate the ACE inhibitory peptide enzymatic hydrolysate obtained in the step (2) by sephadex g-25 column chromatography, elute with deionized water at a flow rate of 0.5mL/min, collect the eluted fraction by detecting absorbance at 280nm, and then further purify the fraction with the highest ACE inhibitory activity by HITRAPDEAE-FF anion exchange;
The conditions for the HITRAPDEAE-FF anion exchange purification are as follows: anion exchange chromatography mobile phase A solution is 0.01mol/L pH7.0 phosphate buffer solution, mobile phase B solution is 0.01mol/L pH7.0 phosphate buffer solution containing 1.0mol/L NaCl, the elution condition is 100% A elution for 20min, then the elution is gradually changed to 100% B in 40min, finally the elution is carried out for 20min by 100% B, the elution flow rate is 1mL/min, and the absorbance is detected at 280nm to collect the elution component;
Further comprising purifying the component having the highest ACE inhibition rate by C18 reverse phase high performance liquid chromatography after the anion exchange purification;
The conditions for purifying the C18 reversed-phase high performance liquid chromatography are as follows: mobile phase a was ultrapure water containing 0.1% tfa, mobile phase B was acetonitrile containing 0.1% tfa, flow rate was 3mL/min, and gradient elution procedure was: ①0-5min：100％B,②5-25min：50％A,50％B,③ 25-32min:100% A; detecting absorbance at 280nm, collecting the eluted components, and freeze-drying and concentrating the collected components to obtain the active peptide composition of the royal jelly with ACE inhibition effect.
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