CN117089590A - Royal jelly protein peptide and preparation method and application thereof - Google Patents
Royal jelly protein peptide and preparation method and application thereof Download PDFInfo
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- CN117089590A CN117089590A CN202311283876.7A CN202311283876A CN117089590A CN 117089590 A CN117089590 A CN 117089590A CN 202311283876 A CN202311283876 A CN 202311283876A CN 117089590 A CN117089590 A CN 117089590A
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- royal jelly
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Classifications
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- 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
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
-
- 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
- A61K38/01—Hydrolysed proteins; Derivatives thereof
- A61K38/012—Hydrolysed proteins; Derivatives thereof from animals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/18—Antioxidants, e.g. antiradicals
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/145—Extraction; Separation; Purification by extraction or solubilisation
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/30—Extraction; Separation; Purification by precipitation
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
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- 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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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
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- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Genetics & Genomics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Dermatology (AREA)
- General Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Analytical Chemistry (AREA)
- Biophysics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Diabetes (AREA)
- Epidemiology (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
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- General Engineering & Computer Science (AREA)
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Abstract
The invention belongs to the technical field of medical biology, and particularly discloses a royal jelly protein peptide, a preparation method and application thereof. The invention also discloses a preparation method of the royal jelly protein peptide, which comprises the following steps: purifying the royal jelly protein by an alkali-dissolution acid-precipitation method; hydrolyzing the royal jelly protein into polypeptide by using trypsin; separating the royal jelly protein peptides with different molecular weight fragments by ultrafiltration technology. The royal jelly protein peptide provided by the invention is a safe and effective inhibitor of advanced glycosylation end products (advanced glycation end products, AGE). According to the invention, experimental researches prove that the royal jelly protein peptide can reduce the AGE level in skin tissues of the aging mice and the AGE level in serum and tissues and organs of the diabetic mice. The royal jelly protein peptide provided by the invention is expected to inhibit the formation of AGE under the bad and disease states of the organism, thereby preventing or treating the tissue injury caused by the AGE.
Description
Technical Field
The invention belongs to the technical field of medical biology, and particularly discloses a royal jelly protein peptide, a preparation method and application thereof.
Background
Royal jelly is also called Lac Regis Apis, lac Regis Apis or Lac Regis Apis, is a mixture secreted by glands such as pharyngeal gland and upper jaw gland of young worker bee, and is specially used for supplying food required for queen bee life. The royal jelly has complex combination and contains various components such as water, protein, carbohydrate, lipid, mineral substances, vitamins and the like, wherein the protein content is obvious, and the royal jelly is a substance with extremely high nutritive value. Royal jelly has various activities such as anti-inflammatory, wound healing promoting, antibacterial, anti-tumor, immunity enhancing and the like, and can be widely applied to the fields of food and medical treatment. The Lac Regis Apis can be used as raw material of health food, and has rich nutrition, and has positive effects in promoting health. In the medical field, royal jelly can be used for preparing medicaments for treating some inflammatory diseases and immune system disorder diseases. In addition, the royal jelly has nourishing and moisturizing effects, and is helpful for improving skin quality.
The bioactive peptide is an oligopeptide product obtained from macromolecular proteins by the technologies of protease hydrolysis, microbial fermentation and the like, has wide sources and various biological functions. At present, the action efficacy, action mechanism and industrialization application of various bioactive peptides have become hot spots for domestic and foreign research. The bioactive peptide has antioxidant, antibacterial, antiinflammatory, blood pressure lowering, blood sugar regulating, intestinal health promoting, physiological function regulating, health promoting, and disease preventing effects. The unique advantages of bioactive peptides make them an important component of new generation functional foods and health care products. Along with the continuous and deep research on bioactive peptides, the industrialized application of the bioactive peptides is increasingly expanded, more and more bioactive peptide products enter the market, and the requirements of consumers on health and nutrition are met.
Advanced glycation end products (advanced glycation end product, AGE) are a class of compounds that result from the nonenzymatic glycosylation of proteins, fats or nucleic acids with reducing sugars. AGE has a variety of structures, is stable and irreversible and can be continuously accumulated in the human body. At present, AGE has been found to have a role in aging that can form crosslinks with fibrous collagen and elastin constituting the skin skeleton structure, affecting its normal function, leading to reduced diffusion of nutrients and metabolic waste, leading to reduced skin elasticity, and producing aged symptoms such as wrinkles, dark yellowing, etc. Thus, anti-skin glycosylation has become a hot spot and important issue in the study of delaying skin aging. Furthermore, in diabetics, the hyperglycemic environment accelerates the saccharification reaction, leading to the formation of large amounts of AGE. The accumulated AGE binds to cell membrane receptors, activating intracellular signaling pathways, resulting in abnormal cell function; it also can promote oxidative stress, inflammatory reaction and fibrosis process, and promote development of diabetic complications such as microangiopathy, nephropathy, neuropathy, cardiovascular disease, etc. In addition to controlling blood glucose levels, AGE formation inhibitors may also be used to mitigate AGE-induced damage. In view of the long-term and sustained nature of aging and diabetes, intervention in this need has been accomplished with long-term, non-toxic products.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a safe and effective AGE formation inhibitor and a preparation method thereof.
It is another object of the present invention to provide the AGE formation inhibitor for preventing or treating AGE-induced glycation damage of tissues and organs and complications thereof in a state of aging and diabetes.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows: a royal jelly protein peptide is provided as an AGE formation inhibitor, which is obtained by biological enzymolysis of royal jelly protein.
The preparation method of the royal jelly protein peptide comprises the following steps:
purification of royal jelly protein: adding 5 times deionized water into lyophilized Lac Regis Apis powder, stirring for dissolving. Adjusting pH to 10.0 with 2M NaOH solution, stirring and extracting for 30min, centrifuging at 5000r/min for 20min, and collecting supernatant; adding 2M HCl solution into the supernatant to adjust pH to 3.5, precipitating at 4deg.C in refrigerator for 24h, centrifuging at 5000r/min for 20min, discarding supernatant, collecting precipitate, and drying to obtain purified Lac Regis Apis protein.
The method for extracting the royal jelly protein by adopting the alkali-dissolution acid precipitation method is easy to operate and low in cost, the step is favorable for guaranteeing the stability and sufficiency of the subsequent enzymolysis process, the influence of impurities on the enzymolysis quality is avoided, and the purity and the yield of the royal jelly protein peptide are improved.
Enzymolysis: weighing 10g of purified royal jelly protein, adding 300mL of deionized water according to a feed liquid ratio of 1:30, uniformly stirring, adjusting the pH to 6.0-9.0 by using a NaOH solution, adding 0.2-3.0% of protease for biological enzymolysis, and stirring at a constant temperature of 25-55 ℃ for 2-8h. Inactivating enzyme at 90deg.C for 15min, standing to room temperature, centrifuging at 10000r/min for 20min, collecting supernatant, and lyophilizing to obtain Lac Regis Apis protein peptide.
The inventors compared AGE inhibitory activities of enzyme hydrolysis products prepared from neutral protease, alkaline protease, pepsin, trypsin, papain, bromelain, composite protease and flavourzyme, preferably pepsin and trypsin, and further, the AGE inhibitory activities of royal jelly protein peptides obtained by trypsin hydrolysis were the best.
It is worth to say that the AGE inhibition activity of the enzymolysis product prepared by the protease is obviously higher than that of the original royal jelly protein, so that the hydrolysis processing of the royal jelly protein by the enzymolysis technology obviously increases the biological function of the royal jelly protein and is possibly related to the release of the effective amino acid sequence.
Preferably, the trypsin is added in an amount of 0.5-2.0% by weight.
Preferably, the enzymolysis temperature is 35-45 ℃.
Preferably, the enzymatic pH is 7.0-8.5.
Preferably, the enzymolysis time is 4-6 hours.
Through the optimization of the enzymolysis process, the AGE inhibition rate of the royal jelly protein peptide can reach more than 60 percent.
Furthermore, the inventor adopts ultrafiltration membranes with different molecular weights to separate zymolytes, and an activity comparison experiment proves that the active peptide with the molecular weight smaller than 3kDa has stronger AGE inhibition effect compared with fragments with other molecular weights.
The drying technology adopted by the invention is freeze drying, and the obtained royal jelly protein peptide has good quality and is favorable for maximally preserving the activity of the protein peptide.
The inventor finds that the AGE level in skin tissues of aging mice can be obviously reduced by using the royal jelly protein peptide as an AGE inhibitor through researches, and shows that the royal jelly protein peptide can be used for preventing or repairing saccharification damage and complications thereof in the aging process.
Such skin glycation injury complications include, but are not limited to: skin dullness, stain deposition, increased wrinkles, thinning of the skin, reduced barrier function, sagging of the skin, enlarged pores, dry roughness, vasodilation, and increased sensitivity.
Further, the inventor finds that the royal jelly protein peptide can be used as an AGE inhibitor to obviously reduce the AGE level in serum and tissues and organs of a diabetic mouse, and shows that the royal jelly protein peptide can be used for preventing or treating saccharification damage and complications thereof in the process of diabetes.
The diabetic saccharification injury complications include, but are not limited to: diabetic eye disease, diabetic neuropathy, diabetic nephropathy, diabetic heart disease, diabetic vascular disease, diabetic metabolic disease, diabetic skin disease.
The invention also provides application of the royal jelly protein peptide in preparing a product for preventing, improving or treating AGE-induced saccharification injury, and the royal jelly protein peptide can be developed into an oral preparation, an external preparation or an injection with a proper carrier or auxiliary materials.
Alternatively, in the above formulation, the oral dosage forms include, but are not limited to, tablets, capsules, oral liquids, enteric-coated tablets, granules, syrups, dripping pills, water-honeyed pills, powders, mixtures, soft extracts, wines, lotions, tinctures, sublingual tablets and the like.
The external preparation forms include, but are not limited to, ointments, suppositories, hard ointments, liniments, eye drops, oils, creams, aerosols, sprays and the like.
Modes of administration include, but are not limited to, subcutaneous, intravenous, intramuscular, intraperitoneal, and the like.
Alternatively, the royal jelly protein peptide provided by the invention can be prepared into corresponding toner, moisturizing cream, facial mask, face cream, cake, bath foam, soap, foundation cream, essence, hand cream, body lotion, hand cleanser, shampoo, hair conditioner, facial cleansing cream, facial cleanser, sun cream, skin cream, vanishing cream, nutrient solution, perfume and the like according to the conventional process of cosmetics, skin care products and daily washing products.
Alternatively, in the above use, the product is a drug, a health product, a functional food or a cosmetic.
Compared with the prior art, the invention has the beneficial effects that:
the technical scheme of the invention provides a royal jelly protein peptide active peptide, a preparation method thereof and application thereof in preparation of an AGE inhibitor. The active peptide can obviously inhibit the formation of AGE, relieves the saccharification damage of tissues and organs, is safe and effective, has small molecular weight, good stability and simple preparation process, is applied to the fields of daily cosmetics, foods and medicines, and has wide market prospect.
Detailed Description
Specific embodiments of the present invention will be further described below with reference to examples, but the practice and protection of the present invention are not limited thereto. It should be noted that the following is performed under conventional conditions or conditions recommended by the manufacturer, unless specific conditions are noted. The raw materials, reagents, etc. used, which are not noted to the manufacturer, are conventional products commercially available.
Example 1: preparation of royal jelly protein peptide
Purification of royal jelly protein: adding 5 times deionized water into lyophilized Lac Regis Apis powder, stirring for dissolving. Adjusting pH to 10.0 with 2M NaOH solution, stirring and extracting for 30min, centrifuging at 5000r/min for 20min, and collecting supernatant; adding 2M HCl solution into the supernatant to adjust pH to 3.5, precipitating at 4deg.C in refrigerator for 24h, centrifuging at 5000r/min for 20min, discarding supernatant, collecting precipitate, and drying to obtain purified Lac Regis Apis protein.
Weighing 10g of purified royal jelly protein, adding 300mL of deionized water according to a feed liquid ratio of 1:30, uniformly stirring, adjusting the pH to 7.0 by using a NaOH solution, adding 1% trypsin for biological enzymolysis, and stirring at a constant temperature of 40 ℃ for 5 hours. Inactivating enzyme at 90deg.C for 15min, standing to room temperature, centrifuging at 10000r/min for 20min, collecting supernatant, and lyophilizing to obtain Lac Regis Apis protein peptide.
Example 2: determination of AGE inhibitory Activity
AGE inhibition was measured as follows: and dissolving the prepared royal jelly protein peptide in PBS solution to dilute the royal jelly protein peptide into a sample to be detected of 2 mg/mL. A reaction solution containing 2.8mg/mL bovine serum albumin (bovine serum albumin, BSA) and 1mM methylglyoxal was prepared with PBS. Then, 1mL of the reaction solution was mixed with 1mL of the sample solution, sealed, and placed in an incubator at 37℃for 7 days to form glycosylation-modified albumin. At the same time, 1mL of the reaction solution was mixed with 1mL of PBS solution to prepare a blank. The glycosylation modified albumin has characteristic absorption spectrum at excitation wavelength of 370nm and emission wavelength of 450nm, and the content of the formed glycosylation modified albumin is measured by adopting a fluorescence spectrum analysis method. The inhibitory activity of the sample on AGE was calculated using the following formula: AGE inhibition (%) = [1- (a sample/a blank) ]x100.
Example 3: effect of different proteases on the inhibitory Activity of the Royal jelly protein peptide AGE
Accurately weighing 10g of royal jelly protein, dissolving in 300mL of deionized water, regulating pH to 7.0, respectively adding 1% of neutral protease, alkaline protease, pepsin, trypsin, papain, bromelain, compound protease and flavourzyme, stirring at 40 ℃ for reaction for 5h, inactivating enzyme at 90 ℃ for 15min after the reaction is finished, standing to room temperature, centrifuging at 10000r/min for 20min, collecting supernatant, and freeze-drying to obtain the royal jelly protein peptide. The inventors compared the AGE inhibition activities of unhydrolyzed royal jelly protein and royal jelly protein peptides prepared by different proteases, and as shown in Table 1, the unhydrolyzed royal jelly protein peptides were weak in activity, the AGE inhibition rate was only 4.9%, and the activity of the royal jelly protein peptides obtained by protease hydrolysis was significantly increased, suggesting that the enzymolysis technology is favorable for releasing active peptide fragments in proteins and improving the AGE inhibition activities thereof. Second, differences in biological activity are exhibited due to differences in cleavage sites of different proteases resulting in differences in the released peptide fragments. The inventor finds that compared with other proteases, the active peptide of the royal jelly prepared by pepsin and trypsin has the strongest activity, and the AGE inhibition rate of the active peptide reaches 46.8% and 56.1% respectively.
TABLE 1 influence of different proteases on the inhibitory Activity of the royal jelly protein peptide AGE
| Enzyme species | AGE inhibition (%) |
| Neutral protease | 25.6 |
| Alkaline protease | 31.2 |
| Pepsin | 46.8 |
| Trypsin, trypsin and its preparation method | 56.1 |
| Papain | 20.7 |
| Bromelain | 15.3 |
| Complex protease | 28.7 |
| Flavoured protease | 27.4 |
| / | 4.9 |
Example 4: effect of different enzyme dosages on the inhibitory Activity of the Royal jelly protein peptide AGE
Weighing 10g of purified royal jelly protein, adding 300mL of deionized water according to a feed liquid ratio of 1:30, stirring uniformly, regulating the pH to 7.0 by using a NaOH solution, respectively adding 0.2%, 0.5%, 1.0%, 1.5%, 2.0%, 2.5% and 3.0% of trypsin for biological enzymolysis, and stirring at a constant temperature of 40 ℃ for 5 hours. Inactivating enzyme at 90deg.C for 15min, standing at room temperature, centrifuging at 10000r/min for 20min, collecting supernatant, and lyophilizing to obtain Lac Regis Apis protein peptide. The inventors compared the AGE inhibition activities of the royal jelly protein peptides prepared with different amounts of trypsin, and as shown in table 2, the amounts of enzyme did affect the activity of the product; when the trypsin is used in an amount of 0.2%, the AGE inhibitory activity of the royal jelly protein peptide is only 40.2%; when the enzyme dosage is 0.5% or more, the AGE inhibition rate of the product can reach more than 50%, and the AGE inhibition rate is increased along with the increase of the enzyme dosage within the range of 0.5-2.0%; when the enzyme amount is more than 2.0%, the activity of the product is not further improved. Therefore, the amount of trypsin is preferably 0.5 to 2.0% based on the activity of the product and the cost of the process.
TABLE 2 Effect of different enzyme dosages on the inhibitory Activity of the royal jelly protein peptide AGE
| Enzyme dosage (%) | AGE inhibition (%) |
| 0.2 | 40.2 |
| 0.5 | 51.6 |
| 1.0 | 55.8 |
| 1.5 | 58.3 |
| 2.0 | 61.2 |
| 2.5 | 60.7 |
| 3.0 | 61.1 |
Example 5: effect of different temperatures on the AGE inhibitory Activity of Lac Regis Apis protein peptide
Weighing 10g of purified royal jelly protein, adding 300mL of deionized water at a feed liquid ratio of 1:30, stirring uniformly, adjusting pH to 7.0 with NaOH solution, adding 1% trypsin for biological enzymolysis, and stirring at constant temperature of 25 ℃, 30 ℃, 35 ℃,40 ℃, 45 ℃,50 ℃ and 55 ℃ for 5 hours respectively. Inactivating enzyme at 90deg.C for 15min, standing at room temperature, centrifuging at 10000r/min for 20min, collecting supernatant, and lyophilizing to obtain Lac Regis Apis protein peptide.
The inventors compared the AGE inhibitory activities of the royal jelly protein peptides prepared under different temperature conditions, and the results are shown in Table 3, and the AGE inhibitory activities of the royal jelly protein peptides prepared at different enzymolysis temperatures are different; AGE inhibitory activity of the royal jelly protein peptide is less than 50% at 25-30deg.C, possibly associated with low temperature inhibition of enzyme activity, which is detrimental to release of the active fragment; when the temperature is in the range of 35-45 ℃, the AGE inhibition rate of the product can reach more than 55 percent; when the temperature is increased, the activity of the product is reduced, possibly related to the high temperature destroying the structure of the enzyme, and reducing the enzymolysis efficiency. Therefore, based on the data, the temperature of enzymolysis is preferably 35-45 ℃.
TABLE 3 Effect of different temperatures on the AGE inhibitory Activity of the Lac Regis Apis protein peptides
| Temperature (. Degree. C.) | AGE inhibition (%) |
| 25 | 36.7 |
| 30 | 42.9 |
| 35 | 55.7 |
| 40 | 56.8 |
| 45 | 55.4 |
| 50 | 52.7 |
| 55 | 50.7 |
Example 6: effect of different pH on royal jelly protein peptide AGE inhibitory Activity
Weighing 10g of purified royal jelly protein, adding 300mL of deionized water according to a feed liquid ratio of 1:30, uniformly stirring, respectively regulating pH to 6.0, 6.5, 7.0, 7.5, 8.0, 8.5 and 9.0 by using NaOH solution, adding 1% of trypsin for biological enzymolysis, and stirring at a constant temperature of 40 ℃ for 5 hours. Inactivating enzyme at 90deg.C for 15min, standing at room temperature, centrifuging at 10000r/min for 20min, collecting supernatant, and lyophilizing to obtain Lac Regis Apis protein peptide. The influence of the enzymolysis pH value on the AGE inhibition rate is shown in Table 4, and the enzymolysis pH value is between 7.0 and 8.5, so that the better AGE inhibition activity of the enzymolysis product can be ensured.
TABLE 4 influence of different pH values on the AGE inhibitory Activity of the Lac Regis Apis protein peptides
Example 7: influence of different enzymolysis time on the inhibition activity of royal jelly protein peptide AGE
Weighing 10g of purified royal jelly protein, adding 300mL of deionized water according to a feed liquid ratio of 1:30, uniformly stirring, adjusting the pH to 7.0 by using a NaOH solution, adding 1% of trypsin for biological enzymolysis, and respectively stirring at a constant temperature of 40 ℃ for 2, 3, 4, 5, 6, 7 and 8 hours. Inactivating enzyme at 90deg.C for 15min, standing at room temperature, centrifuging at 10000r/min for 20min, collecting supernatant, and lyophilizing to obtain Lac Regis Apis protein peptide. The influence of the enzymolysis time on the AGE inhibition rate is shown in Table 5, the AGE inhibition rate increases along with the increase of the enzymolysis time within 2-6h, and the longer enzymolysis time may cause excessive hydrolysis of the royal jelly protein, which is unfavorable for maintaining the activity of the product. Therefore, the enzymolysis treatment time is proper when 4-6 hours are selected.
TABLE 5 influence of different enzymolysis time on the inhibitory Activity of the royal jelly protein peptide AGE
| Time (h) | AGE inhibition (%) |
| 2 | 25.7 |
| 3 | 42.3 |
| 4 | 56.8 |
| 5 | 64.7 |
| 6 | 67.8 |
| 7 | 52.4 |
| 8 | 50.2 |
Example 8: activity comparison of fragments of different molecular weights of Lac Regis Apis protein peptide
The royal jelly protein peptide obtained in example 1 was separated by ultrafiltration membrane, and the following components of the molecular weight fraction were collected: the bioactive peptide with the molecular weight range is obtained after freeze drying, wherein the molecular weight ranges are less than 3kDa, 3-5kDa, 5-10kDa and more than 10 kDa. The inventors evaluated the inhibitory activity of different molecular weight royal jelly protein peptides on the glycosylated end products. As a result, as shown in Table 6, the activities of peptide fragments having different molecular weights were different, and it was found that the smaller the molecular weight, the more active was. The active peptide with the molecular weight less than 3kDa has stronger AGE inhibition effect compared with fragments with other molecular weights, and the inhibition rate can reach 77.9 percent.
TABLE 6 AGE inhibition of Lac Regis Apis peptides of different molecular weights
| Molecular weight | AGE inhibition (%) |
| ＜3kDa | 77.9 |
| 3-5kDa | 47.2 |
| 5-10kDa | 25.7 |
| ＞10kDa | 7.6 |
| Not separated | 56.3 |
Example 9: effect of Lac Regis Apis protein peptide on AGE level in aged mouse skin
To verify the effect of the royal jelly protein peptide of the present invention, the inventors evaluated the in vivo activity of the royal jelly protein peptide having a molecular weight of < 3kDa isolated in example 8 from the royal jelly protein peptide prepared in example 1.
SPF-grade male Kunming mice weighing 22-25g were purchased from the Experimental animal center at the university of south medical science. The feeding conditions were as follows: the temperature is 22+/-2 ℃, the humidity is 55+/-5 percent, and the light is irradiated for 12 hours (7:00-19:00) every day. The mice were randomly divided into a normal control group, a D-galactose model group, a royal jelly protein group (200 mg/kg), a royal jelly protein peptide group (200 mg/kg) and a royal jelly protein peptide (< 3 kDa) (200 mg/kg) group. Except for the normal control group, the skin aging model was constructed by subcutaneously injecting 5% D-galactose solution (0.5 mL/20 g) into the back skin of the other mice, and the normal group was injected with an equal volume of physiological saline for 6 consecutive weeks. The administration group was administered by gavage during the molding period, 1 time a day, and the normal group and the model group were administered with equal volumes of physiological saline. After the administration, the isoflurane anesthetized mice are killed by cervical spining, and the back skin is taken and placed in a refrigerator at-80 ℃ for standby.
Cutting skin tissue of a mouse, adding RIPA lysate, homogenizing for 2min by using an automatic homogenizer, placing on ice for continuous pyrolysis for 30min, centrifuging for 10min at 12000r/min at 4deg.C, collecting supernatant, and detecting AGE content by using ELISA kit. The results are shown in Table 7, in which the AGE content in the skin of mice was reduced by the royal jelly protein, the royal jelly protein peptide and the royal jelly protein peptide (< 3 kDa) as compared with the D-galactose-induced skin aging model group of mice. The comparison shows that the inhibition activity is sequentially that the royal jelly protein peptide (< 3 kDa) > the royal jelly protein peptide > the royal jelly protein, which is consistent with the in vitro result. From these results, it was found that the AGE inhibitory activity of the royal jelly protein peptide was still effective in vivo, and the formation of AGEs in aged skin was inhibited.
TABLE 7 AGE levels in mice skin
| Group of | AGE(ng/mL) |
| Normal group | 60.0±7.96 |
| Model group | 149.1±13.66 * |
| Royal jelly protein | 131.9±24.26 |
| Royal jelly protein peptide | 100.5±13.77 # |
| Royal jelly protein peptide (< 3 kDa) | 71.7±10.25 # |
In contrast to the normal group, * P<0.05; in contrast to the set of models, # P<0.05。
example 10: effect of Lac Regis Apis protein peptide on AGE level in tissue and organ of diabetic mouse
Subsequently, the inventors also evaluated the in vivo activity of the royal jelly protein peptide having a molecular weight of < 3kDa isolated in example 8 from the royal jelly protein peptide prepared in example 1 on an animal model of diabetes.
Construction of a hyperglycemic mouse model: SPF-grade male Kunming mice weighing 22-25g were purchased from the Experimental animal center at the university of south medical science. The feeding conditions were as follows: the temperature is 22+/-2 ℃, the humidity is 55+/-5 percent, and the light is irradiated for 12 hours (7:00-19:00) every day. Mice were intraperitoneally injected with 1% STZ solution (40 mg/kg) after 12h of fasting for 5 consecutive days with 10% sucrose added to the drinking water during molding. Mice were assayed for fasting blood glucose 3 weeks after the first STZ injection, and when the fasting blood glucose was higher than 11.1mmol/L, hyperglycemic modeling was considered successful.
The hyperglycemic mice were randomly divided into model groups, royal jelly proteome (200 mg/kg), royal jelly proteome peptide group (200 mg/kg) and royal jelly proteome peptide (< 3 kDa) (200 mg/kg). The administration group was administered by intragastric administration 1 time a day for 6 consecutive weeks, and the normal group and the model group were administered with equal volumes of physiological saline. After the administration is finished, the isoflurane anesthetizing the mouse and the orbit to obtain blood, collecting the eyeball, the kidney and the skin of the mouse, and storing the mouse in a refrigerator at the temperature of-80 ℃ for later use.
The homogenate of the mouse tissue is prepared by adopting a homogenate technology, and the supernatant is collected by centrifugation. AGE in mouse serum and tissue homogenate supernatants was detected using ELISA kit. As shown in Table 8, the serum, kidney, skin and eyeball AGE levels were reduced in mice compared to the model group. It was also found that the inhibitory activity was in turn a royal jelly protein peptide (< 3 kDa) > a royal jelly protein peptide > a royal jelly protein, which is consistent with the in vitro activity results. From these results, it was found that the royal jelly protein peptide can inhibit the formation of AGE in various tissues of diabetic mice in vivo and alleviate glycation injury.
TABLE 8 levels of AGE in mouse serum and tissues
In contrast to the normal group, * P<0.05; in contrast to the set of models,#P<0.05。
it should be further noted that the above embodiments are only for illustrating the technical solution provided by the present invention, but the present invention is not limited to the above embodiments. Therefore, the technical scheme described in the above embodiment is modified or some of the technical features are replaced equivalently, so that the essence of the corresponding technical scheme does not deviate from the scope of the technical scheme described in the embodiment of the present invention.
Claims (10)
1. A royal jelly protein peptide, characterized in that it is obtained from the biological enzymolysis of a royal jelly protein, and is a safe and effective advanced glycosylation end product (advanced glycation end products, AGE) inhibitor.
2. The method for preparing a royal jelly protein peptide according to claim 1, characterized in that the method comprises the steps of:
step 1): purifying the royal jelly protein by an alkali-dissolution acid-precipitation method;
step 2): hydrolyzing the royal jelly protein into polypeptide using protease;
step 3): separating the royal jelly protein peptides with different molecular weight fragments by ultrafiltration technology.
3. The method for preparing a protein peptide of royal jelly according to claim 2, wherein the protease in step 2) is selected from one or more of the following: neutral protease, alkaline protease, pepsin, trypsin, papain, bromelain, complex protease or flavourzyme, preferably the protease is pepsin and/or trypsin.
4. The method for producing a royal jelly protein peptide according to claim 2 or claim 3, characterized in that the protease is added in the amount of 0.5 to 2.0% by weight in step 2).
5. The method for preparing a royal jelly protein peptide according to claim 2 or claim 3, wherein the enzymolysis temperature of the protease in step 2) is 35-45 ℃, the enzymolysis pH of the protease is 7.0-8.5, and the enzymolysis time of the protease is 4-6 hours.
6. The method for preparing a protein peptide of royal jelly according to claim 2 or claim 3, characterized in that the molecular weight cut-off of the ultrafiltration membrane used in step 3) is < 3kDa.
7. Use of the royal jelly protein peptide of claim 1 or the royal jelly protein peptide prepared by the preparation method of any one of claims 2 to 6 in the preparation of AGE inhibitors.
8. The use according to claim 7, wherein the AGE inhibitor is a skin care product, a functional food, a health care product or a pharmaceutical product.
9. The use according to claim 7, characterized in that it is the prevention or repair of glycation lesions and their complications in skin ageing.
10. The use according to claim 7, wherein the use is for preventing or treating tissue organ saccharification damage and complications thereof in diabetes.
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