CN119219733B - Ascidian-derived tripeptide, preparation method thereof, and application thereof in skin tissue repair - Google Patents
Ascidian-derived tripeptide, preparation method thereof, and application thereof in skin tissue repair Download PDFInfo
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/08—Tripeptides
- C07K5/0802—Tripeptides with the first amino acid being neutral
- C07K5/0812—Tripeptides with the first amino acid being neutral and aromatic or cycloaliphatic
-
- 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
-
- 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
- A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/08—Tripeptides
- C07K5/0821—Tripeptides with the first amino acid being heterocyclic, e.g. His, Pro, Trp
-
- 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
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- General Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Dermatology (AREA)
- Pharmacology & Pharmacy (AREA)
- Veterinary Medicine (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
本发明属于多肽及其应用技术领域，公开了海鞘源三肽及其制备方法和在皮肤组织修复中的应用，所述海鞘源三肽包括以下氨基酸序列肽段中的一种或多种的混合物：FPP、WPG、FMP。本发明还提供了海鞘源三肽的生产工艺，生产工艺包括将海鞘利用酶进行酶解的步骤，并采用滤膜分离。本发明还提供了海鞘源三肽在皮肤组织修复中的应用。该海鞘源三肽可显著促进皮肤细胞增殖和分化，能够刺激皮肤细胞的生长，加速皮肤组织修复。
The present invention belongs to the technical field of polypeptides and their application, and discloses a tripeptide derived from sea squirt, a preparation method thereof, and an application thereof in skin tissue repair. The tripeptide derived from sea squirt includes a mixture of one or more of the following amino acid sequence peptide segments: FPP, WPG, and FMP. The present invention also provides a production process of the tripeptide derived from sea squirt, and the production process includes the steps of enzymolysis of sea squirt using an enzyme, and separation using a filter membrane. The present invention also provides the application of the tripeptide derived from sea squirt in skin tissue repair. The tripeptide derived from sea squirt can significantly promote the proliferation and differentiation of skin cells, can stimulate the growth of skin cells, and accelerate skin tissue repair.
Description
Technical Field
The invention belongs to the field of biological peptides, and in particular relates to an ascidian tripeptide, a preparation method thereof and application thereof in skin tissue repair.
Background
The skin is used as the largest organ of the human body, has the function of protecting the internal tissues of the body, and also participates in physiological activities such as regulating the body temperature, sensing external stimulus and the like. However, skin is susceptible to various injuries in daily life, which may be caused by external forces, chemicals, ultraviolet rays, and the like. Once the skin tissue is damaged, the residual part can be restored to the original form and function by itself, namely regeneration, and the restoration mode has the advantages that the new tissue is autologous, and the body function can be restored to the state before the damage. The damaged area can be repaired by the activities of cell proliferation, differentiation, migration and the like of the tissues and organs of the damaged part, and finally the tissues and organs are restored to the original shape and function. However, the ability to regenerate and repair varies from vertebrate to vertebrate, from animal to animal, and from tissue organ to tissue organ.
Many factors affecting repair of skin lesions are closely related to the extent and extent of the lesion, age, endocrine, pharmaceutical action, nutritional status, and the like, in addition to the regenerative capacity of tissues and cells themselves. Once body tissue is damaged, the treatment environment and method can be optimized by increasing nutrient supply and adopting the measure of drug intervention, and the tissue repair and regeneration can be promoted.
The ascidian is a phylum of chordopoda, which is one of the most major tunicates, and has a strong regeneration ability, but it is not clear that an active substance specifically exerting a regeneration function is contained in the ascidian body, which contains various substances having biological activity, including active peptides. The active peptides have various biological activities, such as anti-tumor, antiviral, antioxidant and the like, and have wide application prospects in the fields of drug development, health-care product development and the like. With the intensive research, scientists have had more insight into the structure of the ascidian-derived active peptide. Most of these active peptides have specific chemical structures and simultaneously have corresponding multiple biological functions. For example, some ecteinascidia-derived active peptides have remarkable antitumor activity, and can inhibit the growth and division of tumor cells, while other active peptides have antiviral, antioxidant, etc. functions.
In the aspect of skin repair polypeptide drug development, many researches have proved that the active peptide can promote tissue repair. If researchers isolate Tylotoin from lizard, the active peptide contains 12 amino acid residues and shows strong activity of promoting wound healing. Meanwhile, there are reports of extracting polypeptides capable of promoting skin repair from the rana huashi, the red salamander and the rana grahami respectively, namely 16 peptide, 13 peptide, 24 peptide and 11 peptide. The active peptides are long peptides, contain more amino acids, have relatively low absorption rate in vivo, and can be influenced by various factors such as gastrointestinal environment, enzyme activity, induction of immune response of organism and the like. The short peptide has a simple structure, can be directly absorbed by a human body, does not need to undergo a complex enzymolysis process after being taken into the body, can be rapidly absorbed in tissue repair, and can rapidly reach the damaged part to exert biological activity.
In conclusion, ascidians have a strong regeneration capacity, and when the body is damaged, the regeneration procedure can be rapidly started to replace or restore damaged or lost cells, tissues and even the whole body part. By researching the regeneration mechanism of the ascidian source active peptide, new ideas and methods can be provided for the biological and medical fields. The research of the preparation method of ecteinascidin, the kind and composition of peptide for promoting skin tissue repair and the research of the inherent biochemical mechanism of active peptide are the cores for developing and applying novel tissue repair active peptide.
Disclosure of Invention
The invention provides an ascidian tripeptide, a preparation method and application thereof in skin tissue repair, wherein the ascidian tripeptide can obviously promote proliferation and differentiation of skin cells, can stimulate growth of the skin cells and accelerate the repair process of skin tissues.
In order to achieve the aim, the invention provides the following technical scheme that the ascidian-derived tripeptide comprises a mixture of one or more of the following amino acid sequence peptide fragments FPP, WPG, FMP.
The invention also provides a production process of the sea squirt-derived tripeptide, which comprises the step of carrying out enzymolysis on sea squirts by using enzymes.
Preferably, the step of enzymolysis adopts compound protease.
Preferably, the enzyme adding amount in the enzymolysis step is 2000U/g.
Preferably, the enzymolysis temperature of the enzymolysis step is 55 ℃.
Preferably, the enzymolysis time of the step of enzymolysis is 2.5 hours.
Preferably, the enzymatic hydrolysate obtained in the enzymatic hydrolysis step is filtered by a 5 μm ultrafiltration membrane and then filtered by a 1000 Da ceramic nanofiltration membrane to obtain the ascidian tripeptide.
The invention also provides application of the sea squirt-derived tripeptide in skin tissue repair.
Compared with the prior art, the invention has the beneficial effects that:
1. The sea squirt source active peptide disclosed by the invention is a peptide substance extracted from sea squirts and having special physiological functions, has the characteristics of low molecular weight, high activity and easy absorption, can directly act on target proteins for regulating and controlling tissue regeneration, and promotes proliferation and differentiation of cells at a damaged part, thereby accelerating tissue repair and regeneration;
2. The three tripeptides disclosed by the invention are rich in proline, can obviously promote proliferation and differentiation of skin cells, reduce in-vivo secretion of pro-inflammatory factors, improve secretion of anti-inflammatory factors, and further accelerate the skin tissue repair process;
3. the sea squirt tripeptide is obtained through an enzymolysis process, the production process is simple to operate, a high-temperature and high-pressure environment is not needed, the energy consumption is low, and the production efficiency is high.
Drawings
FIG. 1 is a graph showing comparison of hydrolysis degrees of sea squirts by different enzymes according to the present invention;
FIG. 2 is a graph showing comparison of ecteinascidin recovery rates from enzymatic hydrolysis of ecteinascidins according to the invention;
FIG. 3 is a mass spectrum analysis chart of the composition and content of ecteinascidins obtained by enzymolysis of the complex protease of the invention;
FIG. 4 is a mass spectrum of tripeptide FPP according to the invention;
FIG. 5 is a mass spectrum of the tripeptide WPG of the invention;
FIG. 6 is a mass spectrum of the tripeptide FMP according to the invention;
FIG. 7 is a graph showing the effect of the ecteinascidity-derived tripeptide on the regeneration area of the tail fin of a zebra fish;
FIG. 8 is a graph of HE slices of the effect of the ecteinascidity-derived tripeptides of the invention on zebra fish tail fin regeneration;
FIG. 9 is a bar graph showing the effect of the ecteinascidity-derived tripeptide on the distance traveled by the tail fin of a zebra fish of the present invention;
FIG. 10 is a bar graph showing the effect of the ecteinascidity-derived tripeptide on the speed of movement of the tail fin of a zebra fish;
FIG. 11 is a bar graph showing the effect of the ecteinascidity-derived tripeptides of the present invention on the regeneration area of zebra fish tail fins;
FIG. 12 is a graph showing the analysis of the repair of skin lesions in mice by the sea squirt-derived tripeptide FPP of the present invention;
FIG. 13 is a graph showing the effect of the ecteinascidia-derived tripeptide FPP of the invention on repair of back wound area in mice;
FIG. 14 is a graph showing the effect of the ecteinascidity-derived tripeptide FPP of the invention on the secretion of the pro-inflammatory factor IL-4 in mice;
FIG. 15 is a graph showing the effect of the ecteinascidity-derived tripeptide FPP of the invention on the secretion of the proinflammatory factor IL-6 in mice;
FIG. 16 is a graph showing the effect of the ecteinascidity-derived tripeptide FPP of the invention on the secretion of the pro-inflammatory factor IL-10 in mice;
FIG. 17 is a graph showing the effect of the ecteinascidity-derived tripeptide FPP of the invention on the secretion of anti-inflammatory factor TNF- α in mice;
FIG. 18 is a graph showing the effect of the ecteinascidity-derived tripeptide FPP of the invention on the secretion of anti-inflammatory factor TGF-beta in mice;
FIG. 19 is a graph showing the effect of the ecteinascidity-derived tripeptide FPP of the invention on the secretion of anti-inflammatory factor IFN-gamma in mice;
FIG. 20 is a graph showing analysis of the site of action ERK1 for promoting tissue repair by the sea squirt-derived tripeptide FPP of the present invention;
FIG. 21 is a graph showing the analysis of the site of action P38a for promoting tissue repair by the sea squirt-derived tripeptide FPP of the invention;
FIG. 22 is a graph showing the analysis of the tissue repair promoting action site Caspase3 of the sea squirt-derived tripeptide FPP of the present invention.
Detailed Description
The following description of the embodiments of the present invention will be made more apparent and fully hereinafter with reference to the accompanying drawings, in which some, but not all embodiments of the invention are shown. The following examples are illustrative only and are not intended to be any limitation on the invention and its application or use. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Referring to FIGS. 1-22, the present invention provides an ascidian-derived tripeptide comprising a mixture of one or more of the following amino acid sequence peptide fragments FPP, WPG, FMP.
The composition of amino acids in the nutrient content of the edible parts of sea squirts was analyzed and the analysis results are shown in table 1.
TABLE 1 amino acid composition of the edible parts of the sea squirt tissue
The amino acid analysis result shows that the edible part of the sea squirt has the largest content of acidic amino acids, in addition, the content of Leu, gly, tau is high, the content of imino acid Pro is also high, and the amino acids are closely related to the repair and regeneration of tissues.
The invention also provides a production process of the ascidian tripeptide, which comprises the step of carrying out enzymolysis on the ascidian by utilizing enzymes.
A process for preparing the tripeptide from sea squirt includes such steps as removing silt from fresh sea squirt (Ciona intestinalis), separating the inner bag from the bag by scissors, and washing. And (3) putting the sea squirt inner bag and ice cubes (the mass ratio is 1:2) together into a stirrer for stirring to form sea squirt meat emulsion. And fully homogenizing the sea squirt meat emulsion and deionized water according to the ratio of 1:3-1:5. Pouring the homogenized sample into a glass sleeve cup of an in-vitro gastrointestinal digestion reactor, putting the glass sleeve cup into a super-constant temperature water bath together, putting an electric stirrer into the in-vitro gastrointestinal digestion reactor for fixation, adding 2000U/g of compound protease when the temperature reaches 55 ℃ and the pH is regulated to 6.5-7.0, starting the electric stirrer for enzymolysis for 2.5 hours, regulating the temperature of the water bath to 100 ℃, and keeping for 10 minutes to fully inactivate the protease. And ultrafiltering the ecteinascidia enzymolysis liquid with a 5 μm filter membrane to obtain a crude filtrate, and filtering with a 1000 Da ceramic nanofiltration membrane to obtain ecteinascidia tripeptide.
The sea squirt source tripeptide production process is adopted, under the condition that other conditions are the same, in the enzymolysis step, firstly, different enzyme types are adopted to prepare sea squirt active peptides, which mainly comprise neutral protease, papain, flavourzyme and compound protease, the hydrolysis degree of the sea squirt by the four enzymes and the recovery rate of the sea squirt protein are compared, the effect of the compound protease on enzymolysis is best, and the compound protease is a special compound enzyme preparation aiming at animal proteolysis, and mainly comprises endoprotease, exoenzyme and flavourzyme.
After the optimal enzyme type-composite protease is determined, the optimal technological parameters in the enzymolysis step are determined by optimization, wherein the enzyme adding amount is 2000U/g, the enzymolysis temperature is 55 ℃ and the enzymolysis time is 2.5 hours.
And carrying out mass spectrum analysis on the composition and the content of the ecteinascidin obtained by enzymolysis of the compound protease, wherein the mass spectrum LC-MS/MS analysis result is shown in figure 3, 2556 active peptide fragments are obtained by total identification, about half of peptide fragments belong to tripeptides and tetrapeptides, and other peptide fragments are distributed among 5-10 peptides.
The obtained active peptide fragments are screened in a molecular butt joint mode to obtain three characteristic peptide fragments rich in proline, the three peptide fragments are found to have strong affinity with core proteins for repairing skin injury, and through mass spectrometry, the amino acid sequences of the three peptide fragments are FPP, WPG and FMP respectively, and specific mass spectrometry results are shown in figures 4, 5 and 6.
The application of the three sea squirt-derived tripeptides in skin tissue repair is demonstrated by the following experiments.
The three peptide fragments of FPP, WPG and FMP are chemically synthesized, and subjected to a zebra fish tail fin damage model experiment, and specifically zebra fish are divided into six groups, wherein the groups are as follows, namely a normal group (C group), a model group (M group), a Vc group (Vc group) with 50 mug/mL, an FPP group (FPP group) with 50 mug/mL, an FMP group (FMP group) with 50 mug/mL, a WPG group (WPG group) with 50 mug/mL, and 30 groups. After grouping, using zebra fish E3 culture solution water as a solvent, adding 3mL of the solution into each hole of a 6-hole plate, setting a normal group by using the solvent, performing zebra fish tail fin excision operation on M groups, vc groups, FPP groups, FMP groups and WPG groups respectively, specifically sucking a juvenile fish onto a glass slide by using a rubber head dropper, then, the surrounding water is removed by a pipette, a solution of tricaine with the concentration of 0.016% is dripped for anesthesia, after about 1min, anesthetic is sucked, and the glass slide is placed under a split microscope, and the razor blade is sterilized by 75% alcohol. After the sterilization was completed, a young fish tail fin removal operation was performed along the end of the spinal column under a 10-fold microscope, and a post-operation photograph was taken after the removal was completed. Finally, the glass slide is taken down, a drop of E3 water culture solution is sucked by a rubber head dropper for cleaning young fish, then the young fish is placed into a 6-hole plate with each group of solutions added in advance, the culture solution is replaced every day, specific experimental results are shown in fig. 7 and 8, and compared with a model group, the three peptide segments can promote the expansion of the regeneration area of the damaged part of the tail fin of the zebra fish, and particularly shown in fig. 11. At the same time, the recovery of the swimming speed, distance and function of the zebra fish tail fin can be promoted, and the zebra fish tail fin is particularly shown in fig. 9 and 10, and particularly tripeptide FPP has the strongest regeneration promoting activity.
The method is characterized in that the active peptide FPP with the strongest activity and different dosages is applied to a model of back skin excision of a mouse, and the contrast effect of a positive medicament pseudo-ginseng injury tablet is increased, wherein the specific experimental process comprises the steps that 20-25 g (8 weeks) of male C57BL/6 mice are subjected to adaptive feeding in a laboratory for 1 week and then are randomly divided into 4 groups (9 groups each) of model groups, pseudo-ginseng injury medicine groups, low-concentration FPP groups (LFPP) and high-concentration FPP groups (HFPP). The model group is filled with normal saline 0.1 mL/g each day, the pseudo-ginseng wound medicine group is filled with pseudo-ginseng wound medicine 0.36 mg/g each day, the LFPP group is filled with low-concentration FPP 0.18 mg/g each day, and the HFPP group is filled with high-concentration FPP 0.36 mg/g each day. Experiments were performed on day 0, day 3, day 7, day 12, 3 mice were randomly harvested from each group after a fasted 12 h to harvest blood from the eyeballs, and serum was isolated and stored at-80 ℃. The skin of the left back wound of the mouse is cut after being sampled by an 8mm punch and stored in a 2 mLEP tube at-80 ℃. The skin of the wound on the right back of the mouse is sampled by an 8mm puncher and then is fixed by 4% paraformaldehyde solution for pathological section. The specific experimental results are shown in fig. 12 and 13, and the results show that the high-dose FPP active peptide has remarkable repair and regeneration activity, and the repair function of the FPP active peptide is enhanced along with the increase of the concentration, so that the active peptide has obvious dose dependency.
In the above experiments, the secretion of pro-inflammatory factors IL-4, IL-6 and IL-10 in mice of different groups and the secretion of anti-inflammatory factors TNF-alpha, TGF-beta and IFN-gamma are detected simultaneously, and the specific results are shown in figures 14-19, wherein the active peptide FPP can reduce the secretion of pro-inflammatory factors IL-4, IL-6 and IL-10 in mice and increase the secretion of anti-inflammatory factors TNF-alpha, TGF-beta and IFN-gamma.
FPP was applied to damaged skin tissue, and proteomics and molecular docking results of skin tissue at different time phases were analyzed to confirm that the action sites of FPP for promoting tissue repair were ERK1, P38a and Caspase3, respectively. Hydrogen bonds and weak non-covalent forces are generated between the FPP active peptide and ARG-370, GLU-344 and ARG-94 of ERK 1. See in particular figures 20-22.
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