Ascidian-derived tripeptide, preparation method thereof, and application thereof in skin tissue repair

Abstract

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

[0001] The invention belongs to the field of biological peptides, and in particular relates to an ascidian-derived tripeptide, a preparation method thereof, and an application thereof in skin tissue repair. Background Art

[0002] As the largest organ in the human body, the skin not only protects the internal tissues of the body, but also participates in physiological activities such as regulating body temperature and sensing external stimuli. However, the skin is susceptible to various injuries in daily life, which may come from various factors such as external forces, chemicals, and ultraviolet rays. Once the skin tissue is damaged, the remaining part can repair itself to its original form and function, which is called regeneration. The advantage of this repair method is that the new tissue is autologous and the body function may be restored to the state before the damage. The tissues and organs in the damaged area will repair the damaged area through activities such as cell proliferation, differentiation, and migration, and eventually restore the tissues and organs to their original form and function. However, the regeneration and repair capabilities vary between different vertebrates, and between different tissues and organs in the same animal.

[0003] There are many factors that affect the repair of skin damage. In addition to the regenerative capacity of tissues and cells themselves, it is also closely related to the degree and scope of damage, age, endocrine, drug effects and nutritional status. Once the body tissue is damaged, the treatment environment and methods can be optimized by increasing nutritional supply and using drug intervention measures to promote tissue repair and regeneration.

[0004] Sea squirts are chordates, Ascidians, and are one of the most important tunicates. Sea squirts have strong regenerative abilities, but the specific active substances that play a role in regeneration are still unclear. Sea squirts contain a variety of biologically active substances, including active peptides. These active peptides have a variety of biological activities, such as anti-tumor, anti-viral, and antioxidant, and have broad application prospects in the fields of drug research and development and health care product development. With the deepening of research, scientists have a deeper understanding of the structure of sea squirt-derived active peptides. Most of these active peptides have special chemical structures and corresponding multiple biological functions. For example, some sea squirt-derived active peptides have significant anti-tumor activity and can inhibit the growth and division of tumor cells; while other active peptides have anti-viral and antioxidant functions.

[0005] In the development of skin repair peptide drugs, many studies have confirmed that active peptides can promote tissue repair. For example, some researchers isolated Tylotoin from lizards. This active peptide contains 12 amino acid residues and shows strong wound healing activity. At the same time, there are reports that peptides that can promote skin repair have been extracted from South China rain frogs, red warty newts, and fingerless disc frogs, which are 16 peptides, 13 peptides, 24 peptides, and 11 peptides, respectively. The active peptides discovered above are all long peptides, containing more amino acids, and are relatively slowly absorbed in the body. They may be affected by a variety of factors, such as gastrointestinal environment, enzyme activity, and induced immune response. Short peptides can be directly absorbed by the human body due to their simple structure. After being ingested into the body, they do not need to undergo a complex enzymatic hydrolysis process, so they can be quickly absorbed in tissue repair, quickly reach the damaged area, and exert biological activity.

[0006] In summary, sea squirts have a strong regenerative ability. When the body is damaged, it can quickly start the regeneration process to replace or restore damaged or missing cells, tissues, and even entire body parts. By studying the regeneration mechanism of sea squirt-derived active peptides, new ideas and methods can be provided for the fields of biology and medicine. Exploring the preparation methods of sea squirt peptides, the types and composition of peptides that promote skin tissue repair, and exploring the intrinsic biochemical mechanism of active peptides are the core of developing and applying new tissue repair active peptides. Summary of the invention

[0007] The invention provides an ascidian tripeptide, a preparation method thereof and application thereof in skin tissue repair. The ascidian tripeptide can significantly promote skin cell proliferation and differentiation, stimulate skin cell growth and accelerate the skin tissue repair process.

[0008] To achieve the above object, the present invention provides the following technical solution: a tripeptide derived from tunicate, wherein the tripeptide derived from tunicate comprises a mixture of one or more of the following amino acid sequence peptide segments: FPP, WPG, and FMP.

[0009] The present invention also provides a production process of the tripeptide derived from sea squirt, which comprises the step of enzymolyzing the sea squirt with an enzyme.

[0010] Preferably, the enzymatic hydrolysis step uses a composite protease.

[0011] Preferably, the amount of enzyme added in the enzymatic hydrolysis step is 2000 U / g.

[0012] Preferably, the enzymatic hydrolysis temperature in the enzymatic hydrolysis step is 55°C.

[0013] Preferably, the enzymatic hydrolysis time of the enzymatic hydrolysis step is 2.5 hours.

[0014] Preferably, the enzymatic hydrolyzate obtained in the enzymatic hydrolysis step is filtered through a 5 μm ultrafiltration membrane, and then filtered through a 1000 Da ceramic nanofiltration membrane to obtain the ascidian-derived tripeptide.

[0015] The invention also provides the use of the ascidian-derived tripeptide in skin tissue repair.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] 1. The sea squirt-derived active peptides disclosed in the present invention are a class of peptide substances with special physiological functions extracted from sea squirts. These peptide substances have the characteristics of low molecular weight, high activity, and easy absorption. They can directly act on the target proteins that regulate tissue regeneration, promote the proliferation and differentiation of cells in the damaged area, and thus accelerate tissue repair and regeneration;

[0018] 2. The three tripeptides disclosed in the present invention are all rich in proline, which can significantly promote the proliferation and differentiation of skin cells, reduce the secretion of pro-inflammatory factors in the body, increase the secretion of anti-inflammatory factors, and further accelerate the skin tissue repair process;

[0019] 3. The ascidian-derived tripeptide of the present invention is obtained by an enzymatic hydrolysis process, and the production process is simple to operate, does not require a high temperature and high pressure environment, consumes less energy, and has high production efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a comparison diagram of the hydrolysis degree of sea squirts hydrolyzed by different enzymes of the present invention;

[0021] Figure 2 This is a comparison chart of the recovery rates of ascidian proteins from ascidian hydrolyzed with different enzymes of the present invention;

[0022] Figure 3 This is a mass spectrometry analysis of the composition and content of the ascidian peptides obtained by enzymatic hydrolysis with the composite protease of the present invention;

[0023] Figure 4 This is the mass spectrum of the tripeptide FPP of the present invention;

[0024] Figure 5 This is the mass spectrum of the tripeptide WPG of the present invention;

[0025] Figure 6 This is the mass spectrum of the tripeptide FMP of the present invention;

[0026] Figure 7 This is a graph showing the effect of the tripeptide derived from sea squirt on the regeneration area of ​​the zebrafish tail fin;

[0027] Figure 8 HE section diagram showing the effect of the tripeptide from sea squirt on the regeneration of zebrafish tail fin;

[0028] Fig. 9This is a bar graph showing the effect of the tripeptide derived from sea squirt on the movement distance of the zebrafish tail fin;

[0029] Fig.10 This is a bar graph showing the effect of the tripeptide derived from sea squirt on the movement speed of the zebrafish tail fin;

[0030] Fig.11 This is a bar graph showing the effect of the tripeptide derived from sea squirt on the regeneration area of ​​the zebrafish tail fin;

[0031] Fig.12 This is an analysis diagram of the repair of mouse skin damage by the ascidian-derived tripeptide FPP of the present invention;

[0032] Fig.13 This is a graph showing the effect of the ascidian-derived tripeptide FPP of the present invention on the repair of the back wound area of ​​mice;

[0033] Fig.14 This is a graph showing the effect of the ascidian-derived tripeptide FPP of the present invention on the secretion of the pro-inflammatory factor IL-4 in mice;

[0034] Fig.15 This is a graph showing the effect of the ascidian-derived tripeptide FPP of the present invention on the secretion of the pro-inflammatory factor IL-6 in mice;

[0035] Fig.16 This is a graph showing the effect of the ascidian-derived tripeptide FPP of the present invention on the secretion of the pro-inflammatory factor IL-10 in mice;

[0036] Fig.17 This is a graph showing the effect of the sea squirt-derived tripeptide FPP of the present invention on the secretion of the anti-inflammatory factor TNF-α in mice;

[0037] Fig.18 This is a graph showing the effect of the sea squirt-derived tripeptide FPP of the present invention on the secretion of the anti-inflammatory factor TGF-β in mice;

[0038] Fig.19 This is a graph showing the effect of the sea squirt-derived tripeptide FPP of the present invention on the secretion of the anti-inflammatory factor IFN-γ in mice;

[0039] Fig. 20 This is an analysis diagram of the ERK1 site of action of the ascidian-derived tripeptide FPP in promoting tissue repair;

[0040] Fig.21 This is an analysis diagram of the P38a site of action of the ascidian-derived tripeptide FPP in promoting tissue repair;

[0041] Fig. 22 This is an analysis diagram of Caspase 3, the site of action of the ascidian-derived tripeptide FPP in promoting tissue repair. DETAILED DESCRIPTION

[0042] The technical scheme of the present invention will be clearly and completely described below in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. The description of the following embodiments is merely illustrative and does not serve as any limitation to the present invention and its application or use. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0043] See also Figure 1-22 The present invention provides a tripeptide derived from tunicate, which includes a mixture of one or more of the following amino acid sequence peptides: FPP, WPG, and FMP.

[0044] The amino acid composition content of the edible part of the tunicate was analyzed, and the analysis results are shown in Table 1.

[0045] Table 1 Amino acid composition of edible parts of ascidian tissues

[0046]

[0047] The results of amino acid analysis show that the edible part of the sea squirt has the highest content of acidic amino acids. In addition, the content of Leu, Gly, and Tau is very high, and the content of the imino acid Pro is also relatively high. These amino acids are closely related to the repair and regeneration of tissues.

[0048] The invention also provides a production process of the tripeptide derived from sea squirt, comprising the step of enzymolyzing the sea squirt with an enzyme.

[0049] The specific production process of the tripeptide from sea squirt includes: using ultrapure water to remove the mud and sand from the purchased fresh Ciona intestinalis, using scissors to separate the inner capsule and the tunicate of the sea squirt, and cleaning the impurities. Put the inner capsule of the sea squirt and ice cubes (mass ratio of 1:2) into a blender and crush them to form sea squirt mince. The sea squirt mince is fully homogenized with deionized water at a ratio of 1:3 - 1:5. Pour the homogenized sample into the glass cup of the in vitro gastrointestinal digestion reactor, put them into the ultra-constant temperature water bath, put the electric stirrer into the in vitro gastrointestinal digestion reactor and fix it. When the temperature reaches 55°C and the pH is adjusted to 6.5-7.0, add 2000 U / g of composite protease, start the electric stirrer for enzymatic hydrolysis for 2.5 hours, adjust the water bath temperature to 100°C, and keep it for 10 minutes to fully inactivate the protease. The ascidian hydrolysate was then ultrafiltered using a 5 μm filter membrane to obtain a coarse filtrate, which was then filtered through a 1000 Da ceramic nanofiltration membrane to obtain the ascidian-derived tripeptide.

[0050] The above sea squirt-derived tripeptide production process is adopted. Under the same other conditions, in the enzymatic hydrolysis step, different enzyme types are first used to prepare sea squirt active peptides, mainly including neutral protease, papain, flavor protease and composite protease. After comparing the hydrolysis degree of sea squirt hydrolyzed by the above four enzymes and the recovery rate of sea squirt protein, it is known that the composite protease has the best enzymatic hydrolysis effect. For details, see Figure 1 and Figure 2 Among them, the composite protease is a special composite enzyme preparation for the hydrolysis of animal protein, which is mainly composed of protein endo-enzyme, exo-enzyme and flavor enzyme.

[0051] After determining the optimal enzyme type - composite protease, through optimization, the optimal process parameters in the enzymatic hydrolysis step were determined to be: enzyme addition amount of 2000U / g, enzymatic hydrolysis temperature of 55℃, and enzymatic hydrolysis time of 2.5 hours.

[0052] The composition and content of the ascidian peptides obtained by the compound protease hydrolysis were analyzed by mass spectrometry. The results of mass spectrometry LC-MS / MS analysis are shown in Figure 3 A total of 2556 active peptides were identified, about half of which were tripeptides and tetrapeptides, and the others were distributed between 5-10 peptides.

[0053] The obtained active peptides were screened by molecular docking to obtain three characteristic proline-rich peptides. It was found that these three peptides had a strong affinity with the core protein of skin damage repair. After mass spectrometry analysis, the amino acid sequences of these three peptides were FPP, WPG and FMP, respectively. The specific mass spectrometry analysis results are shown in Figure 4 , Figure 5 , Figure 6 .

[0054] The application of the above three tripeptides from ascidian origin in skin tissue repair is specifically confirmed through the following experiments.

[0055] The three peptides, FPP, WPG and FMP, were chemically synthesized and used in the zebrafish caudal fin injury model experiment. Specifically, the zebrafish were divided into six groups as follows: normal group (Group C), model group (Group M), 50 μg / mL Vc group (Group Vc), 50 μg / mL FPP group (Group FPP), 50 μg / mL FMP group (Group FMP), and 50 μg / mL WPG group (Group WPG), with 30 fish in each group. After the grouping, zebrafish E3 culture medium water was used as the solvent, and 3 mL of the above solution was added to each well of the 6-well plate. The normal group was set up with the solvent, and the zebrafish tail fin removal surgery was performed on the M group, Vc group, FPP group, FMP group, and WPG group. Specifically, a young fish was sucked onto a glass slide with a rubber-tipped dropper, and then the surrounding water was removed with a pipette. A 0.016% tricaine solution was added for anesthesia. After about 1 minute, the anesthetic was removed, and the glass slide was placed under a stereo microscope. The razor blade was disinfected with 75% alcohol. After disinfection, the tail fin of the young fish was removed along the end of the spine under a 10x microscope. After the removal, postoperative photos were taken. Finally, the glass slide was removed, and a drop of E3 water culture medium was sucked with a rubber-tipped dropper to clean the young fish, and then it was placed in a 6-well plate with the solutions of each group added in advance. The culture medium was replaced regularly every day. The specific experimental results are shown in. Figure 7 and Figure 8 Compared with the model group, these three peptides can promote the expansion of the regeneration area of ​​the zebrafish tail fin injury site. Fig.11 . It will also promote the recovery of the swimming speed, distance and function of the zebrafish tail fin. Fig. 9 and Fig.10 , especially the tripeptide FPP, which has the strongest regeneration activity.

[0056] The most active peptide FPP at different doses was applied to the model of skin excision on the back of mice, and the positive drug Sanqi Shangyao tablets were added as a control. The specific experimental process was as follows: male C57BL / 6 mice 20-25g (8 weeks) were randomly divided into 4 groups (9 mice in each group) after 1 week of laboratory adaptation: model group, Sanqi Shangyao group, low-concentration FPP group (LFPP), and high-concentration FPP group (HFPP). The model group was gavaged with 0.1 mL / g of normal saline every day, the Sanqi Shangyao group was gavaged with 0.36 mg / g of Sanqi Shangyao drug every day, the LFPP group was gavaged with 0.18 mg / g of low-concentration FPP every day, and the HFPP group was gavaged with 0.36 mg / g of high-concentration FPP every day. The experiment was carried out on the 0th day, the 3rd day, the 7th day, and the 12th day. After fasting for 12 hours, 3 mice in each group were randomly selected to remove the eyeballs and collect blood. The serum was separated and stored at -80℃. The skin of the wound on the left side of the mouse back was sampled with an 8 mm punch and cut into pieces, stored in a 2 mL EP tube at -80°C. The skin of the wound on the right side of the mouse back was sampled with an 8 mm punch and fixed with 4% paraformaldehyde solution for pathological sections. Specific experimental results are shown in Fig.12 and Fig.13 The results showed that high doses of FPP active peptide had significant repair and regeneration activity, and its repair function was enhanced with increasing concentration, indicating that the active peptide had obvious dose dependence.

[0057] In the above experiments, the secretion of pro-inflammatory factors IL-4, IL-6, and IL-10, as well as the secretion of anti-inflammatory factors TNF-α, TGF-β, and IFN-γ in mice of different groups were simultaneously detected. The specific results are shown in Figure 14-19 As shown, 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-α, TGF-β and IFN-γ.

[0058] FPP was applied to damaged skin tissue, and the proteomics and molecular docking results of skin tissue at different time stages were analyzed, confirming that the action sites of FPP in promoting tissue repair were ERK1, P38a and Caspase3. FPP active peptides generated hydrogen bonds and weak non-covalent forces with ARG-370, GLU-344 and ARG-94 of ERK1. For details, see Figure 20-22 shown.

Claims

1. Use of a tripeptide derived from sea squirt with an amino acid sequence of one or more of a mixture of FPP, WPG, and FMP in the preparation of a skin tissue repair drug.

Images