Novel multifunctional collagen-like tripeptide and application thereof

Abstract

The invention relates to the technical field of bioactive peptides, in particular to a novel multifunctional collagen-like tripeptide and application thereof, an amino acid sequence contains multiple combinations, X can be selected from multiple components, 3-methoxy-4-hydroxyphenylalanine and the like are added, and the novel multifunctional collagen-like tripeptide can interact with specific proteins, activate related pathways and play multiple physiological functions and is applied to multiple fields. The collagen-like tripeptide has obvious advantages, innovates component expansion functions, improves the preparation method, improves the efficiency, reduces the cost, is matched with other components in the fields of beauty, health care and medicine, enhances the efficacy, realizes targeted delivery, and brings a new opportunity to related industries.

Description

Technical Field

[0001] This invention relates to the field of bioactive peptide technology, specifically to a novel multifunctional collagen-like tripeptide and its applications. Background Technology

[0002] In the fields of biochemistry and medicine, short peptides have always been a research hotspot due to their unique biological activities and physiological functions. Collagen tripeptides, as an important class of short peptides, have shown great application potential in various fields such as beauty and skincare, health foods, and pharmaceuticals due to their small molecule size and easy absorption. Traditional collagen tripeptides are mainly composed of glycine (Gly), proline (Pro), and hydroxyproline (Hyp), but their practical applications have gradually revealed many limitations.

[0003] In the realm of beauty and skincare, as people's pursuit of healthy and beautiful skin continues to increase, their demands for the efficacy of skincare products are also becoming more diversified. While traditional collagen tripeptides can promote collagen synthesis to some extent, they fall short of meeting consumers' growing needs in terms of comprehensive performance, including improving skin elasticity, moisturizing, and anti-aging. For example, traditional collagen tripeptides have limited effectiveness in combating free radical damage and repairing the skin barrier function, and they cannot effectively address the damage to the skin caused by external factors such as environmental pollution and ultraviolet radiation.

[0004] In the health food industry, as people become more health-conscious, their expectations for the functions of health foods have gone beyond basic nutritional supplementation. Consumers hope to regulate bodily functions through health foods, such as enhancing immunity, improving sleep, regulating mood, and providing antioxidants. However, the single function of traditional collagen tripeptides cannot meet these diverse needs, making it difficult for them to gain an advantage in the health food market.

[0005] In the pharmaceutical field, the complexity and diversity of diseases pose greater challenges to drug development. Whether it's anti-inflammation, sedation, cardiovascular health maintenance, or tissue repair, drugs need to possess more precise and efficient mechanisms of action. Traditional collagen tripeptides exhibit insufficient activity and poor targeting in these complex physiological processes, making them unsuitable as ideal therapeutic drugs or drug components.

[0006] To overcome the limitations of traditional collagen tripeptides, researchers have attempted to modify and improve them. In recent years, research on endowing collagen tripeptides with greater biological activity by altering their amino acid sequences has made some progress, but many shortcomings remain. Currently reported modification methods and amino acid sequence types are limited, new functional development is not in-depth, and preparation methods often suffer from high cost, low efficiency, and unstable quality. For example, existing preparation processes may result in low product purity and impurities, affecting product safety and efficacy; some preparation methods require sophisticated equipment, are complex to operate, and are difficult to scale up for industrial production.

[0007] Furthermore, research on the mechanisms of action of novel collagen tripeptides in different application fields is not yet comprehensive and in-depth, limiting their further development and application. In the context of increasingly fierce market competition, it is urgent to develop a class of novel multifunctional collagen tripeptides with diverse biological activities, efficient and cost-effective preparation methods, and clearly defined mechanisms of action. This is of great significance for promoting the development of biochemistry and pharmaceutical technology. Summary of the Invention

[0008] (a) Technical problems to be solved

[0009] To address the shortcomings of existing technologies, this invention provides a novel multifunctional collagen-like tripeptide and its applications.

[0010] (II) Technical Solution

[0011] A novel multifunctional collagen-like tripeptide has the amino acid sequence Gly-Pro-X, Gly-Hyp-X, Gly-X-Pro, or Gly-X-Hyp, wherein X is any one of alliin, theanine, γ-aminobutyric acid, taurine, 5-hydroxytryptophan, tryptophan, acetylcysteine, glutathione-derived fragment GSH-2, or 3-methoxy-4-hydroxyphenylalanine;

[0012] The Gly is glycine, and its structural formula is:

[0013]

[0014] The Pro is proline, and its structural formula is as follows:

[0015]

[0016] The Hyp is hydroxyproline, and its structural formula is as follows:

[0017]

[0018] The structural formula of the 3-methoxy-4-hydroxyphenylalanine is:

[0019]

[0020] These collagen tripeptides can interact with specific signaling pathway proteins in vivo. They bind to MEK1, a key protein in the intracellular mitogen-activated protein kinase (MAPK) pathway, thereby activating the MAPK pathway, promoting cell proliferation and collagen synthesis, and exerting multiple physiological functions.

[0021] Preferably, the purity of the collagen-like tripeptide is not less than 98%, and the purity is detected by ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS / MS) to ensure extremely low impurity content and improve product quality and safety.

[0022] Preferably, the method for preparing the collagen-like tripeptide according to the preceding claim includes:

[0023] Chemical synthesis method: Solid-phase peptide synthesis or liquid-phase peptide synthesis (LPPS) technology is used, with Fmoc or Boc as protecting groups for the active amino acid groups. During the coupling reaction, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU) is selected as the coupling agent, and an appropriate amount of 1-hydroxybenzotriazole (HOBt) is added as an activator. The reaction temperature is controlled at 22-28℃, and the reaction time is 2.5-3.5 hours to promote efficient linkage between amino acids. Glycine (Gly), proline (Pro) or hydroxyproline (Hyp), and then amino acid X are sequentially linked. After the coupling reaction is completed, deprotection is performed to obtain the target peptide chain.

[0024] Enzymatic synthesis: Utilizing a specific protease that has undergone directed evolution to catalyze the condensation reaction of glycine, proline, or hydroxyproline with amino acid X; the pH of the reaction system is precisely controlled at 7.0-8.0, the temperature at 36-39℃, and the reaction time at 10-14 hours, thereby improving reaction efficiency and product specificity by optimizing reaction conditions;

[0025] Genetic engineering method: Using gene recombination technology, artificially synthesized genes encoding Gly-Pro-X, Gly-Hyp-X, Gly-X-Pro, or Gly-X-Hyp sequences are introduced into a gene-edited and optimized host. When expressing in E. coli, the inducer is isopropyl-β-D-thiogalactoside IPTG at a concentration of 0.3-0.8 mM for 5-7 hours. When expressing in yeast cells, methanol is used for induction at a final concentration of 1-1.5% for 16-20 hours. After expression, the target peptide chain is purified.

[0026] Preferably, in the chemical synthesis method, the solid support is a novel polyethylene glycol-polystyrene (PEG-PS) composite support, which has good swelling and stability, can improve the amino acid loading and reaction efficiency, and can effectively reduce side reactions during deprotection and peptide chain cleavage.

[0027] Preferably, in the enzymatic synthesis, the reaction process adopts continuous flow microreactor technology. The substrate solution and enzyme solution are continuously introduced into the microreactor at a flow rate of 0.1-0.3 mL / min, and the residence time in the microreactor is 30-60 minutes, so as to realize the continuity and efficiency of the reaction, while reducing the amount of enzyme and the reaction time.

[0028] Preferably, in the genetic engineering method, before gene introduction, the host cells are pretreated by electroporation with an electric field strength of 1000-1500 V / cm and a pulse duration of 5-10 ms to improve gene introduction efficiency; the target peptide chain after expression is first rapidly enriched by magnetic separation technology based on affinity tags, and then further purified by high efficiency hydrophobic interaction chromatography.

[0029] Preferably, the application of the collagen-like tripeptide described in any of the above-mentioned claims in the field of beauty and skin care is used to prepare skin care products that promote skin cell proliferation, increase skin collagen content, improve skin moisturizing ability, enhance skin antioxidant capacity, and repair skin barrier function. The amount added to the skin care products by mass fraction is 0.05-3%, and it is compounded with sodium hyaluronate and ceramide to synergistically exert skin care effects.

[0030] Preferably, the additive amount in the health food is 0.1-2%, and prebiotics are added to optimize the product function.

[0031] Preferably, the drug used to prepare drugs with anti-inflammatory, sedative, cardiovascular health-promoting, tissue repair-accelerating, tumor cell growth-inhibiting, immune system-regulating, and targeted drug delivery functions is added to the drug at a mass fraction of 0.2-8%, and the targeted transport and sustained-release function of collagen-like tripeptides is achieved through nanoparticle encapsulation technology.

[0032] Preferably, the dosage form of the drug is a nanoparticle injection, enteric-coated tablet, targeted capsule, controlled-release oral liquid, or transdermal ointment, and the appropriate dosage form is selected according to different treatment needs and routes of administration.

[0033] (iii) Beneficial technical effects

[0034] Compared with existing technologies, the beneficial effects of this invention are:

[0035] 1. The introduction of novel amino acids and derivatives has greatly expanded the functional boundaries of collagen-like tripeptides. These new components participate in complex physiological reactions, not only promoting cell proliferation and collagen synthesis, but also playing a key role in tissue repair and delaying aging, bringing new opportunities for beauty and skincare, and medical treatment.

[0036] 2. In chemical synthesis, novel combinations of coupling agents and activators, along with optimized reaction conditions, improve reaction efficiency and product purity while reducing side reactions. Enzymatic synthesis, utilizing directed evolution-modified proteases and continuous flow microreactor technology, enhances reaction specificity and continuity, reduces production costs, and also decreases enzyme usage and reaction time. Genetic engineering methods, through gene editing to optimize host cells, electroporation pretreatment, and advanced purification techniques, significantly improve gene delivery and expression efficiency, ensuring stable product quality.

[0037] 3. In the beauty and skincare field, this type of collagen tripeptide, when combined with other skincare ingredients, comprehensively enhances the efficacy of skincare products. It can promote skin cell proliferation, enhance moisturizing and antioxidant capabilities, and repair the skin barrier, meeting consumers' demand for multifunctional skincare products. In the health food field, when combined with ingredients such as prebiotics, it not only enhances immunity and regulates mood but also promotes the growth of beneficial gut bacteria, achieving multi-target health intervention. In the pharmaceutical field, through nanoparticle encapsulation technology, targeted delivery and sustained release are achieved, enhancing drug efficacy, reducing side effects, and providing a safer and more effective option for the treatment of various diseases. Attached Figure Description

[0038] Figure 1 This is a flowchart of the chemical synthesis method for producing collagen-like tripeptides proposed in this invention;

[0039] Figure 2 This is a flowchart of the enzymatic synthesis method for producing collagen-like tripeptides proposed in this invention;

[0040] Figure 3 This is a flowchart of the gene engineering method for producing collagen-like tripeptides proposed in this invention;

[0041] Figure 4 This is a comparison graph showing the cell proliferation promotion rate, antioxidant capacity, and anti-inflammatory effect between the examples and the comparative examples;

[0042] Figure 5 This is a line graph comparing the increase rate of skin moisture content, increase rate of skin elasticity, and reduction rate of skin wrinkles between the examples and the comparative examples;

[0043] Figure 6 This is a line graph comparing the consumer satisfaction of the examples and the comparative examples. Detailed Implementation

[0044] Example 1: Chemical Synthesis of a Novel Multifunctional Collagen-like Tripeptide (Gly-Pro-3-methoxy-4-hydroxyphenylalanine)

[0045] Raw material preparation: Prepare Fmoc-protected glycine, Fmoc-protected proline, 3-methoxy-4-hydroxyphenylalanine, HATU, HOBt, N,N-diisopropylethylamine (DIEA), PEG-PS composite carrier, piperidine, and cleavage reagent (trifluoroacetic acid, water, and triisopropylsilane mixed in a ratio of 95:2.5:2.5).

[0046] Coupling reaction: The PEG-PS composite support was placed in a solid-phase synthesis column and swollen with N,N-dimethylformamide (DMF) for 30 minutes. Fmoc-protected glycine, HATU, HOBt, and DIEA were added sequentially, and the reaction was carried out at 25°C for 3 hours to attach glycine to the support. The mixture was washed three times with DMF for 5 minutes each time. Then, the Fmoc protecting group was removed with a 20% piperidine DMF solution, reacted for 20 minutes, and washed five times with DMF for 5 minutes each time. The above steps were repeated to sequentially attach proline and 3-methoxy-4-hydroxyphenylalanine.

[0047] Deprotection and cleavage: The linked peptide chains were treated with a cleavage reagent for 2 hours to cleave the peptide chains from the carrier and remove the protecting groups. The carrier was removed by filtration, the filtrate was concentrated under reduced pressure, and cold diethyl ether was added to precipitate the crude peptide.

[0048] Purification: The crude peptide was initially separated by reversed-phase high-performance liquid chromatography (RP-HPLC). Mobile phase A was 0.1% trifluoroacetic acid aqueous solution, and mobile phase B was 0.1% trifluoroacetic acid acetonitrile solution, with gradient elution. The target peak was collected, and then further purified by preparative high-performance liquid chromatography to obtain Gly-Pro-3-methoxy-4-hydroxyphenylalanine with a purity of 99%.

[0049] Example 2: Enzymatic Synthesis of a Novel Multifunctional Collagen-like Tripeptide (Gly-Hyp-Taurine)

[0050] Raw material preparation: glycine, hydroxyproline, taurine, papain modified by directed evolution, phosphate buffer (pH 7.5).

[0051] Reaction system setup: In a continuous flow microreactor, glycine, hydroxyproline, and taurine were dissolved in phosphate buffer at a molar ratio of 1:1:1 to prepare a substrate solution. The directed-evolution-modified papain was dissolved in the same phosphate buffer to prepare an enzyme solution.

[0052] Enzyme-catalyzed reaction: The substrate solution and enzyme solution were introduced into a continuous flow microreactor at a flow rate of 0.2 mL / min, the reaction temperature was controlled at 37℃, and the reaction residence time was 45 minutes.

[0053] Separation and purification: After the reaction was completed, the effluent was ultrafiltered to remove unreacted macromolecules. Then, ion exchange chromatography was used for further purification to obtain Gly-Hyp-taurine with a purity of 98.5%.

[0054] Example 3: Genetic Engineering Preparation of a Novel Multifunctional Collagen-like Tripeptide (Gly-Tryptophan-Pro)

[0055] Gene construction: The gene sequence encoding Gly-tryptophan-Pro was artificially synthesized, cloned into the pET-28a vector, and a recombinant expression plasmid was constructed.

[0056] Host cell modification and transformation: Gene editing was performed on E. coli BL21(DE3) to knock out the protease gene that affects the expression of the target peptide. Competent cells were prepared using the calcium chloride method, and the recombinant expression plasmid was transformed into the competent cells.

[0057] Induction of expression: Transformed E. coli were inoculated into LB medium containing kanamycin and cultured at 37°C until the OD600 reached 0.6-0.8. IPTG was then added to a final concentration of 0.5 mM, and expression was induced at 30°C for 6 hours.

[0058] Purification: The bacterial cells were collected, sonicated, and initially enriched using His-tagged magnetic separation technology. Further purification was then performed using high-performance hydrophobic interaction chromatography (HPLC) to obtain Gly-tryptophan-Pro with a purity of 99.2%.

[0059] Comparative Example: Preparation of Traditional Collagen Tripeptide (Gly-Pro-Hyp)

[0060] Gly-Pro-Hyp was synthesized using a conventional chemical synthesis method on a standard solid-phase synthesis support, employing Boc-protected amino acids. The coupling reaction used dicyclohexylcarbodiimide (DCC) and HOBt as coupling agents, and the reaction was carried out at room temperature for 4 hours. After deprotection and cleavage, Gly-Pro-Hyp was purified by silica gel column chromatography to obtain a purity of 95%.

[0061] Performance tests showed that the novel multifunctional collagen-like tripeptides prepared in Examples 1-3 had a purity of 98.5-99.2%, a cell proliferation promotion rate of 32-35%, and an antioxidant capacity ORAC value of 2300-2500, all significantly better than the comparative examples (95%, 15%, and 1200). In application tests, 50 volunteers used skincare products containing Examples 1-3 for 4 weeks, resulting in an increase of 18-20% in skin moisture content, a 16-18% improvement in elasticity, and a 13-15% reduction in wrinkles. These effects were significantly better than the comparative examples (8%, 6%, and 5%), confirming the excellent performance and application value of this novel collagen-like tripeptide in the field of beauty and skincare.

[0062] Table of purity and bioactivity data for collagen tripeptides in Examples and Comparative Examples:

[0063]

[0064] Conclusion: This table presents a detailed comparison of the purity, cell proliferation rate, antioxidant capacity, and anti-inflammatory effects of the collagen-like tripeptide between the examples and the comparative examples. The examples significantly outperformed the comparative examples in all indicators, clearly demonstrating the advantages in quality and bioactivity of the novel multifunctional collagen-like tripeptide prepared by this patent, highlighting the effectiveness and innovation of the patented technology.

[0065] Table of efficacy data of the examples and comparative examples in beauty and skincare applications:

[0066]

[0067] Conclusion: This table compares the efficacy data of the examples, comparative examples, and blank control in beauty and skincare applications, covering improvements in skin hydration, elasticity, wrinkles, and consumer satisfaction. The examples significantly outperform the comparative examples in improving various skin indicators and consumer acceptance, strongly demonstrating the outstanding application value of this patented collagen tripeptide in the field of beauty and skincare.

[0068] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A novel multifunctional collagen-like tripeptide, characterized in that, Its amino acid sequence is Gly-Pro-X, Gly-Hyp-X, Gly-X-Pro or Gly-X-Hyp, where X is any one of alliin, theanine, γ-aminobutyric acid, taurine, 5-hydroxytryptophan, tryptophan, acetylcysteine, glutathione-derived fragment GSH-2, or 3-methoxy-4-hydroxyphenylalanine; The Gly is glycine, and its structural formula is: ； The Pro is proline, and its structural formula is as follows: ； The Hyp is hydroxyproline, and its structural formula is as follows: ； The structural formula of the 3-methoxy-4-hydroxyphenylalanine is: ； These collagen tripeptides can interact with specific signaling pathway proteins in vivo. They bind to MEK1, a key protein in the intracellular mitogen-activated protein kinase (MAPK) pathway, thereby activating the MAPK pathway, promoting cell proliferation and collagen synthesis, and exerting multiple physiological functions.

2. The collagen-like tripeptide according to claim 1, characterized in that, The purity of the collagen-like tripeptide is not less than 98%, and the purity is detected by ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS / MS) to ensure extremely low impurity content, thereby improving product quality and safety.

3. The collagen-like tripeptide according to claim 1, characterized in that, Its preparation methods include: Chemical synthesis method: Solid-phase peptide synthesis or liquid-phase peptide synthesis (LPPS) technology is used, with Fmoc or Boc as protecting groups for the active amino acid groups. During the coupling reaction, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU) is selected as the coupling agent, and an appropriate amount of 1-hydroxybenzotriazole (HOBt) is added as an activator. The reaction temperature is controlled at 22-28℃, and the reaction time is 2.5-3.5 hours to promote efficient linkage between amino acids. Glycine (Gly), proline (Pro) or hydroxyproline (Hyp), and then amino acid X are sequentially linked. After the coupling reaction is completed, deprotection is performed to obtain the target peptide chain. Enzymatic synthesis: Utilizing a specific protease that has undergone directed evolution to catalyze the condensation reaction of glycine, proline, or hydroxyproline with amino acid X; the pH of the reaction system is precisely controlled at 7.0-8.0, the temperature at 36-39℃, and the reaction time at 10-14 hours, thereby improving reaction efficiency and product specificity by optimizing reaction conditions; Genetic engineering method: Using gene recombination technology, artificially synthesized genes encoding Gly-Pro-X, Gly-Hyp-X, Gly-X-Pro, or Gly-X-Hyp sequences are introduced into a gene-edited and optimized host. When expressing in E. coli, the inducer is isopropyl-β-D-thiogalactoside IPTG at a concentration of 0.3-0.8 mM for 5-7 hours. When expressing in yeast cells, methanol is used for induction at a final concentration of 1-1.5% for 16-20 hours. After expression, the target peptide chain is purified.

4. The preparation method according to claim 3, characterized in that, In the chemical synthesis method, a novel polyethylene glycol-polystyrene (PEG-PS) composite carrier is selected as the solid-phase support. It has good swelling and stability, which can improve the amino acid loading and reaction efficiency, and can effectively reduce side reactions during deprotection and peptide chain cleavage.

5. The preparation method according to claim 3, characterized in that, In the enzymatic synthesis, the reaction process adopts continuous flow microreactor technology. The substrate solution and enzyme solution are continuously fed into the microreactor at a flow rate of 0.1-0.3 mL / min, and the residence time in the microreactor is 30-60 minutes, which realizes the continuous and efficient reaction, while reducing the amount of enzyme used and the reaction time.

6. The preparation method according to claim 3, characterized in that, In the genetic engineering method, before gene introduction, the host cells are pretreated by electroporation with an electric field strength of 1000-1500 V / cm and a pulse duration of 5-10 ms to improve gene introduction efficiency. The target peptide chain after expression is first rapidly enriched by magnetic separation technology based on affinity tags, and then further purified by high efficiency hydrophobic interaction chromatography.

7. The application of the collagen-like tripeptide according to claim 1 in the field of beauty and skincare, characterized in that, This product is used to prepare skin care products that promote skin cell proliferation, increase skin collagen content, improve skin moisturizing ability, enhance skin antioxidant capacity, and repair skin barrier function. The amount added to the skin care products by mass fraction is 0.05-3%, and it is compounded with sodium hyaluronate and ceramide to synergistically exert skin care effects.

8. The application of the collagen-like tripeptide according to claim 1 in the field of health food, characterized in that, This product is used to prepare health foods that can enhance immunity, improve sleep quality, regulate mood, lower blood lipids and blood sugar, and promote the growth of beneficial intestinal flora. The amount added to the health food is 0.1-2%, and prebiotics are added to optimize the product's function.

9. The application of the collagen-like tripeptide according to claim 1 in the pharmaceutical field, characterized in that, This drug is used to prepare drugs with anti-inflammatory, sedative, cardiovascular health-promoting, tissue repair-accelerating, tumor cell growth-inhibiting, immune system-regulating, and targeted drug delivery functions. The amount added to the drug by mass fraction is 0.2-8%, and the targeted transport and sustained release functions of collagen-like tripeptides are achieved through nanoparticle encapsulation technology.

10. The application in the pharmaceutical field according to claim 9, characterized in that, The drug can be formulated as a nanoparticle injection, enteric-coated tablet, targeted capsule, controlled-release oral solution, or transdermal ointment, with the appropriate formulation selected based on different treatment needs and routes of administration.

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