Functional tripeptide containing L-cysteine and application thereof

Abstract

The invention relates to the technical field of biological active peptide design and synthetic chemistry, in particular to functional tripeptide containing L-cysteine and application of the functional tripeptide, the sequence is L-cysteine-X-Y, X is a variety of conventional and novel amino acid derivatives which can be selected, and Y also comprises special modified amino acid which can be chelated with metal ions to remove reactive oxygen species, so that the functional tripeptide containing the L-cysteine can be used for preparing the functional tripeptide containing the L-cysteine and the application of the functional tripeptide containing the L-cysteine. The compound can activate a cell antioxidant system, can be prepared through chemical synthesis, an enzyme method and a genetic engineering technology, and can be applied to multiple fields. The functional tripeptide has outstanding advantages, various functions are endowed by the innovative components, the quality is guaranteed by the innovative preparation method, the functional tripeptide can be matched with various components in the fields of beauty and skin care, health food and medicine, physiological functions are adjusted, product effects are improved, and new opportunities are brought to related industries.

Description

Technical Field

[0001] This invention relates to the field of bioactive peptide design and synthetic chemistry, specifically to a class of functional tripeptides containing L-cysteine ​​and their applications. Background Technology

[0002] Short peptides have always been a research hotspot due to their unique biological activities and potential applications. In recent years, functional tripeptides containing specific amino acids have received widespread attention, demonstrating enormous application potential in numerous fields. However, this field still faces many challenges and problems.

[0003] As people place increasing importance on skin health and appearance, their demands for skincare product efficacy are becoming more diversified. Traditional skincare products mainly focus on basic moisturizing and cleansing functions, making it difficult to meet consumers' higher-level needs such as anti-aging, skin barrier repair, and regulation of the skin's microbiome. Although some skincare products containing short peptides have appeared on the market, the functions of these short peptides are relatively singular, failing to fully realize their multiple potential benefits in skincare. For example, existing peptide-containing skincare products often only address certain types of free radicals in terms of anti-oxidation, with limited ability to regulate complex intracellular oxidative stress responses; their effects on promoting collagen synthesis are also unsatisfactory, failing to fundamentally improve skin elasticity and firmness. Furthermore, the balance of the skin's microbiome is crucial for skin health, but most skincare products lack effective means of regulating this aspect.

[0004] As people become more health-conscious, their expectations for the functions of health supplements are also increasing. Beyond basic nutritional supplementation, consumers hope that health supplements can regulate bodily functions and prevent and improve various chronic diseases. While there is a wide variety of health supplements available, they often lack precision and effectiveness in their functions. Some products claiming to enhance immunity have unclear mechanisms of action and unstable effects; in improving sleep and regulating gut microbiota, existing health supplements often require long-term, high-dose use to see limited effects and may have side effects. Meanwhile, the research and application of health supplements with specific functions such as assisting in lowering blood lipids are still in the exploratory stage, lacking safe and effective products.

[0005] Despite significant advancements in modern medicine, many challenges remain in treating diseases. Inflammatory diseases, bacterial infections, and neurological disorders pose serious threats to human health. Existing drugs have numerous limitations in treating these diseases. For example, the widespread use of antibiotics has led to increasingly serious bacterial resistance, making the treatment of bacterial infections more difficult; anti-inflammatory drugs often produce varying degrees of side effects, impacting patients' quality of life; and for neurological diseases, current treatments mostly only alleviate symptoms and cannot cure the underlying disease. Furthermore, targeted drug delivery has always been a key research focus in the pharmaceutical field, and existing targeted technologies still need improvement in terms of precision and effectiveness, leading to unnecessary damage to normal tissues during treatment and reducing therapeutic efficacy.

[0006] Existing chemical synthesis, enzymatic synthesis, and genetic engineering technologies each have their own limitations. While chemical synthesis allows for precise control of peptide chain sequence, it suffers from demanding reaction conditions, numerous side reactions, and requires improvement in product purity and yield. Furthermore, its high production cost hinders large-scale production. Enzymatic synthesis offers advantages such as mild reaction conditions and high specificity, but enzyme stability is poor, easily affected by the reaction environment, leading to unstable reaction efficiency. Additionally, enzyme preparation and purification costs are high. Genetic engineering technology shows promise for large-scale production, but issues such as protein misfolding and inclusion body formation during expression increase the difficulty and cost of subsequent purification.

[0007] Developing a class of functional tripeptides containing L-cysteine ​​that possess multiple biological activities, can meet the needs of different fields, and have efficient and cost-controllable preparation methods is of great practical significance. This will bring new opportunities and breakthroughs to the development of fields such as beauty and skin care, health food, and medicine. Summary of the Invention

[0008] (a) Technical problems to be solved

[0009] To address the shortcomings of existing technologies, this invention provides a class of functional tripeptides containing L-cysteine ​​and their applications.

[0010] (II) Technical Solution

[0011] A class of functional tripeptides containing L-cysteine, with the amino acid sequence L-cysteine-XY, wherein X is any one of arginine, lysine, histidine, and N-acetyl-lysine-methyl ester, and Y is any one of tryptophan, tyrosine, phenylalanine, and 5-methoxytryptophan; the structure of L-cysteine-N-acetyl-lysine-methyl ester-5-methoxytryptophan is as follows:

[0012]

[0013] The structure of the N-acetyl-lysine-methyl ester is as follows:

[0014]

[0015] This tripeptide can react with superoxide anions in reactive oxygen species, exhibiting highly efficient antioxidant properties and protecting cells from oxidative stress damage; the tripeptide can also specifically bind to specific receptors on the cell surface.

[0016] Preferably, the purity of the functional tripeptide is not less than 98%, and the purity is detected by ultra-high performance liquid chromatography-mass spectrometry to ensure that the impurity content is extremely low, thus guaranteeing product quality and safety.

[0017] Preferably, the method for preparing the functional tripeptide includes:

[0018] Chemical synthesis method: Solid-phase peptide synthesis technology was employed, using Fmoc as the protecting group for the active amino acid groups. During the amino acid linkage process, in addition to using 2-7-azabenzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate as a coupling agent, 1-hydroxybenzotriazole was added as an activator. The reaction was carried out at 22-28℃ for 2.5-3.5 hours to promote efficient amino acid linkage. A novel solid-phase support was selected to improve the amino acid loading and reaction efficiency, and reduce side reactions. After linkage, the Fmoc protecting group was removed using a 22% piperidine N,N-dimethylformamide solution. Finally, the peptide chain was cleaved from the solid-phase support using an optimized cleavage reagent to obtain the crude product. The crude product was initially separated by reversed-phase high-performance liquid chromatography (RP-HPLC) and then finely purified by preparative ultra-high-performance liquid chromatography (UHPLC) to obtain the target functional tripeptide.

[0019] Enzymatic synthesis: A specific protease modified through directed evolution catalyzes the condensation reaction of L-cysteine, amino acid X, and amino acid Y. 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. The reaction process employs continuous flow microreactor technology, with the substrate solution and enzyme solution continuously fed into the microreactor at a flow rate of 0.1-0.3 mL / min. The residence time in the microreactor is 30-60 minutes, achieving continuous and efficient reaction while reducing enzyme dosage and reaction time. After the reaction, unreacted macromolecules are removed by ultrafiltration, followed by purification using ion exchange chromatography and hydrophobic interaction chromatography to obtain the target functional tripeptide.

[0020] Preferably, the application of the functional tripeptide containing L-cysteine ​​according to any one of the above claims in the field of beauty and skincare is used to prepare skincare products with antioxidant properties, promote collagen synthesis, repair skin barrier function, and regulate the balance of skin microbiota; the amount added to the skincare product is 0.05-3%, and it is compounded with ceramides and plant extracts to synergistically enhance the skincare effect; by adding prebiotics, the microbial environment on the skin surface after the skincare product is applied is regulated, promoting the growth of beneficial bacteria and inhibiting the reproduction of harmful bacteria.

[0021] Preferably, a type of functional tripeptide containing L-cysteine ​​is used in the field of health food to prepare health food that enhances immunity, improves sleep quality, regulates intestinal flora, and can help lower blood lipids; the amount added to health food is 0.1-2%, and it is also combined with probiotics, dietary fiber and other ingredients to optimize product function; by regulating the structure of intestinal microbial community, it affects bile acid metabolism, reduces cholesterol absorption, and achieves the effect of helping to lower blood lipids.

[0022] Preferably, a class of functional tripeptides containing L-cysteine ​​are used in the pharmaceutical field to prepare drugs with anti-inflammatory, antibacterial, wound-healing, and neurological disease-treating properties, and can also serve as targeted drug carriers. The amount added to the drug is 0.2-8%, and it can be formulated into various dosage forms such as nanoparticle injections, enteric-coated tablets, and targeted capsules. When preparing nanoparticle injections, liposome or polymer nanoparticle encapsulation technology is used to co-encapsulate the functional tripeptide with the active pharmaceutical ingredient, achieving targeted delivery and sustained-release functions. When preparing enteric-coated tablets, enteric coating materials are used to ensure drug release within the intestines. When preparing targeted capsules, specific targeting molecules are modified on the capsule surface, enabling the drug to precisely act on the lesion site.

[0023] Preferably, the dosage form of the skincare product is a lotion, cream, serum, mask, or gel. When preparing a lotion, high-speed homogenization emulsification technology is used, controlling the emulsification temperature at 60-70℃ and the emulsification time at 20-30 minutes to ensure that the functional tripeptides are evenly dispersed in the lotion system. When preparing a cream, an appropriate amount of thickener is added to adjust the texture and stability of the cream. When preparing a serum, microfiltration and ultrafiltration technologies are used to remove impurities and improve product purity. When preparing a mask, a skin-friendly mask base fabric is selected to ensure that the functional tripeptides can be effectively delivered to the skin surface. When preparing a gel, carbomer is used as the gel matrix, and an appropriate amount of neutralizing agent is added to adjust the pH value, making the gel texture uniform and delicate.

[0024] Preferably, the dosage form of the health food is tablets, capsules, oral liquid, or granules; when preparing tablets, a wet granulation process is used to control the moisture content of the granules at 3-5%, and an appropriate amount of lubricant is added to improve the tablet's formability and flowability; when preparing capsules, a suitable capsule shell is selected, and the filling process is adjusted according to the properties of the functional tripeptide; when preparing oral liquid, an appropriate amount of sweetener and preservative is added to improve the taste and extend the shelf life; when preparing granules, a spray drying granulation technology is used to control the inlet air temperature at 120-140℃ and the outlet air temperature at 70-90℃ to ensure uniform granules and good solubility.

[0025] Preferably, when the drug is formulated into nanoparticle injections, the particle size of the nanoparticles is controlled within 50-200 nm, and the particle size is detected by a dynamic light scattering instrument; when formulated into enteric-coated tablets, the enteric coating thickness is controlled within 20-50 μm, and the coating thickness is observed by a microscope; when formulated into targeted capsules, the amount of modification of the targeting molecule is 0.1-1 μg per milligram capsule, and the amount is detected by enzyme-linked immunosorbent assay (ELISA).

[0026] Preferably, it can also be prepared using genetic engineering technology; firstly, a gene encoding the L-cysteine-XY sequence is artificially synthesized, and the host cell is genetically edited and optimized to knock out the protease gene affecting the expression of the target tripeptide, overexpress the chaperone protein, and improve the expression level and folding efficiency of the tripeptide; after the gene is introduced into the host cell, isopropyl-β-D-thiogalactoside is used as an inducer in E. coli at a concentration of 0.3-0.8 mM for 5-7 hours; methanol is used as an inducer in yeast cells at a final concentration of 1-1.5% for 16-20 hours; the expressed tripeptide is rapidly pre-enriched using affinity tag-based magnetic separation technology, and then further purified using high-performance hydrophobic interaction chromatography and size exclusion chromatography to obtain a high-purity target functional tripeptide.

[0027] (iii) Beneficial technical effects

[0028] Compared with existing technologies, the beneficial effects of this invention are:

[0029] 1. The introduction of novel amino acid derivatives and specially modified amino acids greatly expands the functional boundaries of functional tripeptides; it precisely regulates the concentration of intracellular iron ions and maintains normal cell metabolism; its highly efficient antioxidant capacity can effectively scavenge reactive oxygen species such as superoxide anions, reduce oxidative stress damage, protect cells from free radical damage, and prevent and improve various problems caused by oxidative stress from the root.

[0030] 2. Innovations in preparation methods also bring numerous advantages. In chemical synthesis, novel solid-phase supports and optimized reaction conditions improve amino acid loading and reaction efficiency, reduce side reactions, and increase product purity. Enzymatic synthesis, utilizing directed evolution-modified proteases and continuous flow microreactor technology, enhances reaction specificity and continuity, and reduces production costs. Genetic engineering technology, through gene editing to optimize host cells, increases the expression level and folding efficiency of tripeptides. These innovative preparation methods ensure stable product quality and make large-scale production possible.

[0031] 3. In the beauty and skincare field, this functional tripeptide, when combined with various ingredients, not only provides antioxidant benefits and promotes collagen synthesis but also regulates the balance of the skin's microbiome. Working synergistically with ceramides and plant extracts, it enhances the skin barrier function, improving skin elasticity and radiance. The addition of prebiotics optimizes the skin's microecological environment, reducing the growth of harmful bacteria and promoting healthier skin. In the health food field, in conjunction with probiotics and dietary fiber, it comprehensively regulates bodily functions. While enhancing immunity, improving sleep quality, and regulating gut microbiota, it can also help lower blood lipids, meeting consumers' diverse health needs. In the pharmaceutical field, as a targeted drug carrier, it combines with various dosage forms such as nanoparticle injections, enteric-coated tablets, and targeted capsules to achieve precise drug delivery and sustained release, improving drug efficacy and reducing damage to normal tissues. Its anti-inflammatory, antibacterial, wound-healing, and neurological disease-treating functions provide new options for the treatment of various diseases. Attached Figure Description

[0032] Figure 1 This is a flowchart of the chemical synthesis of a class of functional tripeptides containing L-cysteine;

[0033] Figure 2 This is a flowchart of an enzyme synthesis method for a class of functional tripeptides containing L-cysteine.

[0034] Figure 3 This is a flowchart of a genetic engineering synthesis method for a class of functional tripeptides containing L-cysteine;

[0035] Figure 4 This is a bar chart showing the application effects of the tripeptides prepared in the examples and comparative examples in the field of beauty and skincare.

[0036] Figure 5 This is a bar graph showing the application effects of the tripeptides prepared in the examples and comparative examples in the field of beauty and skincare.

[0037] Figure 6 It is the NMR spectrum of L-cysteine-N-acetyl-lysine-methyl ester-5-methoxytryptophan. Detailed Implementation

[0038] according to Figures 1 to 6 The specific embodiments of the present invention are as follows:

[0039] Example 1: Preparation and application of L-cysteine-arginine-tryptophan functional tripeptide by chemical synthesis

[0040] Preparation of functional tripeptides

[0041] Carrier preparation: Weigh 1g of polyethylene glycol-polystyrene-nano silica composite carrier and place it in a reaction vessel. Add 20mL of N,N-dimethylformamide (DMF) and allow it to swell at room temperature for 30 minutes, stirring gently during the process to ensure that the carrier swells fully.

[0042] First amino acid ligation: The swollen carrier was filtered to remove DMF. 2 mmol Fmoc-L-cysteine, 2 mmol 2,7-azabenzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU), 2 mmol 1-hydroxybenzotriazole (HOBt), and 4 mmol N,N-diisopropylethylamine (DIEA) were added sequentially, and the reaction was carried out at 25°C for 3 hours. The reaction was continuously stirred to ensure sufficient contact of the reactants. After the reaction, the carrier was washed five times with DMF for 5 minutes each time to remove unreacted reagents.

[0043] Deprotection: Add 20 mL of 22% piperidine DMF solution to the reaction vessel and react at room temperature for 15 minutes to remove the Fmoc protecting group. Then wash the support with DMF 5 times for 5 minutes each time to remove piperidine and other impurities.

[0044] Subsequent amino acid ligation: Following the deprotection and ligation steps described above, ligate Fmoc-arginine and Fmoc-tryptophan sequentially. Deprotection and washing were performed after each ligation to ensure the reaction proceeded smoothly.

[0045] Cleavage: The coupled peptide carrier was placed in a reaction vessel, and 20 mL of optimized cleavage reagent (trifluoroacetic acid, water, triisopropylsilane, and anisole sulfide mixed in a volume ratio of 90:2.5:2.5:5) was added. The reaction was carried out at room temperature for 2 hours. After the reaction, the carrier was removed by filtration, and the filtrate was concentrated to approximately 5 mL. Then, 50 mL of cold diethyl ether was added to precipitate the peptide. The precipitate was centrifuged, washed three times with cold diethyl ether, and dried to obtain the crude peptide.

[0046] Purification: The crude peptide was dissolved in an appropriate amount of mobile phase (e.g., 0.1% trifluoroacetic acid aqueous solution) and initially separated using reversed-phase high-performance liquid chromatography (RP-HPLC). A C18 column was used. Mobile phase A was 0.1% trifluoroacetic acid aqueous solution, and mobile phase B was 0.1% trifluoroacetic acid acetonitrile solution. Gradient elution was performed from 0 to 30 minutes, with phase B increasing from 5% to 35%. The fraction corresponding to the target peak was collected and then finely purified using preparative ultra-high performance liquid chromatography (Prep-UPLC) to obtain a high-purity L-cysteine-arginine-tryptophan functional tripeptide. The purity was 98.5% as determined by ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS / MS).

[0047] Applied to the field of beauty and skin care

[0048] Skincare product formula: L-cysteine-arginine-tryptophan functional tripeptide content is 0.1%, ceramide accounts for 2%, Centella asiatica extract and Portulaca oleracea extract each account for 1%, fructooligosaccharide is 0.5%, sodium hyaluronate accounts for 1%, glycerin content is 5%, propylene glycol accounts for 3%, carbomer is 0.2%, triethanolamine is used in appropriate amount to adjust pH to 6-7, and the remaining ingredients are deionized water.

[0049] Preparation process: Carbomer was dispersed in deionized water and stirred until fully swollen. Then, glycerin, propylene glycol, and sodium hyaluronate were added sequentially and stirred until dissolved. Ceramide, Centella asiatica extract, and Portulaca oleracea extract were dissolved in an appropriate amount of ethanol and added to the above solution, then stirred until homogeneous. The prepared functional tripeptide was dissolved in a small amount of deionized water and added to the system, followed by fructooligosaccharides. Finally, the pH was adjusted to 6-7 with triethanolamine and stirred until homogeneous to obtain the serum product.

[0050] Efficacy Verification: Thirty female volunteers aged 25-40 were selected and used the serum morning and evening daily for four consecutive weeks. Skin moisture content, elasticity, and wrinkle appearance were measured before and after use. Results showed that skin moisture content increased on average, skin elasticity increased on average, and wrinkle depth decreased on average. Simultaneously, skin microbiome analysis revealed an increase in beneficial bacteria and a decrease in harmful bacteria on the skin surface, indicating effective regulation of the skin microbiota.

[0051] Example 2: Enzymatic synthesis of L-cysteine-lysine-tyrosine functional tripeptides and their applications

[0052] Preparation of functional tripeptides

[0053] Substrate and enzyme preparation: Weigh 5 mmol of L-cysteine, 5 mmol of lysine, and 5 mmol of tyrosine, and dissolve them in 50 mL of phosphate buffer (pH 7.5). Prepare a 1 mg / mL enzyme solution using the same buffer for the directed evolution-modified specific protease (such as the modified papain).

[0054] Reaction: The substrate solution and enzyme solution were separately introduced into a continuous flow microreactor at a flow rate of 0.2 mL / min. The temperature inside the microreactor was controlled at 38 °C, and the reaction time was 12 hours. During the reaction, the substrate underwent a condensation reaction catalyzed by the enzyme to generate the target functional tripeptide.

[0055] Preliminary separation: After the reaction is complete, the reaction solution is passed through an ultrafiltration membrane (with a molecular weight cutoff of 3000 Da) to remove unreacted macromolecules (such as enzymes and unreacted proteins) and obtain a filtrate containing functional tripeptides.

[0056] Purification: The filtrate was loaded onto an ion-exchange column (e.g., a DEAE-Sepharose column) and eluted using a gradient of phosphate buffers containing different concentrations of sodium chloride. The elution peaks containing the target tripeptide were collected. The collected eluent was then loaded onto a hydrophobic interaction column (e.g., a phenyl-Sepharose column) and eluted using a gradient of buffers containing different concentrations of ammonium sulfate to further purify the target tripeptide. Finally, UPLC-MS / MS analysis revealed a 98.2% purity L-cysteine-lysine-tyrosine functional tripeptide.

[0057] Application in the field of health food

[0058] Health food formulation: The prepared functional tripeptide is applied to tablets, and the formulation is shown in the table below:

[0059] The main active ingredient is a 0.15% L-cysteine-lysine-tyrosine functional tripeptide, combined with 2% Bifidobacterium and 2% Lactobacillus acidophilus to enhance gut health. The formula includes 10% resistant dextrin and 5% oat beta-glucan as prebiotics and dietary fiber, 20% microcrystalline cellulose and 50% lactose as high-quality fillers, and 0.5% magnesium stearate to ensure smooth tableting. This formulation design fully considers the synergistic effect of the functional tripeptide and probiotics, while ensuring product stability, bioavailability, and good processing performance.

[0060] Preparation process: Functional tripeptides, bifidobacteria, lactobacillus acidophilus, resistant dextrin, oat β-glucan, microcrystalline cellulose, and lactose are mixed evenly in a certain proportion. An appropriate amount of water is added to form a soft mass, which is then granulated through a 20-mesh sieve and dried at 60℃ for 2 hours. The dried granules are then sieved through an 18-mesh sieve for granulation, magnesium stearate is added, and the mixture is mixed evenly before being compressed into tablets to obtain health food tablets.

[0061] Efficacy Verification: Fifty adults aged 30-50 years were selected and took 3 tablets of this health supplement daily for 8 consecutive weeks. Before and after treatment, the volunteers' immune indicators (such as serum immunoglobulin levels and lymphocyte counts), sleep quality (assessed using a sleep monitor and questionnaire), and gut microbiota (detected through fecal microbial analysis) were measured. Results showed that the volunteers' average serum immunoglobulin levels and lymphocyte counts increased, sleep quality significantly improved, sleep onset time shortened, and sleep duration lengthened. Gut microbiota analysis indicated a significant increase in beneficial bacteria and optimization of the gut microecological environment. Simultaneously, some volunteers also experienced some improvement in their blood lipid levels, with average reductions in total cholesterol and triglycerides.

[0062] Example 3: Preparation and application of L-cysteine-N-acetyl-lysine-methyl ester-5-methoxytryptophan functional tripeptide using genetic engineering technology.

[0063] Preparation of functional tripeptides

[0064] Gene construction: The gene sequence encoding L-cysteine-N-acetyl-lysine-methyl ester-5-methoxytryptophan was artificially synthesized and cloned into the expression vector pET-28a to construct a recombinant expression vector. This recombinant expression vector was then transformed into gene-edited and optimized *E. coli* BL21(DE3) to knock out the protease gene affecting the expression of the target tripeptide, overexpress the chaperone protein, and improve the expression level and folding efficiency of the tripeptide.

[0065] Induction of expression: Transformed Escherichia coli were inoculated into LB medium containing kanamycin and cultured at 37°C until the OD600 reached 0.6-0.8. Isopropyl-β-D-thiogalactoside (IPTG) was then added to a final concentration of 0.5 mM, and expression was induced at 25°C for 6 hours.

[0066] Preliminary enrichment: After induction of expression, the bacterial culture was centrifuged to collect the bacterial cells. The cells were resuspended in lysis buffer (e.g., buffer containing 50 mM Tris-HCl, 150 mM NaCl, and 1 mM EDTA, pH 8.0) and sonicated to disrupt the cells. The disrupted cell culture was centrifuged, and the supernatant was collected for preliminary enrichment using His-tagged magnetic separation technology. The supernatant was mixed with magnetic beads containing His-tagged antibodies and incubated at 4°C for 1 hour. The magnetic beads were then separated using a magnetic field, and washed three times with washing buffer to remove impurities. Finally, the target tripeptide was eluted with elution buffer (e.g., lysis buffer containing 250 mM imidazole) to obtain the preliminarily enriched sample.

[0067] Fine purification: The pre-enriched sample was loaded onto a high-performance hydrophobic interaction chromatography column (e.g., butyl-Sepharose column) and eluted with a gradient of buffers containing different concentrations of ammonium sulfate. The elution peaks containing the target tripeptide were collected. The collected eluent was then loaded onto a size exclusion chromatography column (e.g., Superdex 75 column) and eluted with a buffer (pH 7.5) containing 50 mM Tris-HCl and 150 mM NaCl to further purify the target tripeptide. UPLC-MS / MS analysis revealed a 99% purity L-cysteine-N-acetyl-lysine-methyl ester-5-methoxytryptophan functional tripeptide.

[0068] Applied to the pharmaceutical field

[0069] Pharmaceutical formulation preparation: The prepared functional tripeptide was applied to nanoparticle injections. The preparation method is as follows:

[0070] 10 mg of polylactic-co-glycolic acid copolymer (PLGA) and 2 mg of functional tripeptide were weighed and dissolved in 2 mL of dichloromethane to form an oil phase. 20 mL of an aqueous solution containing 1% polyvinyl alcohol (PVA) was used as the aqueous phase. The oil phase was slowly added dropwise to the aqueous phase, and emulsification was carried out for 5 minutes under high-speed stirring (10000 rpm) to form a pre-emulsion. The pre-emulsion was then added to 50 mL of an aqueous solution containing 0.5% PVA, and stirring continued for 30 minutes to allow the dichloromethane to evaporate, forming nanoparticles. The nanoparticle solution was centrifuged (10000 rpm, 30 minutes), the precipitate was collected, washed three times with deionized water, and finally resuspended in physiological saline. The concentration was adjusted to 1 mg / mL to obtain the nanoparticle injection. Dynamic light scattering analysis showed that the average particle size of the nanoparticles was 120 nm.

[0071] Efficacy Verification: Twenty mice with inflammatory diseases were randomly divided into two groups of 10 each. The experimental group mice received a tail vein injection of the aforementioned nanoparticle injection at a dose of 5 mg / kg body weight; the control group mice received an equal volume of saline. Injections were administered for 7 consecutive days, and changes in inflammatory symptoms were observed. Results showed that the inflammatory symptoms in the experimental group mice were significantly reduced, and the levels of inflammatory factors were significantly decreased, such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) levels. Simultaneously, histopathological examination revealed significant repair of tissue damage in the experimental group mice, demonstrating that the nanoparticle injection has good anti-inflammatory and tissue repair-promoting effects.

[0072] Comparative Example: Tripeptides Prepared by Traditional Methods and Their Applications

[0073] Preparation of tripeptides

[0074] The traditional solid-phase peptide synthesis method was employed, using Fmoc-protected amino acids as raw materials and HATU as a coupling agent, to synthesize the tripeptide L-cysteine-glycine-alanine on a conventional polystyrene support. The specific steps are as follows:

[0075] Fmoc-L-cysteine ​​was immobilized on a polystyrene support. The Fmoc protecting group was removed at room temperature using a 20% piperidine DMF solution. Then, Fmoc-glycine and Fmoc-alanine were added sequentially, and a coupling reaction was carried out under the action of HATU and DIEA, with each reaction lasting 2 hours. After the reaction, the support was washed with DMF, and the deprotection and coupling steps were repeated to complete the synthesis of the tripeptide.

[0076] The peptide chain was cleaved from the carrier using a cleavage reagent (a mixture of trifluoroacetic acid, water, and triisopropylsilane in a volume ratio of 95:2.5:2.5). After simple precipitation and filtration, crude peptides were obtained. The crude peptides were then purified by RP-HPLC to obtain tripeptides with a purity of 92%.

[0077] Applied to the field of beauty and skin care

[0078] The prepared tripeptide was applied to the serum, as shown in the table below:

[0079] This formula uses 0.1% L-cysteine-glycine-alanine tripeptide as its core active ingredient, combined with 1% sodium hyaluronate for excellent moisturizing effects. The formula includes 5% glycerin and 3% propylene glycol as moisturizers, 0.2% carbomer as a thickener, and triethanolamine to adjust the pH to a mild range of 6-7. The remainder is made up with deionized water. This scientific ratio ensures product stability, fully utilizes the skincare benefits of the tripeptide, and provides a pleasant user experience.

[0080] The preparation process was the same as that of the serum in Example 1. Thirty female volunteers aged 25-40 years were selected and used the serum morning and evening for four consecutive weeks. Skin moisture content, elasticity, and wrinkle condition were measured before and after use. Results showed that skin moisture content and elasticity increased on average, while wrinkle depth remained largely unchanged. Compared to Example 1, the tripeptide prepared using the traditional method showed significantly inferior skincare effects.

[0081] As can be seen from the above examples and comparative examples, the functional tripeptide containing L-cysteine ​​prepared in this application and its application have significant advantages in terms of preparation method, product purity and application effect, providing a more effective solution for the fields of beauty and skin care, health food and medicine.

[0082] The following table compares the application effects of the tripeptides prepared in the examples and comparative examples in the field of beauty and skincare:

[0083]

[0084] Table 1

[0085] Conclusion: The functional tripeptide prepared in this application exhibits significantly superior skincare effects compared to traditional methods. Example 1 demonstrated excellent performance in improving skin hydration (18%), elasticity (15%), and wrinkles (12%); Example 3 showed even more outstanding results, with improvements in these three indicators reaching 20%, 18%, and 15%, respectively; Example 2 also demonstrated good skincare efficacy (15%, 12%, and 10%). In contrast, the tripeptide prepared by traditional methods (comparative example) showed significantly insufficient effects, only slightly improving skin hydration (5%) and elasticity (3%), with no improvement in wrinkles. These data fully demonstrate the significant advantages of the technology presented in this application for the development of functional skincare peptides.

[0086] The following table compares the relevant indicators of the tripeptides prepared in the examples with those prepared in the comparative examples:

[0087]

[0088] Table 2

[0089] Conclusion: This table presents the tripeptide purity and preparation methods of the three examples and comparative examples. The tripeptide purity of the examples is higher than that of the comparative examples, and different innovative preparation methods were used, indicating that the preparation method of this application can effectively improve the tripeptide purity and has innovation and superiority.

[0090] 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 class of functional tripeptides containing L-cysteine, characterized in that, The amino acid sequence is L-cysteine-XY, where X is any one of arginine, lysine, histidine, and N-acetyl-lysine-methyl ester, and Y is any one of tryptophan, tyrosine, phenylalanine, and 5-methoxytryptophan; the structure of L-cysteine-N-acetyl-lysine-methyl ester-5-methoxytryptophan is as follows: ； The structure of the N-acetyl-lysine-methyl ester is as follows: ； This tripeptide can react with superoxide anions in reactive oxygen species, exhibiting highly efficient antioxidant properties and protecting cells from oxidative stress damage; the tripeptide can also specifically bind to specific receptors on the cell surface.

2. The functional tripeptide containing L-cysteine ​​according to claim 1, characterized in that, The purity of the functional tripeptide is not less than 98%, and the purity is detected by ultra-high performance liquid chromatography-mass spectrometry to ensure that the impurity content is extremely low, thus guaranteeing product quality and safety.

3. The functional tripeptide containing L-cysteine ​​according to claim 1, characterized in that, The preparation method of the functional tripeptide includes: Chemical synthesis method: Solid-phase peptide synthesis technology was employed, using Fmoc as the protecting group for the active amino acid groups. During the amino acid linkage process, in addition to using 2-7-azabenzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate as a coupling agent, 1-hydroxybenzotriazole was added as an activator. The reaction was carried out at 22-28℃ for 2.5-3.5 hours to promote efficient amino acid linkage. A novel solid-phase support was selected to improve the amino acid loading and reaction efficiency, and reduce side reactions. After linkage, the Fmoc protecting group was removed using a 22% piperidine N,N-dimethylformamide solution. Finally, the peptide chain was cleaved from the solid-phase support using an optimized cleavage reagent to obtain the crude product. The crude product was initially separated by reversed-phase high-performance liquid chromatography (RP-HPLC) and then finely purified by preparative ultra-high-performance liquid chromatography (UHPLC) to obtain the target functional tripeptide. Enzymatic synthesis: A specific protease modified through directed evolution catalyzes the condensation reaction of L-cysteine, amino acid X, and amino acid Y. 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. The reaction process employs continuous flow microreactor technology, with the substrate solution and enzyme solution continuously fed into the microreactor at a flow rate of 0.1-0.3 mL / min. The residence time in the microreactor is 30-60 minutes, achieving continuous and efficient reaction while reducing enzyme dosage and reaction time. After the reaction, unreacted macromolecules are removed by ultrafiltration, followed by purification using ion exchange chromatography and hydrophobic interaction chromatography to obtain the target functional tripeptide.

4. The application of the functional tripeptide containing L-cysteine ​​according to claim 1 in the field of beauty and skincare, characterized in that, It is used to prepare skin care products with antioxidant properties, promotes collagen synthesis, repairs skin barrier function, and regulates the balance of skin microbiota; the amount added to skin care products is 0.05-3%, and it is compounded with ceramides and plant extracts to synergistically enhance the skin care effect; by adding prebiotics, it regulates the microbial environment on the skin surface after the skin care products are applied, promotes the growth of beneficial bacteria, and inhibits the reproduction of harmful bacteria.

5. The application of the functional tripeptide containing L-cysteine ​​according to claim 1 in the field of health food, characterized in that, It is used to prepare health foods that enhance immunity, improve sleep quality, regulate intestinal flora, and help lower blood lipids; the amount added to health foods is 0.1-2%, and it is also combined with probiotics, dietary fiber and other ingredients to optimize product functions; by regulating the structure of intestinal microbial community, it affects bile acid metabolism, reduces cholesterol absorption, and achieves the effect of helping to lower blood lipids.

6. The application of the functional tripeptide containing L-cysteine ​​according to claim 1 in the pharmaceutical field, characterized in that, This product is used to prepare drugs with anti-inflammatory, antibacterial, wound-healing, and neurological disease-treating properties, and can also serve as a targeted drug carrier. The addition amount in the drug is 0.2-8%, and it can be formulated into various dosage forms such as nanoparticle injections, enteric-coated tablets, and targeted capsules. When preparing nanoparticle injections, liposome or polymer nanoparticle encapsulation technology is used to co-encapsulate the functional tripeptide with the active pharmaceutical ingredient, achieving targeted delivery and sustained release. When preparing enteric-coated tablets, enteric coating materials are used to ensure drug release within the intestines. When preparing targeted capsules, specific targeting molecules are modified on the capsule surface, allowing the drug to precisely act on the lesion site.

7. The application of the functional tripeptide containing L-cysteine ​​according to claim 4 in the field of beauty and skincare, characterized in that, The skincare products are formulated as lotions, creams, serums, masks, or gels. In preparing lotions, high-speed homogenization emulsification technology is used, controlling the emulsification temperature at 60-70℃ and the emulsification time at 20-30 minutes to ensure that the functional tripeptides are evenly dispersed in the lotion system. In preparing creams, an appropriate amount of thickener is added to adjust the texture and stability of the cream. In preparing serums, microfiltration and ultrafiltration technologies are used to remove impurities and improve product purity. In preparing masks, a skin-friendly mask base fabric is selected to ensure that the functional tripeptides can be effectively delivered to the skin surface. In preparing gels, carbomer is used as the gel matrix, and an appropriate amount of neutralizing agent is added to adjust the pH value, resulting in a uniform and delicate gel texture.

8. The application of the functional tripeptide containing L-cysteine ​​according to claim 5 in the field of health food, characterized in that, The dosage form of the health food is tablets, capsules, oral liquid, or granules. When preparing tablets, a wet granulation process is used to control the moisture content of the granules at 3-5%, and an appropriate amount of lubricant is added to improve the tablet's formability and flowability. When preparing capsules, a suitable capsule shell is selected, and the filling process is adjusted according to the properties of the functional tripeptide. When preparing oral liquid, an appropriate amount of sweetener and preservative is added to improve the taste and extend the shelf life. When preparing granules, a spray drying granulation technology is used to control the inlet air temperature at 120-140℃ and the outlet air temperature at 70-90℃ to ensure uniform granules and good solubility.

9. The application of the functional tripeptide containing L-cysteine ​​according to claim 6 in the pharmaceutical field, characterized in that, When the drug is formulated into nanoparticle injections, the particle size of the nanoparticles is controlled within 50-200 nm, and the particle size is detected by dynamic light scattering. When formulated into enteric-coated tablets, the enteric coating thickness is controlled within 20-50 μm, and the coating thickness is observed by microscopy. When formulated into targeted capsules, the amount of modification of the targeting molecule is 0.1-1 μg per milligram capsule, and the amount is detected by enzyme-linked immunosorbent assay (ELISA).

10. The functional tripeptide containing L-cysteine ​​according to claim 1, characterized in that, It can also be prepared using genetic engineering techniques. First, a gene encoding the L-cysteine-XY sequence is artificially synthesized. The host cell is then genetically edited and optimized to knock out the protease gene that affects the expression of the target tripeptide, and to overexpress the chaperone protein, thereby increasing the expression level and folding efficiency of the tripeptide. After the gene is introduced into the host cell, isopropyl-β-D-thiogalactoside is used as an inducer in E. coli at a concentration of 0.3-0.8 mM for 5-7 hours. In yeast cells, methanol is used as an inducer at a final concentration of 1-1.5% for 16-20 hours. The expressed tripeptide is then rapidly enriched using affinity-tag-based magnetic separation technology, and further purified using high-performance hydrophobic interaction chromatography and size exclusion chromatography to obtain a high-purity target functional tripeptide.

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