Small molecule collagen peptide with anti-aging activity and preparation method thereof

Abstract

This invention discloses a small-molecule collagen peptide with anti-aging activity and its preparation method, belonging to the field of functional peptide preparation technology. It is a molecular-level conjugate formed by site-specific covalent coupling of a small-molecule collagen peptide and pyrroloquinoline quinone; the small-molecule collagen peptide is a basic functional component for collagen synthesis and also serves as a targeted delivery carrier for fibroblasts; pyrroloquinoline quinone is a cellular energy activation and antioxidant functional component; pyrroloquinoline quinone is grafted onto the ε-amino site of the lysine residue on the side chain of the small-molecule collagen peptide via an amide bond. This invention, through the site-specific covalent coupling molecular structure design of the small-molecule collagen peptide and pyrroloquinoline quinone, solves the problems of mismatched absorption rates and low effective concentrations in target tissues in existing physical compound formulations; relying on the targeted recognition characteristics of the small-molecule collagen peptide on fibroblasts, it drives the synchronous uptake of pyrroloquinoline quinone by target cells, increasing the effective local dose to the skin.

Description

Technical Field

[0001] This invention relates to the field of functional peptide preparation technology, specifically to a small molecule collagen peptide with anti-aging activity and its preparation method. Background Technology

[0002] With increased public awareness of skincare and health maintenance, and changes in living environment, aging problems such as skin laxity, decreased elasticity, and increased wrinkles are showing a clear trend of affecting younger people, leading to a continuous increase in demand for skin care and maintenance related to aging. The loss of collagen in the dermis and the decline in fibroblast function are the core causes of skin aging. Small molecule collagen peptides, due to their high absorption efficiency and good biocompatibility, have become one of the most widely used active ingredients in the current anti-aging field.

[0003] Small molecule collagen peptides are oligopeptide products prepared by enzymatic hydrolysis and purification from collagen-rich tissues such as animal skin, bones, and scales. Their molecular weight is mostly below 1000 Da, and they are rich in characteristic amino acids such as glycine, proline, and hydroxyproline. Among them, glycyl-prolyl-hydroxyproline tripeptide is the core active sequence, which can be directly absorbed by the human body and act on the dermis to promote collagen synthesis and improve skin elasticity. It is currently widely used in functional foods, health foods, and skin care cosmetics.

[0004] Existing related products are mostly developed for single effects, and still have many shortcomings and deficiencies. Single small molecule collagen peptides can only supplement collagen synthesis substrates and stimulate endogenous collagen secretion. Their own antioxidant capacity is weak, and they cannot effectively eliminate reactive oxygen free radicals generated during aging. They are also unable to inhibit the degradation of dermal collagen by matrix metalloproteinases, and they cannot improve the core cause of cellular aging, namely the decline of mitochondrial function in fibroblasts. Simply supplementing collagen cannot block the continuous degradation of collagen and the process of cellular aging, and the overall anti-aging effect has a natural bottleneck.

[0005] To expand efficacy, current technologies often physically combine collagen peptides with anti-aging ingredients such as pyrroloquinoline quinone. However, this approach only achieves a simple additive effect and cannot achieve true synergistic benefits. While pyrroloquinoline quinone can improve cellular energy metabolism and exert antioxidant effects, its skin targeting is poor when used alone. After oral administration, it is widely distributed throughout the body, with very low effective doses actually concentrated in skin fibroblasts. Furthermore, the absorption and metabolic rates of the two are mismatched. Pyrroloquinoline quinone has a small molecular weight and a fast absorption and metabolism rate, while collagen peptides have a relatively slow absorption and onset of action, making it impossible to reach effective concentrations in the target skin tissue simultaneously, thus hindering the full realization of synergistic effects. At the same time, physical mixing cannot improve the stability of the two components. Collagen peptides are easily hydrolyzed by digestive tract proteases, and pyrroloquinoline quinone is easily inactivated in the acidic environment of the stomach, failing to effectively improve the bioavailability of either.

[0006] In addition, existing modification techniques for collagen peptides often suffer from uncontrollable reactions and easily compromised activity: traditional chemical cross-linking reactions lack site specificity, easily damaging the core active sequence of the peptide chain and leading to decreased biological activity; non-covalent complex systems are prone to dissociation in physiological environments and during digestion, failing to maintain a stable binding structure and making it difficult to sustain the modification effect. Therefore, this paper proposes a small molecule collagen peptide with anti-aging activity and its preparation method to address these issues. Summary of the Invention

[0007] Technical problems to be solved To address the shortcomings of existing technologies, this invention provides a small molecule collagen peptide with anti-aging activity and its preparation method, thus solving the problems mentioned in the background section.

[0008] Technical solution To achieve the above objectives, the present invention is implemented through the following technical solution: a small molecule collagen peptide with anti-aging activity, which is a molecular-level conjugate formed by site-specific covalent coupling of a small molecule collagen peptide and pyrroloquinoline quinone.

[0009] This solution addresses the shortcomings of existing technologies: Existing small-molecule collagen peptides primarily function as substrates for collagen synthesis, but their antioxidant capacity is limited, failing to reduce the degradation and loss of endogenous collagen; when pyrroloquinoline quinone is used alone, its targeting to skin fibroblasts is weak, its distribution in vivo is dispersed, and its effective concentration is low; when the two are mixed physically, their absorption rates differ, preventing them from exerting their effects synchronously within target cells, thus limiting their efficacy. This solution covalently binds the two components into a single molecule, enabling simultaneous delivery and functional complementarity, thus overcoming the functional limitations of individual components.

[0010] Small molecule collagen peptides are fundamental functional components for collagen synthesis and can be specifically recognized and taken up by fibroblasts. The purpose of this component is to allow small molecule collagen peptides to directly provide substrates for dermal collagen synthesis, while simultaneously stimulating the collagen synthesis pathway of fibroblasts and replenishing the matrix components of the dermal layer of the skin.

[0011] Preferably, the small molecule collagen peptides are prepared by targeted enzymatic hydrolysis using deep-sea fish skin as raw material, wherein the deep-sea fish skin is selected from at least one of cod skin and salmon skin. Deep-sea fish skin is chosen as the raw material because it has a high collagen content, relatively controllable levels of impurities and fats, and the extracted collagen has low allergenicity, making it a suitable raw material for preparing active collagen peptides.

[0012] Preferably, the molecular weight of the small molecule collagen peptides is concentrated in the range of 500 Da to 1000 Da, wherein the glycylprolyl hydroxyproline tripeptide accounts for no less than 15% of the total peptide mass, and the peptides in the 500 Da to 1000 Da range account for no less than 80% of the total peptide mass. This molecular weight range is chosen because peptides in this range have high absorption efficiency and can easily cross the intestinal or skin barrier; glycylprolyl hydroxyproline is the core sequence in collagen peptides that stimulates collagen synthesis, and ensuring the proportion of this sequence stabilizes the basic physiological activity of the collagen peptides.

[0013] Pyrroloquinoline quinone is a cellular energy regulation and antioxidant component. The purpose of this component is that it can act on mitochondria within cells, enhancing cellular energy metabolism, while simultaneously scavenging reactive oxygen species and reducing oxidative damage to skin cells.

[0014] Pyrroloquinoline quinone is grafted onto the ε-amino site of lysine residue on the side chain of small collagen peptides via an amide bond, forming a stable covalently bound structure. This grafting site and binding method were chosen because the ε-amino group of lysine is located on the peptide side chain, ensuring that grafting does not disrupt the structure of core active sequences such as glycylprolyl-hydroxyproline, thus preserving the physiological function of the collagen peptide itself. Furthermore, the amide bond is chemically stable and does not easily dissociate during in vivo transport, ensuring that both components reach the target cells simultaneously.

[0015] Preferably, the grafting rate of the conjugate is 2% to 4%; and the purity of the pyrroloquinoline quinone is ≥98%. This grafting rate range is set because if the grafting rate is too low, the amount of bound pyrroloquinoline quinone is insufficient, resulting in insignificant energy regulation and antioxidant effects; if the grafting rate is too high, it will alter the spatial conformation of the collagen peptide, affecting its activity. Limiting the purity of the pyrroloquinoline quinone can reduce the impact of raw material impurities on the reaction process and the activity of the final product.

[0016] Preferably, the residual mass fraction of free pyrroloquinoline quinone in the coupling compound is ≤0.5%. The reason for controlling the residual amount of free monomer is that the absorption rate of unbound free pyrroloquinoline quinone differs from that of the coupling compound, which can lead to asynchronous action; reducing the amount of free residue can ensure the uniformity of the product's action.

[0017] This invention also provides a method for preparing small molecule collagen peptides with anti-aging activity, which includes the following steps: Step 1, preparing a highly active small molecule collagen peptide solution: Deep-sea fish skin is sequentially defatted, deproteinized, and subjected to low-temperature swelling treatment to obtain purified collagen fibers. First, pepsin is added for a first-stage enzymatic hydrolysis to remove collagen terminal peptides. Then, a complex enzyme system composed of collagenase and alkaline protease is added for a second-stage directed enzymatic hydrolysis. After hydrolysis, the enzyme is inactivated by heating to obtain a crude enzymatic hydrolysate. The crude enzymatic hydrolysate is purified by ultrafiltration membrane fractionation, and the permeate is collected to obtain a small molecule collagen peptide solution. The purpose of this step is to prepare small molecule collagen peptides with concentrated molecular weight distribution and high content of active sequences from fish skin raw materials through pretreatment and stepwise enzymatic hydrolysis, providing a qualified base material for subsequent grafting reactions.

[0018] Preferably, in step one, the degreasing treatment involves soaking in a 95% isopropanol solution at a material-to-liquid ratio of 1:8 to 1:12, at room temperature for 12 to 18 hours. The reason for setting up the degreasing step is that fish skin contains a certain amount of fatty tissue, which hinders the contact between enzymes and collagen fibers, affecting enzymatic hydrolysis efficiency and reducing the purity of the final product. Isopropanol can effectively dissolve the oily components in the fish skin, achieving the degreasing effect.

[0019] Preferably, the protein removal process involves soaking the fish skin in a 0.1 mol / L to 0.3 mol / L sodium hydroxide solution for 2 to 4 hours, followed by rinsing with water until neutral. The reason for this protein removal step is that fish skin contains various non-collagenous proteins in addition to collagen. These proteins can affect the activity and purity of the final peptides. A dilute alkaline solution can dissolve and remove most of these proteins, thus improving the purity of the collagen raw material.

[0020] Preferably, the first-stage enzymatic hydrolysis temperature is 35°C to 40°C, the pH value is 2.0 to 3.0, the hydrolysis time is 4 hours to 6 hours, and the amount of pepsin added is 0.5% to 1% of the dry weight of collagen fibers. The reason for setting up the first-stage enzymatic hydrolysis is that natural collagen molecules have telopeptide structures at both ends. Telopeptides have strong antigenicity and maintain the tight structure of the collagen triple helix. Pepsin can specifically cleave the telopeptide regions, releasing soluble collagen molecules and reducing the risk of product sensitization. This temperature and pH are suitable reaction conditions for pepsin, ensuring stable enzyme activity.

[0021] Preferably, the second-stage enzymatic hydrolysis temperature is 50℃ to 55℃, the pH value is 7.0 to 8.0, the hydrolysis time is 3h to 5h, the mass ratio of collagenase to alkaline protease in the complex enzyme system is 1:1 to 2:1, and the total enzyme amount is 1% to 2% of the dry weight of collagen fibers. The reason for setting up a second-stage enzymatic hydrolysis is that collagenase can specifically recognize the peptide bond sequence of collagen and directionally cleave it to obtain peptides containing active sequences; alkaline protease can further hydrolyze large peptides into smaller peptides. The combination of the two allows for precise control of the molecular weight distribution and the proportion of active sequences in the peptides. This temperature and pH are suitable reaction conditions for the complex enzyme system, ensuring enzymatic hydrolysis efficiency.

[0022] Preferably, the ultrafiltration membrane has a molecular weight cutoff of 1000 Da. The reason for setting up ultrafiltration fractionation is that the enzymatic hydrolysate contains peptides of different molecular weights and incompletely hydrolyzed proteins. By passing through the ultrafiltration membrane with a cutoff of 1000 Da, large molecular components can be filtered out, and peptides within the target molecular weight range can be collected, ensuring that the molecular weight distribution of the product is concentrated.

[0023] Step 2, Site-Specific Covalent Grafting: The pH of the small molecule collagen peptide solution was adjusted to 5.5-6.0 using a buffer solution. Pyrroloquinoline quinone was added in proportion and stirred until completely dissolved. Under light-protected and constant-temperature conditions, carbodiimide and hydroxysuccinimide were added sequentially to initiate an amidation reaction. The grafting rate was controlled to the target range by adjusting the reaction time. After the reaction was completed, the pH was adjusted to neutral to terminate the reaction. The purpose of this step is to covalently bind pyrroloquinoline quinone to specific sites of the collagen peptide through a mild amidation reaction, achieving molecular-level coupling while maximizing the preservation of the bioactivity of both components.

[0024] Preferably, in step two, the buffer solution is morpholine ethanesulfonic acid buffer, and the reaction temperature is controlled between 20°C and 25°C. Morpholine ethanesulfonic acid buffer is chosen because it has stable buffering capacity under weakly acidic conditions and does not participate in the amidation reaction, thus avoiding the introduction of additional side reactions. Controlling the reaction temperature within this range ensures a moderate reaction rate, facilitating precise control of the grafting rate, while preventing high temperatures from damaging the active structures of the peptide and pyrroloquinoline quinone.

[0025] Preferably, the molar ratio of pyrroloquinoline quinone to small molecule collagen peptide is 0.05:1 to 0.1:1. This ratio is set to provide the necessary raw materials for the target grafting rate; too low a feed amount will prevent the target grafting rate from being achieved, while too high a feed amount will increase the residue of unreacted monomers, thus increasing the burden on subsequent purification.

[0026] Preferably, the molar ratio of carbodiimide to pyrroloquinoline quinone is 1.2:1 to 1.5:1, and the molar ratio of hydroxysuccinimide to pyrroloquinoline quinone is 1:1 to 1.2:1, with a reaction time of 2 to 4 hours. Carbodiimide and hydroxysuccinimide are used as condensing agents because carbodiimide can activate the carboxyl group of pyrroloquinoline quinone, and hydroxysuccinimide can stabilize the activated intermediate. Their combination can improve the efficiency of the amidation reaction and reduce side reactions. By controlling the reaction time within this range, the grafting rate can be precisely controlled. Too short a time results in incomplete grafting, while too long a time can lead to overgrafting.

[0027] Step 3: Purification and Drying to Obtain the Finished Product: The reaction solution is subjected to nanofiltration to remove unreacted monomers and salt byproducts, followed by vacuum freeze-drying to obtain the small molecule collagen peptide product. The purpose of this step is to remove impurities from the reaction system, obtain a high-purity conjugate product, and preserve the product's bioactivity through low-temperature drying.

[0028] Preferably, in step three, the nanofiltration membrane has a molecular weight cutoff of 200 Da. This molecular weight cutoff is set because unreacted pyrroloquinoline quinone, condensing agent byproducts, and salts are all small molecules. A nanofiltration membrane with a 200 Da cutoff can effectively remove them while retaining the target conjugate components, thus improving product purity.

[0029] Preferably, the cold trap temperature for vacuum freeze drying is -50°C to -60°C, and the vacuum degree is less than 10 Pa. Vacuum freeze drying is used because this drying method is carried out in a low-temperature vacuum environment, where water directly sublimates, preventing the degradation of the peptides and pyrroloquinoline quinones. The resulting product is loose and porous with good resolubility.

[0030] This invention utilizes a site-specific covalently coupled structural design, employing highly active small-molecule collagen peptides as the functional substrate, and grafting pyrroloquinoline quinone onto specific sites on the peptide chain side chain via stable amide bonds. The small-molecule collagen peptides play a fundamental role in collagen synthesis, simultaneously delivering pyrroloquinoline quinone to target cells; pyrroloquinoline quinone then exerts cellular energy regulation and antioxidant effects, compensating for the functional limitations of a single collagen peptide. This entire approach, through molecular structural modification, achieves the simultaneous delivery and functional complementarity of two functional components, resulting in a clearly defined product composition and pathway of action.

[0031] Beneficial effects The present invention has the following beneficial effects: (1) The small molecule collagen peptide with anti-aging activity and its preparation method solve the problems of mismatched absorption rate and low effective concentration in target tissue by designing a molecular structure that specifically covalently couples small molecule collagen peptide with pyrroloquinoline quinone site; relying on the targeted recognition characteristics of small molecule collagen peptide on fibroblasts, it drives pyrroloquinoline quinone to be taken up by target cells at the same time, increases the effective dose of local skin action, and achieves synergistic effect of collagen matrix repair and cell energy regulation.

[0032] (2) The small molecule collagen peptide with anti-aging activity and its preparation method solve the problem of easy degradation and inactivation of single components and low bioavailability under physiological environment through the stable covalent binding mode formed by amide bonds; the steric hindrance of the peptide chain can reduce the destruction of the active structure of pyrroloquinoline quinone by digestive juice, and the grafted pyrroloquinoline quinone can block some of the enzyme cleavage sites of the peptide chain. The two protect each other and jointly improve the retention rate and duration of action during digestion.

[0033] Of course, any product implementing this invention does not necessarily need to achieve all of the advantages described above at the same time. Attached Figure Description

[0034] Figure 1 This is a flowchart of a method for preparing small molecule collagen peptides with anti-aging activity according to the present invention. Detailed Implementation

[0035] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0036] This embodiment provides a small molecule collagen peptide with anti-aging activity and its preparation method. The small molecule collagen peptide is a conjugate formed by site-specific covalent coupling of small molecule collagen peptides derived from cod skin and pyrroloquinoline quinone. The final product has a grafting rate of 4.0% and a free pyrroloquinoline quinone residue of 0.48%. Specifically, peptides with a molecular weight of 500 Da to 1000 Da account for 90% of the total peptide mass, and glycylprolylhydroxyproline tripeptide accounts for 18% of the total peptide mass; the purity of pyrroloquinoline quinone is 99%.

[0037] The preparation method of the small molecule collagen peptides, such as... Figure 1 As shown, it includes the following steps: Step 1: Preparation of a highly active small-molecule collagen peptide solution: Cod skin was defatted with 95% isopropanol solution at a material-to-liquid ratio of 1:12 and soaked at room temperature for 18 hours. After removal, it was soaked in 0.3 mol / L sodium hydroxide solution for 4 hours to remove impurities and proteins, and washed with water until neutral. After low-temperature swelling, purified collagen fibers were obtained. The pH of the collagen fibers was adjusted to 3.0, and pepsin (1% of the dry weight of the collagen fibers) was added. The first stage of enzymatic hydrolysis was carried out at 40℃ for 6 hours to remove collagen terminal peptides. Subsequently, the pH was adjusted to 8.0, and a complex enzyme system (2% of the dry weight of the collagen fibers) was added, with a collagenase to alkaline protease mass ratio of 2:1. The second stage of enzymatic hydrolysis was carried out at 55℃ for 5 hours. The enzyme was then inactivated at 90℃ for 10 minutes to obtain a crude enzymatic hydrolysate. The crude enzyme solution was purified by ultrafiltration using a 1000 Da molecular weight cutoff membrane, and the permeate was collected to obtain a small-molecule collagen peptide solution.

[0038] Step 2, Site-Specific Covalent Grafting: The pH of the small molecule collagen peptide solution was adjusted to 6.0 using morpholine ethanesulfonic acid buffer. Pyrroloquinoline quinone was added at a molar ratio of 0.1:1 to the small molecule collagen peptide, and stirred until completely dissolved. Under light-protected conditions at 25°C, carbodiimide and hydroxysuccinimide were added sequentially, with a molar ratio of 1.5:1 for carbodiimide to pyrroloquinoline quinone and a molar ratio of 1.2:1 for hydroxysuccinimide to pyrroloquinoline quinone, initiating the amidation reaction for 4 hours. After the reaction was completed, the pH was adjusted to 7.0 to terminate the reaction.

[0039] Step 3, purification and drying to obtain the finished product: The reaction solution is passed through a nanofiltration membrane with a molecular weight cutoff of 200 Da to remove unreacted monomers and salt byproducts; then it is subjected to vacuum freeze drying at a cold trap temperature of -60℃ and a vacuum degree of 5 Pa to obtain the small molecule collagen peptide product.

[0040] Example 2

[0041] This embodiment provides a small molecule collagen peptide with anti-aging activity and its preparation method. The small molecule collagen peptide is a conjugate formed by site-specific covalent coupling of small molecule collagen peptides derived from salmon skin and pyrroloquinoline quinone. The final product has a grafting rate of 2.0% and a free pyrroloquinoline quinone residue of 0.35%. Specifically, peptides with a molecular weight of 500 Da to 1000 Da account for 80% of the total peptide mass, and glycylprolylhydroxyproline tripeptide accounts for 15% of the total peptide mass; the purity of pyrroloquinoline quinone is 98%.

[0042] The method for preparing the small molecule collagen peptide includes the following steps: Step 1: Preparation of a highly active small-molecule collagen peptide solution: Salmon skin was defatted with 95% isopropanol solution at a material-to-liquid ratio of 1:8 and soaked at room temperature for 12 hours. After removal, it was soaked in 0.1 mol / L sodium hydroxide solution for 2 hours to remove impurities and proteins, and washed with water until neutral. After low-temperature swelling, purified collagen fibers were obtained. The pH of the collagen fibers was adjusted to 2.0, and 0.5% of the dry weight of the collagen fibers was added with pepsin. The first stage of enzymatic hydrolysis was carried out at 35°C for 4 hours to remove collagen terminal peptides. Subsequently, the pH was adjusted to 7.0, and a complex enzyme system with a total enzyme content of 1% of the dry weight of the collagen fibers was added, wherein the mass ratio of collagenase to alkaline protease was 1:1. The second stage of enzymatic hydrolysis was carried out at 50°C for 3 hours. The enzyme was then inactivated at 90°C for 10 minutes to obtain a crude enzymatic hydrolysate. The crude enzyme solution was purified by ultrafiltration using a 1000 Da molecular weight cutoff membrane, and the permeate was collected to obtain a small-molecule collagen peptide solution.

[0043] Step 2, Site-Specific Covalent Grafting: The pH of the small molecule collagen peptide solution was adjusted to 5.5 using morpholine ethanesulfonic acid buffer. Pyrroloquinoline quinone was added at a molar ratio of 0.05:1 to the small molecule collagen peptide, and stirred until completely dissolved. Under light-protected conditions at 20°C, carbodiimide and hydroxysuccinimide were added sequentially, with a molar ratio of 1.2:1 for carbodiimide to pyrroloquinoline quinone and a molar ratio of 1:1 for hydroxysuccinimide to pyrroloquinoline quinone, initiating the amidation reaction for 2 hours. After the reaction was completed, the pH was adjusted to 7.0 to terminate the reaction.

[0044] Step 3, purification and drying to obtain the finished product: The reaction solution is passed through a nanofiltration membrane with a molecular weight cutoff of 200 Da to remove unreacted monomers and salt byproducts; then it is subjected to vacuum freeze drying at a cold trap temperature of -50℃ and a vacuum degree of 9 Pa to obtain the small molecule collagen peptide product.

[0045] Comparative Example 1 This comparative example provides a control sample whose raw material amounts are exactly the same as those in Example 1. The key difference is that no covalent grafting reaction is performed. Instead, equal amounts of small molecule collagen peptides and pyrroloquinoline quinone are mixed evenly at room temperature in the same morpholine ethanesulfonic acid buffer system. The mixture is then subjected to the same nanofiltration and vacuum freeze-drying processes to obtain a physically mixed sample. All other conditions are exactly the same as those in Example 1.

[0046] Comparative Example 2 This comparative example provides a control sample that uses only the same amount of small molecule collagen peptide as in Example 1, without the addition of pyrroloquinoline quinone. All other conditions are identical to those in Example 1, including buffer conditioning, processing under the same conditions, nanofiltration, and vacuum freeze-drying. Experimental Example 1: In Vitro Anti-aging Activity Verification Test. The core objective of this test was to verify the activity advantages of the covalent conjugate of this invention in terms of antioxidation, cell proliferation, and collagen synthesis. The test items and current implementation standards are as follows: DPPH free radical scavenging rate: Spectrophotometric method was used, with a sample concentration of 1 mg / mL, in accordance with standard GB / T39100-2020; Human skin fibroblast proliferation rate: CCK-8 assay was used, cells were cultured for 24 h, sample concentration was 500 μg / mL, and standard T / SHRH 030-2020 was followed. Type I collagen secretion: The content of type I collagen in the supernatant was detected by enzyme-linked immunosorbent assay (ELISA) after 48 h of cell culture. The increase rate was calculated with the blank control group as the benchmark, and the standard T / SHRH 031-2020 was followed. Test samples: Sample of Example 1, Sample of Example 2, Sample of Comparative Example 1, Sample of Comparative Example 2; The test results are shown in the table below:

[0047] Analysis of test results: Examples 1 and 2 outperformed Comparative Examples 1 and 2 in all three test indicators. Comparative Example 2, being a single collagen peptide, had weak antioxidant capacity and only possessed basic cell proliferation and collagen synthesis-promoting effects. Comparative Example 1, being a physically mixed sample, showed better effects than the single collagen peptide, but significantly lower than the covalently coupled example sample. This is because, under physical mixing, the two components could not be simultaneously taken up by cells, and pyrroloquinoline quinone could not be targeted and enriched in fibroblasts along with the collagen peptide, thus limiting the overall effect. This invention achieves simultaneous delivery and targeted enrichment of the two components through a covalently coupled structure, resulting in superior antioxidant and collagen synthesis-promoting effects compared to simple physical mixing.

[0048] Experiment Example 2: Simulated Digestion Stability Verification Test The core objective of this test is to verify the effect of covalent coupling structure on improving the resistance to enzymatic degradation and stability of the components. The test items and current implementation standards are as follows: Retention rate of pyrroloquinoline quinone after simulated gastrointestinal digestion: The system was digested with simulated gastric juice for 2 hours and simulated intestinal juice for 4 hours in sequence, and the proportion of effective pyrroloquinoline quinone in the system was detected. Collagen peptide anti-enzymatic hydrolysis retention rate: After 4 hours of simulated pancreatic enzyme digestion, the retention rate of target molecular weight peptides from 500 Da to 1000 Da was detected, in accordance with standard GB / T22729-2008. Test samples: The test results for Sample 1, Sample 2, Comparative Example 1, and Comparative Example 2 are shown in the table below:

[0049] Analysis of test results: The digestion retention rate of pyrroloquinoline quinone in the example sample was significantly higher than that in the physically mixed sample of Comparative Example 1, indicating that the steric hindrance of the peptide chain can protect pyrroloquinoline quinone and reduce damage from digestive juices. Simultaneously, the collagen peptide anti-enzymatic retention rate of the example sample was significantly higher than that of Comparative Example 2, indicating that the grafted pyrroloquinoline quinone can block some enzyme cleavage sites, enhancing the anti-enzymatic ability of collagen peptides. The two components mutually protect each other through covalent bonding, and their stability under digestive conditions is superior to that of single components and physically mixed systems.

[0050] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0051] The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the invention to the specific implementations described. Clearly, many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention. The invention is limited only by the claims and their full scope and equivalents.

Claims

1. A small molecule collagen peptide with anti-aging activity, characterized in that, It is a molecular-level conjugate formed by site-specific covalent coupling of small molecule collagen peptides and pyrroloquinoline quinone; Small molecule collagen peptides are the basic functional components of collagen synthesis and also serve as targeted delivery carriers for fibroblasts. Pyrroloquinoline quinone is a cellular energy activating and antioxidant component; Pyrroloquinoline quinone is grafted onto the ε-amino site of lysine residues on the side chain of small collagen peptides via an amide bond, forming a stable covalent bond structure. This allows both to be simultaneously taken up by target cells, achieving a synergistic anti-aging effect of collagen matrix repair and cellular energy rejuvenation.

2. The small molecule collagen peptide with anti-aging activity according to claim 1, characterized in that, The small molecule collagen peptides are prepared by targeted enzymatic hydrolysis using deep-sea fish skin as raw material. The deep-sea fish skin is selected from at least one of cod skin and salmon skin. The molecular weight of the small molecule collagen peptides is concentrated in the range of 500 Da to 1000 Da, of which glycylprolyl hydroxyproline tripeptide accounts for no less than 15% of the total peptide mass, and peptides in the range of 500 Da to 1000 Da account for no less than 80% of the total peptide mass.

3. The small molecule collagen peptide with anti-aging activity according to claim 1, characterized in that, The grafting rate of the coupling compound is 2% to 4%; the purity of the pyrroloquinoline quinone is ≥98%.

4. The small molecule collagen peptide with anti-aging activity according to claim 1, characterized in that, The residual mass fraction of free pyrroloquinoline quinone in the conjugate is ≤0.5%.

5. A method for preparing a small molecule collagen peptide with anti-aging activity, characterized in that, The preparation of the small molecule collagen peptide with anti-aging activity according to any one of claims 1 to 4 includes the following steps: Step 1: Deep-sea fish skin is subjected to defatting, removal of impurities and proteins, and low-temperature swelling treatment to obtain purified collagen fibers. First, pepsin is added for the first stage of enzymatic hydrolysis to remove collagen telopeptides. Then, a complex enzyme system composed of collagenase and alkaline protease is added for the second stage of targeted enzymatic hydrolysis. After the enzymatic hydrolysis is completed, the enzyme is inactivated by heating to obtain crude enzymatic hydrolysate. The crude enzymatic hydrolysate is purified by ultrafiltration membrane fractionation, and the permeate is collected to obtain a small molecule collagen peptide solution. Step 2: Adjust the pH of the small molecule collagen peptide solution to 5.5 to 6.0 using a buffer solution. Add pyrroloquinoline quinone in proportion and stir until completely dissolved. Under light-proof and constant temperature conditions, add carbodiimide and hydroxysuccinimide in sequence to initiate an amidation reaction. Control the grafting rate to 2% to 4% by controlling the reaction time. After the reaction is completed, adjust the pH to neutral to terminate the reaction. Step 3: The reaction solution is subjected to nanofiltration to remove unreacted monomers and salt byproducts, and then subjected to vacuum freeze-drying to obtain the small molecule collagen peptide product.

6. The preparation method according to claim 5, characterized in that, In step one, the deep-sea fish skin is selected from at least one of cod skin and salmon skin; the first stage of enzymatic hydrolysis is carried out at a temperature of 35°C to 40°C, a pH of 2.0 to 3.0, and a hydrolysis time of 4 to 6 hours, with pepsin added at 0.5% to 1% of the dry weight of collagen fibers; the second stage of enzymatic hydrolysis is carried out at a temperature of 50°C to 55°C, a pH of 7.0 to 8.0, and a hydrolysis time of 3 to 5 hours, with the mass ratio of collagenase to alkaline protease in the complex enzyme system being 1:1 to 2:1, and the total amount of enzyme added being 1% to 2% of the dry weight of collagen fibers; the ultrafiltration membrane has a molecular weight cutoff of 1000 Da.

7. The preparation method according to claim 5, characterized in that, In step two, the buffer solution is morpholine ethanesulfonic acid buffer, and the reaction temperature is controlled at 20°C to 25°C; the molar ratio of pyrroloquinoline quinone to small molecule collagen peptide is 0.05:1 to 0.1:1; the molar ratio of carbodiimide to pyrroloquinoline quinone is 1.2:1 to 1.5:1, the molar ratio of hydroxysuccinimide to pyrroloquinoline quinone is 1:1 to 1.2:1, and the reaction time is 2h to 4h.

8. The preparation method according to claim 5, characterized in that, In step one, the degreasing treatment involves soaking in a 95% isopropanol solution at a material-to-liquid ratio of 1:8 to 1:12 for 12 to 18 hours at room temperature; the protein removal treatment involves soaking in a 0.1 mol / L to 0.3 mol / L sodium hydroxide solution for 2 to 4 hours, followed by rinsing with water until neutral.

9. The preparation method according to claim 5, characterized in that, In step three, the nanofiltration membrane has a molecular weight cutoff of 200 Da; the cold trap temperature for vacuum freeze drying is -50°C to -60°C, and the vacuum degree is less than 10 Pa.

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