A composition for improving mitochondrial function and use thereof

Abstract

The application provides a composition for improving mitochondrial function and application thereof, and relates to the technical field of hyaluronic acid grafts.The application first finds that hyaluronic acid oligosaccharide-cyclopentapeptide grafts obtained by covalent combination of hyaluronic acid oligosaccharides and cyclopentapeptides which do not have the effect of improving mitochondrial function alone can effectively improve mitochondria or restore mitochondrial function and cell function, thereby providing a new functional ingredient for developing mitochondrial function regulators, cell function regulators, and products for improving the ability of cells to resist aging.Further, the application also finds that the hyaluronic acid oligosaccharide-cyclopentapeptide grafts combined with ergothioneine can produce a synergistic effect, thereby bringing beneficial effects for further improving mitochondrial function, cell function, and the ability of cells to resist aging.

Description

Technical Field

[0001] This application relates to the field of hyaluronic acid graft technology, and more particularly to a composition for enhancing mitochondrial function and its application. Background Technology

[0002] Mitochondria are organelles within cells. As the cell's "energy factory," they generate ATP through oxidative phosphorylation to power cellular activities and play a vital role in metabolism.

[0003] Recently, aging has been shown to be a cellular response characterized by irreversible cell growth arrest. Studies have found a close correlation between aging and a significant decrease in intracellular mitochondrial membrane potential (ΔΨM), a decrease synchronized with the timeline of cellular senescence. Aging and various chronic disease states are associated with the decline of mitochondrial function, manifested as a decrease in mitochondrial membrane potential and reduced adenosine triphosphate (ATP) production. Mitochondrial dysfunction leads to a loss of ATP synthesis efficiency. Mitochondrial dysfunction results in energy loss, increased fatigue, and loss of alertness that affects cognition and learning. These symptoms are often due to insufficient ATP to maintain healthy cellular functions (such as maintaining ion gradients, enzyme synthesis and activity, muscle synthesis and contractility, cardiac function, and neuronal function), leading to many chronic diseases, including: neurodegenerative diseases (such as Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and multiple sclerosis), cardiovascular diseases (such as congestive heart failure and atherosclerosis), metabolic diseases (such as diabetes), mental illnesses (such as neuroendocrine disorders, depression, autism, bipolar disorder, and schizophrenia), osteoarthritis, chronic fatigue syndrome, Gulf War syndrome, fibromyalgia, and chronic muscle atrophy. Therefore, improving mitochondrial function can improve cellular aging and enhance cellular vitality; healthy mitochondrial function is key to maintaining cell proliferation, renewal, and differentiation.

[0004] Studies by Duchen et al. (Duchen MR, et al. 2000) have shown that a higher mitochondrial membrane potential (ΔΨm) facilitates calcium uptake by mitochondria and activates bioenergy metabolism through its action on tricarboxylic acid (TCA) cycle dehydrogenases. Furthermore, research by Berry et al. (Berry, BJ et al. 2023) found that membrane potential naturally declines with age in *C. elegans*. Optogenetics-based increases in mitochondrial membrane potential improved age-related phenotypes and extended the lifespan of *C. elegans*, directly demonstrating that rescuing age-related decline in mitochondrial membrane potential is sufficient to slow aging and extend healthy lifespan. Therefore, enhancing mitochondrial membrane potential has a significant impact on mitochondrial function and human health.

[0005] To date, there are many active ingredients and products on the market that promote mitochondrial health, such as Coenzyme Q10, MitoQ, PQQ, and EGT. For example, the Novogene Coenzyme Q10 soft capsules from the German company AQUANOVA AG claim to have cardiovascular protection and cellular energy enhancement effects, and clinical studies have also shown that they can promote myocardial health (Staiano C, et al. 2023).

[0006] However, there is still a need for effective and safe mitochondrial function modulators to promote mitochondrial health and enable the body to maintain / restore a healthy state. Summary of the Invention

[0007] This invention is the first to discover that hyaluronic acid oligosaccharides and cyclic pentapeptides, which individually do not have the effect of enhancing mitochondrial membrane potential, can effectively enhance or restore mitochondrial and cellular functions by covalently binding. Further research revealed that when hyaluronic acid oligosaccharides-cyclic pentapeptides are combined with ergothioneine in a certain proportion, a synergistic effect is further produced, bringing beneficial effects for further enhancing mitochondrial and cellular functions.

[0008] On one hand, this application provides a composition comprising a hyaluronic acid oligosaccharide-cyclic pentapeptide graft and ergothioneine. The mass ratio of the hyaluronic acid oligosaccharide-cyclic pentapeptide graft to ergothioneine is 1:5-30:1. The graft is obtained by reacting the hydroxyl group (-OH) at the end of the hyaluronic acid oligosaccharide structure with the amino group (-NH2) in the cyclic pentapeptide structure.

[0009] Furthermore, the structural formula of hyaluronic acid oligosaccharide is shown in formula (I) below: ... (I), In the formula, n is selected from one or more natural numbers from 0 to 19, for example, it can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19.

[0010] Optionally, n is selected from one or more of 0, 1, 2, 3, 4, and 5. That is, the hyaluronic acid oligosaccharide includes one or more of hyaluronic acid disaccharide, hyaluronic acid tetrasaccharide, hyaluronic acid hexasaccharide, hyaluronic acid octasaccharide, hyaluronic acid decasaccharide, and hyaluronic acid dodecasaccharide.

[0011] More preferably, the mass percentage of the hyaluronic acid oligosaccharide with n=1 is 35% or more, for example, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or more preferably, 35%-90%.

[0012] In one specific implementation, the mass percentage of hyaluronic acid oligosaccharides with n=1 is 35-90%, the mass percentage of hyaluronic acid oligosaccharides with n=0 is 1-40%, the mass percentage of hyaluronic acid oligosaccharides with n=2 is 1-50%, the mass percentage of hyaluronic acid oligosaccharides with n=3 is 1-15%, the mass percentage of hyaluronic acid oligosaccharides with n=4 is 0.1-10%, and the mass percentage of hyaluronic acid oligosaccharides with n=5 is 0.01-5%.

[0013] In one specific implementation, the mass percentage of hyaluronic acid oligosaccharides with n=1 is 35-70%, the mass percentage of hyaluronic acid oligosaccharides with n=0 is 5-40%, the mass percentage of hyaluronic acid oligosaccharides with n=2 is 10-50%, the mass percentage of hyaluronic acid oligosaccharides with n=3 is 1-15%, the mass percentage of hyaluronic acid oligosaccharides with n=4 is 0.1-10%, and the mass percentage of hyaluronic acid oligosaccharides with n=5 is 0.01-5%.

[0014] Furthermore, the amino acid sequence of the cyclic pentapeptide includes Cycle(-Phe-Phe-Lys-Tyr-Pro-).

[0015] Optionally, the cyclic pentapeptide has the following molecular structure (II): ... (II).

[0016] Optionally, the hydroxyl group (-OH) at the end of the hyaluronic acid oligosaccharide structure reacts with the amino group (-NH2) on the lysine in the cyclic pentapeptide structure.

[0017] Optionally, the hydroxyl group (-OH) on the uronic acid at the end of the hyaluronic acid oligosaccharide structure reacts with the amino group (-NH2) on the lysine in the cyclic pentapeptide structure.

[0018] Furthermore, the hyaluronic acid oligosaccharide-cyclic pentapeptide graft has the structural formula shown in formula (III): ... (III), In the formula, n is selected from one or more natural numbers from 0 to 19, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19. Optionally, n is selected from one or more of 0, 1, 2, 3, 4.

[0019] The hyaluronic acid oligosaccharide-cyclic pentapeptide graft in this application can be obtained by purchase, for example, from Bloomage Biotechnology Co., Ltd.

[0020] The preparation method of the hyaluronic acid oligosaccharide-cyclic pentapeptide graft in this application has been described in Chinese Patent Application No. 202511761821.1.

[0021] In one optional embodiment, the preparation method of the hyaluronic acid oligosaccharide-cyclic pentapeptide graft includes: dissolving the cyclic pentapeptide in a solvent, adding hyaluronic acid oligosaccharide and an alkaline catalyst to react, wherein the molar mass ratio of the hyaluronic acid oligosaccharide to the cyclic pentapeptide is (1-5):1, the reaction temperature is 40℃-50℃, the reaction time is more than 2 h, and the hyaluronic acid oligosaccharide-cyclic pentapeptide graft (SPHA) is obtained by separation and purification.

[0022] Optionally, the base catalyst includes a tertiary amine base catalyst; more preferably, the base catalyst includes N,N-diisopropylethylamine (DIEA).

[0023] Optionally, those skilled in the art may choose conventional purification methods for separation and purification.

[0024] Furthermore, the mass ratio of the hyaluronic acid oligosaccharide-cyclic pentapeptide graft to ergothionein is 1:5-30:1.

[0025] Optionally, the mass ratio of the hyaluronic acid oligosaccharide-cyclic pentapeptide graft and ergothionein can be any value or range of 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 2:2, 2:3, 2:4, 2:5, 2:6, 2:7, 2:8, 2:9, 3:1, 4:1, 5:1, 6:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, or 30:1.

[0026] Optionally, the mass ratio of the hyaluronic acid oligosaccharide-cyclic pentapeptide graft to ergothionein can be 1:4-19:1.

[0027] Furthermore, the content of the hyaluronic acid oligosaccharide-cyclic pentapeptide graft in the composition may be 0.005%-10%.

[0028] The content of the hyaluronic acid oligosaccharide-cyclic pentapeptide graft in the composition may be any value or range of 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 9.5%, and 10%.

[0029] Furthermore, the content of ergothionein in the composition may be 0.005%-10%.

[0030] The content of ergothionein in the composition may be any value or range of 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%.

[0031] In one embodiment, the composition may further include a physiologically acceptable medium, such as a solvent and / or excipients, the solvent being, for example, water.

[0032] In one embodiment, the composition can be formulated into food, pharmaceutical, or cosmetic products, and excipients suitable for the product can be added. For example, when the composition is formulated into a cosmetic, it may also include emollients, thickeners, chelating agents, UV filters, antioxidants, emulsifiers, fillers, colorants, preservatives, fragrances, and active agents such as moisturizers, anti-aging agents, whitening agents, and / or mixtures thereof.

[0033] The composition of this application can be a composition that enhances mitochondrial function.

[0034] The compositions of this application can be prepared by common methods.

[0035] In one alternative embodiment, the preparation method involves mixing hyaluronic acid oligosaccharide-cyclic pentapeptide graft and ergothioneine.

[0036] One aspect of this application relates to compositions, which are any compositions capable of achieving the effects described in this application. The compositions include, but are not limited to, the simultaneous or sequential use of the components. "Simultaneous use" includes using them together in the same formulation or separately in different formulations. "Sequential use" includes using them sequentially in different formulations, with no restriction on the order of sequential use.

[0037] On the other hand, this application also provides the use of the described composition in the preparation of cosmetics, pharmaceuticals, health products and / or medical devices.

[0038] Optionally, the drug may be a drug for improving mitochondrial dysfunction, including neurodegenerative diseases, cardiovascular diseases, metabolic diseases, mental illnesses, osteoarthritis, chronic fatigue syndrome, Gulf War syndrome, fibromyalgia, and chronic muscle atrophy.

[0039] Alternatively, the neurodegenerative diseases include Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and multiple sclerosis; The cardiovascular diseases mentioned include congestive heart failure and atherosclerosis; The metabolic diseases mentioned include diabetes, etc. The mental illnesses mentioned include neuroendocrine disorders, depression, autism, bipolar disorder, and schizophrenia; The health products mentioned include those that maintain healthy cell function, enhance cell energy, promote myocardial health, boost cell vitality, relieve fatigue, and improve learning ability.

[0040] The cosmetics mentioned include those that enhance cell vitality, improve cell aging, and replenish cell energy, particularly those related to skin cells, such as human skin fibroblasts.

[0041] On the other hand, this application also provides the use of the described composition in the preparation of mitochondrial function regulators and / or cell function regulators.

[0042] Furthermore, the mitochondrial function regulators include regulators that enhance or restore mitochondrial membrane potential (ΔΨM value), ATP content, and / or mitochondrial integrity.

[0043] Optionally, the enhancement or restoration of mitochondrial integrity includes enhancing or restoring mitochondrial DNA (mtDNA) integrity, including inhibiting mitochondrial DNA fragmentation. Optionally, inhibiting mitochondrial DNA fragmentation includes reducing mitochondrial DNA breaks.

[0044] Furthermore, the cell function regulators include regulators that enhance cell viability and / or improve cell aging.

[0045] Optionally, the improvement of cell aging includes regulating the content of β-galactosidase (β-Gal), and more preferably, downregulating the content of β-galactosidase.

[0046] In this application, cell aging is improved by downregulating β-galactosidase content and reducing β-galactosidase activity.

[0047] In one alternative implementation, the cells are human skin fibroblasts.

[0048] On the other hand, this application also provides the use of the composition in enhancing the ability of cells to resist aging.

[0049] Optionally, the anti-aging effect is achieved by regulating mitochondrial function and / or cellular function.

[0050] More preferably, the regulation of mitochondrial function includes increasing or restoring mitochondrial membrane potential (ΔΨM value), ATP content and / or mitochondrial integrity. More preferably, increasing or restoring mitochondrial integrity includes increasing or restoring mitochondrial DNA (mtDNA) integrity, including inhibiting mitochondrial DNA fragmentation. More preferably, inhibiting mitochondrial DNA fragmentation includes reducing mtDNA breaks.

[0051] More preferably, the regulation of cell function includes improving cell viability and / or improving cell aging; more preferably, the improvement of cell aging includes regulating β-galactosidase (β-Gal) content; and more preferably, downregulating β-galactosidase content.

[0052] In one alternative implementation, the cells are human skin fibroblasts.

[0053] On the other hand, this application also provides the application of hyaluronic acid oligosaccharide-cyclic pentapeptide graft in the preparation of mitochondrial function regulators, the preparation of cell function regulators and / or the enhancement of cells' ability to resist aging, characterized in that the graft is obtained by reacting the hydroxyl group (-OH) at the end of the hyaluronic acid oligosaccharide structure with the amino group (-NH2) in the cyclic pentapeptide structure.

[0054] This application is the first to discover that hyaluronic acid oligosaccharide-cyclic pentapeptide grafts (SPHA), obtained by covalently binding hyaluronic acid with cyclic peptides, can effectively enhance mitochondrial membrane potential, restore mitochondrial function, and improve cell vitality. This provides a novel active ingredient for products that regulate mitochondrial membrane potential, improve mitochondrial health, and have anti-aging effects.

[0055] Furthermore, the mitochondrial function regulators include regulators that enhance or restore mitochondrial membrane potential, ATP content, and / or mitochondrial integrity.

[0056] Optionally, the enhancement or restoration of mitochondrial integrity includes enhancing or restoring mitochondrial DNA (mtDNA) integrity, including inhibiting mitochondrial DNA fragmentation; more preferably, inhibiting mitochondrial DNA fragmentation includes reducing mtDNA breaks.

[0057] Furthermore, the cell function regulators include regulators that enhance cell viability and / or improve cell aging.

[0058] Optionally, the improvement of cell aging includes regulating the content of β-galactosidase (β-Gal), and more preferably, downregulating the content of β-galactosidase.

[0059] Optionally, the anti-aging effect is achieved by regulating mitochondrial function and / or cellular function.

[0060] In one alternative implementation, the cells are human skin fibroblasts.

[0061] Further, the amino acid sequence of the cyclic pentapeptide includes Cycle(-Phe-Phe-Lys-Tyr-Pro-); optionally, the hyaluronic acid oligosaccharide-cyclic pentapeptide graft has the structural formula shown in formula (III) below: ... (III), In the formula, n is selected from one or more natural numbers from 0 to 19.

[0062] The present invention has the following beneficial effects: This application is the first to discover that hyaluronic acid oligosaccharide-cyclic pentapeptide grafts obtained by covalently combining hyaluronic acid oligosaccharide and cyclic pentapeptide, which do not have the effect of enhancing mitochondrial function on their own, can effectively enhance or restore mitochondrial function and improve cell vitality, providing a new functional ingredient for the development of products such as mitochondrial function regulators, cell function regulators, and products that enhance the ability of cells to resist aging. Furthermore, this application also discovered that the combination of hyaluronic acid oligosaccharide-cyclic pentapeptide graft and ergothioneine produces a synergistic effect, which brings beneficial effects to further enhance mitochondrial function, cell function, and the ability of cells to resist aging. Attached Figure Description

[0063] The accompanying drawings, which are included to provide a further understanding of this application and form part of this application, illustrate exemplary embodiments of this application and are used to explain this application, but do not constitute an undue limitation of this application. In the drawings: Figure 1 , 2 A bar chart showing the ratio of mitochondrial membrane potential; Figure 3 , 4 A bar chart showing ATP content; Figure 5 , 6 A bar chart showing the ratio of long to short mtDNA fragments; Figure 7 , 8 This is a bar chart showing relative cell viability. Figure 9 , 10 This is a bar chart showing the β-Gal content. Detailed Implementation

[0064] To more clearly illustrate the overall concept of this application, a detailed description is provided below with reference to the accompanying drawings and embodiments. Numerous specific details are set forth in the following description to provide a more thorough understanding of the invention. However, it will be apparent to those skilled in the art that the invention can be practiced without one or more of these details. In other instances, certain technical features well-known in the art have not been described to avoid confusion with the invention.

[0065] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention. Test methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the respective manufacturers.

[0066] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to this application. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof. When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0067] Unless otherwise specified, in the following embodiments, reagents or instruments whose manufacturers are not indicated are all conventional products that can be purchased commercially.

[0068] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention all employ conventional techniques in the fields of microbiology, biochemistry, analytical chemistry, cell culture, and related areas.

[0069] In addition, the "water" mentioned in this invention includes any feasible water that can be used in the art, such as deionized water, distilled water, ion-exchanged water, double-distilled water, high-purity water, and purified water.

[0070] In the following embodiments, unless otherwise specified, % means wt%, i.e., weight percentage.

[0071] Information on the reagents and equipment involved in the following examples is shown in Tables 1 and 2: Table 1

[0072] Table 2

[0073] The above-mentioned SPHA can also be prepared using the method described in Example 1 of Chinese Patent Application No. 202511761821.1, as follows: 1. Feeding: Weigh 5 g of cyclic pentapeptide (SP, phenylalanine-phenylalanine-lysine-tyrosine-proline- (Cycle(-Phe-Phe-Lys-Tyr-Pro-)) and dissolve it evenly in 50 mL of DMSO. Add 4.4 mL of DIEA and 10.44 g of hyaluronic acid oligosaccharide (micro-true HA) powder. After the feeding is completed, stir the reaction solution at 45℃ for 2 h. 2. Central control: After the reaction, the reaction progress is monitored by MS+LC-MS. When the proportion of substrate cyclic pentapeptide is below 5%, it indicates that the reaction is complete and the product mixture (crude SPHA) is obtained. 3. Purification: 5 g of crude SPHA was dissolved in 500 mL of purified water by sonication and filtered through a 0.45 μm filter membrane to obtain the filtrate. The filtrate was loaded onto a chromatographic column and washed with a gradient elution method until all crude product was purified to obtain a qualified SPHA product solution. 4. Freeze-drying: Freeze-dry the qualified SPHA product solution in a hanging bottle to obtain the SPHA sample.

[0074] Example 1 This embodiment explores a composition that enhances mitochondrial function. The composition is prepared as follows: Weigh out SPHA, EGT, HA, or SP, and dissolve them in DMEM-H complete culture medium solution according to the formulation ratio in Table 3. Details are as follows: Weigh 0.05 g of SPHA powder and prepare a working solution Cell-SPHA using DMEM-H complete culture medium solution, wherein the SPHA content is 100 μg / mL.

[0075] Weigh 0.05 g of EGT powder and prepare a working solution Cell-EGT using DMEM-H complete culture medium solution, wherein the EGT content is 100 μg / mL.

[0076] Cell-1, a working solution of SPHA and EGT, was prepared using DMEM-H complete culture medium solution, with SPHA at a concentration of 98 μg / mL and EGT at a concentration of 2 μg / mL.

[0077] Working solutions of SPHA and EGT, Cell-2, were prepared using DMEM-H complete culture medium solution, with SPHA concentration of 95 μg / mL and EGT concentration of 5 μg / mL.

[0078] Working solutions of SPHA and EGT, Cell-3, were prepared using DMEM-H complete culture medium solution, with SPHA concentration of 90 μg / mL and EGT concentration of 10 μg / mL.

[0079] Working solutions of SPHA and EGT, Cell-4, were prepared using DMEM-H complete culture medium solution, with SPHA concentration of 50 μg / mL and EGT concentration of 50 μg / mL.

[0080] Working solutions of SPHA and EGT were prepared in Cell-5 using DMEM-H complete culture medium solution, with SPHA concentration of 20 μg / mL and EGT concentration of 80 μg / mL.

[0081] Working solutions of SPHA and EGT were prepared in Cell-6 using DMEM-H complete culture medium solution, with SPHA concentration of 10 μg / mL and EGT concentration of 90 μg / mL.

[0082] Weigh 0.050 g of HA powder and prepare a working solution Cell-HA using DMEM-H complete culture medium solution, wherein the HA content is 100 μg / mL.

[0083] Weigh 0.05 g of SP powder and prepare a working solution Cell-SP using DMEM-H complete culture medium solution, wherein the SP content is 100 μg / mL.

[0084] Working solutions Cell-1' of HA and SP were prepared using DMEM-H complete culture medium solution, with HA content of 92.5 μg / mL and SP content of 7.5 μg / mL.

[0085] Working solutions Cell-2' of HA and SP were prepared using DMEM-H complete culture medium solution, with HA at a concentration of 58 μg / mL and SP at a concentration of 42 μg / mL.

[0086] Working solutions of HA and SP, Cell-3', were prepared using DMEM-H complete culture medium solution, with HA content of 13.3 μg / mL and SP content of 86.7 μg / mL.

[0087] Zinc α-sulfate was dissolved in DMEM-H complete culture medium solution to prepare a working solution Cell-PC as a positive control, in which the content of zinc α-sulfate was 100 μg / mL.

[0088] The above working solution was used for cytological testing, as follows: 1) Cell seeding: Fibroblasts were seeded into 6-well plates (3 × 10⁻⁶ cells / well). 5 Cells / well were cultured in an incubator (37℃, 5% CO2) for 24 h and divided into normal control group (Cell-BC), model group (Cell-NC), positive control group (Cell-PC) and sample group (Cell-1-6, Cell-1'-3', Cell-SPHA, Cell-EGT, Cell-HA, Cell-SP). 2) Solution preparation: Prepare working solutions according to the test plan (Table 3) and the methods described above.

[0089] 3) Modeling: The culture medium from each well was aspirated, and PBS was added. Except for Cell-BC, all other groups were irradiated with UVB at a dose of 30 mJ / cm². 2 (Equipment power: 1.5 mW / cm) 2 Irradiation time: 20 s.

[0090] 4) Drug administration: Remove PBS, add DMEM-H complete medium to Cell-BC and Cell-NC, and add the working solutions from Table 3 to Cell-PC and the sample groups respectively. After drug administration, place the 6-well plates in an incubator (37℃, 5% CO2) for 24 h. 5) Mitochondrial function and cell function detection 5-1) Mitochondrial function is detected by mitochondrial membrane potential, ATP content and degree of mitochondrial DNA fragmentation. An increase in mitochondrial membrane potential, an increase in ATP content and a decrease in mitochondrial fragmentation all reflect the improvement and / or recovery of mitochondrial function.

[0091] Mitochondrial membrane potential detection: After culturing, incubation tests were performed using a mitochondrial membrane potential detection kit (JC-1). For detecting JC-1 monomers, the excitation wavelength was set to 473 nm and the emission wavelength to 530 nm; for detecting JC-1 polymers, the excitation wavelength was set to 575 nm and the emission wavelength to 590 nm. Quantitative analysis of fluorescence at different wavelengths was performed. Red fluorescence represents healthy mitochondria, and green fluorescence represents unhealthy mitochondria. The ratio of red fluorescence intensity to green fluorescence intensity, i.e., the mitochondrial membrane potential ratio, was calculated. The results are shown below. Figure 1 , Figure 2 As shown in Table 3.

[0092] ATP content detection: After culture, the ATP content was detected using an ATP detection kit and a chemiluminescence analyzer. The results are as follows: Figure 3 , Figure 4 As shown in Table 3.

[0093] mtDNA detection: After culture, mtDNA was extracted according to the BioVision K280-50 kit instructions. The degree of mtDNA fragmentation was assessed by the long / short fragment PCR ratio method. The results are as follows: Figure 5 , Figure 6 As shown in Table 3.

[0094] 5-2) Cell function is detected by cell viability and β-galactosidase (β-Gal) content. In senescent cells, β-Gal content increases and cell viability decreases. Therefore, increased cell viability and decreased β-galactosidase (β-Gal) content both represent the improvement and / or recovery of cell function.

[0095] Cell viability assay: After culture, chemiluminescence immunoassay (CTG) was performed using a cell viability assay kit and an ELISA reader. Data were normalized to the cell viability of the Cell-BC group, and relative cell viability was calculated. The results are as follows: Figure 7 , Figure 8 As shown in Table 4.

[0096] β-galactosidase (β-Gal) content detection: ELISA kits were used to test β-gal content according to the kit instructions. Results are as follows: Figure 9 , Figure 10 As shown in Table 4.

[0097] 6) Data Analysis Results and statistical analysis: t-tests were used for comparisons between groups. A p-value < 0.05 was considered statistically significant.

[0098] Criteria for efficacy assessment: Each group is considered effective if its efficacy is superior to Cell-NC and is statistically significant.

[0099] Synergistic judgment criteria: Synergistic effect is defined as the composition being significantly superior to each monomer; Antagonistic effect is defined as the composition being significantly inferior to each monomer; otherwise, it is defined as additive.

[0100] Table 3

[0101] Note: When performing statistical analysis using the t-test method, compared with the normal control group, a p-value < 0.05 is represented by #, a p-value < 0.01 by ##, and a p-value < 0.001 by ###; compared with the model group, a p-value < 0.05 is represented by *, a p-value < 0.01 by **, and a p-value < 0.001 by ***.

[0102] Table 4

[0103] Note: When performing statistical analysis using the t-test method, compared with the normal control group, a p-value < 0.05 is represented by #, a p-value < 0.01 by ##, and a p-value < 0.001 by ###; compared with the model group, a p-value < 0.05 is represented by *, a p-value < 0.01 by **, and a p-value < 0.001 by ***.

[0104] The model group showed significant differences compared to the normal control group, and the positive control group showed significant differences compared to the model group, indicating that the model was successfully established.

[0105] From Table 3 and Figures 1-6 The results showed that, compared with Cell-SPHA and Cell-1' to Cell-3', Cell-HA, and Cell-SP, HA and SP alone, or the combination obtained by physically mixing the two, did not have the ability to regulate mitochondrial membrane potential, ATP content, and mitochondrial DNA fragmentation. However, SPHA covalently grafted with hyaluronic acid and cyclic peptides could effectively improve mitochondrial membrane potential, ATP content, and mitochondrial DNA fragmentation, thereby enhancing mitochondrial function. Compared with Cell-SPHA, Cell-EGT, and Cell-1-6, the physical mixture of SPHA and EGT in a specific ratio in Cell-2 to Cell-5 groups could produce a synergistic effect in enhancing mitochondrial function.

[0106] From Table 4 and Figures 7-10 The results showed that, compared with Cell-SPHA and Cell-1' to Cell-3', Cell-HA, and Cell-SP, HA and SP alone, or the combination obtained by physically mixing the two, did not have the ability to regulate cell viability and β-Gal content. However, SPHA, which was covalently grafted with hyaluronic acid and cyclic peptides, could effectively regulate cell viability and β-Gal content, thereby improving cell function. Compared with Cell-SPHA, Cell-EGT, and Cell-1 to Cell-6, the physical mixture of SPHA and EGT in a specific ratio in the Cell-2 to Cell-5 group could produce a synergistic effect in improving cell function. The above description is merely an embodiment of this application and is not intended to limit the scope of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the scope of the claims of this application.

Claims

1. A composition, characterized in that, The composition comprises hyaluronic acid oligosaccharide-cyclic pentapeptide graft and ergothioneine. The mass ratio of the hyaluronic acid oligosaccharide-cyclic pentapeptide graft to ergothioneine is 1:5-30:

1. The graft is obtained by reacting the hydroxyl group (-OH) at the end of the hyaluronic acid oligosaccharide structure with the amino group (-NH2) in the cyclic pentapeptide structure.

2. The composition according to claim 1, characterized in that, The amino acid sequence of the cyclic pentapeptide includes Cycle(-Phe-Phe-Lys-Tyr-Pro-); optionally, the hyaluronic acid oligosaccharide-cyclic pentapeptide graft has the structural formula shown in formula (III) below: ……(III), In the formula, n is selected from one or more natural numbers from 0 to 19.

3. The use of the composition according to claim 1 or 2 in the preparation of mitochondrial function regulators and / or cell function regulators.

4. The application according to claim 3, characterized in that, The mitochondrial function regulators include those that enhance or restore mitochondrial membrane potential, ATP content, and / or mitochondrial integrity.

5. The application according to claim 3, characterized in that, The cell function regulators include regulators that improve cell viability and / or improve cell aging.

6. The use of the composition as described in claim 1 or 2 in enhancing the ability of cells to resist aging.

7. The application of hyaluronic acid oligosaccharide-cyclic pentapeptide grafts in the preparation of mitochondrial function regulators, cell function regulators, and / or enhancing cellular resistance to aging, characterized in that, The graft is obtained by reacting the hydroxyl group (-OH) at the end of the hyaluronic acid oligosaccharide structure with the amino group (-NH2) in the cyclic pentapeptide structure.

8. The application according to claim 7, characterized in that, The mitochondrial function regulators include those that enhance or restore mitochondrial membrane potential, ATP content, and / or mitochondrial integrity.

9. The application according to claim 7, characterized in that, The cell function regulators include regulators that improve cell viability and / or improve cell aging.

10. The application according to any one of claims 7-9, characterized in that, The amino acid sequence of the cyclic pentapeptide includes Cycle(-Phe-Phe-Lys-Tyr-Pro-); optionally, the hyaluronic acid oligosaccharide-cyclic pentapeptide graft has the following structural formula: ， In the formula, n is selected from one or more natural numbers from 0 to 19.

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