Oral composition and use thereof

Abstract

The invention provides an oral composition and application thereof. The oral composition comprises hyaluronic acid or salt thereof, N-acetylglucosamine and ergothioneine, wherein the mass ratio of the sodium hyaluronate or the salt thereof to the N-acetylglucosamine to the ergothioneine is 1: (1-10): (0.1-5), the hyaluronic acid or the salt thereof, the N-acetylglucosamine and the ergothioneine in the oral composition are synergistically compounded to achieve a synergistic effect, and the oral composition can be used for moisturizing or cartilage repair.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of oral technology, in particular to an oral composition and use thereof. BACKGROUND

[0002] Osteoarthritis (OA) is a chronic degenerative joint disease that starts with the damage of articular cartilage and eventually leads to joint pain and decreased joint function. Healthy articular cartilage plays a key role in weight-bearing, lubrication and cushioning, and the series of chain reactions caused by cartilage damage not only makes patients feel pain and joint function limited, but also leads to the continuous aggravation of OA disease if not intervened in time.

[0003] Inflammation and metabolic imbalance in cartilage damage promote each other, forming a vicious cycle. When the cartilage is subjected to mechanical stress or inflammatory stimuli (such as trauma, obesity or arthritis), local immune cells (such as macrophages) and chondrocytes themselves release a large amount of pro-inflammatory factors (such as IL-1β, TNF-α and IL-6), which activate the NF-κB and MAPK signaling pathways, prompting chondrocytes to overexpress matrix-degrading enzymes (such as MMP-3, MMP-13 and ADAMTS-5), thereby accelerating the decomposition of collagen II and proteoglycans, and destroying the structural integrity of the cartilage matrix. At the same time, the inflammatory response also induces oxidative stress, producing excess reactive oxygen species (ROS), which further damage the DNA, proteins and lipids of chondrocytes, inhibit their synthetic function, and promote cell apoptosis or aging. On the other hand, metabolic imbalance is manifested as the imbalance between synthesis and decomposition of chondrocytes — the inflammatory microenvironment inhibits the effects of synthetic cytokines such as TGF-β and IGF-1, leading to insufficient regeneration of proteoglycans and collagen; and abnormal intracellular energy metabolism (such as mitochondrial dysfunction) exacerbates the survival pressure of chondrocytes. This persistent inflammation and metabolic disorder not only weakens the self-repairing ability of cartilage, but also expands the damage range, eventually leading to thinning, fissuring or even full-thickness loss of cartilage, and triggering degenerative diseases such as osteoarthritis.

[0004] Dry skin focuses on barrier disruption and dynamic imbalance of water, and with age, the proliferation of epidermal keratinocytes and dermal fibroblasts slows down, and the repair capacity weakens. Senescent cells secrete SASP (senescence-associated secretory phenotype), release pro-inflammatory factors (IL-6, IL-1α), inhibit collagen and hyaluronic acid synthesis, and at the same time reduce sebaceous gland secretion, making the skin surface lack oil protection, further exacerbating dryness.

[0005] Ultraviolet rays, pollution and other external stimuli produce reactive oxygen species (ROS), attack the membrane lipids of keratinocytes, destroy tight junction proteins (such as claudin-1), and weaken the barrier function. At the same time, ROS inhibits the decomposition of filaggrin into natural moisturizing factor (NMF), reduces the water absorption capacity of the stratum corneum, and activates MMPs to degrade collagen and elastic fibers, leading to the collapse of the dermal water storage structure. Under normal circumstances, dermal hyaluronic acid and extracellular matrix (ECM) absorb water through osmotic pressure, maintaining fullness of the skin. However, in aging or photo-damaged skin, hyaluronidase activity is enhanced, hyaluronic acid is degraded, and the dermal water retention capacity is reduced; at the same time, microcirculation disorders lead to insufficient nutrition supply, abnormal differentiation of keratinocytes, and formation of loose and incomplete stratum corneum, which cannot effectively lock water.

[0006] Barrier damage, aging, oxidation and water metabolism disorder form a vicious cycle: barrier damage accelerates water loss, oxidative stress promotes cell aging, and aging weakens the repair capacity, ultimately leading to persistent dryness, roughness and even sensitivity of the skin.

[0007] According to the formation mechanism of cartilage damage and skin dryness and water loss, supplementing the lost endogenous substance hyaluronic acid in the body while strengthening anti-inflammatory and anti-oxidation can improve cartilage damage and skin dryness and water loss from the root. At present, there are various such products on the market, but their true effects are not the same. SUMMARY

[0008] To solve the technical problem of poor effect of improving cartilage damage and skin moisturizing and water supplementing in the prior art, the present application provides an oral composition comprising hyaluronic acid or a salt thereof, N-acetyl glucosamine and ergothioneine. The three substances in the oral composition are synergistically compounded to significantly improve cartilage damage and moisturizing and water supplementing effect.

[0009] The specific technical scheme of the present application is as follows:

[0010] 1. An oral composition comprising hyaluronic acid or a salt thereof, N-acetyl glucosamine and ergothioneine.

[0011] The mass ratio of the sodium hyaluronate or a salt thereof, N-acetyl glucosamine and ergothioneine is 1:1-10:0.1-5.

[0012] 2. The oral composition according to item 1, wherein the molecular weight of the hyaluronic acid or a salt thereof is 20k-2200kDa.

[0013] 3. A product comprising the composition of item 1 or 2 and a physiologically acceptable excipient.

[0014] 4. Use of ergothioneine in improving oral moisturization of N-acetyl glucosamine and hyaluronic acid or a salt thereof, which is for oral administration.

[0015] 5. The use according to item 4, wherein the mass ratio of the sodium hyaluronate or a salt thereof, N-acetyl glucosamine and ergothioneine is 1:1-10:0.1-5.

[0016] 6. The use according to item 4 or 5, wherein the molecular weight of the hyaluronic acid or a salt thereof is 30k-600k Da.

[0017] 7. Use of ergothioneine in improving oral cartilage repair of N-acetyl glucosamine and hyaluronic acid or a salt thereof, which is for oral administration.

[0018] 8. The use according to item 7, wherein the mass ratio of the sodium hyaluronate or a salt thereof, N-acetyl glucosamine and ergothioneine is 1:1-10:0.1-5.

[0019] Effects of the invention

[0020] (1) The present application provides a composition comprising hyaluronic acid or a salt thereof, N-acetyl glucosamine (NAG) and ergothioneine, which are synergistically compounded by hyaluronic acid or a salt thereof, N-acetyl glucosamine (NAG) and ergothioneine, for moisturization or for cartilage repair.

[0021] (2) The raw materials used in the present application are all materials of natural fermentation origin, which are high in safety.

[0022] (3) The present application has low cost, short process cycle, small pollution, low energy consumption, and is easy to realize industrialization. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 Picture of zebrafish tail fin area for water replenishment and moisturization research. DETAILED DESCRIPTION

[0024] The present application will be described in detail below in conjunction with the described embodiments. Although specific embodiments of the present application are shown, it should be understood that the present application can be implemented in various forms and should not be limited by the embodiments described herein. On the contrary, these embodiments are provided to enable a more thorough understanding of the present application and to fully convey the scope of the present application to those skilled in the art.

[0025] It should be noted that some terms are used in the description and claims to refer to particular components. One skilled in the art will understand that the person skilled in the art can use different names to refer to the same component. The description and claims of the present application do not distinguish the components by the difference in the name, but by the difference in the function of the components. As mentioned throughout the description and claims, "including" or "including" is an open term, which should be interpreted as "including but not limited to". The subsequent description of the specification is a preferred embodiment of the present application, but the description is for the purpose of the general principles of the specification, and is not intended to limit the scope of the present application. The scope of protection of the present application is defined by the appended claims.

[0026] The present application provides an oral composition comprising hyaluronic acid or a salt thereof, N-acetyl glucosamine and ergothioneine;

[0027] The mass ratio of the hyaluronic acid or a salt thereof, N-acetyl glucosamine and ergothioneine is 1:1-10:0.1-5.

[0028] The molecular weight of the hyaluronic acid or a salt thereof is 20k-2200k Da.

[0029] The three components in the oral composition described in the present application can synergistically act, and have a synergistic effect on moisturizing or cartilage repair. The composition described in the present application can be administered orally and can be used for moisturizing or cartilage repair.

[0030] For example, the mass ratio (m 透明质酸 Sodium or a salt thereof: m N-The ratio of the hyaluronic acid salt, the acetyl glucosamine and the ergothioneine can be 1:1:0.1, 1:1:0.2, 1:1:0.3, 1:1:0.4, 1:1:0.5, 1:1:0.6, 1:1:0.7, 1:1:0.8, 1:1:0.9, 1:1:1, 1:1:1.5, 1:1:1.5, 1:1:2, 1:1:2.5, 1:1:3, 1:1:3.5, 1:1:4, 1:1:4.5, 1:1:5, 1:1.5:0.1, 1:2:0.1, 1:2.5:0.1, 1:3:0.1, 1:3.5:0.1, 1:4:0.1, 1:4.5:0.1, 1:5:0.1, 1:5.5:0.1, 1:6:0.1, 1:6.5:0.1, 1:7:0.1, 1:7.5:0.1, 1:8:0.1, 1:8.5:0.1, 1:9:0.1, 1:9.5:0.1, 1:10:0.1, 1:1.5:0.2, 1:2:0.2, 1:2.5:0.2, 1:3:0.2, 1:3.5:0.2, 1:4:0.2, 1:4.5:0.2, 1:5:0.2, 1:5.5:0.2, 1:6:0.2, 1:6.5:0.2, 1:7:0.2, 1:7.5:0.2, 1:8:0.2, 1:8.5:0.2, 1:9:0.2, 1:9.5:0.2, 1:10:0.2, 1:1.5:0.5, 1:2:0.5, 1:2.5:0.5, 1:3:0.5, 1:3.5:0.5, 1:4:0.5, 1:4.5:0.5, 1:5:0.5, 1:5.5:0.5, 1:6:0.5, 1:6.5:0.5, 1:7:0.5, 1:7.5:0.5, 1:8:0.5, 1:8.5:0.5, 1:9:0.5, 1:9.5:0.5, 1:10:0.5, and any ratio therebetween.

[0031] In the present application, the hyaluronic acid salt refers to a metal salt of hyaluronic acid, which can be sodium hyaluronate.

[0032] The molecular weight of the hyaluronic acid or salt thereof may be, for example, 20 kDa, 30 kDa, 40 kDa, 50 kDa, 60 kDa, 70 kDa, 80 kDa, 90 kDa, 100 kDa, 150 kDa, 200 kDa, 250 kDa, 300 kDa, 350 kDa, 400 kDa, 450 kDa, 500 kDa, 550 kDa, 600 kDa, 650 kDa, 700 kDa, 750 kDa, 800 kDa, 850 kDa, 900 kDa, 950 kDa, 1000 kDa, 1050 kDa, 1100 kDa, 1150 kDa, 1200 kDa, 1250 kDa, 1300 kDa, 1350 kDa, 1400 kDa, 1450 kDa, 1500 kDa, 1550 kDa, 1600 kDa, 1650 kDa, 1700 kDa, 1750 kDa, 1800 kDa, 1850 kDa, 1900 kDa, 1950 kDa, 2000 kDa, 2050 kDa, 2100 kDa, 2150 kDa, 2200 kDa, or the like.

[0033] In the present application, the determination method of the hyaluronic acid or salt thereof in the composition is not limited, and it can be determined by a method conventional in the art, for example, by a gel permeation chromatography (GPC), a viscosity method, or a laser light scattering method.

[0034] In some embodiments, the content of the hyaluronic acid or salt thereof, N-acetylglucosamine, and ergothioneine in the composition is not limited as long as the ratio of the three satisfies the conditions of the present application.

[0035] The present application provides the use of the above-mentioned composition in the preparation of a moisturizing or cartilage repair product.

[0036] The composition described in the present application has a synergistic effect in moisturizing or cartilage repair, and thus can be used in the preparation of a moisturizing or cartilage repair product.

[0037] The present application provides a moisturizing product comprising the above-mentioned composition.

[0038] In the present application, the content of the composition in the moisturizing product is not limited, and a person skilled in the art can make a conventional selection based on actual needs.

[0039] In the present application, the moisturizing product can further comprise other excipients and / or other active ingredients conventional in the art.

[0040] In particular, the adjuvants can be suitable solvents, propellants, solubilizers, co-solvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow agents, flavorings, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, integrating agents, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, antifoaming agents, thickening agents, inclusion agents, humectants, absorbents, diluents, flocculating and deflocculating agents, filtration aids, release retarders, etc.

[0041] In the present application, no limitation is made to other adjuvants and other ingredients, and a person skilled in the art can make a routine selection based on actual needs.

[0042] Similarly, no limitation is made in the present application to the content of other adjuvants and other active ingredients, and a person skilled in the art can make a routine selection based on actual needs.

[0043] In the present application, the moisturizing product is administered orally.

[0044] In some embodiments, the hyaluronic acid or salt thereof has a molecular weight of 30k-600k Da.

[0045] For example, the hyaluronic acid or salt thereof can have a molecular weight of 30k Da, 40k Da, 50k Da, 60k Da, 70k Da, 80k Da, 90k Da, 100k Da, 110k Da, 120k Da, 130k Da, 140k Da, 150k Da, 160k Da, 170k Da, 180k Da, 190k Da, 200k Da, 210k Da, 220k Da, 230k Da, 240k Da, 250k Da, 260k Da, 270k Da, 280k Da, 290k Da, 300k Da, 310k Da, 320k Da, 330k Da, 340k Da, 350k Da, 360k Da, 370k Da, 380k Da, 390k Da, 400k Da, 410k Da, 420k Da, 430k Da, 440k Da, 450k Da, 460k Da, 470k Da, 480k Da, 490k Da, 500k Da, 510k Da, 520k Da, 530k Da, 540k Da, 550k Da, 560k Da, 570k Da, 580k Da, 590k Da, 600k Da, etc.

[0046] The present application provides a cartilage repair product comprising the above-mentioned composition.

[0047] In the present application, the content of the composition in the cartilage repair product is not limited in the present application, and can be routinely selected by a person skilled in the art based on actual needs.

[0048] In the present application, the cartilage repair product can further comprise other excipients and / or other active ingredients, and again, the excipients and the active ingredients and their respective contents are not limited in the present application, and can be routinely selected by a person skilled in the art based on actual needs.

[0049] In some embodiments, the hyaluronic acid or salt thereof has a molecular weight of 600k-2000k Da.

[0050] For example, the hyaluronic acid or salt thereof can have a molecular weight of 600k Da, 650k Da, 700k Da, 750k Da, 800k Da, 850k Da, 900k Da, 950k Da, 1000k Da, 1050k Da, 1100k Da, 1150k Da, 1200k Da, 1250k Da, 1300k Da, 1350k Da, 1400k Da, 1450k Da, 1500k Da, 1550k Da, 1600k Da, 1650k Da, 1700k Da, 1750k Da, 1800k Da, 1850k Da, 1900k Da, 1950k Da, 2000k Da, etc.

[0051] The cartilage repair product described in the present application is administered orally.

[0052] The present application provides the use of ergothioneine in improving the moisturizing effect of N-acetyl glucosamine and hyaluronic acid or salt thereof, which is for oral administration. In some embodiments, the mass ratio of the sodium hyaluronate or salt thereof, N-acetyl glucosamine and ergothioneine is 1:1-10:0.1-5. In some embodiments, the hyaluronic acid or salt thereof has a molecular weight of 30k-600k Da.

[0053] The present application provides the use of ergothioneine in improving the cartilage repair effect of N-acetyl glucosamine and hyaluronic acid or salt thereof, which is for oral administration. In some embodiments, the mass ratio of the sodium hyaluronate or salt thereof, N-acetyl glucosamine and ergothioneine is 1:1-10:0.1-5. In some embodiments, the hyaluronic acid or salt thereof has a molecular weight of 600k-2000k Da.

[0054] Examples

[0055] The materials used in the test and the test methods are generally and / or specifically described in the present application. In the following examples, % means wt%, i.e. weight percentage, if no other specific description is given. The reagents or instruments used are not specified by the manufacturer, but are conventional reagent products that can be obtained commercially. Sodium hyaluronate (HA), J (HA), N-acetyl glucosamine ACTIVENAG TM (NAG) and ergothioneine (EGT) are all purchased from Huaxibio.

[0056] Preparation of powders in Examples 1-1 to 11-1

[0057] (1) Mixing: sodium hyaluronate J, N-acetyl glucosamine and ergothioneine are mixed uniformly, wherein the total mass of the three substances is 100 g, and the addition ratio of the three substances is as shown in Table 1;

[0058] (2) Sieving: the uniformly mixed material is sieved through a 60-mesh Tyler standard sieve to obtain a uniformly powdered material;

[0059] (3) Sterilization: the material obtained in step (2) is sterilized by ultraviolet light for 30 min;

[0060] (4) Packaging: the sterilized material is packaged, with a dosage of 2.5 g per bag.

[0061] Preparation of powders in Examples 1-2 to 11-2

[0062] (1) Mixing: sodium hyaluronate J, N-acetyl glucosamine and ergothioneine are mixed uniformly, wherein the total mass of the three substances is 100 g, and the addition ratio of the three substances is as shown in Table 2;

[0063] (2) Sieving: the uniformly mixed material is sieved through a 60-mesh Tyler standard sieve to obtain a uniformly powdered material;

[0064] (3) Sterilization: the material obtained in step (2) is sterilized by ultraviolet light for 30 min;

[0065] (4) Packaging: the sterilized material is packaged, with a dosage of 2.5 g per bag.

[0066] Preparation of tablets in Example 12

[0067] (1) Mixing: sodium hyaluronate J, N-acetyl glucosamine, ergothioneine and excipients are mixed uniformly; wherein the total mass of the three substances is 100 g, and the addition ratio of the three substances is as shown in Table 3; J, N-acetyl glucosamine, ergothioneine and excipients are mixed uniformly; wherein the total mass of the three substances is 100 g, and the addition ratio of the three substances is as shown in Table 3; J, N-acetyl glucosamine, ergothioneine and excipients are mixed uniformly; wherein the total mass of the three substances is 100 g, and the addition ratio of the three substances is as shown in Table 3; J, N-acetyl glucosamine and ergothioneine in a mass ratio of 1:5:2, the total mass of the three substances being 42 g, and the excipients consisting of starch 29 g and sodium carboxymethyl cellulose 29 g;

[0068] (2) Wet granulation: after mixing evenly, 10 ml of purified water was added by spraying, and wet granulation was performed;

[0069] (3) Drying: the material after wet granulation was placed in an oven for constant temperature drying at 50°C for 4 h, and after drying, ultraviolet sterilization was performed for 15 min to obtain whole granules;

[0070] (4) Tabletting: the whole granules after drying were tabletted to obtain 80 tablets, each tablet being 1.0 g.

[0071] Preparation of granules in Example 13

[0072] (1) Mixing: sodium hyaluronate or J, N-acetyl glucosamine, ergothioneine and excipients were mixed evenly in a mass ratio of 1:5:2.5; wherein the total mass of the three substances was 1 kg;

[0073] (2) One-step granulation: after mixing evenly, the material was added into a FBM-3 type fluidized bed granulator (equipment capacity 3 L), 30 wt% ethanol-containing aqueous solution was used as a wetting agent, and was sprayed from the top of the fluidized bed granulator, the atomization pressure of the fluidized bed granulator was 0.1 MPa, the air inlet temperature was 60°C, and after treatment for 84 min, 20 mesh and 60 mesh sieves were used for screening to obtain granules between 60 mesh and 80 mesh;

[0074] (3) Sterilization: the material obtained in step (2) was sterilized by ultraviolet light for 30 min;

[0075] (4) Packaging: the material after sterilization was packaged to obtain 180 bags of granules, each bag containing 5 g.

[0076] Preparation of powder in Comparative Example 1-1

[0077] Sodium hyaluronate After ultraviolet sterilization for 30 min, the material was packaged in an aluminum foil bag to obtain a powder, each bag containing 2.5 g, as shown in Table 1.

[0078] Preparation of powder in Comparative Example 2

[0079] N-acetyl glucosamine, after ultraviolet sterilization for 30 min, the material was packaged in an aluminum foil bag to obtain a powder, each bag containing 2.5 g, as shown in Table 1 or 2.

[0080] Preparation of powder in Comparative Example 3

[0081] The ergothioneine is sterilized by ultraviolet rays for 30 minutes, then packed in an aluminum foil bag, and each bag contains 2.5 g. The contents are shown in Table 1 or 2.

[0082] Preparation of the powders of Comparative Examples 1-2

[0083] Sodium hyaluronate The ergothioneine is sterilized by ultraviolet rays for 30 minutes, then packed in an aluminum foil bag, and each bag contains 2.5 g. The contents are shown in Table 2.

[0084] Preparation of the powders of Comparative Examples 4-1 to 5-1, 6, 7-1 to 8-1, 9, 10-1 to 13-1, 14, 15-1 to 16-1, 17, and 18-1 to 19-1

[0085] (1) Mixing: The sodium hyaluronate and the N-acetylglucosamine are mixed uniformly or the sodium hyaluronate and the ergothioneine are mixed uniformly, and the total mass of the two substances is 100 g, and the addition ratio of the two substances is shown in Table 1;

[0086] (2) Sieving: The uniformly mixed material is sieved through a 60-mesh Tyler standard sieve to obtain a uniformly powdered material;

[0087] (3) Sterilization: The material obtained in step (2) is sterilized by ultraviolet rays for 30 minutes;

[0088] (4) Packaging: The sterilized material is packaged, and each bag contains 2.5 g.

[0089] Preparation of the powders of Comparative Examples 4-2 to 5-2, 7-2 to 8-2, 10-2 to 13-2, 15-2 to 16-2, and 18-2 to 19-2

[0090] (1) Mixing: The sodium hyaluronate J and the N-acetylglucosamine are mixed uniformly or the sodium hyaluronate J and the ergothioneine are mixed uniformly, and the total mass of the two substances is 100 g, and the addition ratio of the two substances is shown in Table 2;

[0091] (2) Sieving: The uniformly mixed material is sieved through a 60-mesh Tyler standard sieve to obtain a uniformly powdered material;

[0092] (3) Sterilization: The material obtained in step (2) is sterilized by ultraviolet rays for 30 minutes;

[0093] (4) Packaging: The sterilized material is packaged, and each bag contains 2.5 g.

[0094] Table 1 Ingredients and ratios of examples and comparative examples

[0095]

[0096]

[0097]

[0098] Note: "-" means that the corresponding component is not added; "√" means that the corresponding component is added.

[0099] Table 2. Composition and proportion table of examples and comparative examples (2.5 g / portion)

[0100]

[0101]

[0102]

[0103] Note: "-" means that the corresponding component is not added; "√" means that the corresponding component is added.

[0104] Test Example 1: Moisturizing test

[0105] In the field of skin care, moisturizing is a key step to maintain skin health and barrier function. The stability of skin moisture content not only affects the appearance of the skin, such as smoothness and elasticity, but also is closely related to the barrier function of the skin and the ability to resist external stimuli. Zebrafish, as a new model organism, has transparent embryos, which can be directly observed under a microscope to observe internal structures and physiological changes; its genome has a high similarity of 87% to human genes, and many genes and signaling pathways related to skin development and barrier function are highly conserved between zebrafish and humans; in addition, zebrafish have strong reproductive ability, rapid embryonic development, short experimental period, and low cost, making them suitable for large-scale compound screening and efficacy evaluation. In skin moisturizing research, zebrafish embryos exhibit skin barrier damage and water loss in a high-osmotic environment, similar to human skin dryness. By observing the protective effect of compounds on zebrafish embryos in a high-osmotic environment, the moisturizing efficacy of the compounds can be evaluated.

[0106] I. Experimental method

[0107] The powders prepared in Examples 1-1 to 11-1, Comparative Example 1-1, Comparative Example 2, Comparative Example 3, and Comparative Examples 4-1 to 5-1, Comparative Example 6, Comparative Examples 7-1 to 8-1, Comparative Example 9, Comparative Examples 10-1 to 13-1, Comparative Example 14, Comparative Examples 15-1 to 16-1, Comparative Example 17, and Comparative Examples 18-1 to 19-1 were used as solutes, and the solvent was standard dilution water.

[0108] 1. Zebrafish strain: Wild-type zebrafish of Tübingen (TU) strain is commonly used as a model for zebrafish research.

[0109] 2. Reagents: Potassium chloride (KCl), calcium chloride (CaCl2), magnesium sulfate (MgSO4), sodium chloride (NaCl): used to prepare embryo culture solution (ERS) and high-osmotic ERS to provide the necessary electrolyte environment for zebrafish embryos.

[0110] Paraformaldehyde: used to fix zebrafish embryos for subsequent photographic observation and morphological analysis.

[0111] Phenylthiourea (PTU): a tyrosinase inhibitor that can inhibit the formation of melanin in zebrafish embryos, avoiding the interference of melanin in photographic observation and area measurement.

[0112] Other reagents: paraformaldehyde (analytical pure, used for embryo fixation for subsequent observation and processing), PTU (phenylthiourea, analytical pure, can inhibit melanin production, avoid interference of pigments on observation).

[0113] 3. Instruments and equipment: 6-well plate (special for cell culture, used for culturing zebrafish embryos), stereomicroscope for embryo morphological observation and photography, Image J software for image analysis and data measurement, incubator for maintaining the appropriate environment for embryo culture, etc.

[0114] II. Observation and measurement

[0115] (I) Embryo collection and culture:

[0116] 1. Place zebrafish parents in a ratio of 1:1 or 2:1 in a mating tank in advance, set appropriate light cycle (14 hours light, 10 hours dark), and induce natural mating and spawning.

[0117] 2. After fertilization, collect the embryos in time, wash them several times with embryo culture solution (ERS) to remove impurities and unfertilized eggs. The formula of ERS is: containing appropriate amount of potassium chloride, calcium chloride, magnesium sulfate and sodium chloride, to maintain the ion balance and osmotic pressure required for normal development of embryos, the specific concentration refers to the standard formula of zebrafish embryo culture.

[0118] 3. Place the collected healthy embryos in the incubator under the condition of 28.5℃ and appropriate humidity for normal culture to 48hpf. During the culture process, replace the culture solution regularly to keep the culture solution fresh and clean, avoiding the accumulation of metabolic waste affecting the development of embryos.

[0119] (II) Preparation of high-osmotic ERS and compound-containing solution:

[0120] 1. Preparation of hypertonic ERS: accurately weigh 10 g of sodium chloride, add it to an appropriate amount of ERS, stir to dissolve, then dilute to the desired volume with distilled water, mix thoroughly, and adjust the concentration of sodium chloride to 10 g / L. This is the hypertonic ERS.

[0121] 2. Preparation of compound solutions: according to the experimental design, weigh the required amount of sodium hyaluronate (HA), NAG, and ergothioneine (EGT). For HA, due to its large molecular weight, it needs to be heated or dissolved for a longer time to ensure complete dissolution. NAG and EGT can be directly dissolved in hypertonic ERS, and if necessary, ultrasonic treatment can be used to promote dissolution. Then dilute each compound solution to the desired concentration with hypertonic ERS.

[0122] (III) Embryo grouping and treatment:

[0123] 1. Gently transfer the 48 hpf zebrafish embryos to a 6-well plate using a pipette, placing 30 embryos in each well. During the transfer process, avoid damaging the embryos and try to select embryos with consistent development to reduce experimental errors.

[0124] 2. Carefully aspirate the ERS in the wells using a pipette, taking care not to aspirate the embryos.

[0125] 3. According to the experimental grouping, add 4 mL of hypertonic ERS containing the appropriate concentration of compounds to each well of the experimental group. The model group is added with hypertonic ERS without compounds, and the blank group is added with normal ERS (non-hypertonic). When adding the liquid, slowly add it along the well wall to avoid liquid impact on the embryos.

[0126] 4. Place the 6-well plate in an incubator and continue to culture at 28.5°C for 12 hours. During the culture process, maintain a stable environment in the incubator and avoid frequently opening the door to prevent temperature and humidity fluctuations from affecting the embryos.

[0127] (IV) After the culture ends, observe the zebrafish embryos by taking photos using conventional methods in the field. Use a body microscope and adjust the appropriate magnification (such as 10x or 20x) to take photos of the tail of each embryo under the same lighting conditions. Ensure that the tail of the embryo is in the same plane during photography to avoid measurement errors due to angle problems.

[0128] Image analysis: import the photographed images into Image J software and measure the zebrafish tail fin area using a unified method. The software automatically calculates the area of the outlined region and records the tail fin area data for each embryo. Take the average value as the tail fin area of the sample to improve the accuracy and reliability of the measurement. The statistical part does not include the tail muscle and spine.

[0129] Data collection description: when taking pictures with a stereo microscope, a NIKON SMZ18 stereo microscope is used, equipped with a high-resolution camera, and the picture taking parameters are set as follows: resolution 6000*3984, exposure time 500 ms, to ensure that the pictures are clear, with appropriate contrast, and can accurately reflect the morphology and structure of the caudal fin. Image J is used to measure the area of the zebrafish caudal fin, and the operation is performed by trained experimental personnel, and each sample is measured 3 times. Statistical analysis is performed, and p<0.05 indicates that the difference is statistically significant.

[0130] III. Experimental results and analysis, the results of which are shown in Table 3.

[0131] Table 3: Results of water replenishment and moisturizing of samples (area of zebrafish caudal fin)

[0132]

[0133]

[0134]

[0135] Compared with the model control group (between the two groups), *p<0.05, **p<0.01, ***p<0.001

[0136] The area of the zebrafish caudal fin is used to evaluate the water replenishment and moisturizing effect Based on the transparent nature of the caudal fin and its similarity to human skin. In a high permeability model, the effect of the substance on the area of the zebrafish caudal fin can be used to evaluate the water replenishment and moisturizing effect, and an increase in the area of the caudal fin corresponds to an increase in skin hydration in the human body, and the extracellular matrix swells after absorbing water.

[0137] Under the conditions of this experiment, it can be seen from Table 3 that sodium hyaluronate, N-acetyl glucosamine and ergothioneine all have water replenishment and moisturizing effect, which is manifested by an increase in the area of the zebrafish caudal fin, and the average value of the area of the zebrafish caudal fin in each group is higher than that in the model group, and the results have significant differences.

[0138] Multiple analysis is performed on the average value of the area of the zebrafish caudal fin in each group.

[0139] (1) The results of Example 4-1 compared with Comparative Example 1-1, Comparative Example 2, Comparative Example 3, Comparative Example 4-1 to 5-1 and Comparative Example 6 are shown in Table 4.

[0140] As can be seen from Table 4, under the premise of consistent concentration of the active ingredients, the effect of the combination of sodium hyaluronate, N-acetyl glucosamine and ergothioneine in a ratio of 1:2.5:0.15 is significantly better than that of sodium hyaluronate, N-acetyl glucosamine, ergothioneine alone, and the same ratio of two combinations, and among the three substances, they are complementary and synergistic.

[0141] Table 4 Sample results of water supplement and moisturizing (zebrafish tail fin area)

[0142]

[0143]

[0144] * p < 0.05, ** p < 0.01, *** p < 0.001 compared with Example 4-1 (between two groups)

[0145] (2) Example 7-1 was compared with Comparative Example 1-1, Comparative Example 2, Comparative Example 3, Comparative Example 7-1, Comparative Example 8-1 and Comparative Example 9, respectively, and the results are shown in Table 5.

[0146] As can be seen from Table 5, under the premise of consistent concentration of active ingredients, the effect of sodium hyaluronate, N-acetyl glucosamine and ergothioneine compounded at a ratio of 1:0.5:0.05 was significantly better than that of sodium hyaluronate and N-acetyl glucosamine used alone, but compared with two-by-two combinations, i.e. Comparative Examples 7-1, 8-1 and 9, the tail fin area increased, but there was no significant difference in statistical analysis.

[0147] Table 5 Sample results of water supplement and moisturizing (zebrafish tail fin area)

[0148] Group Concentration Zebrafish caudal fin area Example 7-1 150 μg / ml 0.2811±0.03 Comparative Example 1-1 150 μg / ml 0.2569±0.01* Comparative Example 2 150 μg / ml 0.2551±0.02* Comparative Example 3 150 μg / ml 0.2645±0.03 Comparative Example 7-1 150 μg / ml 0.2759±0.04 Comparative Example 8-1 150 μg / ml 0.2628±0.02 Comparative Example 9 150 μg / ml 0.2618±0.02

[0149] * p < 0.05, ** p < 0.01, *** p < 0.001 compared with Example 7-1 (between two groups)

[0150] (3) Example 8-1 was compared with Comparative Example 1-1, Comparative Example 2, Comparative Example 3, Comparative Example 10-1, Comparative Example 11-1 and Comparative Example 9, respectively, and the results are shown in Table 6.

[0151] As can be seen from Table 6, under the premise of consistent concentration of active ingredients, the effect of sodium hyaluronate, N-acetyl glucosamine and ergothioneine compounded at a ratio of 1:1:0.1 was significantly better than that of sodium hyaluronate, N-acetyl glucosamine, ergothioneine and two-by-two combinations at the same ratio used alone, and among the three substances, they were balanced and complementary, and synergistic.

[0152] Table 6 Sample results of water supplement and moisturizing (zebrafish tail fin area)

[0153]

[0154]

[0155] * p < 0.05, ** p < 0.01, *** p < 0.001 compared with Example 8-1 (between two groups)

[0156] (4) Example 9-1 was compared with Comparative Example 1-1, Comparative Example 2, Comparative Example 3, Comparative Example 12-1, Comparative Example 13-1 and Comparative Example 14 respectively. The results are shown in Table 7.

[0157] As can be seen from Table 7, under the premise of consistent concentration of active ingredients, the effect of sodium hyaluronate, N-acetylglucosamine and ergothioneine in a ratio of 1:5:2 is significantly better than using sodium hyaluronate, N-acetylglucosamine and ergothioneine alone, or in pairs in equal proportions. In the combination of the three substances, they are balanced and complementary, and synergistically enhance each other's effects.

[0158] Table 7. Results of hydration and moisturizing of samples (zebrafish tail fin area)

[0159] Example 9-1 150 μg / ml 0.3022±0.02 Comparative Example 1-1 150 μg / ml 0.2569±0.01*** Comparative Example 2 150 μg / ml 0.2551±0.02*** Comparative Example 3 150 μg / ml 0.2645±0.03*** Comparative Example 12-1 150 μg / ml 0.2774±0.03* Comparative Example 13-1 150 μg / ml 0.2642±0.02*** Comparative Example 14 150 μg / ml 0.2626±0.01***

[0160] Compared with Example 9-1 (between two groups), *p<0.05, **p<0.01, ***p<0.001

[0161] (5) Example 10-1 was compared with Comparative Example 1-1, Comparative Example 2, Comparative Example 3, Comparative Example 15-1, Comparative Example 16-1 and Comparative Example 17 respectively. The results are shown in Table 8.

[0162] As can be seen from Table 8, under the premise of consistent concentration of active ingredients, the effect of sodium hyaluronate, N-acetylglucosamine and ergothioneine in a ratio of 1:10:5 is significantly better than using sodium hyaluronate, N-acetylglucosamine and ergothioneine alone, or in pairs in equal proportions. In the combination of the three substances, they are balanced and complementary, and synergistically enhance the effect.

[0163] Table 8. Results of hydration and moisturizing of samples (cupper fin area of ​​zebrafish)

[0164]

[0165]

[0166] Compared with Example 10-1 (between two groups), *p<0.05, **p<0.01, ***p<0.001

[0167] (6) Example 11-1 was compared with Comparative Example 1-1, Comparative Example 2, Comparative Example 3, Comparative Example 18-1, Comparative Example 19-1 and Comparative Example 14 respectively. The results are shown in Table 9.

[0168] As can be seen from Table 9, under the premise of consistent efficacy ingredient concentration, the effect of the combination of sodium hyaluronate, N-acetyl glucosamine and ergothioneine in a ratio of 1:15:6 is not significantly different from the effects of the combinations of Comparative Examples 1-1, 2, 3 and the combinations of the same ratio two by two.

[0169] Table 9 Results of sample moisturizing (zebrafish tail fin area)

[0170] Example 11-1 150 μg / ml 0.2718±0.03 Comparative Example 1-1 150 μg / ml 0.2569±0.01 Comparative Example 2 150 μg / ml 0.2551±0.02 Comparative Example 3 150 μg / ml 0.2645±0.03 Comparative Example 18-1 150 μg / ml 0.2756±0.03 Comparative Example 19-1 150 μg / ml 0.2640±0.02 Comparative Example 14 150 μg / ml 0.2626±0.01

[0171] *p<0.05, **p<0.01, ***p<0.001 compared with Example 11-1 (between the two groups)

[0172] (7) Comparison of Example 4-1, Example 8-1, Example 9-1, Example 10-1 with Example 7-1, Example 11-1, respectively, and the results are shown in Table 10.

[0173] As can be seen from Table 10, Example 4-1, Example 8-1, Example 9-1, Example 10-1 are significantly different from Example 7-1, Example 11-1, respectively.

[0174] Table 10 Results of sample moisturizing (zebrafish tail fin area)

[0175]

[0176]

[0177] a indicates p<0.05 compared with Example 7-1, b indicates p<0.05 compared with Example 11-1

[0178] (8) Examples 1-1 to 6-1 respectively use sodium hyaluronate with different molecular weights, and after measuring the tail area using Image J, the average tail fin area of each group is calculated. The inhibition rate calculation formula is: inhibition rate (%) = [(experimental group area-model group area) / (blank control group area-model group area)] x 100%, and the results are shown in Table 6. The results show that the inhibition rates of Examples 2-1 to 2-5 can all reach more than 70%, and the peak value appears at a molecular weight of 30 kDa. However, when the molecular weight is 800 kDa or 20 kDa, the inhibition rate drops to below 60%, and the application of 30 kDa-600 kDa HA in moisturizing has a more optimal effect.

[0179] Table 11 Results of sample moisturizing (zebrafish tail fin area)

[0180] Group HA molecular weight (kDa) Ratio Concentration Zebrafish caudal fin area Inhibition rate (%) Example 1-1 20 1:2.5:0.15 150 μg / ml 0.2855±0.02 58 Example 2-1 30 1:2.5:0.15 150 μg / ml 0.3049±0.02 81 Example 3-1 200 1:2.5:0.15 150 μg / ml 0.3037±0.02 79 Example 4-1 400 1:2.5:0.15 150 μg / ml 0.3017±0.02 77 Example 5-1 600 1:2.5:0.15 150 μg / ml 0.3009±0.02 76 Example 6-1 800 1:2.5:0.15 150 μg / ml 0.2812±0.03 53

[0181] From the results of the moisturizing test, it can be seen that, in the composition described in the present application, the use of sodium hyaluronate When the mass ratio of sodium hyaluronate, N-acetyl glucosamine and ergothioneine is 1:(1-10):(0.1-5), especially when the molecular weight of sodium hyaluronate is 30-600 kDa, the zebrafish caudal fin area can be significantly increased, and the moisturizing effect is significantly improved.

[0182] Test Example 2 Cartilage Repair Test

[0183] Zebrafish embryos are transparent, which facilitates real-time observation of the cartilage development process; the pharyngeal and cranial cartilage structure is simple and highly conserved, and has high homology with the core regulatory genes of human cartilage development (such as Sox9 and Col2a1). High-dose Dexamethasone is used to induce zebrafish chondrocyte apoptosis and matrix degradation, simulating the pathological process of human cartilage injury. Alcian Blue can specifically bind to acidic glycosaminoglycans, and the latter is the main component of cartilage matrix. The staining intensity and changes in cartilage morphology can be used to directly evaluate the cartilage repair effect.

[0184] In this study, a Dexamethasone-induced zebrafish cartilage injury model was first established, and the Alcian Blue staining technique was used to evaluate the potential efficacy of sodium hyaluronate The potential efficacy of J, N-acetyl glucosamine (NAG), ergothioneine and other compounds on cartilage repair provides new experimental basis for the development of cartilage repair drugs.

[0185] I. Experimental Methods

[0186] The powders of Examples 1-2 to 11-2, Comparative Example 1-2, Comparative Example 2, Comparative Example 3, and Comparative Examples 4-2 to 5-2, Comparative Example 6, Comparative Examples 7-2 to 8-2, Comparative Example 9, Comparative Examples 10-2 to 13-2, Comparative Example 14, Comparative Examples 15-2 to 16-2, Comparative Example 17, and Comparative Examples 18-2 to 19-2 were prepared as solutes, and the solvent was standard dilution water.

[0187] 1. Zebrafish strain: Tübingen (TU) strain wild-type zebrafish.

[0188] 2. Reagents: Dexamethasone, Trypsin (source leaf, s10031-100g), Alcian Blue, hyaluronic acid J, N-acetyl glucosamine (NAG), ergothioneine.

[0189] Other reagents: Embryo Raising Solution (ERS), 4% paraformaldehyde (PFA, fixative), potassium hydroxide (KOH, for bleaching), glycerol (tissue preservation and transparency), etc.

[0190] 3. Instruments and equipment: 6-well plates (cell culture specific, for culturing zebrafish embryos), stereomicroscope, for embryo morphological observation and photography, Image J software (for image analysis and data measurement), incubator (temperature controllable, to maintain suitable environment for embryo culture), etc.

[0191] II. Observation and measurement

[0192] (I) Zebrafish embryo feeding and grouping

[0193] 1. Zebrafish embryo feeding

[0194] Adult TU zebrafish were mated at a ratio of 1:2 (female:male), and the fertilized embryos were collected and washed with embryo culture solution (ERS, containing 60 mg / L KCl, 140 mg / L CaCl2, 200 mg / L MgSO4, 5 g / L NaCl, pH 7.2-7.4) for 3 times to remove unfertilized eggs and impurities. The embryos were placed in a 28.5°C constant temperature incubator and cultured for 24 hours (24hpf), during which the fresh ERS was replaced every 12 hours to maintain water quality.

[0195] 2. Compound treatment and injury induction

[0196] Drug intervention stage: 24-48hpf: After the ERS was removed, ERS containing the composition (4mL / well) was added, and the culture solution containing the drug was replaced every 12 hours to protect the embryos and enhance their subsequent anti-injury ability.

[0197] 48hpf-62dpf: ERS containing the composition and 7μM dexamethasone was used, the model group was added with 7μM dexamethasone ERS without the composition, and the blank group was added with normal ERS, to continuously induce cartilage injury and observe the repair effect, and the liquid was replaced every 12 hours (to avoid drug concentration decay), and the temperature and humidity were kept constant during the culture process.

[0198] (II) Cartilage staining and sample processing

[0199] 1. Fixation and washing

[0200] After 6 days of culture (6dpf), the embryos were fixed with 4% paraformaldehyde (PFA) at 4°C for 24 hours to ensure the stability of the cartilage structure. After fixation, the embryos were washed with PBS for 3 times, 5 minutes each time, to remove residual fixative.

[0201] 2. Alcian blue staining

[0202] Staining solution preparation: 0.1 mg / mL Alcian blue, prepared fresh before use.

[0203] Staining step: embryos were immersed in the staining solution and stained for 60 minutes at room temperature (adjust the time according to the staining depth to avoid over-staining or under-staining), and slight shaking was performed to ensure uniform staining.

[0204] 3. Rehydration and bleaching

[0205] Gradient rehydration: sequentially immerse the embryos in 90%, 50%, and 30% ethanol solutions for 5 minutes each to gradually remove ethanol and prevent the precipitation of the staining agent.

[0206] Bleaching treatment: mix equal volumes of 1% H2O2 and 1% KOH (prepare fresh), and treat the embryos until the pigment is completely removed (about 30 minutes) to avoid interference with the observation of cartilage by melanin.

[0207] 4. Trypsin digestion and transparency

[0208] Digestion treatment: use a 30% sodium tetraborate solution with 50 mg / mL trypsin to treat the embryos overnight (16-18 hours) to digest the soft tissue and highlight the cartilage structure. Glycerol gradient dehydration: sequentially immerse the embryos in 20%, 50%, 80%, and 100% glycerol (containing 1% KOH) for 1 hour each to replace the water in the tissue and make the cartilage transparent for easy microscopic observation.

[0209] Photographing parameters: take photographs of the ventral side of the embryos under a stereomicroscope (10-20x objective lens) with fixed focal length and exposure time to ensure image consistency.

[0210] Cartilage counting criteria: the normal zebrafish pharyngeal skull cartilage is bilaterally symmetrical, including Meckel's cartilage, palatine cartilage, and gill arch cartilage. Record the number of complete cartilages in each embryo.

[0211] Data calculation

[0212] Cartilage repair rate (%) = [(number of cartilages in the experimental group - number of cartilages in the model group) / (number of cartilages in the blank control group - number of cartilages in the model group)] x 100% (Note: the number of cartilages in the model group is lower than that in the blank control group, and the higher the repair rate indicates the stronger the effect of the compound)

[0213] Statistical analysis

[0214] Set the significance level to P<0.05, and when P<0.05, consider that the difference is statistically significant.

[0215] Three, experimental results and analysis, the results are shown in Table 12.

[0216] Table 12

[0217]

[0218]

[0219] * indicates comparison with model control group (between two groups), *p <0.05, **p <0.01, ***p <0.001

[0220] In the zebrafish gill arch / hyoid cartilage region, the number of complete bifurcated cartilage branches (such as the "Y" shape structure of ceratobranchial cartilage) was counted. The cartilage fracture / absence in the dexamethasone group resulted in a decrease in the number of roots, and the recovery of the number of roots after repair treatment indicated the reconstruction of the structure, reflecting the morphological recovery of the cartilage branches, suggesting cell migration, proliferation and differentiation, and the reconstruction of the three-dimensional structure.

[0221] Under the experimental conditions, it can be seen from Table 12 that sodium hyaluronate J, N-acetylglucosamine and ergothioneine all have cartilage repair effects, which are manifested as an increase in the number of zebrafish cartilage roots, and the average number of zebrafish cartilage in each group is higher than that in the model group, and the results have significant differences.

[0222] The average number of zebrafish cartilage in each group was subjected to multiple analysis.

[0223] (1) The results of comparing Example 3-2 with Comparative Example 1-2, Comparative Example 2-3, Comparative Example 4-2 to 5-2 and Comparative Example 6 are shown in Table 13.

[0224] As can be seen from Table 13, sodium hyaluronate J, N-acetylglucosamine and ergothioneine have better effects than sodium hyaluronate J, N-acetylglucosamine, ergothioneine and the same proportion of two combinations.

[0225] Table 13 Cartilage repair results of samples

[0226]

[0227] * indicates comparison with Example 3-2 (between two groups), *p <0.05, **p <0.01, ***p <0.001

[0228] (2) The results of comparing Example 7-2 with Comparative Example 1-2, Comparative Example 2, Comparative Example 3, Comparative Example 7-2, Comparative Example 8-2 and Comparative Example 9 are shown in Table 14.

[0229] As can be seen from Table 14, sodium hyaluronate J, N-acetylglucosamine and ergothioneine have better effects than sodium hyaluronate J, N-acetylglucosamine and ergothioneine, but compared with Comparative Example 7-2, the statistical analysis of the number of zebrafish cartilage roots had no significant difference.

[0230] Table 14 Cartilage repair results

[0231] Group Concentration Zebrafish cartilage root number Example 7-2 150 μg / ml 12.9412±1.98 Comparative Example 1-2 150 μg / ml 11.1667±1.62** Comparative Example 2 150 μg / ml 9.5909±1.68*** Comparative Example 3 150 μg / ml 8.3333±2.07*** Comparative Example 7-2 150 μg / ml 12.0800±1.26 Comparative Example 8-2 150 μg / ml 11.4615±0.78* Comparative Example 9 150 μg / ml 11.3636±1.05**

[0232] * indicates comparison between Example 7-2 and (between groups), *p <0.05, **p <0.01, ***p <0.001

[0233] (3) Example 8-2 was compared with Comparative Example 1-2, Comparative Example 2, Comparative Example 3, Comparative Example 10-2, Comparative Example 11-2, and Comparative Example 9, respectively, and the results are shown in Table 15.

[0234] As can be seen from Table 15, sodium hyaluronate J, N-acetylglucosamine, and ergothioneine were compounded in the same ratio of 1:5:2. J, N-acetylglucosamine, and ergothioneine were compounded in the same ratio of 1:5:2.

[0235] Table 15 Cartilage repair results

[0236] Group Concentration Zebrafish cartilage root number Example 8-2 150 μg / ml 13.9333±0.88 Comparative Example 1-2 150 μg / ml 11.1667±1.62*** Comparative Example 2 150 μg / ml 9.5909±1.68*** Comparative Example 3 150 μg / ml 8.3333±2.07*** Comparative Example 10-2 150 μg / ml 12.3846±0.87*** Comparative Example 11-2 150 μg / ml 11.3846±0.77*** Comparative Example 9 150 μg / ml 11.3636±1.05***

[0237] * indicates comparison between Example 8-2 and (between groups), *p <0.05, **p <0.01, ***p <0.001

[0238] (4) Example 9-2 was compared with Comparative Example 1-2, Comparative Example 2, Comparative Example 3, Comparative Example 12-2, Comparative Example 13-2, and Comparative Example 14, respectively, and the results are shown in Table 16.

[0239] As can be seen from Table 16, sodium hyaluronate J, N-acetylglucosamine, and ergothioneine were compounded in the same ratio of 1:5:2. J, N-acetylglucosamine, and ergothioneine were compounded in the same ratio of 1:5:2.

[0240] Table 16 Cartilage repair results

[0241] Group Concentration Zebrafish cartilage root number Example 9-2 150 μg / ml 13.3000±0.84 Comparative Example 1-2 150 μg / ml 11.1667±1.62** Comparative Example 2 150 μg / ml 9.5909±1.68*** Comparative Example 3 150 μg / ml 8.3333±2.07*** Comparative Example 12-2 150 μg / ml 12.3077±0.75* Comparative Example 13-2 150 μg / ml 11.3077±0.85*** Comparative Example 14 150 μg / ml 11.3846±0.65***

[0242] * indicates comparison between Example 9-2 and (between groups), *p <0.05, **p <0.01, ***p <0.001

[0243] (5) Example 10-2 was compared with Comparative Example 1-2, Comparative Example 2, Comparative Example 3, Comparative Example 15-2, Comparative Example 16-2, and Comparative Example 17, respectively, and the results are shown in Table 17.

[0244] As can be seen from Table 17, sodium hyaluronate J, N-acetylglucosamine and ergothioneine compounded in a ratio of 1:10:5 are better than sodium hyaluronate alone J, N-acetylglucosamine, ergothioneine and the same ratio of two two-compound.

[0245] Table 17 Cartilage repair results

[0246] Group Concentration Zebrafish cartilage root number Example 10-2 150 μg / ml 13.5000±1.45 Comparative Example 1-2 150 μg / ml 11.1667±1.62*** Comparative Example 2 150 μg / ml 9.5909±1.68*** Comparative Example 3 150 μg / ml 8.3333±2.07*** Comparative Example 15-2 150 μg / ml 12.2308±0.60** Comparative Example 16-2 150 μg / ml 11.1538±0.90*** Comparative Example 17 150 μg / ml 11.2308±0.44***

[0247] * indicates comparison between Example 10-2 and (two groups), *p <0.05, **p <0.01, ***p <0.001

[0248] (6) Example 11-2 was compared with Comparative Example 1-2, Comparative Example 2, Comparative Example 3, Comparative Example 18-2, Comparative Example 19-2 and Comparative Example 14, respectively, and the results are shown in Table 18.

[0249] As can be seen from Table 18, sodium hyaluronate J, N-acetylglucosamine and ergothioneine compounded in a ratio of 1:15:6 are better than sodium hyaluronate alone J, N-acetylglucosamine, ergothioneine, but compared with two two-compound Comparative Example 18-2, there was no significant difference in the statistical analysis of the number of zebrafish cartilage roots.

[0250] Table 18 Cartilage repair results

[0251] Group Concentration Zebrafish cartilage root number Example 11-2 150 μg / ml 13.1500±1.60 Comparative Example 1-2 150 μg / ml 11.1667±1.62*** Comparative Example 2 150 μg / ml 9.5909±1.68*** Comparative Example 3 150 μg / ml 8.3333±2.07*** Comparative Example 18-2 150 μg / ml 12.3846±1.04 Comparative Example 19-2 150 μg / ml 11.2308±0.83*** Comparative Example 14 150 μg / ml 11.3846±0.65***

[0252] * indicates comparison between Example 11-2 and (two groups), *p <0.05, **p <0.01, ***p <0.001

[0253] In summary, the composition described in the present application has synergistic effect, which can significantly increase the area of zebrafish tail fin, i.e. has significant water and moisture retention effect, and can significantly increase the number of zebrafish cartilage roots, i.e. has significant tissue repair, i.e. cartilage repair effect.

[0254] The above is only a preferred embodiment of the present application, and is not intended to limit the present application in other forms. Any skilled person in the art can modify or change the above disclosed technical content to equivalent embodiments. However, any simple modification, equivalent change and modification made on the basis of the technical essence of the present application to the above embodiments, without departing from the technical solution content of the present application, still belongs to the protection scope of the present application.

Claims

1. An oral composition comprising hyaluronic acid or a salt thereof, N-acetyl glucosamine and ergothioneine; wherein the mass ratio of the sodium hyaluronate or a salt thereof, N-acetyl glucosamine and ergothioneine is 1 : 1-10: 0.1-5.

2. The oral composition according to claim 1, wherein the molecular weight of the hyaluronic acid or a salt thereof is 20k-2200kDa.

3. A product comprising the composition of claim 1 or 2 and a physiologically acceptable adjuvant.

4. Use of ergothioneine for enhancing the moisturizing of N-acetyl glucosamine and hyaluronic acid or a salt thereof, for oral use.

5. The use according to claim 4, wherein the mass ratio of the sodium hyaluronate or a salt thereof, N-acetyl glucosamine and ergothioneine is 1 : 1-10: 0.1-5.

6. The use according to claim 4 or 5, wherein the molecular weight of the hyaluronic acid or a salt thereof is 30k-600k Da.

7. Use of ergothioneine for enhancing the cartilage repair of N-acetyl glucosamine and hyaluronic acid or a salt thereof, for oral use.

8. The use according to claim 7, wherein the mass ratio of the sodium hyaluronate or a salt thereof, N-acetyl glucosamine and ergothioneine is 1 : 1-10: 0.1-5.

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