Composition of micromolecular hyaluronic acid or salt thereof as well as preparation method and application of composition

Abstract

The invention discloses a composition of micromolecular hyaluronic acid or salt thereof and a preparation method and application thereof, the composition comprises hyaluronic acid disaccharide or salt thereof, and the hyaluronic acid disaccharide or salt thereof is a mixture of saturated hyaluronic acid disaccharide or salt thereof and unsaturated hyaluronic acid disaccharide or salt thereof. According to the invention, high-concentration high-molecular-weight hyaluronic acid or salt thereof is synergistically degraded into saturated hyaluronic disaccharide or salt thereof and unsaturated hyaluronic disaccharide or salt thereof through double enzymes, the yield is high, an obvious synergistic interaction effect is shown in the aspects of moisturizing and aging resistance, a new idea is provided for preparing hyaluronic disaccharide in academic and industrial circles, and the application prospect is wide. The method has important academic value and economic significance.

Description

TECHNICAL FIELD

[0001] The present application relates to a small molecule hyaluronic acid or salt thereof composition, its preparation method and application, and belongs to the field of bioengineering technology. BACKGROUND

[0002] Hyaluronic acid disaccharide is the smallest structural unit of hyaluronic acid, with a theoretical molecular weight of 397 daltons. According to the 500 dalton rule proposed by researchers, in the field of skin science and skin care products, it is determined that hyaluronic acid disaccharide can quickly pass through the epidermal barrier to the dermis layer to play the role of deep moisturizing and anti-aging.

[0003] Patent CN115386608B discloses a method for preparing a high-activity hyaluronic acid oligosaccharide composition by real-time regulation of enzyme activity. The method needs to prepare an enzyme-promoting agent first, then perform secondary enzymolysis, and subsequently needs to remove the introduced small molecule enzyme-promoting agent impurities by chromatographic purification. The process is complicated and the production cost is high. Patent CN110982862B discloses a method for large-scale preparation of high-purity unsaturated hyaluronic acid disaccharide. The method adopts a two-time enzymolysis and two-time purification strategy, and the process is complicated. Patent CN116179629A discloses a preparation method of unsaturated sodium hyaluronate disaccharide. Although a single enzymolysis is adopted, the enzyme substrate concentration is low and the disaccharide conversion rate is low. It can be seen that most of the existing technologies currently focus on the preparation of unsaturated hyaluronic acid disaccharide, and all of them adopt a single hyaluronidase for enzymolysis. There is no report on the use of multiple hyaluronidases for enzymolysis. SUMMARY

[0004] The inventors have found that the combination of saturated hyaluronic acid disaccharide and unsaturated hyaluronic acid disaccharide has a significant anti-aging and moisturizing effect. Based on this, the present application provides a small molecule hyaluronic acid or salt thereof composition, which contains both saturated hyaluronic acid disaccharide and unsaturated hyaluronic acid disaccharide, and has a synergistic anti-aging and moisturizing effect.

[0005] The small molecule hyaluronic acid or salt thereof composition provided by the present application comprises hyaluronic acid disaccharide or salt thereof, and the hyaluronic acid disaccharide or salt thereof is a mixture of saturated hyaluronic acid disaccharide or salt thereof and unsaturated hyaluronic acid disaccharide or salt thereof.

[0006] Further, the small molecule hyaluronic acid or salt thereof composition described above can only contain saturated hyaluronic acid disaccharide or salt thereof and unsaturated hyaluronic acid disaccharide or salt thereof, or can further contain other components, such as hyaluronic acid tetrasaccharide or salt thereof and hyaluronic acid hexasaccharide or salt thereof.

[0007] Further, the mass ratio of the saturated hyaluronic acid disaccharide or salt thereof and the unsaturated hyaluronic acid disaccharide or salt thereof can be at least 1:0.05-10, 1:0.08-8, 1:0.1-5, 1:0.8-2.2, and specifically, the mass ratio can be specifically 1:0.05, 1:0.06, 1:0.07, 1:0.08, 1:0.09, 1:0.1, 1:0.2, 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, 1:5.5, 1:6, 1:6.5, 1:7, 1:7.5, 1:8, 1:8.5, 1:9, 1:9.5, 1:10, and any ratio therebetween.

[0008] Preferably, the mass ratio can be 1:0.5-2.5. More preferably, the mass ratio can be 1:1-2.

[0009] Further, the reducing end of the saturated hyaluronic acid disaccharide or salt thereof and the unsaturated hyaluronic acid disaccharide or salt thereof can be N-acetylglucosamine, uronic acid.

[0010] Preferably, the reducing end of the saturated hyaluronic acid disaccharide or salt thereof and the unsaturated hyaluronic acid disaccharide or salt thereof can be N-acetylglucosamine.

[0011] The present application also provides a preparation method of a small molecule hyaluronic acid or salt composition, which comprises the step of subjecting high molecular weight hyaluronic acid or salt thereof to double enzyme hydrolysis by using hyaluronidase and hyaluronate lyase.

[0012] Further, the hyaluronidase can be at least endo-beta-N-acetylglucosaminidase (EC 3.2.1.35).

[0013] Preferably, the hyaluronidase can be recombinant Pachycrepocephalus duffi hyaluronidase.

[0014] Further, the hyaluronate lyase (EC 4.2.2.1) can be at least a hyaluronate lyase of microbial origin.

[0015] Preferably, the hyaluronate lyase (EC 4.2.2.1) can be a hyaluronate lyase of bacterial origin.

[0016] Further, the enzyme activity of the hyaluronidase in the enzyme system can be 1x10 5 ~ 3x10 5 U / mL, and specifically can be 1x10 51.5×10 5 2×10 5 2.5×10 5 3×10 5 And the activity of any enzyme within it.

[0017] Preferably, the enzymatic activity of the hyaluronic acid lyase in the enzymatic hydrolysis system is 10-100 U / mL; specifically, it can be 10 U / mL, 11 U / mL, 12 U / mL, 13 U / mL, 14 U / mL, 15 U / mL, 16 U / mL, 17 U / mL, 18 U / mL, 19 U / mL, 20 U / mL, 21 U / mL, 22 U / mL, 23 U / mL, 24 U / mL, 25 U / mL, 26 U / mL, 27 U / mL, 28 U / mL, 29 U / mL, 30 U / mL, 31 U / mL, 32 U / mL, 33 U / mL, 34 U / mL, 35 U / mL, 36 U / mL, 37 U / mL, 38 U / mL, 39U / mL, 40 U / mL, 41 U / mL, 42 U / mL, 43 U / mL, 44 U / mL, 45 U / mL, 46 U / mL, 47 U / mL, 48 U / mL, 49 U / mL, 50 U / mL, 51 U / mL, 52 U / mL, 53 U / mL, 54 U / mL, 55 U / mL, 56 U / mL, 57 U / mL, 58 U / mL, 59 U / mL, 60 U / mL, 61 U / mL, 62 U / mL, 63 U / mL, 64 83 U / mL, 84 U / mL, 85 U / mL, 86 U / mL, 87 U / mL, 88 U / mL, 89 U / mL, 90 U / mL, 91 U / mL, 92 U / mL, 93 U / mL, 94 U / mL, 95 U / mL, 96 U / mL, 97U / mL, 98 U / mL, 99 U / mL, 100 U / mL and any value in between.

[0018] Preferably, during enzymatic hydrolysis, hyaluronic acid hydrolase is added first for enzymatic hydrolysis, and then hyaluronic acid lysin is added for enzymatic hydrolysis.

[0019] Preferably, during enzymatic hydrolysis, hyaluronic acid hydrolase and hyaluronic acid lysin are added simultaneously.

[0020] Further, the concentration of the high molecular weight hyaluronic acid or its salt is at least 2% to 20%; specifically, the concentration can be 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, and any value between these values.

[0021] Furthermore, the molecular weight of the high molecular weight hyaluronic acid or its salt is at least 400 kDa - 1500 kDa; specifically, the molecular weight can be any value between 400 kDa, 500 kDa, 600 kDa, 700 kDa, 800 kDa, 900 kDa, 1000 kDa, 1100 kDa, 1200 kDa, 1300 kDa, 1400 kDa, and 1500 kDa.

[0022] Furthermore, the pH of the enzymatic hydrolysis system is controlled at least between 4.0 and 6.5; specifically, the pH can be any value between 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, and 6.5.

[0023] Furthermore, the enzymatic hydrolysis temperature is controlled at least between 25°C and 40°C; specifically, the temperature can be any value between 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, and so on.

[0024] Furthermore, the enzymatic hydrolysis time should be controlled at least between 12h and 48h; specifically, the time can be any value between 12h, 13h, 14h, 15h, 16h, 17h, 18h, 19h, 20h, 21h, 22h, 23h, 24h, 25h, 26h, 27h, 28h, 29h, 30h, 31h, 32h, 33h, 34h, 35h, 36h, 37h, 38h, 39h, 40h, 41h, 42h, 43h, 44h, 45h, 46h, 47h, and 48h.

[0025] Furthermore, the enzymatic hydrolysis process also includes at least an enzyme inactivation step.

[0026] Furthermore, the enzymatic hydrolysis process also includes at least a filtration step.

[0027] Furthermore, the enzymatic hydrolysis process also includes at least a drying step.

[0028] Furthermore, the enzymatic hydrolysis process also includes at least a separation step.

[0029] Any method applicable to enzyme inactivation, filtration, drying, and separation of hyaluronic acid products in this field is applicable to this technical solution, and no further limitations are made here.

[0030] The present invention also provides a small molecule hyaluronic acid or a salt composition thereof prepared according to the above preparation method.

[0031] Furthermore, the composition of the small molecule hyaluronic acid or its salt includes hyaluronic acid disaccharide or its salt, wherein the hyaluronic acid disaccharide or its salt is a mixture of saturated hyaluronic acid disaccharide or its salt and unsaturated hyaluronic acid disaccharide or its salt.

[0032] Furthermore, the above-mentioned composition of small molecule hyaluronic acid or its salt may contain only saturated hyaluronic acid disaccharide or its salt and unsaturated hyaluronic acid disaccharide or its salt, or may contain other components, such as hyaluronic acid tetrasaccharide or its salt, hyaluronic acid hexasaccharide or its salt.

[0033] Furthermore, the mass ratio of saturated hyaluronic acid disaccharide or its salt to unsaturated hyaluronic acid disaccharide or its salt can be at least 1:0.05-10, 1:0.08-8, 1:0.1-5, or 1:0.8-2.2. Specifically, the mass ratio can be 1:0.05, 1:0.06, 1:0.07, 1:0.08, 1:0.09, 1:0.1, 1:0.2, or 1:0. 3, 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, 1:5.5, 1:6, 1:6.5, 1:7, 1:7.5, 1:8, 1:8.5, 1:9, 1:9.5, 1:10, and any ratios thereof.

[0034] Preferably, the mass ratio can be 1:0.5-2.5.

[0035] More preferably, the mass ratio can be 1:1-2.

[0036] Furthermore, the reducing end of the saturated hyaluronic acid disaccharide or its salt and the unsaturated hyaluronic acid disaccharide or its salt can be N-acetylglucosamine or uronic acid.

[0037] Preferably, the reducing end of the saturated hyaluronic acid disaccharide or its salt and the unsaturated hyaluronic acid disaccharide or its salt can be N-acetylglucosamine.

[0038] The present invention also provides a product comprising the above-described composition of small molecule hyaluronic acid or its salts.

[0039] Furthermore, the product also includes physiologically acceptable excipients.

[0040] Specifically, the excipients may be suitable solvents, propellants, solubilizers, co-solvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, binding agents, penetration promoters, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculants and anti-flocculation agents, filter aids, release inhibitors, etc.

[0041] The product of this application can be prepared using general methods, wherein one or more diluents or carriers may be added. The product of this application can be prepared into any formulation commonly prepared in this field. For example, it can be formulated into creams, lotions, emulsions, masks, foundations, medical devices, hair cosmetics, etc. Specifically, skin lotions, skin softeners, hyaluronic acid injections, skin toners, astringents, lotions, moisturizing lotions, nourishing lotions, massage creams, nourishing creams, moisturizing creams, hand creams, foundations, serums, nourishing essences, masks, soaps, cleansing foams, cleansing milks, cleansing creams, lotions, or shower gels.

[0042] The products of this application may also be mixed with other ingredients commonly formulated in cosmetics, as needed. These may include, for example, oils, moisturizers, surfactants, organic pigments, inorganic pigments, UV absorbers, preservatives, bactericides, antioxidants, plant extracts, pH adjusters, alcohols, colorants, fragrances, blood circulation enhancers, cooling agents, antiperspirants, or may be mixed with purified water.

[0043] The present invention also provides the use of the above-described small molecule hyaluronic acid or its salt composition and / or the small molecule hyaluronic acid or its salt composition prepared according to the above preparation method in the preparation of products that promote CD44 expression and / or products that promote aquaporin AQP3 expression and / or products that promote HAS1 gene expression and / or products that promote HAS2 gene expression and / or products that promote telomere relative length.

[0044] The present invention also provides the application of the above-mentioned small molecule hyaluronic acid or its salt composition and / or the small molecule hyaluronic acid or its salt composition prepared according to the above preparation method in improving the extracellular matrix (ECM).

[0045] The present invention also provides the use of the above-described small molecule hyaluronic acid or its salt composition and / or the small molecule hyaluronic acid or its salt composition prepared according to the above preparation method in the preparation of anti-skin aging products and / or skin moisturizing products and / or anti-wrinkle products and / or products for reducing eye bags and / or products for improving dark circles.

[0046] Furthermore, the improvement of dark circles includes, but is not limited to, vascular dark circles, structural dark circles, and pigmented dark circles, with vascular dark circles being preferred.

[0047] Furthermore, the improvement of dark circles includes, but is not limited to, reducing the melanin MI value, skin color b value, L value, improving microcirculation around the eyes, and inhibiting angiogenesis.

[0048] Furthermore, the anti-wrinkle effects include, but are not limited to, skin anti-wrinkle and improvement of fine lines around the eyes.

[0049] Furthermore, the reduction of eye bags includes, but is not limited to, reducing the area of ​​eye bags, relieving chronic inflammation, and reducing eye bag puffiness.

[0050] The present invention also provides the application of the above-mentioned small molecule hyaluronic acid or its salt composition and / or the small molecule hyaluronic acid or its salt composition prepared according to the above preparation method in cosmetics and pharmaceutical products.

[0051] Furthermore, the applications mentioned above include, but are not limited to, minimally invasive cosmetic skincare products, regular skincare products, hyaluronic acid injections, and dermal fillers.

[0052] The present invention has the following beneficial effects: 1. This invention is the first to combine saturated hyaluronic acid disaccharide with unsaturated hyaluronic acid disaccharide. This combination is significantly better than either a single saturated disaccharide or a single unsaturated disaccharide in promoting CD44 synthesis, showing a significant synergistic effect.

[0053] 2. This invention provides a method for the degradation of hyaluronic acid or its salts using a dual-enzyme approach. By combining the two enzymes, the degradation products simultaneously contain both saturated and unsaturated hyaluronic acid disaccharides. This method achieves high disaccharide yields, reaching 92.95 g / L or higher, with a conversion rate exceeding 90%. This method provides a new approach for the preparation of hyaluronic acid disaccharides in both academia and industry.

[0054] 3. The small molecule hyaluronic acid or its salt composition obtained by double enzymatic hydrolysis in this invention promotes CD44 expression, promotes aquaporin AQP3 expression, promotes HAS1 gene expression, and promotes HAS2 gene expression. 、It exhibits better effects in promoting the relative length of telomeres than the combination of saturated and unsaturated hyaluronic acid disaccharides, with superior performance and more prominent anti-aging, moisturizing, anti-wrinkle, eye bag smoothing, and dark circle improvement effects. Attached Figure Description

[0055] Figure 1 This is a high-performance liquid chromatogram of the enzymatic hydrolysis product in Example 1; Figure 2 The total particle chromatogram of the enzymatic hydrolysis products in Example 1 is shown in the HPLC-MS mass spectrometry. Figure 3 This is an ionic strength graph of the enzymatic hydrolysis products in Example 1; Figure 4 The effect of different samples on CD44 expression in Experiment 1; Figure 5 The effect of different samples on AQP3 expression in Experimental Example 2; Figure 6 The effect of different samples on HAS1 expression in Experiment 3; Figure 7 The effect of different samples on HAS2 expression in Experiment 3; Figure 8 The effect of hyaluronic acid disaccharide on the relative length of telomeres in Experiment Example 4; Figure 9 The difference in skin moisture content before and after using the sample; Figure 10 The difference in the percentage of crow's feet area before and after using the sample; Figure 11 The difference in melanin MI values ​​in the skin before and after using the sample; Figure 12 Skin color L before and after using the sample Value difference; Figure 13 Skin color b before and after using the sample Value difference; Figure 14 The difference in dark circle level scores before and after using the sample; Figure 15 Examples of improved dark circles under the eyes of test subjects; Figure 16 The difference in eye bag volume before and after using the sample; Figure 17 The difference in eye bag grade score before and after using the sample; Figure 18 Examples of improved under-eye bags in test subjects. Detailed Implementation

[0056] The present invention will be further described below with reference to embodiments. It should be understood that the embodiments are only used to further illustrate and explain the present invention, and are not intended to limit the present invention.

[0057] Unless otherwise defined, technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art. While similar or identical methods and materials may be applied in experimental or practical applications, materials and methods are described herein. In case of conflict, the definitions included herein shall prevail. Furthermore, materials, methods, and examples are for illustrative purposes only and are not intended to be limiting. The invention is further described below with reference to specific embodiments, but is not intended to limit the scope of the invention.

[0058] The present invention provides a hyaluronic acid disaccharide or a salt thereof (HA2), wherein the hyaluronic acid disaccharide or salt thereof is a composition of saturated hyaluronic acid disaccharide or salt thereof and unsaturated hyaluronic acid disaccharide or salt thereof.

[0059] Furthermore, the mass ratio of the saturated hyaluronic acid disaccharide or its salt to the unsaturated hyaluronic acid disaccharide or its salt is 1:0.1-5, for example 1:0.1, 1:0.2, 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1.0, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2.0, 1:2.1, 1:2.2, 1:2.3, 1:2.4, 1:2.5, 1:3.0, 1:3.5, 1:4.0, 1:4.5, 1:5.0, preferably 1:0.5-2.5, more preferably 1:1-2.

[0060] The present invention also provides a composition of small molecule hyaluronic acid or its salt, the composition comprising the above-mentioned hyaluronic acid disaccharide or its salt.

[0061] Furthermore, the content of hyaluronic acid disaccharide or its salt in the composition of small molecule hyaluronic acid or its salt is 85wt%-98wt%, for example 85wt%, 86wt%, 87wt%, 88wt%, 89wt%, 90wt%, 91wt%, 92wt%, 93wt%, 94wt%, 95wt%, 96wt%, 97wt%, and 98wt%.

[0062] Furthermore, the reducing end of both the saturated hyaluronic acid disaccharide or its salt and the unsaturated hyaluronic acid disaccharide or its salt is N-acetylglucosamine.

[0063] Furthermore, the hyaluronic acid disaccharide or its salt refers to at least one of the following: hyaluronic acid disaccharide, sodium salt, calcium salt, zinc salt, potassium salt, and manganese salt of hyaluronic acid disaccharide.

[0064] The present invention also provides a feasible method for preparing the composition of the low molecular weight hyaluronic acid or its salt, which includes a step of double enzymatic hydrolysis of high molecular weight hyaluronic acid or its salt using hyaluronic acid hydrolase and hyaluronic acid lysin.

[0065] Furthermore, the hyaluronic acid hydrolase is an endopeptidase β-N-acetylglucosidase (EC3.2.1.35), which can hydrolyze the β-1,4 glycosidic bond of hyaluronic acid to produce a saturated final product with N-acetylglucosamine at the reducing end.

[0066] Furthermore, the hyaluronic acid lyase (EC 4.2.2.1) is a microbial hyaluronic acid lyase, belonging to the first class of hyaluronidases. It can cleave the β-1,4 glycosidic bond of hyaluronic acid, forming an unsaturated bond at the 4,5 glycosidic bond of glucuronic acid, and producing an unsaturated final product with N-acetylglucosamine at the reducing end.

[0067] In one embodiment of the present invention, the hyaluronic acid hydrolase used To Hyaluronic acid hydrolase derived from the giant toothed ant was further prepared using the method described in CN114350691B.

[0068] In one embodiment of the present invention, the hyaluronic acid lysin used is a bacterial hyaluronic acid lysin.

[0069] In one embodiment of the present invention, the hyaluronic acid lyase is a lyase derived from Bacillus subtilis. Preferably, the Bacillus subtilis has the accession number CGMCC No. 5744, the accession date is February 8, 2012, and the accession institution is the China General Microbiological Culture Collection Center (CGMCC). Further, it is prepared using the method in CN103255076B.

[0070] In one embodiment of the present invention, the hyaluronic acid lyase is a lyase derived from Citrobacter freundii. Preferably, the Citrobacter freundii has the accession number CGMCC1.61949 and is deposited at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0071] Furthermore, the molecular weight of the high molecular weight hyaluronic acid or its salt is 400kDa-1500kDa, for example 400kDa, 500kDa, 600kDa, 700kDa, 800kDa, 900kDa, 1000kDa, 1100kDa, 1200kDa, 1300kDa, 1400kDa, and 1500kDa.

[0072] Furthermore, the salt of the high molecular weight hyaluronic acid refers to at least one of its sodium salt, calcium salt, zinc salt, potassium salt, and manganese salt.

[0073] Furthermore, the enzymatic hydrolysis is carried out in an aqueous environment. High molecular weight hyaluronic acid or its salts, hyaluronic acid lysin, and hyaluronic acid hydrolase are dissolved in water. To meet the pH requirements for enzymatic hydrolysis, a pH adjuster can also be added. All these raw materials are combined to form the enzymatic hydrolysis system.

[0074] In one specific embodiment, in the enzymatic hydrolysis system, the concentration of the substrate high molecular weight hyaluronic acid or its salt is controlled at 2wt% to 20wt%, for example, it can be 2%, 5%, 10%, 15%, or 20%. For example, if the enzymatic hydrolysis reaction system formed by mixing hyaluronidase, substrate, water, and pH adjuster is 1 L, the amount of high molecular weight hyaluronic acid or its salt added is 20-200 g.

[0075] In one specific embodiment, the activity of hyaluronic acid hydrolase in the enzymatic hydrolysis system is controlled at 1×10⁻⁶. 5 ~3×10 5 U / mL (i.e., enzyme activity of 1×10⁻⁶ per milliliter of reaction system) 5 ~3×10 5 U), for example, can be 1×10 5 U / mL, 2×10 5 U / mL, 3×10 5 U / mL. The enzyme activity unit (U) of hyaluronic acid hydrolase is defined as the amount of enzyme required per hour to release 1 μg of glucose-reducing sugar equivalent from the hyaluronic acid glycan chain under the conditions of pH 5.5 and 38℃.

[0076] In one specific embodiment, in the enzymatic hydrolysis system, the activity of hyaluronic acid lyase is controlled at 10-100 U / mL (i.e., 10-100 U of enzyme activity per milliliter of reaction system), for example, it can be 10 U / mL, 20 U / mL, 30 U / mL, 40 U / mL, 50 U / mL, 60 U / mL, 70 U / mL, 80 U / mL, 90 U / mL, or 100 U / mL. The enzyme activity unit (U) of hyaluronic acid lyase is defined as the amount of enzyme required to release 1 μg of glucose-reducing equivalent reducing sugar from the hyaluronic acid glycan chain per hour under conditions of pH 5.5 and 38°C.

[0077] In one specific embodiment, the pH in the enzymatic hydrolysis system is 4.0 to 6.5, for example, it can be 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, or 6.5.

[0078] In one specific embodiment, the enzymatic hydrolysis temperature in the enzymatic hydrolysis system is 25℃~40℃, for example, it can be 25℃, 26℃, 27℃, 28℃, 29℃, 30℃, 31℃, 32℃, 33℃, 34℃, 35℃, 36℃, 37℃, 38℃, 39℃, or 40℃.

[0079] In one specific implementation, hyaluronic acid hydrolase is added first, and after a period of enzymatic hydrolysis, hyaluronic acid lysin is added to continue the enzymatic hydrolysis. After adding hyaluronic acid hydrolase, the enzymatic hydrolysis time is generally 4-8 hours.

[0080] In one specific embodiment, in the enzymatic hydrolysis system, enzymatic hydrolysis is stopped when the proportion of hyaluronic acid disaccharide or its salt is greater than or equal to 85 wt%. The total enzymatic hydrolysis time is generally 12 h to 48 h, for example, 12 h, 16 h, 20 h, 24 h, 28 h, 32 h, 36 h, 40 h, 44 h, or 48 h.

[0081] Furthermore, after enzymatic hydrolysis, at least one of the following steps is included: enzyme inactivation, filtration, drying, and separation. For example, after enzyme hydrolysis, enzyme inactivation and filtration yield a solution of small molecule hyaluronic acid or its salt composition. Alternatively, after enzyme hydrolysis, enzyme inactivation, filtration, and drying, a powder of small molecule hyaluronic acid or its salt composition is obtained. Or, after enzyme hydrolysis, enzyme inactivation and filtration, followed by purification to separate hyaluronic acid disaccharide or its salt, and then drying, pure hyaluronic acid disaccharide or its salt is obtained.

[0082] The present invention also provides a small molecule hyaluronic acid or a salt composition thereof prepared according to the above preparation method.

[0083] The present invention also provides the application of the above-described small molecule hyaluronic acid or its salt composition and / or the small molecule hyaluronic acid or its salt composition prepared according to the above preparation method in promoting CD44 expression.

[0084] The present invention also provides the application of the above-mentioned small molecule hyaluronic acid or its salt composition and / or the small molecule hyaluronic acid or its salt composition prepared according to the above preparation method in promoting the expression of aquaporin AQP3.

[0085] The present invention also provides the application of the above-mentioned small molecule hyaluronic acid or its salt composition and / or the small molecule hyaluronic acid or its salt composition prepared according to the above preparation method in promoting HAS1 gene expression.

[0086] The present invention also provides the application of the above-mentioned small molecule hyaluronic acid or its salt composition and / or the small molecule hyaluronic acid or its salt composition prepared according to the above preparation method in promoting HAS2 gene expression.

[0087] The present invention also provides the use of the above-described small molecule hyaluronic acid or its salt composition and / or the small molecule hyaluronic acid or its salt composition prepared according to the above preparation method in promoting the relative length of telomeres.

[0088] The present invention also provides the use of the above-mentioned small molecule hyaluronic acid or its salt composition and / or the small molecule hyaluronic acid or its salt composition prepared according to the above preparation method in the preparation of products for anti-skin aging and / or skin moisturizing and / or anti-wrinkle and / or smoothing eye bags and / or improving dark circles.

[0089] Furthermore, the product in question is a cosmetic.

[0090] Example The materials and test methods used in the following embodiments, comparative examples, and experimental examples of this invention are described in a general and / or specific manner. Unless otherwise specified, % represents wt%, i.e., weight percentage. Reagents or instruments used, unless otherwise specified, are all commercially available conventional reagent products. Commercially available saturated and unsaturated hyaluronic acid disaccharides were purchased from Jinan Glecon Biotechnology Co., Ltd. Hyaluronic acid hydrolase was prepared using the method described in CN114350691B. Hyaluronic acid lyase was obtained by fermentation purification using Bacillus subtilis (accession number CGMCC No. 5744) or Citrobacter freundii (accession number CGMCC 1.61949). Further, the Bacillus subtilis-derived hyaluronic acid lyase was prepared using the method described in CN103255076B.

[0091] In the following examples and comparative examples, the content of degradation products was determined by liquid chromatography under the following chromatographic conditions: Column: Superdex TM 75 chromatographic column (10 × 300 mm, 13 μm) Detector: Ultraviolet-Vis spectrophotometer; Detection wavelength: 200 nm; Column temperature: 25℃; Mobile phase: 5 mM ammonium acetate aqueous solution; Flow rate: 0.3 ml / min; Injection volume: 20 μL; The peak times of each component are shown in Table 1: Table 1 Peak times of each component ×100%.

[0092] Example 1 Set up an enzymatic hydrolysis system of 1000 L, with the enzyme activity of hyaluronic acid hydrolase in the system being 1.47 × 10⁻⁶. 5 Hyaluronic acid hydrolase was added to 853 kg of pure water at a concentration of U / mL, followed by the addition of sodium acetate to a final concentration of 50 mM. The pH was adjusted to 5.53 with acetic acid, and then 100 kg of high molecular weight HA (1000 kDa - 1300 kDa) was added. Enzymatic hydrolysis was carried out at 38℃ and 60 r / min. After 8 h of enzymatic hydrolysis, bacterial hyaluronic acid lyase (from Bacillus subtilis) with a final enzyme activity of 50 U / mL was added, and enzymatic hydrolysis continued under the same conditions. The sample components during the enzymatic hydrolysis process were analyzed by high performance liquid chromatography. After 48 h of enzymatic hydrolysis, the proportion of HA2 (hyaluronic acid disaccharide or its salt) was measured to be ≥85%, at which point the enzymatic hydrolysis was terminated.

[0093] Boil the enzyme hydrolysate for 5 minutes to inactivate the enzyme, then dilute the hydrolysate 100 times with purified water and filter. Analyze the sample components using high-performance liquid chromatography (HPLC). The HPLC results are shown below. Figure 1 As shown, HA2 accounts for 92.95% of the enzymatic hydrolysis products. Enzymatic hydrolysis of 100 g / L of high molecular weight HA will produce 92.95 g / L of HA2, indicating a high HA2 yield.

[0094] The enzymatic hydrolysis products were analyzed by HPLC-MS. The theoretical molecular weight of saturated HA2 was 397 Da, and the theoretical molecular weight of unsaturated HA2 was 379 Da. Mass spectrometry results are as follows: Figure 2 As shown, the mass spectrum peak appearing at 20.5 min in the total particle flux mass spectrometry peak diagram is [M-H] in anion mode. ‑ The values ​​are 396.1 and 378.1, [M-H] 2‑ The values ​​were 793.23 and 757.2, indicating that the HA2 content contained both saturated and unsaturated HA2. Figure 3 Calculations show that the mass ratio of saturated HA2 to unsaturated HA2 is 1:1.14.

[0095] Example 2 Set up an enzymatic hydrolysis system of 1000 L, with the enzyme activity of hyaluronic acid hydrolase in the system being 1.0 × 10⁻⁶. 5Hyaluronic acid hydrolase was added to 900 kg of pure water at a concentration of U / mL, followed by the addition of sodium acetate to a final concentration of 50 mM. The pH was adjusted to 5.0 with acetic acid, and then 80 kg of high molecular weight HA (1000 kDa - 1300 kDa) was added. Enzymatic hydrolysis was carried out at 38℃ and 60 r / min. After 8 h of enzymatic hydrolysis, bacterial hyaluronic acid lyase (from Bacillus subtilis) with a final enzyme activity of 50 U / mL was added, and enzymatic hydrolysis continued under the same conditions. The sample components during the enzymatic hydrolysis process were analyzed by high performance liquid chromatography. Enzymatic hydrolysis was terminated when the HA2 content was ≥85% after 36 h of enzymatic hydrolysis.

[0096] The enzyme hydrolysate was boiled for 5 minutes to inactivate the enzyme, then diluted 100 times with purified water and filtered. The sample components were analyzed using high-performance liquid chromatography (HPLC). The HPLC results showed that HA2 accounted for 88.32% of the hydrolysate, and the hydrolysis of 80 g / L high-molecular-weight HA produced 70.66 g / L of HA2. HPLC-MS analysis of the hydrolysate showed that the mass ratio of saturated HA2 to unsaturated HA2 was 1:1.42.

[0097] Example 3 Set up an enzymatic hydrolysis system of 1000 L, with the enzyme activity of hyaluronic acid hydrolase in the system being 1.0 × 10⁻⁶. 5 Hyaluronic acid hydrolase was added to 900 kg of pure water at a concentration of U / mL, followed by the addition of sodium acetate to a final concentration of 50 mM. The pH was adjusted to 5.0 with acetic acid, and then 100 kg of high molecular weight HA (400 kDa) was added. Enzymatic hydrolysis was carried out at 38℃ and 60 r / min. After 8 h of enzymatic hydrolysis, bacterial hyaluronic acid lyase (from *Citrobacter folliculorum*) with a final enzyme activity of 60 U / mL was added, and enzymatic hydrolysis continued under the same conditions. The sample components during the enzymatic hydrolysis process were analyzed by high performance liquid chromatography. Enzymatic hydrolysis was terminated when the HA2 content was ≥85% after 24 h of enzymatic hydrolysis.

[0098] The enzyme hydrolysate was boiled for 5 minutes to inactivate the enzyme, then diluted 100 times with purified water and filtered. The sample components were analyzed using high-performance liquid chromatography (HPLC). HPLC results showed that HA2 accounted for 94.75% of the hydrolysate, and the hydrolysis of 100 g / L high-molecular-weight HA produced 94.75 g / L of HA2. HPLC-MS analysis of the hydrolysate showed that the mass ratio of saturated HA2 to unsaturated HA2 was 1:1.51.

[0099] Example 4 Set up an enzymatic hydrolysis system of 1000 L, with the enzyme activity of hyaluronic acid hydrolase in the system being 3.0 × 10⁻⁶. 5Hyaluronic acid hydrolase was added to 700 kg of pure water at a concentration of U / mL, followed by the addition of sodium acetate to a final concentration of 50 mM. The pH was adjusted to 5.0 with acetic acid, and then 100 kg of high molecular weight HA (1000 kDa - 1300 kDa) was added. Enzymatic hydrolysis was carried out at 38℃ and 60 r / min. After 4 h of enzymatic hydrolysis, bacterial hyaluronic acid lyase (from *Citrobacter folliculorum*) with a final enzyme activity of 80 U / mL was added, and enzymatic hydrolysis continued under the same conditions. The sample components during the enzymatic hydrolysis process were analyzed by high performance liquid chromatography. Enzymatic hydrolysis was terminated when the HA2 content was ≥85% after 36 h of enzymatic hydrolysis.

[0100] The enzyme hydrolysate was boiled for 5 minutes to inactivate the enzyme, then diluted 100 times with purified water and filtered. The sample components were analyzed using high-performance liquid chromatography (HPLC). The HPLC results showed that HA2 accounted for 85.26% of the hydrolysate, and the hydrolysis of 100 g / L high-molecular-weight HA produced 85.26 g / L of HA2. HPLC-MS analysis of the hydrolysate showed that the mass ratio of saturated HA2 to unsaturated HA2 was 1:1.8.

[0101] Experimental Example 1: Effect of small molecule hyaluronic acid or its salt composition on CD44 expression CD44 proteins are a group of widely distributed, multi-molecular membrane-integrated proteins with high sugar content. CD44 mediates cell-cell and extracellular matrix interactions and is a glycoprotein composed of extracellular, transmembrane, and cytospheric components. Because the N-terminus of the CD44 peptide chain can bind hyaluronic acid, CD44 is also considered a hyaluronic acid receptor, participating in the uptake and degradation of hyaluronic acid.

[0102] Preparation of experimental sample solutions: 0.1% small molecule hyaluronic acid or its salt composition: Take an appropriate amount of the sample from Example 3, prepare a 0.1% (wt%) sample solution with serum-free culture medium, and filter it with a 0.22 μm filter membrane for sterilization. Prepare and use immediately.

[0103] 0.1% saturated and unsaturated disaccharide 1:0.05 mixed sample: Prepare a solution containing 0.095% (wt%) saturated disaccharide and 0.005% (wt%) unsaturated disaccharide using serum-free culture medium, and sterilize by filtration through a 0.22 μm filter membrane. Prepare and use immediately.

[0104] 0.1% saturated and unsaturated disaccharide 1:0.1 mixed sample: Prepare a solution containing 0.09% (wt%) saturated disaccharide and 0.01% (wt%) unsaturated disaccharide using serum-free culture medium, and sterilize by filtration through a 0.22 μm filter membrane. Prepare and use immediately.

[0105] 0.1% saturated and unsaturated disaccharide 1:1 mixed sample: Prepare a solution containing 0.05% (wt%) saturated disaccharide and 0.05% (wt%) unsaturated disaccharide using serum-free culture medium, and sterilize by filtration through a 0.22 μm filter membrane. Prepare and use immediately.

[0106] 0.1% saturated and unsaturated disaccharide 1:1.5 mixed sample: Prepare a solution containing 0.04% (wt%) saturated disaccharide and 0.06% (wt%) unsaturated disaccharide using serum-free culture medium, and sterilize by filtration through a 0.22 μm filter membrane. Prepare and use immediately.

[0107] 0.1% saturated and unsaturated disaccharide 1:2 mixed sample: Prepare a solution containing 0.033% (wt%) saturated disaccharide and 0.067% (wt%) unsaturated disaccharide using serum-free culture medium, and sterilize by filtration through a 0.22 μm filter membrane. Prepare and use immediately.

[0108] 0.1% saturated and unsaturated disaccharide 1:5 mixed sample: Prepare a solution containing 0.017% (wt%) saturated disaccharide and 0.083% (wt%) unsaturated disaccharide using serum-free culture medium, and sterilize by filtration through a 0.22 μm filter membrane. Prepare and use immediately.

[0109] 0.1% saturated and unsaturated disaccharide 1:10 mixed sample: Prepare a solution containing 0.009% (wt%) saturated disaccharide and 0.091% (wt%) unsaturated disaccharide using serum-free culture medium, and sterilize by filtration through a 0.22 μm filter membrane. Prepare and use immediately.

[0110] 0.1% saturated hyaluronic acid disaccharide: Prepare a 0.1% (wt%) solution using fetal bovine serum culture medium, and sterilize by filtration through a 0.22μm filter membrane. Prepare and use immediately.

[0111] 0.1% Unsaturated Hyaluronic Acid Disaccharide: Prepare a 0.1% (wt%) solution using fetal bovine serum culture medium and sterilize by filtration through a 0.22μm filter membrane. Prepare and use immediately.

[0112] Human keratinocytes (HaCaT) in the logarithmic growth phase were harvested, digested with trypsin, and then the cell density was adjusted to 2 × 10⁶ cells / year using serum-free culture medium. 5 Cells were seeded at 1 mL / well in 12-well cell culture plates, with 1 mL of cell suspension per well. The plates were incubated overnight at 37°C with 5% CO2. The old culture medium was discarded. 1 mL of sample solution was added to each well of the experimental group, and 1 mL of fresh fetal bovine serum (FBS) culture medium (purchased from Gibco) was added to the negative control group. The plates were then incubated for another 24 h. The culture medium was discarded from both the negative control and experimental groups. Cells were washed twice with PBS, centrifuged, and the supernatant was discarded. Cells were then cultured at 1 × 10⁻⁶ cells / well. 6Cells were aliquoted into 100 μL PBS buffer at a density of [insert density here], and CD44 antibody (purchased from Proteintech) was added to each tube at 5 μL. The cells were incubated at 4°C in the dark for 20–40 min. The cell suspension was washed with PBS, the supernatant was discarded, and the cells were resuspended in 200–500 μL PBS buffer for flow cytometry analysis. Fluorescence intensity values ​​were recorded, and results were expressed as mean ± deviation. CD44 expression elevation rate = (mean fluorescence intensity of experimental group - mean fluorescence intensity of negative control group) / mean fluorescence intensity of negative control group × 100.

[0113] The results are as follows Figure 4 As shown, compared with the negative control group, neither saturated nor unsaturated hyaluronic acid disaccharide alone had the effect of promoting CD44 expression at a concentration of 0.1%. Mixed samples of saturated and unsaturated disaccharides at ratios of 1:0.05 and 1:10 had no effect of promoting CD44 expression at a concentration of 0.1%. Mixed samples of saturated and unsaturated disaccharides at ratios of 1:0.1, 1:1, 1:1.5, 1:2, and 1:5, as well as the sample in Example 3 of this application, all significantly promoted CD44 expression at the experimental concentration of 0.1%. This indicates that when the ratio of saturated to unsaturated disaccharides is in the range of 1:0.1-5, the effect of promoting CD44 is significantly better than that of saturated and unsaturated disaccharides alone, and the combination of the two has a synergistic effect.

[0114] Note: , indicating that compared to the negative control, p <0.05; , indicating that compared to the negative control, p <0.01; , indicating that compared to the negative control, p <0.001.

[0115] Experimental Example 2: Effect of small molecule hyaluronic acid or its salt composition on the expression of aquaporin AQP3 Hyaluronic acid (HA) is a major component of the extracellular matrix and is responsible for maintaining the skin's moisture content. Aquaporin 3 (AQP3) is the most abundant aquaporin subtype expressed in human skin. It functions to transport small molecules such as water, glycerol, and urea across the membrane, maintaining the hydration levels of the intracellular and extracellular matrix of the skin. It plays a crucial role in skin hydration and is one of the indicators for evaluating the moisturizing efficacy of cosmetics. HA is a natural regulator of AQP3, and HA and AQP3 have a synergistic effect in the skin's extracellular matrix.

[0116] Sample solution preparation: Take an appropriate amount of sample from Example 3, prepare a 0.1% (wt%) sample solution with fetal bovine serum culture medium, and filter it with a 0.22 μm filter membrane for sterilization. Prepare and use immediately.

[0117] Preparation of 2% glycerol glucoside solution: Prepare a 2% (wt%) glycerol glucoside solution using fetal bovine serum culture medium, and sterilize it by filtration through a 0.22 μm filter membrane. Prepare and use immediately.

[0118] HaCaT cells in the logarithmic growth phase were harvested, digested with trypsin, and then the cell density was adjusted to 2 × 10⁶ cells / year using fetal bovine serum culture medium. 5 1 mL of cell suspension was seeded into each well of a 12-well cell culture plate and incubated overnight at 37°C with 5% CO2. The old culture medium was discarded. 1 mL of sample solution was added to each well of the experimental group, 1 mL of fresh serum-free culture medium was added to the negative control group, and 2% glycerol glucoside solution was added to the positive control group. The plates were then incubated for another 48 h. The culture medium was discarded from both the negative control and experimental groups. The plates were first blocked with 1% BSA for 30 min, and then primary antibody (AQP3 antibody, purchased from Abcam) (1 μg / 10⁻¹⁰) was added. 6 Incubate at 20°C for 30 min, shaking once every 10 min; wash three times with PBS, then add secondary antibody (goat anti-rabbit IgG H&L, purchased from Abcam) (5 μg / 1000 ml). 6 Cells were incubated at 20°C for 30 min, agitated every 10 min; after trypsin digestion, they were washed three times with PBS. After resuspending in 200 μL PBS, the cells were analyzed by flow cytometry. Fluorescence intensity values ​​were recorded, and results were recorded as mean ± deviation. AQP3 elevation rate = (mean fluorescence intensity of experimental group - mean fluorescence intensity of negative control group) / mean fluorescence intensity of negative control group × 100.

[0119] The results are as follows Figure 5 As shown, compared with the negative control group, small molecule hyaluronic acid or its salt composition (Example 3) at a concentration of 0.1% significantly promoted AQP3 expression. Note: , indicating that compared to the negative control, p <0.05; , indicating that compared to the negative control, p <0.01; , indicating that compared to the negative control, p <0.001.

[0120] Experimental Example 3: Effects of small molecule hyaluronic acid or its salts on the expression of hyaluronic acid synthase genes HAS1 and HAS2. Hyaluronic acid plays a vital role in maintaining tissue hydration and elasticity. As we age, the expression and activity of hyaluronic acid synthase decrease, leading to a reduction in endogenous HA secretion. This is one of the reasons why skin loses elasticity and wrinkles and other signs of aging appear.

[0121] Sample solution preparation: Take an appropriate amount of the sample from Example 3, prepare a 0.1 wt% sample solution using serum-free DMEM culture medium, and sterilize by filtration through a 0.22 μm filter membrane. Prepare and use immediately. Preparation of TGF-β1 solution: Prepare a 100 ng / mL TGF-β1 solution using serum-free DMEM cell culture medium, and sterilize by filtration through a 0.22 μm filter membrane. Use immediately after preparation.

[0122] HaCaT cells in the logarithmic growth phase were harvested at a concentration of 2 × 10⁻⁶. 5 Cells were seeded at a density of 3 mL / well in 6-well plates using DEME basal medium (purchased from Bloomage Biotechnology (Hainan) Co., Ltd.) supplemented with 10% fetal bovine serum. The cells were incubated at 37°C with 5% CO2 for 24 hours. The old culture medium was discarded. Sample solution was added to the experimental groups, serum-free culture medium to the negative control group, and 100 ng / mL TGF-β1 solution (3 mL / well) to the positive control group. Cells were cultured for another 24 hours. After incubation, each well was washed with 2 mL of pre-cooled PBS. Cell lysis buffer was then prepared using RNA extraction reagent (Thermo Fisher), and total RNA was extracted. The RNA was reverse transcribed into cDNA, and gene expression levels were detected using real-time quantitative PCR. Results were presented as follows: -△△Ct The gene expression levels of each group of cells were obtained by analyzing and quantifying the data.

[0123] △△Ct=(CT 目的基因 -CT 内参 ) 试验组或阳性对照组 -(CT) 目的基因 -CT 内参 ) 阴性对照组 .

[0124] Relative gene expression level (%) = 2 -△△Ct ×100.

[0125] The PCR primer sequences are shown in Table 2.

[0126] Table 2 PCR primer test sequences The results are as follows Figures 6-7 As shown, compared with the negative control group, TGF-β1 significantly increased the relative expression levels of HAS1 and HAS2 genes; the hyaluronic acid disaccharide composition at a concentration of 0.1% significantly promoted the expression of HAS1 and HAS2. Note: , indicating that compared to the negative control, p <0.05; , indicating that compared to the negative control, p <0.01; , indicating that compared to the negative control, p <0.001.

[0127] Experimental Example 4: Effect of small molecule hyaluronic acid or its salt composition on the relative length of telomeres Telomeres play a vital biological role. As we age, telomere DNA naturally shortens during somatic cell division. When telomere length becomes extremely short, telomere function is impaired, leading to genomic instability, cellular senescence, and apoptosis. Somatic telomeres gradually shorten with age; telomerase acts to slow this shortening, while decreased telomerase activity leads to telomere shortening. Telomeres are located at the ends of chromosomes and consist of highly repetitive DNA sequences. Their main function is to protect chromosome ends from degradation and fusion, maintaining genomic stability. Telomere length is closely related to cellular aging and carcinogenesis. This study simulates the aging process by stimulating telomere shortening and cell damage, establishing an aging cell model. After drug administration, gene expression in telomeres of aging cells is measured to evaluate the anti-aging effects of the samples.

[0128] The single-copy gene 36B4 is a gene that exists in a single copy in the genome, and its copy number remains constant in cells. Therefore, the 36B4 gene can be used as an internal control gene to correct for differences in DNA quantity between samples, ensuring the accuracy of quantitative analysis. Specific primers were designed for both telomere genes and the single-copy gene 36B4. These primers can specifically bind to the target DNA sequence, thereby amplifying the corresponding product in the PCR reaction.

[0129] Gene expression levels in cells can be measured using quantitative real-time PCR (qRT-PCR), a method that uses fluorescent chemicals to measure the total amount of product after each polymerase chain reaction (PCR) cycle in DNA amplification. It is a method for quantitative analysis of specific DNA sequences in the sample using internal or external controls. The fluorescence signal intensities (T and S) of telomere genes and the single-copy gene 36B4 reflect their relative copy numbers in the sample. By calculating the T / S ratio, differences in DNA quantity and PCR amplification efficiency between samples can be eliminated, thus obtaining the copy number ratio of telomere genes relative to the single-copy gene 36B4. Since the copy number of a single-copy gene is constant, the T / S ratio actually reflects the relative copy number of telomere genes, i.e., the relative length of telomeres.

[0130] Preparation of experimental samples at different concentrations: Take an appropriate amount of sample from Example 3 and prepare samples of 1.25wt%, 0.625wt%, 0.25wt%, 0.125wt%, and 0.025wt% using complete culture medium (DMEM medium). Prepare and use immediately.

[0131] Take an appropriate amount of the competitor product (unsaturated hyaluronic acid disaccharide) and prepare a 0.4 wt% sample using complete culture medium (DMEM medium). Prepare and use immediately.

[0132] Human fibroblasts (HSF) in logarithmic growth phase were digested and centrifuged, and the cells were prepared into a cell suspension of appropriate density using complete culture medium (DMEM). The suspension was then seeded into 12-well cell culture plates according to the experimental design and incubated at 37°C in a 5% CO2 incubator for 24 ± 2 hours. The culture medium in the 12-well plates was discarded, and the drug administration was performed. Complete culture medium containing different concentrations (1.25%, 0.625%, 0.25%, 0.125%, 0.025%) of the Example 3 sample and 50 μmol / L H2O2 was added to the sample group wells. Complete culture medium containing the competitor (0.4%) and 50 μmol / L H2O2 was added to the competitor group wells. Complete culture medium containing 50 μmol / L H2O2 was added to the blank control group wells. The model group wells were added to the model group wells. The volume added to each well was 1 mL. After drug administration, the 12-well plates were incubated in a CO2 incubator for 24 ± 2 hours. After incubation, discard the original culture medium in the cell culture plate, wash once with DPBS, and transfer the cell plate to the DNA sample preparation room for later use. Extract DNA from the collected cell samples according to the DNA extraction kit instructions, then dilute with ultrapure water as directed before qRT-PCR to detect the expression level of telomere DNA in the cells. Data are expressed as mean ± standard deviation (Mean ± SD), and one-way ANOVA was performed. p Values ​​indicate statistical differences. p <0.05 indicates statistical significance.

[0133] Use 2 -△△Ct qRT-PCR data were processed, and the changes in DNA expression levels of telomeres at the posterior ends of cells treated with different samples were compared to the model group to calculate the relative telomere length of the sample groups. The relative telomere length values ​​of the samples were greater than those of the model group and showed a significant difference compared to the model group. p If the concentration is <0.05, then the sample is considered to have an anti-aging effect.

[0134] The results are as follows Figure 8As shown, the relative telomere lengths of the samples in Example 3 at test concentrations of 1.25%, 0.625%, 0.25%, 0.125%, and 0.025% were 380.29%, 238.69%, 218.17%, 207.41%, and 167.73%, respectively. Compared with the model group, these lengths were significantly increased by 278.87%, 137.80%, 117.35%, 106.63%, and 67.09%, respectively. p <0.01). At a concentration of 0.4%, the monounsaturated disaccharide exhibits a relative telomere length of 201.32%, while the hyaluronic acid disaccharide composition achieves a relative telomere length of 218.17% at a concentration of 0.25%, with the relative telomere length increasing with increasing concentration. Therefore, the enzymatic hydrolysis composition of this invention exhibits anti-aging effects at concentrations of 1.25%, 0.625%, 0.25%, 0.125%, and 0.025%, and these anti-aging effects are superior to those of the monounsaturated disaccharide.

[0135] Experimental Example 5: Human Efficacy of Small Molecule Hyaluronic Acid or its Salt Combinations 1. Prepare the sample and placebo according to the table below: Table 3 Sample and Placebo Information Sheet 2. Sample Usage Instructions Sample group: After cleansing, take an appropriate amount of product into the palm of your hand and apply it evenly to the skin around the eyes (half of the face), massaging gently until fully absorbed. Use once in the morning and once in the evening for 28 consecutive days until the end of the trial.

[0136] Placebo group: After cleansing, take an appropriate amount of product in the palm of your hand and apply it evenly to the skin around the eyes (half of the face), massaging gently until fully absorbed. Use once in the morning and once in the evening for 28 consecutive days until the end of the trial.

[0137] 3. Inclusion criteria for test subjects 1) The subjects were Chinese women aged between 20 and 55 years old (based on the test date); 2) Respondents described themselves as people suffering from sleep deprivation, i.e., those who work shifts or frequently stay up late (staying up late at least three times a week, and at least three of those times being after midnight). 3) Facial features include obvious dark circles (dark circles graded as level 2 or above by a dermatologist), puffy skin around the eyes and fine lines at the corners of the eyes, as well as dull skin tone and sagging. 4) Dermatologists will score the crow's feet according to the "Skin Aging Atlas," with a score of 2 or higher. 5) Dermatologists will score the level of eye bags with reference to the "Evaluation Standard for Human Skin Aging". The score level must be 2 or above. 6) The subjects mainly engage in indoor activities, avoid prolonged exposure to sunlight, have a certain awareness of sun protection, and have no travel plans in the near future; 7) Individuals without serious systemic diseases, immunodeficiency, or autoimmune diseases, and whose test sites have not undergone skin treatments, cosmetic procedures, or other tests that may affect the results; 8) Individuals without active allergic diseases; 9) Individuals without a history of severe allergies.

[0138] 4. Methods 4.1 Experimental Design This study employed a randomized, controlled, and before-and-after comparison experimental design, comprising two groups: a sample group and a placebo group. Moisturizing, anti-wrinkle, dark circle, and eye bag effects were tested at days 0, 14, and 28.

[0139] 4.2 Test Environment Ambient temperature (21.0℃±1.0℃), ambient humidity (50%±10%RH).

[0140] 4.3 Equipment Information Table 4 Equipment Information Table 4.4 Monitoring of adverse symptoms During sample use, monitor and record any skin discomfort symptoms (such as redness, stinging, itching, burning) that occur.

[0141] 5. Results 5.1 Observation of adverse symptoms No adverse symptoms were detected during the test.

[0142] 5.2 Changes in skin moisturizing parameters The results are as follows Figure 9 Compared with the placebo group (difference), the skin moisture content of the sample group increased significantly by 24.61% after 14 days of use (P < 0.001); compared with the placebo group (difference) after 28 days of use (P < 0.001), the skin moisture content of the sample group increased significantly by 23.42% (P < 0.001).

[0143] The sample group can penetrate efficiently and reach the skin base. By upregulating the synthesis of aquaporins, it accelerates water transport; by upregulating the synthesis of endogenous HA, it builds an "endogenous water reservoir" to efficiently replenish water and resist dryness, lock in moisture for a long time and promote repair, and nourish "water-glowing skin" while moisturizing and repairing.

[0144] 5.3 Changes in skin anti-wrinkle parameters The results are as follows Figure 10Compared with the placebo group after 14 days of sample use (difference), the proportion of crow's feet area was significantly reduced by 12.35% (P<0.050); compared with the placebo group after 28 days of sample use (difference), the proportion of crow's feet area was significantly reduced by 17.77% (P<0.010).

[0145] The sample group achieves moisturizing and ECM filling effects through endogenous HA and collagen, thereby improving wrinkles and reducing fine lines.

[0146] 5.4 Examine the product's effect on dark circles. The results are as follows Figures 11-15 : Compared with the placebo group after 14 days of sample use (difference), the skin melanin MI value was significantly reduced by 9.85% (P < 0.001); compared with the placebo group after 28 days of sample use (difference), the skin melanin MI value was significantly reduced by 11.40% (P < 0.001).

[0147] Skin color L was compared with the placebo group after 14 days using the sample (difference). The value increased significantly by 2.43% (P < 0.050); after 28 days of sample use, compared with the placebo group (difference), skin color L... The value increased significantly by 4.60% (P < 0.001).

[0148] Skin color b was compared with the placebo group after 14 days using the sample (difference). The value decreased significantly by 4.22% (P < 0.050); after 28 days of sample use, compared with the placebo group (difference), skin color b The value decreased significantly by 4.72% (P < 0.050).

[0149] Compared with the placebo group (difference), the dark circle score of the sample group was significantly reduced by 16.28% after 28 days (P<0.001).

[0150] In summary, after 28 days of continuous use of the serum containing the sample from Example 3, compared with the placebo group, the change rate of melanin MI value was significantly reduced by 11.40%, the change rate of skin color b value was significantly reduced by 4.72%, the change rate of L value was significantly increased by 4.60%, and the dark circle grade score was reduced by 16.28%, indicating that the composition of this application has the effect of improving dark circles.

[0151] The composition of this application improves dark circles by improving microcirculation and inhibiting angiogenesis, thereby greatly reducing the visual age of the face. Continuous use for 28 days is equivalent to approximately 3 laser treatments.

[0152] 5.5 Examine the effect of the product on eye bags The results are as follows Figures 16-18 : Compared with the placebo group after 14 days of sample use (difference), the volume of eye bags was significantly reduced by 14.47% (P<0.050); compared with the placebo group after 28 days of sample use (difference), the volume of eye bags was significantly reduced by 22.30% (P<0.001).

[0153] Compared with the placebo group (difference), the eye bag grade score was significantly reduced by 22.98% after 28 days of use (P<0.001).

[0154] The composition of this application reduces puffiness under the eyes by soothing chronic inflammation and promoting lymphatic circulation, thus significantly reducing the visual age of the face.

Claims

1. A composition of a small molecule hyaluronic acid or a salt thereof, characterized by: The hyaluronic acid disaccharide or salt thereof is a mixture of saturated hyaluronic acid disaccharide or salt thereof and unsaturated hyaluronic acid disaccharide or salt thereof; preferably, the mass ratio of the saturated hyaluronic acid disaccharide or salt thereof and the unsaturated hyaluronic acid disaccharide or salt thereof is 1:0.1-5, preferably 1:0.5-2.5, and more preferably 1:1-2.

2. The small hyaluronic acid or salt thereof composition according to claim 1, characterized by: The reducing ends of the saturated hyaluronic acid disaccharide or salt thereof and the unsaturated hyaluronic acid disaccharide or salt thereof are both N-acetylglucosamine.

3. A method for producing a small-molecule hyaluronic acid or salt thereof composition, characterized by: The method comprises the step of performing double-enzyme hydrolysis on the high molecular weight hyaluronic acid or salt thereof using a hyaluronate lyase and a hyaluronate lyase.

4. The preparation method according to claim 3, characterized in that: The hyaluronidase is an endo-beta-N-acetylglucosaminidase (EC3.2.1.35) , preferably a recombinant Pachycondyla goeldii hyaluronidase; Preferably, The The hyaluronate lyase (EC 4.2.2.1 ) is preferably a hyaluronate lyase of bacterial origin; Preferably, the hyaluronan hydrolase has an enzyme activity of 1 x 10 5 ~ 3 x 10 5 U / mL in the enzyme system, and the hyaluronan lyase has an enzyme activity of 10-100 U / mL in the enzyme system. Preferably, the hyaluronate lyase and the hyaluronate lyase are added simultaneously for the enzymolysis.

5. The method of claim 3 wherein: The concentration of the high molecular weight hyaluronic acid or salt thereof is 2%-20%. Preferably, the molecular weight of the high molecular weight hyaluronic acid or salt thereof is 400 kDa-1500 kDa.

6. The method of claim 3 wherein: The pH of the enzymolysis system is controlled at 4.0-6.5; preferably, the enzymolysis temperature is controlled at 25°C-40°C; and preferably, the enzymolysis time is controlled at 12h-48h.

7. A small molecular hyaluronic acid or salt composition prepared according to any one of claims 3-6.

8. A product comprising at least the small molecular hyaluronic acid or salt composition of claim 1 or 2 and / or the small molecular hyaluronic acid or salt composition of claim 7 and a physiologically acceptable adjuvant.

9. Use of the small molecular hyaluronic acid or salt composition of claim 1 or 2 and / or the small molecular hyaluronic acid or salt composition of claim 7 in the preparation of a product for promoting the expression of CD44 and / or the expression of aquaporin AQP3 and / or the expression of HAS1 gene and / or HAS2 gene and / or the relative length of telomeres. Preferably, the small molecular hyaluronic acid or salt composition of claim 1 or 2 and / or the small molecular hyaluronic acid or salt composition of claim 7 is used in the preparation of a product for anti-skin aging and / or skin moisturizing and / or anti-wrinkle and / or flattening of eye bags and / or improvement of dark circles.

10. Use of the small molecular hyaluronic acid or salt composition of claim 1 or 2 and / or the small molecular hyaluronic acid or salt composition of claim 7 in cosmetic and pharmaceutical products.

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