A hyaluronic acid oligosaccharide composition and its preparation method and use

Abstract

The present invention provides a hyaluronic acid oligosaccharide composition and its preparation method and use, wherein the hyaluronic acid oligosaccharide composition includes a hyaluronic acid oligosaccharide with a uronic acid structure at the reducing end. The hyaluronic acid oligosaccharide composition can improve the synthesis of ceramide and / or the expression of hyaluronic acid synthase 1 (HAS1) and / or hyaluronic acid synthase 3 (HAS3). The present invention can separate the oligosaccharides obtained by enzymolysis from the enzymolysis system in time by coupling the enzymolysis reaction with ultrafiltration and nanofiltration, reduce the inhibitory effect of oligosaccharides on enzymolysis, improve enzymolysis efficiency, and shorten the enzymolysis time. After enzymolysis, the enzyme can be reused 1-2 times by ultrafiltration to intercept the enzyme, improve the reuse degree of the enzyme, and save the heating enzyme inactivation stage at the same time, reduce the influence of heating on the activity of oligosaccharides, and maximize the retention of the activity of oligosaccharides. Reduce the amount of activated carbon added, reduce the carbon pollution to the operator, and simplify the purification process.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering technology, and in particular, relates to a hyaluronic acid oligosaccharide composition and a preparation method and use thereof. Background Art

[0002] Hyaluronic acid (HA) is an acidic mucopolysaccharide. It is an unbranched high-molecular glycosaminoglycan composed of N-acetylglucosamine and D-glucuronic acid disaccharide repeating units through β-(1→4) glycosidic bonds and β-(1→3) glycosidic bonds. It exists in the interstitial cells of animal tissues and the capsules of some bacteria. Hyaluronic acid is widely used in medicine, cosmetics, food and other fields. Its molecular weight is generally 10 5 ~10 7 Dalton (Da).

[0003] Hyaluronic acid of different molecular weights exhibits different biological activities, and low molecular weight hyaluronic acid even exhibits completely opposite activities to high molecular weight hyaluronic acid. Many literatures have reported on the role of hyaluronic acid in wound repair, especially low molecular weight and hyaluronic acid oligosaccharides as active substances have received more attention. Because the preparation of hyaluronic acid oligosaccharides by acid method, alkali method, etc., if applied to large-scale production, has a higher risk and may cause harm to operators or equipment, the preparation of oligosaccharides by enzymatic cleavage has greater advantages.

[0004] There are few reports on the preparation and efficacy of hyaluronic acid tetrasaccharides with uronic acid as the reducing end, especially hyaluronic acid oligosaccharide mixtures (uronic acid reducing end) containing HA2-12. Patent CN 111040048A discloses an ultra-low molecular weight hyaluronic acid and its preparation method. The patent does not separate the oligosaccharides produced by the enzymatic hydrolysis process in time, and the oligosaccharides present will inhibit the forward reaction of the enzymatic hydrolysis to a certain extent, resulting in the extension of the enzymatic hydrolysis time and increasing the difficulty of later purification. The patent also does not disclose its role in promoting the synthesis of hyaluronic acid synthase and ceramide.

[0005] In addition, the structure of a substance will affect its biological activity, and this is also true for oligosaccharides.

[0006] Non-patent literature 1 discloses that sulfated hyaluronic acid (sHA) has good biological functions, and the specific sulfation pattern plays a key role in regulating the binding pattern between glycosaminoglycans and proteins. It proves that sHA tetrasaccharide with 6-O-sulfation (sHA-6S) plays an important role in promoting the axon growth of rat E18 hippocampal neurons in vitro.

[0007] Non-patent literature 2 uses fluorescence polarization competition assay to analyze the relative binding affinity of synthetic compounds, and reveals that there is interaction between synthetic chondroitin sulfate-like tetrasaccharides and midkine. It can be seen that the difference in polymerization degree or residue of hyaluronic acid oligosaccharides (HAOs) will produce different biologically active functions. This is worth further study. In the prior art, there is no research on the influence of ultra-small molecular weight hyaluronic acid with uronic acid end groups on the synthesis of ceramide and hyaluronidase.

[0008] Non-patent document 3 selectively produces hyaluronic acid oligosaccharides (HAOs) with different degrees of polymerization and reducing ends by changing the hydrochloric acid concentration. The data of molecular docking simulation of HAOs with CD44 and TLR4 using MOE software show that the binding ability of HAOs to CD44 and TLR4 increases with the increase of the degree of polymerization of HAOs. At the same time, the binding ability increases significantly when the reducing end of HAOs is a GlcNAc residue, while the binding ability does not change significantly when the reducing end is a GlcA residue.

[0009] Non-patent literature 1: Unravel a neuroactive sHA sulfation pattern with neurogenesis activity by a library of defined oligosaccharides. Yao, W., Chen, M., Dou, X., Jin, H., Zhang, X., Zhu, Y.,... & Li, Z. (2019). definedoligosaccharides.European journal of medicinal chemistry,163,583-596.

[0010] Non-patent literature 2: Chondroitin Sulfate Tetrasaccharides: Synthesis, Three-Dimensional Structure and Interaction with Midkine. Solera, C., Macchione, G., Maza, S., Kayser, M., Corzana, F., Paz, JLD, & Nieto, PM (2016). Chondroitinsulfate tetrasaccharides: synthesis, three-dimensional structure and interaction with midkine.

[0011] Non-patent document 3: Preparation, Characterization, and Inhibition of Hyaluronic Acid Oligosaccharides in Triple-Negative Breast Cancer. Han, W., Song, L., Wang, Y., Lv, Y., Chen, X., & Zhao, X. (2019). Preparation, characterization, and inhibition of hyaluronic acid oligosaccharides in triple-negative breastcancer.Biomolecules,9(9),436.

[0012] Therefore, it is very meaningful to continue studying HA oligosaccharide products with different structures or compositions or to develop new functions. Summary of the invention

[0013] In view of the problems existing in the prior art, the present invention provides a hyaluronic acid oligosaccharide composition and a preparation method thereof.

[0014] Specifically, the present invention relates to the following aspects:

[0015] 1. A hyaluronic acid oligosaccharide composition, characterized in that the hyaluronic acid oligosaccharide composition comprises a hyaluronic acid oligosaccharide having a structure shown in formula (I):

[0016]

[0017] wherein n is an integer selected from 0 to 5, and X is selected from H, K, Na, Ca or Zn, preferably Na;

[0018] In the hyaluronic acid oligosaccharide composition,

[0019] The mass percentage of hyaluronic acid oligosaccharide with n=1 is 35-70%;

[0020] The mass percentage of hyaluronic acid oligosaccharide with n=0 is 5-40%;

[0021] The mass percentage of hyaluronic acid oligosaccharides with n=2 is 0-50%;

[0022] The mass percentage of hyaluronic acid oligosaccharides with n=3 is 0-15%;

[0023] The mass percentage of hyaluronic acid oligosaccharides with n=4 is 0-10%;

[0024] The mass proportion of hyaluronic acid oligosaccharide with n=5 is 0-5%.

[0025] 2. The hyaluronic acid oligosaccharide composition according to item 1, characterized in that, in the hyaluronic acid oligosaccharide composition,

[0026] The mass percentage of hyaluronic acid oligosaccharide with n=1 is 35-70%;

[0027] The mass percentage of hyaluronic acid oligosaccharide with n=0 is 5-40%;

[0028] The mass percentage of hyaluronic acid oligosaccharides with n=2 is 1-50%;

[0029] The mass percentage of hyaluronic acid oligosaccharides with n=3 is 1-15%;

[0030] The mass proportion of hyaluronic acid oligosaccharides with n=4 is 1-10%;

[0031] The mass proportion of hyaluronic acid oligosaccharides with n=5 is 0.01-1.5%.

[0032] 3. The hyaluronic acid oligosaccharide composition according to item 1, characterized in that, in the hyaluronic acid oligosaccharide composition,

[0033] The mass proportion of hyaluronic acid oligosaccharide with n=1 is 40-60%;

[0034] The mass percentage of hyaluronic acid oligosaccharide with n=0 is 5-15%;

[0035] The mass percentage of hyaluronic acid oligosaccharides with n=2 is 20-40%;

[0036] The mass proportion of hyaluronic acid oligosaccharides with n=3 is 1-10%;

[0037] The mass proportion of hyaluronic acid oligosaccharides with n=4 is 1-5%;

[0038] The mass proportion of hyaluronic acid oligosaccharides with n=5 is 0.01-1.5%.

[0039] 4. The hyaluronic acid oligosaccharide composition according to item 1, characterized in that the weight average molecular weight of the hyaluronic acid oligosaccharide composition is less than or equal to 1 kDa.

[0040] 5. The hyaluronic acid oligosaccharide composition according to item 1, characterized in that the hyaluronic acid oligosaccharide composition is used in cosmetics or health products, and preferably has a mass concentration of 0.001-10% in the cosmetics or health products.

[0041] 6. A method for preparing the hyaluronic acid oligosaccharide composition according to any one of claims 1 to 5, characterized in that the preparation method comprises the following steps:

[0042] The hyaluronic acid is enzymatically hydrolyzed with hyaluronidase.

[0043] Separate the enzymatic reaction products to obtain the product stock solution,

[0044] Concentrating the product stock solution to obtain a product concentrate;

[0045] removing impurities from the product concentrate and drying to obtain a hyaluronic acid oligosaccharide composition,

[0046] Wherein, the hyaluronidase is a hyaluronidase that cleaves β-1,3 glycosidic bonds,

[0047] Preferably, the hyaluronidase is leech hyaluronan hydrolase expressed by Pichia pastoris engineered bacteria.

[0048] Further preferably, the separation is ultrafiltration;

[0049] Further preferably, the concentration is nanofiltration;

[0050] More preferably, activated carbon adsorption is used to remove impurities.

[0051] 7. The method according to item 6 is characterized in that, in the enzymatic hydrolysis reaction, the initial concentration of the hyaluronic acid is 5-150 g / L.

[0052] 8. The method according to item 6 is characterized in that, in the enzymatic hydrolysis reaction, the molecular weight of the hyaluronic acid is 1000-2000 kDa.

[0053] 9. The method according to item 6, characterized in that in the enzymatic hydrolysis reaction, the initial content of the hyaluronidase is 1×10 4 -3×10 5 U / mL.

[0054] 10. The method according to item 6 is characterized in that, in the enzymatic hydrolysis reaction, the initial ratio of the hyaluronidase to the hyaluronic acid is 1-5 U / mg.

[0055] 11. The method according to item 6 is characterized in that the ultrafiltration uses an ultrafiltration membrane with a molecular weight cutoff of 600-1000Da, and the nanofiltration uses a nanofiltration membrane with a molecular weight cutoff of 100-300Da.

[0056] 12. The method according to item 6 is characterized in that the temperature of the enzymatic hydrolysis reaction is 30-46°C and the time is 12-24h.

[0057] 13. The method according to item 6 is characterized in that the drying is spray drying, and the air inlet temperature used is 120°C-160°C, and the air outlet temperature is 60°C-80°C.

[0058] 14. Use of the hyaluronic acid oligosaccharide composition described in any one of items 1 to 5 for promoting the synthesis of ceramide and / or hyaluronic acid synthase 1 (HAS1) and / or hyaluronic acid synthase 3 (HAS3).

[0059] The present invention can separate the oligosaccharides obtained by enzymolysis from the enzymolysis system in time by coupling the enzymolysis reaction with ultrafiltration and nanofiltration, reduce the inhibitory effect of oligosaccharides on enzymolysis, improve the efficiency of enzymolysis, and shorten the enzymolysis time. After enzymolysis, the enzyme can be reused 1-2 times by ultrafiltration to intercept the enzyme, improve the reuse degree of the enzyme, and save the heating enzyme inactivation stage at the same time, reduce the influence of heating on the activity of oligosaccharides, and maximize the activity of oligosaccharides. Reduce the amount of activated carbon added, reduce carbon pollution to the operator, and simplify the purification process. Further, the hyaluronic acid oligosaccharide composition obtained by the present invention can promote the synthesis of ceramide and / or hyaluronic acid synthase 1 (HAS1) and / or hyaluronic acid synthase 3 (HAS3). The present invention has great application value for the industrial production of hyaluronic acid oligosaccharide composition. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 4 is the HPLC chromatogram of hyaluronic acid oligosaccharide in Example 1.

[0061] Figure 2 This is the HPLC chromatogram of hyaluronic acid oligosaccharide in Example 2.

[0062] Figure 3 This is the HPLC chromatogram of hyaluronic acid oligosaccharide in Example 3.

[0063] Figure 4 It is the high performance liquid chromatogram of hyaluronic acid oligosaccharide in comparative example 1.

[0064] Figure 5This is the total ion current peak diagram of the mass spectrum of hyaluronic acid oligosaccharide in Example 2.

[0065] Figure 6 This is the ionic intensity diagram of hyaluronan disaccharide (HA2).

[0066] Figure 7 This is the ionic intensity diagram of hyaluronan tetrasaccharide (HA4).

[0067] Figure 8 This is the ionic intensity diagram of hyaluronan hexasaccharide (HA6).

[0068] Fig. 9 This is the ionic intensity diagram of hyaluronic acid octasaccharide (HA8).

[0069] Fig.10 The ion intensity diagrams are for hyaluronic acid decasaccharide (HA10) and dodecasaccharide (HA12).

[0070] Fig.11 This is the reducing end identification result of the hyaluronic acid oligosaccharide composition. DETAILED DESCRIPTION

[0071] The present invention is further described below in conjunction with examples. It should be understood that the examples are only used to further illustrate and explain the present invention, and are not intended to limit the present invention.

[0072] Unless otherwise defined, the technical and scientific terms in this specification are the same as those commonly understood by those skilled in the art. Although similar or identical methods and materials as described herein can be used in experiments or practical applications, materials and methods are described herein below. In the event of a conflict, the present specification including the definitions therein shall prevail. In addition, materials, methods and examples are for illustration only and are not restrictive. The present invention is further described below in conjunction with specific embodiments, but is not intended to limit the scope of the present invention.

[0073] Hyaluronic acid oligosaccharides or hyaluronic acid oligosaccharides (oligo-HA) are oligosaccharides with a molecular weight of 10 4 A hyaluronic acid molecule fragment with a monosaccharide residue number of 2 to 25 (generally 4 to 16) and less than 1 Da. Oligo-HA is a small molecule polysaccharide, and its properties are very different from those of ordinary hyaluronic acid. Studies have shown that Oligo-HA has biological activities such as antioxidant, immunomodulatory, anti-inflammatory, wound healing, angiogenesis and anti-tumor. More importantly, due to its small molecular size, it can penetrate into the stratum corneum of the skin to exert deep moisturizing and nourishing effects, and can be widely used in cosmetics.

[0074] The present invention provides a hyaluronic acid oligosaccharide composition, wherein the hyaluronic acid oligosaccharide composition comprises a hyaluronic acid oligosaccharide having a structure shown in formula (I):

[0075]

[0076] As can be seen from formula (I), the reducing end of the hyaluronic acid oligosaccharide of the present invention is a uronic acid structure.

[0077] Wherein, n is selected from an integer of 0-5, for example, n can be 0, 1, 2, 3, 4 or 5, and X is selected from H, K, Na, Ca or Zn, preferably Na. In the hyaluronic acid oligosaccharide composition, the mass proportion of hyaluronic acid oligosaccharides of n=1 is 35-70%; the mass proportion of hyaluronic acid oligosaccharides of n=0 is 5-40%; the mass proportion of hyaluronic acid oligosaccharides of n=2 is 0-50%; the mass proportion of hyaluronic acid oligosaccharides of n=3 is 0-15%; the mass proportion of hyaluronic acid oligosaccharides of n=4 is 0-10%; the mass proportion of hyaluronic acid oligosaccharides of n=5 is 0-5%.

[0078] When n=0, the hyaluronic acid oligosaccharide is a disaccharide, when n=1, the hyaluronic acid oligosaccharide is a tetrasaccharide, when n=2, the hyaluronic acid oligosaccharide is a hexasaccharide, when n=3, the hyaluronic acid oligosaccharide is an octasaccharide, when n=4, the hyaluronic acid oligosaccharide is a decasaccharide, and when n=5, the hyaluronic acid oligosaccharide is a dodecasaccharide. Therefore, in the hyaluronic acid oligosaccharide composition of the present invention, the mass proportion of tetrasaccharide is 35-70%; the mass proportion of disaccharide is 5-40%; the mass proportion of hexasaccharide is 0-50%; the mass proportion of octasaccharide is 0-15%; the mass proportion of decasaccharide is 0-10%; the mass proportion of dodecasaccharide is 0-5%.

[0079] Among them, the mass proportion of hyaluronic acid tetrasaccharide is 35-70%, for example, it can be 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, preferably 40-60%.

[0080] The mass proportion of hyaluronic acid disaccharide is 5-40%, for example, it can be 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, preferably 5-15%.

[0081] The mass proportion of hyaluronic acid hexasaccharide is 0-50%, for example, it can be 0%, 1%, 5%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, preferably 1-50%, and more preferably 20-40%.

[0082] The mass proportion of hyaluronic acid octasaccharide is 0-15%, for example, it can be 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, preferably 1-15%, and more preferably 1-10%.

[0083] The mass proportion of hyaluronic acid decasaccharide is 0-10%, for example, it can be 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, preferably 1-10%, and more preferably 1-5%.

[0084] The mass proportion of hyaluronic acid dodecasaccharide is 0-5%, for example, it can be 0%, 0.01%, 0.05%, 0.1%, 1%, 2%, 3%, 4%, 5%, preferably 0.01-1.5%.

[0085] In a specific embodiment, in the hyaluronic acid oligosaccharide composition, the mass proportion of hyaluronic acid disaccharide is 5-40%, the mass proportion of hyaluronic acid tetrasaccharide is 35-70%, the mass proportion of hyaluronic acid hexasaccharide is 0-50%, the mass proportion of hyaluronic acid octasaccharide is 0-15%, the mass proportion of hyaluronic acid decasaccharide is 0-10%, and the mass proportion of hyaluronic acid dodecasaccharide is 0-5%.

[0086] In a specific embodiment, in the hyaluronic acid oligosaccharide composition, the mass proportion of hyaluronic acid disaccharide is 5-40%, the mass proportion of hyaluronic acid tetrasaccharide is 35-70%, the mass proportion of hyaluronic acid hexasaccharide is 1-50%, the mass proportion of hyaluronic acid octasaccharide is 1-15%, the mass proportion of hyaluronic acid decasaccharide is 1-10%, and the mass proportion of hyaluronic acid dodecasaccharide is 0.01-1.5%.

[0087] In a specific embodiment, in the hyaluronic acid oligosaccharide composition, the mass proportion of hyaluronic acid disaccharide is 5-15%, the mass proportion of hyaluronic acid tetrasaccharide is 40-60%, the mass proportion of hyaluronic acid hexasaccharide is 20-40%, the mass proportion of hyaluronic acid octasaccharide is 1-10%, the mass proportion of hyaluronic acid decasaccharide is 1-5%, and the mass proportion of hyaluronic acid dodecasaccharide is 0.01-1.5%.

[0088] In a specific embodiment, in the hyaluronic acid oligosaccharide composition, the mass proportion of hyaluronic acid disaccharide is 5-10%, the mass proportion of hyaluronic acid tetrasaccharide is 45-55%, the mass proportion of hyaluronic acid hexasaccharide is 30-40%, the mass proportion of hyaluronic acid octasaccharide is 2-8%, the mass proportion of hyaluronic acid decasaccharide is 1-2%, and the mass proportion of hyaluronic acid dodecasaccharide is 0.01-1%.

[0089] In a specific embodiment, in the hyaluronic acid oligosaccharide composition, the mass proportion of hyaluronic acid disaccharide is 10%, the mass proportion of hyaluronic acid tetrasaccharide is 49.5%, the mass proportion of hyaluronic acid hexasaccharide is 33%, the mass proportion of hyaluronic acid octasaccharide is 5%, the mass proportion of hyaluronic acid decasaccharide is 1.5%, and the mass proportion of hyaluronic acid dodecasaccharide is 1%.

[0090] In a specific embodiment, in the hyaluronic acid oligosaccharide composition, the mass proportion of hyaluronic acid disaccharide is 9%, the mass proportion of hyaluronic acid tetrasaccharide is 49%, the mass proportion of hyaluronic acid hexasaccharide is 32%, the mass proportion of hyaluronic acid octasaccharide is 7%, the mass proportion of hyaluronic acid decasaccharide is 2%, and the mass proportion of hyaluronic acid dodecasaccharide is 1%.

[0091] Furthermore, the weight average molecular weight of the hyaluronic acid oligosaccharide composition is less than or equal to 1 kDa, for example, it can be 1 kDa, 990 Da, 980 Da, 970 Da, 960 Da, 950 Da, 940 Da, 930 Da, 920 Da, 910 Da, 900 Da, 890 Da, 880 Da, 870 Da, 860 Da, 850 Da, 840 Da, 830 Da, 820 Da, 810 Da, 800 Da, 790 Da, 780 Da, 770 Da, 760 Da, 750 Da, 740 Da, 730 Da, 720 Da, 710 Da, 700 Da.

[0092] The hyaluronic acid oligosaccharide composition of the present invention can promote the expression of ceramide and / or hyaluronic acid synthase 1 and / or hyaluronic acid synthase 3. Among them, ceramide is a type of phospholipid with ceramide as the skeleton, mainly including ceramide phosphorylcholine and ceramide phosphorylethanolamine. Phospholipid is the main component of cell membrane. 40% to 50% of sebum in the stratum corneum is composed of ceramide. Ceramide is the main part of the intercellular matrix and plays an important role in maintaining the balance of moisture in the stratum corneum. Ceramide has a strong ability to associate water molecules, and it maintains skin moisture by forming a mesh structure in the stratum corneum. Therefore, ceramide has the effect of maintaining skin moisture.

[0093] Hyaluronic acid (HA) in the skin is mainly synthesized by hyaluronan synthase (HAS). Three HAS subtypes (HAS1, HAS2, HAS3) have been identified in mammals. HAS participates in the synthesis of hyaluronic acid chains of different molecular weights on the inner side of the plasma membrane, and secretes the synthesized hyaluronic acid into the extracellular matrix. It is reported that the length of hyaluronic acid chains synthesized by HAS1 and HAS2 is similar, about 2000KDa, and the molecular weight of hyaluronic acid synthesized by HAS3 is between 200-300kDa. Extracellular macromolecular HA is hydrolyzed by hyaluronidase or oxidatively degraded to obtain small molecule HA. Small molecule HA can stimulate cell proliferation, initiate signal cascade reactions, and also participate in angiogenesis. Hyaluronic acid of 100-300Ka has the effects of softening keratin, locking water, and three-dimensional moisturizing. Furthermore, the document “Expression of Hyaluronan Synthase and Collagen Type I mRNA by Hyaluronan Tetrasaccharides in Normal Human Dermal Fibroblasts” discloses that hyaluronan tetrasaccharide can promote the expression of HAS1, but has no effect on the expression of HAS2 and HAS3.

[0094] The hyaluronic acid oligosaccharide composition of the present invention can be used in cosmetics or health products, preferably in the cosmetics or health products, the mass concentration is 0.001-10%, for example, it can be 0.001%, 0.005%, 0.01%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%. The mass concentration of the hyaluronic acid oligosaccharide composition in cosmetics or health products can be further adjusted according to the different uses of the hyaluronic acid oligosaccharide composition in cosmetics or health products. For example, when the hyaluronic acid oligosaccharide composition is used to promote ceramide expression in cosmetics or health products, its mass concentration can be 0.001-1%. When the hyaluronic acid oligosaccharide composition is used in cosmetics or health products to promote the expression of hyaluronic acid synthase 1 (HAS1) and / or hyaluronic acid synthase 3 (HAS3), its mass concentration can be 0.01-10%. When the hyaluronic acid oligosaccharide composition is used to promote the expression of hyaluronic acid synthase 1 (HAS1) and / or hyaluronic acid synthase 3 (HAS3) in cells, its mass concentration can be 0.001-1%.

[0095] The present invention also provides a method for preparing a hyaluronic acid oligosaccharide composition, which comprises the following steps: subjecting hyaluronic acid to an enzymatic hydrolysis reaction using hyaluronidase, separating the enzymatic hydrolysis reactants to obtain a product stock solution, and concentrating the product stock solution to obtain a product concentrated solution; removing impurities from the product concentrated solution, and drying to obtain a hyaluronic acid oligosaccharide composition, wherein the hyaluronidase is a hyaluronidase that cleaves β-1,3 glycosidic bonds, preferably, the separation is ultrafiltration; further preferably, the concentration is nanofiltration; further preferably, activated carbon adsorption is used to remove impurities.

[0096] The hyaluronidase used in the preparation method is a hyaluronidase that cleaves β-1,3 glycosidic bonds, ensuring that the reducing end of the oligosaccharide obtained by hyaluronidase cleavage is a uronic acid structure.

[0097] In a specific embodiment, the hyaluronidase is a leech hyaluronan hydrolase expressed by Pichia pastoris, and the preparation method thereof can refer to the method described in CN103695448A.

[0098] Ultrafiltration is a membrane separation technology (UItrafil-tration, referred to as UF). It can purify, separate or concentrate solutions. Ultrafiltration is between microfiltration and nanofiltration, and there is no obvious dividing line between the three. Generally speaking, the pore size of the ultrafiltration membrane is between 0.05μm-1nm, and the operating pressure is 0.1-0.5Mpa. It is mainly used to intercept and remove macromolecular substances such as suspended matter, colloids, particles, bacteria and viruses in water. Ultrafiltration membranes can be divided into organic membranes and inorganic membranes according to the membrane material. According to the appearance of the membrane, it can be divided into: flat plate, tubular, capillary, hollow fiber and porous. At present, household ultrafiltration water purifiers are mostly hollow membranes.

[0099] Nanofiltration (NF) is a pressure-driven membrane separation process between reverse osmosis and ultrafiltration. The pore size of the nanofiltration membrane is in the range of several nanometers. Nanofiltration (NF) is used to separate substances with relatively small molecular weight, such as inorganic salts or small molecular organic substances such as glucose and sucrose, from solvents. Nanofiltration, also known as low-pressure reverse osmosis, is an emerging field of membrane separation technology. Its separation performance is between reverse osmosis and ultrafiltration, allowing some inorganic salts and certain solvents to pass through the membrane, thereby achieving the separation effect.

[0100] By controlling the conditions of the enzymatic hydrolysis reaction, such as the amount of substrate and enzyme, the molecular weight of the substrate, the reaction temperature and reaction time, as well as the different types of membranes used in ultrafiltration and nanofiltration, hyaluronic acid oligosaccharide compositions with different molecular weights and different oligosaccharide proportions can be obtained.

[0101] In a specific embodiment, in the enzymatic hydrolysis reaction, the initial concentration of the hyaluronic acid is 5-150 g / L, for example, 5 g / L, 10 g / L, 20 g / L, 30 g / L, 40 g / L, 50 g / L, 60 g / L, 70 g / L, 80 g / L, 90 g / L, 100 g / L, 110 g / L, 120 g / L, 130 g / L, 140 g / L, 150 g / L. For example, if the reaction system is 1 L, the initial content of the hyaluronic acid is 5-150 g, that is, the content when no hydrolysis occurs is 5-150 g.

[0102] In a specific embodiment, the initial molecular weight of the hyaluronic acid is 1000-2000 kDa, for example, 1000 kDa, 1100 kDa, 1200 kDa, 1300 kDa, 1400 kDa, 1500 kDa, 1600 kDa, 1700 kDa, 1800 kDa, 1900 kDa, or 2000 kDa.

[0103] In a specific embodiment, in the enzymatic hydrolysis reaction, the initial content of the hyaluronidase is 1×10 4 -3×10 5U / mL, for example, 1×10 4 U / mL, 2×10 4 U / mL, 4×10 4 U / mL, 6×10 4 U / mL, 8×10 4 U / mL, 1×10 5 U / mL, 1.5×10 5 U / mL, 2×10 5 U / mL, 2.5×10 5 U / mL, 3×10 5 U / mL. The definition of hyaluronidase activity unit (U) is: the amount of enzyme required to release 1 μg of glucose reducing equivalent from hyaluronic acid sugar chains per hour at pH 5.5 and 38°C.

[0104] In a specific embodiment, in the enzymatic hydrolysis reaction, the initial ratio of the hyaluronidase to the hyaluronic acid is 1-5 U / mg, for example, 1 U / mg, 2 U / mg, 3 U / mg, 4 U / mg, 5 U / mg.

[0105] In a specific embodiment, ultrafiltration uses an ultrafiltration membrane with a molecular weight cutoff of 600-1000Da, for example, the molecular weight cutoff can be 600Da, 700Da, 800Da, 900Da, 1000Da, and nanofiltration uses a nanofiltration membrane with a molecular weight cutoff of 100-300Da, for example, the molecular weight cutoff can be 100Da, 200Da, 300Da.

[0106] In a preferred embodiment, the ultrafiltration uses an ultrafiltration membrane with a molecular weight cutoff of 1000 Da, and the nanofiltration uses a nanofiltration membrane with a molecular weight cutoff of 200 Da.

[0107] The retention molecule described in the present invention is the retention performance of the ultrafiltration membrane expressed by the molecular weight, also known as the cut-off molecular weight. Since it is quite difficult to directly measure the pore size of the ultrafiltration membrane or the nanofiltration membrane, a spherical substance with a known molecular weight is used for measurement. If the retention rate of the membrane for the retained substance is greater than 90%, the retention performance of the membrane is expressed by the molecular weight of the retained substance, which is called the membrane's retention molecular weight. For example, a membrane with a molecular weight cut-off of 10kDa can retain substances with a molecular weight greater than 10kDa, while allowing substances with a molecular weight less than 10kDa to pass through.

[0108] The drying in the method can be carried out by various drying methods in the prior art. In a specific embodiment, the drying is spray drying. The air inlet temperature used in the spray drying is 120°C-160°C, and the air outlet temperature is 60°C-80°C.

[0109] Furthermore, the hyaluronic acid oligosaccharide composition obtained by the method is the above-mentioned hyaluronic acid oligosaccharide composition, that is, the hyaluronic acid oligosaccharide having the structure shown in formula (I):

[0110]

[0111] Wherein, n is an integer selected from 0-5, and X is selected from H, K, Na, Ca or Zn, preferably Na.

[0112] The average molecular weight of the hyaluronic acid oligosaccharide product can be measured by a multi-angle laser light scattering instrument. The identification of various oligosaccharides in the hyaluronic acid oligosaccharide product can be detected by mass spectrometry, and the content of various oligosaccharides can be detected by high performance liquid chromatography.

[0113] The present invention also provides use of the hyaluronic acid oligosaccharide composition in promoting the expression of hyaluronic acid synthase 1 (HAS1) and / or hyaluronic acid synthase 3 (HAS3).

[0114] The hyaluronic acid oligosaccharide composition of the present invention has various types of hyaluronic acid oligosaccharides, such as hyaluronic acid disaccharide, hyaluronic acid tetrasaccharide, hyaluronic acid hexasaccharide, hyaluronic acid octasaccharide, hyaluronic acid decasaccharide, and hyaluronic acid dodecasaccharide, with the reducing end being a uronic acid structure. The oligosaccharide composition has no cytotoxicity and no potential adverse reactions to human skin; it can significantly promote the expression of hyaluronic acid synthase HAS1 and HAS3 in fibroblasts, especially the expression of HAS3; and it can significantly promote the synthesis of ceramide.

[0115] Example

[0116] Example 1 Preparation of hyaluronic acid oligosaccharide composition

[0117] In a 5L reactor, add 4L of water, control the temperature at 40°C, add hyaluronidase (the enzyme is leech hyaluronidase expressed by Pichia pastoris, the preparation method is described in CN 103695448 A) 4×10 8 U, the enzyme activity of the system is 1×10 5 U / mL, add 40g hyaluronic acid (molecular weight 150kDa), after it is completely dissolved, use ultrafiltration to separate the enzymatic reaction to obtain the product stock solution, and use nanofiltration to concentrate the product stock solution to obtain the product concentrate. Keep the system at 40°C and stir the reaction for 18h. The ultrafiltration uses an ultrafiltration membrane with a molecular weight cutoff of 1kDa, and the nanofiltration uses a nanofiltration membrane with a molecular weight cutoff of 200Da. After the reaction is completed, the nanofiltration concentrate is collected, and after adding activated carbon for adsorption, the filtrate is filtered and collected, and spray dried, wherein the inlet temperature is 140°C and the outlet temperature is 70°C. A hyaluronic acid oligosaccharide composition is obtained, and the product yield is 86%.

[0118] The hyaluronic acid oligosaccharide composition was analyzed for its oligosaccharide distribution using a high performance liquid chromatograph, and the specific chromatographic conditions were as follows:

[0119] Column: SUPERDEX 200 10 / 300 GL

[0120] Column temperature: 40°C

[0121] Detector: UV-Vis spectrophotometer

[0122] Mobile phase: 1 mol / L ammonium sulfate solution

[0123] Injection concentration: 0.5%

[0124] Injection volume: 20 μL

[0125] Flow rate: 2ml / min

[0126] Detection wavelength: 200nm

[0127] The results are as follows Figure 1 As shown, in the hyaluronic acid oligosaccharide composition, the mass proportion of hyaluronic acid disaccharide is 9.12%, the mass proportion of hyaluronic acid tetrasaccharide is 47.73%, the mass proportion of hyaluronic acid hexasaccharide is 32.83%, the mass proportion of hyaluronic acid octasaccharide is 6.05%, the mass proportion of hyaluronic acid decasaccharide is 1.49%, and the mass proportion of hyaluronic acid dodecasaccharide is 0.42%.

[0128] The average molecular weight of the hyaluronic acid oligosaccharide composition was measured using a laser light scattering instrument (DAWN HELEOS-II) and was 890 Da.

[0129] Example 2 Preparation of hyaluronic acid oligosaccharide composition

[0130] In a 5L reactor, add 4L of water, control the temperature at 30°C, add hyaluronidase (the enzyme is leech hyaluronidase expressed by Pichia pastoris, the preparation method is described in CN 103695448 A) 4×10 7 U, the enzyme activity of the system is 1×10 4 U / mL, add 5g hyaluronic acid (molecular weight 100kDa), after it is completely dissolved, use ultrafiltration to separate the enzymatic reaction to obtain the product stock solution, and use nanofiltration to concentrate the product stock solution to obtain the product concentrate. Keep the system at 30°C and stir the reaction for 12h. The ultrafiltration uses an ultrafiltration membrane with a molecular weight cutoff of 1kDa, and the nanofiltration uses a nanofiltration membrane with a molecular weight cutoff of 200Da. After the reaction is completed, the nanofiltration concentrate is collected, and after adding activated carbon adsorption, the filtrate is filtered and collected, and spray dried, wherein the inlet temperature is 120°C and the outlet temperature is 60°C. A hyaluronic acid oligosaccharide composition is obtained, and the product yield is 84%.

[0131] The hyaluronic acid oligosaccharide composition was analyzed for oligosaccharide distribution using a high performance liquid chromatograph, and the specific chromatographic conditions were the same as those in Example 1. The results are shown in FIG. Figure 2 As shown, in the hyaluronic acid oligosaccharide composition, the mass proportion of hyaluronic acid disaccharide is 9.35%, the mass proportion of hyaluronic acid tetrasaccharide is 47.84%, the mass proportion of hyaluronic acid hexasaccharide is 32.92%, the mass proportion of hyaluronic acid octasaccharide is 6.04%, the mass proportion of hyaluronic acid decasaccharide is 1.48%, and the mass proportion of hyaluronic acid dodecasaccharide is 0.42%.

[0132] The average molecular weight of the hyaluronic acid oligosaccharide composition was measured by laser light scattering and was 920 Da.

[0133] Example 3 Preparation of hyaluronic acid oligosaccharide composition

[0134] In a 5L reactor, add 4L of water, control the temperature at 46°C, add 1.2×10 hyaluronidase (the enzyme is leech hyaluronidase expressed by Pichia pastoris, the preparation method is described in CN 103695448 A) 9 U, the enzyme activity of the system is 3×10 5 U / mL, add 150g hyaluronic acid (molecular weight 200kDa), after it is completely dissolved, use ultrafiltration to separate the enzymatic reaction to obtain the product stock solution, and use nanofiltration to concentrate the product stock solution to obtain the product concentrate. Keep the system at 46°C and stir the reaction for 24h. The ultrafiltration uses an ultrafiltration membrane with a molecular weight cutoff of 1kDa, and the nanofiltration uses a nanofiltration membrane with a molecular weight cutoff of 200Da. After the reaction is completed, the nanofiltration concentrate is collected, and after adding activated carbon adsorption, the filtrate is filtered and collected, and spray dried, wherein the inlet temperature is 160°C and the outlet temperature is 80°C. A hyaluronic acid oligosaccharide composition is obtained, and the product yield is 87%.

[0135] The hyaluronic acid oligosaccharide composition was analyzed for oligosaccharide distribution using a high performance liquid chromatograph, and the specific chromatographic conditions were the same as those in Example 1. The results are shown in FIG. Figure 3 As shown, in the hyaluronic acid oligosaccharide composition, the mass proportion of hyaluronic acid disaccharide is 8.94%, the mass proportion of hyaluronic acid tetrasaccharide is 52.80%, the mass proportion of hyaluronic acid hexasaccharide is 29.00%, the mass proportion of hyaluronic acid octasaccharide is 4.71%, the mass proportion of hyaluronic acid decasaccharide is 1.5%, and the mass proportion of hyaluronic acid dodecasaccharide is 0.40%.

[0136] The average molecular weight of the hyaluronic acid oligosaccharide composition was measured using a laser light scattering instrument and was found to be 915 Da.

[0137] Comparative Example 1

[0138] In a 5L reactor, add 4L of water, control the temperature at 40°C, add 8×10 hyaluronidase (the enzyme is leech hyaluronidase expressed by Pichia pastoris, the preparation method is described in CN 103695448 A) 8 U, the enzyme activity of the system is 2×10 5 U / mL, add 100g hyaluronic acid (molecular weight 150KDa), after it is completely dissolved, keep the system at 40℃ and stir for 20h. After the reaction is over, collect the reaction solution, add activated carbon for adsorption, filter and collect the filtrate, and spray dry it, wherein the inlet temperature is 140℃ and the outlet temperature is 70℃. The hyaluronic acid hydrolyzate is obtained, and the product yield is 90%.

[0139] The oligosaccharide distribution of the hyaluronic acid hydrolysate was analyzed by high performance liquid chromatography, and the specific chromatographic conditions were the same as those in Example 1. Figure 4 As shown, in the hyaluronic acid oligosaccharide composition, the mass proportion of hyaluronic acid disaccharide is 0.00%, the mass proportion of hyaluronic acid tetrasaccharide is 0.98%, the mass proportion of hyaluronic acid hexasaccharide is 2.10%, the mass proportion of hyaluronic acid octasaccharide is 2.67%, the mass proportion of hyaluronic acid decasaccharide is 3.31%, and the mass proportion of hyaluronic acid dodecasaccharide is 3.97%.

[0140] The average molecular weight of the hyaluronic acid oligosaccharide composition was measured using a laser light scattering instrument and was 7580 Da.

[0141] Test example

[0142] Test Example 1

[0143] The hyaluronic acid oligosaccharide composition obtained in Example 2 was characterized by ESI-MS, and the conditions for mass spectrometry detection were:

[0144] Chromatograph: Waters Acquity UPLC; analytical column: BEH Amide 2.1mm×100mm 1.7μm, column temperature: 45℃, flow rate 0.3mL / min, injection volume 1μL, mobile phase: A: ammonium formate, B: acetonitrile, program elution according to the conditions shown in Table 1, ion mode: ESI. Ion source parameters ESI, nebulizer pressure: 30psi; desolvation gas flow rate 50L / h; temperature 400℃; negative ion mode.

[0145] Table 1

[0146]

[0147] The results are as follows Figure 5-10 As shown, the mass peak [M] at 1.380 min on the mass spectrometry total ion current peak graph was 397.1, which was identified as hyaluronan disaccharide ( Figure 6); the mass peak [M] at 3.649 min on the mass spectrometry total ion current peak graph was 776.2, which was identified as hyaluronic acid tetrasaccharide ( Figure 7 ); the mass peak [M] at 5.612 min on the mass spectrometry total ion current peak graph was 1155.3, which was identified as hyaluronic acid hexasaccharide ( Figure 8 ); the mass peak [M] at 6.724min on the mass spectrometry total ion current peak graph was 1535.5, which was identified as hyaluronic acid octasaccharide ( Fig. 9 ); the mass peak [M] at 7.257min on the mass spectrometry total ion current peak graph was 1913.5, which was identified as hyaluronic acid decasaccharide ( Fig.10 ), the subsequent mass spectrum peak [M] was 2292.6, which was identified as hyaluronic acid dodecasaccharide ( Fig.10 ).

[0148] Experimental Example 2 Identification of reducing ends of hyaluronic acid oligosaccharide compositions

[0149] The reducing end of the hyaluronic acid oligosaccharide composition obtained in Example 2 was identified.

[0150] The reducing end of the hyaluronic acid oligosaccharide composition was determined by colorimetry using the Morgan–Elson reaction. If it turns red, it indicates that the reducing end is N-acetylglucosamine, and if it does not turn red, it indicates that the reducing end is uronic acid.

[0151] Reaction buffer: alkaline boric acid solution (dissolve 1.73 g H 3 BO 3 and 0.78 g KOH, and add one tenth of its volume of 0.8 g / ml K 2 CO 3 . ) p-Dimethylaminobenzaldehyde solution (dissolve 2 grams of p-dimethylaminobenzaldehyde solution in 2.5 ml of concentrated hydrochloric acid and 7.5 ml of glacial acetic acid, and dilute with 4 times the volume of glacial acetic acid before use).

[0152] 400 μl of 10 g / L hyaluronic acid oligosaccharide composition solution was taken, 110 μl of alkaline boric acid solution was added, and the mixture was boiled for 4 min. 1.5 ml of p-dimethylaminobenzaldehyde was added, and the mixture was incubated at 37° C. for 20 min.

[0153] Hyaluronic acid (produced by Bloomage Biotechnology, with N-acetylglucosamine at the reducing end and a molecular weight of 1700 KDa) and hyaluronic acid tetrasaccharide (purchased from Sigma, with N-acetylglucosamine at the reducing end) solutions were used as controls.

[0154] The results are as follows Fig.11 As shown, the hyaluronic acid oligosaccharide composition did not show red color after the Moran-Elson reaction, proving that the reducing end was uronic acid.

[0155] Test Example 3 Cytotoxicity Evaluation and Patch Test

[0156] Cytotoxicity evaluation and patch test of the hyaluronic acid oligosaccharide composition obtained in Example 2

[0157] Cytotoxicity test

[0158] 1) Plate laying: Human epidermal keratinocytes HaCaT in logarithmic growth phase were plated at 5×10 4 The cells were inoculated at a density of 100 μL / mL in a 96-well plate. The culture system was DMEM high-glucose medium supplemented with 10% fetal bovine serum. The inoculated cells were placed in a carbon dioxide incubator at 37°C and 5% CO. 2 Routine culture for 24 hours.

[0159] 2) Sample solution preparation: The sample was prepared into a stock solution of a certain concentration using serum-free culture medium, and sterilized by filtration with a 0.22 μm filter membrane. When used, it was diluted with serum-free culture medium to the required mass concentration of 1%, 0.5% and 0.05% final concentration.

[0160] 3) Adding drugs: After 24 hours of routine culture, discard the old culture medium, replace the experimental group with 100 μL sample, and add the same amount of serum-free culture medium to the normal control group (control), with 6 parallel wells for each level.

[0161] 4) Detection: After 24 hours of continuous culture, the sample solution was discarded, and 10% WST-1 solution prepared with serum-free culture medium was added to each well, and the cells were placed in a cell culture incubator for further incubation for 3 hours. The absorbance was measured at a wavelength of 450 nm using an enzyme marker. The relative proliferation rate (RGR) is the ratio of the absorbance of the experimental group to that of the normal control group. According to the requirements of GB / T16886.5-2017, when the RGR is less than 70%, the sample is considered to be cytotoxic.

[0162] The results are shown in Table 2.

[0163] Table 2 Relative cell proliferation rate (%)

[0164]

[0165]

[0166] Table 2 shows that within the mass concentration range of 0.05%-1% of the hyaluronic acid oligosaccharide composition, the relative cell proliferation rate is much greater than 70%, indicating that the hyaluronic acid oligosaccharide composition has no obvious cytotoxicity.

[0167] Patch test

[0168] 1) Recruit 30 healthy subjects, both male and female. The following subjects should be excluded: a. Those who have used antihistamines in the past week or immunosuppressants in the past month; b. Those who have used any anti-inflammatory drugs on the test site in the past two months; c. Subjects with inflammatory skin diseases that have not been cured clinically; d. Patients with insulin-dependent diabetes; e. Patients with asthma or other chronic respiratory diseases being treated; f. Those who have received anti-cancer chemotherapy in the past 6 months; g. Patients with immunodeficiency or autoimmune diseases; h. Lactating or pregnant women; i. Those who have undergone bilateral mastectomy and bilateral axillary lymph node resection; j. Those whose skin test site has scars, pigments, atrophy, port wine stains or other blemishes that affect the determination of the test results; k. Those who have participated in other clinical trial researchers; l. Those with highly sensitive constitutions; m. Non-voluntary participants or those who cannot complete the specified content according to the experimental requirements.

[0169] 2) Sample preparation

[0170] The hyaluronic acid oligosaccharide composition of Example 2 was diluted with purified water to a mass concentration of 1% before use, and purified water was used as a control.

[0171] 3) Skin patch test: Open the patch tester package, measure 0.025 mL of each prepared sample and add it into the small chamber. Apply the patch tester to the flexed side of the subject's forearm, and gently press with the palm of your hand to evenly apply it to the skin for 24 hours.

[0172] 4) Analysis of results Observe and record the reaction results according to Table 3 30 minutes, 24 hours and 48 hours after removing the spot tester.

[0173] The results are shown in Table 4.

[0174] Table 3 Skin reaction grading standards for skin occlusion patch test

[0175]

[0176]

[0177] Table 4 Patch test results statistics (total number of cases: 30)

[0178] Grading Hyaluronic acid oligosaccharide composition Purified water Level 0 30 30 Level 1 0 0 Level 2 0 0 Level 3 0 0 Level 4 0 0

[0179] The results showed that patch tests of the hyaluronic acid oligosaccharide composition with a mass concentration of 1% had no adverse reactions, indicating that the hyaluronic acid oligosaccharide composition has no potential adverse reactions to human skin.

[0180] Test Example 4 Evaluation of the efficacy and activity of oligosaccharide compositions - Effects on endogenous hyaluronidase and hyaluronic acid

[0181] The expression of hyaluronic acid synthase I (HAS1) and hyaluronic acid synthase III (HAS3) genes and the secretion of hyaluronic acid (HA) in the cells were used to evaluate the moisturizing effect of the sample to be tested. The specific method is:

[0182] Cell seeding: 2.2×10 5 Fibroblasts were seeded into 6-well plates at a seeding density of 100 cells / well and incubated in an incubator (37°C, 5% CO 2 ) and incubate overnight. When the cell plating rate in the 6-well plate reached 50% to 60%, the grouping and administration were carried out, and the concentrations of administration were 0.01 mg / ml, 1 mg / ml and 10 mg / ml, respectively, with 3 replicates in each group. After the administration, the 6-well plate was placed in an incubator (37°C, 5% CO 2 ) and incubate for 24 h. ELISA detection: Detection was performed according to the operating instructions of Hyaluronan Quantikine ELISA Kit. Gene detection: Wash twice with 2 mL / well PBS, add 1 mL RNAiso Plus to each well, blow and lyse the cells, and collect the samples. RNA extraction, reverse transcription and fluorescence quantitative PCR detection were carried out according to the instructions of the kit, using 2- △△CT Method to calculate the results.

[0183] The blank control refers to the addition of no sample, and the rest is carried out simultaneously with the test group. The promoting effect of hyaluronan synthase and the promoting effect of endogenous hyaluronic acid are shown in Table 5 and Table 6 respectively.

[0184] Table 5 Promotion effect of hyaluronan synthase

[0185] Sample name HAS1 HAS3 Blank control 1.01 1.00 Hyaluronic acid oligosaccharide composition (0.01 mg / ml) 1.06 1.15 Hyaluronic acid oligosaccharide composition (1 mg / ml) 3.15 6.15 Hyaluronic acid oligosaccharide composition (10 mg / ml) 14.44 25.22

[0186] Note: Use 2- △△CT Methods When calculating the results and performing statistical analysis using the t-test method, the mRNA amplification fold of the blank control group was normalized.

[0187] Table 6 Promotion effect of endogenous hyaluronic acid

[0188]

[0189] The results show that the hyaluronic acid oligosaccharide composition prepared by the present invention can significantly improve the production of hyaluronic acid synthase HAS1, HAS3, especially HAS3 in fibroblasts. The increase of hyaluronic acid synthase helps to improve the synthesis of endogenous hyaluronic acid in fibroblasts. The detection of endogenous hyaluronic acid in fibroblasts shows that it has indeed increased, significantly increased compared with the blank group. The dermis in human skin contains abundant fibroblasts, and the hyaluronic acid oligosaccharide composition has a very small molecular weight, which shows that the hyaluronic acid oligosaccharide composition has the potential effect of increasing endogenous hyaluronic acid in the dermis of human skin.

[0190] Test Example 5 Evaluation of the efficacy and activity of hyaluronic acid oligosaccharide composition - Effect on ceramide expression

[0191] Based on the 3D epidermal skin model (purchased from Guangdong Boxi Biotechnology Co., Ltd.), in Test Example 2, hyaluronic acid oligosaccharide composition samples (0.01 mg / ml, 1 mg / ml and 10 mg / ml, respectively) were added to the culture medium for 6 consecutive days. The total ceramide content in the skin model was normalized by dividing it by the total water-soluble protein to evaluate the sample's ability to promote the synthesis of ceramide.

[0192] 1. Extraction of water-soluble total protein: Cut the three models in each group into two halves, place 1 / 2 of the halves in a 1.5mL EP tube, add 250μL of 0.2mg / mL proteinase K solution, and place in a 50℃ water bath for 2h. After the water bath, use tweezers to grab the stratum corneum and rinse it in deionized water, and place the stratum corneum in a new EP tube. Add 125μL of protein extract to each tube; ultrasonic oscillate for 30min. After the ultrasonic oscillation, centrifuge at 4℃, 14000rpm for 10min. After the centrifugation, pipette the supernatant in the protein extraction tube into a new EP tube, and use the BCA kit to detect the concentration.

[0193] 2. Use the BCA protein concentration assay kit to detect the protein concentration. After the reaction is complete, read the result at 562 nm on an ELISA reader.

[0194] Preparation of lipid extract: 1. Take 50 mL of methanol and chloroform, mix well; 2. Ceramide extraction: put the remaining 1 / 2 model after protein extraction into the EP tube containing lipid extract, ultrasonically shake in ice water bath for 2 minutes, then switch to a system of methanol: chloroform = 1:2, and ultrasonically shake in ice water bath for 2 minutes; after the end, switch to a system of methanol: chloroform = 2:1, ultrasonically shake in ice bath for two minutes; after the end, remove the model and blow dry the extract with nitrogen. 3. Add a mixed solution of acetonitrile: isopropanol = 1:1 (containing ceramide internal standard) to the nitrogen-dried sample bottle, ultrasonically shake for 10 minutes, shake to dissolve for 30 minutes, transfer to a centrifuge tube, centrifuge at 12000rpm for 10 minutes, take the upper clear liquid for testing.

[0195] The blank control refers to the addition of no sample, and the rest is carried out simultaneously with the experimental group.

[0196] The results are shown in Tables 7, 8 and 9.

[0197] Table 7 Determination results of water-soluble total protein content

[0198] Sample name Total protein content (μg) Decline rate (%) Blank control 193.17 —— Hyaluronic acid oligosaccharide composition (0.01 mg / ml) 188.3 2.5 Hyaluronic acid oligosaccharide composition (1 mg / ml) 152.15 21.2 Hyaluronic acid oligosaccharide composition (10 mg / ml) 132.34 31.5

[0199] The results in Table 7 show that compared with the control group, the water-soluble protein content in the epidermal model using the hyaluronic acid oligosaccharide composition was significantly reduced. This result is speculated to be because the hyaluronic acid oligosaccharide composition can promote the differentiation of the skin model, advance the living cell layer to the stratum corneum, and thicken the stratum corneum, showing a decrease in the total soluble protein content of the living cell layer, thereby reflecting that the sample oligosaccharide composition can enhance the barrier of the epidermal model.

[0200] Table 8 Total content of ceramide

[0201] Sample name Total ceramide content (μg) Improvement rate (%) Blank control 15.398 —— Hyaluronic acid oligosaccharide composition (0.01 mg / ml) 15.732 2.2 Hyaluronic acid oligosaccharide composition (1 mg / ml) 22.531 46.3 Hyaluronic acid oligosaccharide composition (10 mg / ml) 26.562 72.5

[0202] The results in Table 8 show that compared with the control group, the total content of ceramide in the epidermal model treated with the hyaluronic acid oligosaccharide composition was significantly increased, which indicates that the oligosaccharide composition can increase the ceramide content in the epidermal model.

[0203] Table 9 Ratio of total ceramide content to total protein content

[0204]

[0205] The calculation of total ceramide content / total protein content can normalize the ceramide in the skin and can more scientifically reflect the changes in ceramide in the skin. As shown in Table 9, the normalized results show that after adding the oligosaccharide composition, the ceramide content in the skin is significantly increased, indicating that the hyaluronic acid oligosaccharide composition can significantly promote the synthesis of ceramide.

Claims

1. A hyaluronic acid oligosaccharide composition for promoting the synthesis of ceramide and / or hyaluronic acid synthase, characterized in that: The hyaluronic acid oligosaccharide composition comprises hyaluronic acid oligosaccharides having a structure shown in formula (I): Formula (I); wherein n is an integer selected from 0 to 5, and X is selected from H, K, Na, Ca or Zn; In the hyaluronic acid oligosaccharide composition, The mass proportion of hyaluronic acid oligosaccharides with n=1 is 42-52%; The mass proportion of hyaluronic acid oligosaccharide with n=0 is 8-10%; The mass proportion of hyaluronic acid oligosaccharides with n=2 is 27-35%; The mass proportion of hyaluronic acid oligosaccharides with n=3 is 4-7%; The mass proportion of hyaluronic acid oligosaccharides with n=4 is 1-2%; The mass proportion of hyaluronic acid oligosaccharides with n=5 is 0.1-1%; The weight average molecular weight of the hyaluronic acid oligosaccharide composition is less than or equal to 1 kDa.

2. The hyaluronic acid oligosaccharide composition according to claim 1, characterized in that The hyaluronic acid oligosaccharide composition is used in cosmetics.

3. The hyaluronic acid oligosaccharide composition according to claim 2, characterized in that The mass concentration in the cosmetic is 0.001-10%.

4. A method for preparing the hyaluronic acid oligosaccharide composition according to any one of claims 1 to 3, characterized in that: The preparation method comprises the following steps: The hyaluronic acid is enzymatically hydrolyzed with hyaluronidase. Separate the enzymatic reaction products to obtain the product stock solution, Concentrating the product stock solution to obtain a product concentrate; removing impurities from the product concentrate and drying to obtain a hyaluronic acid oligosaccharide composition, Wherein, the hyaluronidase is a hyaluronidase that cleaves β-1,3 glycosidic bonds.

5. The method according to claim 4, characterized in that The hyaluronidase is a leech hyaluronan hydrolase expressed by Pichia pastoris engineered bacteria.

6. The method according to claim 4, characterized in that The separation is ultrafiltration.

7. The method according to claim 4, characterized in that The concentration is nanofiltration.

8. The method according to claim 4, characterized in that Use activated carbon adsorption to remove impurities.

9. The method according to claim 4, characterized in that In the enzymatic hydrolysis reaction, the initial concentration of the hyaluronic acid is 5-150 g / L.

10. The method according to claim 4, characterized in that The molecular weight of the hyaluronic acid is 1000-2000 kDa.

11. The method according to claim 4, characterized in that The initial content of the hyaluronidase is 1×10 4 -3×10 5 U / mL.

12. The method according to claim 4, characterized in that The initial ratio of the hyaluronidase to the hyaluronic acid is 1-5 U / mg.

13. The method according to claim 6, characterized in that Ultrafiltration uses an ultrafiltration membrane with a molecular weight cutoff of 600-1000Da.

14. The method according to claim 7, characterized in that Nanofiltration uses a nanofiltration membrane with a molecular weight cutoff of 100-300Da.

15. The method according to claim 4, characterized in that The temperature of the enzymatic hydrolysis reaction is 30-46° C. and the time is 12-24 h.

16. The method according to claim 4, characterized in that The drying is spray drying, and the air inlet temperature is 120°C-160°C, and the air outlet temperature is 60°C-80°C.

17. Use of a hyaluronic acid oligosaccharide composition in the preparation of cosmetics for promoting the synthesis of ceramide and / or hyaluronic acid synthase 1 (HAS1) and / or hyaluronic acid synthase 3 (HAS3), The hyaluronic acid oligosaccharide composition comprises hyaluronic acid oligosaccharides having a structure shown in formula (I): Formula (I); in, n is an integer selected from 0-5, X is selected from H, K, Na, Ca or Zn; In the hyaluronic acid oligosaccharide composition, The mass proportion of hyaluronic acid oligosaccharides with n=1 is 42-52%; The mass proportion of hyaluronic acid oligosaccharide with n=0 is 8-10%; The mass proportion of hyaluronic acid oligosaccharides with n=2 is 27-35%; The mass proportion of hyaluronic acid oligosaccharides with n=3 is 4-7%; The mass proportion of hyaluronic acid oligosaccharides with n=4 is 1-2%; The mass proportion of hyaluronic acid oligosaccharides with n=5 is 0.1-1%; The weight average molecular weight of the hyaluronic acid oligosaccharide composition is less than or equal to 1 kDa.

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