A Hyaluronic Acid Endogenous Growth Promoter and Its Application

Abstract

This invention discloses a hyaluronic acid endogenous growth promoter, composed of oat β-glucan and mannan, both with a molecular weight less than 10 kDa. The hyaluronic acid endogenous growth promoter is prepared by enzymatic hydrolysis of a mixture of natural oat β-glucan and natural mannan in a mass ratio of 1-7:3-9, or by enzymatic hydrolysis of each separately. This invention also discloses the application of this hyaluronic acid endogenous growth promoter in the preparation of products with skin barrier repair effects. Furthermore, this invention discloses the role of this hyaluronic acid endogenous growth promoter in promoting the mRNA expression of hyaluronic acid synthase, ceramide synthase, and human β-defensin. The hyaluronic acid endogenous growth promoter of this invention promotes the mRNA expression of skin barrier-related genes and thus promotes skin barrier repair.

Description

Technical Field

[0001] This invention relates to the field of natural medicinal chemistry, and more specifically, to a hyaluronic acid endogenous growth promoter and its application. Background Technology

[0002] The skin is the body's first line of defense, playing a vital role in resisting the invasion of harmful external factors, anti-oxidation, and microbial infection. Some exogenous or endogenous factors or diseases can damage the skin barrier, increasing moisture loss and leading to dryness, peeling, and sensitivity. The skin barrier, broadly defined, includes the microbial barrier, physical barrier, pigment barrier, and immune barrier. The skin microbiota is one of the main components of the skin barrier, and its balance is essential for maintaining skin health. Damage to the skin barrier can easily lead to the proliferation of harmful bacteria, which can be countered by promoting the production of endogenous antibacterial components.

[0003] β-defensins are a class of antimicrobial proteins found in various organisms, possessing antimicrobial and antiviral activities. Human β-defensin (HBD), present in the human body, is a cationic antimicrobial peptide with natural antimicrobial activity. There are multiple types of HBD, among which HBD-3 is mainly expressed in tonsils and skin tissue.

[0004] Hyaluronic acid (HA) is an acidic mucopolysaccharide with exceptional water-retention properties, making it the most effective moisturizing substance found in nature. Skin contains a large amount of hyaluronic acid, and its content changes throughout the maturation and aging process of human skin. Hyaluronic acid can improve the nutritional metabolism of skin cells, making the skin soft and smooth, and can also reduce wrinkles, increase skin elasticity, and delay skin aging. Hyaluronic acid synthase (HAS) is an enzyme that plays a crucial role in the synthesis of hyaluronic acid. HAS catalyzes the synthesis of a significant amount of hyaluronic acid, replenishing the body's hyaluronic acid levels at different stages and in different parts of the body.

[0005] Ceramides are a major component of lipids in the stratum corneum of the skin, accounting for approximately 50% of intercellular lipids. Ceramides are linked to proteins on the surface of keratinocytes via ester bonds, playing a role in cell adhesion. A decrease in ceramide content in the stratum corneum reduces the adhesion between keratinocytes, leading to dry, flaky, and scaly skin. The content of ceramides is regulated through complex metabolic pathways, primarily controlled by its synthases and degradative enzymes. There are six isoforms of ceramide synthases, each capable of synthesizing ceramides with different acyl chain lengths. Ceramide synthase 2 (CerS2) is the most widely expressed gene in the CerS family.

[0006] Therefore, studies on the effects of gene expression on hyaluronic acid synthase (HAS), ceramide synthase 2 (CerS2), and human β-defensin (HBD) can be used to identify substances that can repair the skin barrier. Furthermore, zebrafish have an extremely similar genome to humans, with a similarity of up to 87%. In addition, the development and structural function of the tissue barrier in zebrafish embryos are similar to those in mammals, making zebrafish an ideal model for studying the structure and function of tissue barriers.

[0007] Oat beta-glucan is a water-soluble dietary fiber extracted from oat bran. Modern research shows that oat beta-glucan has strong water-holding capacity and a good moisturizing effect on the skin.

[0008] Mannans are highly branched polymers with α-1,6-mannose as the backbone chain. Most, or even all, of the residues are linked by α-1,2 or α-1,3 glycosidic bonds to side chains containing 2 to 5 mannose residues. They are mainly derived from Bletilla striata, konjac, and Dendrobium nobile. Modern research shows that mannans have good water absorption, film-forming, and water retention properties.

[0009] However, there are no reports on the effects of combining small molecule oat β-glucan and small molecule mannan on the skin barrier. Summary of the Invention

[0010] This invention addresses the aforementioned technical problems by proposing a hyaluronic acid endogenous growth promoter, composed of small-molecule oat β-glucan and small-molecule mannan. This promoter effectively promotes the synthesis of hyaluronic acid synthase, ceramide synthase, and human β-defensin, thus playing a significant role in skin barrier repair. Therefore, the first objective of this invention is to provide a hyaluronic acid endogenous growth promoter. The second objective of this invention is to provide an application of this hyaluronic acid endogenous growth promoter.

[0011] To achieve the above objectives, the present invention adopts the following technical solution:

[0012] As a first aspect of the present invention, a hyaluronic acid endogenous growth promoter is provided, which is composed of oat β-glucan and mannan, wherein the molecular weight of oat β-glucan and mannan is less than 10 kDa, and the hyaluronic acid endogenous growth promoter is prepared by enzymatic hydrolysis of natural oat β-glucan and natural mannan in a mass ratio of 1-7:3-9 or by enzymatic hydrolysis of each separately.

[0013] According to the present invention, the specific steps for preparing the hyaluronic acid endogenous growth promoter by mixed enzymatic hydrolysis are as follows: natural oat β-glucan and natural mannan are mixed in a mass ratio of 1-7:3-9, enzymatically hydrolyzed by β-glucanase, and then passed through a 3-10kDa cutoff membrane to obtain small molecule polysaccharides and oligosaccharides. The monosaccharide solution is then removed through a 200-500Da concentrating membrane, and the product is prepared after drying.

[0014] According to the present invention, the activity unit of the β-glucanase is 50,000-200,000 U / g, and its mass is 0.1%-10% of the total mass of the polysaccharide.

[0015] Preferably, the molecular weight cutoff of the retention membrane is 10 kDa, and the molecular weight cutoff of the concentration membrane is 500 Da.

[0016] According to the present invention, the specific steps for preparing the hyaluronic acid endogenous growth promoter by enzymatic hydrolysis are as follows: natural oat β-glucan and natural mannan in a mass ratio of 1-7:3-9 are respectively enzymatically hydrolyzed by β-glucanase, and then passed through a 3-10kDa cutoff membrane to obtain small molecule polysaccharides and oligosaccharides, respectively. The monosaccharide solution is removed by passing through a 200-500Da concentration membrane, and after drying, small molecule oat β-glucan and mannan are obtained respectively. The two are then mixed evenly to prepare the hyaluronic acid endogenous growth promoter.

[0017] According to the present invention, the activity unit of the β-glucanase is 50,000-200,000 U / g, and the amount of enzyme added is 0.1%-10% of the mass of natural oat β-glucan or natural mannan.

[0018] Preferably, the molecular weight cutoff of the retention membrane is 10 kDa, and the molecular weight cutoff of the concentration membrane is 500 Da.

[0019] As a second aspect of the present invention, the application of a hyaluronic acid endogenous growth promoter in the preparation of products with skin barrier repair function.

[0020] Furthermore, the application of a hyaluronic acid endogenous growth promoter in the preparation of cosmetic products or topical formulations with skin barrier repair effects.

[0021] As a third aspect of the present invention, the application of a hyaluronic acid endogenous promoter in promoting the mRNA expression of hyaluronic acid synthase, ceramide synthase, and human β-defensin.

[0022] The beneficial effects of this invention are as follows: The hyaluronic acid endogenous growth promoter of this invention can effectively promote the expression of hyaluronic acid synthase, ceramide synthase, and human β-defensin mRNA, and can be well applied in skin barrier repair. Therefore, it has wide applications in the fields of cosmetics and topical preparations. Attached Figure Description

[0023] Figure 1 A schematic flow chart for preparing a mixed concentrate of small molecule oat β-glucan and small molecule mannan using an online enzymatic hydrolysis-molecular retention unit.

[0024] Figure 2 The bar chart shows the promoting effect of each sample on HAS1 mRNA expression.

[0025] Figure 3 The bar chart shows the promoting effect of each sample on HAS2 mRNA expression.

[0026] Figure 4 The bar chart shows the promoting effect of each sample on HAS3 mRNA expression.

[0027] Figure 5 The bar chart shows the promoting effect of each sample on CerS2 mRNA expression.

[0028] Figure 6 The bar chart shows the promoting effect of each sample on HBD-3 mRNA expression.

[0029] Figure 7 The bar chart shows the effect of each group on promoting the repair of the caudal fin of zebrafish embryos. Detailed Implementation

[0030] The present invention will be further explained and illustrated below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0031] 1. The source of raw materials for the following embodiments:

[0032] Natural oat beta-glucan comes from oats; natural mannan comes from dendrobium, bletilla, konjac, and microbial fermentation, etc.

[0033] 2. Online enzymatic hydrolysis-molecular retention unit:

[0034] Examples 1-3 and Comparative Examples 1 and 2 all employed an online enzymatic hydrolysis-molecular retention unit. Figure 1The diagram shown is a schematic flow chart of the online enzymatic hydrolysis-molecular cutoff unit of this invention. The online enzymatic hydrolysis-molecular cutoff unit consists of an enzymatic hydrolysis tank and a circulating cutoff-concentration membrane unit. In the circulating cutoff-concentration membrane unit, the molecular weight cutoff of the cutoff membrane can be 3-10 kDa, and the molecular weight cutoff of the concentration membrane can be 200-500 Da. In the examples and comparative examples, a cutoff membrane with a molecular weight cutoff of 10 kDa and a concentration membrane with a molecular weight cutoff of 500 Da were used. A natural macromolecular polysaccharide solution and β-glucanase are placed in an enzymatic hydrolysis tank for enzymatic hydrolysis. The hydrolysate is then passed through a 10kDa retention membrane. Polysaccharides, oligosaccharides, and monosaccharides with molecular weights less than 10kDa, as well as water molecules, can pass through this membrane, while polysaccharide molecules with molecular weights greater than 10kDa are retained and returned to the enzymatic hydrolysis tank for further enzymatic hydrolysis. The small molecule solution is then passed through a 500Da concentration membrane. Monosaccharide molecules and water molecules pass through the concentration membrane, while the retained solution is a concentrated solution of polysaccharides or oligosaccharides with molecular weights less than 10kDa that does not contain monosaccharide molecules. The monosaccharide solution that has passed through the concentration membrane is returned to the enzymatic hydrolysis tank to ensure sufficient water volume for continued enzymatic hydrolysis, thus forming a circulating mechanism of the online enzymatic hydrolysis-molecular retention unit.

[0035] Example 1: Preparation of Hyaluronic Acid Endogenous Growth Promoter

[0036] (1) Take 10g of natural oat β-glucan and 90g of natural mannan, mix them evenly, and add 2000g of deionized water;

[0037] (2) Add 0.1g of β-glucanase with an activity unit of 200,000 U / g;

[0038] (3) Prepare a mixed concentrate of polysaccharides and oligosaccharides of oat β-glucan and mannan with a molecular weight of less than 10 kDa by an online enzymatic hydrolysis-molecular retention unit;

[0039] (4) The mixed concentrate obtained in step (3) is dried to obtain 65g of small molecule polysaccharide, which is the endogenous growth promoter of hyaluronic acid.

[0040] Example 2 Preparation of Hyaluronic Acid Endogenous Growth Promoter

[0041] (1) Take 70g of natural oat β-glucan and 30g of natural mannan, mix them evenly, and add 10000g of deionized water;

[0042] (2) Add 10g of β-glucanase with an activity unit of 100,000 U / g;

[0043] (3) Prepare a mixed concentrate of polysaccharides and oligosaccharides of oat β-glucan and mannan with a molecular weight of less than 10 kDa by an online enzymatic hydrolysis-molecular retention unit;

[0044] (4) The mixed concentrate obtained in step (3) is dried to obtain 56g of small molecule polysaccharide, which is the endogenous growth promoter of hyaluronic acid.

[0045] Example 3 Preparation of Hyaluronic Acid Endogenous Growth Promoter

[0046] 1. Preparation of small molecule oat β-glucan with a molecular weight of less than 10 kDa:

[0047] (1) Take 50g of natural oat β-glucan and add 2500g of deionized water to it;

[0048] (2) Add 3.5g of β-glucanase with an activity unit of 50,000 U / g, and use an online enzymatic hydrolysis-molecular retention unit to obtain a mixed concentrate of polysaccharides and oligosaccharides of small molecule oat β-glucan with a molecular weight of less than 10kDa;

[0049] (3) The mixed concentrate obtained in step (2) is dried to obtain small molecule oat β-glucan powder with a molecular weight of less than 10kDa.

[0050] 2. Preparation of small molecule mannans with a molecular weight of less than 10 kDa:

[0051] (1) Take 50g of natural mannan and add 2500g of deionized water to it;

[0052] (2) Add 3.5g of β-glucanase with an activity unit of 50,000 U / g, and use an online enzymatic hydrolysis-molecular retention unit to obtain a mixed concentrate of polysaccharides and oligosaccharides of small molecule mannan with a molecular weight of less than 10kDa;

[0053] (3) The mixed concentrate obtained in step (2) is dried to obtain small molecule mannan powder with a molecular weight of less than 10 kDa.

[0054] 3. Mix the small molecule oat β-glucan powder and small molecule mannan powder obtained above evenly to obtain 66g of product, namely hyaluronic acid endogenous growth promoter.

[0055] Comparative Example 1

[0056] 1. Preparation of small molecule oat β-glucan with a molecular weight of less than 10 kDa:

[0057] (1) Take 50g of natural oat β-glucan and add 2500g of deionized water to it;

[0058] (2) Add 0.025g of β-glucanase with an activity unit of 10,000 U / g, and use an online enzymatic hydrolysis-molecular retention unit to obtain a mixed concentrate of polysaccharides and oligosaccharides of small molecule oat β-glucan with a molecular weight of less than 10kDa;

[0059] (3) The mixed concentrate obtained in step (2) is dried to obtain small molecule oat β-glucan powder with a molecular weight of less than 10kDa.

[0060] 2. Preparation of small molecule mannans with a molecular weight of less than 10 kDa:

[0061] (1) Take 50g of natural mannan and add 2500g of deionized water to it;

[0062] (2) Add 0.025g of β-glucanase with an activity unit of 10,000 U / g, and use an online enzymatic hydrolysis-molecular retention unit to obtain a mixed concentrate of polysaccharides and oligosaccharides of small molecule mannan with a molecular weight of less than 10kDa;

[0063] (3) The mixed concentrate obtained in step (2) is dried to obtain small molecule mannan powder with a molecular weight of less than 10 kDa.

[0064] 3. Mix the small molecule oat β-glucan powder and small molecule mannan powder obtained above evenly to obtain 28g of product.

[0065] Comparative Example 2

[0066] 1. Preparation of small molecule oat β-glucan with a molecular weight of less than 10 kDa:

[0067] (1) Take 50g of natural oat β-glucan and add 2500g of deionized water to it;

[0068] (2) Add 10g of β-glucanase with an activity unit of 300,000 U / g, and use an online enzymatic hydrolysis-molecular retention unit to obtain a mixed concentrate of polysaccharides and oligosaccharides of small molecule oat β-glucan with a molecular weight of less than 10kDa;

[0069] (3) The mixed concentrate obtained in step (2) is dried to obtain small molecule oat β-glucan powder with a molecular weight of less than 10kDa.

[0070] 2. Preparation of small molecule mannans with a molecular weight of less than 10 kDa:

[0071] (1) Take 50g of natural mannan and add 2500g of deionized water to it;

[0072] (2) Add 10g of β-glucanase with an activity unit of 300,000 U / g, and use an online enzymatic hydrolysis-molecular retention unit to obtain a mixed concentrate of polysaccharides and oligosaccharides of small molecule mannan with a molecular weight of less than 10kDa;

[0073] (3) The mixed concentrate obtained in step (2) is dried to obtain small molecule mannan powder with a molecular weight of less than 10 kDa.

[0074] 3. Mix the small molecule oat β-glucan powder and the small molecule mannan powder obtained above evenly to obtain 21g of product.

[0075] The main differences between the above embodiments and comparative examples are listed in Table 1.

[0076] Table 1. Differences between the embodiments and comparative examples

[0077]

[0078] Example 1: Comparison of Product Yields

[0079] The yields of the products in each embodiment and comparative example are shown in Table 2.

[0080] The yield is calculated as follows: W=M n / M0.

[0081] Among them, M n The total mass of small molecule oat β-glucan and mannan with a molecular weight of less than 10 kDa obtained during preparation;

[0082] M0 refers to the total mass of the natural macromolecular oat β-glucan and mannan added.

[0083] Table 2. Product yield

[0084]

[0085] As shown in Table 2, the yields of small molecule oat β-glucan and small molecule mannan prepared by the methods of Examples 1-3 in this invention are all above 50%, which is significantly higher than that of the comparative examples.

[0086] Examples 1 and 2 involve uniformly mixing natural macromolecular oat β-glucan and mannan to prepare a small molecule mixed concentrate with a molecular weight less than 10 kDa. Example 3 involves separately preparing small molecule oat β-glucan and small molecule mannan, and then mixing them uniformly. When the amount of β-glucanase added is 0.1%-10% of the total mass of polysaccharides, and the enzyme activity is in the range of 10,000 U / g-200,000 U / g, there is no significant difference in the yield of the product obtained by preparing the hyaluronic acid endogenous growth promoter of the present invention using the first and second methods.

[0087] The preparation methods used in Comparative Examples 1 and 2 are the same as those used in Example 3, but the amount and activity of the added β-glucanase have been adjusted. It can be seen that both excessive and insufficient amount and activity of the added enzyme will result in a low yield of the product.

[0088] Example 2: Verification of the effect of promoting the expression of genes related to skin barrier repair

[0089] 1. Main reagents:

[0090] DMEM medium, fetal bovine serum (FBS); T25 cell culture flasks and 96-well plates were purchased from Corning Incorporated; cell counting kit (cck)-8 was purchased from DojindoMolecular Technologies; phosphate-buffered saline (PBS, 1X) was purchased from Beyotime.

[0091] Trypsin, total RNA extraction reagent (Trizol), chloroform, isopropanol, ethanol, DEPC water, PrimeScript™ RTMaster Mix (Takara), TB Green Premix Ex Taq™ II (Takara), and primers for each gene.

[0092] 2. Cell lines and cell culture:

[0093] The cell line used in this invention, human immortalized keratinocytes (HaCaT), was derived from Shanghai Skin Disease Hospital. HaCaT cells were cultured in a cell culture incubator (5% CO2, 37°C) using DMEM + 10% FBS medium.

[0094] 3. The effect of the test sample on the expression of relevant barrier genes:

[0095] HaCaT cells were seeded at 200,000 cells per well in a 6-well plate and cultured for 24 hours. Then, culture medium or 2 mL of the sample solution was added and incubated for another 24 hours. Total RNA was extracted from HaCaT cells according to the kit instructions and reverse transcription was performed.

[0096] Genes detected: hyaluronan synthase (HAS, including HAS-1, HAS-2, HAS-3), ceramide synthase (CerS2), and human β-defensin (HBD-3).

[0097] Table 3 Primer List

[0098]

[0099] 4. Effects of different samples on the expression of skin barrier repair-related genes in HaCaT cells

[0100] A mixture of natural macromolecular oat β-glucan and natural macromolecular mannan (mass ratio 1:9) before hydrolysis in Example 1, small-molecule oat β-glucan and small-molecule mannan prepared in Example 3, and mixed products of small-molecule oat β-glucan and small-molecule mannan prepared in Examples 1, 2, and 3 were diluted to a mass fraction of 0.01% and used as test sample solutions to promote the mRNA expression of HAS1, HAS2, HAS3, CerS2, and HBD-3 in HaCaT cells. The relative expression levels of each group are recorded in Tables 4 and 5, and bar charts are plotted.

[0101] Table 4. Effects of each sample on the expression of hyaluronic acid synthase mRNA

[0102]

[0103] As shown in Table 4 Figure 2 , Figure 3 , Figure 4 As shown, at a concentration of 0.01%, the mixture of natural macromolecular polysaccharides, small-molecule oat β-glucan, small-molecule mannan, and the hyaluronic acid endogenous growth promoters prepared in Examples 1-3 all had a certain promoting effect on the mRNA expression of hyaluronic acid synthases HAS1, HAS2, and HAS3. However, the promoting effect of using the two small molecules in combination in Examples 1-3 was significantly stronger than the promoting effect of using them alone, and also significantly stronger than the promoting effect of using the unhydrolyzed macromolecular oat β-glucan and mannan in combination. Furthermore, the hyaluronic acid endogenous growth promoters prepared by enzymatic hydrolysis of the two natural macromolecular polysaccharides in Examples 1 and 2 also had a significantly stronger promoting effect on the expression of hyaluronic acid synthase mRNA than the hyaluronic acid endogenous growth promoters prepared by separately enzymatic hydrolysis of the two small molecules and then mixing them in Example 3. In addition, the promoting effect of Examples 1-3 on the expression of HAS1 mRNA was also significantly stronger than the promoting effect on the expression of HAS2 and HAS3.

[0104] Table 5. Effects of each sample on the expression of ceramide synthase and human β-defensin mRNA

[0105]

[0106] As shown in Table 5 and Figure 5 , Figure 6As shown, at a concentration of 0.01%, each group of samples had a certain promoting effect on the expression of ceramide synthase CerS2 and human β-defensin HBD-3 mRNA. However, the promoting effect of using the two small molecules in combination in Examples 1-3 was significantly stronger than the promoting effect of using them alone, and also significantly stronger than the promoting effect of using the unhydrolyzed natural macromolecules oat β-glucan and mannan in combination. Furthermore, the hyaluronic acid endogenous growth promoter prepared by enzymatic hydrolysis of the two natural macromolecular polysaccharides in Examples 1 and 2 had a significantly stronger promoting effect on the expression of CerS2 and HBD-3 mRNA than the hyaluronic acid endogenous growth promoter prepared by separately enzymatic hydrolysis of the two small molecules and then mixing them in Example 3.

[0107] Example 3: Verification of the Repairing Effect of Hyaluronic Acid Endogenous Growth Promoter – Zebrafish Embryo Tail Fin Repair Test

[0108] 1. Instruments and Equipment

[0109] Zebrafish farming equipment (must be equipped with temperature control, water circulation and filtration devices; farming containers should be made of glass or common food-grade PC material), spawning boxes / tanks, pH meter, dissolved oxygen meter, salinity meter (conductivity meter), analytical balance, microscope (with photographic system, stereo microscope with maximum magnification greater than 80x), 96-well plates, petri dishes, incubator, brine shrimp incubator, pipettes, centrifuge tubes, glass containers (such as beakers, volumetric flasks, etc.), vortex mixer, ultrasonic water bath, centrifuge, low-temperature freezer, adjustable pipettes, pipette tips and laboratory scalpels.

[0110] 2. Preparation of Zebrafish Embryos

[0111] (1) Test organisms: Wild-type AB strain zebrafish (Daniorerio) from reliable sources (e.g., China National Zebrafish Resource Center) were used for spawning tests. Broodstock should have good reproductive capacity – 6-12 months of age is optimal. Sidecrossing should be used as much as possible to maintain genetic diversity. Purebred broodstock should be replaced with a new batch after 5 generations. Broodstock should not have obvious signs of infection or disease and should not have undergone drug treatment within the past 2 months. Broodstock should be acclimatized to the laboratory for at least 14 days before being introduced for spawning tests.

[0112] (2) Aquaculture requirements: The aquaculture water temperature should be controlled at 26℃-28.5℃, and the indoor temperature should be controlled at 20℃-25℃; the stocking density should be controlled at 1-2 fish per liter of water, and a fixed 12h-16h light per day should be provided, and a good filtration system should be maintained; feed at least twice a day, including at least once with brine shrimp (Artemiasalina) larvae, with a feeding interval of more than 3 hours, to avoid overfeeding affecting water quality and cleanliness.

[0113] (3) Spawning requirements: If using a spawning tank to collect fish eggs, place male and female fish in the spawning tank at a ratio of 2:1 1-2 hours before turning off the lights the day before spawning. Since zebrafish sometimes do not spawn, it is recommended to prepare multiple spawning tanks for backup. To avoid genetic bias, mix the fish eggs collected from at least 3 spawning tanks and then select for backup. If using a fish tank to collect fish eggs, place the egg collection box into the fish tank to be collected before turning off the lights the day before spawning or before turning on the lights on the day of spawning. To prevent the fish eggs from being eaten by adult fish, cover the egg collection box with an inert net. Mating, spawning and fertilization are completed within about 30 minutes after turning on the lights, at which time the egg collection box can be removed from the fish tank. After the fish eggs are taken out of the egg collection box, it is recommended to clean the fish embryos with fish embryo culture medium. Select healthy zebrafish embryos and culture them at a density of no more than 1 fish embryo in 200μL of fish embryo culture medium at 28℃±1℃ until 3 days after fertilization.

[0114] 3. Group settings

[0115] The test substance is dissolved directly in fish embryo culture medium to prepare the test solution. The concentration of the test substance in the test solution must ensure that the mortality rate of zebrafish embryos is less than 10%. Multiple concentration groups can be set as needed during testing.

[0116] (1) Blank control group: fish embryo culture medium;

[0117] (2) Positive control group: Rehmannia glutinosa extract solution;

[0118] (3) Test group 1: Fish embryo culture medium containing 1% small molecule oat β-glucan;

[0119] (4) Test group 2: Fish embryo culture medium containing 1% small molecule mannan;

[0120] (5) Test group 3: Fish embryo culture medium containing 1% of the hyaluronic acid endogenous growth promoter obtained in Example 1.

[0121] 4. Experimental Procedure

[0122] (1) Select large zebrafish embryos three days after fertilization under a microscope, anesthetize the zebrafish embryos with tricaine solution, and remove the caudal fin of the zebrafish embryos under a microscope with an experimental scalpel.

[0123] (2) 24 fish embryos were randomly selected and placed in a 96-well plate, with each well containing 1 fish embryo and 0.2 mL of fish embryo culture medium;

[0124] Twenty-four fish embryos were randomly selected and placed in a 96-well plate, with each well containing one fish embryo and 0.2 mL of Rehmannia glutinosa extract solution.

[0125] Twenty-four fish embryos were randomly selected and placed in a 96-well plate, with each well containing one fish embryo and 0.2 mL of test group 1 solution;

[0126] Twenty-four fish embryos were randomly selected and placed in a 96-well plate, with each well containing one fish embryo and 0.2 mL of test group 2 solution;

[0127] Twenty-four fish embryos were randomly selected and placed in a 96-well plate, with each well containing one fish embryo and 0.2 mL of test group 3 solution;

[0128] The 96-well plate was incubated in a constant temperature incubator at 28℃±1℃ for 48±1h.

[0129] (3) Anesthetize the zebrafish with tricaine and take a side view of the tail of the fish embryo under a stereomicroscope.

[0130] 5. Result Calculation and Analysis

[0131] Count the length of the tail fin for each fish embryo and calculate the tail fin repair promotion rate.

[0132] Promotion rate = (SC) / C * 100%,

[0133] In the formula, S represents the average length of the tail fin of fish embryos in each test group and the positive control group;

[0134] The average length of the caudal fin of fish embryos in the C-blank control group.

[0135] The calculated data are shown in Table 6. A bar chart was then created based on this data.

[0136] Table 6. Promotion rate of tail fin repair in each group

[0137]

[0138] From Table 6 and Figure 7 It can be seen that the positive control group, test group 1, and test group 2 all have a promoting effect on the repair of fish tail fins, test group 2 has an inhibitory effect on the repair of fish tail fins, and test group 3 has a stronger promoting effect on the repair of fish tail fins than the positive control group, namely the Rehmannia glutinosa extract solution. This indicates that the hyaluronic acid endogenous growth promoter of the present invention has a significant effect on promoting damage repair.

[0139] In summary, the hyaluronic acid endogenous growth promoter prepared in the embodiments of the present invention has the effect of promoting the synthesis of hyaluronic acid synthase, ceramide synthase and human β-defensin, and has been verified by zebrafish embryo tail fin repair test to have the effect of promoting skin barrier repair.

[0140] The above description is merely an example of the embodiments of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A hyaluronic acid endogenous growth promoter, composed of oat β-glucan and mannan, characterized in that, The molecular weights of both oat β-glucan and mannan are less than 10 kDa. The hyaluronic acid endogenous growth promoter is prepared by enzymatic hydrolysis of natural oat β-glucan and natural mannan in a mass ratio of 1-7:3-9, or by enzymatic hydrolysis of each separately. The preparation of the mixed or separate enzymatic hydrolysis involves enzymatic hydrolysis with β-glucanase, the activity unit of which is 50,000-200,000 U / g, and the amount of enzyme added is 0.1%-10% of the total mass of the polysaccharides. The specific steps for preparing the hyaluronic acid endogenous growth promoter by the aforementioned mixed enzymatic hydrolysis method are as follows: natural oat β-glucan and natural mannan are mixed in a mass ratio of 1-7:3-9, hydrolyzed by β-glucanase, and then passed through a 10kDa retention membrane to obtain small molecule polysaccharides and oligosaccharides. The monosaccharide solution is then removed through a 500Da concentration membrane, and the hyaluronic acid endogenous growth promoter is obtained after drying. The specific steps for preparing the hyaluronic acid endogenous growth promoter by the aforementioned enzymatic hydrolysis method are as follows: Natural oat β-glucan and natural mannan in a mass ratio of 1-7:3-9 are respectively enzymatically hydrolyzed by β-glucanase, and then passed through a 10kDa retention membrane to obtain small molecule polysaccharides and oligosaccharides, respectively. The monosaccharide solution is then removed through a 500Da concentration membrane, and after drying, small molecule oat β-glucan and mannan are obtained, respectively. The two are then mixed evenly to prepare the product.

2. The use of the hyaluronic acid endogenous growth promoter as described in claim 1 in the preparation of a product with skin barrier repair function.

3. The use of the hyaluronic acid endogenous growth promoter as described in claim 1 in the preparation of cosmetic products or topical formulations with skin barrier repair function.

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