WO2023040952A1 - 一种透明质酸寡糖组合物及其制备方法和用途 - Google Patents
一种透明质酸寡糖组合物及其制备方法和用途 Download PDFInfo
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- WO2023040952A1 WO2023040952A1 PCT/CN2022/119005 CN2022119005W WO2023040952A1 WO 2023040952 A1 WO2023040952 A1 WO 2023040952A1 CN 2022119005 W CN2022119005 W CN 2022119005W WO 2023040952 A1 WO2023040952 A1 WO 2023040952A1
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- hyaluronic acid
- oligosaccharide composition
- mass proportion
- oligosaccharides
- acid oligosaccharide
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Classifications
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7016—Disaccharides, e.g. lactose, lactulose
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/702—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
- A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
- A61K31/728—Hyaluronic acid
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
- A61K8/73—Polysaccharides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
Definitions
- the invention belongs to the technical field of bioengineering, and in particular relates to a hyaluronic acid oligosaccharide composition and its preparation method and application.
- Hyaluronic acid is an acidic mucopolysaccharide composed of N-acetylglucosamine and D-glucuronic acid disaccharide repeating units through ⁇ -(1 ⁇ 4) glycosidic bonds and ⁇ -(1 ⁇ 3) Unbranched high-molecular glycosaminoglycans composed of glycosidic bonds exist in the intercellular matrix of animal tissues and the capsule of some bacteria.
- Hyaluronic acid is widely used in medicine, cosmetics, food and other fields, and its molecular weight is generally 10 5 -10 7 Daltons (Da).
- Hyaluronic acid with different molecular weights exhibits different biological activities, and low molecular weight hyaluronic acid even exhibits activities completely opposite to those of 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 have received more attention as active substances. Because the preparation of hyaluronic acid oligosaccharides by acid method and alkali method has a higher risk if it is used in large-scale production, it may cause harm to operators or equipment, and the preparation of oligosaccharides by enzyme digestion has greater advantages.
- Patent CN 111040048A discloses an ultra-low molecular weight hyaluronic acid and its preparation method. This patent does not separate the oligosaccharides produced in the enzymatic hydrolysis process in time, and the existing oligosaccharides will inhibit the forward reaction of enzymatic hydrolysis to a certain extent. , resulting in prolongation of 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.
- Non-Patent Document 1 discloses that sulfated hyaluronic acid (sHA) has good biological functions, and the specific sulfated mode plays a key role in regulating the binding mode between glycosaminoglycans and proteins, which proves that there is The 6-O-sulfated (sHA-6S) sHA tetrasaccharide plays an important role in promoting axonal outgrowth of rat E18 hippocampal neurons in vitro.
- sHA-6S 6-O-sulfated sHA tetrasaccharide plays an important role in promoting axonal outgrowth of rat E18 hippocampal neurons in vitro.
- Non-Patent Document 2 used a fluorescence polarization competition assay to analyze the relative binding affinities of synthetic compounds, and revealed that there was an interaction between the synthetic chondroitin sulfate-like tetrasaccharide and midkine. It can be seen that the difference in the degree of polymerization or residues of hyaluronic acid oligosaccharides (HAOs) will produce different bioactive functions. This is worth studying in depth. In the prior art, there is no research on the influence of ultra-low molecular weight hyaluronic acid with uronic acid end groups on the synthesis of ceramide and hyaluronidase.
- Non-Patent Document 3 selectively produces hyaluronic acid oligosaccharides (HAOs) with different degrees of polymerization and reducing ends by changing the concentration of hydrochloric acid.
- HAOs hyaluronic acid oligosaccharides
- the molecular docking simulation data of HAOs and CD44 and TLR4 using MOE software show that HAOs and CD44 and TLR4 The binding ability of TLR4 increased with the increase of the degree of polymerization of HAOs, and the binding ability increased significantly when the reducing end of HAOs was GlcNAc residues, while there was no significant change when the reducing end was GlcA residues.
- Non-Patent Document 1 Unravel a neuroactive sHA sulfate 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).Unravel a neuroactive sHA sulfate pattern with neurogenesis activity by a library of defined oligosaccharides.European journal of medicinal chemistry,163,583-596.
- Non-Patent Document 2 Chondroitin Sulfate Tetrasaccharides: Synthesis, Three-Dimensional Structure and Interaction with Midkine. Solera, C., Macchione, G., Maza, S., Kayser, M., Corzana, F., Paz, J.L.D., & Nieto , P.M.(2016). Chondroitin sulfate tetrasaccharides: synthesis, three-dimensional structure and interaction with midkine.
- 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 breast cancer. Biomolecules, 9(9), 436.
- the present invention provides a hyaluronic acid oligosaccharide composition and a preparation method thereof.
- the present invention relates to the following aspects:
- a hyaluronic acid oligosaccharide composition is characterized in that, described hyaluronic acid oligosaccharide composition comprises the hyaluronic acid oligosaccharide of structure shown in formula (I):
- n is an integer selected from 0-5, X is selected from H, K, Na, Ca or Zn, preferably Na;
- the hyaluronic acid oligosaccharide composition according to any one of items 1-3, 6, characterized in that, the hyaluronic acid oligosaccharide composition is used in cosmetics or health care products, preferably in the cosmetic or health care products.
- the mass concentration in the product is 0.001-10%.
- Hyaluronic acid is enzymatically hydrolyzed with hyaluronidase
- the hyaluronidase is a hyaluronidase that cleaves ⁇ -1,3 glycosidic bonds
- the hyaluronidase is leech hyaluronan hydrolase expressed by Pichia pastoris engineering bacteria,
- the separation is ultrafiltration
- the concentration is nanofiltration
- activated carbon adsorption is used to remove impurities.
- the initial concentration of the hyaluronic acid is 5-150g/L, preferably, the molecular weight of the hyaluronic acid is 1000-2000kDa Further preferably, the initial content of the hyaluronidase is 1 ⁇ 104-3 ⁇ 105 U/mL.
- the hyaluronic acid oligosaccharide composition described in any one of items 1-3, 6 is effective in promoting ceramide and/or hyaluronan synthase 1 (HAS1) and/or hyaluronan synthase 3 (HAS3) use in the synthesis.
- HAS1 hyaluronan synthase 1
- HAS3 hyaluronan synthase 3
- hyaluronic acid oligosaccharide composition resists skin aging by promoting collagen production and/or epidermal cell proliferation and differentiation.
- the hyaluronic acid oligosaccharide composition is used to prepare health products that promote collagen production, or to prepare health products that protect and strengthen organs, or to prepare Prepare health products for protecting gastric mucosa, or for preparing health products for supplementing calcium, or for preparing joint lubricating injections.
- the oligosaccharides obtained by enzymatic hydrolysis can be separated from the enzymatic hydrolysis system in time, reducing the inhibitory effect of oligosaccharides on enzymatic hydrolysis, improving the efficiency of enzymatic hydrolysis, and shortening the time of enzymatic hydrolysis .
- ultrafiltration to intercept the enzyme and reuse it 1-2 times to improve the reusability of the enzyme. At the same time, it saves the stage of inactivating the enzyme by heating, reduces the influence of heating on the activity of oligosaccharides, and makes the oligosaccharides Sugar activity is maximized.
- the hyaluronic acid oligosaccharide composition obtained in the present invention can promote the synthesis of ceramide and/or hyaluronan synthase 1 (HAS1) and/or hyaluronan synthase 3 (HAS3).
- HAS1 hyaluronan synthase 1
- HAS3 hyaluronan synthase 3
- the hyaluronic acid oligosaccharide composition of the present invention has a small molecular weight and has uronic acid at the reducing end, which can significantly promote the production of collagen in dermal fibroblasts, increase the collagen content in the dermis, and reduce the generation of wrinkles. antiaging activity. At the same time, the unique triple helix structure of collagen can strongly lock 30 times of water, making the skin moist, shiny, and tender for a long time.
- the hyaluronic acid oligosaccharide composition of the present invention can be used to prepare anti-aging and moisturizing cosmetics and medical devices.
- the hyaluronic acid oligosaccharide composition of the present invention has the purpose of promoting collagen production, and can be further used to prepare health products that promote collagen production, or to prepare health products that protect and strengthen organs, or to prepare health products that protect stomach Health products for mucous membranes, or for the preparation of health products supplemented with calcium, or for the preparation of joint lubricating injections.
- Fig. 1 is the high performance liquid phase chromatogram of hyaluronic acid oligosaccharide in embodiment 1.
- Figure 2 is a high performance liquid chromatogram of hyaluronic acid oligosaccharides in Example 2.
- Figure 3 is a high performance liquid chromatogram of hyaluronic acid oligosaccharides in Example 3.
- FIG. 4 is a high performance liquid chromatogram of hyaluronic acid oligosaccharides in Comparative Example 1.
- Example 5 is a mass spectrum total ion current peak diagram of hyaluronic acid oligosaccharides in Example 2.
- Figure 6 is a diagram of the ionic strength of hyaluronic acid disaccharide (HA2).
- Figure 7 is a diagram of the ionic strength of hyaluronate tetraose (HA4).
- Figure 8 is a diagram of the ionic strength of hyaluronic acid hexasaccharide (HA6).
- Figure 9 is a diagram of the ionic strength of hyaluronic acid octaose (HA8).
- Figure 10 is a diagram of the ionic strength of hyaluronic acid decasaccharide (HA10) and dodecasaccharide (HA12).
- Fig. 11 shows the content of collagen I secreted by fibroblasts after using samples 1-1 and 2-1.
- Fig. 12 shows the content of collagen I secreted by fibroblasts after using samples 1, 1-1, 1-2, and 1-3.
- Figure 13 shows the structure of the 3D full-thickness skin model after using sample 1 and sample 2.
- Figure 14 shows the photomicrograph of sample 4 transdermal condition.
- Hyaluronic acid oligosaccharides or hyaluronic acid oligosaccharides are hyaluronic acid with a molecular weight of less than 10 4 Da and a number of monosaccharide residues of 2 to 25 (generally 4 to 16). molecular fragments.
- Oligo-HA is a small molecular polysaccharide, and its properties are very different from ordinary hyaluronic acid. Studies have shown that Oligo-HA has biological activities such as anti-oxidation, immune regulation, anti-inflammation, promotion of wound healing, promotion of angiogenesis and anti-tumor. More importantly, because of its small molecular size, it can penetrate into the stratum corneum of the skin to exert deep moisturizing and moisturizing effects, and can be widely used in cosmetics.
- the present invention provides a hyaluronic acid oligosaccharide composition
- the hyaluronic acid oligosaccharide composition comprises the hyaluronic acid oligosaccharide of the structure shown in formula (I):
- the reducing end of the hyaluronic acid oligosaccharide of the present invention is an uronic acid structure.
- n is selected from an integer of 0-5, for example, n may be 0, 1, 2, 3, 4 or 5, and X is selected from H, K, Na, Ca or Zn, preferably Na.
- 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%.
- the mass proportion of hyaluronic acid tetraose is 35-70%, such as 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%.
- the mass proportion of hyaluronan 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%.
- 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 it is 1-50%, more preferably 20-40%.
- the mass proportion of hyaluronic acid octaose 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%, more preferably 1-10%.
- 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%, more preferably 1-5%.
- the mass proportion of hyaluronic acid dodecose 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% %.
- the mass proportion of hyaluronic acid disaccharide is 5-40%, and the mass proportion of hyaluronic acid tetraose 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 dodecose
- the mass proportion of sugar is 0-5%.
- the mass proportion of hyaluronic acid disaccharide is 5-40%, and the mass proportion of hyaluronic acid tetraose 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 dodecose
- the mass proportion of sugar is 0.01-1.5%.
- the mass proportion of hyaluronic acid disaccharide is 5-15%, and the mass proportion of hyaluronic acid tetraose 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 dodecose
- the mass proportion of sugar is 0.01-1.5%.
- the mass proportion of hyaluronic acid disaccharide is 5-10%, and the mass proportion of hyaluronic acid tetraose 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%
- the mass proportion of hyaluronic acid dodecose The mass proportion of sugar is 0.01-1%.
- the mass proportion of hyaluronic acid disaccharide is 10%
- the mass proportion of hyaluronic acid tetraose is 49.5%
- the mass proportion of hyaluronic acid hexasaccharide The mass proportion of sugar is 33%
- the mass proportion of hyaluronic acid octasaccharide is 5%
- the mass proportion of hyaluronic acid decasaccharide is 1.5%
- the mass proportion of hyaluronic acid dodecose is 1%.
- 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 The mass proportion of sugar is 32%
- the mass proportion of hyaluronic acid octasaccharide is 7%
- the mass proportion of hyaluronic acid decasaccharide is 2%
- the mass proportion of hyaluronic acid dodecose is 1%.
- the weight average molecular weight of the hyaluronic acid oligosaccharide composition is less than or equal to 1kDa, such as 1kDa, 990Da, 980Da, 970Da, 960Da, 950Da, 940Da, 930Da, 920Da, 910Da, 900Da, 890Da, 880Da, 870Da , 860Da, 850Da, 840Da, 830Da, 820Da, 810Da, 800Da, 790Da, 780Da, 770Da, 760Da, 750Da, 740Da, 730Da, 720Da, 710Da, 700Da.
- 1kDa such as 1kDa, 990Da, 980Da, 970Da, 960Da, 950Da, 940Da, 930Da, 920Da, 910Da, 900Da,
- the hyaluronic acid oligosaccharide composition of the present invention can promote the expression of ceramide and/or hyaluronan synthase 1 and/or hyaluronan synthase 3.
- ceramide is a kind of phospholipid with ceramide as the skeleton, mainly including ceramide phosphorylcholine and ceramide phosphoethanolamine.
- Phospholipid is the main component of cell membrane, and 40% to 50% of sebum in the stratum corneum is produced by nerve Composed of amides, ceramides are the main part of the intercellular matrix and play an important role in maintaining the water balance of the stratum corneum. Ceramide has a strong ability to associate water molecules, and it maintains skin moisture by forming a network structure in the stratum corneum. Therefore, ceramides have the effect of maintaining skin moisture.
- Hyaluronic Acid (HA) in the skin is mainly synthesized by hyaluronic acid synthase (Hyaluronan Synthases, HAS).
- HAS hyaluronic acid synthase
- Three HAS subtypes (HAS1, HAS2, HAS3) have been identified in mammals.
- HAS participates in the synthesis of hyaluronic acid chains of different molecular weights inside the plasma membrane, and at the same time secretes the synthesized hyaluronic acid into the extracellular matrix.
- HAS1 and HAS2 synthesized hyaluronic acid chain length is similar, about 2000KDa
- HAS3 catalytic synthesis of hyaluronic acid molecular weight between 200-300kDa.
- Extracellular macromolecular HA is hydrolyzed by hyaluronidase or degraded by oxidation reaction to obtain small molecular HA.
- Small molecule HA can stimulate cell proliferation, initiate signaling cascade reaction, and also participate in angiogenesis.
- 100-300Ka hyaluronic acid has the functions of softening cutin, locking water and three-dimensional moisturizing.
- the hyaluronic acid oligosaccharide composition of the present invention can be used in cosmetics or health products, and the mass concentration in the cosmetics or health products is preferably 0.001-10%, such as 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 its different uses in cosmetics or health products. For example, when the hyaluronic acid oligosaccharide composition is used to promote the expression of ceramide in cosmetics or health care products, its mass concentration can be 0.001-1%.
- hyaluronic acid oligosaccharide composition When the hyaluronic acid oligosaccharide composition is used to promote the expression of hyaluronan synthase 1 (HAS1) and/or hyaluronan synthase 3 (HAS3) in cosmetics or health products, its mass concentration can be 0.01 -10%, when the hyaluronic acid oligosaccharide composition is used to promote the expression of hyaluronan synthase 1 (HAS1) and/or hyaluronan synthase 3 (HAS3) in cells, its mass concentration can be 0.001- 1%.
- the present invention also provides a preparation method of a hyaluronic acid oligosaccharide composition, the preparation method comprising the following steps: performing an enzymolysis reaction on hyaluronic acid with hyaluronidase, separating the enzymolysis reactant to obtain a product stock solution, and The product stock solution is concentrated to obtain a product concentrate; impurities in the product concentrate are removed, and dried to obtain a hyaluronic acid oligosaccharide composition, wherein the hyaluronidase enzyme cleaves ⁇ -1,3 glycosidic bonds
- the hyaluronidase preferably, the separation is ultrafiltration; more preferably, the concentration is nanofiltration; more preferably, activated carbon adsorption is used to remove impurities.
- the hyaluronidase used in the preparation method is a hyaluronidase for enzymatically cutting ⁇ -1,3 glycosidic bonds, so as to ensure that the reducing end of the oligosaccharide obtained by enzymatically cutting the hyaluronid is an uronic acid structure.
- the hyaluronidase is leech hyaluronan hydrolase expressed by Pichia pastoris engineering bacteria. Its preparation method can refer to the method described in CN103695448A.
- ultrafiltration is a membrane separation technology (UItrafil-tration, referred to as UF). Solutions can be purified, separated or concentrated. Ultrafiltration is between microfiltration and nanofiltration, and there is no clear dividing line between the three. Generally speaking, the pore size of the ultrafiltration membrane is between 0.05 ⁇ m-1nm, and the operating pressure is 0.1-0.5Mpa. It is mainly used to intercept and remove macromolecular substances such as suspended solids, colloids, particles, bacteria and viruses in water. Ultrafiltration membranes can be divided into organic membranes and inorganic membranes according to membrane materials. According to the appearance of the membrane, it can be divided into: flat plate, tube, capillary, hollow fiber and porous. At present, household ultrafiltration water purifiers are mostly hollow membranes.
- Nanofiltration 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 weights, 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 to achieve the separation effect.
- the initial concentration of the hyaluronic acid is 5-150g/L, such as 5g/L, 10g/L, 20g/L, 30g/L, 40g/L L, 50g/L, 60g/L, 70g/L, 80g/L, 90g/L, 100g/L, 110g/L, 120g/L, 130g/L, 140g/L, 150g/L.
- the reaction system is 1 L
- the initial content of hyaluronic acid is 5-150 g, that is, the content when no hydrolysis occurs is 5-150 g.
- the initial molecular weight of the hyaluronic acid is 1000-2000kDa, such as 1000kDa, 1100kDa, 1200kDa, 1300kDa, 1400kDa, 1500kDa, 1600kDa, 1700kDa, 1800kDa, 1900kDa, 2000kDa.
- the initial content of the hyaluronidase is 1 ⁇ 10 4 -3 ⁇ 10 5 U/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 hyaluronidase activity unit (U) is defined as: the amount of enzyme required to release 1 ⁇ g of glucose reducing equivalent reducing sugar from the hyaluronic acid sugar chain per hour under the conditions of pH 5.5 and 38°C.
- the initial ratio of the hyaluronidase to the hyaluronic acid is 1-5 U/mg, such as 1 U/mg, 2 U/mg, 3 U/mg , 4U/mg, 5U/mg.
- the ultrafiltration adopts an ultrafiltration membrane with a molecular weight cut-off of 600-1000Da
- the molecular weight cut-off can be 600Da, 700Da, 800Da, 900Da, 1000Da
- the nanofiltration adopts a nanofiltration membrane with a molecular weight cut-off of 100-300Da.
- the molecular weight cut-off can be 100Da, 200Da, 300Da.
- the ultrafiltration adopts an ultrafiltration membrane with a molecular weight cutoff of 1000 Da
- the nanofiltration adopts a nanofiltration membrane with a molecular weight cutoff of 200 Da.
- the cut-off molecule described in the present invention is the cut-off performance of the ultrafiltration membrane represented by molecular weight, also known as cut molecular weight. Since it is quite difficult to directly measure the pore size of ultrafiltration membranes or nanofiltration membranes, spherical substances with known molecular weights are used for determination. If the rejection rate of the membrane to the intercepted substance is greater than 90%, the molecular weight of the intercepted substance is used to represent the interception performance of the membrane, which is called the molecular weight cut-off of the membrane. For example, a membrane with a molecular weight cut-off of 10 kDa can block substances with a molecular weight greater than 10 kDa, while allowing substances with a molecular weight less than 10 kDa to pass through.
- Drying in the method can adopt various drying methods in the prior art.
- the drying employed is spray drying.
- the air inlet temperature used in spray drying is 120°C-160°C, and the outlet air temperature is 60°C-80°C.
- the hyaluronic acid oligosaccharide composition obtained by the method is the above-mentioned hyaluronic acid oligosaccharide composition, that is, the hyaluronic acid oligosaccharide of the structure shown in formula (I):
- n is an integer selected from 0-5, X is selected from H, K, Na, Ca or Zn, preferably Na.
- 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.
- the present invention also provides the use of the above-mentioned hyaluronan oligosaccharide composition in promoting the expression of hyaluronan synthase 1 (HAS1) and/or hyaluronan synthase 3 (HAS3).
- HAS1 hyaluronan synthase 1
- HAS3 hyaluronan synthase 3
- the hyaluronic acid oligosaccharide composition of the present invention has hyaluronic acid disaccharide, hyaluronic acid tetrasaccharide, hyaluronic acid hexasaccharide, hyaluronic acid octaose, hyaluronic acid decaose, hyaluronic acid decaose, hyaluronic acid decaose, hyaluronic acid decaose, and hyaluronic acid Various types of hyaluronic oligosaccharides such as dodecaose.
- the oligosaccharide composition has no cytotoxicity and no potential adverse reaction to human skin; it can significantly promote the expression of hyaluronic acid synthases HAS1 and HAS3 in fibroblasts, especially the expression of HAS3; it can significantly promote the synthesis of ceramide.
- Natural aging is endogenous aging, manifested as skin whitening, fine wrinkles, decreased elasticity, skin relaxation, etc.
- Environmental stimulation is exogenous aging, such as photoaging caused by sun exposure. If the skin is not well maintained or declines with age, dead skin will adhere to the surface of the skin and not fall off, thus causing a series of problems that seriously affect the beauty.
- the present invention provides a use of a hyaluronic acid oligosaccharide composition in anti-skin aging, the hyaluronic acid oligosaccharide composition comprising a hyaluronic acid oligosaccharide of the structure shown in formula (I) :
- n is an integer selected from 0-5;
- X is selected from H, K, Na, Ca or Zn, preferably Na.
- the mass proportion of the oligosaccharide is 0.01-5%.
- the causes of skin aging are mainly reflected in the following points: 1. Changes in the extracellular matrix of the dermis.
- Collagen is the main component of the extracellular matrix of the dermis, accounting for about 90% of the protein in the human dermis.
- the protein in the dermis is mainly composed of type I collagen (80%) and a small amount of type III collagen (10%). Collagen gives the skin its strength and elasticity. As we age, collagen fibers in the skin decrease, causing the skin to lose its elasticity and cause wrinkles.
- Collagen in the dermis is mainly secreted by fibroblasts, and the reduction of collagen secreted by fibroblasts is also one of the major causes of aging and wrinkles.
- Changes in the structure of the basal layer After human skin ages, the structure of the basal layer is loose, and the cell connection is loose, which reduces the material exchange efficiency between the dermis and the epidermis. As a result, epidermal nutrition is insufficient, and epidermal cells cannot proliferate and differentiate normally. In turn, the epidermis becomes thinner and loosens.
- the hyaluronic acid oligosaccharide composition can resist skin aging by promoting collagen production and/or epidermal cell proliferation and differentiation.
- the hyaluronic acid oligosaccharide composition resists skin aging by promoting collagen production.
- the hyaluronic acid oligosaccharide composition resists skin aging by promoting the proliferation and differentiation of epidermal cells.
- the hyaluronic acid oligosaccharide composition can resist skin aging by promoting collagen production and epidermal cell proliferation and differentiation.
- the hyaluronic acid oligosaccharide composition can be directly or indirectly applied to the skin, for example, it can be administered by external application, injection or oral administration.
- the hyaluronic acid oligosaccharide composition can be used in cosmetics and medical devices, and is further suitable for skin.
- the hyaluronic acid oligosaccharide composition can be formulated into cosmetics alone or together with other active ingredients for anti-aging skin.
- the mass concentration of the hyaluronic acid oligosaccharide composition in cells is 0.0001%-0.1%.
- the mass concentration of the hyaluronic acid oligosaccharide composition in the cosmetic or medical device is 0.0001%-5%.
- 0.001%-1% such as 0.0001%, 0.001%, 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3 %, 4%, 5%, preferably 0.001%-1%.
- the present invention also provides the use of the above-mentioned hyaluronic acid oligosaccharide composition in promoting collagen production.
- Visceral organs and tissues all contain collagen. Under the epidermal structure of these organs is collagen. Collagen can protect and strengthen organs. Therefore, the hyaluronic acid oligosaccharide composition can be used to prepare health care products for protecting and strengthening organs. Taste.
- Collagen is the main component of muscle tissue, which can provide necessary nutrients for muscles and protect gastric mucosa. Therefore, the hyaluronic acid oligosaccharide composition can be used to prepare health products for protecting gastric mucosa.
- Collagen also forms the calcium attachment grid, locks bone calcium, and prevents calcium loss, so the hyaluronic acid oligosaccharide composition can be used to prepare calcium-supplementing health products.
- Collagen can also effectively repair articular cartilage, restore the lubrication of the surface of articular cartilage, and reduce friction. Therefore, the hyaluronic acid oligosaccharide composition can be used to prepare joint lubrication injection.
- the mass concentration of the hyaluronic acid oligosaccharide composition in the health care product or injection is 0.0001%-5%, such as 0.0001%, 0.001%, 0.01%, 0.05% , 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, preferably 0.001%-1% .
- Embodiment 1 Preparation of hyaluronic acid oligosaccharide composition
- hyaluronidase the enzyme is leech hyaluronidase expressed by Pichia pastoris engineering bacteria, the preparation method refers to the method 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 hydrolysis reaction product to obtain the product stock solution, use nanofiltration to The product stock solution is concentrated to obtain a product concentrate. Keep the system at 40°C and stir the reaction for 18h.
- ultrafiltration adopts ultrafiltration membrane with molecular weight cut off of 1 kDa
- nanofiltration adopts nanofiltration membrane with molecular weight cut off of 200 Da.
- collect the nanofiltration concentrate add activated carbon for adsorption, filter and collect the filtrate, and carry out spray drying.
- the air inlet temperature is 140°C and the outlet air temperature is 70°C.
- the hyaluronic acid oligosaccharide composition was obtained, and the product yield was 86%.
- the oligosaccharide distribution of the hyaluronic acid oligosaccharide composition is analyzed by high performance liquid chromatography, and the specific chromatographic conditions are:
- UV-Vis spectroscopic detector UV-Vis spectroscopic detector
- the average molecular weight of the hyaluronic acid oligosaccharide composition measured by a laser light scattering instrument is 890Da.
- hyaluronidase the enzyme is leech hyaluronidase expressed by Pichia pastoris engineering bacteria, the preparation method refers to the method 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 enzymolysis reaction product to obtain the product stock solution, use nanofiltration to The product stock solution is concentrated to obtain a product concentrate. Keep the system at 30°C and stir the reaction for 12h.
- the ultrafiltration adopts the ultrafiltration membrane with a molecular weight cut-off of 1 kDa
- the nanofiltration adopts a nanofiltration membrane with a molecular weight cut-off of 200 Da.
- collect the nanofiltration concentrate add activated carbon for adsorption, filter and collect the filtrate, and carry out spray drying.
- the air inlet temperature is 120°C and the outlet air temperature is 60°C.
- the hyaluronic acid oligosaccharide composition was obtained, and the product yield was 84%.
- the oligosaccharide distribution of the hyaluronic acid oligosaccharide composition was analyzed by high performance liquid chromatography, and the specific chromatographic conditions were the same as in Example 1. The results are shown in Figure 2.
- the mass ratio of hyaluronic acid disaccharide is 9.35%
- the mass ratio of hyaluronic acid tetrasaccharide is 47.84%
- the mass ratio of hyaluronic acid hexasaccharide is The mass proportion of hyaluronic acid octaose is 32.92%
- the mass proportion of hyaluronic acid decaose is 6.04%
- the mass proportion of hyaluronic acid decaose is 1.48%
- the mass proportion of hyaluronic acid dodecaose is 0.42%.
- the average molecular weight of the hyaluronic acid oligosaccharide composition measured by a laser light scattering instrument was 920Da.
- hyaluronidase the enzyme is leech hyaluronidase expressed by Pichia pastoris engineering bacteria, the preparation method refers to the method described in CN 103695448A) 1.2 ⁇ 10 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 enzymolysis reaction product to obtain the product stock solution, and use nanofiltration to filter the The product stock solution is concentrated to obtain a product concentrate. Keep the system at 46°C and stir the reaction for 24h.
- the ultrafiltration adopts the ultrafiltration membrane with a molecular weight cut-off of 1 kDa
- the nanofiltration adopts a nanofiltration membrane with a molecular weight cut-off of 200 Da.
- collect the nanofiltration concentrate add activated carbon for adsorption, filter and collect the filtrate, and carry out spray drying, wherein the inlet air temperature is 160°C, and the outlet air temperature is 80°C.
- the hyaluronic acid oligosaccharide composition was obtained, and the product yield was 87%.
- the oligosaccharide distribution of the hyaluronic acid oligosaccharide composition was analyzed by high performance liquid chromatography, and the specific chromatographic conditions were the same as in Example 1. The results are shown in Figure 3.
- 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 octaose is 29.00%
- the mass proportion of hyaluronic acid decaose is 4.71%
- the mass proportion of hyaluronic acid decaose is 1.5%
- the mass proportion of hyaluronic acid dodecaose is 0.40%.
- the average molecular weight of the hyaluronic acid oligosaccharide composition measured by a laser light scattering instrument was 915Da.
- hyaluronidase the enzyme is leech hyaluronidase expressed by Pichia pastoris engineering bacteria, the preparation method refers to the method described in CN 103695448 A) 8 ⁇ 10 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°C and stir for 20h.
- the oligosaccharide distribution of the hyaluronic acid hydrolyzate was analyzed by high performance liquid chromatography, and the specific chromatographic conditions were the same as in Example 1. The results are shown in Figure 4.
- 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 0.00%.
- the mass proportion of hyaluronic acid octaose is 2.10%, the mass proportion of hyaluronic acid decaose is 2.67%, the mass proportion of hyaluronic acid decaose is 3.31%, and the mass proportion of hyaluronic acid dodecaose is 3.97%.
- the average molecular weight of the hyaluronic acid oligosaccharide composition measured by a laser light scattering instrument was 7580Da.
- Pretreatment Dissolve 50 g of high molecular weight hyaluronic acid with a molecular weight of 1000 kDa in 30 parts by weight of water, adjust the pH of the solution to 8.5 with 0.1 mol/L sodium methoxide solution, fully stir and swell at room temperature, and slowly heat up to 90 °C, constant temperature hydrolysis for 4 hours, then extract and suction filter the filter residue, repeat the above extraction operation, and combine the two filtrates for later use;
- step 2) Neutral salt fractional precipitation: the solution obtained in step 1) is heated to 40°C, and 25% of the solution weight is added as a solid magnesium sulfate, stirred and dissolved completely, and then ultrafiltered with a sulfonated polysulfone ultrafiltration membrane of 0.1 ⁇ m. Then add 43% magnesium sulfate solid of solution weight again in the filtrate, stir until completely dissolved and let it stand for precipitation, then filter, ultrafilter, dry, and then add 6 times of purified water to redissolve the dried precipitate ;
- step 2) Purification by filtration: The solution obtained in step 2) is pretreated by ultrafiltration through a sulfonated polysulfone microfiltration membrane with a molecular weight cut-off of 4KDa at a pressure of 0.3 MPa; The sulfonated polysulfone nanofiltration membrane is subjected to nanofiltration and concentration, and the finally obtained concentrated solution is vacuum freeze-dried to obtain a hyaluronic acid oligosaccharide composition.
- the average molecular weight of the hyaluronic acid oligosaccharide composition measured by a laser light scattering instrument (model: DAWN HELEOS-II) is 2682Da.
- the hyaluronic acid oligosaccharide composition obtained in Example 2 was characterized by ESI-MS, and the conditions for mass spectrometry detection were:
- Chromatography instrument Waters Acquity UPLC; analytical column: BEH Amide 2.1mm ⁇ 100mm1.7 ⁇ m, column temperature: 45°C, flow rate 0.3mL/min, injection volume 1 ⁇ L, mobile phase: A: ammonium formate, B: acetonitrile, according to The conditions shown in Table 1 were used for program elution, ion mode: ESI.
- the mass spectrum peak [M] that occurs at 6.724min on the mass spectrum total ion current peak figure is 1535.5, identified as hyaluronic acid octasaccharide (Fig. 9); the mass spectrum that occurs at 7.257min on the mass spectrum total ion current peak figure
- the peak [M] was 1913.5, identified as hyaluronic acid decaose ( FIG. 10 ), and the mass spectrum peak [M] that appeared thereafter was 2292.6, identified as hyaluronic acid dodecose ( FIG. 10 ).
- Example 2 The reducing end identification of the hyaluronic acid oligosaccharide compositions obtained in Example 2 and Comparative Example 2 was carried out.
- the reducing end of the hyaluronic acid oligosaccharide composition was determined colorimetrically using the Morgan–Elson reaction. If it turns red, it proves that the reducing end is N-acetylglucosamine, and if it does not turn red, it proves that the reducing end is uronic acid.
- Reaction buffer alkaline boric acid solution (dissolve 1.73g H3BO3 and 0.78g KOH in 10ml water, add 0.8g ml-1K2CO3 of one tenth of its volume before use.)
- p-dimethylaminobenzaldehyde solution mixture 2 1 g of p-dimethylaminobenzaldehyde solution was dissolved in 2.5ml of concentrated hydrochloric acid and 7.5ml of glacial acetic acid, and diluted with 4 times the volume of glacial acetic acid before use).
- hyaluronic acid molecular weight 1500 KDa
- HA4 hyaluronic acid tetraose
- Plating Human epidermal keratinocytes HaCaT in the logarithmic growth phase were inoculated in a 96-well plate at a density of 5 ⁇ 10 4 /mL, 100 ⁇ L per well, and the culture system was DMEM high-glucose medium supplemented with 10% fetal bovine serum. The inoculated cells were routinely cultured in a carbon dioxide incubator at 37°C and 5% CO 2 for 24 hours.
- sample solution the sample is made into a stock solution of a certain concentration with serum-free culture medium, and sterilized by filtration through a 0.22 ⁇ m filter membrane. %Final concentration.
- RGR relative growth rate
- Table 3 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.
- the hyaluronic acid oligosaccharide composition of Example 2 was diluted with purified water to a mass concentration of 1% before use, and the purified water was used as a control.
- Cell seeding Seed fibroblasts into 6-well plates at a seeding density of 2.2 ⁇ 10 5 cells/well, and incubate overnight in an incubator (37° C., 5% CO 2 ). When the cell plating rate in the 6-well plate reached 50% to 60%, administration was performed in groups, with administration concentrations of 0.01 mg/ml, 1 mg/ml and 10 mg/ml, respectively, and 3 replicate wells were set up for each group. After administration, the 6-well plate was placed in an incubator (37° C., 5% CO 2 ) and incubated for 24 hours.
- ELISA detection detection was performed according to the operating instructions of the Hyaluronan Quantikine ELISA Kit.
- RNAiso Plus RNAiso Plus to each well, lyse the cells by pipetting, and collect the samples. According to the kit instructions, RNA extraction, reverse transcription, and fluorescent quantitative PCR detection were carried out, and the results were calculated using the 2- ⁇ CT method.
- the blank control refers to that no sample is added, and the rest are carried out simultaneously with the test group.
- the promotion effect of hyaluronan synthase and the promotion effect of endogenous hyaluronic acid are shown in Table 6 and Table 7, respectively.
- Hyaluronic acid oligosaccharide composition (0.01mg/ml) 1.06 1.15 Hyaluronic acid oligosaccharide composition (1mg/ml) 3.15 6.15 Hyaluronic acid oligosaccharide composition (10mg/ml) 14.44 25.22
- the 2- ⁇ CT method was used for the calculation of the results, and the t-test method was used for the statistical analysis, and the mRNA amplification factor of the blank control group was normalized.
- the hyaluronic acid oligosaccharide composition prepared by the present invention can significantly improve the production of hyaluronan synthases HAS1 and HAS3, especially HAS3, in fibroblasts.
- the increase of hyaluronan synthase helps to enhance the synthesis of endogenous hyaluronic acid in fibroblasts.
- the detection of endogenous hyaluronic acid in fibroblasts showed that it was indeed increased, which was significantly increased compared with the blank group.
- the dermis in human skin contains abundant fibroblasts, and the molecular weight of the hyaluronic acid oligosaccharide composition is very small, which shows that the hyaluronic acid oligosaccharide composition has the potential of increasing endogenous hyaluronic acid in the dermis of human skin effect.
- Test Example 2 Based on the 3D epidermal skin model (purchased from Guangdong Boxi Biotechnology Co., Ltd.), Test Example 2 added hyaluronic acid oligosaccharide composition samples (0.01mg/ml, 1mg/ml and 10mg/ml respectively) to the culture medium. After 6 days of continuous action, normalize by dividing the total content of ceramide in the skin model by the total water-soluble protein to evaluate the ability of the sample to promote the synthesis of ceramide.
- Water-soluble total protein extraction Cut the three models of each group into two halves, put 1/2 piece in a 1.5mL EP tube, add 250 ⁇ L of 0.2mg/mL proteinase K solution, and place in a 50°C water bath 2h. After the water bath, use tweezers to pick up the cuticle, rinse it in deionized water, and place the cuticle in a new EP tube. Add 125 ⁇ L of protein extract solution to each tube; sonicate for 30 minutes. After the ultrasonic oscillation, centrifuge at 14000rpm for 10min at 4°C. After centrifugation, pipette the supernatant from the protein extraction tube into a new EP tube, and use the BCA kit to detect the concentration.
- the blank control refers to that no sample is added, and the rest are carried out simultaneously with the test group.
- Hyaluronic acid oligosaccharide composition (10mg/ml) 132.34 31.5
- Table 8 show that, compared with the control group, the water-soluble protein content in the epidermal model using the hyaluronic acid oligosaccharide composition is significantly reduced. , making the living cell layer advance toward the stratum corneum, thickening the stratum corneum, and showing a decrease in the total soluble protein content of the living cell layer, thus reflecting that the sample oligosaccharide composition can improve the barrier of the epidermal model.
- the calculation of the total ceramide content/total protein content can normalize the ceramide in the skin and reflect the changes of ceramide in the skin more scientifically. As shown in Table 10, the normalized results showed that after adding the oligosaccharide composition, the ceramide content in the skin was significantly increased, indicating that the hyaluronic acid oligosaccharide composition can significantly promote the synthesis of ceramide.
- Collagen determines the physical properties of human tissues, including skin. Collagen in the dermis is mainly secreted by fibroblasts.
- the present invention evaluates the effect of the hyaluronic acid composition on promoting collagen production by evaluating the effect of the hyaluronic acid composition on the ability of fibroblasts to secrete and express collagen.
- sample 1 and sample 2 Take 20 ⁇ L of sample 1 and sample 2 and add them to 1.98 mL of 10% FBS-DMEM medium respectively to prepare a solution with a final mass concentration of 0.001% to obtain samples 1-1 and 2-1.
- Sample 1 was taken and added to 10% FBS-DMEM medium to prepare solutions with final mass concentrations of 0.001%, 0.01%, and 0.0001%, respectively, to obtain samples 1-1, 1-2, and 1-3.
- Human fibroblasts were plated in a 96-well plate at a density of 10,000/well, and cultured in an incubator for 24 hours. Discard the supernatant, add samples 1, 1-1, 1-2, 1-3, 2-1 into the well plate, 200 ⁇ L per well. Place in the incubator and incubate for 48h. The supernatant was collected, diluted tenfold with DMEM medium, and the collagen I in the supernatant was quantified using the Human Col I Elisa Kit. The results are shown in Figure 11 and Figure 12. Wherein, the control group is the test group without adding any samples.
- samples 1-1 and 2-1 both have a certain effect of promoting collagen production, the effect of promoting collagen production of sample 1-1 with a small molecular weight is more significant.
- sample 1-1 increased the secretion of collagen I by 33%, while sample 2-1 promoted the secretion of collagen I without significant difference compared with the control group.
- sample 1, sample 1-1, sample 1-2, and 1-3 all have a significant effect of promoting collagen production, indicating that the hyaluronic acid oligosaccharide composition of Example 2 has a mass concentration of 0.0001%. -0.1% can promote fibroblasts to secrete collagen. Especially when the mass concentration is 0.001%, the effect is the best.
- Test example 6-2 3D skin model
- the present invention evaluates the effect of the hyaluronic acid composition on the proliferation and differentiation of epidermal cells by evaluating the effect of the hyaluronic acid composition on the tissue structure of the 3D full-thickness skin model.
- the 3D full-thickness skin model is a living tissue similar to the structure of human skin that is reconstructed in vitro from normal human skin cells using tissue engineering technology and appropriate medium. By assessing the tissue structure of the 3D skin model, the anti-aging effects of the samples can be judged.
- the basal layer is the material exchange barrier between the dermis and the epidermis. The more stable the structure of the basal layer, the more nutrients the epidermis can absorb, and the better the proliferation and differentiation of epidermal cells, thereby achieving anti-aging effects.
- tissue into the embedding box respectively put 4% paraformaldehyde, 70% ethanol-water solution, 80% ethanol-water solution, 90% ethanol-water solution, 95% ethanol-water solution, absolute ethanol I, absolute ethanol Gradient dehydration in II, methylcyclohexane I, methylcyclohexane II, 1 hour per tank. After dehydration, the tissues were embedded in paraffin, trimmed, and cut into paraffin sections with a thickness of 5 ⁇ m.
- the above solution was transferred to a centrifuge tube, placed in a centrifuge and centrifuged at a centrifugal force of 4000*g for 10 minutes, the upper layer liquid was discarded, and the fluorescein-labeled Example 2 was removed.
- Example 2 15 mg of fluorescently labeled Example 2 was dissolved in 1 mL of water to prepare Sample 4 with a final mass concentration of 1.5%. With 15mL of 1 ⁇ PBS as the receiving solution, pigskin with a diameter of 25mm was loaded on the manual transdermal instrument. The receiving solution was heated in a water bath at (32.0 ⁇ 0.1)°C, and was equilibrated for 1 hour under constant-speed magnetic stirring (300 ⁇ 5) r/min. Add 500 ⁇ L of sample 4 to the upper layer of skin and cover. Finish after 20h, take off the pigskin and dry.
- sample 4 has fluorescence brightness in the epidermis and dermis, which shows that sample 4 can penetrate the epidermis and dermis well.
- the above results show that Example 2 can penetrate and stay in the dermis and epidermis to play a role.
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Abstract
提供一种透明质酸寡糖组合物及其制备方法和用途,其中所述透明质酸寡糖组合物包括还原端为糖醛酸结构的透明质酸寡糖。该透明质酸寡糖组合物可以提高神经酰胺的合成和或透明质酸合成酶1(HAS1)和/或透明质酸合成酶3(HAS3)的表达,同时可以显著促进真皮成纤维细胞胶原蛋白的生成,使真皮中的胶原蛋白含量增加,减少皱纹的产生,具有一定的抗衰老活性。通过将酶解反应耦联超滤、纳滤可以使酶解得到的寡糖及时从酶解体系中分离出来,减少寡糖对酶解的抑制作用,提高酶解效率,缩短酶解时间。
Description
本发明属于生物工程技术领域,具体地,涉及一种透明质酸寡糖组合物及其制备方法和用途。
透明质酸(hyaluronic acid,HA)是一种酸性黏多糖,是由N-乙酰葡糖胺和D-葡糖醛酸双糖重复单位通过β-(1→4)糖苷键和β-(1→3)糖苷键构成的无分支高分子糖胺聚糖,存在于动物组织细胞间质和某些细菌的荚膜中。透明质酸广泛用于医药、化妆品、食品等领域,分子量一般为10
5~10
7道尔顿(Da)。
不同分子量的透明质酸表现出不同的生物活性,低分子量透明质酸甚至表现出与高分子量透明质酸完全相反的活性。很多文献都对透明质酸在创伤修复中发挥的作用进行了报道,尤其是低分子量及透明质酸寡糖作为活性物质受到更多关注。因利用酸法、碱法等制备透明质酸寡糖如果应用于大规模生产的话,有较高的风险,可能对操作人员或设备造成伤害,利用酶切法制备寡糖有着更大的优势。
对于还原端为糖醛酸的透明质酸四糖,尤其是包含HA2-12的透明质酸寡糖混合物(糖醛酸还原端)制备及功效研究报道较少。专利CN 111040048A公开了一种超低分子量透明质酸及其制备方法,该专利没有及时将酶解过程产生的寡糖分离出来,而这存在的寡糖会对酶解正向反应造成一定的抑制,造成酶解时间的延长,增加了后期纯化的难度。该专利同时也没披露其在促进透明质酸合成酶以及神经酰胺合成方面的作用。
另外,物质的结构对其生物活性会产生影响,寡糖类物质亦是如此。
非专利文献1批露了硫酸化透明质酸(sHA)具有良好的生物学功能,特定的硫酸化模式在调节糖胺聚糖和蛋白质之间的结合模式中起着关键作用,证明了带有6-O-硫酸化(sHA-6S)的sHA四糖,在体外促进大鼠E18海马神经元轴突生长方面具有重要作用。
非专利文献2采用荧光偏振竞争测定来分析合成化合物的相对结合亲和力,并揭示了合成硫酸软骨素样四糖和中期因子之间具有相互作用。可以看出透明质酸寡糖(HAOs)聚合度或者残基的不同,会产生不同的生物活性功能。这是值得去深入研究的。现有技术中,尚未有具有糖醛酸端基的超小分子量透明质酸对神经酰胺和透明质酸酶合成方面影响的研究。
非专利文献3通过改变盐酸浓度,选择性地产生具有不同聚合度和还原端的透明质酸寡糖(HAOs),使用MOE软件对HAOs与CD44和TLR4进行分子对接模拟的数据表明,HAOs与CD44和TLR4的结合能力随着HAOs的聚合度增加而增加,同时当HAOs的还原端为GlcNAc残基时结合能力显着增加,而当还原端为GlcA残基时结合能力没有显著变化。
非专利文献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).Unravel a neuroactive sHA sulfation pattern with neurogenesis activity by a library of defined oligosaccharides.European journal of medicinal chemistry,163,583-596.
非专利文献2:Chondroitin Sulfate Tetrasaccharides:Synthesis,Three-Dimensional Structure and Interaction with Midkine.Solera,C.,Macchione,G.,Maza,S.,Kayser,M.,Corzana,F.,Paz,J.L.D.,&Nieto,P.M.(2016).Chondroitin sulfate tetrasaccharides:synthesis,three-dimensional structure and interaction with midkine.
非专利文献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 breast cancer.Biomolecules,9(9),436.
因此继续研究不同结构或组成HA寡糖产品或者开发新功效是非常有意义的。
发明内容
针对现有技术存在的问题,本发明提供一种透明质酸寡糖组合物及其制备方 法。
具体来说,本发明涉及如下方面:
1.一种透明质酸寡糖组合物,其特征在于,所述透明质酸寡糖组合物包括式(I)所示结构的透明质酸寡糖:
式(I)
其中,n为选自0-5的整数,X选自H、K、Na、Ca或Zn,优选为Na;
在所述透明质酸寡糖组合物中,
n=1的透明质酸寡糖的质量占比为35-70%;
n=0的透明质酸寡糖的质量占比为5-40%;
n=2的透明质酸寡糖的质量占比为0-50%;
n=3的透明质酸寡糖的质量占比为0-15%;
n=4的透明质酸寡糖的质量占比为0-10%;
n=5的透明质酸寡糖的质量占比为0-5%。
2.根据项1所述的透明质酸寡糖组合物,其特征在于,在所述透明质酸寡糖组合物中,
n=1的透明质酸寡糖的质量占比为35-70%;
n=0的透明质酸寡糖的质量占比为5-40%;
n=2的透明质酸寡糖的质量占比为1-50%;
n=3的透明质酸寡糖的质量占比为1-15%;
n=4的透明质酸寡糖的质量占比为1-10%;
n=5的透明质酸寡糖的质量占比为0.01-1.5%。
3.根据项1所述的透明质酸寡糖组合物,其特征在于,在所述透明质酸寡糖组合物中,
n=1的透明质酸寡糖的质量占比为40-60%;
n=0的透明质酸寡糖的质量占比为5-15%;
n=2的透明质酸寡糖的质量占比为20-40%;
n=3的透明质酸寡糖的质量占比为1-10%;
n=4的透明质酸寡糖的质量占比为1-5%;
n=5的透明质酸寡糖的质量占比为0.01-1.5%。
4.根据项1所述的透明质酸寡糖组合物,其特征在于,在所述透明质酸寡糖组合物中,
n=1的透明质酸寡糖的质量占比为35-70%;
n=0的透明质酸寡糖的质量占比为5-40%;
n=2的透明质酸寡糖的质量占比为10-50%;
n=3的透明质酸寡糖的质量占比为1-15%;
n=4的透明质酸寡糖的质量占比为0.1-10%;
n=5的透明质酸寡糖的质量占比为0.01-5%。
5.根据项1所述的透明质酸寡糖组合物,其特征在于,在所述透明质酸寡糖组合物中,
n=1的透明质酸寡糖的质量占比为40-60%;
n=0的透明质酸寡糖的质量占比为5-20%;
n=2的透明质酸寡糖的质量占比为20-40%;
n=3的透明质酸寡糖的质量占比为3-8%;
n=4的透明质酸寡糖的质量占比为1-5%;
n=5的透明质酸寡糖的质量占比为0.01-1.5%。
6.根据项1所述的透明质酸寡糖组合物,其特征在于,所述透明质酸寡糖组合物的重均分子量小于等于1kDa。
7.根据项1-3、6任一项所述的透明质酸寡糖组合物,其特征在于,所述透明质酸寡糖组合物用于化妆品或保健品,优选在所述化妆品或保健品中的质量浓度为0.001-10%。
8.一种如项1-7任一项所述的透明质酸寡糖组合物的制备方法,其特征在于,所述制备方法包括以下步骤:
将透明质酸用透明质酸酶进行酶解反应,
分离酶解反应物以得到产物原液,
对所述产物原液进行浓缩以得到产物浓缩液;
去除所述产物浓缩液中的杂质,干燥得到透明质酸寡糖组合物,
其中,所述透明质酸酶为酶切β-1,3糖苷键的透明质酸酶,
优选,所述透明质酸酶为利用毕赤酵母工程菌表达的水蛭透明质酸水解酶,
进一步优选,所述分离为超滤;
进一步优选,所述浓缩为纳滤;
进一步优选,使用活性炭吸附来去除杂质。
9.根据项8所述的方法,其特征在于,在所述酶解反应中,所述透明质酸的初始浓度为5-150g/L,优选,所述透明质酸的分子量为1000-2000kDa,进一步优选所述透明质酸酶的初始含量为1×104-3×105U/mL。
10.根据项8所述的方法,其特征在于,超滤采用截留分子量为600-1000Da的超滤膜,纳滤采用截留分子量为100-300Da的纳滤膜。
11.项1-3、6中任一项所述的透明质酸寡糖组合物在促进神经酰胺和/或透明质酸合成酶1(HAS1)和/或透明质酸合成酶3(HAS3)的合成中的用途。
12.项4-6中任一项所述的透明质酸寡糖组合物在抗皮肤衰老中的用途。
13.根据项12所述的用途,其特征在于,所述透明质酸寡糖组合物通过促进胶原蛋白生成和/或表皮细胞增殖与分化而抗皮肤衰老。
14.根据项12所述的用途,其特征在于,所述透明质酸寡糖组合物施用于皮肤。
15.根据项12所述的用途,其特征在于,所述施用于皮肤的方式为通过化妆品或医疗器械,所述透明质酸寡糖组合物在所述化妆品或医疗器械中的质量浓度为0.0001%-5%。
16.根据项12所述的用途,其特征在于,所述透明质酸寡糖组合物在所述化妆品或医疗器械中的质量浓度为0.001%-1%。
17.项4-6中任一项所述透明质酸寡糖组合物在促进胶原蛋白生成中的用途。
18.根据项17所述的用途,其特征在于,所述透明质酸寡糖组合物用于制备促进胶原蛋白生成的保健品,或者用于制备保护、强化脏器的保健品,或者用于制备保护胃粘膜的保健品,或者用于制备补充钙质的保健品,或者用于制备关节润滑注射液。
19.根据项18所述的用途,其特征在于,所述透明质酸寡糖组合物在所述保健品或注射液中的质量浓度为0.0001%-5%。
20.根据项19所述的用途,其特征在于,所述透明质酸寡糖组合物在所述保健品或注射液中的质量浓度为0.001%-1%。
本发明通过将酶解反应耦联超滤、纳滤可以使酶解得到的寡糖及时从酶解体系中分离出来,减少寡糖对酶解的抑制作用,提高酶解效率,缩短酶解时间。酶解完后,利用超滤把酶截留下来可以再重复利用1-2次,提高酶的重复利用度,同时省掉了加热灭酶活阶段,减少了加热对寡糖活性的影响,使寡糖的活性得到最大化的保留。减少活性炭加入量,减少了对操作人员的炭污染,简化了纯化工艺。进一步的,本发明获得的透明质酸寡糖组合物可以促进神经酰胺和/或透明质酸合成酶1(HAS1)和/或透明质酸合成酶3(HAS3)的合成。本发明对于透明质酸寡糖组合物工业化生产,具有重大应用价值。
本发明的透明质酸寡糖组合物分子量较小,在还原端具有糖醛酸,可以显著促进真皮成纤维细胞胶原蛋白的生成,使真皮中的胶原蛋白含量增加,减少皱纹的产生,具有一定的抗衰老活性。同时,胶原蛋白独有的三螺旋结构,能强力锁住30倍的水分,使皮肤持久水润、光泽、细嫩人体。本发明的透明质酸寡糖组合物可用于制备抗衰老和保湿的化妆品、医疗器械。本发明的透明质酸寡糖组合物具有促进胶原蛋白生成的用途,可进一步用于制备促进胶原蛋白生成的保健品,或者用于制备保护、强化脏器的保健品,或者用于制备保护胃粘膜的保健品,或者用于制备补充钙质的保健品,或者用于制备关节润滑注射液。
图1为实施例1中透明质酸寡糖的高效液相色谱图。
图2为实施例2中透明质酸寡糖的高效液相色谱图。
图3为实施例3中透明质酸寡糖的高效液相色谱图。
图4为对比例1中透明质酸寡糖的高效液相色谱图。
图5为实施例2中透明质酸寡糖的质谱总离子流峰图。
图6为透明质酸二糖(HA2)的离子强度图。
图7为透明质酸四糖(HA4)的离子强度图。
图8为透明质酸六糖(HA6)的离子强度图。
图9为透明质酸八糖(HA8)的离子强度图。
图10为透明质酸十糖(HA10)和十二糖(HA12)的离子强度图。
图11所示为使用样品1-1、2-1后成纤维细胞分泌胶原蛋白Ⅰ的含量。
图12所示为使用样品1、1-1、1-2、1-3后成纤维细胞分泌胶原蛋白Ⅰ的含量。
图13所示为使用样品1、样品2后3D全层皮肤模型的结构。
图14所示为样品4透皮情况的显微镜下照片。
下面结合实施例进一步说明本发明,应当理解,实施例仅用于进一步说明和阐释本发明,并非用于限制本发明。
除非另外定义,本说明书中有关技术的和科学的术语与本领域内的技术人员所通常理解的意思相同。虽然在实验或实际应用中可以应用与此间所述相似或相同的方法和材料,本文还是在下文中对材料和方法做了描述。在相冲突的情况下,以本说明书包括其中定义为准,另外,材料、方法和例子仅供说明,而不具限制性。以下结合具体实施例对本发明作进一步的说明,但不用来限制本发明的范围。
透明质酸寡糖或透明质酸寡聚糖(oligosaccharides of HA,简称oligo-HA)为分子量在10
4Da以下,单糖残基数量为2~25(一般为4~16)的透明质酸分子片段。oligo-HA属于小分子多糖,其性质与普通透明质酸有很大不同。研究表明,Oligo-HA具有抗氧化、免疫调节、抗炎症和促进伤口愈合、促血管生成和抗肿瘤等生物活性。尤为重要的是,因其分子尺寸较小,可渗入到皮肤角质层发挥深层保湿和滋润的功效,可广泛应用到化妆品中。
本发明提供一种透明质酸寡糖组合物,所述透明质酸寡糖组合物包括式(I)所示结构的透明质酸寡糖:
由式(I)可以看出,本发明的透明质酸寡糖的还原端为糖醛酸结构。
其中,n选自0-5的整数,例如n可以为0、1、2、3、4或5,X选自H、K、Na、Ca或Zn,优选为Na。在所述透明质酸寡糖组合物中,n=1的透明质酸寡糖的质量占比为35-70%;n=0的透明质酸寡糖的质量占比为5-40%;n=2的透明质酸寡糖的质量占比为0-50%;n=3的透明质酸寡糖的质量占比为0-15%;n=4的透明质酸寡糖的质量占比为0-10%;n=5的透明质酸寡糖的质量占比为0-5%。
当n=0时,透明质酸寡糖为二糖,当n=1时,透明质酸寡糖为四糖,当n=2时,透明质酸寡糖为六糖,当n=3时,透明质酸寡糖为八糖,当n=4时,透明质酸寡糖为十糖,当n=5时,透明质酸寡糖为十二糖。所以,在本发明的透明质酸寡糖组合物中,四糖的质量占比为35-70%;二糖的质量占比为5-40%;六糖的质量占比为0-50%;八糖的质量占比为0-15%;十糖的质量占比为0-10%;十二糖的质量占比为0-5%。
其中,透明质酸四糖的质量占比为35-70%,例如可以为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%,优选为40-60%。
透明质酸二糖的质量占比为5-40%,例如可以为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%,优选为5-15%。
透明质酸六糖的质量占比为0-50%,例如可以为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%,优选1-50%,进一步优选20-40%。
透明质酸八糖的质量占比为0-15%,例如可以为0%、1%、2%、3%、4%、5%、6%、7%、8%、9%、10%、11%、12%、13%、14%、15%,优选1-15%,进一步优选1-10%。
透明质酸十糖的质量占比为0-10%,例如可以为0%、1%、2%、3%、4%、 5%、6%、7%、8%、9%、10%,优选1-10%,进一步优选1-5%。
透明质酸十二糖的质量占比为0-5%,例如可以为0%、0.01%、0.05%、0.1%、1%、2%、3%、4%、5%,优选0.01-1.5%。
在一个具体的实施方式中,在所述透明质酸寡糖组合物中,透明质酸二糖的质量占比为5-40%,透明质酸四糖的质量占比为35-70%,透明质酸六糖的质量占比为0-50%,透明质酸八糖的质量占比为0-15%,透明质酸十糖的质量占比为0-10%,透明质酸十二糖的质量占比为0-5%。
在一个具体的实施方式中,在所述透明质酸寡糖组合物中,透明质酸二糖的质量占比为5-40%,透明质酸四糖的质量占比为35-70%,透明质酸六糖的质量占比为1-50%,透明质酸八糖的质量占比为1-15%,透明质酸十糖的质量占比为1-10%,透明质酸十二糖的质量占比为0.01-1.5%。
在一个具体的实施方式中,在所述透明质酸寡糖组合物中,透明质酸二糖的质量占比为5-15%,透明质酸四糖的质量占比为40-60%,透明质酸六糖的质量占比为20-40%,透明质酸八糖的质量占比为1-10%,透明质酸十糖的质量占比为1-5%,透明质酸十二糖的质量占比为0.01-1.5%。
在一个具体的实施方式中,在所述透明质酸寡糖组合物中,透明质酸二糖的质量占比为5-10%,透明质酸四糖的质量占比为45-55%,透明质酸六糖的质量占比为30-40%,透明质酸八糖的质量占比为2-8%,透明质酸十糖的质量占比为1-2%,透明质酸十二糖的质量占比为0.01-1%。
在一个具体的实施方式中,在所述透明质酸寡糖组合物中,透明质酸二糖的质量占比为10%,透明质酸四糖的质量占比为49.5%,透明质酸六糖的质量占比为33%,透明质酸八糖的质量占比为5%,透明质酸十糖的质量占比为1.5%,透明质酸十二糖的质量占比为1%。
在一个具体的实施方式中,在所述透明质酸寡糖组合物中,透明质酸二糖的质量占比为9%,透明质酸四糖的质量占比为49%,透明质酸六糖的质量占比为32%,透明质酸八糖的质量占比为7%,透明质酸十糖的质量占比为2%,透明质酸十二糖的质量占比为1%。
进一步地,所述透明质酸寡糖组合物的重均分子量小于等于1kDa,例如可以为1kDa、990Da、980Da、970Da、960Da、950Da、940Da、930Da、920Da、 910Da、900Da、890Da、880Da、870Da、860Da、850Da、840Da、830Da、820Da、810Da、800Da、790Da、780Da、770Da、760Da、750Da、740Da、730Da、720Da、710Da、700Da。
本发明的透明质酸寡糖组合物可以促进神经酰胺和/或透明质酸合成酶1和/或透明质酸合成酶3的表达。其中,神经酰胺(Ceramide)是以神经酰胺为骨架的一类磷脂,主要有神经酰胺磷酸胆碱和神经酰胺磷酸乙醇胺,磷脂是细胞膜的主要成分,角质层中40%~50%的皮脂由神经酰胺构成,神经酰胺是细胞间基质的主要部分,在保持角质层水分的平衡中起着重要作用。神经酰胺具有很强缔合水分子能力,它通过在角质层中形成网状结构维持皮肤水分。因此,神经酰胺具有保持皮肤水分作用。
皮肤中的透明质酸(Hyaluronic Acid,HA)主要由透明质酸合成酶(Hyaluronan Synthases,HAS)催化合成。已在哺乳动物中鉴定出三种HAS亚型(HAS1,HAS2,HAS3)。HAS参与质膜内侧不同分子量的透明质酸链的合成,同时将合成的透明质酸分泌到细胞外基质中。据报道,HAS1和HAS2合成透明质酸链长度相似,约为2000KDa,HAS3催化合成透明质酸分子量在200-300kDa之间。细胞外的大分子HA被透明质酸酶酶解或发生氧化反应降解可以获得小分子HA。小分子HA能够刺激细胞激增,启动信号级联反应,同时还参与血管生成。100-300Ka的透明质酸具有软化角质、锁水、立体保湿的作用。进一步地,文献“Expression of Hyaluronan Synthase and Collagen Type I mRNA by Hyaluronan Tetrasaccharides in Normal Human Dermal Fibroblasts”披露透明质酸四糖能促进HAS1的表达,但对HAS2和HAS3的表达无影响。
本发明的透明质酸寡糖组合物可用于化妆品或保健品,优选在所述化妆品或保健品中的质量浓度为0.001-10%,例如可以为0.001%、0.005%、0.01%、0.1%、0.5%、1%、2%、3%、4%、5%、6%、7%、8%、9%、10%。可以根据所述透明质酸寡糖组合物在化妆品或保健品中的不同用途进一步调整其在化妆品或保健品中的质量浓度。例如,当所述透明质酸寡糖组合物在化妆品或保健品中用于促进神经酰胺表达时,其质量浓度可以为0.001-1%。当所述透明质酸寡糖组合物在化妆品或保健品中用于促进透明质酸合成酶1(HAS1)和/或透明质酸合成酶3(HAS3)的表达时,其质量浓度可以为0.01-10%,所述透 明质酸寡糖组合物用于细胞中促进透明质酸合成酶1(HAS1)和/或透明质酸合成酶3(HAS3)的表达时,其质量浓度可以为0.001-1%。
本发明还提供一种透明质酸寡糖组合物的制备方法,所述制备方法包括以下步骤:将透明质酸用透明质酸酶进行酶解反应,分离酶解反应物以得到产物原液,对所述产物原液进行浓缩以得到产物浓缩液;去除所述产物浓缩液中的杂质,干燥得到透明质酸寡糖组合物,其中,所述透明质酸酶为酶切β-1,3糖苷键的透明质酸酶,优选,所述分离为超滤;进一步优选,所述浓缩为纳滤;进一步优选,使用活性炭吸附来去除杂质。
制备方法中所使用的透明质酸酶为酶切β-1,3糖苷键的透明质酸酶,保证透明质酸酶切得到的寡糖的还原端为糖醛酸结构。
在一个具体的实施方式中,所述透明质酸酶为利用毕赤酵母工程菌表达的水蛭透明质酸水解酶。其制备方法可以参照CN103695448A所描述的方法。
其中超滤是一种膜分离技术,(UItrafil-tration,简称UF)。能够将溶液净化,分离或者浓缩。超滤是介于微滤与纳滤之间,且三者之间无明显的分界线。一般来说,超滤膜的孔径在0.05μm–1nm之间,操作压力为0.1–0.5Mpa。主要用于截留去除水中的悬浮物、胶体、微粒、细菌和病毒等大分子物质。超滤膜根据膜材料,可分为有机膜和无机膜。按膜的外型,又可分为:平板式、管式、毛细管式、中空纤维和多孔式。目前家用超滤净水器,多以中空膜为主。
纳滤(NF)是一种介于反渗透和超滤之间的压力驱动膜分离过程,纳滤膜的孔径范围在几个纳米左右。纳滤(NF)用于将相对分子质量较小的物质,如无机盐或葡萄糖、蔗糖等小分子有机物从溶剂中分离出来。纳滤又称为低压反渗透,是膜分离技术的一种新兴领域,其分离性能介于反渗透和超滤之间,允许一些无机盐和某些溶剂透过膜,从而达到分离的效果。
通过控制酶解反应的条件,如底物和酶的用量、底物的分子量、反应温度和反应时间等,以及超滤和纳滤所采用的不同型号的膜,可以获得不同分子量,不同寡糖占比的透明质酸寡糖组合物。
在一个具体的实施方式中,在酶解反应中,所述透明质酸的初始浓度为5-150g/L,例如可以为5g/L、10g/L、20g/L、30g/L、40g/L、50g/L、60g/L、70g/L、80g/L、90g/L、100g/L、110g/L、120g/L、130g/L、140g/L、150g/L。 例如,反应体系为1L,则透明质酸的初始含量为5-150g,即没有发生水解时的含量为5-150g。
在一个具体的实施方式中,所述透明质酸的初始分子量为1000-2000kDa,例如可以为1000kDa、1100kDa、1200kDa、1300kDa、1400kDa、1500kDa、1600kDa、1700kDa、1800kDa、1900kDa、2000kDa。
在一个具体的实施方式中,在酶解反应中,所述透明质酸酶的初始含量为1×10
4-3×10
5U/mL,例如可以为1×10
4U/mL、2×10
4U/mL、4×10
4U/mL、6×10
4U/mL、8×10
4U/mL、1×10
5U/mL、1.5×10
5U/mL、2×10
5U/mL、2.5×10
5U/mL、3×10
5U/mL。其中,透明质酸酶活力单位定义(U)为:在pH 5.5和38℃条件下,每小时从透明质酸糖链中释放出1μg葡萄糖还原当量的还原糖所需的酶量。
在一个具体的实施方式中,在酶解反应中,所述透明质酸酶与所述透明质酸的初始比值为1-5U/mg,例如可以为1U/mg、2U/mg、3U/mg、4U/mg、5U/mg。
在一个具体的实施方式中,超滤采用截留分子量为600-1000Da的超滤膜,例如截留分子量可以为600Da、700Da、800Da、900Da、1000Da,纳滤采用截留分子量为100-300Da的纳滤膜,例如截留分子量可以为100Da、200Da、300Da。
在一个优选的实施方式中,超滤采用截留分子量为1000Da的超滤膜,纳滤采用截留分子量为200Da的纳滤膜。
本发明所述的截留分子是使用分子量大小表示的超滤膜的截留性能,又称作切割分子量。由于直接测定超滤膜或纳滤膜的孔径相当困难,所以使用已知分子量的球状物质进行测定。如膜对被截留物质的截留率大于90%时,就用被截留物质的分子量表示膜的截留性能,称为膜的截留分子量。例如截留分子量为10kDa的膜可以截留分子量大于10kDa的物质,而允许分子量小于10kDa的物质通过。
所述方法中的干燥可以采用现有技术中的各种方式的干燥。在一个具体的实施方式中,所采用的干燥为喷雾干燥。喷雾干燥中所采用的进风温度为120℃-160℃,出风温度60℃-80℃。
进一步地,所述方法得到的透明质酸寡糖组合物为上述的透明质酸寡糖组合物,即式(I)所示结构的透明质酸寡糖:
其中,n为选自0-5的整数,X选自H、K、Na、Ca或Zn,优选为Na。
其中,透明质酸寡糖产物的平均分子量可以通过多角度激光光散射仪测得。透明质酸寡糖产物中各种寡糖的鉴定可以采用质谱进行检测,各种寡糖的含量可以采用高效液相色谱进行检测。
本发明还提供上述透明质酸寡糖组合物在促进透明质酸合成酶1(HAS1)和/或透明质酸合成酶3(HAS3)的表达中的用途。
本发明的透明质酸寡糖组合物具有还原端为糖醛酸结构的透明质酸二糖、透明质酸四糖、透明质酸六糖、透明质酸八糖、透明质酸十糖、透明质酸十二糖等多种类型的的透明质寡糖。该寡糖组合物无细胞毒性,对人体皮肤无潜在的不良反应;能显著促进成纤维细胞中透明质酸合酶HAS1、HAS3表达,尤其是促进HAS3的表达;可显著促进神经酰胺的合成。
人类的皮肤随年龄增长自然老化或随着环境刺激而衰老,导致皮肤衰老。自然老化为内源性老化,表现为皮肤变白,出现细小皱纹、弹性下降、皮肤松弛等,环境刺激为外源性老化,如日晒所致的光老化。如果皮肤得不到良好的保养或随着年龄增长而衰退,死皮就会附着在皮肤表面而不脱落,因而造成一系列问题,严重影响美容。
为了解决皮肤衰老的问题,本发明提供一种透明质酸寡糖组合物在抗皮肤衰老中的用途,所述透明质酸寡糖组合物包括式(I)所示结构的透明质酸寡糖:
其中,n为选自0-5的整数;
X选自H、K、Na、Ca或Zn,优选为Na。
在所述透明质酸寡糖组合物中,
n=1的透明质酸寡糖的质量占比为35-70%,n=0的透明质酸寡糖的质量占比为5-40%,n=2的透明质酸寡糖的质量占比为10-50%,n=3的透明质酸寡糖的质量占比为1-15%,n=4的透明质酸寡糖的质量占比为0.1-10%,n=5的透明质酸寡糖的质量占比为0.01-5%。
皮肤衰老的原因主要由以下几点体现:1.真皮层细胞外基质的改变。胶原是真皮细胞外基质的主要成分,在人体真皮中约占蛋白质的90%。胶原蛋白种类较多,常见类型为Ⅰ型、Ⅱ型、Ⅲ型、Ⅴ型和Ⅺ型。真皮中的蛋白主要由Ⅰ型胶原(80%)和少量的Ⅲ型胶原(10%)组成,胶原使皮肤有强度和弹性。随着年龄的增长,皮肤中胶原纤维减少,使皮肤失去弹性,产生皱纹。真皮中的胶原蛋白主要由成纤维细胞分泌,成纤维细胞分泌的胶原蛋白减少也是引起衰老、产生皱纹的一大原因之一。2.基底层结构的改变。人的皮肤在衰老后,基底层结构松弛,细胞连接稀松,使得真皮-表皮间的物质交换效率降低。从而导致表皮营养不足,表皮细胞不能正常地增殖与分化。进而使得表皮变薄,产生松弛等现象。
针对上述皮肤衰老的主要原因,所述透明质酸寡糖组合物可以通过促进胶原蛋白生成和/或表皮细胞增殖与分化而抗皮肤衰老。
在一个具体的实施方式中,所述透明质酸寡糖组合物通过促进胶原蛋白生成而抗皮肤衰老。
在一个具体的实施方式中,所述透明质酸寡糖组合物通过促进表皮细胞增殖与分化而抗皮肤衰老。
在一个具体的实施方式中,所述透明质酸寡糖组合物可以通过促进胶原蛋白生成和表皮细胞增殖与分化而抗皮肤衰老。
在本发明的抗皮肤衰老的用途中,所述透明质酸寡糖组合物可以直接或间接地施用于皮肤,例如可以通过外敷、注射或口服等方式施用。
在本发明的抗皮肤衰老的用途中,所述透明质酸寡糖组合物可用于化妆品、医疗器械,进一步适用于皮肤。例如,所述透明质酸寡糖组合物可以单独或与其他活性成分一起配制成化妆品,用于抗皮肤衰老。
进一步地,当所述透明质酸寡糖组合物用于化妆品、医疗器械时,透明质酸寡糖组合物用于细胞中的质量浓度为0.0001%-0.1%。根据细胞-人体面部皮肤的表面积换算及所述透明质酸寡糖组合物的透皮情况,所述透明质酸寡糖组合物在所述化妆品或医疗器械中的质量浓度为0.0001%-5%,例如可以为0.0001%、0.001%、0.01%、0.05%、0.1%、0.2%、0.3%、0.4%、0.5%、0.6%、0.7%、0.8%、0.9%、1%、2%、3%、4%、5%,优选为0.001%-1%。
本发明还提供上述透明质酸寡糖组合物在促进胶原蛋白生成中的用途。
内脏器官及组织都含有胶原蛋白,在这些脏器表皮结构的下方是胶原蛋白,胶原蛋白能够保护及强化脏器,因此所述透明质酸寡糖组合物可用于制备保护、强化脏器的保健品。
胶原蛋白是肌肉组织的主要成分,可以为肌肉提供所需营养,保护胃黏膜,因此所述透明质酸寡糖组合物可用于制备保护胃粘膜的保健品。
胶原蛋白同时也是组成钙质附着网架,锁定骨钙,防止钙流失,因此所述透明质酸寡糖组合物可用于制备补充钙质的保健品。
胶原蛋白还能有效修复关节软骨,恢复关节软骨表面的润滑,减少摩擦。因此所述透明质酸寡糖组合物可用于制备关节润滑注射液。
在一个具体的实施方式中,所述透明质酸寡糖组合物在所述保健品或注射液中的质量浓度为0.0001%-5%,例如可以为0.0001%、0.001%、0.01%、0.05%、0.1%、0.2%、0.3%、0.4%、0.5%、0.6%、0.7%、0.8%、0.9%、1%、2%、3%、4%、5%,优选为0.001%-1%。
实施例
实施例1透明质酸寡糖组合物制备
于5L反应器中,加入4L水,控制温度40℃,加入透明质酸酶(该酶为利用毕赤酵母工程菌表达的水蛭透明质酸酶,制备方法参照CN 103695448 A中描述的方法)4×10
8U,体系酶活为1×10
5U/mL,加入40g透明质酸(分子量150kDa),待其完全溶解后,采用超滤分离酶解反应物以得到产物原液,采用纳滤对所述产物原液进行浓缩以得到产物浓缩液。保持体系40℃搅拌反应18h。其中超滤采用截留分子量为1kDa的超滤膜,纳滤采用截留分子量为200 Da的纳滤膜。反应结束,收集纳滤浓缩液,加入活性炭吸附后,过滤收集滤液,进行喷雾干燥,其中进风温度140℃,出风温度70℃。得到透明质酸寡糖组合物,产品收率为86%。
将透明质酸寡糖组合物使用高效液相色谱仪分析其寡糖分布,具体的色谱条件为:
色谱柱:SUPERDEX 200 10/300 GL
柱温:40℃
检测器:紫外-可见分光检测器
流动相:1mol/L硫酸铵溶液
进样浓度:0.5%
进样量:20μL
流速:2ml/min
检测波长:200nm
结果如图1所示,在透明质酸寡糖组合物中,透明质酸二糖的质量占比为9.12%,透明质酸四糖的质量占比为47.73%,透明质酸六糖的质量占比为32.83%,透明质酸八糖的质量占比为6.05%,透明质酸十糖的质量占比为1.49%,透明质酸十二糖的质量占比为0.42%。
使用激光光散射仪(DAWN HELEOS-II)测量透明质酸寡糖组合物的平均分子量为890Da。
实施例2透明质酸寡糖组合物制备
于5L反应器中,加入4L水,控制温度30℃,加入透明质酸酶(该酶为利用毕赤酵母工程菌表达的水蛭透明质酸酶,制备方法参照CN 103695448 A中描述的方法)4×10
7U,体系酶活为1×10
4U/mL,加入5g透明质酸(分子量100kDa),待其完全溶解后,采用超滤分离酶解反应物以得到产物原液,采用纳滤对所述产物原液进行浓缩以得到产物浓缩液。保持体系30℃搅拌反应12h。其中超滤采用截留分子量为1kDa的超滤膜,纳滤采用截留分子量为200Da的纳滤膜。反应结束,收集纳滤浓缩液,加入活性炭吸附后,过滤收集滤液,进行喷雾干燥,其中进风温度120℃,出风温度60℃。得到透明质酸寡糖组合物,产品收率为84%。
将透明质酸寡糖组合物使用高效液相色谱仪分析其寡糖分布,具体的色谱条件同实施例1。结果如图2所示,在透明质酸寡糖组合物中,透明质酸二糖的质量占比为9.35%,透明质酸四糖的质量占比为47.84%,透明质酸六糖的质量占比为32.92%,透明质酸八糖的质量占比为6.04%,透明质酸十糖的质量占比为1.48%,透明质酸十二糖的质量占比为0.42%。
使用激光光散射仪测量透明质酸寡糖组合物的平均分子量为920Da。
实施例3透明质酸寡糖组合物制备
于5L反应器中,加入4L水,控制温度46℃,加入透明质酸酶(该酶为利用毕赤酵母工程菌表达的水蛭透明质酸酶,制备方法参照CN 103695448A中描述的方法)1.2×10
9U,体系酶活为3×10
5U/mL,加入150g透明质酸(分子量200kDa),待其完全溶解后,采用超滤分离酶解反应物以得到产物原液,采用纳滤对所述产物原液进行浓缩以得到产物浓缩液。保持体系46℃搅拌反应24h。其中超滤采用截留分子量为1kDa的超滤膜,纳滤采用截留分子量为200Da的纳滤膜。反应结束,收集纳滤浓缩液,加入活性炭吸附后,过滤收集滤液,进行喷雾干燥,其中进风温度160℃,出风温度80℃。得到透明质酸寡糖组合物,产品收率为87%。
将透明质酸寡糖组合物使用高效液相色谱仪分析其寡糖分布,具体的色谱条件同实施例1。结果如图3所示,在透明质酸寡糖组合物中,透明质酸二糖的质量占比为8.94%,透明质酸四糖的质量占比为52.80%,透明质酸六糖的质量占比为29.00%,透明质酸八糖的质量占比为4.71%,透明质酸十糖的质量占比为1.5%,透明质酸十二糖的质量占比为0.40%。
使用激光光散射仪测量透明质酸寡糖组合物的平均分子量为915Da。
对比例1
于5L反应器中,加入4L水,控制温度40℃,加入透明质酸酶(该酶为利用毕赤酵母工程菌表达的水蛭透明质酸酶,制备方法参照CN 103695448 A中描述的方法)8×10
8U,体系酶活为2×10
5U/mL,加入100g透明质酸(分子量150KDa),待其完全溶解后,保持体系40℃搅拌反应20h。反应结束,收集反应液,加入活性炭吸附后,过滤收集滤液,进行喷雾干燥,其中进风温度140℃,出风温度70℃。得到透明质酸水解产物,产品收率为90%。
将透明质酸水解产物使用高效液相色谱仪分析其寡糖分布,具体的色谱条件同实施例1。结果如图4所示,在透明质酸寡糖组合物中,透明质酸二糖的质量占比为0.00%,透明质酸四糖的质量占比为0.98%,透明质酸六糖的质量占比为2.10%,透明质酸八糖的质量占比为2.67%,透明质酸十糖的质量占比为3.31%,透明质酸十二糖的质量占比为3.97%。
使用激光光散射仪测量透明质酸寡糖组合物的平均分子量为7580Da。
对比例2透明质酸寡糖组合物制备
参考CN112646055A中描述的方法制备得到更高分子量的透明质酸寡糖组合物,具体制备方法如下:
1)预处理:将50g分子量为1000kDa的高分子量透明质酸溶于30倍重量份的水中,用0.1mol/L甲醇钠溶液调节溶液pH至8.5,在室温下充分搅拌溶胀,缓慢升温至90℃,恒温水解4h,然后将经浸提抽滤,滤渣重复上述浸提操作,合并两次滤液备用;
2)中性盐分级沉淀:将步骤1)得到的溶液加热至40℃,加入溶液重量的25%的硫酸镁固体,搅拌完全溶解后用0.1μm的磺化聚砜超滤膜进行超滤,接着向滤液中再次加入溶液重量的43%的硫酸镁固体,搅拌至完全溶解后静置沉淀,然后进行过滤、超滤、干燥,再然后将干燥后的沉淀物加入6倍的纯化水复溶;
3)过滤纯化:将步骤2)得到的溶液经截留分子质量为4KDa的磺化聚砜微滤膜在0.3MPa透膜压力下进行超滤预处理;渗出液再经截留分子质量为2KDa的磺化聚砜纳滤膜进行纳滤浓缩,最终所得浓缩液经真空冷冻干燥得到透明质酸寡糖组合物。
使用激光光散射仪(型号:DAWN HELEOS-II)测量透明质酸寡糖组合物的平均分子量为2682Da。
试验例
试验例1
采用ESI-MS对实施例2得到的透明质酸寡糖组合物进行表征,质谱检测的条件为:
色谱仪:Waters Acquity UPLC;分析色谱柱:BEH Amide 2.1mm×100mm1.7μm,柱温:45℃,流速0.3mL/min,进样量1μL,流动相:A:甲酸铵,B:乙腈,按照表1所示的条件进行程序洗脱,离子方式:ESI。离子源参数ESI,雾化器压力:30psi;脱溶剂气体流速50L/h;温度400℃;负离子模式。
表1
结果如图5-10所示,在质谱总离子流峰图上1.380min出现的质谱峰[M]为397.1,鉴定为透明质酸二糖(图6);在质谱总离子流峰图上3.649min出现的质谱峰[M]为776.2,鉴定为透明质酸四糖(图7);在质谱总离子流峰图上5.612min出现的质谱峰[M]为1155.3,鉴定为透明质酸六糖(图8);在质谱总离子流峰图上6.724min出现的质谱峰[M]为1535.5,鉴定为透明质酸八糖(图9);在质谱总离子流峰图上7.257min出现的质谱峰[M]为1913.5,鉴定为透明质酸十糖(图10),在其后出现的质谱峰[M]为2292.6,鉴定为透明质酸十二糖(图10)。
试验例2透明质酸寡糖组合物的还原端鉴定
对实施例2和对比例2得到的透明质酸寡糖组合物进行还原端鉴定。
利用Morgan–Elson反应,根据比色法确定透明质酸寡糖组合物的还原端。如果变红证明还原端为N-乙酰葡糖胺,如果不变红证明还原端为糖醛酸。
反应缓冲液:碱性硼酸溶液(在10ml水中溶解1.73g H3BO3和0.78g KOH,在使用前加入其体积十分之一的0.8g ml-1K2CO3。)对二甲基氨基苯甲醛溶液(将2克对二甲基氨基苯甲醛溶液溶于2.5ml浓盐酸和7.5ml冰醋酸中,使用前用4倍体积冰醋酸稀释)。
取400μl 10g/L的透明质酸寡糖组合物溶液,加入110μl碱性硼酸溶液,煮沸4min,加入了1.5ml的对二甲基氨基苯甲醛,在37℃下孵育20min。
使用透明质酸(分子量1500KDa)、透明质酸四糖(HA4,购自Sigma,还原端为N-乙酰葡糖胺)溶液作为对照。
结果如表2所示,实施例2和对比例2制备的透明质酸寡糖组合物经Moran-Elson反应没有显红色,证明还原端为糖醛酸。
表2
Morgan–Elson反应结果 | |
实施例2 | 不变红 |
对比例2 | 不变红 |
试验例3细胞毒性评价及斑贴实验
对实施例2得到的透明质酸寡糖组合物进行细胞毒性评价及斑贴实验
细胞毒性测试
1)铺板:取对数生长期人表皮角质形成细胞HaCaT,以5×10
4个/mL密度接种于96孔板,每孔100μL,培养体系是DMEM高糖培养液添加10%胎牛血清。接种的细胞置二氧化碳培养箱37℃、5%CO
2常规培养24h。
2)样品溶液配制:样品以无血清培养液配成一定浓度的储备液,并0.22μm滤膜过滤除菌,用时用无血清培养液稀释至所需的质量浓度为1%、0.5%和0.05%终浓度。
3)加药:常规培养24h后,弃去旧培养液,实验组换成100μL样品,正常对照组(control)加入等量无血清培养液,每个水平6个平行孔。
4)检测:继续培养24h后,弃去样品溶液,每孔加入用无血清培养液配制的10%WST-1溶液,放入细胞培养箱中继续孵育3h,于450nm波长处用酶标仪测定吸光度。相对增殖率(RGR)为实验组吸光度与正常对照组吸光度的比值。按照GB/T16886.5-2017的要求,RGR低于70%时,认为样品具有细胞毒性。
结果如表3所示。
表3细胞相对增殖率(%)
表3显示,透明质酸寡糖组合物在0.05%-1%的质量浓度范围内,细胞相对增殖率均远大于70%,表明透明质酸寡糖组合物无明显细胞毒性。
斑贴试验
1)招募健康受试者30名,男女不限。具有下列情况者作应被排除:a近一周使用抗组胺药或近一个月内使用免疫抑制剂者;b近两个月内受试部位应用任何抗炎药物者;c受试者患有炎症性皮肤病临床未愈者;d胰岛素依赖性糖尿病患者;e正在接受治疗的哮喘或其它慢性呼吸系统疾病患者;f在近6个月内接受抗癌化疗者;g免疫缺陷或自身免疫性疾病患者;h哺乳期或妊娠妇女;i双侧乳房切除及双侧腋下淋巴结切除者;j在皮肤待试部位由于瘢痕、色素、萎缩、鲜红斑痣或其它瑕疵而影响试验结果的判定者;k参加其它的临床试验研究者;l体质高度敏感者;m非志愿参加者或不能按实验要求完成规定内容者。
2)样品的制备
实施例2的透明质酸寡糖组合物用纯化水稀释至1%的质量浓度后使用,纯化水作为对照。
3)皮肤斑贴试验撕开斑试器的包装,量取配置好的样品各0.025mL加入小室内。将斑试器贴敷于受试者的前臂屈侧,用手掌轻压使之均匀地贴敷于皮肤上,持续24h。
4)结果分析去除斑试器后30min、24h和48h按表4观察记录反应结果。
得到的结果如表5所示。
表4皮肤封闭斑贴试验皮肤反应分级标准
表5斑贴试验结果统计(总例数:30)
分级 | 透明质酸寡糖组合物 | 纯化水 |
0级 | 30 | 30 |
1级 | 0 | 0 |
2级 | 0 | 0 |
3级 | 0 | 0 |
4级 | 0 | 0 |
结果显示,质量浓度为1%的透明质酸寡糖组合物的斑贴试验均没有不良反应,表明透明质酸寡糖组合物对人体皮肤无潜在的不良反应。
试验例4寡糖组合物功效活性评价-对内源性透明质酸酶及透明质酸的影响
细胞的透明质酸合成酶1(Hylauronic acid synthase I,HAS1)、透明质酸合成酶3(Hylauronic acid synthase III,HAS3)基因表达量和透明质酸(Hylauronic acid,HA)分泌量,评价待测样品的保湿功效。具体方法为:
细胞接种:按2.2×10
5个/孔的接种密度接种成纤维细胞至6孔板,培养箱(37℃、5%CO
2)中孵育过夜。当6孔板中细胞铺板率达到50%~60%时,进行分组给药,给药浓度分别为0.01mg/ml、1mg/ml和10mg/ml,每组设3个复孔。给药完成后将6孔板放置在培养箱(37℃、5%CO
2)中孵育培养24h。ELISA检测:根据Hyaluronan Quantikine ELISA Kit的操作说明书进行检测。 基因检测:2mL/孔PBS清洗两次,每孔加入1mL RNAiso Plus,吹打裂解细胞后,收样。依据试剂盒说明书,开展RNA提取、反转录及荧光定量PCR检测,采用2-
△△CT方法进行结果计算。
空白对照指的是不加入样品,其余同试验组同步进行。透明质酸合成酶的促进效果和内源性透明质酸的促进效果分别如表6和表7所示。
表6透明质酸合成酶的促进效果
样品名称 | HAS1 | HAS3 |
空白对照 | 1.01 | 1.00 |
透明质酸寡糖组合物(0.01mg/ml) | 1.06 | 1.15 |
透明质酸寡糖组合物(1mg/ml) | 3.15 | 6.15 |
透明质酸寡糖组合物(10mg/ml) | 14.44 | 25.22 |
备注:采用2-
△△CT方法进行结果计算,用t-test方法进行统计分析时,将空白对照组mRNA扩增倍数归一处理。
表7内源性透明质酸的促进效果
结果表明,本发明制备的透明质酸寡糖组合物能显著提升成纤维细胞中透明质酸合酶HAS1、HAS3,尤其是HAS3的生成。透明质酸合酶的增加,有助于提升成纤维细胞中内源性透明质酸的合成。通过对成纤维细胞中内源性透明质酸的检测表明,其确实得到了增加,与空白组相比显著增加。人体皮肤中的真皮层含有丰富的成纤维细胞,而透明质酸寡糖组合物分子量非常小,这表明,透明质酸寡糖组合物具有增加人体皮肤真皮层中内源性透明质酸的潜在功效。
试验例5透明质酸寡糖组合物功效活性评价-对神经酰胺表达量的影响
基于3D表皮皮肤模型(购自广东博溪生物科技有限公司),测试实施例2给培养液里面添加透明质酸寡糖组合物样品(分别为0.01mg/ml、1mg/ml和10mg/ml)连续作用6天后,通过皮肤模型中神经酰胺总含量除以水溶性总蛋白来归一化后,以评估样品对神经酰胺的促合成能力。
1.水溶性总蛋白提取:将每组的三个模型分别剪为两半,其中1/2片置于一个1.5mL EP管中,并加入250μL的0.2mg/mL蛋白酶K溶液,50℃水浴2h。水浴结束后用镊子夹取角质层于去离子水中漂洗,将角质层置于新的EP管中。在每个管中加125μL蛋白提取液;超声震荡30min。超声震荡结束后,4℃,14000rpm离心10min。离心结束后,吸取提蛋白管中上清液至新的EP管中,采用BCA试剂盒检测浓度。
2.使用BCA蛋白浓度测定试剂盒对蛋白浓度进行检测,反应完毕后,于酶标仪562nm处读数。
脂质提取液配制:1.量取甲醇,氯仿各50mL,混匀;2.神经酰胺提取:将提取完蛋白剩余的1/2模型放入装有脂质提取液的EP管中,冰水浴超声震荡2min后,过渡到甲醇:氯仿=1:2的体系,再次在冰水浴中超声2min;结束后,过渡到甲醇:氯仿=2:1的体系,冰浴超声两分钟;结束后,夹出模型,提取液氮气吹干。
3.在氮吹干的样品瓶中加乙腈:异丙醇=1:1的混合溶液(含神经酰胺内标),超声10min,震摇溶解30min,转移到离心管中,12000rpm离心10min,取上层清液,待测。
空白对照指的是不加入样品,其余同试验组同步进行。
结果如表8、表9和表10所示。
表8水溶性总蛋白含量测定结果
样品名称 | 蛋白总含量(μg) | 下降率(%) |
空白对照 | 193.17 | —— |
透明质酸寡糖组合物(0.01mg/ml) | 188.3 | 2.5 |
透明质酸寡糖组合物(1mg/ml) | 152.15 | 21.2 |
透明质酸寡糖组合物(10mg/ml) | 132.34 | 31.5 |
表8的结果表明,与对照组相比,使用过透明质酸寡糖组合物的表皮模型中水溶性蛋白含量显著降低,该结果推测可能是因为透明质酸寡糖组合物能够促进皮肤模型分化,使活细胞层向角质层推进,使角质层增厚,表现出活细胞层可溶性总蛋白含量下降,从而反映出样品寡糖组合物能够提升表皮模型屏障。
表9神经酰胺总含量
样品名称 | 神经酰胺总含量(μg) | 提升率(%) |
空白对照 | 15.398 | —— |
透明质酸寡糖组合物(0.01mg/ml) | 15.732 | 2.2 |
透明质酸寡糖组合物(1mg/ml) | 22.531 | 46.3 |
透明质酸寡糖组合物(10mg/ml) | 26.562 | 72.5 |
表9的结果表明,与对照组相比,使用过透明质酸寡糖组合物的表皮模型中神经酰胺的总含量显著提升,该结果说明了寡糖组合物能够提升表皮模型中的神经酰胺含量。
表10神经酰胺总含量与蛋白总含量比值
神经酰胺总含量/蛋白总含量的计算可以将皮肤中的神经酰胺进行归一化,能更科学地反应皮肤中神经酰胺的变化。如表10所示,归一化后的结果表明,加入寡糖组合物后,皮肤中神经酰胺含量得到显著提升,表明透明质酸寡糖组合物可显著促进神经酰胺的合成。
试验例6
称取实施例2和对比例2的透明质酸寡糖组合物各10mg,分散于10mL的DMEM培养基中,0.22μm滤膜除菌过滤,分别得到质量浓度为0.1%的样品1和样品2。
试验例6-1胶原蛋白含量测定
胶原蛋白决定着包括皮肤在内的人体组织的物理特性。真皮中的胶原蛋白主要由成纤维细胞分泌。本发明通过评估透明质酸组合物对成纤维细胞分泌表达胶原蛋白的能力的影响来评估其对促进胶原蛋白生成的影响。
1、样品准备
取20μL样品1和样品2分别加入1.98mL的10%FBS-DMEM培养基中,制成终质量浓度为0.001%的溶液得样品1-1、2-1。
取样品1加入10%FBS-DMEM培养基中,制成终质量浓度分别为0.001%、0.01%、0.0001%的溶液得样品1-1、1-2、1-3。
2、人成纤维细胞胶原Ⅰ分泌实验
将人成纤维细胞以1万/孔的密度铺于96孔板中,置于培养箱中培养24h。弃上清,将样品1、1-1、1-2、1-3、2-1加入孔板中,每孔200μL。置于培养箱中孵育48h。收集上清,将上清用DMEM培养基稀释十倍后,使用Human Col Ⅰ Elisa Kit对上清中的胶原蛋白Ⅰ进行定量。结果如图11和图12所示。其中,对照组为不加入任何样品的试验组。
3、结果显示
如图11所示,虽然样品1-1和2-1均有一定的促进胶原生成的作用,但是小分子量的样品1-1促胶原蛋白生成效果更为显著。其中样品1-1使得胶原蛋白I的分泌量提高33%,而样品2-1促胶原蛋白I的分泌量与对照组相比无显著性差异。
如图12所示,样品1、样品1-1、样品1-2、1-3均有显著的促进胶原生成的作用,说明实施例2的透明质酸寡糖组合物在质量浓度为0.0001%-0.1%时均有促进成纤维细胞分泌胶原的作用。尤其是当质量浓度为0.001%时效果最佳。
试验例6-2 3D皮肤模型
本发明通过评估透明质酸组合物对3D全层皮肤模型的组织结构影响来评 估其对表皮细胞增殖与分化的作用。
3D全层皮肤模型是利用组织工程技术,采用适当的培养基将正常人皮肤细胞在体外重建而成的类似于人体皮肤结构的活性组织。通过评估3D皮肤模型的组织结构可以判断样品在抗衰老方面的作用。基底层为真皮-表皮之间的物质交换屏障,基底层的结构越稳定,表示表皮层可吸收的养分越多,则表皮细胞能更好地增殖分化,从而达到抗衰老的作用。
1、3D全层皮肤模型的构建
通过将胶原与人真皮成纤维细胞组合构建成3D全层皮肤模型的真皮层,再于真皮层上接种人角质形成细胞形成表皮层,得到完整的具有真皮-表皮结构的皮肤模型。再分别加入样品1、样品2对其进行处理。
2、3D全层皮肤模型石蜡组织切片
将组织放入包埋盒中,分别放入4%多聚甲醛、70%乙醇-水溶液、80%乙醇-水溶液、90%乙醇-水溶液、95%乙醇-水溶液、无水乙醇Ⅰ、无水乙醇Ⅱ、甲基环己烷Ⅰ、甲基环己烷Ⅱ中进行梯度脱水,每缸溶液1h。脱水完毕,将组织放入石蜡中包埋,修片,切成厚度为5μm的石蜡切片。
3、3D全层皮肤模型苏木精-伊红染色
将石蜡切片分别放入甲基环己烷Ⅰ、甲基环己烷Ⅱ、无水乙醇、95%乙醇-水溶液、80%乙醇-水溶液、70%乙醇-水溶液、蒸馏水中浸泡10min进行脱蜡、复水后。放入苏木精染液中染色8min后,依次放入盐酸-乙醇溶液中分化2s、氨水溶液中复蓝1min。接着用伊红溶液染色1min,最后放入70%乙醇-水溶液、95%乙醇-水溶液、无水乙醇、甲基环己烷溶液中脱水,中性树胶封片。显微镜下拍照。结果见图13。其中,对照组为不加入任何样品的试验组。
4、结果显示
如图13所示,与对照组和样品2相比,在加入样品1后,3D全层皮肤模型基底层细胞连接紧密,表皮层分化良好、结构完整,棘层和颗粒层结构清晰。说明样品1具有良好的抗衰老作用。
试验例7透明质酸寡糖组合物透皮验证
1.样品荧光标记
取实施例2的透明质酸寡糖组合物2g分散于40mL的水中配制成浓度为 50mg/mL的溶液,加入20mLDMSO溶液混匀。向上述60mL溶液中加入500μL的50mg/mL 5-羟基荧光素溶液,25μL的环己基异腈,100μL的25%乙醛溶液,在室温下搅拌反应5小时。移取40mL荧光标记过的溶液,取饱和氯化钠的冰乙醇溶液上层清液560mL加入,使之产生荧光标记的透明质酸沉淀。将上述溶液转移至离心管中,置于离心机以4000*g的离心力离心10分钟,弃去上层液体,取下荧光素标记的实施例2。
2.荧光标记物透皮
取15mg荧光标记过的实施例2溶解于1mL的水中,制成终质量浓度为1.5%的样品4。以15mL的1×PBS为接收液,25mm直径猪皮装载在手动透皮仪上。接收液在(32.0±0.1)℃水浴加热,在定速磁力搅拌(300±5)r/min条件下平衡1小时。在皮肤上层加入500μL的样品4,加盖。经20h后结束,取下猪皮晾干。
3.猪皮冰冻切片
取晾干的猪皮,置于OCT中于液氮中迅速冷却。置于冰冻切片机中切成厚度为10μM的切片,于荧光显微镜下观察。
4.结果显示
如图14所示,样品4在表皮层和真皮层都有荧光亮度,这说明样品4能很好的渗透表皮层和真皮层。以上结果说明实施例2可渗透并停留在真皮和表皮发挥作用。
Claims (20)
- 根据权利要求1所述的透明质酸寡糖组合物,其特征在于,在所述透明质酸寡糖组合物中,n=1的透明质酸寡糖的质量占比为35-70%;n=0的透明质酸寡糖的质量占比为5-40%;n=2的透明质酸寡糖的质量占比为1-50%;n=3的透明质酸寡糖的质量占比为1-15%;n=4的透明质酸寡糖的质量占比为1-10%;n=5的透明质酸寡糖的质量占比为0.01-1.5%。
- 根据权利要求1所述的透明质酸寡糖组合物,其特征在于,在所述透明质酸寡糖组合物中,n=1的透明质酸寡糖的质量占比为40-60%;n=0的透明质酸寡糖的质量占比为5-15%;n=2的透明质酸寡糖的质量占比为20-40%;n=3的透明质酸寡糖的质量占比为1-10%;n=4的透明质酸寡糖的质量占比为1-5%;n=5的透明质酸寡糖的质量占比为0.01-1.5%。
- 根据权利要求1所述的透明质酸寡糖组合物,其特征在于,在所述透明质酸寡糖组合物中,n=1的透明质酸寡糖的质量占比为35-70%;n=0的透明质酸寡糖的质量占比为5-40%;n=2的透明质酸寡糖的质量占比为10-50%;n=3的透明质酸寡糖的质量占比为1-15%;n=4的透明质酸寡糖的质量占比为0.1-10%;n=5的透明质酸寡糖的质量占比为0.01-5%。
- 根据权利要求1所述的透明质酸寡糖组合物,其特征在于,在所述透明质酸寡糖组合物中,n=1的透明质酸寡糖的质量占比为40-60%;n=0的透明质酸寡糖的质量占比为5-20%;n=2的透明质酸寡糖的质量占比为20-40%;n=3的透明质酸寡糖的质量占比为3-8%;n=4的透明质酸寡糖的质量占比为1-5%;n=5的透明质酸寡糖的质量占比为0.01-1.5%。
- 根据权利要求1所述的透明质酸寡糖组合物,其特征在于,所述透明质酸寡糖组合物的重均分子量小于等于1kDa。
- 根据权利要求1-3、6任一项所述的透明质酸寡糖组合物,其特征在于,所述透明质酸寡糖组合物用于化妆品或保健品,优选在所述化妆品或保健品中的质量浓度为0.001-10%。
- 一种如权利要求1-7任一项所述的透明质酸寡糖组合物的制备方法,其特征在于,所述制备方法包括以下步骤:将透明质酸用透明质酸酶进行酶解反应,分离酶解反应物以得到产物原液,对所述产物原液进行浓缩以得到产物浓缩液;去除所述产物浓缩液中的杂质,干燥得到透明质酸寡糖组合物,其中,所述透明质酸酶为酶切β-1,3糖苷键的透明质酸酶,优选,所述透明质酸酶为利用毕赤酵母工程菌表达的水蛭透明质酸水解酶,进一步优选,所述分离为超滤;进一步优选,所述浓缩为纳滤;进一步优选,使用活性炭吸附来去除杂质。
- 根据权利要求8所述的方法,其特征在于,在所述酶解反应中,所述透明质酸的初始浓度为5-150g/L,优选,所述透明质酸的分子量为1000-2000kDa,进一步优选所述透明质酸酶的初始含量为1×10 4-3×10 5U/mL。
- 根据权利要求8所述的方法,其特征在于,超滤采用截留分子量为600-1000Da的超滤膜,纳滤采用截留分子量为100-300Da的纳滤膜。
- 权利要求1-3、6中任一项所述的透明质酸寡糖组合物在促进神经酰胺和/或透明质酸合成酶1(HAS1)和/或透明质酸合成酶3(HAS3)的合成中的用途。
- 权利要求4-6中任一项所述的透明质酸寡糖组合物在抗皮肤衰老中的用途。
- 根据权利要求12所述的用途,其特征在于,所述透明质酸寡糖组合物通过促进胶原蛋白生成和/或表皮细胞增殖与分化而抗皮肤衰老。
- 根据权利要求12所述的用途,其特征在于,所述透明质酸寡糖组合物施用于皮肤。
- 根据权利要求12所述的用途,其特征在于,所述施用于皮肤的方式为通过化妆品或医疗器械,所述透明质酸寡糖组合物在所述化妆品或医疗器械中的质量浓度为0.0001%-5%。
- 根据权利要求12所述的用途,其特征在于,所述透明质酸寡糖组合物在所述化妆品或医疗器械中的质量浓度为0.001%-1%。
- 权利要求4-6中任一项所述透明质酸寡糖组合物在促进胶原蛋白生成中的用途。
- 根据权利要求17所述的用途,其特征在于,所述透明质酸寡糖组合物用于制备促进胶原蛋白生成的保健品,或者用于制备保护、强化脏器的保健品,或者用于制备保护胃粘膜的保健品,或者用于制备补充钙质的保健品,或者用于制备关节润滑注射液。
- 根据权利要求18所述的用途,其特征在于,所述透明质酸寡糖组合物在所述保健品或注射液中的质量浓度为0.0001%-5%。
- 根据权利要求19所述的用途,其特征在于,所述透明质酸寡糖组合物在所述保健品或注射液中的质量浓度为0.001%-1%。
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