CN115721579A - Composition for promoting long-acting skin moisturizing, application thereof and moisturizing product - Google Patents
Composition for promoting long-acting skin moisturizing, application thereof and moisturizing product Download PDFInfo
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Abstract
The present invention provides a composition for promoting long-lasting moisturization of skin, comprising: acetylated hyaluronic acid or its salt, wherein said acetylated hyaluronic acid or its salt accounts for greater than or equal to 30%, preferably greater than or equal to 40% of the mass ratio in the said composition. The composition contains hyaluronic acid and derivatives thereof with different molecular weights, and through reasonable compounding of a plurality of hyaluronic acids and derivatives thereof, the composition can keep skin moisture to play a physical moisturizing role and can also penetrate into the deep layer of the skin, so that epidermal cells can continuously secrete the hyaluronic acid by promoting the epidermal cells to up-regulate the expression of HAS2, HABP2 and AQP3, the capability and the water content of epidermal bound water are improved, and the biological moisturizing effect is achieved, thereby achieving the effects of long-acting moisturizing and barrier repair.
Description
Technical Field
The invention belongs to the field of material science, and particularly relates to a composition for promoting long-acting skin moisturizing, application thereof and a moisturizing product.
Background
Skin is a natural barrier between the body and the environment, protects the body from the environment by maintaining an effective epidermal barrier, not only can resist external factors, but also can prevent body water loss. Hydration levels not only affect the visible skin condition, such as softness of the skin, but also cellular metabolism within the epidermis, enzyme activity, signal transduction, etc., and thus adequate skin hydration is critical to maintaining skin health, and moisturization is an important component of basic skin care. The water content of the normal stratum corneum is 20-35%, and when the water content of the stratum corneum falls below 10%, the skin barrier function is impaired. Atopic dermatitis, psoriasis, acne, and dry skin are all associated with an impaired skin barrier.
The existing moisturizing compositions generally achieve the purpose of maintaining the skin or increasing the moisture content of the skin by physical moisturizing. The skin moisturizing cream is mainly added with substances with water absorption capacity to improve the moisture content of the skin, or a closed film is formed on the surface of the skin through high-molecular polymer components to slow down the moisture loss on the surface of the skin, and the hydration and the state of the whole skin cannot be changed fundamentally, so that the effect is maintained for a short time.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a composition for promoting long-acting moisture retention of skin and a preparation method thereof.
Specifically, the present invention relates to the following aspects:
1. a composition for promoting long-lasting moisturization of skin comprising:
acetylated hyaluronic acid or a salt thereof,
wherein the mass ratio of the acetylated hyaluronic acid or the salt thereof in the composition is greater than or equal to 30%, preferably greater than or equal to 40%, and more preferably 40% -60%.
2. The composition according to item 1, wherein the composition further comprises:
hydrolyzed hyaluronic acid or its salt,
One or two of low molecular weight hyaluronic acid or a salt thereof.
3. The composition according to item 2, wherein the hydrolyzed hyaluronic acid or the salt thereof is one or more selected from the group consisting of a first hydrolyzed hyaluronic acid or the salt thereof having a molecular weight of 1kDa or less, a second hydrolyzed hyaluronic acid or the salt thereof having a molecular weight of 1.1k to 5kDa, and a third hydrolyzed hyaluronic acid or the salt thereof having a molecular weight of 5.1k to 10kDa.
4. The composition of claim 3, wherein the hydrolyzed hyaluronic acid or salt thereof consists of the first hydrolyzed hyaluronic acid or salt thereof, the second hydrolyzed hyaluronic acid or salt thereof, and the third hydrolyzed hyaluronic acid or salt thereof.
5. The composition according to item 4, wherein the mass ratio of the first hydrolyzed hyaluronic acid or salt thereof is 0.01% to 17.5%, preferably 10% to 15%, the mass ratio of the second hydrolyzed hyaluronic acid or salt thereof is 0.01% to 17.5%, preferably 10% to 15%, and the mass ratio of the third hydrolyzed hyaluronic acid or salt thereof is 0.01% to 17.5%, preferably 10% to 15%.
6. The composition according to item 2, characterized in that it consists of the acetylated hyaluronic acid or salt thereof, the hydrolyzed hyaluronic acid or salt thereof, and the low molecular weight hyaluronic acid or salt thereof, preferably in a mass ratio of 0.03% to 52.5%, preferably 30% to 45%, of the hydrolyzed hyaluronic acid or salt thereof, and 0.01% to 17.5%, preferably 10% to 15%, of the low molecular weight hyaluronic acid or salt thereof in the composition.
7. The composition according to item 2, wherein the low molecular weight hyaluronic acid or salt thereof has a molecular weight of 200k-400kDa.
8. The composition according to item 1, wherein the acetylated hyaluronic acid or salt thereof has a molecular weight of 30k-100kDa.
9. The composition according to any one of claims 1 to 7, wherein the composition further comprises cross-linked hyaluronic acid or a salt thereof.
10. Use of a composition according to any of claims 1 to 9 in the preparation of a composition wherein the epidermal cells express HAS2, HABP2 and/or AQP 3.
11. Use of a composition according to any one of items 1 to 9 in the preparation of a moisturizing composition.
12. A moisturizing product comprising the composition of any one of items 1 to 9.
The composition contains hyaluronic acid and derivatives thereof with different molecular weights, and through reasonable compounding of a plurality of hyaluronic acids and derivatives thereof, the composition can keep skin moisture to play a physical moisturizing role and can also penetrate into the deep layer of the skin, so that epidermal cells can continuously secrete the hyaluronic acid by promoting the epidermal cells to up-regulate the expression of HAS2, HABP2 and AQP3, the capability and the water content of epidermal bound water are improved, and the biological moisturizing effect is achieved, thereby achieving the effects of long-acting moisturizing and barrier repair.
Drawings
Fig. 1 shows the structure of a 3D full-thickness skin model after using examples 1-2 and comparative examples 1-4.
FIG. 2 shows HAS2 expression using the 3D full-thickness skin model after examples 1-2, comparative example 1.
FIG. 3 shows the expression of HABP2 using the 3D full-thickness skin model after examples 1-2 and comparative example 1.
Figure 4 shows AQP3 expression in a 3D full-thickness skin model after use of examples 1-2, comparative example 1.
Detailed Description
The present invention is further illustrated by the following examples, which are intended to be purely exemplary and are not intended to limit the scope of the invention.
Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or experimental applications, the materials and methods are described below. In case of conflict, the present specification, including definitions, will control, and the materials, methods, and examples are illustrative only and not intended to be limiting. The present invention is further illustrated by the following examples, which are not intended to limit the scope of the invention.
The biological moisture retention is embodied by the following three points: 1. the increase in thickness of the stratum corneum and the integrity of the granular layer structure. A healthy and complete stratum corneum with a certain thickness and a well-differentiated stratum granulosum are the good embodiment of the barrier function and are the first defense line for preventing the water loss of the epidermis. 2. Content of glycosaminoglycan in epidermal extracellular matrix. Endogenous hyaluronic acid is the predominant glycosaminoglycan in the epidermal layer, which is the substance in the epidermal layer that binds primarily moisture. An increase in the content of endogenous hyaluronic acid may then laterally reflect an increase in the hydration capacity of the epidermal layer. Hyaluronan synthase 2 (HAS 2), a cell membrane surface secreted protein of keratinocytes and fibroblasts, secretes endogenously synthesized hyaluronic acid, while hyaluronan-binding protein 2 (HABP 2) reflects the endogenous hyaluronic acid content of the skin. 3. The ability of epidermal cells to transport water. Since the epidermal layer has no capillaries, the transport of water and nutrients is mainly dependent on some receptors on the cell membrane surface. Aquaporin 3 (AQP 3) is an aquaporin protein, a cell membrane surface receptor for the transport of water by epidermal cells. The increase of the expression quantity of AQP3 can increase the capacity of transporting and absorbing water of epidermal cells.
Through the three dimensions, the moisture loss of the epidermis layer can be prevented, the skin can continuously drink full moisture, and the drunk moisture is stored, so that the long-acting moisturizing effect is achieved.
Hyaluronic acid is a non-sulfated glycosaminoglycan and is a major component of the extracellular matrix. In human body, 50% of hyaluronic acid is present in the skin, and it constitutes the extracellular matrix of the skin together with collagen and chondroitin sulfate, and can play a role in retaining moisture, maintaining extracellular space and regulating osmotic pressure. Since hyaluronic acid itself contains a large amount of carboxyl groups and hydroxyl groups, it can bind a large amount of moisture to play a role in stably retaining water.
Hyaluronic acid in the skin is mainly synthesized by Hyaluronic Acid Synthase (HAS). Three HAS subtypes have been identified in mammals. HAS is involved in the synthesis of hyaluronic acid chains of different molecular weights inside the plasma membrane, while secreting the synthesized hyaluronic acid into the extracellular matrix. HAS1 and HAS2 were reported to synthesize hyaluronic acid of 20-2000kDa and HAS3 to synthesize hyaluronic acid of 100-1000 kDa. Fibroblasts and keratinocytes primarily utilize HAS2. The content of hyaluronic acid in the epidermis can be reflected by the human hyaluronic acid binding protein 2 (HABP2).
Aquaporins (AQPs) are proteins located on the cell membrane. They form channels in the cell membrane and can control the flow of water through the cell membrane like a "cell pump". To date, 11 types of AQP have been found in humans. Several references have demonstrated that AQP3 plays an important role in the restoration of skin barrier function, and that modulation of AQP3 expression can increase skin moisture levels and improve skin dryness, etc.
By adjusting the three target points, the epidermal cells can continuously secrete hyaluronic acid, the capability of combining water with the epidermis and the water content are improved, and the long-acting moisturizing effect is achieved.
In the prior art, hyaluronic acid has been reported to have a physical moisturizing function, and hyaluronic acid has also been reported to be capable of binding to four receptors in the skin: CD44, TLR2/TLR4, RHAMM and ICAM-1 receptor are combined to regulate the physiological activities and functions of keratinocytes and fibroblasts, but what hyaluronic acid or the combination thereof is used to promote the hyaluronic acid to promote the expression of cell moisturizing related proteins so as to essentially improve the moisturizing capability of epidermis and achieve the effect of biological long-acting moisturizing, and the research is less.
In order to achieve the effect of long-acting moisture retention of skin, the invention provides a composition for promoting long-acting moisture retention of skin, which comprises acetylated hyaluronic acid or a salt thereof, wherein the mass ratio of the acetylated hyaluronic acid or the salt thereof in the composition is greater than or equal to 30%, such as greater than or equal to 35%, such as 30%, 32%, 35%, 38%, 40%, 42%, 45%, 48%, 50%, 52%, 55%, 58%, 60%, 62%, 65%, 68%, 70%, 75%, 80%, preferably greater than or equal to 40%, and more preferably 40% -60%.
The composition may include only acetylated hyaluronic acid or a salt thereof, and may further include one or more of hydrolyzed hyaluronic acid or a salt thereof, low-molecular-weight hyaluronic acid or a salt thereof, and cross-linked hyaluronic acid or a salt thereof.
In a particular embodiment, the composition consists of acetylated hyaluronic acid or salt thereof.
In a particular embodiment, the composition consists of acetylated hyaluronic acid or salt thereof, and hydrolyzed hyaluronic acid or salt thereof.
In a particular embodiment, the composition consists of acetylated hyaluronic acid or salt thereof, and low molecular weight hyaluronic acid or salt thereof.
In a particular embodiment, the composition consists of acetylated hyaluronic acid or salt thereof, and cross-linked hyaluronic acid or salt thereof.
In a preferred embodiment, the composition consists of acetylated hyaluronic acid or salt thereof, hydrolyzed hyaluronic acid or salt thereof, and low molecular weight hyaluronic acid or salt thereof.
Further, in the composition, the mass ratio of the hydrolyzed hyaluronic acid or a salt thereof is 0.03% to 52.5%, and for example, may be 0.03%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 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%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 52.2%, and preferably 30% to 45%. The mass ratio of the low molecular weight hyaluronic acid or a salt thereof is 0.01% to 17.5%, and may be, for example, 0.01%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 17.5%, and preferably 10% to 15%.
In a particular embodiment, the composition comprises acetylated hyaluronic acid or salt thereof, hydrolyzed hyaluronic acid or salt thereof, low molecular weight hyaluronic acid or salt thereof, cross-linked hyaluronic acid or salt thereof.
Wherein, the Acetylated hyaluronic acid (AcHA) is obtained by acetylation reaction of sodium Hyaluronate. The introduction of acetyl brings lipophilicity to hyaluronic acid, enhances the affinity and adsorbability of hyaluronic acid to skin, and improves the moisturizing performance of hyaluronic acid.
In a specific embodiment, the acetylated hyaluronic acid or salt thereof has a molecular weight of 30k-100kDa. For example, the nucleic acid molecules may be 30kDa, 40kDa, 50kDa, 60kDa, 70kDa, 80kDa, 30kDa, 90kDa or 100kDa.
The hydrolyzed hyaluronic acid or the salt thereof is small molecular weight hyaluronic acid obtained by hydrolyzing hyaluronic acid or the salt thereof, and hydrolyzed hyaluronic acid with different molecular weights can be obtained by controlling hydrolysis conditions. The research finds that the physiological action of the hyaluronic acid is closely related to the molecular weight, and the hyaluronic acid with different molecular weights has different biological activities. The hyaluronic acid with small molecular weight has the functions of immunological activity, promoting the proliferation of vascular endothelial cells, reversing the multidrug resistance of tumor cells and the like. Meanwhile, hydrolyzed hyaluronic acid can permeate into the epidermis due to small molecular weight.
Further, the hydrolyzed hyaluronic acid or a salt thereof is one or more selected from the group consisting of a first hydrolyzed hyaluronic acid or a salt thereof having a molecular weight of 1kDa or less, a second hydrolyzed hyaluronic acid or a salt thereof having a molecular weight of 1.1k-5kDa, and a third hydrolyzed hyaluronic acid or a salt thereof having a molecular weight of 5.1k-10 kDa. That is, the hydrolyzed hyaluronic acid or salt thereof may be any one, any two, or three of a first hydrolyzed hyaluronic acid or salt thereof having a molecular weight of 1kDa or less, a second hydrolyzed hyaluronic acid or salt thereof having a molecular weight of 1.1k-5kDa, and a third hydrolyzed hyaluronic acid or salt thereof having a molecular weight of 5.1k-10 kDa. Wherein the first hydrolyzed hyaluronic acid or salt thereof has a molecular weight of 1kDa or less, 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. The molecular weight of the second hydrolyzed hyaluronic acid or its salt is 1.1k-5kDa, and may be, for example, 1.1kDa, 1.5kDa, 2kDa, 2.5kDa, 3kDa, 3.5kDa, 4kDa, 4.5kDa, 5kDa. The molecular weight of the third hydrolyzed hyaluronic acid or its salt is 5.1k-10kDa, and may be 5.1kDa, 5.5kDa, 6kDa, 6.5kDa, 7kDa, 7.5kDa, 8kDa, 8.5kDa, 9kDa, 9.5kDa, or 10kDa, for example.
In a specific embodiment, the hydrolyzed hyaluronic acid or salt thereof consists of the first hydrolyzed hyaluronic acid or salt thereof.
In a specific embodiment, the hydrolyzed hyaluronic acid or salt thereof consists of the second hydrolyzed hyaluronic acid or salt thereof.
In a specific embodiment, the hydrolyzed hyaluronic acid or salt thereof consists of the third hydrolyzed hyaluronic acid or salt thereof.
In a specific embodiment, the hydrolyzed hyaluronic acid or salt thereof consists of the first hydrolyzed hyaluronic acid or salt thereof, and the second hydrolyzed hyaluronic acid or salt thereof.
In a specific embodiment, the hydrolyzed hyaluronic acid or salt thereof consists of the first hydrolyzed hyaluronic acid or salt thereof, and the third hydrolyzed hyaluronic acid or salt thereof.
In a specific embodiment, the hydrolyzed hyaluronic acid or salt thereof consists of the second hydrolyzed hyaluronic acid or salt thereof, and the third hydrolyzed hyaluronic acid or salt thereof.
In a preferred embodiment, the hydrolyzed hyaluronic acid or salt thereof consists of the first hydrolyzed hyaluronic acid or salt thereof, the second hydrolyzed hyaluronic acid or salt thereof, and the third hydrolyzed hyaluronic acid or salt thereof.
In a particular embodiment, the first hydrolyzed hyaluronic acid or salt thereof is present in the composition in a mass ratio of 0.01% to 17.5%, for example, 0.01%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 17.5%, preferably 10% to 15%; the second hydrolyzed hyaluronic acid or a salt thereof may be 0.01% to 17.5% by mass, for example, 0.01%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 17.5%, preferably 10% to 15%; the mass ratio of the third hydrolyzed hyaluronic acid or a salt thereof is 0.01% to 17.5%, and may be, for example, 0.01%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 17.5%, and preferably 10% to 15%.
In a specific embodiment, the low molecular weight hyaluronic acid or salt thereof has a molecular weight of 200k-400kDa. For example, 200kDa, 210kDa, 220kDa, 230kDa, 240kDa, 250kDa, 260kDa, 270kDa, 280kDa, 290kDa, 300kDa, 310kDa, 320kDa, 330kDa, 340kDa, 350kDa, 360kDa, 370kDa, 380kDa, 390kDa, 400kDa.
In a specific embodiment, the composition consists of 12.5% of the first hydrolyzed hyaluronic acid or salt thereof, 12.5% of the second hydrolyzed hyaluronic acid or salt thereof, 12.5% of the third hydrolyzed hyaluronic acid or salt thereof, 50% of the acetylated hyaluronic acid or salt thereof, and 12.5% of the low molecular weight hyaluronic acid or salt thereof, by mass of the composition. Wherein the molecular weight of the first hydrolyzed hyaluronic acid or the salt thereof is 1kDa, the molecular weight of the first hydrolyzed hyaluronic acid or the salt thereof is 2.6kDa, the molecular weight of the third hydrolyzed hyaluronic acid or the salt thereof is 7kDa, the molecular weight of the acetylated hyaluronic acid or the salt thereof is 30kDa, and the molecular weight of the low molecular weight hyaluronic acid or the salt thereof is 373kDa.
In a specific embodiment, the composition consists of 15% of the first hydrolyzed hyaluronic acid or salt thereof, 15% of the second hydrolyzed hyaluronic acid or salt thereof, 15% of the third hydrolyzed hyaluronic acid or salt thereof, 40% of the acetylated hyaluronic acid or salt thereof, and 15% of the low molecular weight hyaluronic acid or salt thereof, by weight of the composition. Wherein the molecular weight of the first hydrolyzed hyaluronic acid or the salt thereof is 1kDa, the molecular weight of the first hydrolyzed hyaluronic acid or the salt thereof is 2.6kDa, the molecular weight of the third hydrolyzed hyaluronic acid or the salt thereof is 7kDa, the molecular weight of the acetylated hyaluronic acid or the salt thereof is 30kDa, and the molecular weight of the low molecular weight hyaluronic acid or the salt thereof is 373kDa.
In a specific embodiment, the composition consists of 10% of the first hydrolyzed hyaluronic acid or salt thereof, 10% of the second hydrolyzed hyaluronic acid or salt thereof, 10% of the third hydrolyzed hyaluronic acid or salt thereof, 60% of the acetylated hyaluronic acid or salt thereof, and 10% of the low molecular weight hyaluronic acid or salt thereof, by mass of the composition. Wherein the molecular weight of the first hydrolyzed hyaluronic acid or the salt thereof is 1kDa, the molecular weight of the first hydrolyzed hyaluronic acid or the salt thereof is 2kDa, the molecular weight of the third hydrolyzed hyaluronic acid or the salt thereof is 7kDa, the molecular weight of the acetylated hyaluronic acid or the salt thereof is 30kDa, and the molecular weight of the low molecular weight hyaluronic acid or the salt thereof is 373kDa.
In a specific embodiment, the composition consists of 12.5% of the first hydrolyzed hyaluronic acid or salt thereof, 12.5% of the second hydrolyzed hyaluronic acid or salt thereof, 12.5% of the third hydrolyzed hyaluronic acid or salt thereof, 50% of the acetylated hyaluronic acid or salt thereof, and 12.5% of the low molecular weight hyaluronic acid or salt thereof, by mass of the composition. Wherein the molecular weight of the first hydrolyzed hyaluronic acid or salt thereof is 900Da, the molecular weight of the first hydrolyzed hyaluronic acid or salt thereof is 3kDa, the molecular weight of the third hydrolyzed hyaluronic acid or salt thereof is 9kDa, the molecular weight of the acetylated hyaluronic acid or salt thereof is 50kDa, and the molecular weight of the low molecular weight hyaluronic acid or salt thereof is 250kDa.
The cross-linked hyaluronic acid in the invention is the hyaluronic acid with ultrahigh molecular weight formed by the cross-linking reaction of hyaluronic acid, is a compact three-dimensional net structure, uniformly covers the surface of the skin to form a breathable film, can enhance the barrier function of the skin, reduces the water evaporation in the skin, and forms a more stable protective film on the surface of the skin with more lasting effect.
The invention also provides a moisturizing product comprising the composition. The different types of hyaluronic acid described above in the composition of the moisturizing product can be added simultaneously or separately to the moisturizing product. Further, the moisturizing product can be supplemented with other ingredients according to different needs and applications.
The invention also provides the application of the composition in preparing a moisturizing composition.
The invention also provides the use of the above composition in the preparation of a composition for promoting expression of HAS2, HABP2 and/or AQP3 in epidermal cells.
The results of the influence of the composition on the tissue structure of a 3D full-thickness skin model show that the composition can obviously increase the thickness of the stratum corneum, reduce the water loss, improve the hydration of the epidermis and keep the skin in a healthy state; can promote epidermal cells to express HAS2, HABP2 and AQP3, further promote epidermal cells to generate and secrete hyaluronic acid, improve the epidermal hydration capability and achieve the effect of long-acting moisture preservation.
Examples
The experimental methods used in the following examples are all conventional methods unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available, unless otherwise specified, and hyaluronic acid used is commercially available from Huaxi Biotech Ltd.
Example 1
Hydrolyzed sodium hyaluronate (trade name: nissum 1000 (Hybloom) with molecular weight of 1kDa is weighed TM ) 12.5g of hydrolyzed sodium hyaluronate with a molecular weight of 2.6kDa (trade name: ximin TM Super-active hyaluronic acid) 12.5g, hydrolyzed sodium hyaluronate with a molecular weight of 7kDa (trade name: nano
) 12.5g of acetylated sodium hyaluronate with a molecular weight of 30kDa (trade name: hymagic TM -AcHA) 50g, low molecular sodium hyaluronate with molecular weight 373kDa (trade name: hybloom TM Low molecular sodium hyaluronate) 12.5g.
And (3) uniformly stirring the weighed components to obtain the hyaluronic acid composition.
Example 2
Sodium acetylated hyaluronate (trade name: hymagic) having a molecular weight of 30kDa was weighed TM -AcHA)100g。
Thus obtaining the hyaluronic acid composition.
Comparative example 1
The molecular weight was 1kDa (trade name: nissum 1000 (Hybloom) TM ) 50g of hydrolyzed sodium hyaluronate, a hydrolyzed sodium hyaluronate with a molecular weight of 2.6kDa (trade name: ximin TM Super-active hyaluronic acid) 12.5g, hydrolyzed sodium hyaluronate with a molecular weight of 7kDa (trade name: nano
) 12.5g of acetylated sodium hyaluronate with a molecular weight of 30kDa (trade name: hymagic TM -AcHA) 12.5g, low molecular sodium hyaluronate with molecular weight 373kDa (trade name: hybloom TM Low molecular sodium hyaluronate) 12.5g.
And (3) uniformly stirring the weighed components to obtain the hyaluronic acid composition.
Comparative example 2
The molecular weight is 1kDa (trade name: microtrue 1000 (Hybloom) TM ) 12.5g of hydrolyzed sodium hyaluronate, a molecular weight of 2.6kDa (trade name: ximin drug TM Super-active hyaluronic acid) 50g, hydrolyzed sodium hyaluronate with a molecular weight of 7kDa (trade name: nano
) 12.5g of acetylated sodium hyaluronate with a molecular weight of 30kDa (trade name: hymagic TM -AcHA) 12.5g, low molecular sodium hyaluronate with molecular weight 373kDa (trade name: hybloom TM Low molecular sodium hyaluronate) 12.5g.
And uniformly stirring the weighed components to obtain the hyaluronic acid composition.
Comparative example 3
The molecular weight is 1kDa (trade name: microtrue 1000 (Hybloom) TM ) 12.5g of hydrolyzed sodium hyaluronate, a molecular weight of 2.6kDa (trade name: ximin TM Super-active hyaluronic acidAcid) 12.5g, hydrolyzed sodium hyaluronate with a molecular weight of 7kDa (trade name: nano
) 50g of acetylated sodium hyaluronate with a molecular weight of 30kDa (trade name: hymagic TM -AcHA) 12.5g, low molecular sodium hyaluronate with molecular weight 373kDa (trade name: hybloom TM Low molecular sodium hyaluronate) 12.5g.
And uniformly stirring the weighed components to obtain the hyaluronic acid composition.
Comparative example 4
The molecular weight was 1kDa (trade name: nissum 1000 (Hybloom) TM ) 12.5g of hydrolyzed sodium hyaluronate, a molecular weight of 2.6kDa (trade name: ximin TM Super-active hyaluronic acid) 12.5g, hydrolyzed sodium hyaluronate with a molecular weight of 7kDa (trade name: nano
) 12.5g of acetylated sodium hyaluronate with a molecular weight of 30kDa (trade name: hymagic TM -AcHA) 12.5g, low molecular sodium hyaluronate with molecular weight 373kDa (trade name: hybloom TM Low molecular sodium hyaluronate) 50g.
And uniformly stirring the weighed components to obtain the hyaluronic acid composition.
The conditions of the above examples and comparative examples are shown in table 1.
TABLE 1 compositions and contents (g) of the various examples and comparative examples
Test examples
The 3D full-layer skin model is an active tissue which is similar to a human skin structure and is formed by reconstructing normal human skin cells in vitro by using a tissue engineering technology and adopting a proper culture medium, and can be used for screening active substances. The beneficial active substances for the skin can be primarily screened by exploring the influence of the active substances on the tissue structure of a skin model.
Test example 1
The efficacy of the composition was assessed by assessing its histological effect on a 3D full-thickness skin model.
1. Sample preparation
The compositions prepared in examples 1-2 and comparative examples 1-4 were dissolved in a certain amount of DMEM medium to obtain hyaluronic acid composition-medium solutions (samples 1-6) at a final concentration of 0.2%.
2. Construction of 3D full-layer skin model
The collagen and human dermal fibroblasts are combined to construct a dermis layer of a 3D full-layer skin model, and then the human keratinocytes are inoculated on the dermis layer to form an epidermis layer, so that the complete skin model with a dermis-epidermis structure is obtained. Samples 1-6 were then added and treated, with the control group being untreated.
3. 3D full-layer skin model paraffin tissue section
Placing the tissue into an embedding box, and respectively placing the tissue into 4% paraformaldehyde, 70% ethanol-water solution, 80% ethanol-water solution, 90% ethanol-water solution, 95% ethanol-water solution, absolute ethanol I, absolute ethanol II, methylcyclohexane I and methylcyclohexane II for gradient dehydration, wherein the solution is placed in each cylinder for 1h. After dehydration, the tissue was embedded in paraffin, trimmed and cut into paraffin sections of 5 μm thickness.
4. 3D full-thickness skin model hematoxylin-eosin staining
And (3) respectively putting the paraffin sections into methylcyclohexane I, methylcyclohexane II, absolute ethyl alcohol, 95% ethanol-water solution, 80% ethanol-water solution, 70% ethanol-water solution and distilled water, soaking for 10min, dewaxing and rehydrating. Placing into hematoxylin staining solution for staining for 8min, sequentially placing into hydrochloric acid-ethanol solution for differentiation for 2s, and rewetting in ammonia water solution for 1min. Dyeing with eosin solution for 1min, dewatering in 70% ethanol-water solution, 95% ethanol-water solution, anhydrous ethanol, and methylcyclohexane solution, and sealing with neutral gum. Take pictures under microscope. The results are shown in FIG. 1.
5. Results display
When the moisture content of the skin rises, the skin barrier is enhanced and the skin is in a healthy, active state. The individual structures in the skin also tend to a well-differentiated state. Therefore, the influence of the sample on the hydration state of the 3D full-thickness skin model can be judged by evaluating the thicknesses of the epidermis and the cuticle layer of the loaded 3D full-thickness skin model and the differentiation state of each structure.
By analyzing the structure of the 3D full-thickness skin model (shown in figure 1) after the sample is added, the embodiment, especially the embodiment 1, enables the 3D full-thickness skin model to be well differentiated, the stratum corneum, the granular layer and the acanthosphere to be well differentiated, the structure is clear, the epidermis thickness is increased, and the stratum corneum thickness is increased compared with the contrast. Meanwhile, the basal layer has a complete structure and cells are arranged closely. Examples, especially example 1, significantly increased stratum corneum thickness, reduced moisture loss, improved epidermal hydration, and skin health.
Test example 2
The efficacy of the compositions was assessed by assessing the amount of expression of HAS2, HABP2, AQP3 in a 3D full-thickness skin model.
1. Sample preparation
The compositions prepared in examples 1, 2 and comparative example 1 were dissolved in a certain amount of DMEM medium to obtain hyaluronic acid composition-medium solutions (samples 1, 2, 3) having a final concentration of 0.2%.
2. Construction of 3D full-layer skin model
The collagen and human dermal fibroblasts are combined to construct a dermis layer of a 3D full-layer skin model, and then the human keratinocytes are inoculated on the dermis layer to form an epidermis layer, so that the complete skin model with a dermis-epidermis structure is obtained. Samples 1, 2, and 3 were then added and treated, with the control group being untreated.
3. Frozen tissue section of 3D full-layer skin model
And (3) placing the 3D full-thickness skin model in an OCT tissue embedding medium, and adding liquid nitrogen to rapidly cool. The embedded tissue block was placed in a cryomicrotome and cut into sections of 5 μm thickness.
4. Immunofluorescence staining method for ice-cutting tissue section of 3D full-thickness skin model
The sections were fixed with ice methanol at-20 ℃ and washed three times with PBS and incubated with blocking solution at room temperature for 1h. After the incubation, the corresponding primary antibody is added, the incubation is carried out overnight at 4 ℃, the next day, the primary antibody is washed with PBS for three times, the secondary antibody is added, the incubation is carried out for 1.5 hours at the room temperature in a dark place, finally, the nuclear sealing piece is dyed with DAPI, and then, the picture is observed and taken under a fluorescence microscope. The results are shown in FIGS. 2, 3 and 4.
5. Results display
Next, the effects on long-lasting moisturization of skin were investigated by the expression of epidermal HAS2, HABP2 and AQP3 in comparative example 1, example 2 and comparative example 1. HAS2, HABP2 and AQP3 in FIGS. 2, 3 and 4 are the staining of the target protein, respectively. DAPI is nuclear staining, representing the cellular localization of 3D full-thickness skin model structures. Merge is the superposition of target protein staining and nuclear staining. In FIGS. 2, 3 and 4, HAS2 and AQP3 are shown to be expressed by epidermal cell membrane, and HABP2 is shown to be expressed by epidermal extracellular matrix, which is consistent with the localization reported in the literature.
Fig. 2 shows that the fluorescence expression of HAS2 in the epidermis of the example, especially in example 1, is brighter than that of HAS2 in the epidermis of the comparative example 1, and the fluorescence expression area in a single cell is larger and uniform, i.e. HAS2 expression is significant, which indicates that example 1 can significantly promote the synthesis and secretion of hyaluronic acid by human epidermal cells.
The expression of HABP2 in fig. 3 is also consistent with this result. Example 1 has a stronger expression of HABP2 than the horny layer in comparative example 1 and example 2.
Fig. 4 shows that AQP3 fluorescence expression is brighter in epidermal layer and cell AQP3 expression is strong in basal layer in example 1, especially in example 1, compared with that in comparative example 1, which shows that example 1 can remarkably promote human epidermal cell to express AQP3 and regulate epidermal hydration balance.
The above results show that example 1 can promote epidermal cells to express HAS2, HABP2 and AQP3, further promote epidermal cells to generate and secrete hyaluronic acid, improve epidermal hydration capacity and achieve long-acting moisturizing effect. As can be seen by comparing example 1 and example 2, the long-lasting moisturizing ability of acetylated sodium hyaluronate through compounding 4 other different sodium hyaluronate (example 1) is significantly greater than the total concentration of acetylated sodium hyaluronate (example 2) used.
Claims (10)
1. A composition for promoting long-lasting moisturization of skin, said composition comprising:
acetylated hyaluronic acid or a salt thereof,
wherein the mass ratio of the acetylated hyaluronic acid or the salt thereof in the composition is more than or equal to 30%, and preferably 40% -60%.
2. The composition as claimed in claim 1, further comprising:
hydrolyzed hyaluronic acid or its salt,
One or two of low molecular weight hyaluronic acid or salt thereof;
preferably, the hydrolyzed hyaluronic acid or salt thereof is one or more selected from the group consisting of a first hydrolyzed hyaluronic acid or salt thereof having a molecular weight of 1kDa or less, a second hydrolyzed hyaluronic acid or salt thereof having a molecular weight of 1.1k-5kDa, and a third hydrolyzed hyaluronic acid or salt thereof having a molecular weight of 5.1k-10 kDa.
3. The composition of claim 2, wherein the hydrolyzed hyaluronic acid or salt thereof consists of the first hydrolyzed hyaluronic acid or salt thereof, the second hydrolyzed hyaluronic acid or salt thereof, and the third hydrolyzed hyaluronic acid or salt thereof.
4. The composition according to claim 3, wherein the first hydrolyzed hyaluronic acid or salt thereof is present in a mass ratio of 0.01% to 17.5%, preferably 10% to 15%, the second hydrolyzed hyaluronic acid or salt thereof is present in a mass ratio of 0.01% to 17.5%, preferably 10% to 15%, and the third hydrolyzed hyaluronic acid or salt thereof is present in a mass ratio of 0.01% to 17.5%, preferably 10% to 15%.
5. The composition according to claim 2, characterized in that it consists of the acetylated hyaluronic acid or salt thereof, the hydrolyzed hyaluronic acid or salt thereof, and the low molecular weight hyaluronic acid or salt thereof, preferably in a mass ratio of 0.03% to 52.5%, preferably 30% to 45%, of the hydrolyzed hyaluronic acid or salt thereof, and 0.01% to 17.5%, preferably 10% to 15%, of the low molecular weight hyaluronic acid or salt thereof.
6. The composition of claim 2, wherein the low molecular weight hyaluronic acid or salt thereof has a molecular weight of 200k-400kDa.
7. The composition according to claim 1, wherein the acetylated hyaluronic acid or salt thereof has a molecular weight of 30k-100kDa.
8. The composition according to any one of claims 1 to 7, wherein the composition further comprises cross-linked hyaluronic acid or a salt thereof.
9. Use of a composition according to any one of claims 1 to 8 for the preparation of a composition for promoting expression of HAS2, HABP2 and/or AQP3 in epidermal cells.
10. A moisturizing product comprising the composition of any one of claims 1 to 8.
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