Application of succinyl glycan riclin in preparation of products for resisting skin aging

Abstract

The invention discloses an application of succinyl glycan riclin in preparation of a product for resisting skin aging. According to the present invention, the succinyl glycan riclin can significantly alleviate the D-gal induced skin cell aging symptom by adjusting the oxidative stress; the succinyl glycan riclin is safe and non-toxic, small in dosage and good in stability, and has a wide application prospect in preparation of anti-skin aging products.

Description

Technical Field

[0001] The present invention belongs to the technical field of anti-skin aging drugs and relates to the application of succinoglycan riclin in the preparation of anti-skin aging products. Background Art

[0002] As the main barrier for the human body to contact with the external environment, the skin is easily exposed to environments such as ultraviolet (UV) rays, pollutants, and tobacco smoke for a long time, and a large amount of free radicals are likely to be generated. These free radicals can attack lipids, proteins, and DNA in the skin, resulting in damage to cell structure and function. For example, free radicals can destroy collagen and elastic fibers in the skin, causing the skin to lose elasticity and firmness, thus forming wrinkles and sagging. In addition, free radicals can also induce inflammatory reactions, further exacerbating skin damage. Therefore, oxidative stress is considered to be one of the main causes of skin aging. Currently, the drugs used for anti-skin aging are mainly based on two mechanisms: promoting epidermal cell renewal to increase the synthesis of collagen, such as retinol, and neutralizing free radicals to reduce oxidative stress, such as vitamin E. However, taking these drugs will cause different adverse reactions. Taking retinol at too high a concentration will cause skin irritation, erythema, and peeling, and taking vitamin E may cause allergic reactions. Therefore, developing new anti-skin aging drugs and providing more feasible options for patients have important practical significance.

[0003] At present, some polysaccharides can be used to improve skin aging in mice through pathways such as immunomodulation and oxidative stress. Ganoderma lucidum is a well-known medicinal fungus, and its polysaccharide components are considered to have a wide range of biological activities, including anti-tumor, anti-inflammatory, and antioxidant. Ganoderma lucidum polysaccharide can improve the antioxidant capacity of the skin by reducing the content of malondialdehyde in the skin and increasing the activities of superoxide dismutase and glutathione peroxidase. However, due to the complex extraction and purification processes of Ganoderma lucidum polysaccharide, the production cost is high and the product quality is unstable. ([1] Hu S, Huang J, Pei S, et al. Ganoderma lucidum polysaccharide inhibits UVB-induced melanogenesis by antagonizing cAMP / PKA and ROS / MAPK signaling pathways. J Cell Physiol. 2019 May; 234(5): 7330 - 7340. doi: 10.1002 / jcp.27492. Epub 2018 Oct 26. PMID: 30362532.)

[0004] The inventors' previous research reported succinoglycan riclin and its preparation method, and pointed out that this succinoglycan has anti-tumor activity and anti-inflammatory activity ([2] Y. Yang, X. Sun, Y. Zhao, et al, Anti-tumor activity and immunogenicity of a succinoglycan riclin, Carbohydr. Polym. 2021 Mar 1; 255:117370). Patent application CN 111286466 A also discloses the preparation method and anti-inflammatory activity of this succinoglycan riclin. The inventors' previous research also reported that succinoglycan riclin has excellent moisturizing properties and can protect the skin from UVB damage by enhancing collagen production ([3] Lu W, Kong C, Cheng S, et al. Succinoglycan riclin relieves UVB-induced skin injury with anti-oxidant and anti-inflammatory properties. Int J Biol Macromol. 2023 Apr 30; 235:123717. doi: 10.1016 / j.ijbiomac.2023.123717. Epub 2023 Feb 17. PMID: 36806772). However, whether succinoglycan riclin has anti-skin aging treatment has not been reported yet. Summary of the Invention

[0005] The present invention provides the use of succinoglycan riclin in the preparation of anti-skin aging products.

[0006] The structural formula of the succinoglycan riclin described in the present invention is:

[0007]

[0008] R = OCCH 2 CH 2 COOH or H, where n = 1 to 2000.

[0009] The anti-skin aging product described in the present invention is a composition or a product.

[0010] The composition described in the present invention includes but is not limited to skin care products, pharmaceutical compositions, etc.

[0011] The products described in the present invention include but are not limited to medical dressings, medical devices, biomaterials, tissue engineering products, etc.

[0012] The dosage forms of the pharmaceutical composition of the present invention include, but are not limited to, topical preparations, such as topical lotions, creams, etc., oral preparations, such as oral liquids, capsules, etc., and injection preparations, such as injection solutions, powder for injection, etc.

[0013] The administration methods of the pharmaceutical composition of the present invention can be topical, oral or injection. When administered by injection, it can be through intravenous, intraperitoneal, subcutaneous or intramuscular injection routes.

[0014] The administration objects of the anti-skin aging product of the present invention can be any animals. These animals include humans and non-human animals, such as pets or livestock, etc.

[0015] The administration concentration and dosage of the anti-skin aging product of the present invention depend on the age, health and weight of the recipient, treatment frequency, administration route, etc.

[0016] In the specific embodiment of the present invention, in the anti-skin aging product, the administration concentration of succinoglycan riclin is 50 μg / ml to 200 μg / ml.

[0017] The pharmaceutical carriers used in the pharmaceutical composition of the present invention can be the pharmaceutical carriers commonly used in the art, such as isotonic physiological saline solution, isotonic glucose solution, or isotonic solution containing a buffer system, such as PBS buffer solution, etc.

[0018] In the specific embodiment of the present invention, the pharmaceutical carrier used is PBS solution.

[0019] Compared with the prior art, the present invention has the following remarkable advantages:

[0020] The present invention first discovers that succinoglycan riclin can treat D-gal-induced skin cell aging; in the animal skin aging model experiment, succinoglycan riclin realizes anti-skin aging treatment by adjusting oxidative stress. Succinoglycan riclin is safe, non-toxic, has a small dosage and good stability, and has broad application prospects in the preparation of anti-skin aging products. Description of the Drawings

[0021] Figure 1 It is a statistical chart of cell viability of the experimental group and the control group acting on senescent skin cells respectively.

[0022] Figure 2 It is a live / dead cell fluorescence image of succinoglycan riclin on senescent cells.

[0023] Figure 3 It is a diagram showing the effects of succinoglycan riclin on the CAT and SOD levels of senescent cells, where A is the index change diagram of CAT in cells and B is the index change diagram of SOD in cells.

[0024] Figure 4 It is a diagram showing the effect of succinoglycan riclin on the ROS level in senescent cells.

[0025] Figure 5 It is about the biochemical indexes of succinoglycan riclin in senescent mice. Among them, A is the diagram showing the index change of CAT in mice, B is the diagram showing the index change of SOD in mice, and C is the diagram showing the index change of SOD in mice.

[0026] Figure 6 It is a diagram showing the skin inflammation response indexes of succinoglycan riclin in senescent mice. Among them, A, B, and C are the diagrams showing the effects on the levels of TNF-α, IL-1β, and IL-6 respectively. Detailed implementation mode

[0027] Unless otherwise specified, the methods used in the following examples are the commonly used methods in this field. All raw materials used in the following examples are commercially available products unless otherwise specified. Unless otherwise defined, all technical and scientific terms used in the present invention have the meanings commonly understood by those of ordinary skill in the art to which the present invention belongs.

[0028] The succinoglycan riclin in the present invention is prepared by conventional methods known in the art. Specifically, it can be prepared according to the methods taught in R. Cheng, L. Wang, J. Li., et al., J. Appl. Microbiol., 127(6): 1716 - 1726. (2019) and Patent Application CN 111286466 A.

[0029] The present invention will be further described in detail below with reference to the examples and the drawings.

[0030] Example 1

[0031] Model study on riclin improving D - galactose (D - gal)-induced skin cell senescence

[0032] (1) Establishment of cell model

[0033] The human immortalized keratinocyte (HACAT) cell line was purchased from the Cell Resource Center of the Chinese Academy of Sciences (Shanghai, China). HACAT cells were cultured in DMEM (high glucose) medium. The medium was supplemented with 10% heat-inactivated fetal bovine serum, 50 mM β - mercaptoethanol, and 1% penicillin / streptomycin. The cells were maintained in a humid environment (5% CO 2 ) and passaged every 2 - 3 days at 37°C. Cells in the normal control group (Ctrl group) were added with PBS, and cells in the experimental group were added with D - gal to induce cell senescence.

[0034] (2) Experimental grouping

[0035] One day after establishing the D-gal model, the experimental groups were divided into 4 groups: D-gal model group (Model), without adding any reagents; Low-riclin group (LR): adding 50 μg / mL riclin solution; Medium-riclin group (MR): adding 100 μg / mL riclin solution; High-riclin group (HR): adding 200 μg / mL riclin solution. The succinoglycan riclin solution was prepared by dissolving riclin in PBS at room temperature and sterilizing it by air filtration.

[0036] (3) MTT assay for cytotoxicity

[0037] One day after the experiment, an MTT cell proliferation and cytotoxicity detection kit was used to detect the in vitro cytotoxicity of the control group and the experimental groups.

[0038] (4) Live / dead cell fluorescence images

[0039] One day after the experiment, the cells were stained with Calcein AM / PI dye for 30 min, and then observed and photographed under a fluorescence microscope.

[0040] (5) Determination of antioxidant activity

[0041] One day after the experiment, the cells were disrupted by sonication. The levels of SOD and CAT in the cells were determined according to the kit procedure.

[0042] (6) Determination of ROS content

[0043] One day after the experiment, the cells cultured in the 6-well plate were added to 1 mL of DCFH-DA probe with a concentration of 10 M and incubated at 37 °C for 60 minutes. After washing twice with PBS, the 6-well plate was photographed under a fluorescence microscope.

[0044] (7) Result analysis

[0045] As Figure 1 shown, the experimental results showed that compared with the Model group, after treatment with riclin, the cytotoxicity was improved and the cell viability increased.

[0046] As Figure 2 shown, the experimental results showed that compared with the Model group, after treatment with riclin, the cell survival was higher than that of the Model group.

[0047] As Figure 3 shown, the experimental results showed that compared with the Model group, after treatment with riclin, the levels of CAT and SOD in the cells were increased.

[0048] As Figure 4 shown, the experimental results indicate that compared with the Model group, after treatment with riclin, the ROS value in cells was significantly reduced. These above results suggest that riclin treatment improved the relevant characteristics of cells in the D-gal aging model, and was non-toxic and harmless, preventing cell aging by adjusting cellular oxidative stress.

[0049] Experimental Example 2

[0050] Model study on riclin improving D-gal-induced animal skin aging

[0051] (1) Establishment of animal model

[0052] The experimental animals were female C57BL / 6 mice, 6 - 8 weeks old, weighing 18 - 20 g. They were housed under standard experimental conditions with a 12-hour light - 12-hour dark cycle and free access to water and food. The mice were randomly divided into 4 groups. Three of the groups were subcutaneously injected with D-gal solution (dissolving D-gal in 0.1 mL PBS solution at 1000 mg / kg) daily for 8 weeks. All treatment groups were administered once a day by gavage starting from the 3rd week for 6 weeks. The remaining group was subcutaneously injected with 0.1 mL PBS solution for 8 weeks as the negative control group, and was gavaged with PBS solution daily starting from the 3rd week for 6 weeks.

[0053] (2) Experimental grouping

[0054] Through drug administration treatment, the mice were randomly divided into 4 groups: The first group was the negative control group (Ctrl), which was gavaged with PBS solution daily starting from the 3rd week for 6 weeks; the second group was the model group (Model), without any drug administration treatment; the third group was the Low-riclin group (LR), which was gavaged with 50 μg / mL riclin solution daily starting from the 3rd week for 6 weeks; the fourth group was the High-riclin group (HR): which was gavaged with 100 μg / mL riclin solution daily starting from the 3rd week for 6 weeks. The succinoglycan riclin solution was prepared by dissolving riclin in PBS at room temperature and sterilizing it using air filtration.

[0055] (3) Biochemical analysis

[0056] After sacrificing the mice, the dorsal skin tissue samples were defatted, weighed and then cut into pieces. For every 0.5 g of wet skin sample, 4.5 mL of pre-cooled physiological saline was added to prepare a 10% homogenate. Then the homogenate was centrifuged at 4000 g for 10 minutes at 4℃, and the supernatant was collected to detect the redox state related to aging. The levels of SOD, CAT and ROS in cells were measured according to the kit procedures.

[0057] (4) Determination of skin inflammatory factors

[0058] After sacrificing the mice, TNF-α, IL-1β, and IL-6 in the skin of different mice were measured.

[0059] (5) Result analysis

[0060] As Figure 5 shown, the experimental results showed that compared with the Model group, after treatment with riclin, the originally decreased CAT and SOD values in the mice increased, and the originally increased ROS value decreased.

[0061] As Figure 6 shown, the experimental results showed that compared with the Model group, after treatment with riclin, the elevated TNF-α, IL-1β, and IL-6 index levels in the mice all decreased, and the inflammatory response in the mice was controlled.

Claims

1. The use of succinoglycan riclin in the preparation of anti-skin aging products, characterized in that: The structural formula of the succinoglycan riclin is: , R = OCCH2CH2COOH or H, where n = 1~2000.

2. The use according to claim 1, characterized in that: Anti-aging skin products are compositions or preparations.

3. The use according to claim 2, characterized in that: The composition is a skin care product or a pharmaceutical composition.

4. The use according to claim 2, characterized in that: The products are medical dressings, medical devices, biomaterials or tissue engineering products.

5. The use according to claim 3, characterized in that: The dosage form of the pharmaceutical composition is a topical preparation, an oral preparation or an injection preparation.

6. The use according to claim 5, characterized in that: Topical preparations are topical ointments or creams, oral preparations are oral liquids or capsules, and injectable preparations are injectable solutions or powder injections.

7. The use according to claim 3, characterized in that: The pharmaceutical composition can be administered topically, orally or by injection. When administered by injection, it can be administered by intravenous, intraperitoneal, subcutaneous or intramuscular injection.

8. The use according to claim 1, characterized in that: The anti-aging skin products are administered to humans or non-human animals.

9. The use according to claim 1, characterized in that: In anti-aging products, the application concentration of succinoglycan riclin is 50 μg / ml~200 μg / ml.

10. The use according to claim 3, characterized in that: The pharmaceutical carrier used in the pharmaceutical composition is an isotonic physiological saline solution, an isotonic glucose solution, or an isotonic solution containing a buffer system.

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