Application of composition in preparation of anti-inflammatory product

Abstract

The invention discloses application of a composition in preparation of anti-inflammatory products, and relates to the technical field of biological medicines and cosmetics. The invention specifically discloses an application of a composition containing benzylidene dimethoxy dimethyl indanone and phenylethyl resorcinol in preparation of an anti-inflammatory product. The benzylidene dimethoxy dimethyl indanone and the phenylethyl resorcinol have a synergistic effect in the aspect of inhibiting expression of inflammatory factors and COX-2, especially have a remarkable regulation and improvement effect on inflammatory reaction caused by UVB, and have the potential of being applied to relieving anti-inflammatory products (especially after-sun repair products).

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and cosmetics, and in particular to the application of a composition in the preparation of anti-inflammatory products. Background Technology

[0002] Ultraviolet radiation can be classified into three categories based on wavelength. Medium-wave ultraviolet (UVB) radiation, with wavelengths between 290-320 nm, can cause skin wrinkles, sagging, pigmentation, and inflammatory responses, making it a common cause of photodamage and a major contributing factor to skin cancer. Studies have shown that UVB can induce the secretion of various cytokines in keratinocytes, including tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-8 (IL-8), which are closely related to inflammatory activity. In the process of UVB damaging keratinocytes, COX-2 plays a crucial role in the occurrence and development of inflammation. COX-2 is a subtype of cyclooxygenase and a key rate-limiting enzyme in the breakdown of arachidonic acid to generate various endogenous prostaglandins (PGs). Under normal circumstances, various tissue cells hardly express COX-2. When the body is stimulated by UVB, COX-2 levels increase dramatically at a rate of 8-10 times, causing the synthesis and accumulation of PGs at the damaged sites. PGs can synergistically promote the increase of vascular permeability and chemotaxis of other inflammatory mediators, causing lesions such as inflammation, tissue destruction, and proliferation in surrounding tissues, thus promoting inflammatory responses and tissue damage. Therefore, it is very important to provide a composition that can effectively improve inflammatory responses. Summary of the Invention

[0003] The present invention aims to at least solve one of the technical problems existing in the prior art. To this end, the present invention proposes the use of a composition in the preparation of anti-inflammatory products.

[0004] The use of a composition according to a first aspect of the present invention in the preparation of an anti-inflammatory product, said composition comprising benzenemethylenedimethoxydimethylindanone and phenylethyl resorcinol.

[0005] According to some embodiments of the present invention, the mass ratio of benzenemethylenedimethoxydimethylindanone to phenylethyl resorcinol is 1:(0.1-5). For example, it can be 1:0.1, 1:0.2, 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5 or 1:5.

[0006] According to some embodiments of the present invention, the mass ratio of benzenemethylenedimethoxydimethylindanone to phenylethyl resorcinol is 1:(4-5).

[0007] According to some embodiments of the present invention, the mass ratio of benzenemethylenedimethoxydimethylindanone to phenylethyl resorcinol is 1:(0.8-1.2).

[0008] According to some embodiments of the present invention, the amount of benzenemethylenedimethoxydimethylindanone used is 0.5 μg / mL to 7 μg / mL; the amount of phenylethyl resorcinol used is 0.1 μg / mL to 25 μg / mL. For example, the dosage of the benzenemethylenedimethoxydimethylindanone can be 0.5 μg / mL, 0.8 μg / mL, 1 μg / mL, 1.5 μg / mL, 2 μg / mL, 2.5 μg / mL, 3 μg / mL, 3.5 μg / mL, 4 μg / mL, 4.5 μg / mL, 5 μg / mL, 5.5 μg / mL, 6 μg / mL, 6.5 μg / mL, or 7 μg / mL; the dosage of the phenylethyl resorcinol can be 0.1 μg / mL, 0.2 μg / mL, 0.3 μg / mL, 0.4 μg / mL, 0.5 μg / mL, 0.6 μg / mL, 0.7 μg / mL, 0.8 μg / mL, 0.9 μg / mL, 1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL, 5 μg / mL, 6 μg / mL, or 7 μg / mL. μg / mL, 8 μg / mL, 9 μg / mL, 10 μg / mL, 11 μg / mL, 12 μg / mL, 13 μg / mL, 14 μg / mL, 15 μg / mL, 16 μg / mL, 17 μg / mL, 18 μg / mL, 19 μg / mL, 20 μg / mL, 21 μg / mL, 22 μg / mL, 23 μg / mL, 24 μg / mL or 25 μg / mL.

[0009] According to some embodiments of the present invention, the amount of benzenemethylenedimethoxydimethylindanone used is 0.5 μg / mL to 6 μg / mL; the amount of phenylethyl resorcinol used is 0.1 μg / mL to 25 μg / mL.

[0010] According to some embodiments of the present invention, the amount of benzenemethylenedimethoxydimethylindanone used is 4 μg / mL-6 μg / mL; the amount of phenylethyl resorcinol used is 4 μg / mL-4.5 μg / mL.

[0011] According to some embodiments of the present invention, the amount of benzenemethylenedimethoxydimethylindanone used is 0.8 μg / mL-1.2 μg / mL; the amount of phenylethyl resorcinol used is 0.1 μg / mL-0.3 μg / mL.

[0012] According to some embodiments of the present invention, the product is a pharmaceutical, cosmetic, or reagent kit.

[0013] According to some embodiments of the present invention, the anti-inflammatory product is used to improve the upregulation of inflammatory cytokine gene expression levels and / or COX-2 gene expression levels resulting from an inflammatory response. The inflammatory response includes photodamage-induced inflammation. The photodamage-induced inflammation includes UVB exposure-induced inflammation.

[0014] According to some embodiments of the present invention, the inflammatory cytokines include at least one of IL-8 and IL-6.

[0015] According to some embodiments of the present invention, the anti-inflammatory product includes cosmetically or pharmaceutically acceptable excipients.

[0016] According to some embodiments of the present invention, the pharmaceutically acceptable excipients include at least one of diluents, wetting agents, binders, disintegrants, lubricants, flavoring agents, solvents, emulsifiers, antioxidants, preservatives, and pH adjusters.

[0017] According to some embodiments of the present invention, the cosmetic excipients acceptable to use include at least one of chelating agents, moisturizers, thickeners, emulsifiers, pH adjusters, antioxidants, skin conditioning agents, emollients, and preservatives.

[0018] According to some embodiments of the present invention, the anti-inflammatory product is a drug; the dosage form of the drug includes at least one of tablets, capsules, granules, pills, powders, elixirs, suspensions, solutions, and ointments.

[0019] According to some embodiments of the present invention, the anti-inflammatory product is a cosmetic; the cosmetic is a gel, lotion, essence, emulsion, cream or mask.

[0020] The present invention has at least the following beneficial effects: Benzyl dimethoxydimethyl indanone and phenylethyl resorcinol have a synergistic effect in inhibiting inflammatory factors and COX-2 expression, especially in regulating and improving the inflammatory response induced by UVB, and have the potential to be applied to soothing and anti-inflammatory products (especially after-sun repair products).

[0021] Other features and advantages of the invention will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the invention. Detailed Implementation

[0022] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0023] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0024] When a numerical range is disclosed herein, the range is considered continuous and includes the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe a feature or characteristic, the ranges may be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are incorporated.

[0025] Unless otherwise specified, "room temperature" in this invention means (25±5)℃.

[0026] Example 1 This example provides a composition obtained by mixing benzenemethylenedimethoxydimethylindanone and phenylethyl resorcinol; The mass ratio of benzenemethylenedimethoxydimethylindanone to phenylethyl resorcinol was 1:0.214 (corresponding to a molar ratio of 1 mg:1 μmol).

[0027] Example 2 This example provides a composition obtained by mixing benzenemethylenedimethoxydimethylindanone and phenylethyl resorcinol; The mass ratio of benzenemethylenedimethoxydimethylindanone to phenylethyl resorcinol was 1:2.143 (corresponding to a molar ratio of 1 mg:10 μmol).

[0028] Example 3 This example provides a composition obtained by mixing benzenemethylenedimethoxydimethylindanone and phenylethyl resorcinol; The mass ratio of benzenemethylenedimethoxydimethylindanone to phenylethyl resorcinol was 1:4.285 (corresponding to a molar ratio of 1 mg:20 μmol).

[0029] Comparative Example 1 This example provides a composition containing only benzenemethylenedimethoxydimethylindanone.

[0030] Comparative Example 2 This example provides a composition containing only phenylethyl resorcinol.

[0031] Comparative Example 3 This example provides a composition that differs from Example 1 only in that benzenemethylenedimethoxydimethylindanone is replaced with an equal mass of glucosylrutin.

[0032] Comparative Example 4 This example provides a composition that differs from Example 1 only in that phenylethyl resorcinol is replaced with an equimolar amount of 4-butylresorcinol.

[0033] Test case Keratinocytes are the most abundant cell type in the epidermis (comprising over 90%) and are the primary target of UVB radiation. When UVB damages keratinocytes, they release large amounts of inflammatory factors (such as IL-8 and IL-6), initiating inflammatory and immune responses in the skin. Furthermore, UVB-induced expression of COX-2 and PEG2 is closely related to the occurrence of cancer and inflammation. UVB can directly penetrate the epidermis and be absorbed by the DNA, proteins, and cell membranes of skin cells, especially keratinocytes, leading to DNA damage, the production of large amounts of reactive oxygen species, and triggering cellular stress and apoptosis. IL-8 is a key chemokine in the UVB-induced skin inflammation pathway. Driven by pathways such as NF-κB and MAPK, damaged keratinocytes synthesize and secrete large amounts of IL-8, thereby exacerbating skin inflammation. COX-2 plays a major role in the rate-limiting enzyme reaction of arachidonic acid to PGsd. In the process of UVB damage to keratinocytes, COX-2 plays an important role in the occurrence and development of inflammation. UVB exposure increases COX-2 expression through the MAPK or PI3K / Akt signaling pathway, leading to aggravated skin cell apoptosis and promoting the synthesis and accumulation of prostaglandins (PGs). PGs can synergistically promote the increase of other inflammatory mediators, increase vascular permeability, and promote inflammatory responses and tissue damage. By irradiating cells with UVB to induce a photodamage-induced inflammatory cell model, the expression of inflammatory factors and COX-2-related genes in keratinocytes can be measured to evaluate the soothing and anti-inflammatory efficacy of test substances.

[0034] (1) Using DMSO as a solvent, prepare 100 mM stock solutions of benzyl dimethoxydimethyl indanone and glucosyl rutin, respectively, and prepare 10 mg / mL stock solutions of phenylethyl resorcinol and 4-butyl resorcinol. Then, according to the concentration required by the examples and comparative examples, use complete culture medium (DMEM medium + 10 (v / v)% FBS + double antibiotics; the same below) as a solvent to prepare complete culture solutions containing different concentrations of drugs.

[0035] (2) Hacat cells were cultured in complete culture medium at 37°C in a carbon dioxide incubator with 5% CO2, and passaged every 2-3 days. Hacat cells in the logarithmic growth phase were collected to obtain a Hacat single-cell suspension, and the cell density was adjusted to 1×10⁻⁶ cells / cells. 5 Cells / mL. Take a standard 12-well plate, seed each well with 1 mL of cell suspension, and incubate at 37℃ and 5% CO2 for 24 h. Discard the old culture medium in the 12-well plate, and add 1 mL of complete culture medium containing different concentrations of drug to each well (sample group). Replace the blank group and model group with fresh complete culture medium. Each group has 3 replicates as parallel groups. After incubating at 37℃ and 5% CO2 for 24 h, replace the culture medium in each well with DPBS. The sample group and model group are irradiated with UVB at 8 mJ / cm². 2 (0.2 mV / cm) 2 (Irradiation intensity), then discard the DPBS, add the original culture medium back into the wells, and continue to incubate at 37°C and 5% CO2 for 24 hours. Discard the supernatant, wash the cells three times with DPBS, remove the DPBS, and leave the cells for subsequent experiments.

[0036] (3) RNA extraction: Add 500 μL of TRNzol Universal total RNA extraction buffer (Tiangen, DP424-100) to each well of a 12-well plate to lyse cells. Add chloroform (1:5 volume ratio of total RNA extraction buffer to the cell lysis buffer), shake thoroughly, and let stand at room temperature for 5 min. Centrifuge the extract at 12000 rpm for 15 min at 4℃. Transfer the colorless supernatant to a new 1.5 mL centrifuge tube, add an equal volume of isopropanol, mix well, and let stand at room temperature for 10 min. Centrifuge the mixture at 12000 rpm for 10 min at 4℃ and discard the supernatant. Add 500 µL of 75 (v / v)% ethanol aqueous solution, centrifuge the mixture at 7500 rpm for 5 min at 4℃, discard the supernatant, and air dry at room temperature for 1-2 min. Add an appropriate amount of DEPC water to fully dissolve the RNA precipitate.

[0037] (4) cDNA synthesis: The RNA extracted in step (2) was reverse transcribed into cDNA using a reverse transcription kit (HiScript® II Q RT SuperMix for qPCR(+gDNA wiper), purchased from Nanjing Novizan Biotechnology Co., Ltd.) according to the kit instructions.

[0038] (5) qRT-PCR detection: qRT-PCR detection was performed using a real-time fluorescence quantitative PCR kit (ChamQ™ Universal SYBR® qPCRMaster Mix, purchased from Nanjing Novizan Biotechnology Co., Ltd.). The detection primers are shown in Table 1. The mixture was prepared according to the system shown in Table 2, and the qRT-PCR reaction was performed. The reaction procedure is shown in Table 3. The average Ct values ​​of the internal reference gene and the target gene were taken to obtain the Ct value. 内参 and Ct 目的 Calculate ΔCt (ΔCt=Ct) 目的 -Ct 内参 ), ΔΔCt (ΔΔCt=ΔCt) 样品组 -ΔCt 对照组 ) and relative expression level (relative expression level = 2) (-ΔΔCt) Finally, the relative expression levels of each gene were normalized to obtain the gene expression downregulation rate.

[0039] Table 1

[0040] Table 2

[0041] Table 3

[0042] The results are shown in Table 4.

[0043] Table 4

[0044] The compositions of Examples 1-3 significantly downregulated the gene expression levels of inflammatory cytokines IL-8 and COX-2 in UVB-induced keratinocytes, and also downregulated the gene expression level of inflammatory cytokine IL-6. These effects were significantly superior to the compositions of Comparative Examples 1-4, and the differences were statistically significant. P <0.05). Low concentrations of phenylethyl resorcinol exhibit pro-inflammatory effects, while only high concentrations show some anti-inflammatory effects. Even when low concentrations of phenylethyl resorcinol are used in combination with benzylidene dimethoxydimethyl indanone, the anti-inflammatory effect of benzylidene dimethoxydimethyl indanone is significantly enhanced. This indicates that phenylethyl resorcinol and benzylidene dimethoxydimethyl indanone have a synergistic effect in soothing and reducing inflammation.

[0045] The embodiments of the present invention have been described in detail above with reference to the examples. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention.

Claims

1. The use of a composition in the preparation of an anti-inflammatory product, characterized in that, The composition includes benzenemethylenedimethoxydimethylindanone and phenylethyl resorcinol.

2. The application according to claim 1, characterized in that, The mass ratio of benzenemethylenedimethoxydimethylindanone to phenylethyl resorcinol is 1:(0.1-5).

3. The application according to claim 1, characterized in that, The mass ratio of benzyl dimethoxydimethyl indanone to phenylethyl resorcinol is 1: (4-5); preferably, the mass ratio of benzyl dimethoxydimethyl indanone to phenylethyl resorcinol is 1: (0.8-1.2).

4. The application according to claim 1, characterized in that, The amount of benzenemethylenedimethoxydimethylindanone used is 0.5 μg / mL to 7 μg / mL; the amount of phenylethyl resorcinol used is 0.1 μg / mL to 25 μg / mL.

5. The application according to claim 1, characterized in that, The amount of benzenemethylenedimethoxydimethylindanone used is 4 μg / mL to 6 μg / mL; the amount of phenylethyl resorcinol used is 4 μg / mL to 4.5 μg / mL.

6. The application according to claim 1, characterized in that, The anti-inflammatory product is used to improve the upregulation of inflammatory cytokine gene expression levels and / or COX-2 gene expression levels produced by the inflammatory response.

7. The application according to claim 1, characterized in that, The product is a drug, cosmetic, or reagent kit.

8. The application according to claim 7, characterized in that, The anti-inflammatory products include cosmetically or pharmaceutically acceptable excipients; Preferably, the pharmaceutically acceptable excipients include at least one of diluents, wetting agents, binders, disintegrants, lubricants, flavoring agents, solvents, emulsifiers, antioxidants, preservatives, and pH adjusters; Preferably, the cosmetic excipients acceptable to use include at least one of chelating agents, moisturizers, thickeners, emulsifiers, pH adjusters, antioxidants, skin conditioning agents, emollients, and preservatives.

9. The application according to claim 7, characterized in that, The anti-inflammatory product is a drug; the dosage form of the drug includes at least one of tablets, capsules, granules, pills, powders, elixirs, suspensions, solutions, and ointments.

10. The application according to claim 7, characterized in that, The anti-inflammatory product is a cosmetic; the cosmetic is a gel, lotion, essence, emulsion, cream, or mask.

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