A composition with anti-inflammatory and firming anti-wrinkle effects and its use

Abstract

The application discloses a composition with anti-inflammatory and firming and anti-wrinkle effects, which is composed of benzaldehyde dimethyl indenone and ginsenoside, wherein the total saponin content of the ginsenoside is greater than or equal to 80%, and the content of ginsenoside C-Mx and CK in the total saponin content is greater than or equal to 95%. In the composition, the benzaldehyde dimethyl indenone and the ginsenoside have a synergistic effect, and the anti-UVB-induced skin inflammation and the anti-UVA-induced skin aging are simultaneously achieved, so that the dual effects of anti-inflammation and firming and anti-wrinkle are achieved. Compared with single components, the anti-inflammatory effect of the composition is stronger, and the improvement effect on the firming and anti-wrinkle related genes in the aging cells under UVA irradiation is better, and these results all reflect the excellent synergistic effect of the composition. In addition, the raw material composition preparation method is simple and easy to implement, the raw materials are easy to obtain, and large-scale production can be implemented.

Description

Technical Field

[0001] This invention belongs to the field of cosmetic technology, specifically relating to a composition that has both anti-inflammatory and firming / anti-wrinkle effects and its application. Background Technology

[0002] Photoaging of the skin is mainly induced by ultraviolet radiation, and is characterized by increased inflammation, collagen degradation and oxidative stress. In the long term, it can lead to changes in appearance such as skin laxity and wrinkles.

[0003] Ultraviolet (UV) radiation is divided into three bands: long-wave UVA (320-400 nm), medium-wave UVB (290-320 nm), and short-wave UVC (100-290 nm). Due to the blocking effect of the atmosphere, the UV components that can penetrate the ozone layer and cause skin damage are UVB and UVA. UVA radiation can induce the excessive production of reactive oxygen species (ROS), activate matrix metalloproteinases, and lead to collagen degradation, thereby causing skin aging phenomena such as wrinkles and sagging. UVB, on the other hand, has strong mutagenic and carcinogenic properties, can reach the superficial dermis, induce erythema and sunburn, and is associated with the occurrence of skin tumors, posing a potential threat to human health.

[0004] Currently, strategies for protecting against and repairing photoaging mostly focus on single-pathway interventions, such as using anti-inflammatory agents or antioxidants (e.g., vitamin C derivatives). However, single ingredients often struggle to comprehensively address the concurrent inflammation and structural damage associated with photoaging, and long-term use may lead to side effects or limited efficacy. Furthermore, existing technologies lack a combination of complex active ingredients capable of simultaneously and effectively inhibiting inflammatory pathways and enhancing the skin's own resistance to photodamage. It is necessary to overcome the limitations of current technologies by combining different compounds to develop a multi-functional composition. Therefore, developing a safe and stable composition with synergistic anti-inflammatory and anti-photoaging functions targeting multiple sites has become a pressing technical challenge in this field. Summary of the Invention

[0005] The present invention aims to solve the problems of limited target and insufficient synergy of single active ingredients in the skin UV protection of the prior art, and provides an anti-photoaging skin care composition with multiple compound effects.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a composition having both anti-inflammatory and firming and anti-wrinkle effects, which is composed of benzenemethylenedimethoxydimethylindanone and ginsenosides, wherein the total saponin content of the ginsenosides is ≥80%, and ginsenosides C-Mx and CK account for ≥95% of the total saponin content.

[0007] Preferably, benzenemethylenedimethoxydimethylindanone and ginsenosides can be in any ratio.

[0008] Preferably, the mass ratio of benzenemethylenedimethoxydimethylindanone to ginsenoside is (0.001~1):(0.005~0.5).

[0009] More preferably, the mass ratio of benzenemethylenedimethoxydimethylindanone to ginsenoside is (0.01~1):(0.005~0.2).

[0010] Preferably, the ginsenosides contain ≥76.55% C-Mx and ≥18.55% CK.

[0011] Secondly, the present invention provides the use of the composition in the preparation of cosmetics for anti-inflammatory and firming / anti-wrinkle purposes.

[0012] Fourthly, the present invention provides a cosmetic product containing the aforementioned composition.

[0013] Thirdly, the present invention provides the use of the composition in the preparation of cosmetics for anti-photoaging.

[0014] Benzylidenedimethoxydimethylindanone (INCI name: Benzylidenedimethoxydimethylindanone, CAS: 924626-15-3, EC: 486-080-1) is an isopentenol ester that has AhR antagonistic effects to block skin damage caused by UVB, such as reducing the expression of collagen degradation signal MMP1 and inflammatory molecule COX2, and has potential applications in sun protection and anti-photoaging.

[0015] Ginsenosides are key active ingredients in ginseng, and they can be classified into dammarane and oleanane types based on differences in glycoside structure. Ginsenosides can play a multidimensional role in anti-aging, neuroprotection, cardiovascular disease intervention, and tumor prevention by enhancing antioxidant enzyme activity, maintaining mitochondrial homeostasis, inhibiting excessive ROS production, and regulating multiple signaling pathways.

[0016] The composition provided by this invention exhibits a significantly superior synergistic effect between the two components compared to individual monomers. It not only inhibits the expression of IL-6, COX-2, and TNF-α genes in inflammatory cells, demonstrating excellent anti-inflammatory efficacy, but also effectively improves the expression of COL-1, COL-2, and MMP-1 genes in cells after UVA irradiation, resulting in firming and anti-wrinkle effects. This comprehensive enhancement of anti-UV damage simultaneously achieves both anti-UVB-induced skin inflammation and anti-UVA-induced skin aging, providing a dual effect of anti-inflammatory and firming / anti-wrinkle benefits. Compared to single components, the composition exhibits stronger anti-inflammatory efficacy and better improvement in firming and anti-wrinkle-related genes in UVA-irradiated aging cells, all demonstrating the excellent synergistic effect of the composition. Furthermore, the preparation method of the raw material composition of this invention is simple and easy to implement, the raw materials are readily available, and it can be mass-produced. Attached Figure Description

[0017] Figure 1 The HPLC chromatogram of ginsenoside raw material is shown. Detailed Implementation

[0018] To facilitate understanding of the present invention, a more complete description will be given below with reference to specific embodiments. Preferred embodiments of the invention are shown in the accompanying drawings. However, the invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the invention.

[0019] Unless otherwise defined, all 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 pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0020] It should be noted that, unless otherwise specified, the experimental methods used in the following embodiments and comparative examples are all conventional methods. Unless otherwise specified, the raw materials used in the technical solutions provided by this invention are all prepared by conventional means or purchased through commercial channels. The ginsenosides used in the embodiments of this invention were purchased from Guangzhou Nuowei Biotechnology Co., Ltd. (item number: 1000003250). HPLC analysis showed that the total saponin content in the ginsenoside raw material was ≥80%, and ginsenosides C-Mx and CK accounted for ≥95% of the total saponin content. Figure 1 (See Table 1). Among them, the C-Mx content is ≥76.55%, and the CK content is ≥18.55%.

[0021] Table 1. HPLC results of ginsenosides

[0022] Example 1 This embodiment provides a composition, the preparation method of which includes the following steps: Benzyl dimethoxydimethyl indanone and ginsenosides were mixed in distilled water. The amount of Benzyl dimethoxydimethyl indanone added to the water was 0.001 μg / mL, and the concentration of ginsenosides in the water was 0.005 μg / mL.

[0023] Example 2 This embodiment provides a composition, the preparation method of which includes the following steps: Benzyl dimethoxydimethyl indanone and ginsenosides were mixed in distilled water. The amount of Benzyl dimethoxydimethyl indanone added to the water was 0.001 μg / mL, and the concentration of ginsenosides in the water was 0.05 μg / mL.

[0024] Example 3 This embodiment provides a composition, the preparation method of which includes the following steps: Benzyl dimethoxydimethyl indanone and ginsenosides were mixed in distilled water. The amount of Benzyl dimethoxydimethyl indanone added to the water was 0.001 μg / mL, and the concentration of ginsenosides in the water was 0.5 μg / mL.

[0025] Example 4 This embodiment provides a composition, the preparation method of which includes the following steps: Benzyl dimethoxydimethyl indanone and ginsenosides were mixed in distilled water. The amount of Benzyl dimethoxydimethyl indanone added to the water was 0.01 μg / mL, and the concentration of ginsenosides in the water was 0.005 μg / mL.

[0026] Example 5 This embodiment provides a composition, the preparation method of which includes the following steps: Benzyl dimethoxydimethyl indanone and ginsenosides were mixed in distilled water. The amount of Benzyl dimethoxydimethyl indanone added to the water was 0.1 μg / mL, and the concentration of ginsenosides in the water was 0.005 μg / mL.

[0027] Example 6 This embodiment provides a composition, the preparation method of which includes the following steps: Benzyl dimethoxydimethyl indanone and ginsenosides were mixed in distilled water. The concentration of Benzyl dimethoxydimethyl indanone in the water was 1 μg / mL, and the concentration of ginsenosides in the water was 0.2 μg / mL.

[0028] Comparative Example 1 This comparative example provides a single component containing only benzenemethyldimethoxydimethylindanone. The amount of benzenemethyldimethoxydimethylindanone added to water is 0.001 μg / mL.

[0029] Comparative Example 2 This comparative example provides a single component containing only benzenemethyldimethoxydimethylindanone. The amount of benzenemethyldimethoxydimethylindanone added to water is 0.01 μg / mL.

[0030] Comparative Example 3 This comparative example provides a single component containing only benzenemethyldimethoxydimethylindanone. The amount of benzenemethyldimethoxydimethylindanone added to water is 0.1 μg / mL.

[0031] Comparative Example 4 This comparative example provides a single component containing only benzenemethyldimethoxydimethylindanone. The amount of benzenemethyldimethoxydimethylindanone added to water is 1 μg / mL.

[0032] Comparative Example 5 This comparative example provides a single component containing only ginsenosides. The amount of ginsenosides added to water is 0.005 μg / mL.

[0033] Comparative Example 6 This comparative example provides a single component containing only ginsenosides. The amount of ginsenosides added to water is 0.05 μg / mL.

[0034] Comparative Example 7 This comparative example provides a single component containing only ginsenosides. The amount of ginsenosides added to water is 0.2 μg / mL.

[0035] Comparative Example 8 This embodiment provides a composition, the preparation method of which includes the following steps: Glucosylrutin and ginsenosides were mixed in distilled water. The amount of glucosylrutin added to the water was 0.001 μg / mL, and the concentration of ginsenosides in the water was 0.005 μg / mL.

[0036] Comparative Example 9 This embodiment provides a composition, the preparation method of which includes the following steps: Glucosylrutin and ginsenosides were mixed in distilled water. The amount of glucosylrutin added to the water was 0.01 μg / mL, and the concentration of ginsenosides in the water was 0.005 μg / mL.

[0037] Comparative Example 10 This embodiment provides a composition, the preparation method of which includes the following steps: Glucosylrutin and ginsenosides were mixed in distilled water. The amount of glucosylrutin added to the water was 1 μg / mL, and the concentration of ginsenosides in the water was 0.2 μg / mL.

[0038] Comparative Example 11 This embodiment provides a composition, the preparation method of which includes the following steps: Benzyl dimethoxydimethyl indanone and ginsenoside RG3 were mixed in distilled water. The amount of Benzyl dimethoxydimethyl indanone added to the water was 0.001 μg / mL, and the concentration of ginsenoside RG3 in the water was 0.005 μg / mL.

[0039] Comparative Example 12 This embodiment provides a composition, the preparation method of which includes the following steps: Benzyl dimethoxydimethyl indanone and ginsenoside RG3 were mixed in distilled water. The amount of Benzyl dimethoxydimethyl indanone added to the water was 0.01 μg / mL, and the concentration of ginsenoside RG3 in the water was 0.005 μg / mL.

[0040] Comparative Example 13 This embodiment provides a composition, the preparation method of which includes the following steps: Benzyl dimethoxydimethyl indanone and ginsenoside RG3 were mixed in distilled water. The concentration of Benzyl dimethoxydimethyl indanone in the water was 1 μg / mL, and the concentration of ginsenoside RG3 in the water was 0.2 μg / mL.

[0041] Experiment 1: UVB Irradiation of Human Keratinocytes Inflammation Experiment 1.1 Experimental Principle Ultraviolet (UV) radiation is a major factor causing skin damage and disrupting the skin barrier function. Epidemiological studies show that approximately 80% of exposure-related skin diseases are caused by photodamage from UV radiation. Sunlight's UV radiation can be divided into short-wave ultraviolet (UVC), medium-wave ultraviolet (UVB), and long-wave ultraviolet (UVA), with wavelength ranges of 200-290 nm, 290-320 nm, and 320-400 nm, respectively. Of these, UVA and UVB are the main types that affect the human body, accounting for approximately 95% and 4%-5% of the total UV radiation reaching the Earth's surface, respectively. UVB exposure is extremely harmful to the skin, often accompanied by erythema or sunburn, blistering, and even visual impairment. Intense or repeated UVB exposure can lead to immunosuppression and permanent eye damage. UVB can penetrate the epidermis to reach the basal layer and can be absorbed by epidermal cells. It can directly act on epidermal DNA, causing gene damage, producing cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts, activating the p53 protein pathway, and leading to keratinocyte apoptosis and skin redness and swelling, resulting in sunspots.

[0042] The inflammatory response induced by cellular exposure to UVB irradiation is primarily mediated by several factors, including tumor necrosis factor-α (TNF-α), PEG2 produced by cyclooxygenase, NO produced by nitric oxide synthase, and other factors such as IL-6 and IL-1. These inflammatory factors are mainly produced by keratinocytes, the main constituent cells of the epidermis and the primary target of UVB irradiation, primarily regulated by NF-κB. UVB irradiation-induced expression of COX-2 and PEG2 is also closely related to the occurrence of cancer and inflammation. Therefore, by irradiating cells with UVB to induce an inflammatory cell model, the anti-UVB efficacy of the test substance can be evaluated by measuring the expression of genes related to inflammatory factors in keratinocytes.

[0043] 1.2 Experimental Methods 1.2.1 Cell Culture HaCaT cells were cultured in DMEM complete medium (DMEM medium + 10% FBS + antibiotics) in a carbon dioxide incubator at 37°C and 5% CO2, and passaged every 2-3 days.

[0044] HaCaT cells in the logarithmic growth phase were selected, digested with 0.25% wt trypsin, and then prepared into a density of 1×10⁶ cells / mL using DMEM complete medium. 5Cell suspensions of 10 cells / mL were seeded into 12-well plates, 1 mL per well, and incubated at 37°C with 5% CO2 for 24 h. DMEM medium containing different concentrations of the test substance was added to each well, and a blank control group (without the test substance) was set up, 1 mL per well. After incubation at 37°C with 5% CO2 for 24 h, the supernatant was discarded, and the cells were washed three times with DPBS. The DPBS was then removed, and the cells were used for subsequent experiments.

[0045] 1.2.2 Extraction of cellular RNA Lyse cells by adding cell lysis buffer to each well of a six-well plate. Add chloroform (1:5 volume ratio to lysis buffer) to the lysis buffer, vortex thoroughly, incubate at room temperature for 5 min, then centrifuge at 12000 rpm for 15 min at 4°C. Transfer the colorless supernatant to a new 1.5 mL centrifuge tube, add an equal volume of isopropanol, mix well, and incubate at room temperature for 10 min. Centrifuge at 12000 rpm for 10 min at 4°C, and discard the supernatant. Add 500 µL of 75% v / v ethanol. Centrifuge at 7500 rpm for 5 min at 4°C, discard the supernatant, and allow to air dry at room temperature for 1–2 min. Add an appropriate amount of DEPC water to fully dissolve the RNA precipitate.

[0046] 1.2.3 cDNA Synthesis The reverse transcription kit used in this experiment was HiScript® II Q RT SuperMix for qPCR (+gDNA wiper), purchased from Nanjing Novizan Biotechnology Co., Ltd. The procedure was performed according to the kit instructions.

[0047] 1.2.4 qRT-PCR Prepare the mixture according to the system in Table 1 below.

[0048] Table 1. qRT-PCR reaction system

[0049] Then, qPCR was performed according to the kit instructions.

[0050] 1.2.5 Result Calculation Gene expression inhibition rate = (relative expression level of UVB irradiated group - relative expression level of sample) / relative expression level of UVB irradiated group × 100%.

[0051] 1.3 Experimental Results Table 2. Interleukin-6 (IL-6) gene expression inhibition rate

[0052] As the main component of the epidermis, keratinocytes, upon exposure to UVB radiation, secrete various inflammatory factors such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), mediating inflammatory responses, regulating immune responses, and inducing apoptosis. The production of cellular inflammatory factors helps promote the body's immune response, but overexpression can trigger adverse reactions. When inflammatory factors are overexpressed, they can induce skin inflammation and accelerate cell damage, leading to adverse skin manifestations such as redness, swelling, and fever. IL-6 is a cytokine with broad biological activity; excessive secretion of IL-6 can cause inflammatory responses. Excessive IL-6 production or persistent activation of its signaling pathways can lead to chronic inflammation and tissue damage, and is closely related to numerous diseases.

[0053] The inhibition rate of IL-6 gene expression in each sample group was calculated by comparing it with the relative expression level of IL-6 in the model group (UVB irradiation group). The results of Examples 1, 3, 4, and 5 in Table 2 show that the combination of benzyldimethoxydimethylindanone and ginsenosides effectively inhibited UVB irradiation-induced IL-6 gene expression. Compared to the UVB irradiation group, its inhibition rate of IL-6 gene expression reached 31.63%~49.30%, indicating that the combination of benzyldimethoxydimethylindanone and ginsenosides can effectively inhibit cellular IL-6 gene expression and suppress cellular inflammatory responses. Furthermore, compared to Comparative Examples 1, 2, 3, 5, and 6, the combination of benzyldimethoxydimethylindanone and ginsenosides showed a higher inhibition rate of cellular IL-6 gene expression than either benzyldimethoxydimethylindanone or ginsenoside alone, demonstrating the excellent synergistic effect of the combination.

[0054] In comparisons of Example 1 (inhibition rate 32.45%) with Comparative Examples 8 (inhibition rate < 0) and 11 (inhibition rate < 0), and Example 4 (inhibition rate 41.91%) with Comparative Examples 9 (inhibition rate < 0) and 12 (inhibition rate < 0), replacing ginsenosides with ginsenoside RG3 and with glucosylrutin, all showed that replacing any component in the combination of ginsenosides and ginsenosides with other components significantly reduced the inhibitory effect on cellular IL-6 gene expression. This result demonstrates the uniqueness of the combination of ginsenosides and ginsenosides. When the skin is exposed to ultraviolet radiation, pollutants, irritants, or damage, IL-6 levels rise rapidly, exacerbating the inflammatory response. This composition can effectively inhibit the overexpression of cellular IL-6 and control skin inflammation, thus possessing potential for application in cosmetics.

[0055] Table 3. Cyclooxygenase 2 (COX-2) gene expression inhibition rate

[0056] During UV irradiation, the skin generates a large amount of reactive oxygen species (ROS), causing oxidative stress. Intermediates from these ROS can affect the expression level of cyclooxygenase-2 (COX-2), which is closely related to inflammatory processes and the development of epidermal tumors. COX-2 plays a crucial role in the occurrence and development of inflammation during UVB damage to keratinocytes. 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 express almost no COX-2. When the body is stimulated by inflammatory factors, COX-2 levels increase sharply, leading to the synthesis and accumulation of inflammatory PGs at the damaged site. PGs can synergistically promote the increase of vascular permeability and chemotaxis of other inflammatory mediators, causing inflammation, tissue destruction, and proliferation in surrounding tissues, thus promoting inflammatory responses and tissue damage.

[0057] The inhibition rate of COX-2 gene expression in each sample group was calculated by comparing it with the relative COX-2 expression level in the model group (UVB irradiation group). The results of Examples 1, 2, 4, and 5 in Table 3 show that the combination of benzyldimethoxydimethylindanone and ginsenosides effectively inhibited COX-2 gene expression induced by UVB irradiation. Compared to the UVB irradiation group, its inhibition rate of COX-2 gene expression reached 27.40%~35.08%, indicating that the combination of benzyldimethoxydimethylindanone and ginsenosides can effectively inhibit the expression of COX-2 gene in cells and suppress inflammatory responses. Furthermore, compared to Comparative Examples 1, 2, 3, and 5, the combination of benzyldimethoxydimethylindanone and ginsenosides showed a higher inhibition rate of COX-2 gene expression in cells compared to either benzyldimethoxydimethylindanone or ginsenoside alone, demonstrating the excellent synergistic effect of the combination.

[0058] In the comparison results of Example 1 (inhibition rate 27.40%) with Comparative Examples 8 (inhibition rate < 0) and 11 (inhibition rate < 0), and Example 4 (inhibition rate 29.46%) with Comparative Examples 9 (inhibition rate < 0) and 12 (inhibition rate 0.35%), replacing the ginsenoside raw material used in this invention with ginsenoside RG3 and replacing benzyl dimethoxydimethyl indanone with glucosylrutin, all showed that replacing any component in the combination of benzyl dimethoxydimethyl indanone and ginsenoside significantly reduced the inhibitory effect on COX-2 gene expression in cells. This result also demonstrates the uniqueness of ginsenosides in this composition. COX-2, when activated by ultraviolet light, catalyzes the production of prostaglandins (such as PGE2), directly leading to vasodilation, skin redness, and pain. Therefore, inhibiting the overexpression of COX-2 can reduce the production of inflammatory mediators at the source and alleviate the symptoms of skin inflammation. Thus, this composition has the potential for application in cosmetics.

[0059] Table 4. Tumor necrosis factor α (TNF-α) gene expression inhibition rate

[0060] Tumor necrosis factor (TNF-α) further increases the number of neutrophils in the skin, stimulates local inflammatory response, dilates skin capillaries, and increases the permeability of vascular endothelial cells, leading to rashes, petechiae, and other skin conditions.

[0061] The inhibition rate of TNF-α gene expression in each sample group was calculated by comparing it with the relative expression level of TNF-α in the model group (UVB irradiation group). The results of Examples 1, 2, 3, and 4 in Table 4 show that the combination of benzyldimethoxydimethylindanone and ginsenosides effectively inhibited UVB irradiation-induced TNF-α gene expression. Compared to the UVB irradiation group, its inhibition rate of TNF-α gene expression reached 56.78%~68.88%, indicating that the combination of benzyldimethoxydimethylindanone and ginsenosides can effectively inhibit cellular TNF-α gene expression and suppress cellular inflammatory responses. Furthermore, compared to Comparative Examples 1, 2, 3, 5, and 6, the combination of benzyldimethoxydimethylindanone and ginsenosides showed a higher inhibition rate of cellular TNF-α gene expression than either benzyldimethoxydimethylindanone or ginsenoside alone, demonstrating the excellent synergistic effect of the combination.

[0062] In the comparison results of Example 1 (inhibition rate 56.78%) with Comparative Examples 8 (inhibition rate < 0) and 11 (inhibition rate < 0), and Example 4 (inhibition rate 57.44%) with Comparative Examples 9 (inhibition rate < 0) and 12 (inhibition rate < 0), replacing the ginsenoside raw material used in this invention with ginsenoside RG3 and replacing benzyl dimethoxydimethyl indanone with glucosylrutin, all showed that replacing any component in the combination of benzyl dimethoxydimethyl indanone and ginsenoside significantly reduced the inhibitory effect on the expression of the TNF-α gene in cells. This result demonstrates the uniqueness of the combination of benzyl dimethoxydimethyl indanone and ginsenoside. When the skin is stimulated by ultraviolet rays, TNF-α is rapidly produced in the skin. It can not only directly cause skin inflammation, but also activate the MAPK cell pathway to significantly upregulate the expression of matrix metalloproteinases (such as MMP-1), thereby degrading type I collagen, the supporting structure of the skin, leading to skin aging. This composition can effectively inhibit the expression of TNF-α in cells, and therefore has the potential to be used in cosmetics.

[0063] The above experimental results all indicate that the combination of benzenemethylenedimethoxydimethylindanone and ginsenosides has excellent synergistic anti-inflammatory effects, promotes the inhibition of gene expression of inflammatory factors in UVB-irradiated cells, significantly enhances anti-inflammatory efficacy, and is more conducive to the application of the compound in cosmetics.

[0064] Experimental Example 2: Photoaging Experiment of Fibroblasts Irradiated by UVA 2.1 Experimental Principle Skin aging is characterized by skin atrophy due to loss of elasticity and slowed metabolic activity. Environmental damage, particularly exposure to ultraviolet radiation (photodamage), contributes to the intrinsic aging process. Fibroblasts, large spindle-shaped cells typically found in intercellular tissue, are the main components of the skin. They produce and arrange the extracellular matrix in the dermis and communicate with other cells, playing a crucial role in regulating skin physiology. Simultaneously, they synthesize collagen (COL) and elastin (ELN) fibers, as well as the extracellular matrix, in the reticular and dense connective tissue. In aging skin, the number of fibroblasts producing collagen and elastin fibers decreases, leading to wrinkle formation. A major characteristic of skin aging is the breakdown of the dermal collagen matrix. This breakdown is caused by the action of specific enzymes, matrix metalloproteinases (MMPs), which impair the structural integrity of the dermis. Fibroblasts that produce and organize the collagen matrix cannot attach to the broken collagen. Loss of attachment prevents fibroblasts from receiving mechanical information from their support structure, causing them to break down. In aging skin, collapsed fibroblasts produce low levels of collagen and high levels of collagen-degrading enzymes. This imbalance accelerates the aging process, creating a self-perpetuating, endless, and harmful cycle.

[0065] Human skin consists of two layers: the epidermis and the dermis. Long-wave ultraviolet (UVA) radiation penetrates longer than medium-wave ultraviolet (UVB) radiation. UVA not only penetrates the epidermis, damaging keratinocytes, but can also be absorbed by human skin fibroblasts (HSF), causing damage to the entire dermis. A significant portion of UVA can completely penetrate the dermis, causing photoaging, manifested as skin damage, sagging, and wrinkles. Therefore, irradiating cells with UVA to induce an aging cell model can be used to evaluate the anti-photoaging efficacy of test substances.

[0066] 2.2 Experimental Methods 2.2.1 Cell Culture Human fibroblast (HSF) cells were cultured in DMEM complete medium (DMEM medium + 10% FBS + antibiotics) in a carbon dioxide incubator at 37°C and 5% CO2, and passaged every 2-3 days.

[0067] HSF cells in logarithmic growth phase were selected, digested with 0.125% wt trypsin, and then prepared into a density of 7.5 × 10⁶ cells / year in DMEM complete medium. 4 Cell suspensions of 10 cells / mL were seeded into 12-well plates, 1 mL per well, and incubated at 37°C in a 5% CO2 incubator for 24 h. The old culture medium in the 12-well plates was discarded, and 1 mL of complete culture medium containing different concentrations of the drug (sample group) was added to each well. The blank group and model group had fresh culture medium, with each group having 3 replicates. After incubation at 37°C in 5% CO2 for 24 h, the culture medium in the wells was replaced with DPBS. Except for the blank group, the other groups were irradiated with UVA at 10 J / cm². 2 (6 mV / cm) 2 (Irradiation intensity), then discard the DPBS, add the original culture medium to the well, continue culturing at 37°C for 24 h, discard the supernatant, wash the cells 3 times with DPBS, remove the DPBS, and leave the cells for subsequent experiments.

[0068] 2.2.2 Extraction of cellular RNA Lyse cells in each well of a 12-well plate by adding cell lysis buffer. Add chloroform (1:5 volume ratio to lysis buffer) to the lysis buffer, vortex thoroughly, incubate at room temperature for 5 min, then centrifuge at 12000 rpm for 15 min at 4°C. Transfer the colorless supernatant to a new 1.5 mL centrifuge tube, add an equal volume of isopropanol, mix well, and incubate at room temperature for 10 min. Centrifuge at 12000 rpm for 10 min at 4°C, and discard the supernatant. Add 500 µL of 75% v / v ethanol. Centrifuge at 7500 rpm for 5 min at 4°C, discard the supernatant, and allow to air dry at room temperature for 1–2 min. Add an appropriate amount of DEPC water to fully dissolve the RNA precipitate.

[0069] 2.2.3 cDNA Synthesis The reverse transcription kit used in this experiment was HiScript® II Q RT SuperMix for qPCR (+gDNA wiper), purchased from Nanjing Novizan Biotechnology Co., Ltd. The procedure was performed according to the kit instructions.

[0070] 2.2.4 qRT-PCR Prepare the mixture according to the system in Table 5 below: Table 5. qRT-PCR reaction system

[0071] Then, qPCR was performed according to the kit instructions.

[0072] 2.2.5 Result Calculation COL-1 / 3 gene expression enhancement rate = (relative expression level of sample - relative expression level of UVA irradiated group) / relative expression level of UVA irradiated group × 100%.

[0073] MMP-1 gene expression enhancement rate = (relative expression level of UVA irradiated group - relative expression level of sample) / relative expression level of UVA irradiated group × 100%.

[0074] 2.3 Experimental Results Table 6. Increased expression rate of type I collagen (COL-1) gene

[0075] UVA, or long-wave ultraviolet radiation, can penetrate the epidermis, damaging keratinocytes, and is also absorbed by dermal fibroblasts, causing damage to the entire dermis. A significant portion of UVA can completely penetrate the dermis, causing photoaging, manifesting as skin damage, sagging, and wrinkles. Type I collagen (COL-1) is mainly found in the skin, bones, muscles, and other connective tissues, playing a crucial role in maintaining tissue structure integrity and elasticity. When COL-1 is specifically degraded, the normal structure of collagen and elastin fibers in the body is disrupted, leading to signs of aging such as wrinkles.

[0076] The inhibition rate of COL-1 gene expression in each sample group was calculated by comparing it with the relative COL-1 expression level in the model group (UVA irradiation group). Table 6 shows the effect of each sample on the mRNA expression level of the COL-1 gene in the UVA-induced aging model. The data in the table show that after treatment in Example 6, the cells significantly promoted COL-1 gene expression compared to the UVA irradiation group, with an increase rate of 67.31%. This indicates that the combination of benzyl dimethoxydimethyl indanone and ginsenosides can effectively promote COL-1 gene expression in cells after UVA irradiation. The promoting effects of Comparative Examples 4 and 7 on COL-1 were much lower than those in Example 4, indicating that the combination has a higher increase rate in COL-1 gene expression compared to either benzyl dimethoxydimethyl indanone or ginsenosides alone, demonstrating the excellent synergistic effect of the combination.

[0077] In the comparison of Example 6 (67.31% improvement rate) with Comparative Example 10 (11.47% improvement rate) and Comparative Example 13 (improvement rate < 0%), glucosylrutin was used to replace benzyldimethoxydimethylindanone, and ginsenoside RG3 was used to replace ginsenosides, respectively. The results showed that replacing either benzyldimethoxydimethylindanone or ginsenosides with other components significantly reduced the effect on the expression of the COL-1 gene in cells. This result demonstrates the uniqueness of the benzyldimethoxydimethylindanone or ginsenoside combination.

[0078] Table 7. Increased expression rate of type III collagen (COL-3) gene

[0079] Type III collagen (COL-3) is an important structural protein in the human body, mainly distributed at the junction of the epidermis and dermis (microcollagen layer), blood vessels, and internal organs, forming a dense, sparse network structure. This protein has excellent elasticity and extensibility, and plays a vital role in maintaining skin suppleness, promoting scarless healing, and locking in moisture.

[0080] The inhibition rate of COL-3 gene expression in each sample group was calculated by comparing it with the relative COL-3 expression level in the model group (UVA irradiation group). Table 7 shows the effect of each sample on the mRNA expression level of COL-3 gene in the UVA-induced aging model. The data in the table show that after treatment in Example 6, the cells significantly promoted COL-3 gene expression compared to the UVA irradiation group, with an increase rate of 24.78%. This indicates that the combination of benzyl dimethoxydimethyl indanone and ginsenosides can effectively promote COL-3 gene expression in cells after UVA irradiation. Comparative Examples 4 and 7 did not show any promoting effect on COL-3, indicating that the combination of benzyl dimethoxydimethyl indanone and ginsenosides, which were initially ineffective, showed an effect of increasing COL-3 gene expression in cells after combination. This result demonstrates the excellent synergistic effect of the composition.

[0081] In the comparison of Example 6 (24.78% improvement rate) with Comparative Example 10 (4.16% improvement rate) and Comparative Example 13 (improvement rate < 0%), glucosylrutin was used to replace benzyldimethoxydimethylindanone, and ginsenoside RG3 was used to replace ginsenosides, respectively. The results showed that replacing either benzyldimethoxydimethylindanone or ginsenosides with other components significantly reduced the effect on the expression of the COL-3 gene in cells. This result demonstrates the uniqueness of the benzyldimethoxydimethylindanone or ginsenoside combination. This composition can effectively promote the expression of the COL-1 and COL-3 genes in cells, stimulate collagen synthesis in the skin, thereby improving wrinkles, and therefore has the potential for application in cosmetics.

[0082] Table 8. Inhibition rate of matrix metalloproteinase 1 (MMP-1) gene expression

[0083] Matrix metalloproteins (MMPs) are a group of important proteolytic enzymes. When MMPs are overexpressed, they specifically degrade extracellular matrix components, disrupting the normal structure of collagen and elastic fibers, leading to signs of aging such as wrinkles. Among them, MMP-1 is the only MMP that can break down intact collagen fibers.

[0084] The inhibition rate of MMP-1 gene expression in each sample group was calculated by comparing it with the relative expression level of MMP-1 in the model group (UVA irradiation group). Table 8 shows the effect of each sample on the mRNA expression level of the MMP-1 gene in the UVA-induced aging model. The data in the table show that after treatment in Example 6, the cells showed a significant inhibitory effect on MMP-1 gene expression compared to the UVA irradiation group, with an inhibition rate of 68.10%. This indicates that the combination of benzyl dimethoxydimethyl indanone and ginsenosides can effectively inhibit UVA-induced MMP-1 gene expression in cells. The inhibitory effects of Comparative Examples 4 and 7 on MMP-1 were much lower than those in Example 4, indicating that the composition has a higher inhibition rate on MMP-1 gene expression in cells compared to either benzyl dimethoxydimethyl indanone or ginsenoside alone, demonstrating the excellent synergistic effect of the composition.

[0085] In the comparison of Example 6 (inhibition rate 68.10%) with Comparative Examples 10 (inhibition rate 57.72%) and 13 (inhibition rate 17.63%), glucosylrutin was used to replace benzyldimethoxydimethylindanone, and ginsenoside RG3 was used to replace ginsenosides, respectively. The results showed that replacing any component in the benzyldimethoxydimethylindanone and ginsenoside combination with other components significantly reduced the inhibitory effect on the expression of the MMP-1 gene in cells. This result demonstrates the uniqueness of the benzyldimethoxydimethylindanone and ginsenoside combination. MMP-1 can degrade collagen, the supporting structure of the skin, leading to wrinkles and skin laxity. This composition can effectively inhibit the expression of the MMP-1 gene in cells, thereby protecting or delaying the loss of collagen in the skin, maintaining skin elasticity, and reducing wrinkle formation, thus possessing potential for application in cosmetics.

[0086] In summary, based on the results of Examples 1, 2, 3, 4, 5, and 6, the composition formulated with benzyl dimethoxydimethyl indanone or ginsenosides not only inhibits the expression of IL-6, COX-2, and TNF-α genes in inflammatory cells, exhibiting excellent anti-inflammatory effects, but also effectively improves the expression of COL-1, COL-2, and MMP-1 genes in cells after UVA irradiation, demonstrating firming and anti-wrinkle effects. Comparison with the comparative examples also shows that compared to single components, the composition has stronger anti-inflammatory effects and a better effect on improving firming and anti-wrinkle related genes in UVA-irradiated aging cells. These results demonstrate the excellent synergistic effect of the composition. The combination of anti-inflammatory and firming / anti-wrinkle effects of this composition indicates its great potential for application in cosmetics.

[0087] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Therefore, any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A composition possessing both anti-inflammatory and firming / anti-wrinkle effects, characterized in that, The composition consists of benzenemethylenedimethoxydimethylindanone and ginsenosides, wherein the total ginsenoside content is ≥80%, and ginsenosides C-Mx and CK account for ≥95% of the total ginsenoside content.

2. The composition according to claim 1, characterized in that, The ratio of benzenemethylenedimethoxydimethylindanone and ginsenosides is any.

3. The composition according to claim 1, characterized in that, The mass ratio of benzenemethylenedimethoxydimethylindanone to ginsenosides is (0.001~1): (0.005~0.5).

4. The composition according to claim 1, characterized in that, The mass ratio of benzenemethylenedimethoxydimethylindanone to ginsenosides is (0.01~1): (0.005~0.2).

5. The composition according to any one of claims 1 to 4, characterized in that, The ginsenosides contain C-Mx content ≥76.55% and CK content ≥18.55%.

6. Use of the composition according to any one of claims 1 to 5 in the preparation of an anti-inflammatory and firming anti-wrinkle cosmetic.

7. Use of the composition according to any one of claims 1 to 5 in the preparation of cosmetics for anti-photoaging.

8. A cosmetic product comprising the composition as described in any one of claims 1 to 5.

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