A composition of ganoderma lucidum fermentation extract and its application in skin antioxidant, immunomodulation, anti-aging and anti-wrinkle skin tender

Abstract

The application discloses a synergistic ganoderma lucidum fermentation extract composition and application thereof in skin antioxidation, immunoregulation, anti-aging and anti-wrinkle skin tendering. The composition provided by the application contains the following raw materials in percentage by weight: 0.1-10% of ganoderma lucidum fermentation extract, 0.01-10% of magnesium ascorbyl phosphate, 0.01-5% of glycyrrhiza extract, 0.1-5% of tocopherol, 0.05-3% of boswellic acid extract, 0.01-3% of asiaticoside, 0.01-0.5% of coffee acid, and the balance of solvent. The composition provided by the application realizes three-effect synergism of "immune homeostasis-antioxidation repair-antioxidation" through specific component cooperation, can significantly improve skin inflammation, stabilize the skin immune microenvironment, delay skin aging, and has important application value in the development of skin antioxidation, immunoregulation, anti-aging and anti-wrinkle skin tendering products.

Description

Technical Field

[0001] This invention relates to the field of cosmetic technology, and more specifically, to a composition that enhances Ganoderma lucidum fermentation extract and its application in skin anti-oxidation, immune regulation, anti-aging, and anti-wrinkle skin rejuvenation. Background Technology

[0002] Reishi mushroom, a traditional medicinal and edible fungus, has fermented extracts (filtrates after fermentation by yeast and other microorganisms) rich in active ingredients such as reishi polysaccharides and reishi triterpenes. These extracts show potential in regulating immunity and providing antioxidant effects, and also hold broad promise for skin care in the cosmetics field. For example, existing technologies disclose that the fermented filtrate of sessile reishi mycelium can promote a 16.67% increase in the expression of scutellarin, enhance skin barrier function, and rapidly reduce redness by 8.81% within 15 minutes.

[0003] However, the application of Ganoderma lucidum fermentation extract alone in skin care has the following limitations: (1) Ganoderma lucidum polysaccharides can activate immune cells, but lack fine regulation of subsequent immune responses, and may not play an ideal "stabilizing" role in the state of skin immune imbalance; (2) Its active ingredient Ganoderma lucidum triterpenes are effective in inhibiting upstream inflammatory pathways (such as NF-κB) and activating endogenous antioxidant defenses (such as Nrf2 pathway), but have weak ability to clear specific downstream inflammatory mediators that have been released in large quantities, and have limited ability to directly neutralize free radicals.

[0004] Besides being used alone, Ganoderma lucidum fermented extract is often combined with other ingredients to achieve synergistic effects. For example, it has been publicly disclosed that Ganoderma lucidum fermented extract can be combined with Tibetan cypress extract to synergistically enhance the skin's antioxidant, anti-oxidative, and moisturizing properties. However, the effect of this combination in enhancing the efficacy of Ganoderma lucidum fermented extract is quite limited. Currently, there is still a need for more and more effective products containing Ganoderma lucidum fermented extract to significantly enhance its efficacy in skin immune regulation, anti-oxidation, and other antioxidant functions.

[0005] Therefore, developing more products that can enhance the efficacy of Ganoderma lucidum fermentation extract, elevating its effects from "one-way activation" to "two-way intelligent regulation," and strengthening it from "upstream inhibition" to "full-process control," while significantly improving its bioavailability, and providing fundamental support for anti-wrinkle and skin rejuvenation through repairing the skin barrier and increasing skin hydration, has significant industrial value. Therefore, this invention application is filed. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to overcome the shortcomings of the prior art. The present invention provides a composition for enhancing Ganoderma lucidum fermentation extract and its application in skin anti-oxidation, immune regulation, anti-aging and anti-wrinkle skin rejuvenation.

[0007] The first objective of this invention is to provide a composition that enhances the effects of Ganoderma lucidum fermentation extract.

[0008] A second object of the present invention is to provide applications of the above-described composition.

[0009] A third objective of this invention is to provide a cosmetic product.

[0010] The above-mentioned objective of this invention is achieved through the following technical solution: This invention provides a composition for enhancing Ganoderma lucidum fermented extract, comprising the following raw materials by weight percentage: 0.1-15% Ganoderma lucidum fermented extract, 0.01-10% magnesium ascorbate phosphate, 0.01-5% licorice (Glycyrrhiza uralensis) extract, 0.1-5% tocopherol, 0.05-3% frankincense (Boswellia carterii) resin extract, 0.01-3% asiaticoside, 0.01-0.5% caffeic acid, and the balance being solvent.

[0011] The composition provided by this invention uses Ganoderma lucidum fermentation extract as the core, supplemented with magnesium ascorbate phosphate, licorice extract, tocopherol, frankincense resin extract, asiaticoside, and caffeic acid. Each component has a synergistic effect in three aspects: immune homeostasis, antioxidant repair, and antioxidant activity. Licorice extract can gently regulate immunity and reduce the local irritation that Ganoderma lucidum fermentation extract may cause. Frankincense resin extract and asiaticoside reduce inflammation and promote collagen synthesis by inhibiting MAPK / NF-κB signaling and activating the TGF-β / Smad pathway, thus shortening the time for redness and injury recovery. Magnesium ascorbate phosphate, tocopherol, and caffeic acid work together to scavenge free radicals and enhance antioxidant levels. The composition provided by this invention has antioxidant, immunomodulatory, anti-aging, and anti-wrinkle skin-rejuvenating effects, and can significantly enhance the effects of Ganoderma lucidum fermented extract. It has better effects than the composition alone and after replacing other components. It is suitable for maintaining skin homeostasis and delaying aging. It can be used to prepare more and more efficient products containing Ganoderma lucidum fermented extract for skin immunomodulation, anti-oxidation and anti-oxidation. It can also be used to prepare medical polymer materials, medical consumables and other daily chemical materials for skin immunomodulation.

[0012] Preferably, the composition contains the following raw materials by weight percentage: 1-10% Ganoderma lucidum fermentation extract, 0.1-5% magnesium ascorbate phosphate, 0.01-2.5% licorice extract, 0.1-2% tocopherol (vitamin E), 0.05-1% frankincense resin extract, 0.01-1% asiaticoside, 0.01-0.1% caffeic acid, and the balance being solvent.

[0013] More preferably, the composition contains, by weight percentage, the following ingredients: 5-10% Ganoderma lucidum fermentation extract, 1-5% magnesium ascorbate phosphate, 0.1-2.5% licorice extract, 0.5-2% tocopherol (vitamin E), 0.5-1% frankincense resin extract, 0.5-1% asiaticoside, 0.03-0.1% caffeic acid, and the balance being solvent.

[0014] Preferably, the Ganoderma lucidum fermentation extract is a purple Ganoderma lucidum yeast fermentation extract; specifically, it is the supernatant of the fermentation broth of yeast-fermented purple Ganoderma lucidum, preferably using yeast / purple Ganoderma lucidum fermentation filtrate.

[0015] Preferably, the yeast is brewer's yeast.

[0016] Preferably, the purple Ganoderma is Yangzi No. 1.

[0017] Preferably, the fermentation conditions are 25~35℃ for 48~72 hours.

[0018] As a specific optional implementation scheme, the preparation method of the yeast / Ganoderma lucidum fermentation filtrate is as follows: fresh Ganoderma lucidum is sliced, dried, crushed and sterilized, and activated brewer's yeast seed liquid is added at an inoculation amount of 1~5wt%, and fermented at 25~35℃ for 48~72h; after fermentation, the supernatant is collected after sonication, filtration and centrifugation.

[0019] Specifically, the Ganoderma lucidum fermentation extract used in this invention can be obtained directly from commercially available products or prepared using conventional methods in the art. Commercially available product source: yeast / Ganoderma lucidum fermentation filtrate, production batch number: GDYD251201, manufacturer: Guangdong Yuandu Biotechnology Co., Ltd.

[0020] As a more preferred embodiment, the present invention provides a method for preparing Ganoderma lucidum fermentation extract, comprising the following steps: (1) Culture of strains: Saccharomyces cerevisiae (CICC 1001) was inoculated into YPD liquid medium and cultured at 30℃ and pH 6.3-6.7 until the mid-to-late logarithmic phase (5-6 h). (2) Ganoderma lucidum pretreatment: Fresh purple Ganoderma lucidum (Yangzi No. 1) was sliced, dried at 60℃, pulverized, and sterilized at 121℃ for 20 min; (3) Fermentation culture: Inoculate the yeast seed liquid into YPD medium at an inoculation amount of 3%, culture at 30℃ for 5-6 hours, add Ganoderma lucidum powder at an inoculation amount of 10%, and continue fermentation at 30℃ for 72 hours; (4) Extraction and purification: After fermentation, sonicate for 10 min, filter with double-layer non-woven fabric, centrifuge the filtrate at 5℃ and 9000rpm for 15 min, and collect the supernatant to obtain the fermentation extract.

[0021] This invention provides the use of the above-mentioned compound in the preparation of skin antioxidant, immune-modulating and / or anti-aging products.

[0022] Preferably, the product is a pharmaceutical or cosmetic product.

[0023] The present invention provides a cosmetic comprising the above-described composition.

[0024] Preferably, the cosmetic product further contains a cosmetically acceptable carrier or excipient.

[0025] More preferably, the cosmetic also contains one or more of the following: moisturizer, preservative cosolvent, stabilizer, pH adjuster, thickener, emulsifier, and emollient.

[0026] Preferably, the cosmetically acceptable carrier or excipient includes 2-20% moisturizer, 0.1-2% preservative co-solvent, and 0.01-1% stabilizer.

[0027] More preferably, the humectant is one or more of panthenol, sodium hyaluronate, and 1,3-propanediol; the preservative co-solvent is p-hydroxyacetophenone / ethylhexylglycerin; and the stabilizer is xanthan gum.

[0028] More preferably, the moisturizer comprises 1-5% panthenol, 0.1-1% sodium hyaluronate, and 1-10% 1,3-propanediol.

[0029] More preferably, the cosmetic also contains 1-5% panthenol, 0.1-1% sodium hyaluronate, 1-10% 1,3-propanediol, 0.1-1% xanthan gum, and 0.1-2% p-hydroxyacetophenone / ethylhexylglycerin.

[0030] More preferably, the mass ratio of p-hydroxyacetophenone / ethylhexylglycerin is (1~3):1.

[0031] More preferably, the mass ratio of p-hydroxyacetophenone to ethylhexylglycerin is 2:1.

[0032] Preferably, the dosage form of the cosmetic is a serum, lotion, cream, gel, or mask.

[0033] The present invention also provides a method for preparing the above-mentioned cosmetic, comprising the following steps: (1) The aqueous phase raw materials: solvent, panthenol, sodium hyaluronate, xanthan gum, magnesium ascorbate phosphate, 1,3-propanediol, p-hydroxyacetophenone / ethylhexylglycerin are heated and stirred until completely dissolved to form the aqueous phase; (2) Melt the oil phase raw material: tocopherol (vitamin E) by heating to obtain the oil phase; (3) Control the system temperature at 60~68 ℃, and under high-speed homogenization conditions of 4000~5000 r / min, slowly add the oil phase to the aqueous phase, homogenize and emulsify for 8~10 min until the emulsion is fine and uniform and there is no obvious oil phase precipitation, thus forming a stable emulsion system. (4) After the homogenized solution has cooled to below 40 °C, add Ganoderma lucidum fermentation extract, licorice extract, frankincense resin extract, asiaticoside and caffeic acid in sequence, and stir evenly; (5) Adjust the pH to 5.5~6.0 and discharge the material.

[0034] Preferably, citric acid or triethanolamine is used to adjust the pH.

[0035] Furthermore, the present invention provides the following applications of the preparation of the cosmetic: (1) Relieves skin inflammation and improves discomfort such as redness, sensitivity, and stinging; (2) Regulates skin immune function, enhances skin resistance, and stabilizes sensitive skin; (3) Neutralize free radicals, reduce oxidative damage, and delay skin photoaging and natural aging; (4) Comprehensively improves skin barrier function and enhances overall skin health and radiance.

[0036] The present invention has the following beneficial effects: The composition of Ganoderma lucidum fermented extract provided by this invention is specifically formulated around the core functions of Ganoderma lucidum fermented extract, aiming to create a synergistic effect of "primary and secondary" pathways such as immune regulation and anti-oxidation, thereby comprehensively improving the efficacy of skin care. Through the scientific combination of Ganoderma lucidum fermented extract and licorice extract, a balanced regulatory system for skin immunity is constructed. On the one hand, the system utilizes licorice extract to inhibit excessive immune response and soothe inflammation, helping to improve redness and stinging in sensitive skin; on the other hand, it uses Ganoderma lucidum fermented extract to enhance the skin's basic immune defense function and stabilize immune cell activity, thereby improving the skin's tolerance to external stimuli, while also working synergistically with frankincense. The resin extract and asiaticoside reduce inflammation and promote collagen synthesis by inhibiting MAPK / NF-κB signaling and activating the TGF-β / Smad pathway, thus shortening the time for redness and damage recovery. Combined with magnesium ascorbate phosphate, tocopherol, and caffeic acid to scavenge free radicals and enhance antioxidant levels, this combination achieves a synergistic effect of "immune homeostasis-antioxidant repair-antioxidation," significantly improving skin inflammation, stabilizing the skin's immune microenvironment, and delaying skin aging. It exhibits anti-wrinkle and skin-rejuvenating effects and can be used to prepare more products that enhance the efficacy of Ganoderma lucidum fermentation extract. It has significant application value in the development of skin antioxidant, immune-regulating, anti-aging, and anti-wrinkle skin-rejuvenating products. Attached Figure Description

[0037] Figure 1 The prepared Ganoderma lucidum fermentation filtrate.

[0038] Figure 2 The results are for cell viability assay at a concentration of 100 μg / mL.

[0039] Figure 3 The results are for the determination of IL-6 content (pg / mL).

[0040] Figure 4 The results are for the determination of IL-8 content (pg / mL).

[0041] Figure 5 The results are for the determination of TNF-α content (pg / mL).

[0042] * in the figure indicates that the difference between groups is statistically significant. P <0.05, ** indicates that the difference is highly statistically significant. P <0.01), *** indicates that the difference is highly statistically significant ( P <0.001), ns indicates that the difference between groups is not statistically significant ( P ≥0.05). Detailed Implementation

[0043] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0044] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0045] The method for preparing the Ganoderma lucidum fermentation extract (or commercially available yeast / Ganoderma lucidum fermentation filtrate product) used in the examples is as follows: (1) Culture of strains: Saccharomyces cerevisiae (CICC 1001) was inoculated into YPD liquid medium and cultured at 30℃ and pH 6.3-6.7 until the mid-to-late logarithmic phase (5-6h). (2) Ganoderma lucidum pretreatment: Fresh purple Ganoderma lucidum (Yangzi No. 1) was sliced, dried at 60℃, pulverized, and sterilized at 121℃ for 20 min; (3) Fermentation culture: Inoculate the yeast seed liquid into YPD medium at an inoculation amount of 3%, culture at 30℃ for 5-6 hours, add Ganoderma lucidum powder at an inoculation amount of 10%, and continue fermentation at 30℃ for 72 hours; (4) Extraction and purification: After fermentation, sonicate for 10 min, filter with double-layer non-woven fabric, centrifuge the filtrate at 5℃ and 9000 rpm for 15 min, and collect the supernatant to obtain the fermentation extract, such as... Figure 1 As shown.

[0046] The licorice (Glycyrrhiza uralensis) extract, frankincense (Boswellia carterii) resin extract, magnesium ascorbate phosphate, tocopherol (vitamin E), asiaticoside, caffeic acid, etc. used in the examples are all commercially available cosmetic-grade standard raw materials.

[0047] Example: Preparation of different Ganoderma lucidum compositions Based on the weight percentage and the proportions of each component in Table 1, the main active ingredients are: Ganoderma lucidum fermentation extract, Glycyrrhiza uralensis extract, Boswellia carterii resin extract, Centella asiatica extract, magnesium ascorbate phosphate, tocopherol (vitamin E), and caffeic acid.

[0048] It also contains cosmetically acceptable carriers or excipients: humectants (panthenol, sodium hyaluronate, 1,3-propanediol), preservatives and solubilizers (p-hydroxyacetophenone / ethylhexylglycerin complex, mass ratio 2:1), and stabilizers (xanthan gum). Different compositions containing Ganoderma lucidum fermentation extract were prepared according to the following preparation process for later use.

[0049] The specific preparation process steps are as follows: (1) The aqueous phase raw materials: deionized water, panthenol, sodium hyaluronate, xanthan gum, magnesium ascorbate phosphate, 1,3-propanediol, p-hydroxyacetophenone / ethylhexylglycerin are heated and stirred until completely dissolved to form the aqueous phase; (2) Melt the oil phase raw material: tocopherol (vitamin E) by heating to obtain the oil phase; (3) Control the system temperature at 65 ℃, and under high-speed homogenization conditions of 4000~5000 r / min, slowly add the oil phase to the aqueous phase, homogenize and emulsify for 8~10 min until the emulsion is fine and uniform and there is no obvious oil phase precipitation, thus forming a stable emulsion system. (4) Cool the homogenized system to below 40 °C, and add Ganoderma lucidum fermentation extract, licorice extract, frankincense resin extract, asiaticoside and caffeic acid in sequence, and stir evenly; (5) Adjust the pH to 5.5~6.0 (the pH can be adjusted by pH adjusters such as citric acid or triethanolamine), and the product is ready for discharge.

[0050] Table 1. Composition ratios of different compositions

[0051] Comparative example: According to the preparation method provided in the examples, different comparative compositions were prepared with reference to the component amounts in Table 2 for each comparative example, and were prepared for later use. The active ingredient substitution settings were as follows: in Comparative Example 6, Ganoderma lucidum fermentation extract was replaced with Tremella fuciformis extract; in Comparative Example 7, Glycyrrhiza uraensis extract was replaced with Chrysanthemum morifolium flower extract; in Comparative Example 8, Boswellia carterii resin extract was replaced with dipotassium glycyrrhizate; in Comparative Example 9, asiaticoside was replaced with β-glucan; and Comparative Example 11 contained only excipient components.

[0052] Table 2 Comparison of raw material usage for each comparative example

[0053] Test Example 1: In vitro immunomodulatory effect assay An inflammatory cell model was induced using Escherichia coli lipopolysaccharide (LPS). Experimental samples were prepared using the compositions described in the examples and comparative examples. PBMC cells were cultured and treated. Finally, the expression levels of interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) in the supernatant were detected by enzyme-linked immunosorbent assay (ELISA). Specific experimental methods were as follows: (1) Preparation of PBMCs: Heparin-anticoagulated peripheral blood from healthy donors was centrifuged using Histopaque-1077 density gradient centrifugation to harvest PBMCs. The cells were resuspended in RPMI-1640 + 10% FBS + 1% double antibody and counted. The concentration was adjusted to 2×10⁻⁶. 6 cells / mL, for later use.

[0054] (2) Seeding in well plates: Take a 96-well plate and add 100 μL of cell suspension (4 × 10⁻⁶ cells per well) to each well. 5 Mononuclear cells were allowed to adhere to the wall by standing at 37°C and 5% CO2 for 2 hours.

[0055] (3) LPS immune activation model: 20 μL of the composition (100 μg / mL) was added to the well of the sampled cells and pre-incubated for 1 h; then 20 μL of LPS (30 ng / mL) was added, followed by 60 μL of culture medium, for a final volume of 200 μL; the LPS model group used culture medium instead of samples.

[0056] (4) CsA immunosuppression model: 20 μL of the composition (100 μg / mL) was added to the well of the sampled cells and pre-incubated for 1 h; then 20 μL of CsA (20 μg / mL) was added, and 60 μL of culture medium was added; the CsA model group used culture medium instead of samples.

[0057] (5) Culture and sample collection: After each group was cultured at 37°C and 5% CO2 for 18h, it was centrifuged at 4°C and 300g for 10min. 80μL of supernatant was taken from each well and stored at -80°C. The levels of IL-6, TNF-α and IL-10 were detected according to the ELISA kit instructions.

[0058] The results are shown in Tables 3 and 4. Compared with the model group, Comparative Example 11, which contained no active ingredients, had almost no regulatory effect on inflammatory and anti-inflammatory factors. In Comparative Example 1, the use of Ganoderma lucidum fermentation extract alone significantly reduced the levels of IL-6 and TNF-α pro-inflammatory factors and increased the expression of IL-10 anti-inflammatory factor in the LPS model, while also restoring the abnormal cytokine secretion in the CsA model. In Comparative Examples 2 and 3, the combination of Ganoderma lucidum fermentation extract with licorice extract, or asiaticoside and tocopherol, respectively, further significantly reduced the levels of pro-inflammatory factors, significantly increased the expression of IL-10 anti-inflammatory factor, and significantly restored the abnormal cytokine secretion in the CsA model. In Comparative Example 4, the effect of other components combined with Ganoderma lucidum fermentation extract was significantly worse than that of Comparative Example 1, without the addition of Ganoderma lucidum fermentation extract. In Comparative Example 5, the combination of licorice extract with pro-inflammatory and anti-inflammatory factors showed poor results, indicating that the combination of licorice extract and Ganoderma lucidum fermentation extract has a certain synergistic effect.

[0059] In Example 1, the content of Ganoderma lucidum fermentation extract and other components was reduced, but compared with the model group, it significantly reduced the levels of IL-6 and TNF-α pro-inflammatory factors in the LPS model, increased the expression of IL-10 anti-inflammatory factor, and restored the abnormal cytokine secretion in the CsA model. Its effect was comparable to that of Comparative Example 1, which contained only a medium concentration of Ganoderma lucidum fermentation extract. That is, it can still have a good effect under the premise of reducing the dosage of each group, and its effect is better than some comparative examples.

[0060] Meanwhile, data analysis of Examples 2 and Comparative Examples 1-5 showed that the combination of Ganoderma lucidum fermentation extract with licorice extract, asiaticoside, tocopherol, frankincense resin extract, magnesium ascorbate phosphate, and caffeic acid exhibited a highly significant synergistic effect. Furthermore, comparative analysis of data from Examples 2 and Comparative Examples 6-11 revealed that the highly significant effects of the compositions in this application are directly related to the selection of specific components.

[0061] Table 3 Results of the in vitro immune-modulating LPS immune activation model

[0062] Table 4 Results of the in vitro CsA immune activation model

[0063] Note: In the table, ① LPS model (overactivated immune system): the lower the values ​​of IL-6 and TNF-α (pro-inflammatory factors), the better the effect; the higher the value of IL-10 (anti-inflammatory factor), the better the effect; ② CsA model (infirm immune system): the higher the values ​​of IL-6 and TNF-α (pro-inflammatory factors), the better the effect of reversing immunosuppression; the closer the value of IL-10 (anti-inflammatory factor) is to the blank level, the better the effect of immune homeostasis repair.

[0064] Test Example 2: In vitro anti-inflammatory and antioxidant experimental test 1. Anti-inflammatory activity assay (1) Assay of HaCat activity in human immortalized epidermal keratinocytes: HaCat cells, immortalized human epidermal keratinocytes in the logarithmic growth phase, were harvested at a rate of 3 × 10⁻⁶. 4 Cells were seeded at a concentration of [number] cells / mL in 96-well plates, with 100 μL of culture medium (DMEM medium containing a mixture of 10% fetal bovine serum and 1% penicillin / streptomycin) per well. Cells were incubated at 37°C and 5% CO2 for 24 h. The supernatant was discarded, and 100 μL of complete culture medium containing 100 μg / mL of the drug was added to each well. The control group received the same volume of culture medium without the drug, while the blank group received only 100 μL of culture medium without cell seeding. Three replicates were set for each group, and cell viability was measured using the CCK-8 assay. Cells were then incubated at 37°C and 5% CO2 for another 48 h. 10 μL of CCK-8 solution was added to each well, and after 4 h of incubation, the absorbance at 450 nm was measured. The blank well was used to zero the absorbance, and cell viability was determined and calculated.

[0065] Cell viability is calculated using the following formula: Cell viability (%) = (Sample A - Blank A) / (Control A - Blank A) × 100%; Wherein, Sample A, Control A, and Blank A represent the absorbance of the sample group, control group, and blank group, respectively.

[0066] The results of cell viability assays are shown in Table 5 and Figure 2 As shown, the addition of each component slightly affects cell viability, but the impact is minimal. It can be seen that at a concentration of 100 μg / mL, each composition group has no significant inhibitory effect on HaCat cell activity, demonstrating good biocompatibility. While replacing the Tremella fuciformis extract in Comparative Example 6 resulted in lower irritation and milder properties, its active ingredient (Tremella fuciformis polysaccharide) primarily contributes to moisturizing and basic antioxidant effects, lacking the ability to deeply stimulate cell metabolism and immune pathways. Therefore, it cannot provide cells with the same intensity of metabolic drive, and the cell viability test results after replacement were slightly lower than in the examples.

[0067] Table 5. Cell viability test results

[0068] (2) Establishment of a lipopolysaccharide (LPS)-induced HaCat cell inflammation model: HaCaT cells in logarithmic phase were maintained at 1×102 4 Cells were evenly seeded at a density of 100 μL of cell suspension per well in 96-well plates and incubated for 24 h. The supernatant was removed, and LPS solutions of 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 6 μg / mL, and 10 μg / mL were added, with the experiment repeated 6 times. After another 24 h of incubation, cell viability was assessed using the CCK-8 assay, and the level of the inflammatory factor IL-6 was measured using a kit. Considering both cell viability and IL-6 levels, a 6-h stimulation with 1 μg / mL LPS solution was ultimately selected as the optimal condition for establishing a cellular inflammation model.

[0069] (3) ELISA method for detecting inflammatory factors in HaCaT cells: HaCaT cells in the logarithmic growth phase were injected with 3.0 × 10⁻⁶ cells. 5 Cells were evenly seeded at a density of cells / well in 6-well plates and incubated for 24 hours. A blank control group, an LPS group, a sample group, and a positive control group were set up. The LPS modeling condition was 1.0 μg / mL for 6 hours. The blank control group consisted of HaCaT cells cultured in serum-free DMEM medium for 24 hours. The sample group consisted of cells modeled with LPS followed by a 24-hour LPS treatment. The concentration and amount of samples in each group were kept consistent. The inflammatory factors IL-6, IL-8, and TNF-α in each group were detected according to the ELISA kit method, with the assay repeated 5 times. The ELISA kit was purchased from Beijing Solarbio Science & Technology Co., Ltd.

[0070] The measurement results are shown in Table 6 and Figures 3-5 As shown, the results indicate that, compared to the model group, Comparative Example 11, which lacks active ingredients, had almost no regulatory effect. Comparative Example 1, using only Ganoderma lucidum fermentation extract, significantly inhibited the release of IL-6, IL-8, and TNF-α factors. In Comparative Examples 2 and 3, combining Ganoderma lucidum fermentation extract with licorice extract, or asiaticoside and tocopherol, respectively, further inhibited the release of IL-6, IL-8, and TNF-α factors. In Comparative Example 4, without the addition of Ganoderma lucidum fermentation extract, the effect of the other components was significantly worse than that of Comparative Example 1. In Comparative Example 5, the effect of combining licorice extract with other components was poor, showing the synergistic effect of licorice extract on Ganoderma lucidum fermentation extract. Among these, Example 1 showed an effect comparable to that of a higher concentration of Comparative Example 1, demonstrating good efficacy even with reduced dosage ratios of each component. The composition shown in the embodiments can effectively regulate the release of skin-related mediators, improve abnormal skin stress, and soothe, relieve external stimuli, and repair the skin barrier. It can be used not only for daily care and stabilization of sensitive skin, but also to regulate the skin microenvironment and repair the skin barrier.

[0071] In each comparative example, due to the absence of the core Ganoderma lucidum fermentation extract or key compound components, the regulatory effect was much weaker than in Examples 2 and 3. This confirms that licorice extract, frankincense resin extract, asiaticoside, magnesium ascorbate phosphate, tocopherol, and caffeic acid can significantly enhance the effect of Ganoderma lucidum fermentation extract. After scientific compounding, each component produces a significant synergistic effect, which can improve abnormal skin stress state by regulating the release of skin-related factors.

[0072] Table 6 Results of intracellular inflammatory factor assay in HaCaT cells

[0073] 2. Antioxidant activity assay (1) Determination of DPPH free radical scavenging ability: Prepare a sample solution with a concentration of 250 μg / mL. Remove the DPPH reagent from the -20℃ freezer and equilibrate at 37℃ for at least 20 minutes until the reagent melts. Add 100 mL of anhydrous ethanol to the reagent bottle and shake vigorously to fully dissolve the reagent, obtaining a DPPH solution. Take 1 mL of the DPPH solution and add 1 mL of anhydrous ethanol and 1 mL of the sample solution to each solution, mixing them one by one. Then, take 1 mL of anhydrous ethanol and mix it with 1 mL of each sample solution of the same concentration, making a final solution volume of 2 mL. Shake thoroughly and let it stand at room temperature in the dark for 30 minutes to allow for complete reaction. Then, measure the absorbance at 517 nm, using vitamin C as a positive control sample.

[0074] Calculate the DPPH clearance rate (%) using the following formula: DPPH clearance rate (%) = [1 - (A1 - A2) / A0] × 100; Where A1 is the absorbance measured after the sample solution and DPPH solution are mixed and reacted, A2 is the absorbance measured after the sample solution and anhydrous ethanol are mixed and reacted, and A0 is the absorbance measured after the anhydrous ethanol and DPPH solution are mixed and reacted.

[0075] The test results are shown in Table 7. Comparative Example 1, which uses only Ganoderma lucidum fermentation extract, has a moderate DPPH scavenging ability, but it is better than Comparative Example 4 and Comparative Example 5, which use other components. The synergistic effect of combining licorice extract with other components is not obvious. In Comparative Example 2 and Comparative Example 3, the DPPH scavenging rate can be improved by combining Ganoderma lucidum fermentation extract with licorice extract, or asiaticoside and tocopherol, respectively, but the improvement effect is limited.

[0076] The effect of Example 1 was comparable to that of Comparative Example 1, which had a higher concentration, meaning it still achieved good results even with reduced dosages in each component. Examples 2 and 3 exhibited excellent DPPH scavenging ability, with Example 3 showing the highest scavenging rate, approaching that of the positive control vitamin C. The scavenging rates of the comparative examples were significantly lower than that of Example 2 due to the absence of the core Ganoderma lucidum fermentation extract or other components, resulting in incomplete ingredient formulation. Comparative Example 11, lacking active ingredients, showed poor scavenging ability. This demonstrates that the scientific combination of Ganoderma lucidum fermentation extract with licorice extract, frankincense resin extract, asiaticoside, magnesium ascorbate phosphate, tocopherol, and caffeic acid forms a synergistic antioxidant system, significantly enhancing the ability to scavenge free radicals.

[0077] Table 7 DPPH Scavenging Ability Results

[0078] (2) Measurement of ROS content in HaCaT cells damaged by UVB: a. Experimental Materials and Grouping ① Cell line: HaCaT cells were cultured in DMEM complete culture medium containing 10% fetal bovine serum, 100 U / mL penicillin and 100 μg / mL streptomycin, and passaged in a 37℃, 5% CO2 incubator. Cells in the logarithmic growth phase were used for experiments.

[0079] ② Group setup: The experiment was randomly divided into 3 groups, namely: Blank control group (Control group): cultured in complete culture medium only, without UVB irradiation or drug treatment; UVB irradiation group (model group): A cell damage model was established by UVB irradiation and cultured in complete culture medium only. Drug administration group: After UVB irradiation, the same 1 wt% concentration of the composition solution was administered for culture.

[0080] b. Experimental methods ① Cell seeding and culture: HaCaT cells in logarithmic growth phase were harvested and the cell density was adjusted to 1×10⁻⁶ cells using complete culture medium. 5 Cells / mL were seeded at 100 μL per well in a 96-well cell culture plate and cultured at 37°C in a 5% CO2 incubator. Further processing was carried out when the cell confluence reached about 70%.

[0081] ② Pre-treatment: Add the corresponding concentration of the composition solution (diluted with complete culture medium) to each well of the drug treatment group, and add the same amount of complete culture medium to the normal group and the UVB irradiation group. All groups are pre-cultured in a 37℃, 5% CO2 incubator for 24h.

[0082] ③ UVB irradiation treatment: Discard all culture medium from the 96-well plate, gently wash the cells twice with PBS buffer, and add 100 μL of PBS to each well to cover the cell surface. Irradiate using a UVB phototherapy device (wavelength 311 nm) at a dose of 60 mJ / cm². 2 During irradiation, keep the culture plate horizontal to prevent cells from drying out.

[0083] ④ Subsequent culture and ROS detection: After irradiation, discard the PBS in the dishes, add 2 mL of complete culture medium to each dish, and continue incubation at 37℃ and 5% CO2 for 24 h. Intracellular ROS levels were detected by flow cytometry.

[0084] Formula for calculating ROS inhibition rate: Inhibition rate (%) = (1 - T / C) × 100%; Where T is the three-time average of the relative fluorescence intensity of ROS in the drug-treated group; C is the three-time average of the relative fluorescence intensity of ROS in the model group.

[0085] The results are shown in Table 8. The results indicate that all compositions in the examples effectively inhibited UVB-induced ROS generation, with Example 3 showing the best inhibition effect. The comparative examples, lacking the core Ganoderma lucidum fermentation extract or key compound components, exhibited significantly lower ROS inhibition rates. This confirms that the scientific combination of Ganoderma lucidum fermentation extract with licorice extract, frankincense resin extract, asiaticoside, magnesium ascorbate phosphate, tocopherol, and caffeic acid produces a significant synergistic effect against photooxidation. The complete formulation system is the core for achieving efficient ROS scavenging; replacing other components with similar effects cannot achieve the same result, and Comparative Example 11, lacking active ingredients, showed the worst inhibition effect. Furthermore, replacing caffeic acid with other components with similar antioxidant activity (such as chlorogenic acid) reduced the antioxidant effect of the composition, while also demonstrating a synergistic effect with magnesium ascorbate phosphate and tocopherol.

[0086] Table 8. Results of ROS inhibition rate

[0087] Test Example 3: Effects on skin barrier repair 1. Skin moisture content and transepidermal water loss rate test (1) Experimental conditions: Volunteers with mild to moderately sensitive skin who had no serious systemic diseases, immunodeficiency or autoimmune diseases, no allergies to cosmetics, no acute inflammation, had not used hormone drugs or immunosuppressants, had not participated in other clinical trials, and had not applied topical preparations were selected. They were randomly divided into groups of 15, and each group received the samples obtained from the examples and comparative examples.

[0088] (2) Experimental methods: The test sample was used twice a day (once in the morning and once in the evening, with the same concentration). During the test, subjects avoided excessive exposure to ultraviolet light. Before use and 4 weeks after use, skin moisture content and transepidermal water loss rate were measured using a skin moisture meter (Corneomete CM825) to assess the skin's moisturizing effect after using the sample.

[0089] The test results are shown in Table 9. The results show that the compositions of the examples can increase skin hydration and reduce transepidermal water loss, exhibiting a good repairing effect on the skin barrier. After 4 weeks of product use, all examples significantly increased skin hydration and reduced transepidermal water loss, with Example 3 showing the best effect. In each comparative example, due to the lack of the core Ganoderma lucidum fermented extract or key compound components, the barrier repair effect was significantly weaker than that of the examples. This confirms that the scientific combination of Ganoderma lucidum fermented extract and each compound component produces a significant synergistic effect in skin barrier repair, better enhancing the efficacy of Ganoderma lucidum fermented extract. Comparative Example 11, lacking active ingredients, showed almost no effective improvement.

[0090] Table 9 Results of Skin Moisture Content and Transepidermal Water Loss (TEWL) Rate Tests

[0091] 2. Erythema Index Measurement The experimental method was the same as described above, with the test sample used twice daily (once in the morning and once in the evening). During the testing period, subjects avoided excessive ultraviolet radiation exposure. The erythema index (EI) was measured using a testing instrument before use and 4 weeks after use.

[0092] The test results are shown in Table 10. The results indicate that after 4 weeks of product use, the compositions of Examples 2-3 all significantly reduced the EI value, with Example 3 showing the best effect. The comparative examples, lacking the core Ganoderma lucidum fermented extract or key compound components, had significantly weaker redness-reducing effects than Examples 2-3. Comparative Example 11, which lacked active ingredients, had extremely weak redness-reducing effects. However, Example 1, with a significantly reduced proportion of active ingredients, still achieved an effect comparable to the high concentrations of active ingredients in Comparative Examples 1 and 2. This confirms that the scientific combination of Ganoderma lucidum fermented extract and various compound components has a significant synergistic effect, significantly enhancing the overall effect. The complete formula is the core of repairing erythema on sensitive skin.

[0093] Table 10 Results of Erythema Index (EI) Test

[0094] In summary, the enhanced Ganoderma lucidum fermentation extract composition provided by this invention, through precise mechanism design and ingredient compatibility, achieves a comprehensive enhancement and intelligent upgrade of the efficacy of the core active ingredients of Ganoderma lucidum. This composition exhibits significant advantages in regulating skin immune balance, multi-pathway synergistic antioxidant activity, and repairing the skin barrier. Experimental data confirms that it can effectively increase skin hydration, reduce transepidermal water loss, and alleviate erythema reactions, providing an innovative solution for developing high-end functional skincare products with antioxidant, immunomodulatory, anti-aging, and anti-wrinkle skin-rejuvenating effects.

[0095] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A composition for enhancing the effects of Ganoderma lucidum fermentation extract, characterized in that, The ingredients are as follows by weight percentage: 0.1-10% Ganoderma lucidum fermentation extract, 0.01-10% magnesium ascorbate phosphate, 0.01-5% licorice extract, 0.1-5% tocopherol, 0.05-3% frankincense resin extract, 0.01-3% asiaticoside, 0.01-0.5% caffeic acid, and the balance being solvent.

2. The composition according to claim 1, characterized in that, The composition contains the following raw materials by weight percentage: 1-10% Ganoderma lucidum fermentation extract, 0.1-5% magnesium ascorbate phosphate, 0.01-2.5% licorice extract, 0.1-2% tocopherol, 0.05-1% frankincense resin extract, 0.01-1% asiaticoside, 0.01-0.1% caffeic acid, and the balance being solvent.

3. The composition according to claim 1 or 2, characterized in that, The Ganoderma lucidum fermentation extract is a purple Ganoderma lucidum yeast fermentation extract.

4. The use of the composition according to any one of claims 1 to 3 in the preparation of products for skin anti-oxidation, immune regulation, anti-aging and / or anti-wrinkle and skin rejuvenation.

5. A cosmetic product, characterized in that, It comprises the composition according to any one of claims 1 to 3.

6. The cosmetic product according to claim 5, characterized in that, It also contains cosmetically acceptable carriers or excipients.

7. The cosmetic product according to claim 6, characterized in that, The cosmetically acceptable carriers or excipients include one or more of the following: humectants, preservatives, cosolvents, stabilizers, pH adjusters, thickeners, emulsifiers, and skin moisturizers.

8. The cosmetic product according to claim 6, characterized in that, The cosmetically acceptable carrier or excipient comprises, by weight percentage, 2-20% moisturizer, 0.1-2% preservative and cosolvent, and 0.01-1% stabilizer.

9. The cosmetic product according to claim 8, characterized in that, The humectant is one or more of panthenol, sodium hyaluronate, and 1,3-propanediol; the preservative co-solvent is p-hydroxyacetophenone / ethylhexylglycerin; and the stabilizer is xanthan gum.

10. The cosmetic product according to claim 9, characterized in that, The moisturizer comprises panthenol 1-5%, sodium hyaluronate 0.1-1%, and 1,3-propanediol 1-10%.

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