Application of combination of ganoderma lucidum extract and ergothioneine in preparation of product for repairing mitochondrial injury of skin cells

Abstract

The invention provides a combined application of a ganoderma lucidum extract and ergothioneine in preparation of a product for repairing mitochondrial injury of skin cells. According to the invention, the ganoderma lucidum extract and the ergothioneine in a mass ratio of 1: (0.5-3.5) are combined for use, so that the skin cell mitochondrial injury can be effectively repaired, and the phenomena of skin barrier injury, wrinkle formation, hyperpigmentation and the like caused by the skin cell mitochondrial injury are further improved, that is, aging caused by the skin cell mitochondrial injury is delayed.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology. More specifically, it relates to the application of Ganoderma lucidum extract combined with ergothioneine in the preparation of products for repairing mitochondrial damage in skin cells. Background Technology

[0002] As the largest organ in the human body, the skin is maintained by the coordinated efforts of various cell types. Keratinocytes (which form the epidermal barrier), fibroblasts (which secrete collagen and elastin fibers), and melanocytes (which regulate pigmentation) are the core representatives of skin cells. Damage to these cells can directly lead to aging phenotypes such as skin barrier impairment, wrinkle formation, and pigmentation.

[0003] Oxidative damage, telomere shortening, and mitochondrial damage are three key types of skin cell damage, with the difficulty of intervention increasing in that order. This is because: intervention for oxidative damage typically involves single-target strategies such as scavenging free radicals, inhibiting oxidase activity, and chelating metal ions to reduce oxidative stress-induced cell damage; the regulatory pathways for telomere shortening focus on a few core targets such as telomerase activation, antioxidant protection, and telomere-binding protein repair, and the mechanisms of action of these targets are relatively well understood; while intervention for mitochondrial damage requires addressing a multi-level interactive network involving structure (e.g., permeability transition pore abnormalities), function (inactivation of the respiratory chain complex), dynamics (fusion / division imbalance), and signaling (ROS-mediated inflammation). Furthermore, tissue-specific differences exist between targets (e.g., different cell types show significant differences in sensitivity to mitochondrial damage) and complex feedback regulation, leading to scientific challenges such as needing interventions tailored to different tissues and developing multi-target synergistic strategies, thus increasing the complexity exponentially. Summary of the Invention

[0004] This invention aims to provide the application of Ganoderma lucidum extract combined with ergothioneine in the preparation of products that repair mitochondrial damage in skin cells, thereby improving the skin barrier damage, wrinkle formation, pigmentation and other phenomena caused by mitochondrial damage in skin cells, that is, delaying aging caused by mitochondrial damage in skin cells.

[0005] The primary objective of this invention is to provide the application of Ganoderma lucidum extract combined with ergothioneine in the preparation of products for repairing mitochondrial damage in skin cells.

[0006] The second objective of this invention is to provide the application of Ganoderma lucidum extract combined with ergothioneine in the preparation of products for repairing skin barrier damage caused by mitochondrial damage to skin cells.

[0007] A third objective of this invention is to provide the application of Ganoderma lucidum extract combined with ergothioneine in the preparation of products for removing wrinkles caused by mitochondrial damage to skin cells.

[0008] The fourth objective of this invention is to provide the application of Ganoderma lucidum extract combined with ergothioneine in the preparation of products that inhibit pigmentation caused by mitochondrial damage in skin cells.

[0009] The fifth objective of this invention is to provide the application of Ganoderma lucidum extract combined with ergothioneine in the preparation of products that delay aging caused by mitochondrial damage in skin cells.

[0010] The sixth object of the present invention is to provide a composition.

[0011] The seventh objective of this invention is to provide a product.

[0012] The eighth objective of this invention is to provide a cosmetic product.

[0013] The above-mentioned objective of this invention is achieved through the following technical solution: This invention combines Ganoderma lucidum extract and ergothioneine in a mass ratio of 1:0.5–3.5, which can effectively repair mitochondrial damage in skin cells, thereby improving skin barrier impairment, wrinkle formation, and pigmentation caused by mitochondrial damage, thus delaying aging caused by mitochondrial damage in skin cells. Therefore, this invention provides the application of Ganoderma lucidum extract and ergothioneine in a mass ratio of 1:0.5–3.5 in any of the following: (1) Application in the preparation of products for repairing mitochondrial damage in skin cells; (2) Application in the preparation of products for repairing skin barrier damage caused by mitochondrial damage to skin cells; (3) Application in the preparation of products for removing wrinkles caused by mitochondrial damage to skin cells; (4) Application in the preparation of products that inhibit pigmentation caused by mitochondrial damage to skin cells; (5) Application in the preparation of products that delay aging caused by damage to mitochondria in skin cells.

[0014] Optionally, the skin cells are one or more of the following: keratinocytes, fibroblasts, and melanocytes. For example, it can repair etoposide-induced mitochondrial damage in keratinocytes.

[0015] Optionally, the mass ratio of the Ganoderma lucidum extract to ergothioneine is 1:0.5-2.

[0016] Preferably, the mass ratio of the Ganoderma lucidum extract to ergothioneine is 1:2. At this mass ratio, the Ganoderma lucidum extract and ergothioneine in the composition of this invention exert a synergistic effect in repairing mitochondrial damage in skin cells, better improving skin barrier damage, wrinkle formation, pigmentation, and other phenomena caused by mitochondrial damage in skin cells, thus better delaying aging caused by mitochondrial damage in skin cells.

[0017] Ganoderma lucidum extract is available commercially or can be prepared using conventional methods in the art, such as water extraction of Ganoderma lucidum.

[0018] Optionally, the water is prepared in advance, and the Ganoderma lucidum is also pulverized.

[0019] Optionally, the ratio of water used for the water extraction to Ganoderma lucidum is 7.8–8.2 mL: 1 g.

[0020] Optionally, the water extraction is a segmented water extraction, that is: first soaking at 20-30 ℃ for 1.5-2.5 h, and then heating and soaking at 95-100 ℃ for 1.5-2.5 h.

[0021] Optionally, the water is further dried after extraction.

[0022] Further, optionally, before drying, the product is concentrated and frozen, for example, concentrated at a pressure of -0.09 to -0.08 MPa and a temperature of 54 to 56 °C, and then frozen at -22 to -18 °C.

[0023] Alternatively, the drying process may be freeze-drying.

[0024] Furthermore, the freeze-drying is carried out at a temperature of -70 to -60 °C and a vacuum degree of 0.04 to 0.05 Pa.

[0025] Based on this, the present invention also provides a composition comprising Ganoderma lucidum extract and ergothioneine in a mass ratio of 1:0.5 to 3.5.

[0026] Optionally, the mass ratio of the Ganoderma lucidum extract to ergothioneine is 1:0.5-2.

[0027] Preferably, the mass ratio of the Ganoderma lucidum extract to ergothioneine is 1:2.

[0028] The preparation method of the composition of the present invention is very simple, requiring only the mixing of each raw material.

[0029] In addition, the present invention also provides a product containing the above composition and a cosmetic.

[0030] Preferably, the product or cosmetic also contains excipients.

[0031] The present invention has the following beneficial effects: This invention combines Ganoderma lucidum extract with ergothioneine at a mass ratio of 1:0.5 to 3.5, which can effectively repair mitochondrial damage in skin cells, thereby improving skin barrier damage, wrinkle formation, pigmentation and other phenomena caused by mitochondrial damage in skin cells, thus delaying aging caused by mitochondrial damage in skin cells. Attached Figure Description

[0032] Figure 1 This is a flow cytometry result image of the blank group in the test case.

[0033] Figure 2 This is a flow cytometry result graph of the model group in the test case.

[0034] Figure 3 This is a flow cytometry result of control group 1 in the test case.

[0035] Figure 4 This is a flow cytometry result of control group 2 in the test case.

[0036] Figure 5 This is a flow cytometry result of control group 3 in the test case.

[0037] Figure 6 This is a flow cytometry result of control group 4 in the test case.

[0038] Figure 7 This is a flow cytometry result of control group 5 in the test case.

[0039] Figure 8 This is a flow cytometry result of experimental group 1 in the test case.

[0040] Figure 9 This is a flow cytometry result of experimental group 2 in the test case.

[0041] Figure 10 This is a flow cytometry result of experimental group 3 in the test case.

[0042] Figure 11 This is a flow cytometry result image of the blank group in the comparative test case.

[0043] Figure 12 This is a flow cytometry result image of the model group in the comparative test cases.

[0044] Figure 13 This is a flow cytometry result image of the sample group in the comparative test case.

[0045] Figure 14 This is a statistical result of the proportion of cells with low membrane potential in the three groups of comparative test cases.

[0046] In the figure, "JC-1 Red" represents JC-1 red fluorescence, and "JC-1 Green" represents JC-1 green fluorescence. Detailed Implementation

[0047] The present invention will be further illustrated below with reference to 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.

[0048] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0049] The extraction method of Ganoderma lucidum extract in this embodiment of the invention includes the following steps: S1. Water Extraction: First, pulverize the Ganoderma lucidum in a pulverizer, then place it in an extraction tank, add distilled water at 25 ℃ (making the water-to-Ganoderma lucidum ratio 8 mL:1 g), soak for 2 h, then heat at 100 ℃ for 2 h, and filter to obtain the extract and residue. Transfer the extract to a concentration tank, and repeat the extraction once with the residue as described above, then combine the extracts.

[0050] S2. Concentration: Set the temperature of the concentration tank to 55 ℃ and the pressure to -0.09~-0.08 MPa, and concentrate until the system becomes viscous, then remove it.

[0051] S3. Freeze-drying: Place the concentrated product in a -20 ℃ freezer. After the concentrate solidifies, transfer it to a freeze dryer and freeze-dry at a cold trap of -65 ℃ and a vacuum of 0.05 Pa. The endpoint of freeze-drying is defined as follows: weigh 2 g of the freeze-dried product into a weighing bottle and dry it in an oven at 105 ℃ for 5 h. The difference between two consecutive weighings should not exceed 5 mg.

[0052] Example 1 A composition Ganoderma lucidum extract and ergothioneine were mixed at a mass ratio of 1:2.

[0053] Example 2 A composition Ganoderma lucidum extract and ergothioneine were mixed at a mass ratio of 1:1.

[0054] Example 3 A composition Ganoderma lucidum extract and ergothioneine were mixed at a mass ratio of 1:0.5.

[0055] Test case Mitochondria, acting as the cell's "powerhouse," drive the reflux of hydrogen ions through the high potential difference between their inner and outer membranes, coupling ADP to ATP, thus providing energy for cellular metabolism. A decrease in mitochondrial membrane potential indicates mitochondrial damage. JC-1 is an ideal fluorescent probe widely used to detect mitochondrial membrane potential, capable of detecting the membrane potential of cells, tissues, or purified mitochondria. When the mitochondrial membrane potential is high, JC-1 aggregates in the mitochondrial matrix, forming J-aggregates and producing red fluorescence; when the mitochondrial membrane potential is low, JC-1 cannot aggregate in the mitochondrial matrix, remaining a monomer and producing green fluorescence. Therefore, the transition from red to green fluorescence represents a decrease in mitochondrial membrane potential, while enhanced green fluorescence indicates a low mitochondrial membrane potential.

[0056] This test case experiment was divided into 10 groups, with 3 parallel samples in each group. The specific treatment for each group is as follows: (1) Blank group: Human skin keratinocytes (HaCaT) in the logarithmic growth phase in DMEM high glucose medium were evenly seeded into 12-well plates at a density of 200,000 cells / mL, 1 mL per well. After the cells adhered, the original medium was discarded, and 1 mL of DMEM high glucose medium was added. The cells were cultured at 37 ℃ and 5% CO2 for 2 days. The supernatant was then aspirated, the cells were washed once with PBS, and then 500 μL of trypsin was added for digestion for 5 min. Then, 500 μL of DMEM high glucose medium was added to stop the digestion and the cell suspension was collected. After centrifuging the cell suspension, resuspend it in 500 μL of DMEM high-glucose medium, then add 500 μL of JC-1 staining working solution (dilute JC-1 by adding 1 mL of JC-1 staining buffer to every 5 µL of JC-1 (200×) and mix thoroughly by pipetting). Incubate at 37 ℃ for 20 min, then centrifuge at 4 ℃ and 600 g for 3.5 min to precipitate the cells. Discard the supernatant, wash twice with JC-1 staining buffer, and finally resuspend the cells in 500 μL of JC-1 staining buffer.

[0057] (2) Model group: Human skin keratinocytes (HaCaT) in the logarithmic growth phase in DMEM high-glucose medium were evenly seeded into 12-well plates at a density of 200,000 cells / mL, 1 mL per well. After the cells adhered, the original medium was discarded, and 1 mL of DMEM high-glucose medium containing 1 μM etoposide was added. The cells were cultured at 37 ℃ and 5% CO2 for 2 days. The supernatant was then aspirated, the cells were washed once with PBS, and then 500 μL of trypsin was added for digestion for 5 min. Then, 500 μL of DMEM high-glucose medium was added to stop the digestion and the cell suspension was collected. After centrifuging the cell suspension, resuspend it in 500 μL of DMEM high-glucose medium, then add 500 μL of JC-1 staining working solution (dilute JC-1 by adding 1 mL of JC-1 staining buffer to every 5 µL of JC-1 (200×) and mix thoroughly by pipetting). Incubate at 37 ℃ for 20 min, then centrifuge at 4 ℃ and 600 g for 3.5 min to precipitate the cells. Discard the supernatant, wash twice with JC-1 staining buffer, and finally resuspend the cells in 500 μL of JC-1 staining buffer.

[0058] (3) Control group 1: Human skin keratinocytes (HaCaT) in the logarithmic growth phase in DMEM high glucose medium were evenly seeded into 12-well plates at a density of 200,000 cells / mL, 1 mL per well. After the cells adhered, the original medium was discarded, and 1 mL of DMEM high glucose medium containing 1 μM etoposide and 0.2 mg / mL Ganoderma lucidum extract was added. The cells were cultured at 37 ℃ and 5% CO2 for 2 days. The supernatant was then aspirated, the cells were washed once with PBS, and then 500 μL of trypsin was added for digestion for 5 min. Then, 500 μL of DMEM high glucose medium was added to stop the digestion and the cell suspension was collected. After centrifuging the cell suspension, resuspend it in 500 μL of DMEM high-glucose medium, then add 500 μL of JC-1 staining working solution (dilute JC-1 by adding 1 mL of JC-1 staining buffer to every 5 µL of JC-1 (200×) and mix thoroughly by pipetting). Incubate at 37 ℃ for 20 min, then centrifuge at 4 ℃ and 600 g for 3.5 min to precipitate the cells. Discard the supernatant, wash twice with JC-1 staining buffer, and finally resuspend the cells in 500 μL of JC-1 staining buffer.

[0059] (4) Control group 2: Human skin keratinocytes (HaCaT) in the logarithmic growth phase in DMEM high glucose medium were evenly seeded into 12-well plates at a density of 200,000 cells / mL, 1 mL per well. After the cells adhered, the original medium was discarded, and 1 mL of DMEM high glucose medium containing 1 μM etoposide and 0.4 mg / mL Ganoderma lucidum extract was added. The cells were cultured at 37 ℃ and 5% CO2 for 2 days. The supernatant was then aspirated, the cells were washed once with PBS, and then 500 μL of trypsin was added for digestion for 5 min. Then, 500 μL of DMEM high glucose medium was added to stop the digestion and the cell suspension was collected. After centrifuging the cell suspension, resuspend it in 500 μL of DMEM high-glucose medium, then add 500 μL of JC-1 staining working solution (dilute JC-1 by adding 1 mL of JC-1 staining buffer to every 5 µL of JC-1 (200×) and mix thoroughly by pipetting). Incubate at 37 ℃ for 20 min, then centrifuge at 4 ℃ and 600 g for 3.5 min to precipitate the cells. Discard the supernatant, wash twice with JC-1 staining buffer, and finally resuspend the cells in 500 μL of JC-1 staining buffer.

[0060] (5) Control group 3: Human skin keratinocytes (HaCaT) in the logarithmic growth phase in DMEM high glucose medium were evenly seeded into 12-well plates at a density of 200,000 cells / mL, 1 mL per well. After the cells adhered, the original medium was discarded, and 1 mL of DMEM high glucose medium containing 1 μM etoposide and 0.1 mg / mL ergothioneine was added. The cells were cultured at 37 ℃ and 5% CO2 for 2 days. The supernatant was then aspirated, the cells were washed once with PBS, and then 500 μL of trypsin was added for digestion for 5 min. Finally, 500 μL of DMEM high glucose medium was added to stop the digestion and the cell suspension was collected. After centrifuging the cell suspension, resuspend it in 500 μL of DMEM high-glucose medium, then add 500 μL of JC-1 staining working solution (dilute JC-1 by adding 1 mL of JC-1 staining buffer to every 5 µL of JC-1 (200×) and mix thoroughly by pipetting). Incubate at 37 ℃ for 20 min, then centrifuge at 4 ℃ and 600g for 3.5 min to precipitate the cells. Discard the supernatant, wash twice with JC-1 staining buffer, and finally resuspend the cells in 500 μL of JC-1 staining buffer.

[0061] (6) Control group 4: Human skin keratinocytes (HaCaT) in the logarithmic growth phase in DMEM high glucose medium were evenly seeded into 12-well plates at a density of 200,000 cells / mL, 1 mL per well. After the cells adhered, the original medium was discarded, and 1 mL of DMEM high glucose medium containing 1 μM etoposide and 0.2 mg / mL ergothioneine was added. The cells were cultured at 37 ℃ and 5% CO2 for 2 days. The supernatant was then aspirated, the cells were washed once with PBS, and then 500 μL of trypsin was added for digestion for 5 min. Finally, 500 μL of DMEM high glucose medium was added to stop the digestion and the cell suspension was collected. After centrifuging the cell suspension, resuspend it in 500 μL of DMEM high-glucose medium, then add 500 μL of JC-1 staining working solution (dilute JC-1 by adding 1 mL of JC-1 staining buffer to every 5 µL of JC-1 (200×) and mix thoroughly by pipetting). Incubate at 37 ℃ for 20 min, then centrifuge at 4 ℃ and 600g for 3.5 min to precipitate the cells. Discard the supernatant, wash twice with JC-1 staining buffer, and finally resuspend the cells in 500 μL of JC-1 staining buffer.

[0062] (7) Control group 5: Human skin keratinocytes (HaCaT) in the logarithmic growth phase in DMEM high glucose medium were evenly seeded into 12-well plates at a density of 200,000 cells / mL, 1 mL per well. After the cells adhered, the original medium was discarded, and 1 mL of DMEM high glucose medium containing 1 μM etoposide and 0.4 mg / mL ergothioneine was added. The cells were cultured at 37 ℃ and 5% CO2 for 2 days. The supernatant was then aspirated, the cells were washed once with PBS, and then 500 μL of trypsin was added for digestion for 5 min. Finally, 500 μL of DMEM high glucose medium was added to stop the digestion and the cell suspension was collected. After centrifuging the cell suspension, resuspend it in 500 μL of DMEM high-glucose medium, then add 500 μL of JC-1 staining working solution (dilute JC-1 by adding 1 mL of JC-1 staining buffer to every 5 µL of JC-1 (200×) and mix thoroughly by pipetting). Incubate at 37 ℃ for 20 min, then centrifuge at 4 ℃ and 600g for 3.5 min to precipitate the cells. Discard the supernatant, wash twice with JC-1 staining buffer, and finally resuspend the cells in 500 μL of JC-1 staining buffer.

[0063] (8) Experimental Group 1: Human skin keratinocytes (HaCaT) in the logarithmic growth phase in DMEM high glucose medium were evenly seeded into 12-well plates at a density of 200,000 cells / mL, 1 mL per well. After the cells adhered, the original medium was discarded, and 1 mL of DMEM high glucose medium containing 1 μM etoposide and 0.6 mg / mL of the composition obtained in Example 1 was added. The cells were cultured at 37°C and 5% CO2 for 2 days. The supernatant was then aspirated, the cells were washed once with PBS, and then 500 μL of trypsin was added for digestion for 5 min. Then, 500 μL of DMEM high glucose medium was added to stop the digestion and the cell suspension was collected. After centrifuging the cell suspension, resuspend it in 500 μL of LMEM high-glucose medium, then add 500 μL of JC-1 staining working solution (dilute JC-1 by adding 1 mL of JC-1 staining buffer to every 5 µL of JC-1 (200×) and mix thoroughly by pipetting). Incubate at 37 ℃ for 20 min, then centrifuge at 4 ℃ and 600 g for 3.5 min to precipitate the cells. Discard the supernatant, wash twice with JC-1 staining buffer, and finally resuspend the cells in 500 μL of JC-1 staining buffer.

[0064] (9) Experimental Group 2: Human skin keratinocytes (HaCaT) in the logarithmic growth phase in DMEM high-glucose medium were evenly seeded into 12-well plates at a density of 200,000 cells / mL, 1 mL per well. After the cells adhered, the original medium was discarded, and 1 mL of DMEM high-glucose medium containing 1 μM etoposide and 0.4 mg / mL of the composition obtained in Example 2 was added. The cells were cultured at 37°C and 5% CO2 for 2 days. The supernatant was then aspirated, the cells were washed once with PBS, and then 500 μL of trypsin was added for digestion for 5 min. Then, 500 μL of DMEM high-glucose medium was added to stop the digestion and the cell suspension was collected. After centrifuging the cell suspension, resuspend it in 500 μL of LMEM high-glucose medium, then add 500 μL of JC-1 staining working solution (dilute JC-1 by adding 1 mL of JC-1 staining buffer to every 5 µL of JC-1 (200×) and mix thoroughly by pipetting). Incubate at 37 ℃ for 20 min, then centrifuge at 4 ℃ and 600 g for 3.5 min to precipitate the cells. Discard the supernatant, wash twice with JC-1 staining buffer, and finally resuspend the cells in 500 μL of JC-1 staining buffer.

[0065] (10) Experimental Group 3: Human skin keratinocytes (HaCaT) in the logarithmic growth phase in DMEM high glucose medium were evenly seeded into 12-well plates at a density of 200,000 cells / mL, 1 mL per well. After the cells adhered, the original medium was discarded, and 1 mL of DMEM high glucose medium containing 1 μM etoposide and 0.3 mg / mL of the composition obtained in Example 3 was added. The cells were cultured at 37°C and 5% CO2 for 2 days. The supernatant was then aspirated, the cells were washed once with PBS, and then 500 μL of trypsin was added for digestion for 5 min. Then, 500 μL of DMEM high glucose medium was added to stop the digestion and the cell suspension was collected. After centrifuging the cell suspension, resuspend it in 500 μL of LMEM high-glucose medium, then add 500 μL of JC-1 staining working solution (dilute JC-1 by adding 1 mL of JC-1 staining buffer to every 5 µL of JC-1 (200×) and mix thoroughly by pipetting). Incubate at 37 ℃ for 20 min, then centrifuge at 4 ℃ and 600 g for 3.5 min to precipitate the cells. Discard the supernatant, wash twice with JC-1 staining buffer, and finally resuspend the cells in 500 μL of JC-1 staining buffer.

[0066] According to the instructions of the JC-1 mitochondrial membrane potential detection kit, JC-1 fluorescence in 10 groups was detected by flow cytometry. The proportion of cells with low membrane potential was calculated according to the formula: "Percentage of cells with low membrane potential (%) = Number of cells with low mitochondrial membrane potential / Total number of cells × 100%". Based on the intermediate-effect principle (Chou-Talalay combination index method), dose-response curves (C-fa curves) and combination index curves (fa-CI curves) under different effects were plotted using CompuSyn statistical software. The effect and nature of the interaction between the two components, Ganoderma lucidum extract and ergothioneine, were quantitatively evaluated from the relationship between the effect and the combination index. A combination index CI=1 indicates an additive effect between the two components, CI>1 indicates an antagonistic effect, and CI<1 indicates a synergistic effect. The results are as follows: Figures 1-10 As shown in Table 1. Among them, Figure 1 This is a flow cytometry result image of the control group. Figure 2 This is a flow cytometry result image of the model group. Figure 3 This is a flow cytometry result image of control group 1. Figure 4 This is a flow cytometry result image of control group 2. Figure 5 This is a flow cytometry result image of control group 3. Figure 6 This is a flow cytometry result image of control group 4. Figure 7 This is a flow cytometry result image of control group 5. Figure 8 This is a flow cytometry result image of experimental group 1. Figure 9This is a flow cytometry result image of experimental group 2. Figure 10 This is a flow cytometry result image of experimental group 3.

[0067] Table 1

[0068] In Table 1, "**" indicates that the results of the experimental group were significantly different from those of the model group (P < 0.01). 。

[0069] It is evident that, compared to the control group, etoposide induction weakened the red fluorescence and enhanced the green fluorescence of the model group JC-1, indicating a significant decrease in the mitochondrial membrane potential of its skin cells. In contrast, experimental groups 1-3, treated with the compositions of Examples 1-3, significantly alleviated the etoposide-induced decrease in mitochondrial membrane potential, effectively repairing mitochondrial damage and thus improving skin barrier impairment, wrinkle formation, and pigmentation caused by mitochondrial damage, thereby delaying aging caused by mitochondrial damage. Furthermore, the combined index (CI) of Ganoderma lucidum extract and ergothioneine in experimental group 1 was less than 1, indicating that when the mass ratio of Ganoderma lucidum extract to ergothioneine is 1:2, the two can exert a synergistic effect in repairing mitochondrial damage, better improving skin barrier impairment, wrinkle formation, and pigmentation caused by mitochondrial damage, thus better delaying aging caused by mitochondrial damage.

[0070] Comparative test cases Adrenal medullary chromaffin cells (PC12) in the logarithmic growth phase were taken in DMEM high glucose medium and evenly seeded into 12-well plates at a density of 100,000 cells / mL, 1 mL per well. After the cells adhered, the original medium was discarded and the cells were divided into three groups: (1) 1 mL of DMEM high glucose medium was added to the blank group; (2) 1 mL of DMEM high glucose medium containing 1 μM etoposide was added to the model group; and (3) 1 mL of DMEM high glucose medium containing 1 μM etoposide and the combination obtained in Example 1 at 0.6 mg / mL was added to the sample group.

[0071] After culturing the three groups at 37 ℃ and 5% CO2 for 2 days, the supernatant was discarded, the cells were washed once with PBS, and then 500 μL of trypsin was added for 5 min of digestion. Digestion was then terminated with 500 μL of DMEM high-glucose medium, and the cell suspension was collected. The cell suspension was centrifuged and resuspended in 500 μL of DMEM high-glucose medium, followed by 500 μL of JC-1 staining working solution (JC-1 was diluted at a ratio of 1 mL JC-1 staining buffer per 5 µL of JC-1 (200×) and repeatedly pipetteted to mix). The cells were incubated at 37 ℃ for 20 min, then centrifuged at 4 ℃ and 600 g for 3.5 min to precipitate the cells. The supernatant was discarded, and the cells were washed twice with JC-1 staining buffer. Finally, 500 μL of JC-1 staining buffer was added to resuspend the cells.

[0072] According to the instructions for the JC-1 mitochondrial membrane potential detection kit, JC-1 fluorescence in the three groups was detected by flow cytometry. The percentage of cells with low membrane potential was calculated using the formula: "Percentage of cells with low membrane potential (%) = Number of cells with low mitochondrial membrane potential / Total number of cells × 100%". The results are shown in Table 2. Figures 11-14 As shown, where, Figure 11 This is a flow cytometry result image of the control group. Figure 12 This is a flow cytometry result image of the model group. Figure 13 This is a flow cytometry result image of the sample group. Figure 14 These are the statistical results of the proportion of cells with low membrane potential in the three groups.

[0073] Table 2

[0074] In Table 2, "####" indicates that the results of the model group are highly significant compared with the blank group (P < 0.0001). 。

[0075] As can be seen, compared with the blank group, etoposide induction weakened the red fluorescence and enhanced the green fluorescence of the model group JC-1, indicating a significant decrease in the mitochondrial membrane potential of its adrenal medullary chromaffin cells. The sample group treated with the composition of Example 1 could slightly alleviate the decrease in mitochondrial membrane potential of adrenal medullary chromaffin cells induced by etoposide, but there was no significant difference between the sample group and the model group. This indicates that the composition of Example 1 has no significant effect on improving mitochondrial damage in adrenal medullary chromaffin cells. It also shows that different types of cells have significantly different sensitivities to mitochondrial damage, and the intervention method cannot be simply transferred.

[0076] 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. The use of Ganoderma lucidum extract and ergothioneine in combination in the preparation of a product for repairing mitochondrial damage of skin cells, characterized in that, The mass ratio of the Ganoderma lucidum extract to ergothioneine is 1:0.5-3.

5.

2. The use of the extract of Ganoderma lucidum in combination with ergothioneine in the preparation of a product for repairing skin barrier damage caused by mitochondrial damage of skin cells, characterized in that, The mass ratio of the Ganoderma lucidum extract to ergothioneine is 1:0.5-3.

5.

3. Use of the extract of Ganoderma lucidum and ergothioneine in the preparation of a product for removing wrinkles caused by mitochondrial damage in skin cells, characterized in that, The mass ratio of the Ganoderma lucidum extract to ergothioneine is 1:0.5-3.

5.

4. Use of Ganoderma lucidum extract in combination with ergothioneine for the preparation of a product for inhibiting pigmentation caused by mitochondrial damage in skin cells, characterized in that, The mass ratio of the Ganoderma lucidum extract to ergothioneine is 1:0.5-3.

5.

5. Use of Ganoderma lucidum extract in combination with ergothioneine for the preparation of a product for delaying aging caused by mitochondrial damage in skin cells, characterized in that, The mass ratio of the Ganoderma lucidum extract to ergothioneine is 1:0.5-3.

5.

6. Use according to any one of claims 1 to 5, characterized in that, The mass ratio of the Ganoderma lucidum extract to ergothioneine is 1:0.5-2.

7. A composition characterized in that, The mass ratio of the Ganoderma lucidum extract to ergothioneine is 1:0.5-3.

5.

8. A product characterized by, The composition of claim 7.

9. A cosmetic product, characterized by, The composition of claim 7.

10. The product according to claim 8 or the cosmetic product according to claim 9, characterized in that, The composition of claim 7. The composition of claim 7.

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