TW202106325A - Use ofrosa rugosaextract for manufacturing composition inhibiting skin aging - Google Patents

Abstract

The present disclosure provides a use ofRosa rugosa extract for manufacturing a composition for inhibiting skin aging, where the composition is used for inhibiting the production of melanin caused by blue light illumination, inhibiting the production of reactive oxygen species on the skin cells caused by blue light illumination, promoting the antioxidation ability for skin cells, and promoting the activity of Mitochondria.

Description

Use of double petal rose extract for preparing composition for inhibiting skin aging

This case is about the application of double-petal rose extract, especially the use of a double-petal rose extract to prepare a composition for inhibiting skin aging.

The skin provides human individuals with the first stage of protection against environmental factors such as ultraviolet (UV) radiation, pathogens and friction in the sun. The skin includes the epidermis layer, the dermis layer mainly composed of connective tissue, and the subcutaneous tissue from the outside to the inside.

The skin will age normally with age, resulting in various damaged skin conditions. For example, decreased skin elasticity, insufficient water retention (dryer, dull skin), large pores, wrinkles, spots on the skin and/or overall dullness, sallow and other aging conditions.

However, under certain environmental factors, it will accelerate the process of skin aging. For example, common natural factors such as ultraviolet rays in sunlight. When exposed to ultraviolet rays, ultraviolet rays will accelerate the generation of the aforementioned skin aging conditions.

In addition, because in modern life, people use electronic products with screens for a significant increase in time. The visible light (hereinafter referred to as blue light) with a shorter wavelength and higher energy emitted by the screen will also affect the skin for a long time It will accelerate the generation of the aforementioned skin aging conditions. Therefore, for skin aging, blue light has also become a factor that cannot be ignored.

In view of this, the present invention provides a use of a double-petal rose extract, which is used to prepare an anti-aging composition. Among them, the double petal rose extract can slow down the skin damage caused by ultraviolet or blue light to provide skin protection against ultraviolet or blue light, thereby slowing down skin aging, thereby maintaining or improving the appearance of the skin.

In some embodiments, a use of a double-petal rose extract is to prepare a composition for inhibiting the production of melanin caused by blue light.

In some embodiments, a use of double petal rose extract is to prepare a composition that inhibits the generation of reactive oxygen species (ROS) caused by blue light in skin cells.

In some embodiments, a use of double petal rose extract is to prepare a composition for enhancing the antioxidant capacity of skin cells.

In some embodiments, a use of double petal rose extract is to prepare a composition for enhancing mitochondrial activity.

In some embodiments, the aforementioned composition is a pharmaceutical composition.

In some embodiments, the aforementioned composition is a food composition or a skin care composition.

In some embodiments, the aforementioned composition is an internal composition or an external composition.

Since this case proves that double petal rose extract can inhibit the production of free radicals and inhibit melanin production, double petal rose extract can effectively prevent various skin aging phenomena caused by ultraviolet or blue light, and provide skin protection against ultraviolet and blue light.

In addition, this case also confirmed that double petal rose extract can enhance the activity of mitochondria. According to this, the double petal rose extract can not only passively reduce the loss of the skin affected by free radicals, but also actively enhance the activity of mitochondria and make the skin healthier.

The following will describe some specific implementation aspects of this case. Without departing from the spirit of this case, this case can still be practiced in many different forms, and the scope of protection should not be limited to the conditions specified in the specification.

Regarding the concentration symbol "wt%" used in this article usually refers to weight percent concentration, and the concentration symbol "vol%" usually refers to volume percent concentration.

In the diagram, "*" represents the p-value is less than 0.05, "**" represents the p-value is less than 0.01, and "***" represents the p-value is less than 0.001. The more "*", the more significant the statistical difference.

In this case, Excel software was used for statistical analysis. Data mean ± standard deviation (SD), said difference between the groups in a Student's t-test (student's t -test) was analyzed.

The values used in this article are approximate values, all experimental data are expressed in the range of plus or minus 10%, the best is in the range of plus or minus 5%.

In some embodiments, the double rose extract obtained from the buds of Rosa rugosa may have at least one of the following abilities: inhibiting the generation of free radicals, inhibiting the production of melanin, and enhancing the activity of mitochondria. Therefore, the double-petal rose extract can be used to prepare a composition having at least one of the following abilities: inhibit free radical generation, inhibit melanin production, and enhance mitochondrial activity.

In some embodiments, the double-petal rose extract can effectively prevent skin aging caused by ultraviolet or blue light, so as to provide skin protection against ultraviolet and blue light, thereby slowing down skin aging caused by ultraviolet or blue light, thereby maintaining or Improve skin appearance.

In some embodiments, the double petal rose extract effectively prevents skin aging caused by ultraviolet light or blue light by at least one of the following effects: inhibiting the production of melanin caused by blue light, and inhibiting the reactive oxygen species of skin cells caused by blue light. The production of substances, enhance the antioxidant capacity of skin cells and enhance the activity of mitochondria.

Here, ultraviolet light or blue light will promote the production of reactive oxygen species in skin cells. Because the active oxygen substances are highly active, they will react with the collagen molecules in the skin to break down the collagen. Since collagen is like a "scaffold" in the skin, it is used to give support and elasticity to the skin. When the skin lacks collagen, it will accelerate the aging of the skin, causing the skin to have reduced elasticity and insufficient water retention (the skin is relatively dry) , Dullness), enlarged pores, and wrinkles and other aging phenomena. On the other hand, ultraviolet light or blue light can also induce melanin cells in the skin to produce melanin, causing the skin to produce spots and/or the overall dullness or sallow aging.

In other words, double petal rose extract can effectively prevent at least one of the following skin aging phenomena: inhibit skin elasticity reduction, prevent insufficient water retention (skin is dry and dull), inhibit large pores, inhibit wrinkles, inhibit skin spots, and prevent skin Dull or sallow.

In some embodiments, a double-petaled rose refers to a rose with more than 17 petals, especially a rose with 17-25 petals. In some embodiments, the rose petals may be purple petals Rose (Rosa rugosa cv.plena) and / or the white rose petals (Rosa rugosa cv. Albo-plena ).

In some embodiments, the aforementioned composition may be a pharmaceutical composition, a skin care product composition, or a food composition.

Among them, the aforementioned pharmaceutical composition contains an effective content of double petal rose extract.

In some embodiments, the pharmaceutical composition may further include a pharmaceutically acceptable carrier that is widely used in pharmaceutical manufacturing technology. For example, a pharmaceutically acceptable carrier may include one or more of the following carriers: emulsifier, suspending agent, decomposer, disintegrating agent, dispersing agent ), binding agent, excipient, stabilizing agent, chelating agent, diluent, gelling agent, preservative, Wetting agent, lubricant, absorption delaying agent, liposome and the like. The selection and quantity of these carriers can be selected by those skilled in the art according to the situation.

In some embodiments, the pharmaceutically acceptable carrier may further comprise any one of the following solvents: water, normal saline (normal saline), phosphate buffered saline (PBS), aqueous solution containing alcohol (aqueous solution containing alcohol) and any other suitable solvents.

In some embodiments, the pharmaceutical composition can be manufactured into a pharmaceutical composition suitable for enterally, parenterally, orally, or topically using techniques well known to those skilled in the art ( topically) the dosage form for administration. The dosage form of the pharmaceutical composition may be, for example, injection (for example, sterile aqueous solution or dispersion), sterile powder, external preparation or other analog.

In some embodiments, the pharmaceutical composition can be administered by any of the following parenteral routes: subcutaneous injection, intraepidermal injection, intradermal injection And intralesional injection.

In some embodiments, the pharmaceutical composition can be manufactured into an external preparation suitable for topical application to the skin using techniques well known to those skilled in the art. For example, the external preparation can be any of the following, but not limited to: emulsion, gel, ointment, cream, patch, liniment (Liniment), powder (powder), aerosol (aerosol), spray (spray), emulsion (lotion), emulsion (serum), paste (paste), foam (foam), drops (drop), suspension (Suspension), ointment (salve) and bandage (bandage).

In some embodiments, the external preparation is made by mixing an effective content of double petal rose extract with a base known to those skilled in the art.

In some embodiments, the substrate may include one or more of the following additives: water, alcohols, glycols, hydrocarbons (such as petroleum jelly) and white petrolatum (White petrolatum,)], wax (such as paraffin and yellow wax), preserving agents, antioxidants, surfactants, absorption enhancers enhancers, stabilizing agents, gelling agents [such as Carbopol®974P (carbopol®974P), microcrystalline cellulose and carboxymethylcellulose], active Active agents, humectants, odor absorbers, fragrances, pH adjusting agents, chelating agents, emulsifiers, occlusive agents ( occlusive agents, emollients, thickeners, solubilizing agents, penetration enhancers, anti-irritants, colorants, and propellants ( propellants) and so on. The selection and quantity of these additives fall within the scope of professionalism and routine technology of those who are familiar with this technology.

Wherein, the aforementioned skin care product composition contains an effective content of double rose extract.

In some embodiments, the skin care composition may further include an acceptable adjuvant that is widely used in skin care product manufacturing technology. For example, acceptable adjuvants may include one or more of the following adjuvants: solvents, gelling agents, active agents, preservatives, antioxidants, screening agents, chelating agents, surfactants, coloring agents ), thickening agent, filler, fragrance and odor absorber. The selection and quantity of these reagents can be appropriately adjusted according to actual needs.

In some embodiments, the skin care composition can be manufactured into a form suitable for skincare or makeup using techniques well known to those skilled in the art. Among them, the type suitable for skin care or makeup can be any of the following, but not limited to: aqueous solution (aqueous solution), water-alcohol solution (aqueous-alcohol solution), oily solution (oily solution), water Oil-in-water type, water-in-oil type or compound emulsion, gel, ointment, cream, mask, patch, patch ), liniment, powder, aerosol, spray, lotion, emulsion, paste, foam, dispersion, drops, mousse, sunblock, tonic water, foundation ), makeup remover products, soap and other body cleansing products, etc.

In some embodiments, the skin care composition may further include one or more of the following external use agents with known activity: whitening agents (such as tretinoin, tretinoin), Catechin, kojic acid, arbutin and vitamin C], humectants, bactericides, ultraviolet absorbers, plant extracts [such as aloe extract )], skin nutrients, anesthetics, anti-acne agents, antipruritics, analgesics, antidermatitis agents, anti-drugs Antihyperkeratolytic agents, anti-dry skin agents, antipsoriatic agents, antiaging agents, antiwrinkle agents, antiseborrheic agents ), wound-healing agents, corticosteroids and hormones. The selection and quantity of these topical agents fall within the scope of professionalism and routine techniques of those who are familiar with this technique.

Wherein, the aforementioned food composition contains an effective content of double petal rose extract.

In some embodiments, the food composition can be administered to an individual orally. Among them, the form of the food composition can be powder, granule, solution, colloid or paste. The individual can be a human.

In some embodiments, the food composition may be a food product or food additive.

In some embodiments, food additives can be added during the preparation of raw materials by conventional methods, or added during the production process of food, and be formulated with any edible material to be ingested by humans and non-human animals. product.

In some embodiments, the food product may be, but is not limited to: beverages, fermented foods, bakery products, health foods, or dietary supplements.

In some embodiments, the "double rose extract" can also be an extract obtained by extracting the buds of double rose using a suitable extraction solvent at a specific extraction temperature and an appropriate extraction time. For example, the buds of a double rose can be soaked in water (ie extraction solvent) for an appropriate soaking time (ie extraction time) at a specific extraction temperature, and then cooled to 25 ℃-30 ℃ after soaking to obtain a double rose Petal extract.

In some embodiments, the weight ratio of the extraction solvent to the buds of the double rose can also be 1:5-1:15. The specific extraction temperature can be 50-80°C. The appropriate extraction time can be 1-3 hours. Among them, if the extraction solvent is too little or the extraction time is too short, the extraction efficiency will be significantly reduced; if the extraction time is too long, the active ingredients in the extract may be degraded.

In some embodiments, the buds of the double-petaled rose can be whole buds, or buds separated into fragments, granules, or powders after physical pretreatment. Among them, the physical pretreatment may include at least one of the following: shredding, shearing, mashing, and grinding.

In some embodiments, the buds of the double-petaled rose are soaked in water for an appropriate soaking time to form a first extract containing solids. The first extract is filtered to remove solid impurities, and the double rose extract is obtained after filtration. In other words, depending on actual needs, the double rose extract can be the first extract before filtration or the filtrate after filtration.

In some embodiments, the filtered filtrate can be concentrated under reduced pressure, and the double rose extract can be obtained after concentration under reduced pressure. According to actual needs, the double-petal rose extract can be the filtrate before concentration under reduced pressure, or the concentrated solution after concentration under reduced pressure. In some embodiments, the filtered filtrate may be concentrated under reduced pressure at 45°C to 70°C.

Unless otherwise stated, the experimental procedures in the following examples are carried out at room temperature (25 ℃-30 ℃) and normal pressure (1 atm).

[Example 1] Preparation of double rose extract

Materials and instruments 1. Double rose (origin: Iran) 2. Centrifuge (brand/model: Thermo Scientific Heraeus Fresco 17) 3. Thickener (brand/model: BUCHI -Rotavapor R-100)

Experimental steps 1. Mix the water and the buds of double-flowered roses at a weight ratio of 1:10, and soak them at 65±5°C for about 2 hours to form the first extract containing solids. 2. Cool the first extract to room temperature. 3. Centrifuge the cooled first extract in a centrifuge for about 10 minutes, and take the supernatant after centrifugation (hereinafter referred to as the first extraction supernatant). 4. Filter the first extraction supernatant with a 400 mesh filter to remove fine solids. 5. The filtered first extraction supernatant is concentrated under reduced pressure with a concentrator at 60 ℃ ± 5 ℃, until the degree of Brix of the concentrate (Degrees Brix) is 10 ± 0.5, the concentration is stopped to obtain a double Rose extract. In other words, the Brix value of double rose extract is 10±0.5.

[Example 2] Effectiveness quantitative experiment-determination of polyphenol content

Materials and instruments 1. Gallic acid (purchased from Sigma, product model G7384). 2. Folin-Ciocalteu’s phenol reagent (purchased from Merck, product model 1.09001). 3. Sodium carbonate (purchased from Sigma, product model 31432). 4. Enzyme Immunoassay Analyzer (BioTek Epoch)

First, prepare a standard solution and draw a standard curve. Dissolve 10 mg of gallic acid in water and quantify it to 10 mL with water in a measuring flask to obtain a stock solution of gallic acid. Here, the stock solution of gallic acid can be stored at -20 ℃ for later use. After that, the stock solution was diluted 10 times to a concentration of 100 μg/mL to obtain an initial solution of gallic acid (that is, containing 1000 ppm of gallic acid). The initial solution of unused gallic acid can be stored at -20 ℃. Then, prepare 0 μg/mL, 20 μg/mL, 40 μg/mL, 60 μg/mL, 80 μg/mL, and 100 μg/mL standard solutions of gallic acid in glass test tubes according to Table 1 below.

Table I Concentration (μg/mL) 0 20 40 60 80 100 Initial solution (μL) 0 20 40 60 80 100 Water (μL) 100 80 60 40 20 0

After that, take 100 μL of the standard solution of each concentration into the glass test tube, add 500 μL of the phoschorol reagent to each glass test tube, mix the two evenly and let it stand for 3 minutes. Then add 400 μL of 7.5% sodium carbonate to each glass test tube, mix the solution in each glass test tube uniformly and react for 30 minutes to obtain a standard reaction solution. Then, after the standard reaction solution is vortexed to ensure that there are no bubbles, 200 μL of the standard reaction solution is taken to measure the absorbance at 750 nm, and a standard curve is drawn.

In addition, the double rose extract obtained in Example 1 was diluted 10 times with water to obtain a diluted solution. Take 100 μL of the diluted solution into a glass test tube. After that, add 500 μL of phoschorol reagent to the glass test tube to mix with the diluted solution and let it stand for 3 minutes. Then add 400 μL of 7.5% sodium carbonate to the glass test tube, mix the solution in the glass test tube uniformly and react for 30 minutes to 1 hour to obtain the reaction solution to be tested. Then, after vortexing the reaction solution to ensure that there is no air bubbles, take 200 μL of the reaction solution to be tested and measure the absorbance at 750 nm with an enzyme immunoassay.

Then, the absorbance value of the reaction solution to be tested is converted into a concentration by the internal difference method of the standard curve, and then multiplied by the dilution factor to obtain the total polyphenol content of the double rose extract.

Here, the total polyphenol content of the double rose extract is 3500 ppm (after the extract is diluted 1:10, the final result is 3500 ppm).

[Example 3] Cell test-skin cell mitochondrial activity test

In order to explore the effect of double rose extract on the mitochondrial function of skin cells, this example uses flow cytometry to evaluate human skin fibroblast (Human skin fibroblast) CCD-966sk after being treated with double rose extract. Changes in thread activity.

Materials and equipment 1. Cell line: Human skin fibroblasts CCD-966sk (Biological Resources Conservation and Research Center (BCRC), No. 60153), hereinafter referred to as CCD-966sk cells. 2. Cell culture medium: basic medium containing 10 vol% FBS (fetal bovine serum, purchased from Gibco, product number 10437-028). Eagle's minimum essential medium (Eagle's minimum essential medium (MEM), purchased from Gibco, product number 15188-319). Among them, the basic medium is made up of Eagle's minimum essential medium (Eagle's minimum essential medium (MEM), purchased from Gibco, product number 15188-319) with additional ingredients to contain 1 mM sodium pyruvate (sodium pyruvate, purchased from Gibco, product No. 11360-070), 1.5 g/L sodium bicarbonate (sodium bicarbonate, purchased from Sigma, product number 31432-500G), and 0.1 mM non-essential amino acid solution, purchased from Gibco, product number 11140 -050). 3. Phosphate buffered saline solution (PBS solution): purchased from Gibco, product number 10437-028. 4. Mitochondrial membrane potential detection kit (BD ^TM MitoScreen (JC-1) kit, model 551302). Among them, the mitochondrial membrane potential detection kit has JC-1 dye (lyophilized) and 10X analysis buffer. Before use, 10X assay buffer was diluted 10 times with 1X PBS to form 1X assay buffer. Add 130 μL of dimethyl sulfoxide (DMSO; purchased from Sigma, product number: D4540) to JC-1 stain (lyophilized) to form a JC-1 stock solution. Then, dilute the JC-1 stock solution with 1X analysis buffer to form the JC-1 working solution (JC-1 mitochondrial specific stain). The dilution ratio is 1:100 from JC-1 stock solution to 1X analysis buffer. 5. Trypsin (Trypsin-EDTA): 10X Trypsin-EDTA (purchased from Gibco; product number 15400-054) diluted 10 times with 1X PBS solution. 6. Flow cytometry: purchased from BD Pharmingen, model BDTM Accuri C6 Plus.

Experimental steps

The experiment will be divided into two groups: the experimental group and the control group (the group without double rose extract), and each group will be subjected to three repeated experiments: 1. The CCD-966sk cells are divided into 1×10 ⁵ cells per well. Method, inoculate in a 6-well culture dish containing 2 mL of cell culture medium per well. 2. Replace the medium in each well of the culture plate with 2 mL of experimental medium. Among them, the experimental medium of the experimental group was 10 μL of the double rose extract obtained in Example 1, and 2 ml of medium was added to obtain a cell culture medium containing 5 μL/mL of the double rose extract obtained in Example 1. The experimental medium of the control group was a pure cell culture medium (that is, without double-petal rose extract). 3. Place the culture plate in 5% CO ₂ , 37 ℃, and incubate for 24 hours. 4. Remove the experimental medium from the culture plate and rinse twice with 1 mL of 1X PBS solution. 5. Add 200 μL trypsin to each well and react for 5 minutes in the dark. After the reaction, add 6 mL of cell culture medium to stop the reaction. Collect the cells and cell culture medium in each well into a corresponding 15 mL centrifuge tube, and centrifuge the tube containing the cells and cell culture medium at 400 xg for 10 minutes. 6. After centrifugation, remove the supernatant, and re-dissolve it with 1 mL of 1X PBS or transfer the cell pellet to a 1.5 mL test tube to form a cell suspension. 7. Centrifuge the tube containing the cell suspension at 400 xg for 5 minutes. 8. After centrifugation, remove the supernatant from each test tube and add 100 μL of JC-1 working reagent to each test tube. 9. Vortex the cell pellets and JC-1 working reagent in each test tube evenly and incubate for 15 minutes in the dark. 10. After 15 minutes, centrifuge each tube at 400 xg for 5 minutes. 11. After centrifugation, remove the supernatant in each test tube, re-dissolve the cell pellet in each test tube with 1 mL of 1X PBS solution, and centrifuge at 400 xg for 5 minutes. 12. After centrifugation, remove the supernatant from each test tube, re-dissolve the cell pellet in each test tube with 1 mL of 1X PBS solution, and centrifuge at 400 xg for 5 minutes. 13. After centrifugation, remove the supernatant from each tube and resuspend the cells in 500 μL of 1X PBS containing 2% FBS to obtain the cell sap to be tested. 14. Measure the membrane potential of cell mitochondria in the liquid to be tested in each well with a flow cytometer for mitochondrial activity analysis. Since the experiment is performed twice, the results of the two repeated experiments in each group are averaged to obtain the average value, and then the average value of the control group is used as the relative JC-1 aggregation amount of 100%, and the average value of the experimental group is converted into a relative value. The accumulation of JC-1 is shown in Figure 1.

It can be seen from the results in Figure 1 that with the control group as the 100% reference standard, the experimental group has been treated with double petal rose extract, and its relative JC-1 aggregation is about 155%. In other words, compared with the control group, the mitochondrial activity of human skin fibroblasts in the experimental group was significantly increased (about 1.5 times). It shows that double petal rose extract can enhance the mitochondrial activity of skin cells and achieve the effect of enhancing skin cell activity.

[Example 4] Cell test-Double rose extract inhibits ROS generation (blue light irradiation)

Here, the fluorescent probe DCFH-DA was combined with a flow cytometer to measure the changes in the content of reactive oxygen species in CCD-966sk cells after being treated with double rose extract and irradiated with blue light.

Materials and instruments 1. Cell line: Human skin fibroblast CCD-966sk (BCRC, No. 60153). 2. Cell culture medium: Containing 1:1 ratio of DMEM culture medium (Dulbecco's Modified Eagle Medium, purchased from Gibco, product number 12100-046) and Hans F12 culture medium (Ham's F12 Nutrient Mixture, purchased from Gibco, product number 12500- 026), to which 0.5 mM sodium pyruvate (purchased from Gibco, product number 11360-070) and 15 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, HEPES) and 10 vol% FBS (purchased from Gibco, product number 10437-028). 4. DCFH-DA solution: Dissolve 2,7-dichloro-dihydro-fluorescein diacetate (DCFH-DA; product code SI-D6883-50MG, purchased from Sigma) in DMSO To prepare a 5 mg/mL DCFH-DA solution. 5. Flow cytometer: purchased from Beckman, product model 660519. 6. Blue light box (wavelength 400 nm-500 nm).

Experimental steps

The experiment will be divided into three groups: experimental group, control group (no double rose extract and blue light irradiation group), and control group (no double rose extract, but blue light irradiation group) Two repeated experiments were performed in each group: 1. Inoculate CCD-966sk cells in a 6-well culture dish containing 2 mL of medium ^{per well at 2×10 5 cells per well.} 2. Place the culture plate in 5% CO ₂ and 37 ℃ for 24 hours. 3. Remove the cell culture medium from each well of the culture plate. 4. Add 2mL of experimental medium to each well of the culture plate and incubate at 37°C for 1 hour. The experimental medium of the experimental group was 10 μL of the double rose extract obtained in Example 1, and 2 ml of medium was added to obtain a cell culture medium containing 5 μL/mL of the double rose extract obtained in Example 1. The experimental medium of the control group was a pure cell culture medium (that is, without double-petal rose extract). The experimental medium of the control group was a simple cell culture medium (that is, without double-petal rose extract). 5. Add 2 μL of cell culture medium containing 5 mg/mL DCFH-DA solution to each well, and allow DCFH-DA to treat the cells for 15 minutes. 6. After the DCFH-DA treatment, each group was incubated under the following conditions for 15 minutes. Experimental group: The 6-well culture plate treated with DCFH-DA was moved to a blue light box and exposed to blue light for 15 minutes at room temperature. Control group: The 6-well culture plate treated with DCFH-DA was placed in a dark place and allowed to stand at room temperature for 15 minutes. Control group: Move the 6-well culture plate treated with DCFH-DA to a blue light box and let it be irradiated with blue light for 15 minutes at room temperature. 7. After 15 minutes, rinse each well twice with 1 mL of 1X PBS solution. 8. Add 200 μL of trypsin to each well and react for 5 minutes in the dark. After the reaction, add 6 mL of cell culture medium to stop the reaction. 9. Collect the cells and cell culture medium from each well into the corresponding 1.5 mL centrifuge tube, and centrifuge the tube containing the cells and cell culture medium at 400 xg for 10 minutes. 10. After centrifugation, remove the supernatant, and re-dissolve the cell pellet into a cell suspension with 1X PBS solution. 11. Centrifuge the cell suspension at 400 xg for another 10 minutes. 12. After centrifugation, remove the supernatant and resuspend the cells in 1 mL of 1X PBS solution in the dark to obtain the cell liquid to be tested. 13. Use a flow cytometer to detect the fluorescent signal of DCFH-DA in the cell liquid to be tested in each well. The excitation wavelength for fluorescence detection is 450-490 nm, and the emission wavelength is 510-550 nm. As DCFH-DA enters the cell, it will be firstly hydrolyzed to DCFH (dichlorodihydroluciferin), and then oxidized by reactive oxygen species into DCF (dichloroluciferin) that emits green fluorescence, and is treated by DCFH-DA The fluorescence intensity of the cells can reflect the content of reactive oxygen species in the cells, and by this means the ratio of the number of cells with highly expressed reactive oxygen species to the number of original cells can be known. Since the experiment is performed twice, the measurement results of the two repeated experiments in each group are averaged to get the average value, and then the average value of the control group is taken as 100% of the relative ROS generation amount, and the control group and the experimental group are averaged The value is converted to relative ROS generation, as shown in Figure 2.

Experimental results

As shown in Figure 2, comparing the control group and the control group, it can be seen that after blue light irradiation, there is a higher relative ROS generation (high fluorescence performance), that is, compared to the control group, the control group has a significant increase in ROS generation (about 5.4 Times); It shows that blue light irradiation does cause the production of reactive oxygen species in the cells, which in turn causes subsequent damage to skin fibroblasts. On the other hand, comparing the control group and the experimental group, when the cells are treated with double rose extract, the relative ROS production is significantly reduced by about 21% compared with the control group; it shows that double rose extract can effectively reduce The production or accumulation of reactive oxygen species in cells. In other words, double rose extract can be used as a scavenger of active oxygen species. In other words, the double-petal rose extract can reduce the oxidative damage caused by blue light and ultraviolet rays by reducing the content of reactive oxygen species in the cells.

[Example 5] Cell experiment-double rose extract reduces melanin production

Here, an enzyme-linked immunosorbent assay reader was used to measure the changes in melanin content of melanoma cells B16F10 after being treated with double rose extract and irradiated with blue light.

Materials and instruments 1. Cell line: mouse melanoma cell B16F10 (ATCC CRL-6475), purchased from American Type Culture Collection (ATCC), hereinafter referred to as B16F10 cell. 2. Cell culture medium: DMEM culture medium (Dulbecco's Modified Eagle Medium, purchased from Gibco, product number 12100-046), with 1 vol% penicillin-streptomycin (Penicillin-streptomycin, purchased from Gibco, product No. 15140122) and 10 vol% FBS (purchased from Gibco, product number 10437-028). 3. Phosphate buffered saline solution (PBS solution): purchased from Gibco, product number 10437-028. 4. Prepare a 1N NaOH solution with double distilled water. NaOH is purchased from Sigma, product number 221465. 5. Enzyme immunoassay analyzer: purchased from BioTek, product model FLx 800. 6. Blue light box: wavelength 400 nm-500 nm.

Experimental steps

The experiment will be divided into three groups: experimental group, control group (group without double rose extract and blue light irradiation), and control group (group without double rose extract but blue light irradiation) Groups, each group performed three repeated experiments: 1. B16F10 cells were seeded in a 6-well culture dish containing 3 mL of cell culture medium ^{per well at 1.5×10 5 cells per well.} 2. Place the culture plate in 5% CO ₂ , 37 ℃ environment, and incubate for 24 hours. 3. Then, without disturbing the attached cells, remove the cell culture medium from each well. 4. Add 3 mL of experimental medium to each well of the culture plate and incubate at 37°C for 1 hour. The experimental medium of the experimental group was 10 μL of the double rose extract obtained in Example 1, and 2 ml of medium was added to obtain a cell culture medium containing 5 μL/mL of the double rose extract obtained in Example 1. The experimental medium of the control group was a pure cell culture medium (that is, without double-petal rose extract). The experimental medium of the control group was a simple cell culture medium (that is, without double-petal rose extract). 5. After 1 hour of incubation, each group was incubated for 3 hours under the following conditions. Experimental group: The 6-well culture plate was moved to a blue light box, and it was exposed to blue light for 3 hours at room temperature. Control group: Move the 6-well culture plate to a dark room and let it stand at room temperature for 3 hours. Control group: Move the 6-well culture plate to the blue light box and let it be irradiated with blue light for 3 hours at room temperature. 6. After 3 hours of incubation, incubate each group of culture plates at 37°C for 48 hours. 7. Then, remove the medium and rinse twice with 1 mL of 1X PBS solution. 8. Add 200 μL of trypsin to each well of the culture plate and react at room temperature for 3 minutes to make the cells fall off the culture plate, and collect the cells in each well with the cell culture solution to the corresponding 1.5 mL centrifugation test tube. Then, centrifuge each tube at 400 xg for 5 minutes, and remove the supernatant after centrifugation. 9. After re-dissolving the cell pellet with 1X PBS solution, centrifuge again at 400 xg for 10 minutes. After centrifugation again, the supernatant was removed, and the cells were resuspended in 200 μL of 1X PBS solution to obtain a cell suspension. 10. Freeze the cell suspension in each centrifuge tube under liquid nitrogen for 10 minutes, and then put it at room temperature for about 30 minutes to completely thaw. 11. After completely thawing, centrifuge each tube at 12,000 xg for 30 minutes. 12. After centrifugation, the supernatant was removed, and the cell pellet was resuspended in 120μL of 1 N sodium hydroxide solution, and then the centrifuge tubes were dried at 60 ℃ for 1 hour to obtain the samples to be tested. 13. Transfer 100 μL of the sample to be tested into a 96-well plate, and use an ELISA plate reader to measure the optical density of the cell solution at 450 nm (hereinafter referred to as OD ₄₅₀ value). Since the experiment is performed in three replicates, the measured values of each group of three replicate experiments are averaged to obtain the OD ₄₅₀ value of each group.

Experimental results

Experimental group, control group, and the relative expression levels of melanin-based control group calculated according to the following equation: Relative expression levels of melanin (%) = (OD ₄₅₀ values in each group / OD ₄₅₀ value of the control group) × 100%, as shown in FIG .

As shown in Figure 3, comparing the results of the control group and the control group, after blue light irradiation, the relative melanin expression in the cells will increase significantly (about 1.8 times); it shows that blue light irradiation does promote the production of melanoma cells Melanin, which will cause spots on the skin or make the skin dull overall. On the other hand, comparing the results of the control group and the experimental group, it can be seen that when the cells are treated with double rose extract, their relative melanin expression under blue light is lower than that of cells without double rose extract (decreased About 72%); It shows that double petal rose extract can effectively reduce the melanin produced by melanocytes due to blue light.

[Example 6] Cell experiment-double rose extract inhibits ROS generation (hydrogen peroxide treatment)

Here, the fluorescent probe DCFH-DA was used in conjunction with a flow cytometer to measure the changes in the content of reactive oxygen species in CCD-966sk cells after being treated with double rose extract and reacted with hydrogen peroxide.

Materials and equipment 1. Cell line: human skin fibroblast CCD-966sk (BCRC No. 60153). 2. Cell culture medium: basic medium containing 10 vol% FBS (purchased from Gibco, product number 10437-028). Eagle's minimal minimal medium (purchased from Gibco, product number 15188-319). Among them, the basic medium is made up of Eagle's minimum essential medium (Eagle's minimum essential medium (MEM), purchased from Gibco, product number 15188-319) with additional ingredients to contain 1 mM sodium pyruvate (sodium pyruvate, purchased from Gibco, product No. 11360-070), 1.5 g/L sodium bicarbonate (sodium bicarbonate, purchased from Sigma, product number 31432-500G), and 0.1 mM non-essential amino acid solution, purchased from Gibco, product number 11140-050). 3. Phosphate buffered saline solution (PBS solution): purchased from Gibco, product number 10437-028. 4. DCFH-DA solution: Dissolve dichlorodihydroluciferin diacetate (product number SI-D6883-50MG, purchased from Sigma) in dimethyl sulfoxide to prepare 5 mg/mL DCFH- DA solution. 5. Hydrogen peroxide (H ₂ O ₂ ): purchased from Sigma. 6. Flow cytometer: purchased from Beckman, product model 660519.

Experimental steps

The experiment will be divided into three groups: experimental group, control group (no double rose extract and no hydrogen peroxide treatment group), and control group (no double rose extract, but hydrogen peroxide treatment group) Two repeated experiments were performed in each group: 1. Inoculate CCD-966sk cells in a 6-well culture dish containing 2 mL of cell culture medium ^{per well at 2×10 5 cells per well.} 2. Place the culture plate in 5% CO ₂ , 37 ℃, and incubate for 24 hours. 3. Remove the cell culture medium. 4. Add 2 mL of experimental medium to each well of the culture plate and incubate at 37 ℃ for 1 hour. The experimental medium of the experimental group was 10 μL of the double rose extract obtained in Example 1, and 2 ml of medium was added to obtain 2 mL of cell culture medium supplemented with 5 μL/mL of the double rose extract obtained in Example 1. The experimental medium of the control group was a simple 2 mL cell culture medium (that is, no double-petal rose extract). The experimental medium of the control group was a simple 2 mL cell culture medium (that is, no double-petal rose extract). 5. Add 2 μL of DCFH-DA solution at a concentration of 5 mg/mL to the cell culture medium in each well, and allow DCFH-DA to treat the cells for 15 minutes. _{6. After DCFH-DA treatment, add H 2} O _{2 to} the experimental medium of the experimental group and the experimental medium of the control group respectively, and react at 37 ℃ for 1 hour. Specifically, 35% wt of hydrogen peroxide is first diluted to 100 mM (10 μL of hydrogen peroxide is added to 990 μL of double distilled water), and then 20 μL of 100 mM hydrogen peroxide is added to a 2 mL cell culture dish. 7. After the reaction, rinse each well with 1 mL of 1X PBS solution twice. 8. Add 200 μL of trypsin to each well and react for 5 minutes in the dark. After the reaction, add 6 mL of cell culture medium to stop the reaction. 9. Collect the cells and cell culture medium in each well into a corresponding 1.5 mL centrifuge tube, and centrifuge the centrifuge tube containing the cells and culture medium at 400 xg for 10 minutes. 10. After centrifugation, remove the supernatant and re-dissolve the cell pellet with 1X PBS solution. 11. Centrifuge at 400 xg for another 10 minutes. 12. After centrifugation, remove the supernatant and resuspend the cells in 1 mL of 1X PBS solution in the dark to obtain the cell liquid to be tested. 13. Use a flow cytometer to detect the fluorescent signal of DCFH-DA in the cell liquid to be tested in each well. The excitation wavelength for fluorescence detection is 450-490 nm, and the emission wavelength is 510-550 nm. Since DCFH-DA enters the cell, it is firstly hydrolyzed to DCFH, and then oxidized by reactive oxygen species to DCF that emits green fluorescence. The fluorescence intensity of cells treated with DCFH-DA can reflect the content of reactive oxygen species in the cell, and From this, the ratio of the number of cells with high expression of reactive oxygen species in the cell to the number of original cells is known. Since the experiment is performed twice, the measurement results of the two repeated experiments in each group are averaged to get the average value, and then the average value of the control group is taken as 100% of the relative ROS generation amount, and the control group and the experimental group are averaged The value is converted to relative ROS generation, as shown in Figure 4.

Experimental results

As shown in Figure 4, comparing the results of the control group and the control group, it can be seen that after hydrogen peroxide treatment, the relative ROS production (high fluorescence performance) will greatly increase; it shows that hydrogen peroxide treatment does cause the production of reactive oxygen species in the cells. This in turn causes subsequent damage to skin fibroblasts. On the other hand, comparing the results of the control group and the experimental group shows that when the cells are treated with double rose extract, the relative ROS production is significantly reduced by about 97%, even lower than the control group; it shows that double rose extract can be Effectively reduce the production or accumulation of reactive oxygen species in cells. In other words, double rose extract can be used as a scavenger of active oxygen species. In other words, double rose extract can reduce the oxidative damage caused by reactive oxygen species by reducing the content of reactive oxygen species in cells.

Based on the foregoing, this case proves that the double-petal rose extract can inhibit the production of free radicals and/or inhibit the production of melanin. Therefore, the double-petal rose extract can effectively prevent various skin aging phenomena caused by ultraviolet or blue light, thereby providing the skin with UV or blue light Protection. In addition, this case also confirmed that double petal rose extract can enhance the activity of mitochondria. According to this, the double petal rose extract can not only passively reduce the loss of the skin affected by free radicals, but also actively enhance the activity of mitochondria and make the skin healthier.

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Figure 1 is a comparison chart of the relative JC-1 aggregation results between the control group and the experimental group. Figure 2 is a comparison chart of the relative amount of reactive oxygen species (ROS) produced in the control group, the control group and the experimental group. Figure 3 is a comparison chart of the results of the melanin expression of the control group, the control group and the experimental group. Figure 4 is a comparison chart of the results of the proportion of cells with high ROS performance in the control group and the experimental group.

Claims (8)

A use of a double-petal rose extract for preparing a composition for inhibiting melanin production caused by blue light. A use of a double rose extract for preparing a composition that inhibits the generation of reactive oxygen species (ROS) caused by blue light in skin cells. A use of a double-petal rose extract for preparing a composition for improving the antioxidant capacity of skin cells. An application of a double rose extract for preparing a composition for enhancing mitochondrial activity. The use according to any one of claim 1 to claim 4, wherein the composition is a pharmaceutical composition. The use according to any one of claim 1 to claim 4, wherein the composition is a food composition or a skin care product composition. The use according to any one of claim 1 to claim 4, wherein the composition is an oral composition. The use according to any one of claim 1 to claim 4, wherein the composition is a composition for external use.

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