CN112386633A - Use of rose juice with multiple petals in preparing composition for inhibiting skin aging - Google Patents
Use of rose juice with multiple petals in preparing composition for inhibiting skin aging Download PDFInfo
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- CN112386633A CN112386633A CN202010820559.4A CN202010820559A CN112386633A CN 112386633 A CN112386633 A CN 112386633A CN 202010820559 A CN202010820559 A CN 202010820559A CN 112386633 A CN112386633 A CN 112386633A
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- rose juice
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-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
Abstract
The invention provides application of rose juice with double petals in preparing a composition for inhibiting skin aging, wherein the composition is used for inhibiting melanin generation caused by blue light, inhibiting active oxygen substance generation of skin cells caused by blue light, and improving the oxidation resistance and the mitochondrial activity of the skin cells.
Description
Technical Field
The application relates to application of rose juice with double petals, in particular to application of the rose juice with the double petals in preparing a composition for inhibiting skin aging.
Background
The skin provides a first stage of protection for human subjects against environmental factors such as Ultraviolet (UV) radiation, pathogens, friction in the sun. The skin comprises, in order from the outside to the inside, an epidermal layer, a dermal layer mainly composed of connective tissue, and a subcutaneous tissue.
Skin ages normally with age, resulting in various impaired skin conditions. For example, the skin has aging symptoms such as decreased skin elasticity, insufficient water retention (dry and dull skin), large pores, wrinkles, blotches and/or dullness on the whole, and yellow wax.
However, under certain environmental factors, the process of skin aging is accelerated. For example, natural factors such as ultraviolet rays in sunlight are generally common. When exposed to ultraviolet light, the ultraviolet light causes the aforementioned skin aging conditions to accelerate.
In addition, since the time of using electronic products with screens is greatly increased in modern life, the visible light (hereinafter referred to as blue light) emitted by the screens and having shorter wavelength and higher energy also leads to the accelerated generation of the above-mentioned skin aging state under the condition of long-time irradiation of skin. Therefore, blue light also becomes a factor that cannot be ignored for skin aging.
Disclosure of Invention
In view of the above, the present invention provides a use of rose juice with double petals, which is used for preparing a composition for resisting skin aging. The rose juice with multiple petals can relieve damage of skin caused by ultraviolet rays or blue light, so that the skin can be protected against the ultraviolet rays or the blue light, skin aging is relieved, and skin appearance is maintained or improved.
In some embodiments, use of a heavy-petal rose juice for preparing a composition for inhibiting melanin production by blue light.
In some embodiments, use of a heavy-petal rose juice is for preparing a composition for inhibiting the production of Reactive Oxygen Species (ROS) by skin cells due to blue light.
In some embodiments, the use of a rose juice from the rose of the double petal type for preparing a composition for increasing the antioxidant capacity of skin cells.
In some embodiments, use of a rose juice of the double petal type for preparing a composition for increasing mitochondrial activity.
In some embodiments, the aforementioned composition is a pharmaceutical composition.
In some embodiments, the aforementioned composition is a food composition or a cosmetic composition.
In some embodiments, the aforementioned composition is an internal composition or an external composition.
The double-petal rose juice can effectively prevent various skin aging phenomena caused by ultraviolet rays or blue light and provide the skin with protection against ultraviolet rays and blue light because the double-petal rose juice can inhibit the generation of free radicals and melanin.
In addition, the present application also proves that the rose juice with double petals can improve the activity of mitochondria. Therefore, the rose juice with the double petals can passively reduce the loss of skin affected by free radicals, and can actively improve the activity of mitochondria so as to make the skin healthy.
The invention is described in detail below with reference to the drawings and specific examples, but the invention is not limited thereto.
Drawings
Figure 1 is a graph comparing the results of relative JC-1 aggregation for control versus experimental groups.
FIG. 2 is a graph comparing the results of the control group, the control group and the experimental group with respect to the amount of Reactive Oxygen Species (ROS) produced.
FIG. 3 is a graph comparing the results of melanin expression in the control group, the control group and the experimental group.
FIG. 4 is a graph comparing the results of high ROS expression in the control and experimental groups.
Detailed Description
Some embodiments of the present disclosure will be described below. The present disclosure may be embodied in many different forms without departing from the spirit thereof, and the scope of protection should not be limited to the details set forth in the specification.
As used herein, the concentration symbol "wt%" generally refers to weight percent concentration, while the concentration symbol "vol%" generally refers to volume percent concentration.
In the figures ". x" represents a p value of less than 0.05, ". x" represents a p value of less than 0.01, and ". x" represents a p value of less than 0.001. As more ". sup.", the statistical differences are represented more significantly.
Statistical analysis was performed using Excel software. Data are expressed as mean ± Standard Deviation (SD) and differences between groups were analyzed by student's t-test (student's t-test).
As used herein, the numerical values are approximate and all experimental data are shown to be within a range of plus or minus 10%, and more preferably within a range of plus or minus 5%.
In some embodiments, the rose juice obtained from the buds of a rose with heavy petals (Rosa rugosa) can have at least one of the following capabilities: inhibiting free radical generation, inhibiting melanin generation, and increasing mitochondrial activity. Thus, the heavy-petal rose juice can be used to prepare a composition having at least one of the following capabilities: inhibiting free radical generation, inhibiting melanin generation, and increasing mitochondrial activity.
In some embodiments, the rose juice can effectively prevent skin aging caused by ultraviolet light or blue light, so as to provide protection to the skin against ultraviolet light and blue light, and further slow down skin aging caused by ultraviolet light or blue light, thereby maintaining or improving the appearance of the skin.
In some embodiments, the rose juice is effective for preventing skin aging caused by ultraviolet or blue light by at least one of: inhibiting melanin generation caused by blue light, inhibiting active oxygen substance generation of skin cells caused by blue light, and improving oxidation resistance and mitochondrial activity of skin cells.
In this way, both ultraviolet and blue light can promote skin cells to generate active oxygen substances. And because the active oxygen substance has high activity, the active oxygen substance can react with collagen molecules in the skin to further crack the collagen. Since collagen, such as "scaffold" in the skin, is used to provide support and elasticity to the skin, skin aging is accelerated in the absence of collagen, resulting in skin aging phenomena such as decreased elasticity, insufficient water retention (dry and dull skin), enlarged pores, and wrinkles. On the other hand, ultraviolet or blue light also induces melanocytes in the skin to produce melanin, which causes skin to have aging phenomena such as spots and/or overall darkness or yellow wax.
In other words, the rose juice of the double petal type is effective in preventing at least one of the following skin aging phenomena: inhibiting decrease of skin elasticity, preventing insufficient water retention (dry and lusterless skin), inhibiting pore enlargement, inhibiting wrinkle generation, inhibiting skin speckle, and preventing skin darkness or yellow wax.
In some embodiments, a heavy petal rose refers to a rose having more than 17 petals, particularly a rose having 17-25 petals. In some embodiments, the rose with heavy petals can be purple flower rose (Rosa rugosa cv. plina) and/or white flower rose (Rosa rugosa cv. albo-plina).
In some embodiments, the composition can be a pharmaceutical composition, a cosmetic composition, or a food composition.
Wherein the pharmaceutical composition comprises an effective amount of rose juice of the double petal type.
In some embodiments, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier (pharmaceutically acceptable carrier) that is widely used in pharmaceutical manufacturing technology. For example, a pharmaceutically acceptable carrier can comprise one or more of the following carriers: emulsifying agents (emulsifying agent), suspending agents (suspending agent), disintegrating agents (disintegrant), disintegrating agents (disintegrating agent), dispersing agents (dispersing agent), binding agents (binding agent), excipients (excipient), stabilizing agents (stabilizing agent), chelating agents (chelating agent), diluents (diluent), gelling agents (gelling agent), preservatives (preserving), wetting agents (wetting agent), lubricants (lubricating agent), absorption delaying agents (absorbing delaying agent), liposomes (liposome), and the like. The choice and amount of such carriers can be readily selected by one of ordinary skill in the art.
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 alcohol-containing solution (aqueous solution stabilizing alcohol), and any other suitable solvent.
In some embodiments, the pharmaceutical composition may be formulated into a dosage form suitable for enteral (enterally), parenteral (parenterally), oral, or topical (topically) administration using techniques well known to those skilled in the art. The dosage form of the pharmaceutical composition may be, for example: injections (injection) [ e.g., sterile aqueous solution (sterile aqueous solution) or dispersion (dispersion) ], sterile powders (sterile powder), external preparations (external preparation), or the like.
In some embodiments, the pharmaceutical composition may be administered by any of the parenteral routes (parenteral routes) described below: subcutaneous injection (subecanal injection), intradermal injection (intraepithelial injection), and intralesional injection (intralesion).
In some embodiments, the pharmaceutical composition can be manufactured into an external preparation (external preparation) suitable for topical application to the skin using techniques well known to those skilled in the art. For example, the external agent may be any of the following, but is not limited thereto: creams (lotions), liniments (liniments), powders (powders), aerosols (aerogels), sprays (sprays), emulsions (positions), serums (serums), pastes (pastes), foams (foams), drops (drops), suspensions (suspensions), ointments (salves), and bandages (bandages).
In some embodiments, the external preparation is prepared by mixing an effective amount of the heavy-petal rose juice with a base (base) as is well known to those skilled in the art.
In some embodiments, the substrate may comprise additives (additives) of one or more of the following: water, alcohols, glycols, hydrocarbons such as petroleum jelly and white petrolatum]Wax (wax) [ such as Paraffin and yellow wax (yellow wax)]Preserving agents (preserving agents), antioxidants (antioxidants), surfactants (surfactants), absorption enhancers (absorption enhancers), stabilisers (stabilizing agents), gelling agents (gelling agents) [ such as
974P(
974P), microcrystalline cellulose (microcrystalline cellulose) and carboxymethyl cellulose (carboxymethyl cellulose)]Active agents (actives), moisturizers (humectants), odor absorbers (odor absorbers), perfumes (fragrans), pH adjusting agents (pH adjusting agents), chelating agents (chelating agents), emulsifiers (emulsifiers), occlusive agents (occlusive agents), softeners (emulsifiers), thickeners (thickeners), solubilizing agents (solubilizing agents), penetration enhancers (penetration enhancers), anti-irritants (anti-irritants), colorants (colorants), and propellants (propellants). The selection and amounts of such additives are within the skill and routine skill of those skilled in the art.
Wherein the cosmetic composition comprises an effective amount of rose juice.
In some embodiments, the care composition may further comprise an acceptable adjuvant (acceptable adjuvant) that is widely used in care manufacturing technology. For example, an acceptable adjuvant may comprise one or more of the following adjuvants: solvents, gelling agents, active agents, preservatives, antioxidants, screening agents, chelating agents, surfactants, colouring agents, thickening agents, fillers, fragrances and odour absorbers. The selection and the amount of the reagents can be properly adjusted according to actual requirements.
In some embodiments, the cosmetic composition may be manufactured in a form suitable for skin care (skincare) or make-up (makeup) using techniques well known to those skilled in the art. Among them, the form suitable for skin care or make-up may be any one of the following, but is not limited thereto: aqueous solutions (aqueous solutions), aqueous-alcoholic solutions (aqueous-alcoholic solutions), oily solutions (oil solutions), emulsions in the form of oil-in-water type, water-in-oil type or compound type, gels, ointments, creams, masks (masks), patches (packs), liniments, powders, aerosols, sprays, lotions, serums, pastes, foams, dispersions, drops, mousses (mousses), sunblocks, lotions (toiletries), foundations (foundations), make-up removal products (makeup remover products), soaps (soaps), and other body cleansing products (body cleansing products).
In some embodiments, the cosmetic composition may further comprise one or more of the following known active topical agents (external use agents): whitening agents (whitening agents) [ such as vitamin a acid (tretinoin), catechin (catechin), kojic acid, arbutin and vitamin C ], moisturizing agents, bactericides (bacteriodes), ultraviolet absorbers (ultravirosomes), plant juices (plant extracts) [ such as aloe vera juice (aloe extract) ], skin nutrients (skin nutrients), anesthetics (anesthesics), antiacne agents (anti-acne agents), antipruritics (antipruritics), analgesics (analgesis), anti-dermatitis agents (anti-dermatitis agents), anti-hypercholesterolitics (anti-xerosis agents), anti-dry skin agents (anti-dry skin agents), antiperspirants (antipsoriatics), anti-aging agents (anti-aging agents), anti-aging agents (anti-inflammation agents), anti-wrinkle agents (anti-inflammation agents), anti-aging agents (anti-wrinkle agents), anti-aging agents (anti-seborrheic agents), anti-aging agents (anti-corticosteroid), and anti-corticosteroid (anti-aging agents). The selection and amounts of such agents for external use are within the skill and routine skill of those skilled in the art.
Wherein the food composition comprises an effective amount of rose juice from the cloves.
In some embodiments, the food composition may be administered to the subject orally. Wherein the food composition is in the form of powder, granule, solution, colloid, or paste. The subject may be a human.
In some embodiments, the food composition may be a food product or food additive.
In some embodiments, the dietary supplement may be formulated with any edible material into a food product for ingestion by humans and non-human animals by conventional methods either during raw material preparation or during food preparation.
In some embodiments, the food product may be, but is not limited to: beverages (leafages), fermented foods (fermented foods), bakery products (bakery products), health foods (health foods) or dietary supplements (dietary supplements).
In some embodiments, the "rose double petal juice" may also be an extract obtained by extracting the buds of the rose double petals using a suitable extraction solvent at a specific extraction temperature for a suitable extraction time. For example, the buds of the rose with double petals can be soaked in water (i.e., extraction solvent) at a specific extraction temperature for a suitable soaking time (i.e., extraction time), and cooled to 25 ℃ to 30 ℃ after soaking to obtain the rose petal juice with double petals.
In some embodiments, the weight ratio of the extraction solvent to the buds of the rose with double petals can also be 1:5 to 1: 15. The specific extraction temperature may be 50-80 deg.C. Suitable extraction time may be 1-3 hours. Wherein, if the extraction solvent is too little or the extraction time is too short, the extraction efficiency is obviously reduced; if the extraction time is too long, the effective components in the juice may be degraded.
In some embodiments, the buds of the double-petal rose can be intact buds or buds separated into pieces, granules or powder after physical pretreatment. Wherein the physical pretreatment may include at least one of: cutting, shearing, mashing and grinding.
In some embodiments, the buds of the rose with double petals are soaked in water for a suitable soaking time to form a first extract solution containing solids. And filtering the first extracting solution to remove solid impurities, and filtering to obtain the rose juice with double petals. In other words, the rose juice of the double petal can be the first extract before filtration or the filtrate after filtration according to actual requirements.
In some embodiments, the filtered filtrate may be further concentrated under reduced pressure to obtain a rose juice. According to actual requirements, the rose juice with double petals can be filtrate before reduced pressure concentration or concentrated solution after reduced pressure concentration. In some embodiments, the filtered filtrate may be concentrated under reduced pressure at 45 ℃ to 70 ℃.
The experimental procedures in the following examples were carried out at room temperature (25 ℃ C. -30 ℃ C.) and normal pressure (1atm), unless otherwise specified.
[ example 1] preparation of Rose juice with heavy petals
Materials and instruments
1. Rose with heavy petals (producing area: Iran)
2. Centrifuge (Brand/model: Thermo Scientific Heraeus Fresco 17)
3. Thickener (Brand of the plant/type: BUCHI-rotavaper R-100)
Experimental procedure
1. Mixing water and buds of rose with multiple petals at a weight ratio of 1:10, and soaking at 65 + -5 deg.C for about 2 hr to form a first extractive solution containing solids.
2. Cooling the first extract to room temperature.
3. The cooled first extract was centrifuged by a centrifuge for about 10 minutes, and a supernatant (hereinafter referred to as a first extract supernatant) was obtained after the centrifugation.
4. The first extract supernatant was filtered through a 400 mesh sieve to remove fine solids.
5. Concentrating the filtered first extractive supernatant at 60 + -5 deg.C under reduced pressure by a concentrator until the Brix value (Degrees Brix) of the concentrated solution is 10 + -0.5 to obtain rose juice. In other words, the brix value of the heavy-petal rose juice is 10 ± 0.5.
[ example 2] Effect quantitative test-measurement of Polyphenol content
Materials and instruments
1. Gallic acid (gallic acid, available from Sigma, product number G7384).
2. Folin-Ciocalteu's phenol reagent (available from Merck, product No. 1.09001).
3. Sodium carbonate (sodium carbonate, available from Sigma, product model 31432).
4. Enzyme immunity analyzer (BioTek Epoch)
First, a standard solution is prepared and a standard curve is drawn. 10mg of gallic acid was dissolved in water and quantified to 10mL with water in a measuring flask to obtain a stock solution of gallic acid (stock solution). Here, a stock solution of gallic acid can be stored at-20 ℃ for later use. Thereafter, the stock solution was diluted 10-fold to a concentration of 100. mu.g/mL to obtain an initial solution of gallic acid (i.e., containing 1000ppm of gallic acid). The initial solution of unused gallic acid can be stored at-20 ℃. Next, 0. mu.g/mL, 20. mu.g/mL, 40. mu.g/mL, 60. mu.g/mL, 80. mu.g/mL and 100. mu.g/mL of gallic acid standard solutions were prepared in glass tubes according to the following Table one, respectively.
Watch 1
Then, 100. mu.L of each concentration of the standard solution was put into a glass tube, 500. mu.L of the foscarnet reagent was added to each glass tube, and the two were mixed uniformly and left to stand for 3 minutes. Then, 400. mu.L of 7.5% sodium carbonate was further added to each glass tube, and the solution in each glass tube was mixed well and reacted for 30 minutes to obtain a standard reaction solution. Next, after the standard reaction solution was ensured to be bubble-free by vortexing (vortex), 200. mu.L of the standard reaction solution was taken to measure absorbance at 750nm, and a standard curve was drawn.
In addition, the double-petal rose juice obtained in example 1 was diluted 10-fold with water to obtain a diluted solution. 100 μ L of the diluted solution was taken into a glass test tube. Thereafter, 500. mu.L of the foscarnol reagent was added to the glass test tube to be uniformly mixed with the diluted solution and left to stand for 3 minutes. Then, 400. mu.L of 7.5% sodium carbonate was added to the glass tube, and the solution in the glass tube was mixed uniformly and reacted for 30 minutes to 1 hour to obtain a reaction solution to be measured. Then, after the reaction solution to be measured was vortexed to ensure that no bubble was present, 200. mu.L of the reaction solution to be measured was used to measure the absorbance at 750nm using a enzyme immunoassay analyzer.
And then converting the light absorption value of the reaction solution to be detected into concentration by utilizing a standard curve interpolation method, and multiplying the concentration by the dilution multiple to obtain the total polyphenol content of the rose juice with double petals.
In this way, 3500ppm of total polyphenol content of the rose juice with double petals (3500 ppm of the final result after 1:10 dilution of the juice) can be obtained.
[ example 3] cell assay-assay for the mitochondrial Activity of skin cells
To investigate the effect of rose juice on the mitochondrial function of skin cells, this example uses a flow cytometer to evaluate the change of mitochondrial activity of Human skin fibroblasts (Human skin fibroblast) CCD-966sk treated with rose juice.
Materials and instruments
1. Cell lines: human dermal fibroblast cells CCD-966sk (purchased from Taiwan center for biological resources preservation and research (BCRC), No.60153), hereinafter referred to as CCD-966sk cells.
2. Cell culture medium: basal medium containing 10 vol% FBS (total bone serum, from Gibco, product No. 10437-. Eagle's Minimal Essential Medium (MEM), available from Gibco, product No. 15188-. Wherein the basal medium is prepared from Eagle's minimal essential medium (MEM, product No. 15188-.
3. Phosphate buffered saline (PBS solution): purchased from Gibco, product No. 10437-.
4. Mitochondrial membrane potential detection kit (BDTM mitoScreen (JC-1) kit, model 551302). Wherein the mitochondrial membrane potential detection kit comprises JC-1 dye (lyophilized) and 10 Xanalysis buffer. Prior to use, 10X assay buffer was diluted 10-fold with 1X PBS to form 1X assay buffer. 130 μ L of dimethyl sulfoxide (DMSO; from Sigma, product number: D4540) was added to JC-1 dye (lyophilized) to form a JC-1 stock solution. The JC-1 stock solution was then diluted with 1 Xassay buffer to form a JC-1working solution (JC-1working solution; i.e., JC-1 mitochondrial specific stain). Dilution ratio JC-1 stock solution to 1 Xassay buffer was 1: 100.
5. Trypsin (Trypsin-EDTA): 10 XTrypsin-EDTA (purchased from Gibco; product No. 15400-.
6. Flow cytometer (Flow cytometry): purchased from BD Pharmingen, model BDTM Accuri C6 Plus.
Experimental procedure
The experiment will be carried out in two groups, an experimental group and a control group (group without the addition of rose juice with heavy petals), each group being subjected to three replicates:
1. CCD-966sk cells were plated at 1X 10 per well5In this manner, the cells were seeded in 6-well culture plates containing 2mL of cell culture medium per well.
2. The medium in each well of the plate was changed to 2mL of experimental medium. Wherein, the experimental culture medium of the experimental group was 10. mu.L of the rose juice of the double petal obtained in example 1, and 2mL of the culture medium was added to obtain a cell culture medium containing 5. mu.L/mL of the rose juice of the double petal obtained in example 1. The experimental medium for the control group was pure cell culture medium (i.e., without rose double petal sap).
3. The culture plate was incubated at 37 ℃ with 5% CO2 for 24 hours.
4. The experimental medium in the culture dish was removed and rinsed 2 times with 1mL of 1X PBS solution.
5. 200 μ L of trypsin was added to each well and reacted in the dark for 5 minutes. After the reaction, 6mL of cell culture medium was added to terminate the reaction. The cells and cell culture medium in each well were collected into respective 15mL centrifuge tubes, and the centrifuge tubes containing the cells and cell culture medium were centrifuged at 400xg for 10 minutes.
6. After centrifugation, the supernatant was removed and the cell pellet was either re-lysed with 1mL of 1 XPBS solution or transferred to a 1.5mL tube to form a cell suspension.
7. The tube containing the cell suspension was centrifuged at 400Xg for 5 minutes.
8. After centrifugation, the supernatant in each tube was removed and 100. mu.L of JC-1working reagent was added to each tube.
9. The cell pellet in each tube was vortexed uniformly with JC-1working reagent and incubated for 15 minutes in the dark.
After 10.15 minutes, each tube was centrifuged at 400Xg for 5 minutes.
11. After centrifugation, the supernatant in each tube was removed, the cell pellet in each tube was redissolved in 1mL of 1X PBS solution and centrifuged at 400xg for 5 minutes.
12. After centrifugation, the supernatant in each tube was removed, the cell pellet in each tube was redissolved in 1mL of 1X PBS solution and centrifuged at 400xg for 5 minutes.
13. After centrifugation, the supernatant in each tube was removed and the cells were resuspended in 500 μ L of 1X PBS containing 2% FBS to obtain a test cell fluid.
14. And measuring the membrane potential of the cell mitochondria in the fine liquid to be detected of each hole by using a flow cytometer so as to analyze the activity of the mitochondria. Since the experiment was conducted in duplicate, the results of duplicate experiments for each group were averaged to obtain an average value, and then the average value of the experimental group was converted to a relative JC-1 aggregation amount by taking the average value of the control group as a relative JC-1 aggregation amount of 100%, as shown in FIG. 1.
As can be seen from the results in FIG. 1, the experimental group had an aggregation of about 155% relative to JC-1 after the treatment with the control group as the reference standard of 100%. In other words, the mitochondrial activity of human dermal fibroblasts in the experimental group was significantly increased (about 1.5-fold) compared to the control group. The rose juice with double petals can improve the activity of the mitochondria of skin cells so as to achieve the effect of improving the activity of the skin cells.
[ example 4] cell assay-inhibition of ROS production by Rose juice with double petals (blue light irradiation)
In this case, the change of the active oxygen species content of CCD-966sk cells after being treated with the double-petal rose sap and irradiated with blue light was measured by using a fluorescent probe DCFH-DA in combination with a flow cytometer.
Materials and instruments
1. Cell lines: human dermal fibroblasts CCD-966sk (BCRC, No. 60153).
2. Cell culture medium: comprises the following components in percentage by weight: 1 ratio of DMEM Medium (Dulbecco's Modified Eagle Medium, available from Gibco, product No. 12100-046) and Han's F12 Medium (Ham's F12Nutrient mix, available from Gibco, product No. 12500-026) to which were added 0.5mM sodium pyruvate (available from Gibco, product No. 11360-070) and 15mM 4-hydroxyethylpiperazine ethanesulfonic acid (4- (2-hydroxyhexyl) -1-piperazineethanesulfonic acid, HEPES) and 10 vol% FBS (available from Gibco, product No. 37-104028).
DCFH-DA solution: dichlorodihydrofluorescein diacetate (2, 7-dichoro-dihydro-fluoroescein diacetate, DCFH-DA; product number SI-D6883-50MG from Sigma) was dissolved in DMSO to prepare a 5MG/mL solution of DCFH-DA.
5. Flow cytometry: available from Beckman, product model 660519.
6. Blue light box (wavelength 400nm-500 nm).
Experimental procedure
The experiment will be carried out in three groups, an experimental group, a control group (the group without the addition of the heavy petal rose juice and without the blue light irradiation), and a control group (the group without the addition of the heavy petal rose juice and with the blue light irradiation), each of which is subjected to two replicates:
1. CCD-966sk cells were plated at 2X 10 per well5In this manner, the culture medium was inoculated into 6-well plates containing 2mL of the medium per well.
2. The culture plate was incubated at 37 ℃ with 5% CO2 for 24 hours.
3. The cell culture medium in each well of the culture tray was removed.
4. 2mL of the experimental medium was added to each well of the plate and incubated at 37 ℃ for 1 hour.
Experimental culture Medium for Experimental group 10. mu.L of the rose juice of double petal obtained in example 1 was added to 2mL of the culture medium to obtain a cell culture medium containing 5. mu.L/mL of the rose juice of double petal obtained in example 1.
The experimental medium for the control group was pure cell culture medium (i.e., without rose double petal sap).
The experimental medium of the control group was pure cell culture medium (i.e., without rose juice with double petals).
5. To each well, 2. mu.L of cell culture medium containing 5mg/mL of DCFH-DA solution was added, and the cells were treated with DCFH-DA for 15 minutes.
6. After DCFH-DA treatment, each group was incubated for 15 minutes under the following conditions.
Experimental groups: DCFH-DA treated 6-well plates were transferred to a blue light box and allowed to receive blue light for 15 minutes at room temperature.
Control group: the 6-well culture plate after DCFH-DA treatment was placed in the dark and allowed to stand at room temperature for 15 minutes.
Control group: DCFH-DA treated 6-well plates were transferred to a blue light box and allowed to receive blue light for 15 minutes at room temperature.
After 7.15 minutes, each well was rinsed 2 times with 1mL of 1 XPBS solution.
8. 200 μ L of trypsin was added to each well and reacted in the dark for 5 minutes. After the reaction, 6mL of cell culture medium was added to terminate the reaction.
9. The cells and cell culture medium in each well were individually collected into a corresponding 1.5mL centrifuge tube, and the centrifuge tube containing the cells and cell culture medium was centrifuged at 400xg for 10 minutes.
10. After centrifugation, the supernatant was removed and the cell pellet was redissolved in 1X PBS solution as a cell suspension.
11. The cell suspension was centrifuged at 400Xg for 10 minutes.
12. After centrifugation, the supernatant was removed and the cells were resuspended in 1mL of 1 XPBS solution in the dark to obtain the test cell fluid.
13. Detecting DCFH-DA fluorescence signals in the test cell fluid of each well by using flow cytometry. The excitation wavelength for fluorescence detection is 450-490nm, and the emission wavelength is 510-550 nm. Since DCFH-DA is hydrolyzed into DCFH (dichlorodihydrofluorescein) and then oxidized into DCF (dichlorofluorescein) capable of emitting green fluorescence by the active oxygen substance after entering the cells, the fluorescence intensity of the DCFH-DA treated cells can reflect the content of the active oxygen substance in the cells, and thus the ratio of the number of the cells highly expressed by the active oxygen substance in the cells to the original number of the cells can be known. Since the experiment was conducted in duplicate, the measurement results of duplicate experiments of each group were averaged to obtain an average value, and then the average values of the control group and the experimental group were converted into relative ROS production amounts by taking the average value of the control group as 100%, as shown in fig. 2.
Results of the experiment
As shown in fig. 2, the control group and the control group showed higher relative ROS production (high fluorescence), i.e. the ROS production of the control group was greatly increased (about 5.4 times) relative to the control group; it shows that blue light irradiation does lead to the generation of reactive oxygen species in the cells, which in turn causes subsequent damage to the skin fibroblasts. On the other hand, as can be seen from the comparison of the control group and the experimental group, after the cells were treated with the rose juice, the relative amount of ROS produced was significantly reduced by about 21% compared to the control group; it was shown that rose juice from the double petal was effective in reducing the production or accumulation of reactive oxygen species in the cells. In other words, the rose juice of the double petal can be used as a scavenger of active oxygen substances. That is, the rose juice with double petals can reduce the content of active oxygen substances in cells and reduce the oxidative damage of the cells caused by blue light, ultraviolet rays and the like.
[ example 5] cell assay-Rose juice with double petals reduced melanogenesis
Herein, the change of melanin content of melanoma cells B16F10 after being treated with a heavy rose sap and irradiated with blue light was measured by an ELISA reader (enzyme-linked immunosorbent assay).
Materials and instruments
1. Cell lines: mouse melanoma cells B16F10(ATCC CRL-6475), purchased from the American Type Culture Collection (ATCC), hereinafter referred to as B16F10 cells.
2. Cell culture medium: DMEM Medium (Dulbecco's Modified Eagle Medium, available from Gibco, product No. 12100-046) to which 1 vol% of a Penicillin-streptomycin two-in-one antibiotic (Penicilin-streptomycin, available from Gibco, product No. 15140122) and 10 vol% of FBS (available from Gibco, product No. 10437-028) were added.
3. Phosphate buffered saline (PBS solution): purchased from Gibco, product No. 10437-.
4. A1N NaOH solution was prepared from redistilled water, wherein NaOH was purchased from Sigma, product No. 221465.
5. Enzyme immunoassay appearance: purchased from BioTek, product model FLx 800.
6. Blue light box: the wavelength is 400nm-500 nm.
Experimental procedure
The experiment will be carried out in three groups, an experimental group, a control group (the group to which no heavy rose sap is added and which is not subjected to blue light irradiation), and a control group (the group to which no heavy rose sap is added but which is subjected to blue light irradiation), each of which is subjected to three replicates:
1. B16F10 cells were plated at 1.5X 10 per well5In this manner, the cells were seeded in 6-well culture plates containing 3mL of cell culture medium per well.
2. The culture dish was incubated at 37 ℃ with 5% CO2 for 24 hours.
3. Then, the cell culture medium was removed from each well without disturbing the attached cells.
4. 3mL of the experimental medium was added to each well of the plate and incubated at 37 ℃ for 1 hour.
Experimental culture Medium for Experimental group 10. mu.L of the rose juice of double petal obtained in example 1 was added to 2mL of the culture medium to obtain a cell culture medium containing 5. mu.L/mL of the rose juice of double petal obtained in example 1.
The experimental medium for the control group was pure cell culture medium (i.e., without rose double petal sap).
The experimental medium of the control group was pure cell culture medium (i.e., without rose juice with double petals).
5. After 1 hour of culture, each group was cultured under the following conditions for 3 hours.
Experimental groups: the 6-well plate was transferred to a blue light box and allowed to receive blue light irradiation for 3 hours at room temperature.
Control group: the 6-well plate was transferred to a dark room and allowed to stand at room temperature for 3 hours.
Control group: the 6-well plate was transferred to a blue light box and allowed to receive blue light irradiation for 3 hours at room temperature.
6. After 3 hours of culture, each group of culture plates was cultured at 37 ℃ for 48 hours.
7. Then, the medium was removed and rinsed 2 times with 1mL of 1X PBS solution.
8. 200. mu.L of trypsin was added to each well of the culture dish and reacted at room temperature for 3 minutes to detach the cells from the culture dish, and the cells in each well were collected in a cell culture medium into a corresponding 1.5mL centrifuge tube. Then, each centrifuge tube was centrifuged at 400Xg for 5 minutes, and the supernatant was removed after centrifugation.
9. After the cell pellet was reconstituted with 1 XPBS solution, it was centrifuged again at 400Xg for 10 minutes. After re-centrifugation, the supernatant was removed and the cells were resuspended in 200. mu.L of 1 XPBS solution to give a cell suspension.
10. The cell suspension from each centrifuge tube was frozen in liquid nitrogen for 10 minutes and then left at room temperature for about 30 minutes to completely thaw.
11. After complete thawing, each centrifuge tube was centrifuged at 12,000Xg for 30 minutes.
12. After centrifugation, the supernatant was removed, and after resuspending the cell pellet with 120. mu.L of 1N sodium hydroxide solution, each of the centrifuged tubes was subjected to dry bath at 60 ℃ for 1 hour to obtain a sample to be assayed.
13. 100 μ L of the sample to be detected was transferred to a 96-well plate, and the optical density (hereinafter referred to as OD450 value) of the cell solution at 450nm was measured using an ELISA plate reader. Since the experiments were performed in triplicate, the measurements from each set of triplicate experiments were averaged to obtain the OD450 value for each set.
Results of the experiment
The relative melanin expression of the experimental group, the control group, and the control group was calculated according to the following formula: the relative melanin expression (%) — (each group OD450 value/control group OD450 value) × 100% is shown in fig. 3.
As shown in fig. 3, from the results of comparing the control group with the control group, it can be seen that the relative melanin expression in the cells is significantly increased (about 1.8 times) after the blue light irradiation; it was shown that blue light irradiation did stimulate melanoma cells to produce melanin, which resulted in skin blotches or overall darkness. On the other hand, comparing the results of the control group and the experimental group, it can be seen that the relative melanin expression in blue light is lower (about 72% lower) than that of the cells without the rose juice treatment; it was shown that the rose juice of the double petal was effective in reducing melanin produced by melanocytes due to blue light.
[ example 6] cell assay-inhibition of ROS production by Rose Bengal juice (Hydrogen peroxide treatment)
In this case, a fluorescent probe DCFH-DA was used in conjunction with a flow cytometer to determine the change in the active oxygen species content of CCD-966sk cells after treatment with the double-petal rose sap and hydrogen peroxide reaction.
Materials and instruments
1. Cell lines: human dermal fibroblasts CCD-966sk (BCRC No. 60153).
2. Cell culture medium: basal medium containing 10 vol% FBS (purchased from Gibco, product No. 10437-. Eagle's minimal medium (purchased from Gibco, product No. 15188-. Wherein the basal medium is prepared from Eagle's minimal essential medium (MEM, product No. 15188-.
3. Phosphate buffered saline (PBS solution): purchased from Gibco, product No. 10437-.
DCFH-DA solution: dichlorodihydrofluorescein diacetate (product number SI-D6883-50MG from Sigma) was dissolved in dimethyl sulfoxide to prepare a 5MG/mL solution of DCFH-DA.
5. Hydrogen peroxide (H2O 2): purchased from Sigma.
6. Flow cytometry: available from Beckman, product model 660519.
Experimental procedure
The experiment is divided into three groups, namely an experiment group, a control group (a group without adding the rose juice with the double petals and without being treated by hydrogen peroxide) and a control group (a group without adding the rose juice with the double petals and treated by hydrogen peroxide), wherein the two groups are subjected to two repeated experiments:
1. CCD-966sk cells were plated at 2X 10 per well5In this manner, the cells were seeded in 6-well culture plates containing 2mL of cell culture medium per well.
2. The culture plate was incubated at 37 ℃ with 5% CO2 for 24 hours.
3. The cell culture medium was removed.
4. 2mL of the experimental medium was added to each well of the plate and incubated at 37 ℃ for 1 hour.
Experimental culture Medium for Experimental group 10. mu.L of the double-petal rose juice obtained in example 1 was added to 2mL of the culture medium to obtain 2mL of the cell culture medium supplemented with 5. mu.L/mL of the double-petal rose juice obtained in example 1.
The experimental medium for the control group was pure 2mL of cell culture medium (i.e. without rose double petal sap).
The experimental medium for the control group was 2mL of cell culture medium alone (i.e., without the rose double petal sap).
5. Add 5mg/mL DCFH-DA solution 2 u L to each hole in the cell culture medium, allow DCFH-DA to treat cells for 15 minutes.
6. After DCFH-DA treatment, H2O2 was added to the experimental medium of the experimental group and the experimental medium of the control group, respectively, and reacted at 37 ℃ for 1 hour. Specifically, 35% wt of hydrogen peroxide was diluted to 100mM (10. mu.L of hydrogen peroxide was added to 990. mu.L of redistilled water), and then 20. mu.L of 100mM hydrogen peroxide was added to 2mL of cell culture plates.
7. After reaction, each well was rinsed 2 times with 1mL of 1 XPBS solution.
8. 200 μ L of trypsin was added to each well and reacted in the dark for 5 minutes. After the reaction, 6mL of cell culture medium was added to terminate the reaction.
9. The cells and cell culture medium in each well were collected into individually corresponding 1.5mL centrifuge tubes, and the centrifuge tubes containing the cells and medium were centrifuged at 400xg for 10 minutes.
10. After centrifugation, the supernatant was removed and the cell pellet was back-lysed with 1X PBS solution.
11. Centrifuge at 400Xg for 10 min.
12. After centrifugation, the supernatant was removed and the cells were resuspended in 1mL of 1 XPBS solution in the dark to obtain the test cell fluid.
13. Detecting DCFH-DA fluorescence signals in the test cell fluid of each well by using flow cytometry. The excitation wavelength for fluorescence detection is 450-490nm, and the emission wavelength is 510-550 nm. Since DCFH-DA enters into cells and is hydrolyzed into DCFH, and then oxidized into DCF emitting green fluorescence by the reactive oxygen species, the fluorescence intensity of DCFH-DA treated cells can reflect the content of reactive oxygen species in the cells, and thus the ratio of the number of cells highly expressed by the reactive oxygen species in the cells to the original number of cells can be obtained. Since the experiment was conducted in duplicate, the measurement results of duplicate experiments of each group were averaged to obtain an average value, and then the average values of the control group and the experimental group were converted into relative ROS production amounts by taking the average value of the control group as 100%, as shown in fig. 4.
Results of the experiment
As shown in fig. 4, the results of comparison between the control group and the control group showed that the amount of ROS produced (high fluorescence) was significantly increased after the hydrogen peroxide treatment; it shows that hydrogen peroxide treatment can indeed lead to the production of reactive oxygen species in the cells, which in turn can cause subsequent damage to skin fibroblasts. On the other hand, the results of comparing the control group with the experimental group show that, after the cells are treated by the rose juice with double petals, the relative ROS production is obviously reduced by about 97 percent, even lower than that of the control group; it was shown that rose juice from the double petal was effective in reducing the production or accumulation of reactive oxygen species in the cells. In other words, the rose juice of the double petal can be used as a scavenger of active oxygen substances. That is, the rose juice of the double petal can reduce the oxidative damage of the cells caused by the active oxygen substances by reducing the content of the active oxygen substances in the cells.
In conclusion, the present application proves that the rose juice with double petals can inhibit the generation of free radicals and/or melanin, so that the rose juice with double petals can effectively prevent various skin aging phenomena caused by ultraviolet rays or blue light, and further provides the protection of the skin against the ultraviolet rays or the blue light. In addition, the present application also proves that the rose juice with double petals can improve the activity of mitochondria. Therefore, the rose juice with the double petals can passively reduce the loss of skin affected by free radicals, and can actively improve the activity of mitochondria so as to make the skin healthy.
The present invention is capable of other embodiments, and various changes and modifications may be made by one skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (8)
1. Use of a rose juice from Rosa bivalvata for preparing a composition for inhibiting melanin production caused by blue light is provided.
2. Use of a rose juice from Rosa bivalvata for preparing a composition for inhibiting the production of reactive oxygen species by skin cells due to blue light.
3. Use of a rose juice from Rosa bivalvis for preparing a composition for improving the antioxidant capacity of skin cells.
4. Use of rose juice with double petals for preparing composition for improving activity of mitochondria is provided.
5. The use according to any one of claims 1 to 4, wherein the composition is a pharmaceutical composition.
6. Use according to any one of claims 1 to 4, wherein the composition is a food composition or a care composition.
7. Use according to any one of claims 1 to 4, wherein the composition is an oral composition.
8. Use according to any one of claims 1 to 4, wherein the composition is a topical composition.
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| CN106360178A (en) * | 2016-08-31 | 2017-02-01 | 云南玖香鲜花生物科技股份有限公司 | Fresh flower petal juice and making method thereof |
| CN106726975A (en) * | 2016-12-28 | 2017-05-31 | 广州环亚化妆品科技有限公司 | A kind of blue light protection component and preparation method thereof and the application in cosmetics |
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| US20070178176A1 (en) * | 2006-02-02 | 2007-08-02 | Chithan Kandaswami | Composition and method for promoting weight loss |
| JP2007330124A (en) * | 2006-06-13 | 2007-12-27 | Kenko Tsusho Kk | Health food composition for preventing and ameliorating obesity and lifestyle-related disease |
| CN103609985B (en) * | 2013-10-25 | 2015-05-13 | 晶叶（青岛）生物科技有限公司 | Fermentation method for production of nutritionally balanced natural fruit enzyme |
| CN104839841A (en) * | 2015-03-30 | 2015-08-19 | 孙村镇中药材种植技术协会 | Weight losing promoting partially fermented paullinia cupana fruit and Chinese wolfberry fruit juice and preparation method thereof |
| CN106754031A (en) * | 2016-12-20 | 2017-05-31 | 潜山县天柱山糯米封缸酒业有限公司 | A kind of preparation technology of Guarana glutinous rice wine |
| CN110051572B (en) * | 2018-01-19 | 2022-03-15 | 百岳特生物科技（上海）有限公司 | Pomegranate fermentation product and use thereof |
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| CN106360178A (en) * | 2016-08-31 | 2017-02-01 | 云南玖香鲜花生物科技股份有限公司 | Fresh flower petal juice and making method thereof |
| CN106726975A (en) * | 2016-12-28 | 2017-05-31 | 广州环亚化妆品科技有限公司 | A kind of blue light protection component and preparation method thereof and the application in cosmetics |
| CN109430831A (en) * | 2018-10-31 | 2019-03-08 | 吉林省恒实传食品科技发展有限公司 | A kind of beautifying face and moistering lotion anti-aging health care's food formula and preparation method thereof |
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| CN110075024A (en) * | 2019-04-29 | 2019-08-02 | 广州科恩生物技术有限公司 | A kind of anti-blue light composition and preparation method thereof |
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