Use of raspberry juice for the preparation of a composition for reducing oxidative damage caused by reactive oxygen species induced by hydrogen peroxide in cells

Abstract

The present invention provides a use of a raspberry juice for preparing a composition for reducing oxidative damage caused by reactive oxygen species induced by hydrogen peroxide in cells, wherein the raspberry juice is obtained by soaking raspberry fruits in an aqueous solvent for 0.5 to 3 hours, and the volume ratio of the aqueous solvent to the raspberry fruits is 5 to 20: 1 to 5.

Description

[0001] This application is a divisional application filed by the applicant based on the parent application (application number: 202110964611.8, invention title: use of strawberry juice in the preparation of a composition for conditioning the skin). Technical Field

[0002] This case relates to the use of Rubus fruticosus juice, and more particularly to the use of Rubus fruticosus juice in the preparation of compositions for reducing oxidative damage to cells caused by reactive oxygen species induced by hydrogen peroxide. Background Technology

[0003] With environmental changes, ultraviolet radiation and air pollution are becoming increasingly severe. Ultraviolet radiation from sunlight is a major cause of skin damage, aging, and tanning. Long-term exposure to sunlight can even lead to the production of melanin, forming spots that are difficult to fade. In addition, ultraviolet radiation from sunlight can stimulate the production of reactive oxygen species (ROS, also known as free radicals) in skin cells, resulting in skin aging, loss of radiance, dryness, and even redness.

[0004] In addition, skin cells gradually age, skin texture becomes rougher and wrinkles appear. As the metabolic rate of skin cells slows down, the skin's recovery speed becomes slower. In addition, long-term exposure to external environmental stimuli such as air pollution leads to the accumulation of a large amount of reactive oxygen species and melanin, which cannot be completely metabolized in time, resulting in the skin condition not being improved.

[0005] Most cosmetics, skincare products, and health foods are made from chemical ingredients. Long-term use is not only harmful to human health, but also irritates sensitive skin and exacerbates redness. In addition, the manufacturing process is environmentally and ecologically unfriendly, causing pollution and even entering the ecosystem through drainage systems.

[0006] To address the aforementioned issues, those skilled in the art urgently need to develop skin-conditioning compositions that combat ultraviolet radiation, provide antioxidant protection, inhibit melanin production, reduce red spots on the skin, soothe redness, and enhance the skin's moisturizing ability, in order to benefit the vast population with such needs. Summary of the Invention

[0007] In view of this, the object of the present invention is to provide the use of Rubus fruticosus juice for preparing a composition that reduces oxidative damage to cells caused by reactive oxygen species induced by hydrogen peroxide, wherein the Rubus fruticosus juice is obtained by soaking Rubus fruticosus fruit in an aqueous solvent for 0.5 to 3 hours, and the volume ratio of the aqueous solvent to the Rubus fruticosus fruit is 5 to 20: 1 to 5.

[0008] In some embodiments, the volume ratio of the aqueous solvent to the dewberry fruit is 5:1.

[0009] In some embodiments, the dewberry fruit is soaked in an aqueous solvent at 75°C to 95°C.

[0010] In some embodiments, the cells are skin cells.

[0011] In some embodiments, the composition is a reactive oxygen species scavenger.

[0012] In some embodiments, the composition is further used to reduce intercellular spaces to protect the dermis.

[0013] In some embodiments, the composition is used to achieve the ability to protect the dermis by increasing the gene expression levels of the keratin 10 (KRT10) gene and the keratin 1 (KRT1) gene in cells.

[0014] Another object of the present invention is to provide a use of Rubus fruticosus juice in the preparation of a skin-conditioning composition, which reduces the hemoglobin content of the skin, soothes redness, improves the skin's moisturizing ability, increases skin radiance, and delays skin aging. The composition inhibits melanin production and reduces red spots on the skin by reducing the expression of melanin-related genes in cells. The composition also reduces oxidative damage caused by reactive oxygen species in cells, thus achieving an antioxidant effect, protecting skin cells, and achieving antioxidant, whitening, and red spot reduction effects.

[0015] Use of a Rubus fruticosus juice for preparing a skin-conditioning composition, wherein the Rubus fruticosus juice is obtained by extraction with an aqueous solvent.

[0016] In some embodiments, the total polyphenol content of the Rubus fruticosus juice is between 8,000 and 11,000 ppm.

[0017] In some embodiments, the total flavonoid content of the dewberry juice is between 1,000 and 3,000 ppm.

[0018] In some embodiments, the composition is used to reduce oxidative damage caused by reactive oxygen species in cells.

[0019] In some embodiments, the composition is used to reduce the hemoglobin content in the skin.

[0020] In some embodiments, the composition is used to inhibit melanin production.

[0021] In some embodiments, the composition is used to inhibit melanin production by reducing the expression of melanin-related genes in cells.

[0022] In some embodiments, the melanin-related genes include: tyrosinase-associated protein-1 (TYRP1), tyrosinase (TYR), microphthalmia-associated transcription factor (MITF), and melanocortin receptor 1 (MC1R).

[0023] In some embodiments, the composition is used to reduce red spots on the skin.

[0024] In some embodiments, the composition is used to enhance the skin's moisturizing ability.

[0025] In some embodiments, the composition is used to enhance the skin's moisturizing ability by increasing the expression of moisturizing-related genes in cells.

[0026] In some embodiments, the composition is further prepared into a skin care composition, a health food composition, a cosmetic composition, or a topical skin agent.

[0027] In summary, the berry juice of any embodiment can reduce oxidative damage to cells caused by reactive oxygen species induced by hydrogen peroxide. Therefore, berry juice can effectively achieve antioxidant effects as a reactive oxygen species scavenger. Furthermore, berry juice can also reduce intercellular spaces to protect the dermis and prevent skin damage and inflammation.

[0028] In addition, the dewberry juice reduces the hemoglobin content in the skin, soothes redness, improves the skin's moisturizing ability, and increases the skin's radiance. Attached Figure Description

[0029] Figure 1 This is a comparison chart of the proportion of cells highly expressing reactive oxygen species (ROS) in the blank control group, control group, and experimental group. ###p value < 0.001, compared to the blank control group; ***p value < 0.001, compared to the control group.

[0030] Figure 2 This is a comparison graph showing the expression levels of moisturizing-related genes between the control group and the experimental group. ***p value < 0.001, compared to the control group.

[0031] Figure 3 This is a comparison chart of the expression levels of melanin-related genes between the control group and the experimental group. **p value < 0.01, ***p value < 0.001, compared to the control group.

[0032] Figure 4This is a comparative graph showing the melanin content results of the blank control group, the control group, and the experimental group. ***p value < 0.001, compared to the blank control group.

[0033] Figure 5A and Figure 5B The images show a comparison of the number of UV-induced red spots on the skin of subjects after continuous consumption of the beverage containing the experimental group at weeks 0, 4, and 8, as well as images taken by a detection device. The number of UV-induced red spots on the subjects before application is taken as 100%. *p value < 0.1, compared to week 0.

[0034] Figure 6A and Figure 6B The data are the skin hemoglobin index of the subjects after continuous ingestion of the beverage containing the experimental group at weeks 0, 4, and 8, and the images captured by the detection device. The skin hemoglobin index of the subjects before application is 100%. *p value < 0.1, ***p value < 0.001, compared to week 0.

[0035] Figure 7A and Figure 7B The data includes changes in skin radiance of subjects after continuous consumption of the beverage containing the experimental group at weeks 0, 4, and 8, as well as images captured by a detection device. Skin radiance of the subjects before application was taken as 100%. *p value < 0.1, compared to week 0. Detailed Implementation

[0036] The following describes some specific implementations of this case. Without departing from the spirit of this case, it can be implemented in many different forms, and the scope of protection should not be limited to the conditions specifically stated in the specification.

[0037] This study used Excel software for statistical analysis. Data are expressed as mean ± standard deviation (SD), and differences between groups were analyzed using Student's t-test.

[0038] The values ​​used in this article are approximate, and all experimental data are expressed within a range of ±10%, with the optimal range being ±5%.

[0039] As used herein, the term "juice" refers to a product prepared by extraction. The juice may be in the form of a solution dissolved in a solvent, or it may be a concentrate or essence containing little or no solvent.

[0040] Blackberry (scientific name: Rubus fruticosus, also known as blackberry) is the fruit of a shrub belonging to the genus Rubus in the family Rosaceae.

[0041] As used in this article, “dewberry” usually refers to plant fruit, which may include raw fruit, dried fruit, or fruit processed by other physical means to facilitate processing, and may further include whole fruit, chopped, diced, ground, milled, or otherwise processed to affect the size and integrity of the raw material.

[0042] In some embodiments, "dewberry juice" may be the juice obtained by pressing the fruit of the dewberry. For example, in some embodiments, the dewberry fruit is crushed, squeezed, and pulp and fine suspended matter are removed, and then concentrated to a certain sugar content. In other embodiments, the dewberry juice may also undergo other procedures during preparation that do not substantially affect its efficacy as described herein, such as fermentation, to add flavor or for other uses.

[0043] In some embodiments, "dewberry juice" may also be an extract obtained by extracting dewberry fruit using a suitable extraction solvent. For example, dewberries can be soaked in water at room temperature for an appropriate time for extraction, and then filtered to remove solid impurities to obtain dewberry fruit extract.

[0044] In some embodiments, the juice extracted from dewberry fruit (sometimes referred to as "dewberry juice") can inhibit free radical generation, reduce hemoglobin levels in the skin, inhibit melanin production, reduce the number of red spots on the skin, and improve the skin's moisturizing ability. Therefore, dewberry juice can be used to prepare skin conditioning compositions that inhibit free radical generation, reduce hemoglobin levels in the skin, inhibit melanin production, reduce the number of red spots on the skin, and improve the skin's moisturizing ability.

[0045] In some embodiments, the aforementioned composition may be a pharmaceutical composition, a skincare composition, a food composition, or a health food composition.

[0046] Pharmaceutical compositions can be manufactured using techniques known to those skilled in the art into dosage forms suitable for enterally, parentally, or topically administered. Examples include: injections [e.g., sterile aqueous solutions or dispersions], sterile powders, external preparations, or other similar formulations.

[0047] Pharmaceutical compositions may further comprise a pharmaceutically acceptable carrier that is widely used in pharmaceutical manufacturing techniques. For example, a pharmaceutically acceptable carrier may include one or more of the following: 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 are to be chosen by those skilled in the art as appropriate.

[0048] Taking oral dosage forms as an example, the bioactive substance or pharmaceutical composition can be provided in a dosage form suitable for oral administration using any suitable method. Liquid dosage forms suitable for oral administration include syrups, oral solutions, suspensions, elixirs, etc., while solid dosage forms suitable for oral administration include powders, granules, lozenges, sugar-coated lozenges, enteric-coated lozenges, chewable lozenges, effervescent lozenges, film-coated lozenges, capsules, long-acting sustained-release lozenges, etc. The bioactive substance or pharmaceutical composition provided according to the present invention may contain any pharmaceutically acceptable carrier that will not adversely affect the desired efficacy of the active ingredient (i.e., Bifidobacterium lactis TCI604 and / or its metabolites). For example, but not limited to, examples of pharmaceutically acceptable carriers for the aforementioned liquid dosage forms include: water, saline, glucose (dextrose), glycerol, ethanol or analogues thereof, oils (e.g., olive oil, castor oil, cottonseed oil, peanut oil, corn oil, and wheat germ oil), glycerol, polyethylene glycol, and combinations thereof; examples of pharmaceutically acceptable carriers for the aforementioned solid dosage forms include: cellulose, starch, kaolinite, bentonite, sodium citrate, gelatin, agar, carboxymethyl cellulose, gum arabic, alginate, glyceryl monostearate, calcium stearate, and combinations thereof.

[0049] In some embodiments, a pharmaceutically acceptable carrier may comprise one of the following solvents: water, normal saline, phosphate-buffered saline (PBS), an aqueous solution containing alcohol, and any other suitable solvent.

[0050] In some embodiments, the pharmaceutical composition may be administered via any of the following parenteral routes: subcutaneous injection, intraepidermal injection, intradermal injection, and intralesional injection.

[0051] In some embodiments, the pharmaceutical composition may be formulated into a topical preparation suitable for application to the skin using techniques known to those skilled in the art. Examples include, but are not limited to, any of the following: emulsion, gel, ointment, cream, patch, liniment, powder, aerosol, spray, lotion, serum, paste, foam, drop, suspension, salve, and bandage.

[0052] In some embodiments, the external formulation is prepared by mixing a pharmaceutical composition with a base known to those skilled in the art.

[0053] In some embodiments, the substrate may contain one or more of the following additives: water, alcohols, glycols, hydrocarbons [such as petroleum jelly and white petrolatum], waxes [such as paraffin and yellow wax], preserving agents, antioxidants, surfactants, absorption enhancers, stabilizing agents, gelling agents [such as Carbopol® 974P, microcrystalline cellulose, and carboxymethylcellulose], active agents, humectants, odor absorbers, fragrances, pH adjusting agents, and chelating agents. The additives include agents, emulsifiers, occlusive agents, softeners, thickeners, solubilizing agents, penetration enhancers, anti-irritants, colorants, and propellants. The selection and quantity of these additives fall within the scope of professional expertise and routine practice of those familiar with this technology.

[0054] In some embodiments, the skincare product may contain an acceptable adjuvant that is widely used in skincare product manufacturing techniques. For example, the acceptable adjuvant may include one or more of the following: solvents, gelling agents, surfactants, preservatives, antioxidants, screening agents, chelating agents, surfactants, coloring agents, thickening agents, fillers, fragrances, and odor absorbers. The selection and quantity of these agents can be appropriately adjusted according to actual needs.

[0055] In some embodiments, the skincare product may be manufactured in a form suitable for skincare or makeup using techniques known to those skilled in the art, and may be any of, but not limited to, the following: an aqueous solution, an aqueous-alcohol solution or an oily solution, an oil-in-water type, a water-in-oil type or a combination of emulsions, gels, ointments, creams, masks, patches, packs, liniments, powders, aerosols, sprays, lotions, emulsions, pastes, foams, dispersions, drops, mousses, sunblocks, sunscreen serums, sunscreen creams, sunscreen sprays, tonic water, foundations, makeup remover products, soaps, and other body cleansing products.

[0056] In one embodiment of the invention, cosmetic compositions and personal care products are provided, comprising a color-changing composition, such as a pigment abrasive containing pigment particles in a carrier and a styrene-maleic anhydride copolymer. The cosmetic composition can be any cosmetic or personal care product form that provides color to human skin. For example, the cosmetic composition can be, but is not limited to, lipstick, lip gloss, moisturizing lipstick, nail polish, foundation, face powder, base powder, concealer, blush, eyeshadow, eyeliner, mascara, or bronzer. The personal care product can be any suitable form that imparts color to human skin. For example, personal care products can include day creams or lotions, night creams or lotions, sunscreens, creams or oils and other SPF products, moisturizers, ointments, creams, gels, body lotions, artificial browning compositions, hair removal products, etc.

[0057] The cosmetic compositions and personal care products of the present invention are applied to the human skin system, including skin, lips, nails, hair, and other keratinized surfaces. As used herein, the term "keratinized surface" refers to portions of the human skin system containing keratin, including but not limited to the skin, lips, hair (including scalp, eyelashes, eyebrows, facial hair, and body hair) of mammals, preferably humans, and nails (toenails, nails, nail cuticles, etc.).

[0058] Cosmetics or personal care products that can display color changes are applied to any area of ​​the skin, preferably the face, neck, hands, feet, or other areas of the body, such as the walls, legs, and back.

[0059] In some embodiments, the skincare product may also be used in combination with one or more of the following known active external use agents: whitening agents [such as tretinoin, catechin, kojic acid, arbutin, and vitamin C], moisturizers, bactericides, ultraviolet absorbers, plant extracts [such as aloe extract], skin nutrients, anesthetics, anti-acne agents, antipruritics, analgesics, antidermatitis agents, antihyperkeratolytic agents, anti-dry skin agents, antipsoriatic agents, antiaging agents, antiwrinkle agents, and antiseborrheic agents. Topical agents, wound-healing agents, corticosteroids, and hormones. The selection and quantity of these topical agents fall within the scope of the professional competence and routine techniques of those skilled in this field.

[0060] In some embodiments, the pharmaceutical composition may be used as a food additive, added by conventional methods during the preparation of raw materials or during the production of food, to formulate a food product for human and non-human animal consumption with any edible material.

[0061] In some embodiments, the types of food may include, but are not limited to: beverages, fermented foods, bakery products, health foods, and dietary supplements.

[0062] The bioactive substance or pharmaceutical composition provided according to the present invention can be in any suitable form without particular limitation, depending on the intended use and the appropriate dosage form. For example, but not limited to, the bioactive substance or pharmaceutical composition can be administered orally to the individual in need. Depending on the form of use and purpose, a pharmaceutically acceptable carrier can be selected to provide the bioactive substance or pharmaceutical composition. The carrier is well known to those skilled in pharmaceutical technology and includes excipients, diluents, adjuvants, stabilizers, absorption enhancers, disintegrants, solubilizers, emulsifiers, antioxidants, binders, binders, thickeners, dispersants, suspending agents, lubricants, hygroscopic agents, etc.

[0063] The food composition provided by the present invention can be a beverage, solid food, or semi-solid food, and can be provided in the form of health food, health care food, functional food, nutritional supplement, or special nutritional food. For example, but not limited to, the food composition can be dairy products, processed meat products, bread, pasta products, biscuits, ice cream, lozenges, capsules, fruit juices, teas, sparkling water, alcoholic beverages, sports drinks, nutritional drinks, infant weaning foods, etc. Preferably, the food composition is provided in the form of health food or health care food.

[0064] Furthermore, depending on the form of use and requirements, any suitable food additives may be included in the food composition provided according to the present invention. Examples include, but are not limited to, preservatives, bactericides, antioxidants, bleaching agents, color-retaining agents, leavening agents, nutritional additives, coloring agents, flavoring agents (e.g., sweeteners), thickeners, binding agents, food industry chemicals, emulsifiers, and agents for quality improvement, brewing, and food manufacturing.

[0065] The recommended dosage, usage standards and conditions for specific groups (e.g., pregnant women, children), or suggestions for taking it together with other foods or medicines can be indicated on the outer packaging of health foods, health care foods, functional foods, nutritional supplements or special nutritional foods provided according to the present invention, so that users can take them on their own without any safety concerns without the guidance of a doctor, pharmacist or relevant personnel.

[0066] Unless otherwise specified, the experimental procedures in the following examples are performed at room temperature (25±5℃) and normal pressure (1atm).

[0067] [Example 1]: Preparation of juice from Rubus fruticosus

[0068] First, the fruits of Rubus fruticosus (from Chile) were cut into 12mm pieces. Then, the Rubus fruticosus were simultaneously homogenized and extracted using water, alcohols, hydrous alcohols, or combinations thereof as extraction solvents. The extraction temperature was between 75°C and 95°C, preferably 80°C to 90°C. The volume ratio of extraction solvent to Rubus fruticosus was between 5 and 20:1 to 5, preferably 5:1. The extraction temperature was between 50°C and 100°C, preferably 80°C to 90°C. In this embodiment, the extraction time was 0.5 to 3 hours, preferably 1 hour.

[0069] After cooling to room temperature, the berry juice obtained from the above extraction steps can be further centrifuged at 800-1300 rpm at 15-25°C for 5-10 minutes to obtain a supernatant. The supernatant can be filtered through a 400-mesh filter to remove residual solids. Further concentration under reduced pressure at 55-65°C yields a concentrated product. In one embodiment, to obtain solid berry juice, the solvent in the aforementioned concentrated berry juice can be removed by spray drying, thus obtaining berry juice powder. In another embodiment, 2.5 g of the above-mentioned concentrated berry juice is added to 50 mL of water or a beverage to obtain a berry juice beverage with a concentration of 0.05 mg / mL and a sugar content of 5.0 ± 0.3 °Bx.

[0070] [Example 2]: Detection of total flavonoids and total polyphenols in dewberry juice

[0071] The analytical method for detecting the total flavonoid content was mainly based on the method described in Zhishen Jia et al., (1999), Food Chemistry, 64: 555-559, with modifications. 200 μL of appropriately diluted sample was added to 1000 μL of H2O, mixed thoroughly, and then 200 μL of 5% sodium citrate was added. After standing for 6 minutes, 200 μL of 10% aluminum nitrate was added, and the mixture was allowed to stand for another 6 minutes. Finally, 2 mL of 4% sodium hydroxide and 1.4 mL of H2O were added, mixed thoroughly, and analyzed at 500 nm. A standard curve was plotted using rutin as a standard. In this embodiment, the total flavonoid content of the berry juice in Example 1 was between 1000 and 3000 ppm, preferably between 2000 and 3000 ppm, specifically 2248.9 ppm.

[0072] The analytical method for detecting the total polyphenol content is as follows: each sample is diluted 10-fold with water, and 100 mL is taken into a centrifuge tube. Next, 500 μL of Folin-Ciocalteu phenol reagent is added to the centrifuge tube, mixed with the diluted sample, and allowed to stand for 3 minutes. Then, 400 μL of 7.5% sodium carbonate is added, mixed well, and allowed to stand for 30 minutes to obtain the test reaction solution. After a second standing period, 200 μL of the test reaction solution is taken into a 96-well plate, and the absorbance of the test reaction solution at 750 nm is measured. Furthermore, a standard curve is prepared using gallic acid as a standard. In this embodiment, the total polyphenol content of the berry juice in Example 1 is between 8000 and 11000 ppm, preferably between 9000 and 10000 ppm, specifically 9364.6 ppm.

[0073] [Example 3]: Cell Experiment - Dewberry Juice Inhibits the Generation of Reactive Oxygen Species (ROS)

[0074] Here, the changes in reactive oxygen species content in human dermal fibroblasts CCD-966sk after treatment with a dewberry extract composition were determined using the fluorescent probe DCFH-DA in conjunction with flow cytometry.

[0075] Materials and Instruments

[0076] 1. Cell line: Human skin fibroblasts CCD-966sk (BCRC, No. 60153), hereinafter referred to as CCD-966sk cells.

[0077] 2. Culture medium: Basal medium containing 10 vol% FBS (fetal bovine serum, purchased from Gibco). The basal medium was prepared by adding components to Eagle's minimum essential medium (MEM, purchased from Gibco, product number 15188-319) to include 1 mM sodium pyruvate (purchased from Gibco), 1.5 g / L sodium bicarbonate (purchased from Sigma), and 0.1 mM non-essential amino acid solution (purchased from Gibco).

[0078] 3. Phosphate-buffered saline (PBS) solution: purchased from Gibco, product number 10437-028.

[0079] 4. DCFH-DA solution: Dissolve 2,7-dichloro-dihydro-fluorescein diacetate (DCFH-DA; product number SI-D6883, purchased from Sigma) in dimethyl sulfoxide (DMSO, purchased from Sigma, product number SI-D6883-50MG) to prepare a 5 mg / mL DCFH-DA solution.

[0080] 5. Flow cytometry, Beckman, Catalog No. 660519.

[0081] 6. Hydrogen peroxide (H2O2): Purchased from Sigma-Aldrich, product model 95299-1L.

[0082] 7. Trypsin-EDTA: 10X Trypsin-EDTA (purchased from Gibco) diluted 10 times with 1X PBS solution.

[0083] 8. Berry juice: The berry juice used in this experiment was obtained through Example 1 above.

[0084] Experimental steps

[0085] The experiment will be conducted in three groups: an experimental group, a blank control group (a group that did not add berry juice or undergo hydrogen peroxide treatment), and a control group (a group that did not add berry juice but underwent hydrogen peroxide treatment). Each group will undergo two replicate experiments.

[0086] 1. Pack CCD-966sk cells at a density of 1 × 10⁶ cells per well. 5 The culture was inoculated in a 6-well culture dish containing 2 mL of culture medium per well using a single method.

[0087] 2. Place the culture dish in 5% CO2 at 37°C and incubate for 24 hours.

[0088] 3. Remove the culture medium.

[0089] 4. Add 2 mL of experimental culture medium to each well of the culture dish and incubate at 37 °C for 1 hour.

[0090] The experimental group used a 2 mL cell culture medium containing 5 μL of berry juice (i.e., the berry juice accounted for 0.25% of the cell culture medium volume).

[0091] The control group used a simple 2 mL cell culture medium (i.e., without dewberry juice).

[0092] The control group received 2 mL of simple cell culture medium (i.e., without dewberry juice).

[0093] 5. Add 2 μL of DCFH-DA solution with a concentration of 5 μg / mL to the cell culture medium in each well and treat the cells with DCFH-DA for 15 minutes.

[0094] 6. After DCFH-DA treatment, H2O2 was added to the experimental culture medium of the experimental group and the experimental culture medium of the control group, and the reaction was carried out at 37 ℃ for 1 hour. Specifically, 35% wt hydrogen peroxide was first diluted to 100 mM (10 μL of hydrogen peroxide was added to 990 μL of double-distilled water), and then 20 μL of 100 mM hydrogen peroxide was added to a 2 mL cell culture dish.

[0095] 7. After the reaction, rinse each well twice with 1 mL of 1X PBS solution.

[0096] 8. Add 200 μL of trypsin to each well and react in the dark for 5 minutes. After the reaction, add 6 mL of cell culture medium to stop the reaction.

[0097] 9. Collect the cells and cell culture medium from each well into individual 1.5 mL centrifuge tubes, and centrifuge the tubes containing cells and culture medium at 400 xg for 10 minutes.

[0098] 10. After centrifugation, remove the supernatant and rehydrate the cell pellet with 1X PBS solution.

[0099] 11. Centrifuge at 400 xg for 10 minutes.

[0100] 12. After centrifugation, remove the supernatant and resuspend the cells in 1 mL of 1X PBS solution in the dark to obtain the cell solution to be tested.

[0101] 13. Flow cytometry was used to detect the fluorescence signal of DCFH-DA in the cell culture of each well. The excitation wavelength for fluorescence detection was 450-490 nm, and the emission wavelength was 510-550 nm. Since DCFH-DA is first hydrolyzed into DCFH (dichlorodihydrofluorescein) after entering the cell, and then oxidized by reactive oxygen species (ROS) to DCF (dichlorofluorescein) which emits green fluorescence, the fluorescence intensity of cells treated with DCFH-DA reflects the content of ROS in the cell, and thus indicates the proportion of cells with high ROS expression to the original cell count. Because the experiment was performed in duplicate, the measurement results of the duplicate experiments in each group were averaged to obtain the mean value. Then, the mean value of the control group was taken as 100% of the relative ROS generation. The mean values ​​of the control group and the experimental group were converted to the relative ROS generation, as follows: Figure 1 As shown.

[0102] Experimental results

[0103] like Figure 1 As shown in the comparison results between the control and experimental groups, the relative ROS generation increased significantly by approximately 280% after hydrogen peroxide treatment, as indicated by high fluorescence expression. This demonstrates that hydrogen peroxide treatment does indeed lead to the production of reactive oxygen species within cells, subsequently causing damage to skin fibroblasts. On the other hand, the comparison results between the control and experimental groups show that after treatment with berry juice, the relative ROS generation decreased significantly by approximately 29%, even lower than the control group. This indicates that berry juice can effectively reduce the production or accumulation of reactive oxygen species within cells. In other words, berry juice can act as a reactive oxygen species scavenger. Specifically, berry juice can reduce oxidative damage to cells caused by reactive oxygen species by lowering the intracellular reactive oxygen species content.

[0104] [Example 4] Cell Experiment - Dewberry Juice Increases the Expression Level of Moisturizing Genes

[0105] This embodiment uses an RNA extraction kit, reverse transcriptase, KAPA SYBR® FAST qPCR reagent kit, and a quantitative PCR instrument. The experimental method is the same as in Example 3. The changes in moisturizing genes in human primary skin keratinocytes after treatment with dewberry juice were measured.

[0106] For example, moisturizing-related genes include the KRT10 gene (Gene ID: 3858), the KRT1 gene (Gene ID: 3848), and the SMPD1 gene (Gene ID: 6609).

[0107] Keratin 10 (KRT10) and keratin 1 (KRT1) are responsible for encoding keratin, the main building block of the skin, while sphingomyelin phosphodiesterase 1 (SMPD1) and tyrosinase-related protein-1 (TYRP1) are responsible for encoding sphingomyelin phosphodiesterase, which converts sphingomyelin into ceramides.

[0108] Materials and Instruments

[0109] Cell line: Human primary epidermal keratinocytes HPEK-50 (CELLnTEC, Switzerland, HPEK-50).

[0110] Culture medium: Keratinocyte-SFM (purchased from Thermo, catalog number 17005042).

[0111] RNA extraction reagent kit (purchased from Geneaid, Taiwan, Lot No. FC24015-G).

[0112] Reverse transcriptase (SuperScript® III Reverse Transcriptase) (Invitrogen, USA, Product No. 18080-051).

[0113] The target gene primers for measurement include the SRD5A1 gene, SRD5A2 gene, AR gene, KROX20 gene, SCF gene, VEGF gene, IGF1 gene, TGF-B gene, and an internal control group (TBP gene).

[0114] KAPA SYBR® FAST qPCR kit (purchased from Sigma, USA, serial number 38220000000).

[0115] ABI StepOnePlus™ Real-Time PCR system (Thermo Fisher Scientific, USA).

[0116] Dewberry juice: The dewberry juice used in this experiment was obtained through Example 1 above.

[0117] Experimental steps

[0118] First, take 1.5 x 10 5 Primary human keratinocytes were cultured in six-well cell culture dishes containing 2 ml of the above-mentioned culture medium per well and cultured at 37°C for 24 hours. The primary human keratinocytes cultured in each well were then divided into a control group and an experimental group (two groups in total) according to the following test conditions.

[0119] Test conditions

[0120] The control group used 2 mL of culture medium to culture primary human keratinocytes without adding any other additives. The experimental group used 2 mL of culture medium containing 0.25 mg / mL of styrax berry juice prepared as described in the above example to culture primary human keratinocytes for 24 hours, with each group performed in triplicate.

[0121] Human primary keratinocytes (control and experimental groups) were treated and their cell membranes were lysed separately using cell lysis buffer to create two cell solutions. RNA was then extracted from the cell solutions of all four groups using an RNA extraction kit (Geneaid, Taiwan, Lot No. FC24015-G). Subsequently, 1000 nanograms (ng) of the extracted RNA from each group was used as a template and processed using SuperScript. ® Reverse transcriptase III (purchased from Invitrogene, USA, catalog number 18080-051) was used to reverse transcribe the extracted RNA into the corresponding cDNA. Then, using ABI StepOnePlus... TM ABIStepOnePlus Real-Time PCR System TM A real-time PCR system (Thermo Fisher Scientific, USA), KAPASYBR FAST (Sigma, USA, catalog number 38220000000), and the primers listed in Table 1 (SEQ ID NO: 1-8) were used to perform quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) on cDNA from two groups to observe the expression levels of KRT10, KRT1, and SMPD1 genes in primary human keratinocytes. The instrument settings for the qRT-PCR were 95°C for 20 seconds, followed by 95°C for 3 seconds, 60°C for 30 seconds, repeated 40 times, and gene quantification was performed using the 2-ΔCt method. Thus, by performing qRT-PCR on cDNA, the mRNA expression levels of KRT10, KRT1, and SMPD1 genes can be indirectly quantified, thereby inferring the expression levels of the proteins encoded by these genes.

[0122] Table 1:

[0123]

[0124] *F stands for FORWARD (forward), and R stands for REVERSE (reverse).

[0125] It should be noted that the relative gene expression of KRT10, KRT1, and SMPD1 genes shown in the graphs described below are presented as relative ploidy rates. The standard deviations were calculated using the STDEV formula in Excel, and a one-tailed Student's t-test was used in Excel to analyze whether there were statistically significant differences. In the graphs, "*" represents a p-value less than 0.05, "**" represents a p-value less than 0.01, and "***" represents a p-value less than 0.001. More "*" characters indicate a more statistically significant difference.

[0126] Please see Figure 2 The expression level of the TBP gene in the control group was considered as 1-fold. The expression levels of the KRT10 gene in the experimental group were 31.6-fold, the KRT1 gene in the experimental group were 34.7-fold, and the SMPD1 gene in the experimental group were 9.9-fold compared to the control group. This indicates that when human melanoma cells were treated with 0.25 mg / mL of berry juice, the expression levels of the KRT10 and KRT1 genes were positively correlated with keratin formation. Keratin contains natural moisturizing factors (NMF), which reduce intercellular spaces, resist external stimuli, protect the dermis, and prevent skin damage and inflammation. The results are as follows... Figure 2 As shown, the berry juice of the present invention can enhance the gene expression of moisturizing genes (KRT10 gene, KRT1 gene and SMPD1 gene) in cells, thereby giving the berry juice a moisturizing effect on the skin.

[0127] [Example 5] Cell Experiment - Dewberry Juice Reduces Melanin Gene Expression

[0128] This embodiment uses an RNA extraction kit, reverse transcriptase, KAPA SYBR® FAST qPCR reagent kit, and a quantitative PCR instrument to determine the changes in whitening genes in human melanoma cells after treatment with dewberry juice.

[0129] For example, melanin-related genes include the TYR gene (Gene ID: 7299), the TYRP1 gene (Gene ID: 7306), the MITF gene (Gene ID: 4286), and the MC1R gene (Gene ID: 4157).

[0130] Microphthalamia-associated transcription factor (MITF) is a transcription factor required for the normal development of melanocytes in the skin. It regulates the expression of genes of several other proteins involved in melanin formation, such as tyrosinase (TYR) and tyrosinase-related protein-1 (TYRP1). The melanocortin 1-receptor (MC1R) gene is a key gene in the process of melanin synthesis in human skin.

[0131] Therefore, in this embodiment, the TYR gene, TYRP1 gene, MITF gene and MC1R gene are used as the analysis targets.

[0132] Materials and Instruments

[0133] 1. Cell line: Human melanoma cell A375.S2 (ATCC, number CRL-1872).

[0134] 2. Culture medium: MEM-NAA medium (Gibco, No. 41500-03) containing 10% fetal bovine serum (FBS) (GIBCO, No. 10438-026, USA), 1% antibiotic-antimycotic (Gibco, No. 15240-062), and 1mM sodium pyruvate (Gibco, No. 11360-070).

[0135] RNA extraction reagent kit (purchased from Geneaid, Taiwan, Lot No. FC24015-G).

[0136] 4. Reverse transcriptase (SuperScript® III Reverse Transcriptase) (Invitrogen, USA, Product No. 18080-051).

[0137] 5. Measurement target gene primers, including the TYRP1 gene, TYR gene, MITF gene and MC1R gene, as well as the internal control group (GAPDH gene).

[0138] 6. KAPA SYBR® FAST qPCR reagent kit (purchased from Sigma, USA, serial number 38220000000).

[0139] 7. ABI StepOnePlus™ Real-Time PCR system (Thermo Fisher Scientific, USA).

[0140] 8. Berry juice: The berry juice used in this experiment was obtained through Example 1 above.

[0141] Experimental steps

[0142] First, take 1.5 x 10 5 Human melanoma cells were cultured in six-well cell culture dishes containing 2 ml of the above-mentioned culture medium per well at 37°C for 24 hours. The cultured human melanoma cells in each well were then divided into a control group and an experimental group (two groups in total) according to the following test conditions.

[0143] Test conditions:

[0144] The control group used only 2 mL of culture medium to culture human melanoma cells without adding any other additives. The experimental group used 2 mL of culture medium containing 0.125 mg / mL of safberry juice prepared as described in the previous example to culture human melanoma cells for 24 hours.

[0145] Human melanoma cells (control and experimental groups) were lysed separately using cell lysis buffer to create two cell solutions. RNA was then extracted from the cell solutions using an RNA extraction kit (Geneaid, Taiwan, Lot No. FC24015-G). Subsequently, 1000 nanograms (ng) of the extracted RNA from each group was used as a template, and the extracted RNA was reverse transcribed into cDNA using SuperScript® III reverse transcriptase (Invitrogene, USA, catalog number 18080-051). The expression levels of TYRP1, TYR, MITF, and MC1R genes in human melanoma cells from the three groups were then observed using an ABI StepOnePlus™ Real-Time PCR system (Thermo Fisher Scientific, USA), KAPASYBR FAST (Sigma, USA, catalog number 38220000000) and the primers listed in Table 2 (SEQ ID NO: 9-18). The instrument settings for the quantitative real-time reverse transcription polymerase chain reaction were: 95°C for 20 seconds, followed by 95°C for 3 seconds, 60°C for 30 seconds, repeated 40 times. Gene quantification was performed using the 2-ΔCt method. Therefore, by using cDNA to perform quantitative real-time reverse transcription polymerase chain reaction, the mRNA expression levels of TYRP1, TYR, MITF, and MC1R genes can be indirectly quantified, thereby inferring the expression levels of the proteins encoded by TYRP1, TYR, MITF, and MC1R genes.

[0146] Table 2:

[0147]

[0148] *F stands for FORWARD (forward), and R stands for REVERSE (reverse).

[0149] It should be noted that the relative gene expression of TYR, TYRP1, MITF, and MC1R genes shown in the graphs described below are presented as relative ploidy rates. The standard deviations were calculated using the STDEV formula in Excel, and a one-tailed Student's t-test was used in Excel to analyze whether there were statistically significant differences. In the graphs, "*" represents a p-value less than 0.05, "**" represents a p-value less than 0.01, and "***" represents a p-value less than 0.001. More "*" characters indicate a more statistically significant difference.

[0150] Please see Figure 3 The expression level of the GAPDH gene in the control group was considered as 1. The expression levels of the TYR gene in the experimental group relative to the control group were 0.12-fold, TYRP1 gene 0.86-fold, MITF gene 0.75-fold, and MC1R gene 0.65-fold. This represents an 88% reduction in TYR gene expression, a 14% reduction in TYRP1 gene expression, a 25% reduction in MITF gene expression, and a 35% reduction in MC1R gene expression compared to the control group. Therefore, when human melanoma cells were treated with 0.125 mg / mL of berry juice, the results showed that, since MC1R and MITF genes are positively correlated with melanin formation, the effects were as follows. Figure 3 As shown, the berry juice of the present invention can inhibit the gene expression of TYR, TYRP1, MC1R and MITF genes in melanoma cells, thereby giving the berry juice an inhibitory effect on melanin formation.

[0151] [Example 6] Cell Experiment - Dewberry Juice Reduces Melanin Production

[0152] Here, the changes in melanin content of melanoma cell line B16F10 after treatment with dewberry juice were determined using an ELISA reader (enzyme-linked immunosorbent assay reader).

[0153] Materials and Instruments

[0154] 1. Cell line: Mouse melanoma cells B16F10, purchased from the American Type Culture Collection (ATCC®, No. 6475), hereinafter referred to as B16F10 cells.

[0155] 2. Culture medium: Dulbecco's modified minimal essential medium (DMEM, purchased from Gibco, Cat. 12100-038) was supplemented with additional components to contain 1 vol% of antibiotic solution (Antibiotic Antimycotic Solution, purchased from Gibco, 15240-062) and 10 vol% of FBS (fetal bovine serum, purchased from Gibco, 10437-028).

[0156] 3. Phosphate-buffered saline (PBS) solution: purchased from Gibco, product number 10437-028.

[0157] 4. Prepare a 1N NaOH solution (purchased from Sigma, product number 221465) using double-distilled water.

[0158] 5. ELISA reader (purchased from BioTek, product number FLx 800).

[0159] 6. Berry juice: The berry juice used in this experiment was obtained through Example 1 above.

[0160] Experimental steps

[0161] The experiment will be conducted in three groups: an experimental group, a control group, and a control group. Each group will undergo a triple-replication experiment.

[0162] 1. Spread B16F10 cells at a density of 1.5 × 10⁶ cells per well. 5 Inoculate in a 6-well culture dish containing 3 mL of culture medium per well using a single method.

[0163] 2. Place the culture dish in a 5% CO2, 37℃ environment and incubate for 24 hours.

[0164] 3. Then, without disturbing the attached cells, remove the culture medium from each well.

[0165] 4. This experiment consisted of a control group, a control group, and an experimental group. Each group received 2 mL of fresh DMEM culture medium. The control group received an additional dose of kojic acid (0.25 mg / mL), which is widely recognized as having the effect of reducing melanin production. The experimental group received an additional dose of dewberry juice (0.25 mg / mL). Each group was tested in triplicate and incubated at 37°C for 24 hours.

[0166] 5. After the reaction was completed, the experimental group and the control group were moved to a blue light chamber and exposed to blue light at room temperature (25±5℃) for 3 hours. Meanwhile, the control group was moved to a dark room and allowed to stand at room temperature for 3 hours.

[0167] 6. After the reaction, the cells were cultured at 37°C for 48 hours.

[0168] 7. Then, remove the culture medium and wash the cells twice with PBS solution.

[0169] 8. Add 200 μl of trypsin-EDTA (10X), purchased from Gibco; product number 15400-054, to each well and react for 3 minutes. After the reaction, add 6 mL of culture medium solution to stop the reaction. Then collect the suspended cells and culture medium from each well into the corresponding 15 mL centrifuge tube, and centrifuge each tube at 400 xg for 5 minutes to precipitate the cells.

[0170] 9. After washing the precipitated cells twice with PBS solution, resuspend the cells in 200 μL of PBS solution.

[0171] 10. Freeze the cell suspension in liquid nitrogen for 10 minutes, then leave it at room temperature for about 30 minutes until completely thawed.

[0172] 11. After completely thawing, centrifuge the test tube at 12,000g for 3 minutes.

[0173] 12. Remove the supernatant, resuspend the cell pellet in 120 μL of 1N sodium hydroxide solution, and then incubate the test tube at 60°C for 1 hour to obtain the sample to be tested.

[0174] 13. Transfer 100 μL of the sample to be tested into a 96-well plate and use an ELISA reader to measure the absorbance of the cell solution at 450 nm.

[0175] Experimental results

[0176] The relative melanin expression levels of the experimental and control groups were calculated using the following formula: Relative melanin expression level (%) = (OD of each group) 450 Value / Control Group OD 450 (Value) × 100%. Since the experiment was conducted in triplicate, the average of the results from the three replicates is displayed. Figure 4 .

[0177] like Figure 4As shown, the treatment with the berry juice of this invention can reduce melanin production. Specifically, the melanin production in the experimental group (berry juice) was 78.81% lower than that in the control group, while the melanin production in the control group was 67.42% lower. The experimental group showed a melanin production reduction of approximately 21.19% compared to the control group, and the effect of the berry juice in this invention is similar to that of kojic acid in reducing melanin production. Therefore, the berry juice prepared in this embodiment of the invention can indeed effectively reduce melanin produced by melanocytes, and thus can be used as an ingredient in compositions for reducing dark spots, improving skin radiance, and whitening the skin.

[0178] The above cell experiments demonstrate that dewberry juice can indeed reduce the production of reactive oxygen species (ROS) in dermal fibroblasts, preventing skin damage caused by ultraviolet radiation. Furthermore, dewberry juice can reduce the expression levels of melanin-related genes (TYRP1, MC1R, and MITF genes) and inhibit melanin production, reducing dark spots caused by blue light and maintaining fair skin.

[0179] To verify the efficacy of wild dewberry juice on the human body, the following human experiments were conducted.

[0180] [Example 7] Detection of the relative amount of skin ultraviolet red spots (UV spots)

[0181] Facial skin was photographed using UV light from the VISIA Complexion Analysis System (Canfield Scientific, USA). Ultraviolet light is absorbed by melanin, increasing the visibility of pigmentation spots and detecting melanin spots in the epidermis that are invisible to the naked eye. Higher measurement values ​​indicate more UV-induced pigmentation spots.

[0182] The berry juice in this study was prepared into a berry juice beverage by mixing it with water to provide subjects with an appropriate and effective dose. In this embodiment, seven subjects (typical men and women aged 20-65 years) consumed the prepared berry juice beverage at a daily intake of one bottle (50 mL) with a concentration of 0.005 mg / mL for eight weeks. Before the start of the experiment (week 0, before consumption), at week 4, and at week 8, the number and area of ​​red spots on the subjects' cleansed facial skin were measured using a full-face skin analysis system (7th Generation VISIA Complexion Analysis System; Canfield, USA).

[0183] The number of red spots on the subjects' skin and the actual changes in appearance are shown in [the figures]. Figure 5A as well as Figure 5B . Figure 5AThe values ​​measured by the instrument based on the number and area of ​​red spots before the subjects consumed the berry juice beverage were taken as 100%, and the values ​​measured based on the number and area of ​​red spots were recorded 4 weeks and 8 weeks after the subjects consumed the berry juice beverage. Figure 5B These are images of one of the subjects' facial skin taken with a testing device at weeks 0, 4, and 8.

[0184] like Figure 5A As shown, after subjects consumed berry juice for 4 weeks, a reduction in the number / area of ​​red spots could be observed. After 4 weeks of continuous consumption, the number of red spots on the facial skin decreased to 2.4% of the original amount, and after 8 weeks of continuous consumption, the number of red spots on the facial skin decreased to 4.1% of the original amount. Figure 5B The actual footage also shows that as the period of consuming berry juice increased, the number and area of ​​red spots on the skin decreased significantly, with an improvement rate of 86%, demonstrating its ability to resist the formation of pigmentation.

[0185] [Example 8] Detection of relative hemoglobin levels in skin redness

[0186] The facial skin of the subjects in Example 7 above was photographed using the RBX polarized light technology of the VISIA Complexion Analysis System (Canfield Scientific, USA) to detect deep skin blood vessels or hemoglobin. The higher the measured value, the more severe the skin redness.

[0187] like Figure 6A As shown, after subjects consumed berry juice for 4 weeks, a decrease in the relative amount of hemoglobin could be observed. After 4 weeks of continuous consumption, the hemoglobin index of facial skin dropped to 7.4% of the original level, and after 8 weeks of continuous consumption, the hemoglobin index of facial skin dropped to 9.3% of the original level. Figure 6B The actual footage also shows that as the period of consuming the berry juice drink increased, the hemoglobin index on the skin decreased significantly, with 100% of the participants showing improvement. The results indicate that the berry juice drink of this invention can effectively improve individual skin redness, maintain stable skin health, and has a good skin-soothing effect.

[0188] [Example 9] Skin Radiance Detection

[0189] The Glossymeter GL200 (C+K Multi Probe Adapter System, Germany) probe measures skin radiance by the direct and diffuse reflection of light incident on the skin surface of the subject in Example 7. Higher measurement values ​​indicate more radiant skin.

[0190] like Figure 7A As shown, after subjects consumed berry juice for 4 weeks, an increase in radiance could be observed. After 4 weeks of continuous consumption, the radiance of facial skin increased to 4.2% of its original value, and after 8 weeks of continuous consumption, the hemoglobin index of facial skin increased to 9.4% of its original value. Figure 7B The actual footage also shows that as the period of consumption of the berry juice drink increased, the skin's radiance increased significantly, with 86% of the participants showing improvement. These results indicate that the berry juice drink of this invention has the effect of brightening the skin.

[0191] In summary, this study demonstrates that berry juice can inhibit free radical production and achieve this by reducing the expression levels of melanin-related genes in cells (e.g., tyrosinase-associated protein-1 (TYRP1), tyrosinase (TYR), microphthalmia-associated transcription factor (MITF), and melanocortin receptor 1 (MC1R)). Therefore, berry juice can effectively prevent various skin aging phenomena caused by ultraviolet radiation, providing protection against ultraviolet and blue light. Furthermore, this study also demonstrates that berry juice can reduce red spots caused by ultraviolet radiation, soothe skin redness, increase skin radiance, and enhance skin's moisturizing ability. Accordingly, berry juice can provide overall antioxidant, whitening, and radiant skin, while preventing ultraviolet damage and aging.

[0192] Of course, the present invention may have other various embodiments. Without departing from the spirit and essence of the present invention, those skilled in the art can make various corresponding changes and modifications according to the present invention, but these corresponding changes and modifications should all fall within the protection scope of the claims of the present invention.

Claims

1. The use of a dewberry juice in the preparation of a composition for reducing oxidative damage to cells caused by reactive oxygen species induced by hydrogen peroxide, characterized in that: The dewberry juice is obtained by soaking dewberry fruits in an aqueous solvent for 0.5 to 3 hours, and the volume ratio of the aqueous solvent to the dewberry fruits is 5 to 20: 1 to 5.

2. The use as described in claim 1, characterized in that: The volume ratio of the aqueous solvent to the berries is 5:

1.

3. The use as described in claim 1, characterized in that: The dewberry fruit was soaked in an aqueous solvent at 75°C to 95°C.

4. The use as described in claim 1, characterized in that: The total flavonoid content of the dewberry juice is between 1000 and 3000 ppm.

5. The use as described in claim 1, characterized in that: The total polyphenol content of the dewberry juice is between 8,000 and 11,000 ppm.

6. The use as described in claim 1, characterized in that: The cells in question are skin cells.

7. The use as described in claim 1, characterized in that: The composition is a reactive oxygen species scavenger.

8. The use as described in claim 6, characterized in that: The composition is further used to reduce intercellular spaces to protect the dermis.

9. The use as described in claim 8, characterized in that: The composition is used to achieve the ability to protect the dermis by increasing the gene expression levels of the keratin 10 (KRT10) gene and the keratin 1 (KRT1) gene in cells.

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