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WO2024181725A1 - Centella asiatica rouge fermenté avec monascus et son utilisation - Google Patents

Centella asiatica rouge fermenté avec monascus et son utilisation Download PDF

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Publication number
WO2024181725A1
WO2024181725A1 PCT/KR2024/002258 KR2024002258W WO2024181725A1 WO 2024181725 A1 WO2024181725 A1 WO 2024181725A1 KR 2024002258 W KR2024002258 W KR 2024002258W WO 2024181725 A1 WO2024181725 A1 WO 2024181725A1
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sample
monascus
fermented
present
skin
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English (en)
Korean (ko)
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주철규
김유정
지하영
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Preparation or treatment thereof
    • A23L2/38—Other non-alcoholic beverages
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
    • A61K8/9783—Angiosperms [Magnoliophyta]
    • A61K8/9789—Magnoliopsida [dicotyledons]
    • 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
    • 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
    • A61Q19/02—Preparations for care of the skin for chemically bleaching or whitening the skin
    • 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

Definitions

  • the present invention relates to a purslane fermented with Hong Aspergillus oryzae. More specifically, it relates to the anti-oxidation, skin whitening, skin wrinkle improvement, cell regeneration, cytotoxicity relief, or anti-inflammatory uses of a purslane fermented with Hong Aspergillus oryzae.
  • oxidation of proteins can damage the skin's connective tissues such as collagen, hyaluronic acid, elastin, proteoglycan, and fibronectin, which not only affect skin tissue but also cause deterioration of health or disease due to decreased immune function, mutations due to DNA mutation, etc., which can affect the health of the entire body.
  • wrinkles occur when the synthesis of collagen and elastin, which maintain elasticity in the skin's dermis layer, decreases, causing structural changes in which the skin becomes thinner and less elastic as skin aging progresses.
  • collagen which keeps the skin's dermis matrix elastic
  • elastin which plays a role in supporting the collagen by weaving it together, is involved, and the oxidation of these proteins is the main cause of wrinkles caused by skin aging.
  • Pigmentation is another major symptom caused by skin aging, and various pigments that affect skin color and pigmentation are involved in this process.
  • melanin is known to play a role in protecting the skin from damage by UV rays by absorbing or scattering UV rays.
  • age group J Invest Dermatol. 2022 Sep;142(9):2521-2523.e1.
  • melanocytes begin to age from the late 40s, and as the number of aged melanocytes increases, the number of other skin aging-related cells also increases.
  • melanin has become known to be an important factor in pigmentation caused by skin aging.
  • the inventors of the present invention have conducted a variety of studies on methods for producing and compositions of various plant extracts containing natural substances safe for the human body as main ingredients, and have endeavored to find a method for enhancing the antioxidant, skin whitening, wrinkle improvement, cell regeneration, cytotoxicity relief, or anti-inflammatory effects of Centella asiatica , which are well known for their efficacy and effects.
  • the purpose of the present invention is to provide a composition containing a purslane fermented with Hongguk fungus.
  • Another object of the present invention is to provide an antioxidant, skin whitening, skin wrinkle improvement, cell regeneration, cytotoxicity relief or anti-inflammatory use of a purslane fermented with Hongguk fungus.
  • the purslane fermented with Monascus aureus is a fermented product and is red in color.
  • the term "red purslane” used in the present invention means a product obtained by fermenting the raw material of purslane ( Centella asiatica ) with Monascus aureus ( Monascus ). In the present specification, the "red purslane” may be used interchangeably with “fermented purslane.”
  • raw material used in the present invention refers to a raw material prior to processing such as fermentation or extraction.
  • the term "fermentation” used in the present invention means a process of decomposing or changing a raw material or an organic material using microorganisms such as bacteria, yeast, etc., or enzymes possessed by microorganisms.
  • the fermentation may be, for example, liquid-phase fermentation or solid-phase fermentation.
  • the fermentation may be pre-fermentation or post-fermentation.
  • the pre-fermentation is a method of fermenting a raw material or an organic material by inoculating a microorganism into the raw material or an organic material without processing the raw material or the organic material by heat treatment or the like
  • the post-fermentation is a method of fermenting a raw material or an organic material by inoculating a microorganism into the raw material or the organic material processed by heat treatment or the like.
  • Centella asiatica is a perennial herb of the Apiaceae family, Umbelliferous order, and Dicotyledonous group, and is a plant often referred to by the common name CICA.
  • CICA common name
  • Monascus is a fungus belonging to the Monascaceae genus of the Hemiascomycetaceae family, and is called “Monascus” because its mycelia are red. It has been used in traditional foods in Korea, China, Hong Kong, Japan, Taiwan, etc. for a long time, and is mainly used for coloring and preservative purposes in alcohol, vegetables, and processed meat products.
  • any type of bacteria used in the fermentation may be used, as long as it is a red yeast strain belonging to the genus Monascus.
  • the red yeast fungus can be Monascus purpureus , Monascus ruber, Monascus rubervan Tieghem , Monascus fuliginosus Sato, Monascus pilosus , Monascus purpurens, Monascus pilosus Sato , Monascus anka , Monascus barykery or Monascus albidus Sato .
  • the above-mentioned herb raw material may be used without limitation, including cultivated, collected, or commercially available materials.
  • the Hongguk fungus may be used without limitation, including purchased commercially available products or cultured products.
  • the red wormwood fermented with the Hong Guk-myun according to the present invention can exhibit antioxidant, skin whitening, wrinkle improvement, cell regeneration, cytotoxicity relief, or anti-inflammatory effects.
  • antioxidant used in the present invention means suppressing oxidative stress. Specifically, it means all actions that suppress cellular oxidation of reactive oxygen species (ROS) generated by oxidative stress caused by intracellular metabolism or ultraviolet rays, etc., or remove reactive oxygen species already generated.
  • ROS reactive oxygen species
  • skin whitening used in the present invention means any action that suppresses or inhibits the production or synthesis of melanin in the skin or decomposes melanin already produced to make the skin even or brighter.
  • improvement of skin wrinkles means alleviating wrinkles already formed by suppressing or inhibiting the formation of wrinkles on the skin or reducing the number or depth of wrinkles, and also includes suppressing skin aging through improvement of wrinkles.
  • cell regeneration used in the present invention refers to a series of processes in which cells around a cell or part of a tissue are divided and proliferated to reconstruct the tissue when the cell or part of the tissue is damaged due to trauma, disease, aging, etc.
  • anti-inflammation used in the present invention means suppressing inflammation or removing inflammation that has already occurred.
  • the inflammation is an immunological defense mechanism that attempts to recover and regenerate damaged areas when the body is damaged by various factors, and is induced by the action of inflammatory cytokines or hormones.
  • the fermented product of the present invention provides an antioxidant effect by reducing free radicals or active oxygen, provides an improvement in wrinkles and cell regeneration effects by reducing the activity of elastase or promoting the production of collagen, and can alleviate cytotoxicity or provide anti-inflammatory effects by alleviating cytotoxicity caused by ultraviolet rays or suppressing the expression of inflammatory cytokines.
  • Another aspect of the present invention provides a composition comprising a purslane fermented with Hong Guk-myun.
  • each term has the same meaning as described above unless specifically mentioned.
  • a cosmetic composition comprising red ginseng fermented with Hongguksu is provided.
  • each term has the same meaning as described above unless specifically mentioned.
  • the content of the effective ingredient included in the cosmetic composition according to the present invention can be suitably determined by considering various factors such as the purpose of use, period of use, type of formulation, route of administration, skin condition of the subject, etc.
  • the content of the red scutellaria baicalensis can be 0.0001 to 100 wt%, for example, 0.01 to 5 wt%, based on the total weight of the cosmetic composition.
  • subject as used herein includes a human or any non-human animal, which non-human animal may be a vertebrate, such as a primate, dog, cow, horse, pig, rodent, such as a mouse, rat, guinea pig, and the like. As used herein, the “subject” is used interchangeably with “individual” and “patient”.
  • the term "administration" means physically introducing the composition into a subject using any of a variety of methods and delivery systems known to those of ordinary skill in the art.
  • the administration may be performed, for example, orally, or intravaginally, intravenously, intramuscularly, subcutaneously, intraperitoneally, spinally, or other parenteral administration, such as by injection or infusion, but is not limited thereto.
  • the number of times the administration may be performed, for example, once, multiple times, and/or over one or more extended periods of time.
  • the dosage may vary depending on the age, sex, and weight of the subject, and specifically, depending on the symptoms of the subject, the composition of the present invention may be administered once or several times daily at 0.1 to 100 mg/kg, or at intervals of several days to several months. In addition, the dosage may be increased or decreased depending on the route of administration, the severity of symptoms, sex, body weight, age, etc.
  • the cosmetic composition according to the present invention can be manufactured in the form of general emulsified formulations and solubilized formulations, etc., using conventionally known manufacturing methods.
  • the cosmetic composition of the present invention can be manufactured in various formulations such as patches, ointments, skin adhesive gels, creams, packs, toners, essences, sprays, masks, foundations, makeup bases, cleansers, water (W) type, oil (O) type, silicone (S) type, oil-in-water (O/W) type, water-in-oil (W/O) type, water-in-silicon (W/S) type, silicone-in-water (S/W) type, solid, and liquid, and conventionally used cosmetic manufacturing methods can be applied.
  • the cosmetic composition according to the present invention may further contain additional components in addition to the fermented product of the purslane.
  • the additional components are not limited as long as they do not offset or reduce the efficacy of the fermented product, and may include, for example, conventional components typically used to add or enhance the function of cosmetics.
  • excipients, carriers, etc. typically used in the field of cosmetics may be further contained.
  • components typically used to add or enhance the function of cosmetics may also be added.
  • the composition may include one or more aqueous additives selected from stabilizers, emulsifiers, thickeners, moisturizers, liquid crystal film strengtheners, pH regulators, antibacterial agents, water-soluble polymers, film-forming agents, metal ion sequestrants, amino acids, organic amines, polymer emulsions, pH regulators, skin nutrients, antioxidants, antioxidant agents, preservatives, fragrances, etc.; and one or more oily additives selected from fats, waxes, hydrocarbon oils, higher fatty acid oils, higher alcohols, synthetic ester oils, and silicone oils.
  • aqueous additives selected from stabilizers, emulsifiers, thickeners, moisturizers, liquid crystal film strengtheners, pH regulators, antibacterial agents, water-soluble polymers, film-forming agents, metal ion sequestrants, amino acids, organic amines, polymer emulsions, pH regulators, skin nutrients, antioxidants, antioxidant agents, preservatives, fragrances, etc.
  • the cosmetic composition according to the present invention can be formulated containing a cosmetically or dermatologically acceptable medium or base. It can be provided in any formulation suitable for topical application, for example, in the form of a solution, a gel, a solid, a pasty anhydrous product, an emulsion obtained by dispersing an oil phase in an aqueous phase, a suspension, a microemulsion, a microcapsule, a microgranule or an ionic (liposome) and nonionic vesicular dispersion, or in the form of a cream, a skin, a lotion, a powder, an ointment, a spray or a concealer stick. It can also be used in the form of a foam or in the form of an aerosol composition further containing a compressed propellant.
  • the cosmetic composition according to the present invention may contain adjuvants commonly used in the fields of cosmetology or dermatology, such as fatty substances, organic solvents, solubilizers, thickeners, gelling agents, emollients, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, surfactants, water, ionic or nonionic emulsifiers, fillers, sequestering agents, chelating agents, preservatives, vitamins, blocking agents, humectants, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles or any other ingredients commonly used in cosmetics.
  • the adjuvants are introduced in amounts commonly used in the fields of cosmetology or dermatology.
  • a food composition comprising red ginseng fermented with Hong Guk-myun is provided.
  • each term has the same meaning as described above unless specifically mentioned.
  • the above food may be a health functional food.
  • the above “health functional food” refers to a food manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. using raw materials or ingredients that have functionality useful to the human body.
  • “functionality” can be interpreted as obtaining a useful effect for health purposes such as regulating nutrients or physiological actions for the structure and function of the human body.
  • the food composition according to the present invention can be manufactured by a method commonly used in the art, and can be manufactured by adding raw materials and ingredients commonly added in the art during the manufacturing process.
  • the food composition according to the present invention contains a natural plant extract as an effective ingredient, there are no side effects or resistance that may generally occur when taken for a long period of time. Therefore, the food composition according to the present invention can be used alone, but it can also be used simultaneously or sequentially with a cosmetic composition, a quasi-drug composition, a pharmaceutical composition, and/or another composition or another treatment according to the present invention for the purpose of maximizing the effect of improving skin condition.
  • the content of the fermented product of purslane included as an effective ingredient in the food composition according to the present invention can be appropriately determined depending on the purpose of use, period of use, skin condition of the subject, etc.
  • the food composition according to the present invention may further include additional ingredients in addition to the effective ingredient to enhance efficacy.
  • the additional ingredient may be an ingredient or agent that exhibits a skin condition improvement effect, as long as it does not offset or reduce the efficacy of the fermented product of the present invention.
  • the food composition may be in the form of a pill, tablet, granule, powder, capsule, liquid, etc.
  • the type of the food is not particularly limited.
  • the food that may include the fermented product of the herb includes dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, ice cream, various soups, beverages, tea, drinks, alcoholic beverages, vitamin complexes, etc., and includes all foods in the conventional sense.
  • the food composition of the present invention may contain various flavoring agents or natural carbohydrates used in conventional foods as additional ingredients.
  • the natural carbohydrates may be monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol.
  • a natural sweetener such as thaumatin and stevia extract, or a synthetic sweetener such as saccharin and aspartame may be used.
  • a pharmaceutical composition for anti-oxidation, skin whitening, skin wrinkle improvement, cell regeneration, cytotoxicity relief or anti-inflammation comprising red ginseng fermented with Hong Guk-myun.
  • each term has the same meaning as described above unless specifically mentioned.
  • the pharmaceutical composition of the present invention may be used for the treatment or prevention of wounds, and may be used for the treatment or prevention of inflammatory diseases.
  • the inflammatory diseases may be, for example, dermatitis, atopy, allergy, psoriasis, scleroderma, fibromyalgia, gastritis, gastric ulcer, pharyngitis, periodontitis, etc., but are not limited thereto.
  • treatment means any action that improves symptoms of a skin condition by administering a composition according to the present invention.
  • prevention means any act of suppressing or delaying the symptoms of a skin pigmentation disorder by administering a composition according to the present invention.
  • the content of the effective ingredient included in the pharmaceutical composition according to the present invention can be appropriately determined according to the skin symptoms, the degree of symptom progression, the condition, age, sex, etc. of the subject.
  • the red scutellaria baicalensis of the present invention can be included in an amount of about 0.0001 to 100 wt% based on the total weight of the pharmaceutical composition.
  • the pharmaceutical composition according to the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of medicines.
  • the carriers, excipients and diluents that may be contained in the pharmaceutical composition may be, for example, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil, but are not limited thereto.
  • the pharmaceutical composition according to the present invention may be an oral administration formulation.
  • Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations may be prepared by mixing the fermented plant of the above-mentioned purslane with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc.
  • Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups, etc., and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, may be included.
  • the pharmaceutical composition according to the present invention can be formulated into a suitable dosage form for topical administration using techniques well known to those skilled in the art, and the dosage form can include an external preparation, an effervescent tablet, a suppository, and the like.
  • the pharmaceutical composition of the present invention can be formulated into an external preparation by mixing the fermented product of the purslane with a base well known and commonly used in the art.
  • the external preparation can be an emulsion, a gel, an ointment, a cream, a patch, a liniment, a powder, an aerosol, a spray, a lotion, a serum, a paste, a foam, an injection, a suspension, a tincture, and the like.
  • the pharmaceutical composition of the present invention may be administered in combination with other therapeutic agents.
  • the pharmaceutical composition of the present invention and the other therapeutic agents may be administered simultaneously, sequentially, or separately.
  • the other therapeutic agents may be drugs such as compounds, proteins, etc. that have antioxidant, skin whitening, skin wrinkle improvement, cell regeneration, cytotoxicity mitigation, or anti-inflammatory effects, but are not limited thereto.
  • the pharmaceutical composition of the present invention may be formulated to be administered simultaneously, sequentially, or separately with other therapeutic agents.
  • the pharmaceutical composition and the other therapeutic agent may be administered simultaneously as one formulation, or may be administered simultaneously, sequentially, or separately as separate formulations.
  • the pharmaceutical composition of the present invention and the other therapeutic agent may be formulated separately in separate containers, or may be formulated together in the same container.
  • the pharmaceutical composition of the present invention and the other therapeutic agent may be the same or different from each other in terms of the pharmaceutically effective amount, administration time, administration interval, administration route, treatment period, etc.
  • Another aspect of the present invention provides a method for anti-oxidation, skin whitening, skin wrinkle improvement, cell regeneration, cytotoxicity relief or anti-inflammation, comprising the step of administering to a subject a composition comprising a fermented plant of Hong Guk-myun.
  • each term has the same meaning as described above unless specifically mentioned.
  • the composition of the present invention may be administered to a subject simultaneously, sequentially or separately with another component exhibiting antioxidation, skin whitening, skin wrinkle improvement, cell regeneration, cytotoxicity relief or anti-inflammation effects.
  • the "simultaneous" administration means administering the composition and the other component at one time as one formulation, or means administering the composition and the other component at one time as separate formulations, in which case the administration route of the composition and the administration route of the other component may be different from each other.
  • the “sequential” administration means administering the composition and the other component relatively continuously, allowing the minimum possible time as the time consumed for the administration interval.
  • the "separate” administration means administering the composition and the other component at a certain time interval. The method of administering the composition and the other component may be appropriately selected by an expert in the art in consideration of the efficacy, side effects, etc. for the subject.
  • Another aspect of the present invention provides the antioxidant, skin whitening, skin wrinkle improvement, cell regeneration, cytotoxicity relief or anti-inflammatory uses of the purslane fermented with Hong Guk-myun.
  • each term has the same meaning as described above unless specifically mentioned.
  • Another aspect of the present invention provides a method for producing a fermented product of purslane.
  • each term has the same meaning as described above unless specifically mentioned.
  • the method for producing a fermented product of purslane according to the present invention comprises a step of fermenting purslane with red yeast.
  • the fermenting step may be fermenting at a temperature of 10°C to 50°C for 24 hours or more by mixing with a solvent, for example, fermenting at a temperature of 20°C to 30°C for 24 to 96 hours, or for example, fermenting at a temperature of 24°C for 72 hours.
  • the above manufacturing method may further include a step of extracting the obtained fermented product with an extraction solvent.
  • extract used in the present invention means a result obtained by extracting a herb with an appropriate extraction solvent, and may include an extract obtained by an extraction treatment process, a diluted or concentrated extract, a dried product obtained by drying the extract, or a purified product or fraction obtained by purifying or fractionating these.
  • the extract according to the present invention can be prepared using a general extraction method known in the art, and for example, methods such as hot water extraction, hot water extraction, cold infusion extraction, reflux cooling extraction, ultrasonic extraction, and high-temperature pressure extraction can be used.
  • the extraction solvent used to prepare the extract according to the present invention may be at least one selected from the group consisting of water, ethanol, methanol, butanol, methylpropanediol, propanediol, butylene glycol, hexanediol, propylene glycol, chloroform, ethyl acetate, dichloromethane, hexane, petroleum ether and diethyl ether, and preferably, water, for example, purified water, may be used as the extraction solvent.
  • the extraction solvent may be used in an amount of 0.01 to 100 times the weight of the total extract.
  • the extract according to the present invention may include not only extracts obtained by the above-described extraction solvent, but also extracts obtained by conventional purification, enzymatic treatment, and fermentation processes.
  • extracts obtained by various purification and extraction methods that can be additionally performed, such as extraction using reduced pressure using carbon dioxide, high temperature, or ultrasonic waves, and extracts obtained by fermentation products using natural conditions or various microorganisms, may also be included in the extract of the present invention.
  • the manufacturing method may further include a step of filtering the obtained fermented product and then drying it to obtain a composition.
  • the purslane and the Hongguk fungus may be mixed in a weight ratio of 0.1 to 10: 0.1 to 10, and preferably, may be mixed in a weight ratio of 1:1.
  • the red wormwood fermented with the Hong Guk-myun according to the present invention provides antioxidant, skin whitening, skin wrinkle improvement, cell regeneration, cytotoxicity relief or anti-inflammatory effects. In addition, it can effectively alleviate skin aging that can occur from various causes.
  • the composition of the present invention can be applied to various fields that require prevention or alleviation of skin aging, and can be used in various ways in cosmetics, quasi-drugs, foods, and medicines.
  • Figure 1 shows red yeast fermented with Monascus .
  • Figure 2 shows the manufacturing process of the fermented herbicide using Honggukgyun.
  • Washed and dried purslane in amounts of 1 wt%, 3 wt%, 5 wt%, 10 wt%, 20 wt%, 50 wt%, and 80 wt% respectively was mixed with 1 wt% dried Hongsukmyun, added to purified water, and fermented at 24°C for 72 hours. Afterwards, the fermented red purslane was taken out through a strainer and the moisture was completely removed using a dry oven (Hanbaek Science, KR). These were designated as Sample 1, Sample 2, Sample 3, Sample 4, Sample 5, Sample 6, and Sample 7, respectively, and used in the following tests.
  • Washed and dried bottle gourd in amounts of 1 wt%, 3 wt%, 5 wt%, and 10 wt% respectively was mixed with 1 wt% dried Lactobacillus and placed in purified water to ferment.
  • the fermented bottle gourd was then taken out on a sieve and the moisture was completely removed using a dry oven (Hanbaek Science, KR). These were designated as Sample 9, Sample 10, Sample 11, and Sample 12, respectively, and used in the following tests.
  • Washed and dried purslane in amounts of 1 wt%, 3 wt%, 5 wt%, and 10 wt% respectively was mixed with 1 wt% dried Saccharomyces fungus and placed in purified water to ferment.
  • the fermented purslane was then taken out through a strainer and the moisture was completely removed using a dry oven (Hanbaek Science, KR). These were designated as Sample 13, Sample 14, Sample 15, and Sample 16, respectively, and used in the following tests.
  • Washed and dried purslane in amounts of 1 wt%, 3 wt%, 5 wt%, and 10 wt% respectively was mixed with 1 wt% dried Bifidobacterium and placed in purified water to ferment.
  • the fermented purslane was then taken out through a strainer and the moisture was completely removed using a dry oven (Hanbaek Science, KR). These were designated as Sample 17, Sample 18, Sample 19, and Sample 20, respectively, and used in the following tests.
  • Washed and dried bottle gourd in amounts of 1 wt%, 3 wt%, 5 wt%, and 10 wt% respectively was mixed with 1 wt% dried black spore ( Aspergillus ) fungus and fermented in purified water. Afterwards, the fermented bottle gourd was taken out through a strainer and the moisture was completely removed using a dry oven (Hanbaek Science, KR). These were designated as Sample 21, Sample 22, Sample 23, and Sample 24, respectively, and used in the following tests.
  • the above complex lactic acid bacteria are Lactobacillus acidophilus, Lactobacillus brebis CD2, Lactobacillus casei , Lactobacillus hilgardii, Lactobacillus mali , Lactobacillus nagelii , Lactobacillus plantarium , Lactobacillus reuteri , Lactobacillus Rhamnosus , Lactobacillus satsumensis , Lactobacillus Thermophilus , Bacillus A mixture of Bacillus amylolyticus , Bacillus cereus, Bacillus licheniformis, Bacillus polymyxa, Bacillus subtilis , Bacillus sterothemophilus , Bifidobacterium bifidum
  • the Monascus pilosus strain was inoculated into Mizutani broth and cultured in a shaking incubator at 30°C to prepare a starter. 6.6 wt% of the prepared starter was inoculated into Mizutani broth and cultured under aeration to obtain a fermented product. Thereafter, the fermented product was treated at 121°C in an autoclave (Ilsin, KR), sterilized and filtered, and referred to as 'Sample 30' and used in the following test.
  • Ilsin, KR autoclave
  • the free radical scavenging test is a test that utilizes the fact that the absorbance of stable DPPH (2,2-diphenyl-1-picryl-hydrazyl radical) shows maximum absorbance at 540 nm. As the purple DPPH is scavenged by the sample and the free radical scavenging rate increases, it becomes transparent, and as a result, the absorbance at a wavelength of 540 nm decreases.
  • Example 1 1 ml of the sample of Example 1 diluted to an appropriate concentration in methanol was mixed with 1 ml of 0.1 mM DPPH (Sigma) solution, left at 37°C for 15 minutes, and the absorbance (540 nm) was measured using a microplate reader (UVT-06685, Thermo max, USA).
  • the absorbance of Examples 2 to 8 in which the ratio of Centella asiatica was changed was also measured using the same method, and Comparative Examples 1 to 20 in which other strains were used instead of Hongguksu and Comparative Examples 21 and 22 in which the post-fermentation method was used were also measured using the same method.
  • control group was measured in the same way by adding 1 ml of DPPH and 1 ml of methanol, and the color correction values for the sample and the control group were obtained by adding 1 ml of methanol and 1 ml of the sample, respectively.
  • the free radical scavenging rate was calculated using the following mathematical formula 1, and the results are shown in Table 1 below.
  • Free radical scavenging rate (%) 100 - ((absorbance of sample / absorbance of control) * 100)
  • the oxygen radical scavenging test is a test that measures the change in absorbance when Nitroblue tetrazolium (NBT) is oxidized by oxygen radicals generated by the enzymatic reaction of xanthine/xanthine oxidase (Sigma), and is characterized by being able to determine the oxygen radical scavenging ability.
  • NBT Nitroblue tetrazolium
  • Example 1 First, 2.4 ml of sodium carbonate, 0.1 ml of xanthine (Sigma), 0.1 ml of EDTA (ethylene-diaminetetraacetic acid), 0.1 ml of BSA (bovine serum albumin, Sigma), 0.1 ml of NBT, and 0.1 ml of the sample of Example 1 were mixed and voltexed using a voltex mixer (Type 37600 Mixer, Mini mix, USA). After leaving it at 25°C for 10 minutes, 0.1 ml of xanthine oxidase was added and reacted at 25°C for 20 minutes.
  • a voltex mixer Type 37600 Mixer, Mini mix, USA
  • control group was measured in the same way by using distilled water instead of the sample solution, and by using distilled water instead of the xanthien oxidase solution, color correction values for the sample and control groups were obtained.
  • Oxygen scavenging rate (%) 100 - ((absorbance of sample / absorbance of control) * 100)
  • the red ginseng fermented with Hong Guo Fungus showed significantly superior antioxidant activity compared to the unfermented ginseng, and showed remarkable effects at all ratios regardless of the ginseng content.
  • the free radical and reactive oxygen scavenging rates were high compared to the ginseng fermented with other fungi species as well as the ginseng fermented by the post-fermentation method or the Monascus phyllosus culture filtrate, proving its superior antioxidant activity.
  • Tyrosinase is an enzyme that promotes the oxidation process of tyrosine in the body and helps produce melanin. When melanin is formed in melanocytes and moves to the upper layer of the skin, the skin turns dark or freckles and blemishes are created.
  • control group was measured in the same way by adding a buffer solution instead of the sample solution, and arbutin (Sigma), known to have an excellent whitening effect, was used as a positive control group.
  • color correction values for the sample and control groups were obtained by adding a buffer solution instead of tyrosinase.
  • the tyrosinase activity inhibition rate was calculated using the following mathematical formula 3, and the results are shown in Table 3 below.
  • Tyrosinase inhibition rate 100 - ((absorbance of sample / absorbance of control) * 100)
  • the red ginseng fermented with Hong Guo Fungus showed a significantly superior whitening effect compared to the unfermented ginseng, and showed a remarkable effect at all ratios regardless of the ginseng content.
  • the tyrosinase activity inhibition rate was high compared to not only the ginseng fermented with other strains but also arbutin (positive control), proving its excellent whitening effect.
  • Elastin is a component that constitutes the dermis matrix layer in the body. As the skin ages, elastin decomposes and the dermis matrix layer collapses, forming wrinkles. Elastase is an enzyme that delays the decomposition of elastin.
  • N-succinyl-(alanine) 3 -p -nitroaniline Sigma
  • a substrate of elastase was used to measure the color change caused by the decomposition of p-nitroaniline.
  • control group was measured in the same way by using distilled water instead of the sample solution, and retinol (>99%, Sigma), which is known to have an excellent wrinkle improvement effect, was used as a positive control group.
  • distilled water was used instead of elastase to obtain color correction values for the sample and control groups, respectively.
  • the inhibition rate of elastase activity was calculated using the following mathematical formula 4, and the results are shown in Table 4 below.
  • the red ginseng fermented with Hong Guk-myun showed a significantly better wrinkle improvement effect than the unfermented ginseng, and showed a remarkable effect at all ratios regardless of the ginseng content.
  • the elastase activity inhibition rate was higher than that of ginseng fermented with other strains as well as retinol (positive control), proving its excellent wrinkle improvement effect.
  • the collagen synthesis ability was measured.
  • Collagen is a component that accounts for 80-90% of the dermis layer of the skin in a living body.
  • the dermis layer becomes thinner, causing wrinkles and loss of elasticity. Therefore, if the production of new collagen increases, it can promote the regeneration of skin cells, increasing the density and thickness of the dermis layer and improving skin elasticity.
  • Each sample prepared as an example and comparative example before the test was mixed with 5.0 wt% of ethyl acetate (>99%, Daejung, KR), extracted for 1 hour, filtered through a 0.45 ⁇ m filter, and the solvent was removed using a rotary evaporator.
  • the mixture was diluted with 1,000 ppm of DMSO, which had no effect on the test, and used in the efficacy evaluation test.
  • human dermal fibroblasts were seeded in a 24-well microplate ( 1x105 cells/well) and cultured in a CO2 incubator at 37°C, 5% concentration, for 24 hours.
  • the samples of the examples and comparative examples were cultured in serum-free DMEM medium for 24 hours. Afterwards, they were transferred to a 96-well plate and coated at 4°C for 24 hours, washed with washing buffer (PBS-T; 0.05% Tween 20 in phosphate buffered saline), and blocking solution (5% skim milk, Fluka) was added and blocked at 37°C for 1 hour.
  • washing buffer PBS-T; 0.05% Tween 20 in phosphate buffered saline
  • blocking solution 5% skim milk, Fluka
  • control group was the absorbance of the reaction of the cell culture medium that was not treated with the sample, and ascorbic acid, known to be essential for collagen synthesis, was set as a positive control group. The experiment was repeated three times, and the average value was calculated and compared.
  • Collagen synthesis activity was calculated using the following mathematical formula 5, and the results are shown in Table 5 below.
  • Collagen synthesis activity rate (%) 100 + ((absorbance of control group - absorbance of cell culture medium of sample) / absorbance of control group) * 100
  • the red ginseng fermented with Hong Guo Mung Bean showed a significantly superior collagen synthesis effect compared to the general ginseng that was not fermented, and showed a remarkable effect at all ratios regardless of the ginseng content.
  • the collagen synthesis activity rate was high compared to the ginseng fermented with other strains as well as ascorbic acid (positive control), proving an excellent cell regeneration effect.
  • Each sample manufactured as an example or comparative example before the test was mixed with 5.0 wt% of n-butanol (>99.5%, Daejung, KR), extracted for 1 hour, filtered through a 0.45 ⁇ m filter, and the solvent was removed using a rotary evaporator.
  • the extract was diluted with 1,000 ppm of DMSO, which has no effect on the test, and used in the efficacy evaluation test.
  • human dermal fibroblasts were seeded in a 24-well microplate (1 x 10 5 cells/well) and cultured in a CO 2 incubator at 37°C and 5% concentration for 24 hours. After washing each well with PBS, 500 ⁇ l of PBS was added to each well, and 10 mJ/cm2 of UVB light was irradiated using an ultraviolet B (UVB) lamp (F15T8, UV B 15 W, Sankyo Dennki, Japan). Thereafter, the PBS was removed, and 1 ml of DMEM supplemented with 10% FBS was added. The samples of the examples and comparative examples to be evaluated were each treated, and cultured for 24 hours.
  • UVB ultraviolet B
  • the medium was removed, and 500 ⁇ l of DMEM supplemented with 10% FBS and 60 ⁇ l of MTT solution (2.5 mg/ml) were added to each well, and cultured in a CO 2 incubator at 37°C for 2 hours. Afterwards, the medium was removed, and 500 ⁇ l of 0.04 N isopropanol-HCl was added to lyse the cells. Finally, 100 ⁇ l of the supernatant of the lysed cells was transferred to a 96-well plate, and the absorbance (565 nm) was measured using a microplate reader (UVT-06685, Thermo max, USA).
  • the cell viability was calculated using the following mathematical formula 6, and the cytotoxicity relief rate by ultraviolet rays was calculated using the following mathematical formula 7. The results are shown in Table 6.
  • the red ginseng fermented with Honggukgyun showed a significantly superior cytotoxicity mitigation effect compared to the general ginseng that was not fermented, and showed a remarkable effect at all ratios regardless of the ginseng content.
  • the cytotoxicity mitigation rate was higher compared to ginseng fermented with other strains, proving an excellent mitigation effect against cytotoxicity caused by ultraviolet rays.
  • the inhibition rate of the expression of inflammatory cytokines expressed due to ultraviolet irradiation was measured.
  • human fibroblasts were seeded in a 24-well microplate (5 x 10 4 cells/well) and allowed to attach for 24 hours. After each well was washed with PBS, 500 ⁇ l of PBS was added to each well, and 10 mJ/cm2 of UVB was irradiated using an ultraviolet B (UVB) lamp (Model: F15T8, UV B 15 W, Sankyo Dennki, Japan). Thereafter, PBS was removed, and 350 ⁇ l of serum-free DMEM was added. The samples of the examples and comparative examples to be evaluated were respectively treated, and then cultured for 5 hours. After 5 hours, the medium was removed, and IL-1 ⁇ in 150 ⁇ l of the culture supernatant was quantified.
  • UVB ultraviolet B
  • the amount of inflammatory cytokine IL-1 ⁇ was quantified using enzyme-linked immunosorbent assay.
  • the IL-1 ⁇ production rate was calculated using the following mathematical formula 8, and the results are shown in Table 7 below.
  • the red ginseng fermented with Hong Guk-myun showed a significantly better inflammatory cytokine reduction effect than the general ginseng that was not fermented, and showed a remarkable effect at all ratios regardless of the ginseng content.
  • the inflammatory cytokine expression inhibition rate was higher than that of ginseng fermented with other strains, proving its excellent anti-inflammatory effect.
  • examples of the formulations of the present invention include flexible toners, astringent toners, nourishing toners, massage creams, essences, and packs, but the formulations of the compositions of the present invention should not be construed as being limited thereto, and changes made by those skilled in the art are possible within the scope of the present invention.

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Abstract

La présente invention concerne Centella asiatica fermenté avec Monascus. Plus spécifiquement, la présente invention concerne des utilisations de Centella asiatica rouge fermenté avec Monascus pour l'antioxydation, le blanchiment de la peau, la réduction des rides de la peau, la régénération cellulaire, la réduction de la cytotoxicité ou l'anti-inflammation. La composition selon la présente invention peut être utilisée dans divers domaines tels que les cosmétiques, les quasi-médicaments, les aliments et les médicaments.
PCT/KR2024/002258 2023-02-27 2024-02-21 Centella asiatica rouge fermenté avec monascus et son utilisation Pending WO2024181725A1 (fr)

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