KR102216210B1 - Solid fermentation composition manufactured by rumex coreanus and vegetable worms and functional cosmetic composition comprising the same - Google Patents

Abstract

The present invention relates to a solid fermentation product obtained by inoculating a cordyceps sinensis (vegetable worms) seed on a Rumex coreanus plant, and a functional cosmetic composition comprising the same, and more specifically, to a sterilized cordyceps sinensis It relates to a solid fermentation product obtained by inoculating and solid fermentation and a functional cosmetic composition for antioxidant, anti-inflammatory, hair loss prevention, skin aging prevention, and skin regeneration promotion comprising the same. The solid fermented product of the present invention is a composition derived from a natural product that does not have cytotoxicity, and is excellent in antioxidant, anti-inflammatory, hair loss prevention, skin aging inhibition, and skin regeneration promotion. It is very useful in the cosmetic industry because it can be applied as a functional cosmetic composition for aging action, particularly, skin regeneration and wrinkle improvement.

Description

Solid fermented product of Cordyceps sinensis and functional cosmetic composition containing the same {SOLID FERMENTATION COMPOSITION MANUFACTURED BY RUMEX COREANUS AND VEGETABLE WORMS AND FUNCTIONAL COSMETIC COMPOSITION COMPRISING THE SAME}

The present invention relates to a solid fermentation product obtained by inoculating a cordyceps sinensis (vegetable worms) seed on a Rumex coreanus plant, and a functional cosmetic composition comprising the same, and more specifically, to a sterilized cordyceps sinensis It relates to a solid fermentation product obtained by inoculating and solid fermentation and a functional cosmetic composition for antioxidant, anti-inflammatory, hair loss prevention, skin aging prevention, and skin regeneration promotion comprising the same.

The skin is a part of the body that is directly exposed to the external environment, and in everyday life, skin troubles and irritation are caused by exposure to ultraviolet radiation caused by sunlight exposure, air pollutants and various bacteria caused by yellow sand or automobile smoke, etc. Will accelerate. Such aging of the skin decreases the elasticity of the skin in the appearance of the skin to form wrinkles, or form spots and senile spots, which induces pigmentation of the skin.

In general, skin aging has various theories such as somatic mutation, excessive generation of free radicals, autoimmune reactions, accumulation of toxic substances, errors generated during gene replication, and modification of dermal proteins, among which aging. As the theory that the causes of various diseases such as and adult diseases are caused by reactive oxygen species is recognized, research to develop natural antioxidants that can control reactive oxygen species derived from oxygen or nitrogen is actively progressing. have.

Reactive oxygen species, commonly known as noxious oxygen, are peroxidative lipids such as peroxide anion radicals, hydrogen peroxide, hydroxy radicals, and free radicals generated here as the most stable form of triplet oxygen is reduced. It occurs even under normal conditions, but antioxidant enzymes such as superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPx) are present to inhibit the generation of excessive reactive oxygen species.

However, when these reactive oxygen species are abnormally excessively generated by stress and external environmental factors and are not erased and exist in the body, oxidative stress due to free radicals is applied to the living body, resulting in DNA, RNA, protein, and It is presumed to cause cell membrane and cell structure damage, cancer, tissue damage, and aging.

In order to suppress the acceleration of various diseases and aging rapidly increasing in modern times, studies on antioxidants that can control or eliminate these reactive oxygen species are being actively conducted. Representatively, studies on the efficacy of natural antioxidants such as tocopherol, ascorbic acid, carotenoids, flavonoids, glutathione, etc. and their stabilization technology, and synthetic antioxidants, butylated hydroxy toluene (BHT), butylated hydroxy anisole Various antioxidants such as (BHA) are known, and studies on many other antioxidants are continuously being conducted.

Among these antioxidants, tocopherol has a relatively low antioxidant effect, and synthetic antioxidants such as BHA and BHT, which have excellent effects, are used in medicine and food, but there are points on mutagenicity and toxicity, and ascorbic acid has stability. As the secured derivatives and formulations are being developed, there is an urgent need to develop more stable and effective natural antioxidant materials.

In order to solve this problem, many studies have been conducted on cosmetic compositions using extracts that have relatively low toxicity, safe and effective effects on the skin among phytochemicals that are in the spotlight recently, but obtained by general extraction methods. The resulting ingredients show insignificant limitations in their effects, so the development of a material that maximizes efficacy is required.

For example, in the case of a natural extract through fermentation, the sugar component present in the natural product is used as a carbon source in the metabolism of microorganisms and converted into alcohol, without using an organic solvent that is harmful to the human body. Therefore, the active ingredient in the natural product, especially flavonoids , Polyphenols, catechins, etc. are saccharified to exist as stable compounds, so the extraction rate of the active ingredient content is increased through fermentation, and sugar is excluded, so excellent skin efficacy is expected by the activated active ingredient.

Cordyceps is a word that means that it stays in the state of an insect in winter and becomes a mushroom in summer. Spores and hyphae of Cordyceps sinensis, a type of fungus, invade the body of a living insect and remain dormant during winter. It is a strange mushroom that kills the insect in summer, uses the material in the host as a nutrient source to create an endobacterial nucleus, and then forms a fruiting body outside the body of the insect.

About 750 kinds of cordyceps have been discovered worldwide so far, and they are classified into more than 100 genera such as Cordyceps spp., Paecilomyces spp, and Isaria spp. do.

According to Chinese old books, cordyceps not only has excellent effects on cancer, liver disease, lung disease, brain disease, circulatory disease, heart disease, diabetes, etc., but also in modern practical Chinese medicine, pulmonary tuberculosis, elderly debilitating chronic tuberculosis anemia, weakness, etc. Has been shown to cure. Recently, the tumor suppression rate is 83%, which is reported to be effective as an anti-cancer effect and antidote to drug addiction, and it has a natural healing power, so it is reported that there is a recovery effect when physical consumption is large due to severe exercise.

On the other hand, Cruciferus is a perennial plant with thick rhizomes. The thick purple stem stands upright and grows to a height of about 60cm, and leaves longer than 30cm are located opposite each other and are lanceolate close to an elongated oval shape. The base of the leaf is round, the tip is dull, and there are many wrinkles on the leaf body. The small flowers bloom in a long root-like shape. They are arranged in a round shape with layers, and there are no petals, and the flower is composed of 6 calyxes, 6 stamens, and a pistil divided into three branches. The diameter of the flower is about 4mm and its color is green, and it is known as a wild grass that bears seeds with 4 wings after the flower blooms.

Sorujagi is distributed all over the country and grows in slightly humid land in the field. It is used mainly for medicinal purposes by removing leaves, stems, and fine roots by drying them in early autumn.

The rhizome contains anthraquinon derivatives Chrysophanol and Emodin, and has effects such as diuresis, hemostasis, and stool pain. Applicable diseases include constipation, indigestion, jaundice, bloody stool, It is also widely used in the treatment of uterine bleeding, scabies, boils, rheumatism, mechanical insects, and genital eczema.

KR 10-0638053 B KR 10-1124968 B KR 10-0858628 B

The present invention was devised to solve the problems of the prior art as described above, and the problem to be solved in the present invention is from a natural product without cytotoxicity, which is excellent in antioxidant, anti-inflammatory, hair loss prevention, skin aging inhibition, and skin regeneration promotion. It is intended to provide a solid fermented product and a functional cosmetic composition using the same.

In order to solve the above problems, the present invention provides a solid fermented product of Cordyceps sinensis, prepared by inoculating and culturing the cordyceps mycelium in sterilized Soryu (Rumex coreanus).

The fungus is coordinating sepseu sinen system (Cordyceps sinensis), Cody sepseu Milla other leases (Cordyceps militaris), Cody sepseu Scarab Cola (Cordyceps scarabaecola), Cody sepseu press Tansu (Cordyceps nutans), Cody sepseu seupeko Seppala (Cordyceps sphecocephala) , Cordyceps tricentri ( Cordyceps tricentri ), Paecilomyceps tenuipes ( Paecilomyceps tenuipes ) and Paecilomyces japonica ( Paecilomyces japonica ) is preferably selected from the group consisting of.

The cultivation is preferably carried out for 10 to 30 days under conditions of 20 to 35 °C, pH 4 to 7.5, and relative humidity of 40 to 80%.

In addition, the present invention provides a functional cosmetic composition comprising the solid fermented product of the present invention Soruga- Cordyceps sinensis.

The functionality is preferably selected from the group consisting of antioxidant, anti-inflammatory, hair loss prevention, skin aging prevention, and skin regeneration promotion.

The solid fermented product of the present invention is a composition derived from a natural product that does not have cytotoxicity, and is excellent in antioxidant, anti-inflammatory, hair loss prevention, skin aging inhibition, and skin regeneration promotion. It is very useful in the cosmetic industry because it can be applied as a functional cosmetic composition for aging action, particularly, skin regeneration and wrinkle improvement.

1 is a graph showing changes in the expression pattern of inflammatory factor-related genes by treatment with solid fermented extract of Cordyceps sinensis of the present invention.
Figure 2 is a graph showing the 5α-reductase inhibitory activity by treatment with solid fermented extract of Cordyceps sinensis of the present invention.

Hereinafter, the present invention will be described in detail.

As a result of repeating research to develop a material for a functional cosmetic composition using natural products, the inventors of the present invention have inoculated the mycelium of Cordyceps sinensis into the Soruga and cultivate them according to the solid culture method. It has been confirmed that it has antioxidant activity, anti-inflammatory activity, hair loss prevention, skin aging prevention and skin regeneration activity, and proposes that the solid fermented product of the present invention can be used as a functional cosmetic composition.

Accordingly, the present invention provides a solid fermented product of Cordyceps sinensis, which is prepared by inoculating and culturing Cordyceps mycelium in sterilized Soryu (Rumex coreanus).

Cordyceps sinensis of the present invention is a fungus belonging to the ascomycetes ergotaceae cordyceps family, specifically, Cordyceps spp., Paecilomyces spp., Isaria spp., and phytocodys It is classified into over 100 genera including Phytocordyceps spp., and about 750 species are known worldwide.

In the present invention, all of these cordyceps can be used, and preferably, Cordyceps sinensis , Cordyceps militaris , Cordyceps scarabaecola , Cordyceps scarabaecola , and Cordyceps nutans . Cordyceps nutans ), Cordyceps sphecocephala , Cordyceps tricentri , Paecilomyceps tenuipes and Paecilomyces japonica , and are selected from the group consisting of , More preferably, it is Paecilomyces japonica , also named snowflake cordyceps japonica .

Cordyceps mycelium of the present invention can be prepared by culturing Cordyceps spp.

The cordyceps seed bacteria can be maintained by periodically subculturing until use.As a preferred embodiment, after inoculation of Cordyceps sinensis mycelium in PDA (Potato Dextrose Agar) medium, passages are maintained at about 25°C temperature at about 4 weeks intervals. I can.

The seed culture is a process of culturing the cordyceps mycelium spawn in storage prior to the main culture. In a preferred embodiment, the seed culture may be obtained as a liquid culture by inoculating the seed bacteria, that is, the mycelium, in a culture medium for mushroom mycelia, and culturing it with rotational shaking.

The Sorubarb plant of the present invention can be used by drying the collected Sorubarb plant. It is possible to use the whole of the plant, but preferably, the root is used.

The sterilization of the sorghum can be performed by washing the prepared sorghum with water, dehydrating and drying it, and applying various known sterilization methods. In a preferred embodiment, the sterilization may be autoclaved at 121° C. for 30 minutes.

The method of inoculating the cordyceps mycelium of the present invention into the sterilized Sorubarb is to aseptically inoculate a certain amount of the liquid culture obtained by the above-described cordyceps spp.

After inoculation of Cordyceps mycelium, a culture process is performed, and the culture conditions are preferably temperature 20 to 35°C, pH 4 to 7.5, relative humidity 40 to 80%, and a culture period of 10 to 30 days, as a preferred embodiment. , It can be incubated for 30 days in an incubator with 80% relative humidity at a temperature of 25°C.

The fermentation is completed Sorupia-Cordyceps sinensis solid fermented product has a shape in which the cordyceps mycelium is entirely covered on the surface of Sorupilla, from which it is possible to prepare an extract of a solid fermentation product of Sorupia-Cordyceps sinensis.

The solvent used for extraction may be water and/or lower alcohol, preferably a mixed solvent of water and ethanol. As a preferred embodiment, about 5 to 20 times the volume of ethanol (10 to 60% (v/v)) was added to the obtained solid fermented product of Soruga- Cordyceps, and shaken for 1 to 5 hours, and the supernatant was centrifuged. It can be taken or immersed for 24 to 96 hours and then filtered under reduced pressure. The process may be performed 1 to 5 times, and preferably, extraction may be repeated 3 times.

It is possible to prepare the solid fermented product of the present invention, Cordyceps sinensis, or an extract thereof by the specific manufacturing process as described above, and it can be applied as a material of the functional cosmetic composition described below.

Accordingly, the present invention provides a functional cosmetic composition comprising the solid fermented product of the present invention Soruga- Cordyceps sinensis.

The functional means an activity selected from the group consisting of antioxidant, anti-inflammatory, hair loss prevention, skin aging prevention, and skin regeneration promotion.

The main component of the cosmetic composition of the present invention, the solid fermented product of Cordyceps sinensis may be contained in an amount of 0.01 to 50% by weight, preferably 0.1 to 20% by weight, more preferably 1 to 10% by weight, based on the total weight of the composition. . In this content range, appropriate formulation stability can be ensured, and desired effects of antioxidant, anti-inflammatory, hair loss prevention, skin aging prevention, and skin regeneration promotion can be expected.

In addition to the solid fermented product of the present invention, the composition of the present invention includes known natural product extracts or other extracts for the effects of antioxidant, anti-inflammatory, hair loss prevention, skin aging prevention, and skin regeneration promotion within a range that does not inhibit the effective activity of the present invention, in addition to the solid fermented product of Cordyceps sinensis. Ingredients may be additionally included and used.

In addition, the composition includes ingredients commonly added to cosmetic compositions, for example, fatty substances, organic solvents, solubilizers, thickening agents, gelling agents, emollients, antioxidants, suspending agents, stabilizers, foaming agents, Cosmetics such as fragrances, surfactants, water, ionic or nonionic emulsifiers, fillers, sequestering and chelating agents, preservatives, vitamins, blockers, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic actives, or It may be molded into a specific formulation, including an adjuvant or carrier commonly used in the field of dermatology.

The cosmetic composition of the present invention may be prepared in any formulation conventionally prepared in the art, for example, solution, suspension, emulsion, paste, gel, cream, lotion, powder, pack, soap, surfactant- It may be formulated as containing cleansing, oil and spray, but is not limited thereto. More specifically, flexible lotion, nutrition lotion, nutrition cream, massage cream, essence, eye cream, powder foundation, emulsion foundation, wax foundation, cleansing cream, cleansing foam, cleansing water, pack, spray, powder, pact, lip gloss It can be prepared in formulations such as rose, lipstick, shadow, shampoo and conditioner.

When the formulation of the present invention is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, or zinc oxide may be used as carrier components. I can.

When the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component. In particular, in the case of a spray, additional chlorofluorohydrocarbon, Propellants such as propane/butane or dimethyl ether.

When the formulation of the present invention is a solution or emulsion, a solvent, a solubilizing agent or an emulsifying agent is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylglycol oil, glycerol aliphatic ester, polyethylene glycol or fatty acid ester of sorbitan.

When the formulation of the present invention is a suspension, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, an ethoxylated isostearyl alcohol, a suspending agent such as polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, and crystallites Sex cellulose, aluminum metahydroxide, bentonite, agar or tracant, and the like can be used.

When the formulation of the present invention is a surfactant containing cleansing, as a carrier component, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide Ether sulfates, alkylamidobetaines, fatty alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives, or ethoxylated glycerol fatty acid esters may be used.

Hereinafter, the present invention will be described in more detail through preferred embodiments. The following examples describe one preferred embodiment of the present invention, and the scope of the present invention is not limited and interpreted by the following examples.

[Example]

1. Preparation of solid fermented extract of Soruga- Cordyceps sinensis

Snowflake Cordyceps ( Paecilomyces Japonica ) was cultured using a PDA (Potato Dextrose Agar, Difco, Co., USA) medium at 25℃ using a strain being passed from Palmbios Co., Ltd., and passaged every 4 weeks while subcultured at 4℃. Used while being stored in. After inoculating the mycelium passaged in 100ml mushroom mycelium solid medium (1 cm × 1 cm, 3 pieces) in a 500 ml flask, 7 in a rotating shaking incubator (Daihan Labtech Co. Korea, 25±1℃, 130rpm). It was cultured for a day and used as a liquid seed.

Sorujak was purchased from the Daegu Herbal Medicine Market, washed with water before use to remove impurities, and then dehydrated. 100g of Sorujak root was put in a 3L culture vessel, autoclaved at 121℃ for 30 minutes, then cooled at room temperature, and the prepared liquid seed bacteria 60 ml was aseptically inoculated into 100 g of sterilized Sorbus. Fermentation was carried out for 30 days in an incubator at 25° C. and 80% relative humidity. After fermentation, 10 times the volume of 60% ethanol was added and shaken for 2 hours, immersed for 72 hours, centrifuged at 10,000 rpm for 10 minutes, and the supernatant was taken, and after repeating this 3 times, the extract was concentrated under reduced pressure to form a powder. It was prepared and used.

2. Activity evaluation

2.1. Antioxidant activity

Antioxidant activity of the prepared solid fermented extract of Sorugak-cordyceps sinensis was evaluated. The prepared solid fermented extract (100 μg/ml) of Cordyceps sinensis was evaluated by measuring DPPH (1,1-diphenyl-2-picryl hydrazyl) radical scavenging activity and FRAP activity.

In the case of DPPH scavenging ability, 380 μl of a 2x10 ^-4 M DPPH solution dissolved in 99.5% ethanol was added to 20 μl of a sample diluted to various concentrations, and then reacted at 37° C. for 30 minutes, and then a microplate reader (Asys Hitech, Expert96, Asys Co., Austria) was used to measure the absorbance. DPPH free radical scavenging activity was expressed as a percentage as shown in Table 1 below.

DPPH radical scavenging activity (% of control) Whisperer Solid fermentation of Soryu-Congchunghacho Average Deviation Average Deviation 0.08 0.02 0.16 0.02

As shown in Table 1, it was confirmed that the DPPH radical scavenging activity was increased in the solid fermentation of Soruga- Cordyceps sinensis of the present invention compared to the case of using the original Sorubarb.

FRAP activity was carried out according to a known method for the prepared solid fermentation product (100 μg/ml) of Cordyceps sinensis. Specifically, the FRAP assay is a representative antioxidant measurement method that measures the ferric reducing ability of a compound. When the ferric tripyridyltriazine (FeIII-TPTZ) complex was converted into a blue ferrous tripyridyltriazine (FeII-TPTZ) complex at pH 3.6, the ferric reducing ability of the compound to be searched was confirmed by measuring the absorbance. . As a reaction solution for the experiment, acetate buffer (pH 3.6, 300mM): 10 mM TPTZ (2,4,6-tripyridyl-s-triazine): 20 mM FeCl ₃ 6H ₂ O 10: 1: 1 Mix in proportions and make right before the experiment. After mixing the reaction solution (190 μL) and the extract (10 μL), absorbance was measured at 590 nm for about 10 minutes at 150 second intervals.

FRAP (% of control) Whisperer Solid fermentation of Soryu-Congchunghacho Average Deviation Average Deviation 0.93 0.04 1.34 0.02

As shown in Table 2, it was confirmed that the FRAP was increased in the solid fermentation of Soruga- Cordyceps sinensis of the present invention compared to the case of using the original Sorubarb.

2.2. Anti-inflammatory activity

Inflammation is a defense mechanism in the body against various stimuli such as physical damage or metabolic abnormalities. An appropriate inflammatory reaction is an indispensable reaction to protect a living body, but an excessive and inappropriate inflammatory reaction causes necrosis of cells and tissues and various chronic diseases. In addition, inflammation can occur in various organs in the body, and in the case of chronic inflammatory diseases, it is known that there is a possibility of developing cancer because it is closely related to carcinogenesis.

Lipopolysaccharide (LPS), an inflammation-inducing substance, stimulates Toll-like receptor 4 on the surface of macrophages to induce activation of mitogen-activated protein kinase (MAPK), a lower cell signaling pathway. Among MAPKs, extracellular signal-regulated kinase (ERK) and p38 are well known to be involved in LPS-stimulated iNOS induction and NO production in macrophages. Nitric oxide (NO), one of the inflammatory regulators, is known to have a harmful effect on the human body when excessive amounts are present, and thus act as an important cause of degenerative diseases such as meningitis, Alzheimer's disease and Parkinson's disease, as well as inflammatory reactions and cell damage.

In this experiment, Griess reagent assay was performed to measure anti-inflammatory activity. The concentration of NO was measured using a griess reagent by measuring the concentration of nitrite in the culture medium. Raw 264.7 cells were adjusted to 5×10 ⁴ cells/ml using DMEM medium, inoculated into 12 well plates, and cultured in a 5% CO ₂ incubator for 24 hours before. Cells were treated with 1 μg/ml of LPS, and 1 hour later, 100 μg/ml of extract was treated and cultured for 24 hours. After obtaining the supernatant of the culture medium, it reacted with the griess reagent, and the absorbance was measured at 540 nm with an ELISA reader, and the NO production rate was expressed as a percentage.

NO generation (% of control) Whisperer Solid fermentation Average Deviation Average Deviation 17.68 0.03 9.37 0.02

As shown in Table 3, it was confirmed that the amount of NO produced was significantly reduced compared to the case of using the raw material of Soruba in the solid fermentation of Soruga- Cordyceps sinensis of the present invention.

2.3. Expression of immune-related genes

Inflammatory reaction is a mechanism that attempts to repair and regenerate the damaged area when invasion that causes any organic change, such as physical action, chemical substance, or bacterial infection, is applied to a living body or tissue. Once irritation is applied, vascular active substances such as inflammatory components are locally released and vascular permeability increases, causing inflammation, but persistent inflammatory reactions rather promote mucosal damage, and as a result, cause various diseases in some cases. .

On the other hand, macrophages are known to be involved in various host reactions such as acquired immunity as well as innate immunity, and are known to be involved in maintaining homeostasis, and during inflammatory reactions, they produce nitric oxide (NO) and cytokine, playing an important role in bioprotection in the early stages of infection. do.

In the case of nitric oxide synthase (NOS) in mammalian cells, there are three similar forms: neuronal NOS (nNOS), endothelial NOS (eNOS), and inducinle NOS (iNOS). Among them, iNOS is particularly involved in the inflammatory response.

nNOS and eNOS are always expressed, and iNOS is expressed when stimulated by interferon-γ, lipopolysaccharide (LPS), and various inflammatory cytokines. In addition, NO regulates acute and chronic inflammatory responses.

PGE2 is produced from arachidonic acid by cyclooxygenase (COX). COX was mainly studied in the early 1990s, but there are also two similar forms. COX-1 is expressed in almost all tissues, and produces prostagrandin to regulate physiological functions such as regulating blood flow to the kidneys and protecting the cells of the stomach.

Conversely, in the case of COX-2, it is expressed in macrophages that respond to the stress of various factors such as infection or damage by microorganisms. In other words, iNOS and COX-2 expression and NO, prostaglandin E2 (PGE2) are representative inflammatory factors of immune cells.

In addition, tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-), which are proinflammatory cytokines as inflammatory factors. 1) etc. are included.

In this experiment, RAW 264.7 cells were adjusted to 5×10 ⁴ cells/ml using DMEM medium, then inoculated into 12 well plates, and cultured in a 5% CO ₂ incubator for 24 hours. Cells were treated with 1 μg/ml LPS, and then 1 hour later, 10 μg/ml extract/sample was treated and cultured for 24 hours. In order to confirm the expression of anti-inflammatory related factors, analysis was performed by reverse transcription-polymerase chain reaction (RT-PCR).

RNA was isolated/quantified by treating TRIzol reagent (Invitrogen Co., Carlsbad, CA, USA) at 4 ℃ for 1 hour, and then each primer, DEPC water, and ONE-STEP RT-PCR PreMix Kit (Intron, Korea) And amplified using a Mastercycler gradient (Eppendorf, Hamburg, Germany). In order to check the quantitative difference of each PCR product, 1% agarose gel was made with 1X TAE buffer, and DNA gel loading solution was mixed and loaded with the PCR product corresponding to each primer per well, followed by electrophoresis at 50 V. After DNA separation by electrophoresis, the gel was stained with ethidium bromide (EtBr), and the difference in expression under UV was confirmed, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control.

As shown in FIG. 1, as a result of LPS treatment in RAW264.7 cells, it was confirmed that TNF-α, COX-2, IL-1β, and IL-6, which are inflammation-related factors, are increased. On the other hand, when comparing the case of treating the original Sorubarb and the case of treating the solid fermented extract of the present invention, it was confirmed that the expression level of the genes was significantly reduced in the solid fermented extract of the present invention.

2.4. 5α-reductase assay

Hair loss is caused by various causes such as tinea head, traumatic alopecia, hereditary baldness, drug side effects, androgenic alopecia, alopecia areata. In particular, androgenic hair loss appears as a general circular hair loss form, and is the most typical form of hair loss.

Typical mammalian androgen hormones include testosterone and dihydrotestosterone, and it has been reported that dihydrotestosterone is more common in hair loss patients. Testosterone is converted to dihydrotestosterone by 5α-reductase, and this study attempted to determine the degree of its inhibition.

The inhibitory effect of 5α-Reductase activity was evaluated by direct isolation of 5α-reductase from rat prostate. As a specific method, a 7-week-old SD rat male was sacrificed to take a prostate, which was homogenized using liquid nitrogen. The homogenized tissue is protein lysed with cold lysis buffer (20 mM potassium phosphate (pH 6.6), 0.32 M sucrose, 1 mM dithiothreitol (DTT), 0.2 mM phenylmethyl-sulfonylfluoride (PMSF))), and the homogenized tissue is 10 mM Tris. -HCl buffer (pH 7.0) was added at a three-fold ratio, and the supernatant was harvested by centrifugation, and then centrifuged again, and the intermediate steam was used as 5α-Reductase.

Working solution was prepared with 90 μL of 10 mM Tris-HCl buffer (pH 6.0), 50 μL of 20 μM testosterone, 2 mM NADPH, 10 μL, and 50 μL of 100 mg/mL enzyme. The addition amount of the sample was treated with 1% concentration of the total reaction solution to measure the degree of inhibition. The enzyme reaction was incubated for 1 h at 37°C, and the amount of change in absorbance thereof was measured under UV-spectrophotometric 340 nm conditions.

The experimental results are shown in Fig. 2, finasteride was used at concentrations of 10 and 100 μM as a positive control, and an inhibitory activity of about 20% was confirmed at 100 μM. On the other hand, in the case of the original Sorubarb, the enzyme activity was increased compared to the control at a concentration of 100 μg/ml, but in the case of the solid fermented extract of the present invention, it was confirmed that the enzyme activity decreased by about 40% compared to the control.

2.5. Cytotoxicity test (MTT assay)

In order to evaluate the cytotoxicity of the solid fermentation of Soruga- Cordyceps sinensis, after measuring the MTT assay using BJ cells, cell viability (%) was measured, and the results are shown in Table 4.

Sample concentration (μg/ml) Whisperer Solid fermentation 25 77.93 87.57 50 58.44 70.23 100 36.59 50.67

As shown in Table 4, when the concentration of the sample was 100 μg/ml, the cell viability decreased to less than 50% in the case of the original Sorubarb. In addition, the cell viability tended to decrease with increasing concentration of the original Sorubarb, but in the case of the solid fermented Soruga- Cordyceps sinensis, it showed a cell viability of 50% or more even at a high concentration of 100 μg/ml.

Claims (5)

A functional cosmetic composition for antioxidant comprising a solid fermented product of Cordyceps sinensis-Cordyceps sinensis produced by inoculating and culturing the sterilized Soryu ( Rumex coreanus ). The method of claim 1, wherein the fungus is coordinated sepseu sinen sheath (Cordyceps sinensis), coordination sepseu Millar other less (Cordyceps militaris), coordination sepseu Scarab coke (Cordyceps scarabaecola), coordination sepseu press Tansu (Cordyceps nutans), coordination sepseu seupeko Seppala (Cordyceps sphecocephala), coordination sepseu tree Sentry (Cordyceps tricentri), L indeed My process Te sister Fes (Paecilomyceps tenuipes) and L indeed antioxidant functionality for which being selected from the group consisting of my process japonica (Paecilomyces japonica) Cosmetic composition. The functional cosmetic composition for antioxidants according to claim 1, wherein the culture is performed for 10 to 30 days under conditions of 20 to 35°C, pH 4 to 7.5, and 40 to 80% relative humidity. delete delete

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