Beta-glucan derivatives that promote the proliferation of skin epidermal cells through fibrillin-increasing effects

Abstract

To provide a beta-glucan derivative exhibiting an activation action of an epidermal cell via a fibrillin increase action.SOLUTION: A beta-glucan derivative is an amphipathic low-molecular compound composed of one molecule of beta-glucan, one molecule of polyphenol, and three molecules of cysteine. A method for manufacturing consists of a production process that uses a natural product fermented by Lactobacillus reuteri using a fermentation liquid obtained by fermenting Acai palm fruit and rice bran with Monascus purpureus as a culture medium. The obtained beta-glucan derivative exhibits a fibrillin-increase effect, which is confirmed at the mRNA and protein levels. Furthermore, it was found to have a protective effect on cells by preventing gene oxidation. This beta-glucan derivative is applied to cosmetics, foods, etc.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This invention relates to a beta-glucan derivative that activates skin epidermal cells through its fibrillin-increasing effect. [Background technology]

[0002] Skin is an organ that covers the surface of the body and has been attracting attention in the fields of drug penetration and beauty. In particular, various research and inventions have been conducted on the proliferation of epidermal cells in relation to regenerative medicine. In addition to growth factors such as EGF, extracellular matrices such as fibronectin and fibrillin are involved in the proliferation of epidermal cells.

[0003] Inventions related to skin cell proliferation include an invention relating to a composition containing a lipoamino acid derivative of the dipeptide carnosine that stimulates the formation of one or more extracellular matrix components in skin or mucus, and describes the proliferation of extracellular matrix and epidermal cells (see, for example, Patent Document 1). Another invention relates to an activator of stem cells and / or progenitor cells, which proposes an activator of stem cells and / or progenitor cells expressing CD34 or nestin that contains batroxobin (see, for example, Patent Document 2).

[0004] Furthermore, there is a new biological material derived from Wharton's jelly of the human umbilical cord, which has been described as a differentiated mesenchymal stem cell or a mesenchymal stem cell differentiated into other cell lines (see, for example, Patent Document 3).

[0005] However, these inventions only have a slight effect on activating skin epidermal cells, and since they are chemically synthesized or derived from humans, there is a risk of viral infection. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Patent No. 6326601 [Patent Document 2] Patent No. 5602364 [Patent Document 3] Patent No. 5427237 Summary of the Invention [Problem to be solved by the invention]

[0007] Existing substances have a mild effect on activating skin epidermal cells, which limits their industrial use. In addition, chemically synthesized substances have safety issues, limiting their use.

[0008] Therefore, there is a demand for natural products that have fewer side effects and exhibit excellent skin epidermal cell activation effects. [Means for solving the problem]

[0009] In order to achieve the above object, the invention described in claim 1 relates to a beta-glucan derivative having the effect of activating skin epidermal cells through the fibrillin-increasing effect shown in the following formula (1).

[0010] [ka] [Effects of the Invention]

[0011] The present invention, configured as described above, has the following advantages.

[0012] The beta-glucan derivative described in claim 1 has an excellent effect of activating skin epidermal cells through its fibrillin-increasing effect.

[0013] DETAILED DESCRIPTION OF THE INVENTION

[0014] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, preferred embodiments of the present invention will be described in detail.

[0015] The beta-glucan derivative that activates skin epidermal cells through the fibrillin-increasing effect has a structure represented by the following formula (1).

[0016] [ka]

[0017] Fibrillin is a glycoprotein with a molecular weight of approximately 350,000, which exists in the periphery of collagenase-resistant elastic fibers with a diameter of 20 nm or less, and is involved in maintaining the structure and proliferation of cells.

[0018] As shown in the above formula (1), the beta-glucan derivative, which activates skin epidermal cells through its fibrillin-increasing effect, is composed of one beta-glucan molecule, one polyphenol side chain molecule, and three cysteine ​​molecules.

[0019] This beta-glucan derivative has the chemical formula C63H97O39N3S3 and is composed of 63 carbon atoms, 97 hydrogen atoms, 39 oxygen atoms, 3 nitrogen atoms, and 3 sulfur atoms.

[0020] These molecules and their bonds are all natural types that exist in nature, and the molecules are bonded together via ester bonds or the like.

[0021] This beta-glucan derivative is produced by chemical synthesis using polyphenols and cysteine ​​as raw materials. However, this chemical synthesis results in a large amount of raw material loss and high production costs, limiting its industrial use. Chemically synthesized beta-glucan derivatives with high purity are used to obtain analytical standards and trace samples.

[0022] Analyzing the structure of this beta-glucan derivative is preferable because it allows identification of the active ingredient and can be used as an indicator of the content of the active ingredient when used in products or formulations for sale.

[0023] As an example of structural analysis of this beta-glucan derivative, when a chemically synthesized high-purity (purity 93% or higher) standard was used and analyzed by 500 MHz H-NMR in C5D5N, peak positions were observed at 0.91, 0.95, 1.10, 1.30, 1.52, 1.57, 1.76, 2.56, 2.72, 3.36, 4.45, 5.03, 5.22, 5.25, 5.35, 6.03, and 6.11 ppm.

[0024] Furthermore, 500MHz H-NMR analysis of C5D5N showed the following peaks: 9.1, 19.0, 19.1, 19.2, 19.3, 19.9, 21.4, 23.6, 25.5, 26.5, 27.3, 28.1, 32.5, 36.4, 37.5, 38.5, 39.3, 42.0, 44.7, 45.3, 49.8, 51.8, 52.7, 63.0, 63.8, 64.4, 64.5, 67.0, 68.1, 69.4, 70.2, 71.4, 72.6, 73.0, 74.6, 75.0, 76.0, 77.0, 78.0, 79.0, 80.0, 81.0, 82.0, 83.0, 84.0, 85.0, 86.0, 87.0, 88.0, 89.0, 90.0, 91.0, 92.0, 93.0, 94.0, 95.0, 96.0, 97.0, 98.0, 99.0, 100.0, 101.0, 102.0, 103.0, 104.0, 105.0, 106.0, 107.0, 108.0, 109.0, 110.0, 111.0, 112.0, 113.0, 114.0, 115.0, 116.0, 117.0, 11 Peaks were observed at 0.3, 71.6, 73.1, 73.3, 73.8, 74.0, 74.4, 75.5, 76.9, 77.1, 77.6, 78.9, 79.4, 79.5, 79.8, 80.3, 80.9, 83.3, 87.1, 89.0, 90.7, 101.2, 103.4, 104.7, 104.9, 105.0, 108.2, 112.2, 119.2, 137.4, 137.8 and 178.3 ppm.

[0025] Furthermore, this beta-glucan derivative was analyzed by high-performance liquid chromatography. Among these, the constituent beta-glucan is a type of polysaccharide that exhibits excellent functions such as acting on cell membrane receptors, immunomodulating effects, anti-inflammatory effects, antitumor effects, fat burning effects, neuroprotective effects, hair growth effects, and antibacterial effects.

[0026] In this beta-glucan derivative, the beta-glucan is ester-bonded to three cysteine ​​molecules at the hydroxyl group at position 6. The ester bond is formed between the hydroxyl group at position 6 and the carboxylic acid of the cysteine.

[0027] This component, beta-glucan, is a naturally occurring compound. The component beta-glucan shown here consists of six glucose molecules linked together by beta-1,4 bonds.

[0028] Furthermore, this beta-glucan is ether-bonded to a polyphenol at its 1-position. The polyphenol present here is a polyphenol with two dihydroxybenzenes. The hydroxyl groups of this polyphenol have excellent antioxidant properties, scavenging reactive oxygen species and free radicals generated in the body and stabilizing the structure.

[0029] This beta-glucan derivative has hydrophobicity due to the beta-glucan backbone, water solubility due to the hydroxyl group of the gallic acid polyphenol, and basicity due to guanine, and exhibits amphiphilicity and pH buffering ability, which enhances absorption.

[0030] The component cysteine ​​has an SH group and exhibits reducing properties. Furthermore, it is a raw material for keratin, forming the cytoskeleton of skin cells and the extracellular matrix on the surface of the cell membrane, strengthening epidermal cells.

[0031] In addition to its antioxidant properties, the polyphenol portion of this beta-glucan derivative protects genes by protecting DNA molecules from aging, oxidation, active oxygen, and ultraviolet rays. This contributes to gene stabilization. It also exhibits radical scavenging properties, eliminating radicals. Radicals break down proteins such as collagen and elastin, so its radical scavenging properties are also desirable for tissue protection.

[0032] Furthermore, since the hydroxyl group of beta-glucan is weakly acidic, it has high acid resistance and is resistant to gastric acid when orally ingested, which is preferable as it increases the absorption rate of the beta-glucan derivative into the body.Furthermore, since it is neutral, it is not irritating to the skin when applied to the skin, which is preferable from the standpoint of safety.

[0033] This beta-glucan derivative is amphiphilic, so it can easily penetrate into cells and reach the nuclear membrane, and it is preferable that it acts directly on genes.

[0034] Beta-glucan derivatives also increase fibrillin in skin epidermal cells. The mechanism of fibrillin increase is that beta-glucan derivatives bind to the surface of the epidermal cell membrane, stimulating the cell membrane and thereby stimulating fibrillin production.

[0035] Furthermore, due to its hydrophobicity, it penetrates the nuclear membrane and activates skin epidermal cells. The mechanism of this cell activation is the activation of DNA polymerase at the genetic level and the activation of EGF (Epidermal Growth Factor) receptors.

[0036] In other words, DNA polymerase is a key enzyme that replicates DNA, increases nucleic acids, and causes cell proliferation. Beta-glucan derivatives activate DNA polymerase, which activates skin epidermal cells.

[0037] Furthermore, the mechanism by which beta-glucan derivatives activate EGF receptor reactivity is by increasing receptor affinity, and by altering the three-dimensional structure of the EGF receptor, enhancing EGF receptor reactivity. It is also desirable that FGF receptors are simultaneously activated. In other words, it is desirable to see a synergistic effect when used in combination with EGF.

[0038] Beta-glucan derivatives also induce the activation of 8-oxoguanine DNA glycosylase or 8-hydroxyldeoxyguanine DNA glycosidase (both abbreviated as OGG1).

[0039] DNA polymerase is an enzyme that amplifies and repairs genes. One method of repairing genetic damage is a DNA polymerase-mediated repair process known as SOS repair. This repair process involves altering bases and adducts, and involves cutting and replicating DNA strands.

[0040] OGG1 is a process that removes oxidized bases such as 8OHdG and incorporates normal bases. This beta-glucan derivative acts on the active center of OGG1, enhancing its activity. This gene repair activity is favorable because it has been shown to have a synergistic effect with EGF.

[0041] This beta-glucan derivative activates epidermal cells, even in all epidermal cells that have genes in their nuclei. Furthermore, it can repair genetic damage caused by all substances, including reactive oxygen species, free radicals, ultraviolet rays, chemicals, side effects of medicines, metals, and aging. For example, it can repair genetic damage in neural epidermal cells and activate them.

[0042] Furthermore, beta-glucan derivatives are preferred because they easily pass through the stratum corneum membrane of the skin, maintaining the barrier function of the stratum corneum and thus maintaining the health and beauty of the skin.Furthermore, these beta-glucan derivatives are preferred because they pass through the cell membrane and activate epidermal cells within skin cells, promoting cell regeneration and function.

[0043] In plants, this beta-glucan derivative penetrates the plant cell wall and cell membrane to enter the plant cell, promoting skin epidermal cells, flowering, fruiting, and leaf growth, thereby extending the plant's lifespan. In other words, it acts as a plant activator.

[0044] Furthermore, this beta-glucan derivative is amphiphilic and can be incorporated into both water-soluble lotions and oily creams, and it is also advantageous that this beta-glucan derivative acts to repair genes, promotes cell proliferation, and promotes normal collagen and elastin production, thereby promoting skin cell function.

[0045] For example, it activates nerve cells. Neurons are susceptible to genetic damage caused by reactive oxygen species and amyloid beta protein in dementia and Alzheimer's disease, and the genes are difficult to repair. Therefore, activating nerve cells with this beta-glucan derivative and restoring nerve function is desirable for the purpose of preventing and recovering from neurological diseases.

[0046] It is also preferable to enhance neurotransmission by promoting the release of neurotransmitters from nerve endings, and to enhance muscle contraction by increasing the activity of nerves and muscles by enhancing the release of acetylcholine from the nerve terminals of motor neurons.

[0047] Furthermore, it is preferable that the beta-glucan derivatives increase the proliferation of epidermal cells and enhance the production of collagen and elastin, which is advantageous in that they can be used in cosmetics.

[0048] This beta-glucan derivative preferably exhibits a cardiotonic effect by activating myocardial cells and activating cardiac activity even in the case of coronary artery infarction or ischemia during myocardial infarction.

[0049] In particular, in blood vessels at the infarcted site, this beta-glucan derivative promotes angiogenesis, improves blood flow, and lowers blood pressure.

[0050] Furthermore, this beta-glucan derivative is preferable for athletes and those who want to build muscle during exercise, as it activates energy production at the genetic level in muscle cells.

[0051] This beta-glucan derivative is broken down in the body by esterases in the kidneys and liver and excreted in the urine. It is broken down into its constituent components, highly safe beta-glucan, polyphenols, and cysteine. Therefore, this beta-glucan derivative does not accumulate in the body, is broken down by enzymes in the body, and the decomposition products are natural products, making it highly safe.

[0052] Furthermore, the beta-glucan moiety has a plant activating effect, which promotes plant growth. In other words, the ability of this beta-glucan derivative to promote plant growth is desirable from the viewpoint of industrial application.

[0053] Furthermore, when a plant is infected with bacteria or viruses, its genes may be damaged. It is desirable to activate and proliferate epidermal cells to resist such genetic damage.

[0054] This beta-glucan derivative is preferably obtained by fermenting natural products due to its high safety. For example, this beta-glucan derivative can be obtained by fermenting acai palm or rice bran with lactic acid bacteria. Among lactic acid bacteria, Lactobacillus reuteri is preferred in terms of production efficiency. Lactobacillus reuteri is a Gram-positive lactobacillus that is normally present in humans and animals, and is present in the intestines as a human intestinal bacterium or on the skin. On the skin, it is a beneficial bacterium with anti-inflammatory effects. Furthermore, it is found in the culture medium of Galactomyces candidus, a type of filamentous fungus, as well as in primordial tissues such as reishi mushrooms, cordyceps sinensis, Agaricus blazei Murill, orchid flowers and stems, rose bushes, citrus fruits such as kumquats, plant shoots, and buds. Furthermore, it is also preferable to use Lactobacillus reuteri as a beta-glucan derivative, which is derived from natural products and is highly safe, by fermenting a fermentation liquid obtained by fermenting eucalyptus fruit and rice bran with Monascus bacteria as a medium, and purifying the resulting fermentation liquid.

[0055] This beta-glucan derivative can be extracted from the above-mentioned culture medium or plant. However, purification requires large amounts of raw materials and uses organic solvents, limiting its industrial use. In particular, it is preferable to identify the desired beta-glucan derivative, which exhibits skin epidermal cell activation via fibrillin-increasing activity, by a production method that involves fermenting acai palm fruit and rice bran with Monascus bacteria and purifying the fermented liquid obtained by fermenting Lactobacillus reuteri in a medium. This fermentation production method, unlike chemical synthesis, is a naturally occurring production method, and impurities are also naturally occurring, making it preferable from the standpoint of safety.

[0056] Specifically, this beta-glucan derivative is obtained by fermenting Lactobacillus reuteri in a medium prepared by fermenting acai palm fruit and rice bran with Monascus spp., followed by purification. This bacterium has a long history of use and is highly safe. In particular, Seti Co., Ltd.'s high-concentration Lactobacillus reuteri has a high concentration of 100 billion cfu / g, making it easy to use and preferable for manufacturing.

[0057] The above-mentioned culture method is preferred because it has been used as a food and produces a large amount of beta-glucan derivative. When the obtained beta-glucan derivative is to be used as a pharmaceutical material, it is preferable to purify the target beta-glucan derivative to increase its purity, as this increases the purity of the target beta-glucan derivative and allows impurities to be removed.

[0058] As pharmaceuticals, they are used as parenteral preparations such as injections, oral preparations, or liniments, and as quasi-drugs, they are incorporated into tablets, capsules, drinks, soaps, liniments, gels, toothpaste, etc. Oral preparations include tablets, capsules, powders, syrups, drinks, etc. When incorporated into the tablets and capsules, they can be used together with binders, excipients, bulking agents, lubricants, sweeteners, flavoring agents, etc. The tablets can also be coated with shellac, sugar, etc.

[0059] In the case of the capsules, the above ingredients may further contain a liquid carrier such as oil or fat. In the case of the syrups and drinks, sweeteners, preservatives, coloring agents, flavoring agents, etc. may be added.

[0060] Examples of parenteral preparations include external preparations such as ointments, creams, and solutions, as well as injections. Vaseline, paraffin, oils and fats, lanolin, macrogold, etc. are used as base materials for external preparations, and they can be made into ointments, creams, etc. by conventional methods.

[0061] Injectables include liquids and freeze-dried formulations, which are dissolved in distilled water for injection or physiological saline under sterile conditions before use.

[0062] As a food preparation, it is used in beauty foods to activate skin epidermal cells, and is preferably used as a health food in nutritional functional foods and foods for specified health uses.

[0063] When the obtained food preparation is used for pets or livestock animals such as dogs and cats, it is used as feed or pet supplements to restore skin luster and coat and prevent hair loss.

[0064] It can be used as a cosmetic product in the usual way together with surfactants, solvents, thickeners, excipients, etc. For example, it can be in the form of a cream, hair gel, face wash, beauty serum, lotion, etc.

[0065] The cosmetic preparation may be in any form, and may be used in the form of a solution, cream, paste, gel, solid, or powder.

[0066] The produced cosmetics are used for beauty purposes such as restoring and increasing damaged epidermal cells, increasing collagen and elastin, and maintaining skin.

[0067] Furthermore, this beta-glucan derivative can be used in toothpastes, mouthwashes, toothpastes, etc., with the aim of maintaining the function of genetically damaged gingival cells.

[0068] In plants, it can be used as a plant activator to promote germination, growth, fruiting, and increase yield by repairing genetic disorders.

[0069] This plant activator can be used to activate rare roses, orchids, and flowers, and stabilize the cultivation of leaves, vegetables, and grains. It can also be used to cultivate vegetables, fruits, and leaves in plant factories, improving cultivation efficiency. When using flowers, roses, orchids, and edelweiss are preferred.

[0070] Below, we will explain one method for producing beta-glucan derivatives, which involves fermenting Lactobacillus reuteri using a fermentation liquid obtained by fermenting asai palm fruit and rice bran with Monascus bacteria as a medium, and purifying the resulting fermentation liquid.

[0071] The raw materials are Lactobacillus reuteri, acai palm fruit, rice bran and Monascus mold.

[0072] To prepare a fermentation medium for Lactobacillus reuteri, acai palm fruit and rice bran are fermented with Monascus bacteria to produce a fermentation liquid.

[0073] Acai palm fruit is the fruit of the acai palm, a palm plant with the scientific name Euterpe oleracea. It can be produced in either Brazil or Thailand, but Brazilian acai is preferred due to its consistent quality. CANA CORPORATION's freeze-dried powder of Brazilian acai palm fruit is high quality and easy to use. Rice bran is the edible part obtained by peeling off part of the outer shell of threshed brown rice during rice milling. Rich in nutrients, it has long been used in Japan as a fermentation medium for pickles and other foods. Japanese-grown, pesticide-free rice bran is particularly preferred for its safety. Rice bran produced at Harada Natural Farm (Kameoka City, Kyoto Prefecture) is particularly preferred due to its high quality. Monascus purpureus is a type of filamentous fungus. It is a useful fermentation fungus and has long been used for tofu and food coloring. The Monascus mold produced by Benikoji Honpo Co., Ltd. (Maizuru City, Kyoto Prefecture) is preferred because of its stable quality.

[0074] The acai palm fruit and rice bran are preferably ground into powder with a particle size of 3 micrometers or less, as this facilitates the cultivation process. The ground material is then sterilized in an autoclave and fermented in a fermentation tank with Monascus mold. The preferred amounts of rice bran and Monascus mold added are 1 to 5 weights per weight of acai palm fruit, and 0.001 to 0.1 weights per weight of Monascus mold. The fermentation temperature is preferably 32 to 37°C, and the fermentation time is preferably 12 to 24 hours.

[0075] The resulting fermentation broth is filtered, and the resulting filtrate is heated at 100°C for 10 to 20 minutes and then naturally cooled for sterilization. This fermentation filtrate is used as a culture medium for Lactobacillus reuteri.

[0076] Lactobacillus reuteri, scientifically known as Lactobacillus reuteri, is a beneficial microorganism that is also present in the human body. It is sold as a high-concentration Lactobacillus reuteri strain by Seti Co., Ltd. (Chiyoda-ku, Tokyo) as a raw material for fermentation.

[0077] 0.001 to 0.1 weight of Lactobacillus reuteri is added to 1 weight of the fermentation filtrate, and the mixture is fermented in a fermentation tank at 35 to 38°C for 24 to 48 hours.

[0078] Furthermore, pre-culturing Lactobacillus reuteri is preferable because it shortens the culture time.

[0079] It is preferable to control the culture process by quantifying the desired beta-glucan derivative by HPLC or other methods and confirming the proliferation of the fungus, as this allows for the production of the desired beta-glucan derivative in which polyphenols and cysteine ​​are bound.

[0080] Extraction of the culture with aqueous ethanol is preferred because it allows for efficient recovery of the product, sterilization of the bacteria, and ease of carrying out the next step. Ultrasonic treatment of the resulting culture is also preferred because it allows the product to be dispersed in the solvent, facilitating the separation of the target substance. Concentration by freeze-drying or other methods is also preferred because it allows the following steps to be carried out in a short time.

[0081] It is preferable to separate and purify the desired beta-glucan derivative from the culture, as this will result in a highly pure substance. This purification method preferably utilizes a purification procedure such as a separation resin.

[0082] For example, the desired beta-glucan derivative can be obtained by separation and fractionation using a separation carrier or resin. Porous polysaccharides, silicon oxide compounds, polyacrylamide, polystyrene, polypropylene, styrene-vinylbenzene copolymers, and the like, whose surfaces are coated as described below, are used as separation carriers or resins. Particle sizes of 0.1 to 300 μm are preferred; the finer the particle size, the more accurate the separation, but the longer the separation time.

[0083] For example, reversed-phase carriers or resins whose surfaces are coated with hydrophobic compounds are used to separate highly hydrophobic substances. Those coated with cationic substances are suitable for separating anionically charged substances. Those coated with anionic substances are also suitable for separating cationically charged substances. When coated with specific antibodies, they are used as affinity carriers or resins that separate only specific substances.

[0084] Affinity carriers or resins are used for the specific preparation of antigens by utilizing antigen-antibody reactions, while partitioning carriers or resins, such as silica gel (Merck), are used for the isolation of substances when there is a difference in the partition coefficient between the substance and the separation solvent.

[0085] Among these, adsorbent carriers or resins, distributive carriers or resins, molecular sieve carriers or resins, and ion exchange carriers or resins are preferred from the viewpoint of reducing production costs. Furthermore, reversed-phase carriers or resins and distributive carriers or resins are more preferred from the viewpoint of a large difference in distribution coefficient with respect to the separation solvent.

[0086] When an organic solvent is used as the separation solvent, a carrier or resin resistant to the organic solvent is used, and carriers or resins used in pharmaceutical or food manufacturing are preferred.

[0087] From these viewpoints, Diaion (manufactured by Mitsubishi Chemical Corporation) and XAD-2 or XAD-4 (manufactured by Rohm and Haas) are more preferred as adsorption carriers, Sephadex LH-20 (manufactured by Amersham Pharmacia) as molecular sieve carriers, silica gel as partition carriers, IRA-410 (manufactured by Rohm and Haas) as ion exchange carriers, and DM1020T (manufactured by Fuji Silysia Chemical Industries, Ltd.) as reverse phase carriers.

[0088] Of these, Diaion, Sephadex LH-20 and DM1020T are more preferred.

[0089] The obtained extract is dissolved in a solvent to swell the separation carrier or resin before separation. The amount of the solvent is preferably 2 to 30 times the weight of the extract, more preferably 4 to 20 times, from the viewpoint of separation efficiency. The separation temperature is preferably 10 to 30°C, more preferably 12 to 25°C, from the viewpoint of substance stability.

[0090] The separation solvent is water, a water-containing lower alcohol, a hydrophilic solvent, or a lipophilic solvent. Examples of lower alcohols include methanol, ethanol, propanol, and butanol, with ethanol being preferred as it is used for food.

[0091] When Sephadex LH-20 is used, the separation solvent is preferably a lower alcohol, and when silica gel is used, the separation solvent is preferably chloroform, methanol, acetic acid, or a mixture thereof.

[0092] When Diaion and DM1020T are used, the separation solvent is preferably a lower alcohol such as methanol or ethanol, or a mixture of a lower alcohol and water.

[0093] It is preferable to collect a fraction containing the beta-glucan derivative, remove the solvent by drying or vacuum drying, and obtain the desired beta-glucan derivative as a powder or a concentrated liquid, since this eliminates the influence of the solvent.

[0094] Furthermore, it is preferable to carry out the final extraction using edible oils or oils used in cosmetics, since the resulting beta-glucan derivative is converted to a fat-soluble form and therefore has affinity for fat-soluble solvents. For example, extraction with soybean oil, rice bran oil, grapeseed oil, olive oil, or jojoba oil is preferable.

[0095] In addition, powdering the beta-glucan derivative is preferable for preservative purposes. The above-described embodiment will be specifically explained below using examples and test examples. Note that these are merely examples, and conditions can be changed within reasonable limits depending on the material, raw materials, and specimen. [Example]

[0096] 1 kg of freeze-dried powder of acai palm fruit (Brazilian, imported by CANA CORPORATION) and 1 kg of rice bran powder (Harada Natural Farm, Kameoka City, Kyoto Prefecture) were crushed in a grinder and then sterilized in an autoclave (SDL-320, Tomy) at 120°C for 15 minutes.

[0097] This was added to a sterilized 40-liter round fermentation tank (manufactured by Endo Scientific Co., Ltd.), to which 50 g of fermented liquid pre-cultured with Monascus mold produced by Benikoji Honpo Co., Ltd. (Maizuru City, Kyoto Prefecture) was added, followed by fermentation at 35-36°C for 15 hours.

[0098] During the fermentation process, the fermented liquid was sampled while bubbling and stirring with aeration. After fermentation was completed, the fermented liquid was removed from the fermentation tank. The fermented liquid was roughly filtered through a filter cloth, and the filtrate was then filtered through a Buchner filter lined with diatomaceous earth and filter paper (Toyo Roshi). The resulting filtrate was heated at 100°C for 15 minutes. After natural cooling, this was used as the fermentation filtrate.

[0099] 10 g of Lactobacillus reuteri (manufactured by Seti Co., Ltd.) was added to 1 kg of the fermentation filtrate, and the mixture was fermented in a clean fermentation tank at 36°C for 40 hours.

[0100] During the fermentation process, the fermentation liquid was sampled while bubbling and stirring with aeration, and the production of the target beta-glucan derivative was detected. After fermentation was completed, the fermentation liquid was removed from the fermentation tank. The fermentation liquid was roughly filtered through a filter cloth, and the filtrate was then filtered through a Buchner filter lined with diatomaceous earth and filter paper (Toyo Roshi). The resulting filtrate was heated at 100°C for 15 minutes. After natural cooling, the filtrate was used as a culture liquid containing the target beta-glucan derivative. This was designated Sample 1. The production volume of Sample 1 was 0.74 kg.

[0101] Furthermore, a purified product was obtained for structural analysis and experimental purposes. Specifically, 2 L of purified water containing 5% ethanol was added to 100 g of the beta-glucan derivative-containing sample 1 described above, and the mixture was loaded onto a glass column (Endo Scientific) packed with 500 g of Diaion (AMP03 type, Mitsubishi Chemical) suspended in 5% ethanol.

[0102] This was washed by adding 10 L of 5% ethanol, and then 1 L of 25% ethanol was added. The desired beta-glucan derivative was eluted by adding 1 L of 65% ethanol, and the eluate was concentrated using an evaporator. This purification process was repeated five times, and the ethanol portion of the purified beta-glucan derivative was removed by vacuum distillation to obtain an aqueous solution. This was then dried under vacuum to obtain 5.1 g of purified beta-glucan derivative, designated Sample 2. The yield was approximately 5%, which was sufficient for purification from a natural product, confirming that this manufacturing method is an excellent method for producing the desired beta-glucan derivative.

[0103] The test methods and results for the structural analysis of beta-glucan derivatives are described below. (Test Example 1)

[0104] The sample 2 obtained as described above was dissolved in ethanol and analyzed by high performance liquid chromatography with a mass spectrometer (HPLC, Shimadzu Corporation). The purity was found to be 99.3%.

[0105] When sample 2 was analyzed using a nuclear magnetic resonance spectrometer (500 MHz, solvent C5D5N, manufactured by Bruker), the target beta-glucan derivative consisting of one beta-glucan molecule, one polyphenol molecule, and three cysteine ​​molecules was detected in sample 2.

[0106] That is, when analyzed by 500 MHz H-NMR in C5D5N, peak positions were observed at 0.91, 0.95, 1.10, 1.30, 1.52, 1.57, 1.76, 2.56, 2.72, 3.36, 4.45, 5.03, 5.22, 5.25, 5.35, 6.03, and 6.11 ppm.

[0107] Furthermore, analysis of 13C-NMR in C5D5N revealed the following: 9.1, 19.0, 19.1, 19.2, 19.3, 19.9, 21.4, 23.6, 25.5, 26.5, 27.3, 28.1, 32.5, 36.4, 37.5, 38.5, 39.3, 42.0, 44.7, 45.3, 49.8, 51.8, 52.7, 63.0, 63.8, 64.4, 64.5, 67.0, 68.1, 69.4, 70.2, 70. Peaks were observed at 3, 71.6, 73.1, 73.3, 73.8, 74.0, 74.4, 75.5, 76.9, 77.1, 77.6, 78.9, 79.4, 79.5, 79.8, 80.3, 80.9, 83.3, 87.1, 89.0, 90.7, 101.2, 103.4, 104.7, 104.9, 105.0, 108.2, 112.2, 119.2, 137.4, 137.8, and 178.3 ppm.

[0108] The chart below shows the results of the 13C-NMR analysis (the horizontal axis is in ppm, and the vertical axis is in peak intensity). JPEG0007755819000003.jpg1375

[0109] The above analysis results showed that the sample exhibited the same structure as the chemically synthesized standard. In other words, Sample 2 was confirmed to be the desired beta-glucan derivative, consisting of one beta-glucan molecule, one polyphenol molecule, and three cysteine ​​molecules. The substance was determined to be C63H97O39N3S3, consisting of 63 carbon atoms, 97 hydrogen atoms, 39 oxygen atoms, 3 nitrogen atoms, and 3 sulfur atoms.

[0110] The following describes a proliferation test using human skin epidermal cells. This test method is a reproducible, standard method that can biochemically verify the effects of ingredients. (Test Example 2)

[0111] Human epidermal cells (epidermal-derived, Epider Cell) purchased from Kurabo were used. 1,000 cells cultured in MEM medium (Sigma) containing 5% fetal bovine serum were seeded onto a 35 mm culture dish (Falcon) and cultured at 37°C under 5% carbon dioxide. After washing with culture medium, the skin epidermal cells were seeded onto a plate (Falcon). This was irradiated with 280 nm ultraviolet light for 1 hour using an ultraviolet irradiation device (Quark Technology). Sample 1, Sample 2, and EGF (Funakoshi, epidermal growth factor) as a positive control were added to a final concentration of 0.1 mg / ml. This was cultured for 48 hours before testing.

[0112] After collecting the culture medium, the proliferation rate of the epidermal cells was counted using the trypan blue method. Then, a suspension of epidermal cells was prepared. mRNA was extracted from the suspension using a nucleic acid extraction kit (Funakoshi). Fibrillin mRNA was quantified by RT-PCR according to standard methods. Furthermore, the amount of fibrillin contained in the cells was quantified spectrophotometrically using ELISA (anti-fibrillin antibody, Funakoshi, code AG-20B-0073-C100) and anti-mouse IgG antibody. The antigen used was fibrillin (Asproson, code AG-20B-0073-C010) manufactured by Funakoshi.

[0113] At the same time, the amount of 8-OHdG in the cell suspension was quantified using an ELISA kit (Japan Institute for the Control of Aging). This is an ELISA kit that uses a monoclonal antibody specific to 8-OHdG.

[0114] The average value was calculated using five petri dishes and compared with the solvent control group.

[0115] As a result, the addition of 0.1 mg / ml of sample 1 increased the number of skin epidermal cells to an average of 188% compared to the solvent control group. Furthermore, sample 2 increased the number to 267%. Meanwhile, EGF increased the number to 180%. Consequently, samples 1 and 2 exhibited superior epidermal cell activation effects to EGF.

[0116] The fibrillin mRNA expression level (copy number) in the above cells was 20 copies in the solvent control group, 50 copies in the sample 1 treatment group, 180 copies in the sample 2 treatment group, and 23 copies in the EGF treatment group. This shows that samples 1 and 2 increased fibrillin at the mRNA level.

[0117] Furthermore, fibrillin was quantified by ELISA, and the addition of 0.1 mg / ml of sample 1 increased the amount of fibrillin to an average of 198% compared to the solvent control group. Sample 2 also increased the amount to 349%, while EGF increased it to 176%. These results demonstrate that samples 1 and 2 exhibited a greater fibrillin-increasing effect than EGF.

[0118] The amount of 8OHdG in the cells was 540 ng in the solvent control group, 155 ng in the sample 1 treatment group, 78 ng in the sample 2 treatment group, and 509 ng in the EGF treatment group.

[0119] 8OHdG is a mutated state in which genes have been modified by reactive oxygen species, and indicates genetic damage. Samples 1 and 2 had low levels of 8OHdG, and were superior to EGF. This indicates the gene repair effect of samples 1 and 2 on skin epidermal cells.

[0120] On the other hand, in a skin irritation experiment using EpiSkin (manufactured by SkinEthic), an artificial skin, as part of a safety test, no irritation was observed with the addition of Sample 1 and Sample 2, confirming safety. This method has been established as an alternative method for evaluating skin irritation without using animals. [Industrial Applicability]

[0121] The beta-glucan derivative obtained by the present invention activates epidermal cells and enhances their cellular function, thereby improving the cosmetic quality of life of the nation, increasing the healthy working population, and reducing medical costs.

[0122] The beta-glucan derivative obtained by the present invention can be used as a functional food product because it is produced by a culture method, and will contribute to the development of the food industry and the culture industry.

Claims

[Claim 1] A beta-glucan derivative that exhibits the effect of activating skin epidermal cells through the fibrillin-increasing effect shown in the following formula (1). 【Chemical 1】

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