HAS3 mRNA expression enhancer

Abstract

To provide an HAS3 mRNA expression promoter having excellent action.SOLUTION: The present disclosure provides an HAS3 mRNA expression promoter containing a fermented product of rice with Saccharomyces veronae as an active ingredient.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to an agent for promoting HAS3 mRNA expression.

Background Art

[0002] Hyaluronic acid is a kind of mucopolysaccharide, which has the function of holding cells by filling the spaces between cells. Furthermore, it has many functions such as retaining moisture in the intercellular spaces, imparting lubricity and flexibility to tissues, and resisting external forces such as mechanical damage. If the production of hyaluronic acid can be promoted by promoting the expression of hyaluronic acid synthase 3 (HAS3) involved in the synthesis of epidermal hyaluronic acid, it is considered that skin aging symptoms such as rough skin, wrinkles, dullness, change in texture, decrease in elasticity, and decrease in moisturizing function can be prevented, treated or improved.

[0003] To date, as substances having an action of promoting the expression of hyaluronic acid synthase 3 (HAS3) mRNA, a glycyrrhiza leaf extract (see Patent Document 1) and the like are known.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] An object of the present invention is to provide an agent for promoting HAS3 mRNA expression having excellent effects.

Means for Solving the Problems

[0006] In order to solve the above problems, the HAS3 mRNA expression promoting agent of the present invention is characterized by comprising a fermentation product by Saccharomyces cerevisiae of rice as an active ingredient. ​​

[0007] According to the present invention, by using a fermented product of rice with Saccharomyces verona as an active ingredient, it is possible to provide a HAS3 mRNA expression promoter with excellent efficacy. [Modes for carrying out the invention]

[0008] [HAS3 mRNA expression promoter] The HAS3 mRNA expression promoter of this embodiment contains a fermented product of rice with Saccharomyces verona as an active ingredient. The fermented product of rice with Saccharomyces verona of this embodiment is obtained by fermenting rice with Saccharomyces verona.

[0009] Herein, unless otherwise specified, “fermented rice with Saccharomyces verona” includes, but is not limited to, any of the following: the fermentate obtained by fermenting rice with Saccharomyces verona, the diluted or concentrated fermentate, the dried product obtained by drying the fermentate, or the crude or refined products thereof.

[0010] In the fermented rice product using Saccharomyces verona according to this embodiment, the rice used as the fermentation raw material is obtained from the grass plant Oryza sativa. Rice is cultivated throughout Japan and is readily available from these regions. There are no particular restrictions on the fermentation raw material, and it can be appropriately selected depending on the purpose, but examples include paddy rice, brown rice, polished rice, or mixtures thereof, with polished rice being preferred among these.

[0011] The rice fermented product with Saccharomyces verona according to this embodiment is obtained by fermenting rice with Saccharomyces verona. The fermentation process can be carried out, for example, by dispersing rice in water and inoculating it with fermenting microorganisms.

[0012] Before inoculating with Saccharomyces verona, it is preferable to perform a treatment to decompose the starch contained in the rice and / or the cell walls of the rice, from the viewpoint of improving fermentation efficiency.

[0013] Starch decomposition treatments include, for example, enzyme treatment and inoculation with Aspergillus oryzae, with enzyme treatment being particularly preferred. Examples of starch-degrading enzymes include amylases such as α-amylase, β-amylase, glucoamylase, and isoamylase, but from the viewpoint of improving fermentation efficiency, α-amylase and glucoamylase are particularly preferred, and glucoamylase is even more preferred. On the other hand, cell wall decomposition treatments are preferably performed using enzymes such as cellulase and xylanase.

[0014] The resulting processed liquid may be subjected to desired means such as cooling, heating, pH adjustment, or sterilization to inactivate enzymes and koji mold, or it may be inoculated with microorganisms that carry out fermentation as is.

[0015] The microorganism that carries out fermentation is Saccharomyces veronae (also known as Lachancea thermotolerans).

[0016] The fermentation process using the above microorganisms can be carried out, for example, by placing the fermentation raw materials and the above microorganisms in a fermentation tank and treating them at a temperature range of 20 to 40°C, preferably 25 to 35°C, for 12 to 72 hours, preferably 18 to 48 hours. Furthermore, if the pH of the reaction solution is adjusted to 5.0 to 7.0, preferably 6.0 to 7.0, at the start of the fermentation process, the fermentation process will proceed favorably. This fermentation process is terminated by subjecting the fermentation solution to a desired means such as cooling, heat sterilization, or filtration to inactivate the fermentation bacteria.

[0017] The fermentation liquid obtained in this way may be used as is as a fermented product, or it may be diluted or concentrated as appropriate before being used as a fermented product. Furthermore, the concentrate may be further dried or subjected to crude purification.

[0018] The fermented product of rice by Saccharomyces verona obtained in this way has an excellent effect of promoting HAS3 mRNA expression, and thus can be used as an active ingredient of a HAS3 mRNA expression promoter. The HAS3 mRNA expression promoter according to this embodiment can be used in a wide range of applications such as pharmaceuticals, quasi-drugs, cosmetics, food and drink products, etc.

[0019] The HAS3 mRNA expression promoter of this embodiment may consist only of the fermented product of rice by Saccharomyces verona, or may be a formulation of the fermented product of rice by Saccharomyces verona.

[0020] The HAS3 mRNA expression promoter of this embodiment can be formulated into any dosage form such as powder, granule, tablet, liquid, etc. according to a conventional method using a pharmaceutically acceptable carrier such as dextrin, cyclodextrin, and other optional auxiliaries. At this time, as the auxiliaries, for example, excipients, binders, disintegrants, lubricants, stabilizers, flavoring and odor-correcting agents, etc. can be used. The HAS3 mRNA expression promoter can be used by being blended with other compositions (for example, external preparations for skin, oral compositions, etc.), and can also be used as an ointment, external liquid preparation, patch, etc.

[0021] When formulating the HAS3 mRNA expression promoter of this embodiment, the content of the fermented product of rice by Saccharomyces verona is not particularly limited and can be appropriately set according to the purpose.

[0022] In addition, the HAS3 mRNA expression promoter of this embodiment can, if necessary, be used as an active ingredient by blending other natural extracts, etc. having the effect of promoting HAS3 mRNA expression together with the fermented product of rice by Saccharomyces verona.

[0023] As a method of administering the HAS3 mRNA expression promoter of the present embodiment to a patient, examples include transdermal administration and oral administration. Depending on the type of disease, a method suitable for its prevention, treatment, etc. may be appropriately selected. Also, the dosage of the HAS3 mRNA expression promoter of the present embodiment may be appropriately increased or decreased depending on the type of disease, severity, individual differences of the patient, administration method, administration period, etc.

[0024] The HAS3 mRNA expression promoter of the present embodiment can promote the expression of hyaluronan synthase 3 (HAS3) through the HAS3 mRNA expression promoting action of the fermented product by Saccharomyces cerevisiae of rice, which is an active ingredient. Therefore, it can be used for the prevention, treatment or improvement of skin aging symptoms such as skin roughness, wrinkles, dullness, change in texture, decrease in elasticity and decrease in moisturizing function. The HAS3 mRNA expression promoter of the present embodiment can be used for all applications that are meaningful in exerting the HAS3 mRNA expression promoting action in addition to these applications.

[0025] Also, since the HAS3 mRNA expression promoter of the present embodiment has an excellent HAS3 mRNA expression promoting action, it is suitable for blending into, for example, skin external preparations, oral compositions, etc. In this case, the fermented product by Saccharomyces cerevisiae of rice may be blended as it is, or a formulated HAS3 mRNA expression promoter from the fermented product by Saccharomyces cerevisiae of rice may be blended.

[0026] Here, the skin external preparation is not limited to its classification and widely includes skin cosmetics, quasi-drugs, pharmaceuticals, etc. that are used transdermally. Specifically, for example, ointments, creams, emulsions, lotions, beauty essences, lotions, gels, beauty oils, packs, foundations, lip creams, bath agents, hair tonics, hair lotions, shampoos, rinses, soaps, body shampoos, etc. can be mentioned.

[0027] Oral compositions refer to substances that pose little risk to human health and are ingested orally or via the gastrointestinal tract in normal social life, and are not limited to the administrative classifications of food and beverages, pharmaceuticals, quasi-drugs, etc. Therefore, "oral compositions" in this embodiment broadly include general foods, animal feed, health foods, functional foods (foods for specified health uses, nutritional functional foods, foods with functional claims), quasi-drugs, pharmaceuticals, etc., that are ingested orally.

[0028] Furthermore, since the HAS3 mRNA expression promoter of this embodiment has excellent HAS3 mRNA expression promoting activity, it can also be suitably used as a reagent for research on the mechanisms of action.

[0029] Although the HAS3 mRNA expression promoter of this embodiment is preferably applied to humans, it can also be applied to animals other than humans (e.g., mice, rats, hamsters, dogs, cats, cattle, pigs, monkeys, etc.) as long as the respective effects are achieved. [Examples]

[0030] The present invention will be specifically described below with reference to test examples, but the present invention is not limited in any way to the following examples.

[0031] [Production Example] Production of rice fermented with Saccharomyces verona

[0032] 0.1 kg of polished white rice flour was mixed with 1 kg of water and dispersed. After sterilization at 110°C for 5 minutes, glucoamylase was added and the mixture was reacted at 60°C for 1 hour.

[0033] Subsequently, Saccharomyces verona (manufactured by Akita Konno Shoten Co., Ltd.) was inoculated into the resulting enzyme-treated solution and fermented at 30°C for 24 hours. The resulting fermented liquid was sterilized at 110°C for 5 minutes. The resulting culture solution was filtered through diatomaceous earth, and the filtrate was concentrated and dried to obtain a fermented product of rice with Saccharomyces verona (approximately 64 g, Sample 1).

[0034] [Test Example 1] Hyaluronic acid synthase 3 (HAS3) mRNA expression promotion test The fermented product of rice with Saccharomyces verona (Sample 1) was tested for HAS3 mRNA expression promotion as follows.

[0035] Normal human neonatal epidermal keratinocytes (NHEKs) were pre-cultured in normal human epidermal keratinocyte growth medium (KGM), and the cells were harvested by trypsin treatment. The harvested cells were 20 × 10⁻⁶ 4 After diluting with KGM medium to achieve a cell density of cells / mL, seed 2 mL each into 35 mm petri dishes (40 × 10 4 Cells were cultured overnight in a petri dish at 37°C under 5% CO2 conditions. After culturing, the culture medium was replaced with normal human epidermal keratinocyte basal medium (KBM, the same as the above KGM medium but without added growth factors (hEGF, BPE, insulin)), and the cells were cultured for a further 24 hours.

[0036] After 24 hours of incubation, the culture medium was removed, and 2 mL of the test sample (Sample 1, see Table 1 below for sample concentration) dissolved in KBM medium was added to each petri dish. The samples were then incubated at 37°C and 5% CO2 for 24 hours. As a control, the same incubation was performed using KBM medium without any sample added. After incubation, the culture medium was removed, and total RNA was extracted using ISOGEN II (Nippon Gene Co., Ltd., Cat. No. 311-07361). The amount of RNA in each sample was measured using a spectrophotometer, and the total RNA was adjusted to 200 ng / μL.

[0037] Using this total RNA as a template, the mRNA expression levels of HAS3 and the internal standard GAPDH were measured. Detection was performed using a real-time PCR instrument, Smart Cycler (Cepheid), and a real-time 2-step RT-PCR reaction with the TaKaRa SYBR Prime Script RT-PCR kit (Perfect Real Time) (Takara Bio, code No. RR063A). The HAS3 mRNA expression level was corrected using the GAPDH value based on total RNA standards prepared from cells cultured with and without the test sample. From the obtained values, the HAS3 mRNA expression enhancement rate (%) was calculated using the following formula.

[0038] HAS3 mRNA expression enhancement rate (%) = A / B × 100 Each term in the formula represents the following: A: Correction value when adding test sample B: Corrected value without sample addition The results are shown in Table 1.

[0039] [Table 1]

[0040] As shown in Table 1, the fermented product of rice with Saccharomyces verona (Sample 1) exhibited excellent HAS3 mRNA expression-promoting activity.

[0041] [Formulation example 1] A cream with the following composition was manufactured by conventional methods. Rice fermented with Saccharomyces verona (production example) 0.05g Sophora flavescens extract 0.1g Scutellaria baicalensis extract 0.1g Liquid paraffin 5.0g 4.0g of bleached beeswax Squalane 10.0g Cetanol 3.0g Lanolin 2.0g Stearic acid 1.0g Polyoxyethylene sorbitan oleate (20E.O.) 1.5g Glyceryl monostearate 3.0g Oil-soluble licorice extract 0.1g 1,3-Butylene glycol 6.0g Methyl parahydroxybenzoate 1.5g Fragrance 0.1g Purified water, remaining amount (assuming a total volume of 100g)

[0042] [Formulation example 2] An emulsion was prepared by conventional methods according to the following composition. Fermented rice product with Saccharomyces verona (production example) 0.01g Jojoba oil 4.00g 1,3-Butylene glycol 3.00g Arbutin 3.00g Polyoxyethylene cetyl ether (20 E.O.) 2.50g Olive oil 2.00g Squalane 2.00g Cetanol 2.00g Glyceryl monostearate 2.00g Polyoxyethylene sorbitan oleate (20E.O.) 2.00g Methyl parahydroxybenzoate 0.15g Stearyl glycyrrhetinate 0.10g Scutellaria baicalensis extract 0.10g Dipotassium glycyrrhizinate 0.10g Ginkgo biloba extract 0.10g Conchiolin 0.10g Phellodendron bark extract 0.10g Chamomile extract 0.10g Fragrance 0.05g Purified water, remaining amount (assuming a total volume of 100g)

[0043] [Formulation example 3] A beauty serum with the following composition was manufactured by conventional methods. Fermented rice product with Saccharomyces verona (production example) 0.01g Chamomile extract 0.1g Carrot extract 0.1g Xanthan gum 0.3g Hydroxyethylcellulose 0.1g Carboxyvinyl polymer 0.1g 1,3-Butylene glycol 4.0g Dipotassium glycyrrhizinate 0.1g Glycerin 2.0g Potassium hydroxide 0.25g Fragrance 0.01g Preservative (methyl parahydroxybenzoate) 0.15g Ethanol 2.0g Purified water, remaining amount (assuming a total volume of 100g)

[0044] [Formulation example 4] Tablets having the following composition were manufactured by conventional methods. Fermented rice product with Saccharomyces verona (production example) 5.0 mg Dolomite (containing 20% ​​calcium and 10% magnesium) 83.4 mg Casein phosphopeptide 16.7 mg Vitamin C 33.4mg Maltitol 136.8mg Collagen 12.7mg Sucrose fatty acid ester 12.0 mg

[0045] [Formulation example 5] An oral liquid formulation having the following composition was manufactured by a conventional method. <Composition per ampoule (100 mL)> Fermented rice product with Saccharomyces verona (production example) 0.3% by mass Sorbitol 12.0% by mass Sodium benzoate 0.1% by mass Fragrance 1.0% by mass Calcium sulfate 0.5% by mass Purified water remainder (100% by mass) [Industrial applicability]

[0046] The HAS3 mRNA expression promoter according to the present invention can make a significant contribution to the prevention, treatment, or improvement of skin aging symptoms such as rough skin, wrinkles, dullness, changes in skin texture, decreased elasticity, and decreased moisturizing function.

Claims

[Claim 1] A HAS3 mRNA expression promoter characterized by containing a fermented product of polished rice with Saccharomyces veronae as an active ingredient.

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