JP2021193142A - Expression promoter of type 17 collagen and method for producing expression promoter of type 17 collagen - Google Patents

Abstract

To provide an expression promoter of Type 17 collagen using a berry-based extract having an action of suppressing hair loss and thinning of hair.SOLUTION: The expression promoter comprises a black raspberry extract as an active ingredient.SELECTED DRAWING: None

Description

The present invention relates to a method for producing an expression-promoting agent for type 17 collagen and an expression-promoting agent for type 17 collagen.

Oxygen is essential for most organisms, but a few percent of the oxygen taken up by the body becomes active oxygen, which is used to protect against bacterial infections. However, excess active oxygen needs to be eliminated by in vivo antioxidant enzymes and foreign antioxidants in foods in order to maintain homeostasis.

Further, when excessive active oxygen is produced by ultraviolet rays, smoking, exhaust gas, air pollution, stress, etc., it causes oxidative damage to living tissues (see, for example, Non-Patent Document 1). As a result, it causes lifestyle-related diseases, age-related diseases, cancer, dementia, etc., and it is said that 90% or more of the diseases have a causal relationship with overproduced active oxygen (for example, non-). See Patent Document 2).

Therefore, along with the activation of Superoxide dismutase (SOD) in the body, antioxidant substances in foods have attracted attention, and the antioxidant capacity measurement method (ORAC; Oxygen Radical Absorbance Capacity) developed by the United States Department of Agriculture (USDA) has been established. , It is widespread (see, for example, Non-Patent Document 3). In addition, 12 items of berry-based foods are present in the foods having a high ORAC value, that is, the best 100 foods having a high antioxidant capacity (see, for example, Non-Patent Document 4).

Hair loss occurs with aging and hair decreases, but many analyzes have been conducted on the mechanism of hair loss and proliferation of dermal papilla cells. It has been clarified that the hair loss phenomenon is caused by the decomposition of type 17 collagen in hair follicle stem cells and the miniaturization of hair follicles (see, for example, Non-Patent Document 5). Further, it is considered that if Hsp47, which is a heat shock protein having a protective action dedicated to collagen, is highly expressed, the decomposition of type 17 collagen is suppressed, and as a result, hair loss is suppressed.

The reductase 5α-reductase converts testosterone (male hormone) to dihydrotestosterone (DHT). Here, DHT is the most potent androgen that promotes hair loss and thinning of male hair (see, for example, Non-Patent Document 6). Therefore, it is considered that hair loss can be prevented by inhibiting the enzymatic activity of 5α-reductase.

Oberdanner, C. ROS and Antioxidant Systems in Apoptosis. VDM Verlag, 2008. ISBN 978-3836482110 Slavin JL et al. Health benefits of fruits and vegetables. Adv Nutr. 2012. 1; 3 (4): 506-516 Cao G et al. Oxygen-radical absorbance capacity assay for antioxidants. Free Radical Biol. Med. 1993. 14: 303-311 "Top 100 List of Foods with High ORAC Value (High Antioxidant Effect)" [online] (Searched June 30, 2017) http://www.anteaoxidant.com/orac_top100.html Matsumura H, Mohri Y, Binh NT, Morinaga H, Fukuda M, Ito M, Kurata S, Hoeijmakers J, Nishimura EK. Hair follicle aging is driven by transepidermal elimination of stem cells via COL17A1 proteolysis. 2016. 5; 351. Ulrike Blume-Peytavi; David A. Whiting; Ralph M. Trueb). Hair Growth and Disorders. Springer Science & Business Media. (2008) p368-370.

An object of the present invention is to provide a method for producing a type 17 collagen expression promoter and a method for producing a type 17 collagen expression promoter using a berry-based extract having an action of suppressing hair loss and thinning of hair. That is.

The present inventors focused on berry-based foods as foods having antioxidative activity, investigated the correlation between berry-based foods and the action of suppressing hair loss, and as a result, black raspberry extracts were the above-mentioned purposes. It was discovered that the above was achieved, and the present invention was completed.

That is, according to the present invention.
(1) An expression promoter for type 17 collagen containing black raspberry extract as an active ingredient,
(2) The method for producing a type 17 collagen expression promoter according to (1), which comprises a freezing step of freezing black raspberry fruits, a thawing step of thawing the black raspberry fruits, and the thawing step. A stirring step of adding a solvent to the thawed black raspberry fruit and stirring, an extraction step of allowing the black raspberry fruit and the solvent stirred by the stirring step to stand and extracting, and removing the solvent to evaporate the solvent. A method for producing a type 17 collagen expression promoter including a step is provided.

According to the present invention, an agent for promoting the expression of type 17 collagen using a berry-based extract having an action of suppressing hair loss and thinning of hair having an action of suppressing hair loss and A method for producing an expression-promoting agent for type 17 collagen can be provided.

It is a graph which shows the result of examination of the growth promotion effect of a dermal papilla cell. It is a graph which shows the measurement result of Hsp47 and 17 type collagen m-RNA levels in dermal papilla cells. It is a graph which shows the result of examination of the 5α-reductase inhibitory activity.

Hereinafter, the black raspberry extract of the present invention will be described. The black raspberry extract of the present invention has an action of promoting the proliferation of dermal papilla cells. In addition, the black raspberry extract of the present invention has an effect of suppressing hair loss.

Black raspberry, which is the raw material of black raspberry extract, belongs to the genus Rubus in the Rosaceae family. Further, as the black raspberry extract of the present invention, an extract mainly from the fruit of black raspberry is preferably used.

The method for extracting the black raspberry extract from the fruit of black raspberry is not particularly limited, but solvent extraction, supercritical extraction and the like can be used.

The extraction solvent used in the case of solvent extraction is not particularly limited, and water, an organic solvent and a mixed solvent thereof can be used. As the organic solvent, alcohols such as methanol, ethanol, n-propanol, isopropanol and n-butanol; glycols such as ethylene glycol and propylene glycol, ethyl acetate, acetone and the like can be used. One type of these organic solvents may be used alone, or two or more types may be used in combination. Further, an aqueous solvent such as water and an organic solvent may be mixed and used.

Among these, an ethanol aqueous solution in which ethanol or ethanol and water are mixed is preferable, and the concentration of ethanol in the ethanol aqueous solution is preferably 1 to 80 v / v% (EtOH), more preferably 10 to 60 v / v% (EtOH). ), More preferably 20-40 v / v% (EtOH).
When performing supercritical extraction, it is preferable to use carbon dioxide in a supercritical state.

Further, as an extraction method in the case of solvent extraction, stirring extraction, immersion extraction, shaking extraction, reflux extraction, ultrasonic extraction and the like can be used. Among these, stirring extraction is preferable. The method of stirring and extracting is not particularly limited, but stirring using a stirrer, stirring using a blender, and the like can be performed.

When performing stirring extraction, preferably several minutes to 60 minutes, more preferably 30 minutes to 60 minutes while stirring at a stirring speed of preferably 500 to 20000 rpm, more preferably 1000 to 10000 rpm, still more preferably 6000 to 10000 rpm. Extract. In addition, it is preferable to perform static extraction after stirring. More specifically, it is preferable to homogenize at 6000 to 10000 rpm for 30 to 60 minutes using a homogenizer, and then allow it to stand for 24 hours for extraction. Solid-liquid separation can be performed by allowing the mixture to stand still and performing extraction.

Further, the temperature at the time of solvent extraction may be room temperature or heating conditions. Further, the pressure at the time of solvent extraction may be normal pressure or may be under pressure.
Further, the extract obtained by solvent extraction may be subjected to a treatment such as filtration, if necessary.

Further, the extract obtained by solvent extraction may be dried to form a powder. As a drying method, a known method can be used, but a method such as spray drying, vacuum drying, freeze drying and the like can be used.
As the black raspberry extract, a commercially available product or a ready-made product in the form of a liquid or powder may be used.

The black raspberry extract of the present invention can be blended in foods, beverages and the like. Examples of foods include breads, noodles, confectionery, processed meat products, processed seafood products, frozen foods, jellies, ice creams, dairy products, and various seasonings. In addition to general foods, it can also be added to foods for specified health use, quasi-drugs, health foods, and supplements. Examples of the beverage include soft drinks, milk beverages, alcoholic beverages, tea, tea beverages, coffee, fruit juice beverages, carbonated beverages, mineral waters, fruit / vegetable beverages and the like.
Further, the food or beverage containing the black raspberry extract of the present invention may be in the same form as an orally administered preparation such as tablets, capsules and syrup.

When producing foods and beverages containing the black raspberry extract of the present invention, sweeteners, colorants, preservatives, thickeners, stabilizers are required as long as they do not interfere with the effects of the present invention. , Gelling agents, antioxidants, coloring agents, bleaching agents, emulsifiers, leavening agents, acidulants, brighteners, fragrances and other additives, solvents and oils may be added. One type of these additives may be used alone, or two or more types may be used in combination.

The ratio of the black raspberry extract blended in the food or beverage can be appropriately adjusted according to the purpose of use, but the proportion of the black raspberry extract blended in the food or beverage is preferably 0. It is .0001 to 80% by weight, more preferably 0.003 to 50% by weight, still more preferably 0.005 to 30% by weight.
The black raspberry extract of the present invention is useful for suppressing hair loss.

Hereinafter, the present invention will be described with reference to examples, but the present invention is not limited thereto. Unless otherwise specified, parts and% in this embodiment are based on weight. In the examples and comparative examples, the evaluation of the antioxidant effect by the ORAC value, the growth promoting effect of the dermal papilla cells, the measurement of the m-RNA level of Hsp47 and 17 type collagen in the dermal papilla cells, and the enzyme activity inhibitory effect of 5α reductase. Study was carried out.

About the sample (preparation of the sample)
As samples, three kinds of berry-based samples (Black raspberry (Rosaceae, Rubus) (hereinafter, may be referred to as "BR"), Blueberry (Ericaceae, Vaccinium) (hereinafter, may be referred to as "BB"). And Blabina (registered trademark), which is a powder formulation containing an extract of raspberry (Rubus genus) (hereinafter, may be referred to as "RB") and an extract of black raspberry, was used.

(Preparation of extract)
Black raspberry, blueberry and raspberry extracts were obtained by the following methods.
First, black raspberry, blueberry and raspberry fruits were stored in a frozen state. Next, each fruit was thawed and cut. Weigh black raspberries, blueberries and raspberries (about 100 g), add 5 times the amount of 30 v / v% (EtOH) ethanol aqueous solution (about 500 mL), and use a blender to stir and extract at 8000 rpm for 30 minutes. gone. Further, the mixture was allowed to stand at room temperature for 24 hours for static extraction, and filtered through 150 mesh to obtain ethanol extracts of the above black raspberries, blueberries and raspberries, respectively. Since the ORAC, which will be described later, is measured in an ethanol-dissolved state, the measurement was performed using an ethanol extract.
For Blabina, Blabina was dissolved in 30 v / v% (EtOH) so as to be 9 mg / mL.

(Preparation of extract)
The ethanol components of the ethanol extracts for black raspberries, blueberries and raspberries obtained in the preparation of the above extracts and the ethanol solutions of Blabina were evaporated and removed using an evaporator to obtain an extract. The ethanol extracts of black raspberries, blueberries and raspberries contained a 9 mg / mL extract.

Measurement and evaluation (evaluation of antioxidant activity by ORAC value)
The ORAC values of the three berry-based samples (black raspberry, blueberry and raspberry ethanol extracts) obtained above were measured. The results are shown in Table 1. For Blabina, the ORAC value was not measured.

The ORAC values of all three berry samples were higher than those of general antioxidant foods (see Non-Patent Document 4 for the ORAC values of general antioxidant foods). Among the three berry-based samples, blueberry showed the highest value, but since this is the result of measurement using an ethanol extract, it is not possible to mention the water-soluble component.

Further, since ethanol of about 0.1% or more has an effect on cells, it is not appropriate to use an ethanol extract using a 30 vol% ethanol aqueous solution when performing measurement and evaluation using cells. Therefore, in the following studies using dermal papilla cells, samples obtained by evaporating and removing the ethanol component from the ethanol extract were used (see "Preparation of extract" above).

(Effect of promoting proliferation of dermal papilla cells)
As the dermal papilla cells used in the experiment, human dermal papilla cells were used. For human hair papilla cells, Human Follicle Dermal Papilla Cells; C-12071 (purchased from PromoCell) was used, and a special medium (Follicle Dermal Papilla Cell Growth Medium Supplement Pack; purchased from C-39620, PromoCell) was used. Then, the cells were cultured under the conditions of 37 ° C. and 5% CO _2. Immediately before the experiment, cells were collected with a 0.25% Trypsin / EDTA solution, and the number of cells was calculated by Countess (Invitrogen) after a trypan blue staining treatment. Then, the seeds were sown on a 24-well plate or a 96-well plate, precultured for 24 hours, and then used for the experiment.

In addition, the three berry-based (black raspberry, blueberry and raspberry) extracts and the ethanol-removed extract of Blabina obtained in the preparation of the above extracts were each dissolved in ultrapure water to a concentration of about 11.5 mg / mL. , Specimens were obtained. Each sample was then serially diluted (100,000-fold to 10-fold diluted) and added to each dermal papilla cell.

Then, each of the above four kinds of samples was serially diluted to the dermal papilla cells, and the number of cells after 48 hours of culture was calculated by Countess (Invitrogen). The results are shown in FIG. In all four species, the effect of promoting the proliferation of dermal papilla cells in a concentration-dependent manner was observed to be about 3 to 20% higher than that of Control.

(Measurement of m-RNA levels of Hsp47 and type 17 collagen in dermal papilla cells)
As the dermal papilla cells used in the experiment, the same human dermal papilla cells used for the above-mentioned dermal papilla cell proliferation promoting effect were used, and the treatment before the experiment was also performed with the dermal papilla cell proliferation promoting effect. It was done in the same way as the processing.

The three berry-based (black raspberry, blueberry and raspberry) extracts and the ethanol-removed extract of Blabina obtained in the preparation of the above extracts were each dissolved in ultrapure water to a concentration of about 11.5 mg / mL as a sample. Got Each sample was then serially diluted (100,000-fold to 10-fold diluted) and added to each dermal papilla cell. Then, the mRNA levels of Hsp47 and type 17 collagen at 1 hour after the sample was added to the dermal papilla cells were measured by a quantitative PCR method.

The specific procedure of the quantitative PCR method was as follows. Total RNA was extracted from the dermal papilla cells to which the sample was added using Trizol reagent (ambion). The extracted RNA was reverse transcribed into cDNA according to the method of PrimeScript RT Master Mix (Takara) and amplified by SYBR Premix EX Taq II (Takara). As the PCR reaction solution, 50μL (25μL SYBR Green Mix ( 2x), 1μL cDNA, 2μL primer pair mix (5 pmol/μL each primer), 22μL H 2 O) using the PCR reaction liquid, the reaction to 95 ° C. A cycle of 30 seconds was performed for one cycle, and a cycle of 5 seconds at 95 ° C. and a cycle of 30 seconds at 60 ° C. was performed under the conditions of 50 cycles. Relative quantification was performed by the comparative Ct method (ΔΔCt method). The Primers of various targets and housekeeping genes (GAPDH) used for RT-PCR are shown in Table 2.

In addition, FIG. 2 shows the results 1 hour after each sample was added to the dermal papilla cells. A high value of 2 or more as the DeltaDeltaCt value was judged to be a significant difference. Regarding the result of the dilution ratio of BR at 10 times, it is considered that the DeltaDeltaCt value decreased due to the variation or too darkness.

From the results shown in FIG. 2, it was found that all four types of specimens tended to induce the expression of type 17 collagen in a concentration-dependent manner in conjunction with Hsp47, but for Blabina and BR, the inducing ability was higher than that of the other two types. It turned out to show.

(5α-reductase inhibitory activity)
As the dermal papilla cells used in the experiment, the same human dermal papilla cells used for the above-mentioned dermal papilla cell proliferation promoting effect were used, and the treatment before the experiment was also performed with the dermal papilla cell proliferation promoting effect. It was done in the same way as the processing.

The three berry-based (black raspberry, blueberry and raspberry) extracts and the ethanol-removed extract of Blabina obtained in the preparation of the above extracts were each dissolved in ultrapure water to a concentration of about 11.5 mg / mL as a sample. Got Each sample was then serially diluted (100,000-fold to 10-fold diluted) and added to each dermal papilla cell.

When 5α-reductase, which is a reductase that metabolizes testosterone to dihydrotestosterone, is inhibited, dihydrotestosterone production is suppressed as a result. Therefore, the amount of dihydrotestosterone (DHT) produced was measured by the ELISA method. The method was based on the DHT ELISA Kit (E18-220; IMMUNOSPEC). The results are shown in FIG. In FIG. 3, 20 pg / mL (BR, 10-fold diluted addition, 1 hour value), which is the maximum inhibitory value for the untreated DHT value of 140 pg / mL, was set as a 100% inhibitory value and displayed as% of inhibition.
From FIG. 3, it was confirmed that BR showed the strongest suppression and Blabina was similar to it, and the effect was continuously observed from 1 to 12 hours in a concentration-dependent manner.

Claims (2)

An expression promoter for type 17 collagen containing black raspberry extract as an active ingredient. The method for producing an expression-promoting agent for type 17 collagen according to claim 1.
The freezing process to freeze the fruits of black raspberries,
The thawing step of thawing the black raspberry fruit and
A stirring step of adding a solvent to the black raspberry fruit thawed in the thawing step and stirring the mixture, and a stirring step.
An extraction step in which the black raspberry fruit and the solvent stirred by the stirring step are allowed to stand for extraction.
A method for producing a type 17 collagen expression promoter, which comprises a solvent removing step of evaporating the solvent.

Images