JP6618961B2 - Activity inhibitor, whitening agent containing activity inhibitor, method for producing activity inhibitor, and method for producing food and drink - Google Patents

Description

  The present invention relates to a black raspberry extract, a whitening agent and food and drink containing the black raspberry extract, a method for producing the black raspberry extract, and a whitening method using the black raspberry extract.

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

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

  Therefore, with the activation of superoxide dismutase (SOD) in the body, antioxidants in foods have attracted attention, and a method for measuring antioxidant power (ORAC: Oxygen Radical Absorbance Capacity) developed by the US Department of Agriculture (USDA) has been established. It is widespread (see Non-Patent Document 3, for example). In addition, twelve berry foods are present in foods having a high ORAC value, that is, a food best 100 having a high antioxidant power (see, for example, Non-Patent Document 4).

  The synthesis of melanin pigments that cause pigmentation in the skin is facilitated by various exogenous and endogenous stimuli. For example, when exposed to ultraviolet rays, stress, air pollution, etc. in sunlight, active oxygen is generated on the surface of the skin, and the melanocytes are activated by the stimulation, thereby increasing the expression of tyrosinase. The tyrosinase thus expressed metabolizes tyrosine, and melanin pigment is produced through several stages. It has been reported that the production of melanin pigment is suppressed by capturing this active oxygen with an antioxidant (see, for example, Non-Patent Document 5). Moreover, if the activity of tyrosinase can be inhibited in melanocytes, the skin whitening effect can also be expected.

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 Values (High Antioxidant Action)” [online] (searched June 30, 2017) http://www.anteaoxidant.com/orac_top100.html Choi MY1, Song HS, Hur HS, Sim SS.Whitening activity of luteolin related to the inhibition of cAMP pathway in alpha-MSH-stimulated B16 melanoma cells. Arch Pharm Res. 2008 Sep; 31 (9): 1166-71.

  An object of the present invention is to use a berry black raspberry extract having an action of suppressing the production of melanin pigment, a whitening agent and food and drink containing the black raspberry extract, a method for producing the black raspberry extract, and a black raspberry extract. Is to provide a whitening method.

  The inventors of the present invention have focused on berry foods as foods having antioxidant power, and examined the correlation between the berry foods and the action of suppressing the production of the melanin pigment. As a result, the extract of black raspberry Has achieved the above object, and the present invention has been completed.

That is, according to the present invention,
(1) Black raspberry extract having an inhibitory effect on the activity of tyrosinase enzyme in melanocytes,
(2) A whitening agent comprising the black raspberry extract according to (1),
(3) Food and drink containing the black raspberry extract according to (1),
(4) A method for producing a black raspberry extract according to (1), wherein a freezing step of freezing black raspberry fruit, and a thawing step of thawing the black raspberry fruit,
A black raspberry comprising a stirring step of adding a solvent to the black raspberry fruit thawed in the thawing step and stirring, and an extraction step of performing extraction by allowing the black raspberry fruit and solvent stirred in the stirring step to stand A method for producing the extract,
(5) A whitening method using the black raspberry extract according to (1) is provided.

  According to the present invention, a black raspberry extract, a whitening agent and food and drink containing the black raspberry extract, a method for producing the black raspberry extract, and a whitening method using the black raspberry extract can be provided.

It is a graph which shows the examination result of the activity inhibitory effect of the tyrosinase enzyme in a melanocyte.

  Hereinafter, the black raspberry extract of the present invention will be described. The black raspberry extract of the present invention has an inhibitory effect on tyrosinase enzyme activity in melanocytes.

Black raspberry, which is a raw material for black raspberry extract, belongs to the genus Raspberry. In addition, as the black raspberry extract of the present invention, an extract mainly from black raspberry fruit is preferably used.
The method for extracting the black raspberry extract from the fruit of black raspberry is not particularly limited, and solvent extraction, supercritical extraction, and the like can be used.

  It does not specifically limit as an extraction solvent used when performing solvent extraction, Water, an organic solvent, and these mixed solvents 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. These organic solvents may be used alone or in combination of two or more. Further, an aqueous solvent such as water and an organic solvent may be mixed and used.

Among these, ethanol and an ethanol aqueous solution in which ethanol and water are mixed are preferable. 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 to 40 v / v% (EtOH).
In addition, when performing supercritical extraction, it is preferable to use the carbon dioxide made into the supercritical state.

  In addition, as the extraction method when performing solvent extraction, stirring extraction, immersion extraction, shaking extraction, reflux extraction, ultrasonic extraction, or the like can be used. Among these, stirring extraction is preferable. Although the method of stirring extraction is not specifically limited, stirring using a stirring bar, stirring using a blender, etc. can be performed.

  Moreover, when performing stirring extraction, Preferably it is 500 minutes to 20000 rpm, More preferably, it is 1000 to 10000 rpm, More preferably, it is a few minutes to 60 minutes, More preferably, it is 30 minutes or more, stirring at the stirring speed of 6000 to 10000 rpm. Extract for 60 minutes. In addition, it is preferable to stand still and to perform stationary extraction after stirring. More specifically, it is preferable to perform homogenization using a homogenizer at 6000 to 10000 rpm for 30 to 60 minutes, and then leave it for 24 hours to perform extraction. In addition, solid-liquid separation can be performed by leaving still and extracting.

Moreover, the temperature in the case of performing solvent extraction may be room temperature or under heating conditions. Further, the pressure for solvent extraction may be normal pressure or under pressure.
Further, the extract obtained by solvent extraction may be subjected to a treatment such as filtration as necessary.

Alternatively, the extract obtained by solvent extraction may be dried to form a powder. As a drying method, a known method can be used, and techniques such as spray drying, vacuum drying, freeze drying, and the like can be used.
In addition, as a black raspberry extract, you may use the commercial item made into liquid or powder form, or a ready-made product.

The black raspberry extract of this invention can be mix | blended with food, a drink, etc. 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 drink include soft drinks, milk drinks, alcoholic beverages, tea, tea drinks, coffee, fruit juice drinks, carbonated drinks, mineral waters, fruit / vegetable drinks, and the like.
In addition, foods and beverages containing the black raspberry extract of the present invention may be in the same form as oral administration preparations such as tablets, capsules and syrups.

  In addition, when producing foods and beverages containing the black raspberry extract of the present invention, sweeteners, coloring agents, preservatives, thickeners, stable, as long as they do not interfere with the effects of the present invention Agents, gelling agents, antioxidants, color formers, bleaching agents, emulsifiers, swelling agents, sour agents, brighteners, fragrances, additives, solvents, and oils may be added. These additives may be used alone or in combination of two or more.

The proportion of black raspberry extract blended in the food or beverage can be adjusted as appropriate according to the intended use, but the proportion of black raspberry extract blended in the food or beverage is preferably 0. 0.0001 to 80% by weight, more preferably 0.003 to 50% by weight, and still more preferably 0.005 to 30% by weight.
The black raspberry extract of the present invention is useful for obtaining a skin whitening effect.

  Hereinafter, the present invention will be described with reference to examples, but the present invention is not limited thereto. In the examples, parts and% are based on weight unless otherwise specified. In Examples and Comparative Examples, the antioxidant activity was evaluated by ORAC values, and the activity inhibitory effect of tyrosinase enzyme in melanocytes was examined.

Sample (preparation of sample)
As samples, three types of berry samples (black raspberry (Rosaceae genus Strawberry) (hereinafter sometimes referred to as “BR”)), blueberries (Azalea family genus Genus) (hereinafter sometimes referred to as “BB”). And Blabina (registered trademark), which is a powder formulation in which an extract of raspberry (Rosaceae genus Raspberry) (hereinafter sometimes referred to as “RB”) and a black raspberry extract were blended.

(Preparation of extract)
Black raspberry, blueberry and raspberry extracts were obtained by the following procedure.
First, black raspberry, blueberry and raspberry fruits were each stored in a frozen state. Next, each fruit was thawed and cut. Black raspberry, blueberry and raspberry were weighed (about 100 g), 5 times the volume of 30 v / v% (EtOH) ethanol aqueous solution (approximately 500 mL) was added, and the mixture was extracted with stirring at 8000 rpm for 30 minutes using a blender. went. Furthermore, it left still at room temperature for 24 hours, static extraction was performed, it filtered with 150 mesh, and the ethanol extract of the said black raspberry, blueberry, and raspberry was obtained, respectively. Since ORAC measurement described later is performed in an ethanol-dissolved state, the measurement was performed using an ethanol extract.
In addition, about Blabina, Blabina was dissolved in 30 v / v% (EtOH) so that it might become 9 mg / mL.

(Preparation of extract)
The ethanol components were evaporated from the black raspberry, the blueberry and the raspberry obtained in the preparation of the extract, and the ethanol solution of Blabina, respectively, using an evaporator to remove the ethanol component. The ethanol extract of black raspberry, blueberry and raspberry contained 9 mg / mL extract (extract).

Measurement and evaluation ( Evaluation of antioxidant effect by ORAC value)
The ORAC values of the three berry specimens obtained above (black raspberry, blueberry and raspberry ethanol extract) were measured. The results are shown in Table 1. For Blabina, the ORAC value was not measured.

  Compared with a general antioxidant food, the ORAC values of three kinds of berry specimens all showed high values (for the ORAC value of a general antioxidant food, see Non-Patent Document 4 above). Among the three berry samples, blueberry showed the highest value, but since this is the result of measurement using an ethanol extract, it cannot mention water-soluble components.

  In addition, when the ethanol concentration is about 0.1% or more, there is an influence on the cells. Therefore, when performing measurement and evaluation using cells, it is not appropriate to use an ethanol extract using a 30 vol% ethanol aqueous solution. . Therefore, in the examination of the activity inhibition effect of tyrosinase enzyme in the following melanocytes, specimens obtained by evaporating and removing the ethanol component from the ethanol extract using an evaporator were used (see “Preparation of Extract” above).

(Inhibition of tyrosinase enzyme activity in melanocytes)
Melanocytes were used as cells used in the experiment. As melanocytes, Normal Human Epidermal Melanocytes, juvenile foreskin (C-12400, purchased from PromoCell), and 37 ° C, 5% using a special medium (Melanocyte Growth Medium Supplement Pack; C-39410, purchased from PromoCell) Culturing was performed under the condition of% 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 trypan blue staining. Thereafter, the cells were seeded in a 24-well plate or a 96-well plate, pre-cultured for 24 hours, and used for the experiment.

  In addition, the three berry (black raspberry, blueberry and raspberry) extracts obtained in the preparation of the extract and the ethanol-extracted extract of Blabina were each dissolved in ultrapure water to make about 11.5 mg / mL. A sample was obtained. Next, each specimen was serially diluted (diluted from 100,000 times to 10 times) and added to melanocytes.

  Next, 33.3 μL of 2.5 mM Tyrosine aqueous solution was added to 60 μL of 0.1 M phosphate buffer (pH 6.5) (25 ° C.). Thereto, 3.3 μL of an L-tyrosine substrate solution was added and stirred, and 3.3 μL of an aqueous tyrosinase solution (about 1300 units / mL) was added to start the enzyme reaction. The inside of the plate reader was set to 25 ° C., and the enzyme reaction was performed.

  The enzyme activity was evaluated using the absorbance (475 nm) of DOPA chromium synthesized by tyrosinase as an index. As positive control, vitamin C 20 mg / mL was used (for example, Non-Patent Document 5 and Matsuki M, Watanabe T, Ogasawara A, Mikami T, Matsumoto T. Inhibitory mechanism of melanin synthesis by glutathione. [Article in Japanese ] Yakugaku Zasshi. 2008 Aug; 128 (8): 1203-7 12.).

The results are shown in FIG. 50% Inhibition (IC50) of DOPA chromium was approximately 100000 times diluted with positive control (vitamin-C), 50000 times diluted with Blabina, 10000 times diluted with BR, 200 times diluted with BB, and 100 times diluted with RB. . This shows that Blabina and BR have inhibitory activity several hundred times or more compared with BB and RB.

Claims (5)

  An inhibitor of tyrosinase enzyme activity in melanocytes containing black raspberry extract as an active ingredient.   The activity inhibitor according to claim 1, wherein the black raspberry extract is an extract from black raspberry fruit.   A whitening agent comprising the activity inhibitor according to claim 1 or 2. A method for producing an activity inhibitor according to claim 1 or 2,
A freezing process of freezing black raspberry fruits;
A thawing step of thawing the fruit of the black raspberry;
A stirring step of adding a solvent to the fruit of the black raspberry thawed in the thawing step and stirring;
A method for producing an activity inhibitor comprising the extraction step of performing extraction by allowing the black raspberry fruit and solvent stirred in the stirring step to stand. The manufacturing method of the food / beverage including the process of mix | blending the activity inhibitor obtained by the manufacturing method of Claim 4 with a foodstuff or a drink.

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