JP7662301B2 - Methods, compositions and preparations using young apple extract - Google Patents

Description

The present invention relates to methods, compositions, and agents using young apple extract.

Apples are a highly nutritious food that are enjoyed around the world. In apple cultivation, in order to harvest fruit with high commercial value, the fruit is thinned out, and many of the fruits are picked while still immature. Immature fruits are not suitable for consumption due to their strong sour and astringent taste, and are sometimes discarded. From the perspective of effective resource utilization, attempts are being made to utilize young apples industrially (see Patent Document 1).

JP 2013-209302 A

It is believed that young apples have great potential for further industrial use.
An object of the present invention is to provide a new use of young apple extract.

The gist of the present invention is as follows.
The methods of the present invention are a method for restoring bound water and/or free water in epidermal and/or stratum corneum keratin, a method for inhibiting changes in the α-helical structure of epidermal and/or stratum corneum keratin, and a method for restoring changes in the α-helical structure of epidermal and/or stratum corneum keratin, which use a composition containing young apple extract.
The composition of the present invention is a skin composition containing the young apple extract used in the method of the present invention.
The agent of the present invention is an agent for inhibiting changes in the α-helical structure of epidermal and/or stratum corneum keratin, an agent for recovering changes in the α-helical structure of epidermal and/or stratum corneum keratin, and an agent for improving the moisture retention ability of the epidermis and/or stratum corneum, which contains an extract from young apple as an active ingredient.

The present invention provides a new use for young apple extract.

1 is a chart of θobs (mdeg) versus wavelength (nm) when CD spectrum measurement was performed on a control A sample (no young apple extract added) in an experiment for CD spectrum measurement. The crosses and the curve connecting them show the chart before heating, and the black circles and the curve connecting them show the chart after heating and cooling. 2 is a chart of θobs (mdeg) versus wavelength (nm) when CD spectrum measurement was performed on the Example A sample (with young apple extract added) in a CD spectrum measurement experiment. The cross marks and the curve connecting them show the chart before heating, and the black circles and the curve connecting them show the chart after heating and cooling. FIG. 3 is a diagram showing the change in θobs (θ222,obs) (mdeg) at a wavelength of 222 nm in the charts of the control A sample and the example A sample shown in FIGS. 1 and 2 before and after heating and cooling. 4 is a chart of θ222, obs (mdeg) versus temperature (° C.) when CD spectra were measured for a Control A sample (no young apple extract added) and an Example A sample (young apple extract added) in an experiment to measure CD spectra. The crosses and the curve connecting them show the chart for the Control A sample, and the black circles and the curve connecting them show the chart for the Example A sample. FIG. 5 is a graph showing peak areas (arbitrary units) obtained by performing microscopic IR measurement on an untreated sample, a Control B sample (no young apple extract added), and an Example B sample (young apple extract added) in an experiment using microscopic IR measurement.

The following describes in detail the form for carrying out the present invention (hereinafter referred to as the "present embodiment") with reference to the drawings. However, the present invention is not limited to the following description, and can be carried out in various modifications within the scope of the gist of the present invention.

(method)
The method of the present embodiment is a method using a composition containing a young apple extract, and more specifically, may be a method for restoring bound water and/or free water in epidermal and/or stratum corneum keratin, a method for inhibiting changes in the α-helical structure of epidermal and/or stratum corneum keratin, or a method for restoring changes in the α-helical structure of epidermal and/or stratum corneum keratin. The method of the present embodiment is used in a manner other than medical practice.
The composition used in the method of the present embodiment may be the composition of the present embodiment described below.

The young apple extract used in the method of this embodiment may be an extract of young apples of various varieties (scientific name: Malus pumila), which are plants of the genus Malus and the family Rosaceae.

Here, the "variety" of apples may be any variety, and may be an early, mid-season, or late variety. Examples of varieties include Fuji, Tsugaru, Orin, Jonagold, Hokuto, Mutsu, Chiaki, Delicious, Kogyoku, Sansa, Akibagare, Youkou, Shinano Sweet, Sekaiichi, Asahi, Indo, Granny Smith, Kunimitsu, Star King, Tsugaru, and Hack9.

The "young fruit" of an apple refers to an immature fruit at any stage after fruit set and before ripening. For example, apple fruit thinning is usually carried out in the period from May to June, approximately 20 to 45 days after fruit set, and the young fruit with a diameter of approximately 2 to 5 cm that is picked can be included in the "young fruit" of this embodiment.
In this case, the "young fruit" may include all or a part of the immature fruit, specifically, the peel, pulp, puree, juice, and seeds.

The term "extract" may refer to an extract obtained by removing solid matter from an extract obtained by soaking young apples in an extraction solvent, or may refer to an extract obtained by removing solid matter from the juice contained in young apples. The "extract" may be an extract obtained by concentrating or drying the extract or juice from which the solid matter has been removed, or may be a liquid product obtained by drying to a solid, or may be dried by a method such as spray drying to obtain a powder.

An example of a method for extracting from young apples will be described below.
The young apples to be subjected to extraction may be fresh or processed. They may be used immediately after thinning, or may be used after some time has passed since thinning, as long as they are not decayed. Furthermore, the young apples may be pretreated, such as cut and crushed, before being subjected to extraction, so that the extraction can be carried out efficiently. The pretreatment is not particularly limited, but low-temperature crushing or freeze-crushing is preferred.
The extraction solvent is not particularly limited, but examples thereof include water, lower alcohols (methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, etc.), polyhydric alcohols (glycerin, propylene glycol, 1,3-butylene glycol (BG), etc.), ketones (acetone, methyl ketone, etc.), ethers (diethyl ether, tetrahydrofuran, etc.), esters such as ethyl acetate; hexane, etc., and among these, water, lower alcohols, and polyhydric alcohols are preferred in terms of usability and safety. These may be used alone or in combination of two or more.
The conditions for extraction can be appropriately designed by a person skilled in the art; for example, young apples may be immersed in an extraction solvent at room temperature (1 to 30° C.) or heated conditions (50 to 80° C.) for a period of from 1 hour to several months, with low-temperature conditions (1 to 15° C.) for 1 to 10 days being particularly preferred.
The mass ratio of young apples to extraction solvent may be appropriately designed, and for example, the lower limit of the amount of extraction solvent is not particularly limited as long as it is an amount that allows the raw material to be sufficiently immersed per 100 parts by mass of young apples, and is preferably 50 parts by mass or more, more preferably 100 parts by mass or more, and more preferably 200 parts by mass or more. The upper limit of the amount of extraction solvent can also be determined from an economical viewpoint, and can be, for example, 10,000 parts by mass or less, preferably 5,000 parts by mass or less, and more preferably 1,000 parts by mass or less per 100 parts by mass of raw material.
When water is used as the extraction solvent, the extraction may be carried out by steam distillation. Solid matter may be removed by a conventional method.
The solids concentration of the resulting young apple extract after removal of the solvent may be 0.1 to 10% by mass.

A method for utilizing the juice of young apples will be described below by way of example.
The fruit juice may be obtained by crushing washed young apples as the raw material at a pH of 3.2 to 4.6, preferably 3.4 to 4.3 (pH measurement temperature: 20°C), treating the resulting crushed material with pectinase for clarification, and removing solids.

Compositions containing young apple extracts favorably maintain the bound water and/or free water contained in the epidermis and/or stratum corneum. The inventors have found that young apple extracts have the effect of restoring the bound water and/or free water in epidermal and/or stratum corneum keratin. Young apple extracts can also suppress the decrease in bound water and/or free water in epidermal and/or stratum corneum keratin, restore the total amount of water in epidermal and/or stratum corneum keratin, and suppress the decrease in the total amount of water.
The mechanism by which the above-mentioned effect on bound water and/or free water is exerted is believed to be as follows.
In animals, including humans and non-humans, the epidermis and stratum corneum of the skin contain keratin, a protein that constitutes the cytoskeleton. The two-dimensional structure of keratin is mainly an α-helical structure, and maintaining the amount of α-helical structure is essential for maintaining the cytoskeleton of the epidermis and/or stratum corneum in an optimal state. The inventors have obtained results suggesting that young apple extract has the effect of stabilizing the hydration structure of keratin, making it easier to maintain the α-helical structure. More specifically, as described in the Examples below, young apple extract has the effect of inhibiting changes in the α-helical structure of keratin derived from human epidermis, and also has the effect of recovering changes in the α-helical structure.
The above findings of the inventors can be said to be new discoveries made through the present invention.

(Composition)
The composition of the present embodiment contains an extract of young apple.
The young apple extract used in the composition of this embodiment may be the same as the young apple extract used in the method of this embodiment.

The composition of this embodiment may contain additives other than the young apple extract as necessary.

Such additives include cell activators, antioxidants, moisturizers, UV protection agents, solvents (water, alcohols, etc.), oils, surfactants, thickeners, powders, chelating agents, pH adjusters, emulsifiers, stabilizers, colorants, gloss agents, flavorings, odorants, excipients, binders, disintegrants, lubricants, diluents, osmotic pressure adjusters, fragrances, etc.

The composition of the present embodiment may be prepared in various forms, for example, a liquid, a milky lotion, a cream, a solid, a gel, a paste, or the like.
The compositions may also be prepared in various formulations, such as oil-based systems, water-in-oil emulsion systems, and oil-in-water emulsion systems.
Specifically, the composition can be various cosmetics, such as skin care cosmetics, such as lotion, milky lotion, cream, beauty serum, cosmetic oil, lip balm, hand cream, facial cleanser, and cleansing agent; makeup cosmetics, such as foundation, makeup base, blusher, eye shadow, mascara, eyeliner, eyebrow, overcoat agent, lipstick, and lip gloss; cosmetics for the scalp or hair, such as hair tonic, hair cream, shampoo, rinse, conditioner, and hair styling products; and massage cosmetics.

The composition of this embodiment may be used as a cosmetic, a quasi-drug, or a medicine.

The method for producing the composition of the present embodiment is not particularly limited, and may be a method commonly used in the art.
The composition of this embodiment is preferably used in the method of this embodiment.

(Agent)
The agent of the present embodiment contains young apple extract as an active ingredient, and examples of the agent include an agent for inhibiting changes in the α-helical structure of epidermal and/or stratum corneum keratin, an agent for recovering changes in the α-helical structure of epidermal and/or stratum corneum keratin, an agent for improving the moisture retention ability of the epidermis and/or stratum corneum, an agent for recovering damage to the epidermis and/or stratum corneum, and an agent for inhibiting damage to the epidermis and/or stratum corneum.
As mentioned above, and as described in the Examples below, young apple extract has the effect of recovering and suppressing changes in the α-helix structure of keratin derived from human epidermis, and is therefore expected to have the effect of recovering from and/or suppressing stimuli and damage caused by heat, oxidation, ultraviolet rays, etc. by inhibiting and recovering from changes in the α-helix structure of epidermal and/or stratum corneum keratin.

The composition of this embodiment may be used as the agent of this embodiment.

In this embodiment, young apple fruit extract can also be used to produce the above-mentioned agents of this embodiment.

The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following examples.

The materials used in the examples and comparative examples described below are described below.
[Young apple extract]
A 13-fold amount of 20% (w/w) 1,3-butylene glycol aqueous solution was added to young (immature) fruits of Hakkuna variety grown in Hokkaido within 20 mm in diameter and within one month after flowering, and extraction was carried out at low temperature (1 to 15° C.) for 2 days. The solid content concentration after removing the solvent was 0.3% by mass.

<1. CD spectrum measurement>
<Measurement method>
Measurement equipment: a circular dichroism spectrometer J-720 (JASCO Corporation) and a Peltier-type thermostatic cuvette holder PTC-423L (JASCO Corporation) were used, and a quartz cell for a spectrophotometer with an optical path length of 1 mm was used. The measurement conditions were as follows:
Measurement cell: quartz 1 mm cell Measurement wavelength: 190-250 nm
Sensitivity: Standard Temperature range: 20.0 to 98.0°C
Heating rate: 1.0 ° C. / min
Measurement sample: Human epidermis-derived keratin (manufactured by SIGMA-ALDRICH) was thoroughly dialyzed against _a 5 mM _Na2PO4 / _NaHPO4 buffer, 50 mM NaCl, pH 7.4 buffer, and then diluted with the same buffer to a measurable concentration. The control A sample was simply diluted with the buffer, whereas the example A sample containing young apple extract had the components added to the solution at 1% by volume.
Measurement and analysis conditions: Each sample was measured according to the procedure below. The regeneration rate of the α-helix structure after heating and cooling was calculated by comparing (1) with (4), and the thermal denaturation temperature was obtained by (2).
(1) CD spectrum measurement is performed at wavelengths of 190 to 250 nm at 20.0° C.
(2) The wavelength was fixed at 222 nm, and the same sample was heated from 20.0 to 98.0°C at a rate of 1.0°C/min. (3) After the measurement at 98.0°C was completed, the sample was left to stand for 10 minutes and then cooled to 20.0°C using a Peltier temperature controller.
(4) After standing at 20.0° C. for 10 minutes, the CD spectrum is measured again at 20.0° C. at a wavelength of 190 to 250 nm.

<Measurement results>
1 is a chart of θobs (mdeg) versus wavelength (nm) when CD spectrum measurement was performed on a control A sample (no young apple extract added) in an experiment for CD spectrum measurement. The crosses and the curve connecting them show the chart before heating, and the black circles and the curve connecting them show the chart after heating and cooling.

Figure 2 is a chart of θobs (mdeg) versus wavelength (nm) when CD spectrum measurement was performed on the Example A sample (with young apple extract added) in a CD spectrum measurement experiment. The cross marks and the curve connecting them show the chart before heating, and the black circles and the curve connecting them show the chart after heating and cooling.

Figure 3 shows the change in θobs (θ222, obs) (mdeg) at a wavelength of 222 nm before heating and after heating and cooling in the charts of the control A sample and the example A sample shown in Figures 1 and 2.

Keratin proteins in their native state contain a large amount of α-helical structures, and the content of α-helical structures in CD measurements can be determined from the peak intensity at a wavelength of 222 nm.

As shown in Figure 2, when comparing the peak intensity at 222 nm at the completion of heating (97.6-98.0°C) under measurement and analysis conditions (2) and during measurement under (4), in Control A, 84.7% of the peak intensity that decreased during heating was restored by cooling after heating, whereas in Example A, the recovery rate increased to 94.5% by adding the young apple extract prepared above (containing 1% by volume of the original solution). In other words, it can be said that approximately 64% of the α-helix structure that does not regenerate by simple heating and cooling alone was regenerated by adding the young apple extract.

As shown in FIG. 3, the amount of change in θ222,obs in the Example A sample was about 34% of the amount of change in θ222,obs in the Control A sample.
These results indicate that heating in the presence of young apple extract has the effect of inhibiting structural changes in human epidermis-derived keratin, and/or that cooling in the presence of young apple extract has the effect of restoring the structure of human epidermis-derived keratin.

4 is a chart of θ222, obs (mdeg) versus temperature (° C.) when CD spectra were measured for a Control A sample (no young apple extract added) and an Example A sample (young apple extract added) in an experiment to measure CD spectra. The crosses and the curve connecting them show the chart for the Control A sample, and the black circles and the curve connecting them show the chart for the Example A sample.
4 shows thermal denaturation curves obtained by measuring samples while heating them from 20.0 to 98.0° C. with a fixed measurement wavelength of 222 nm. The thermal denaturation temperatures (° C.) were calculated from the inflection points of the thermal denaturation curves of Control A and Example A sample to which young apple extract was added, and were 39.28° C. for Control A and 48.92° C. for Example A sample to which young apple extract was added. This indicates that the addition of young apple extract improves the thermodynamic stability of the α-helix structure of keratin protein.

<2. Microscopic IR Measurement>
<Measurement method>
Measurement device: SPring-8 BL43IR, humidity control device for creating an environment with a humidity of 30% RH was used.
Measurement sample: the stratum corneum was peeled off from commercially available human skin, and a 10 mm x 10 mm square human skin-derived stratum corneum sheet was treated under each of the conditions shown below.
(1) Untreated sample: No particular treatment was performed.
(2) (Control B sample): Damage treatment was performed by immersing in _a 3% _H2O2 aqueous solution (pH 10.4) for 20 minutes, followed by washing with water seven times, and then drying in a 25°C environment for 24 hours.
(3) (Example B sample): After the above-mentioned damage treatment, the sample was immersed in an aqueous solution of young apple extract (containing 1% by mass of the original solution) for 3 hours, and then dried in an environment of 25° C. for 24 hours.
Measurement and analysis conditions: Aperture size was approximately 10 μm x 10 μm, with 10 μm steps (vertical and horizontal intervals between each aperture were both 10 μm), and measurements were taken at 20 locations with X = 5 and Y = 4, with 512 cumulative measurements. Baseline correction was performed on the measurement results, and the peak areas of total water (wavenumber 3620-3100 cm ^-1 ), bound water (wavenumber 3420-3245 cm ^-1 ), and free water (wavenumber 3620-3420 cm ^-1 ) normalized by the peak area of the amide I mode derived from the amide bond of the protein (wavenumber 1806-1600 cm ^-1 ) were calculated.

<Measurement results>
FIG. 5 is a graph showing peak areas (arbitrary units) obtained by performing microscopic IR measurement on an untreated sample, a Control B sample (no young apple extract added), and an Example B sample (young apple extract added) in an experiment using microscopic IR measurement.

Table 1 shows the numerical data for each peak area shown in Figure 5.

Water peaks (peaks derived from OH groups) in IR measurements are roughly classified into two types: bound water, which is weakly bound to the structure and does not easily increase or decrease due to external influences such as humidity, and free water, which easily increases or decreases due to the influence of humidity, etc.
In the Control B sample, which was subjected to oxidative damage treatment, both the bound water and free water were reduced compared to the untreated sample, but in the Example B sample to which young apple extract was added, both the bound water and free water were shown to recover to levels close to those of the untreated sample. In addition, the total amount of water in the Example B sample was also restored compared to the Control B sample. In other words, it was shown that the young apple extract essentially improves the moisture content in the stratum corneum, which is reduced by oxidative damage.

The present invention provides a new use for young apple extract.

Claims (2)

A method for restoring bound water lost in oxidatively damaged epidermis and/or stratum corneum keratin to the state before oxidative damage, using a composition containing young apple extract (excluding medical procedures and cases for moisturizing and maintaining moisture ). A moisture retention function improver containing young apple extract as the active ingredient, which restores the bound water lost in oxidatively damaged epidermis and/or stratum corneum keratin to the state before oxidative damage (excluding cases of moisturizing or retaining moisture) .

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