JP7408269B2 - Melanosome degradation promoter - Google Patents

Description

The present technology relates to a melanosome decomposition promoter.

Melanin is a brown to black pigment produced by enzymes from the amino acid tyrosine.
Melanin is widely found in the animal, plant, and fungal kingdoms, and in animals, it is mostly produced by melanocytes (pigment cells) located in the basal layer of the lowest layer of the epidermis of the skin and the matrix of hair.
Melanin is one of the factors that determines skin color in humans, and is said to play an important role in protecting the body from the harmful effects of ultraviolet rays. However, excessive production of melanin is thought to result in dark skin, and phenomena such as age spots, freckles, and dullness caused by its uneven distribution are sometimes considered to be cosmetic defects or skin aging.

The following processes (a) to (d) have been elucidated as melanin production mechanisms. (a) Melanocyte activating factors are produced from keratinocytes by ultraviolet rays and stress, and these factors stimulate melanocytes present in the basal layer of the skin. (b) Tyrosinase, a melanin-producing enzyme, is produced in excess in activated melanocytes, and melanin is produced by an oxidation reaction using tyrosine as a starting material in the intracellular organelle melanosome. (c) This melanosome moves to the end of the melanocyte dendrite and is delivered to keratinocytes (keratinocytes) and hair matrix cells. (d) Melanosomes are transported into these cells and accumulate, eventually changing body color and hair color to black. Areas of skin that have locally turned black are called spots and freckles.
In the case of the skin in particular, as metabolic capacity declines due to aging, the metabolism called skin turnover also declines, making it difficult for melanosomes containing melanin to be excreted from the body, resulting in an increase in age spots and freckles.

Conventionally, many searches have been made for tyrosinase inhibitors to suppress the production of melanin as a measure against age spots and freckles.
For example, Patent Document 1 discloses that an ethanol extract of Secang has a tyrosinase inhibitory effect. Further, Patent Document 2 discloses that an ethanol extract of horseradish leaves has a tyrosinase inhibitory effect.
However, tyrosinase inhibitors suppress melanin before it is produced, so they are only used to prevent age spots and freckles.・Freckles cannot be reduced. However, there are many people who wish to reduce the spots and freckles that have already formed, and there is a need for a new approach to whitening agents or spots and freckles improving agents.

Japanese Patent Application Publication No. 9-87197 JP2006-232806A JP2010-280720A

Therefore, in view of the actual situation, the main purpose of the present technology is to provide a new melanosome decomposition promoter.

Therefore, the present inventors investigated an experimental model in which melanosomes containing already produced melanin were delivered into keratinocytes and accumulated therein. Then, the present inventors adopted an experimental model using keratinocyte-phagocytosed melanosomes and a fluorescence observation microscope, and investigated substances that promote melanosome degradation.
As a result of extensive studies, the present inventors have found that the Caprifoliaceae honeysuckle (Lonicera japonica Thunberg) plant or its extracts (particularly the goldenrod or its extracts) have the effect of promoting the decomposition of melanosomes within keratinocytes. They discovered this and completed the present invention.

Note that honeysuckle (Lonicera japonica Thunberg), which belongs to the Caprifoliaceae family, is known to have diuretic and detoxifying effects, but it is not known to have a melanosome decomposition promoting effect.
In addition, Patent Document 3 states that 50% by weight ethanol extract of Kingfisher has an effect of enhancing melanin production in a B-16 melanoma cell test, and that it was found to be effective in improving gray hair in about half of male test subjects with gray hair. has been done. However, it was completely unexpected to discover that the goldenrod extract of the present technology has the effect of reducing age spots and freckles that have already formed.

That is, the present technology provides a melanosome decomposition promoter containing a Lonicera japonica Thunberg plant of the Caprifoliaceae family or an extract thereof as an active ingredient.
Furthermore, the melanosome decomposition promoter of the present technology may be, but is not particularly limited to, flower buds or the extract thereof.
Furthermore, in the melanosome decomposition promoter of the present technology, the melanosome decomposition promotion may be melanosome decomposition promotion within keratinocytes.

The present technology can provide a new melanosome degradation promoter.
Note that the effects described here are not necessarily limited, and may be any of the effects described in the present technology.

FIG. 1A (left) is a fluorescence observation image after melanosomes were engulfed by keratinocytes, washed, and treated with an antibody for fluorescence observation before addition of the test substance (before addition of the test substance). Figure 1B (middle) is a fluorescence observation image of keratinocytes that have phagocytosed melanosomes, after being washed, cultured for 24 hours in a medium containing water without the addition of the test substance, and then treated with an antibody for fluorescence observation ( Test substance addition (none): water (solvent)). Figure 1C (right) is a fluorescence observation image of keratinocytes phagocytosed by melanosomes, washed, cultured for 24 hours in a medium supplemented with the test substance (Kingica extract), and further treated with an antibody for fluorescence observation ( Addition of test substance (with): Goldfish extract).

The plants used in the present technology include Lonicera japonica Thunberg of the Caprifoliaceae family and its related plants, which are also called honeysuckle as well as honeysuckle. Its related plants are plants that have almost the same efficacy as honeysuckle (Lonicera japonica Thunberg) and are distributed as herbal medicine honeysuckle (especially goldenrod, ginseng, etc.), such as L.confusa (Sweet). DC., L. mamckii MAXIM, L. chinensis WASTON, L. similis HEMSL, L. tragophylla HEMSL, L. pampaninii LEVL, L. hypoglauca MIQ, L. hypoglauca macranthoides HAND MAZZ, etc.
In the present technology, honeysuckle (Lonicera japonica Thunberg) and its related plants are collectively referred to as honeysuckle (Lonicera japonica Thunberg) plants (hereinafter also referred to as "honeysuckle plants").
The honeysuckle plant in this technology is native to Japan, the Korean Peninsula, China, Taiwan, etc. The flower buds are also called goldflowers, and the stems and/or leaves are also called goldenrods. King lily and ginseng have been used as herbal medicines for a long time. King lily is known to have anti-inflammatory, detoxifying, diuretic, and antibacterial effects.

Any part of the honeysuckle plant may be used, and for example, one or more parts selected from flower buds, roots, stems, stems, leaves, spores, etc. can be used. These parts can be used after being subjected to appropriate treatments such as drying, shredding, squeezing, crushing, or fermentation. Further, extraction may be performed after performing such processing.
Among these, it is preferable to use one or more of flower buds, stems, and leaves, and flower buds are more preferable.

The honeysuckle plant extract of the present technology can be obtained by extracting from the honeysuckle plant. Honeysuckle plant extract is preferred because the extraction can further enhance the efficacy of the ingredient.

The method for extracting the honeysuckle plant is not particularly limited. Extraction may be carried out using an extraction solvent by immersing the honeysuckle plant at a constant temperature (low temperature, room temperature, or heating) for a predetermined period of time.
The extraction solvent is not particularly limited, and for example, one or more of water; alcohols; ketones such as acetone; ethers such as ethyl ether; and esters such as ethyl acetate can be used.
Examples of the alcohols include lower monohydric alcohols such as methanol and ethanol; liquid polyhydric alcohols such as glycerin, propylene glycol, butanediol, and the like.
The alcohols are preferably monohydric alcohols and dihydric alcohols, and the alcohol preferably has about 1 to 5 carbon atoms.
Among these solvents, water and/or alcohols (preferably having 1 to 4 carbon atoms) are preferred. The alcohol is preferably one or more selected from methanol, ethanol, propylene glycol (1,2-propanediol), and butylene glycol (1,3-butanediol). Furthermore, water is more preferable as the solvent.
Further, in the case of hydrous alcohol, the alcohol concentration (V/V) is preferably 20 to 80% by volume.

As an example of a preferred method for extracting the honeysuckle plant, the honeysuckle plant is extracted with water, alcohol, or a water-alcohol mixture at room temperature (for example, about 5 to 40°C) or heated (about 40 to 40°C). An example of this is a method in which extraction is carried out at a temperature of about 100°C for 5 minutes to 5 days. Among these, water extraction (especially hot water extraction) is preferred.
The extraction temperature is preferably 60 to 100°C, more preferably 75 to 95°C. Further, in the case of heating extraction, the extraction period is preferably about 30 minutes to 3 hours.

The honeysuckle plant or its extract of the present technology may be used as an active ingredient as it is, or may be subjected to known separation and purification methods as appropriate to remove impurities such as impurities without impairing the efficacy or to enhance the efficacy. It may also be processed. For example, if necessary, the product may be used after further purification treatment such as distillation of the extraction solvent, filtration, deodorization using an ion exchange resin, decolorization, etc. Alternatively, only a highly active fraction can be used using separation means such as liquid chromatography.

The extract of the honeysuckle plant of the present technology can be used as it is, either as an extract alone or as a mixture of extracts obtained by different extraction methods, or it can be diluted, concentrated, or dried to form a liquid or powder. It can also be used in the form of a paste or a paste.

As shown in the Examples below, in a model test for promoting melanosome decomposition within keratinocytes, it was found that the King Ginger extract of the present technology has an effect of promoting melanosome decomposition.

Unlike conventional melanin production inhibitors (e.g., tyrosinase enzyme inhibitors), the honeysuckle plant or goldberry plant of the present technology (hereinafter also referred to as "the plant of the present technology") or the extract is different from conventional melanin production inhibitors (e.g., tyrosinase enzyme inhibitors). It can promote the decomposition of stored melanin granules and melanosomes. The plant of the present technology or the extract thereof can reduce age spots and freckles by promoting the decomposition of melanosomes in keratinocytes, which are the cause of age spots and freckles.
Although the mechanism of action is currently being elucidated, melanosomes are decomposed into many tiny particles by this technology, and these many tiny particles are dispersed and scattered within the cell. It is believed that this dilutes the age spots and freckles that have already formed on the skin, ultimately reducing the number of age spots and freckles.
In this way, the present technology can reduce existing age spots and freckles in humans or animals, and also makes it possible to aim for skin whitening. For this reason, it is desirable that the subjects of the present technology are those who expect improvement in skin whitening and improvement in age spots and freckles through the action of promoting the decomposition of melanosomes once generated. Further, it is desirable that the present technology improves whitening or reduces age spots and freckles for those who already have age spots and freckles.

When a tyrosinase inhibitor is used as in the past, it takes about 1 to 2 months for keratinocytes, which have a reduced melanin content due to suppression of melanin production, to turnover from the basal layer to the surface and be excreted. For this reason, the whitening effect of tyrosinase inhibitors takes time and is considered to be slow-acting. Furthermore, once melanosomes reach keratinocytes, they are beyond the reach of tyrosinase inhibitors.
In contrast, the plants of the present technology or their extracts are effective against melanosomes that are passed from melanocytes to keratinocytes and accumulated within the keratinocytes. This technology can promote the decomposition of melanosomes present in the basal layer, the spinous layer, and the granular layer that are in the process of turnover, so the whitening effect of this technology can be said to be more immediate than that of tyrosinase inhibitors. For this reason, it is desirable that the plants or their extracts of the present technology be targeted at people who expect early whitening effects and effects on improving age spots and freckles.

Therefore, the plant of the present technology or the extract thereof has an effect of promoting melanosome decomposition. Further, the plant of the present technology or the extract thereof can also be used as a melanosome decomposition promoter, a composition for improving age spots and freckles, and a whitening cosmetic.
Furthermore, the plant of the present technology or the extract concerned can prevent, improve, and/or treat various conditions related to melanosome production (e.g., increased age spots and freckles, uneven color, increased accumulation of melanosomes in keratinocytes, etc.). can be used for.
Furthermore, the plant of the present technology or the extract thereof can be included as an active ingredient in a melanosome decomposition promoter, a composition for improving age spots and freckles, a whitening cosmetic, and the like.
The plants of the present technology or various preparations containing the extracts can be used for the prevention, improvement, and/or treatment of "increased age spots and freckles, uneven color, increased accumulation of melanosomes in keratinocytes," etc., as described above. In addition, it can be used as an active ingredient in human or veterinary pharmaceuticals, quasi-drugs, external skin preparations, cosmetics, foods, pharmaceutical compositions, food compositions, and the like. Further, the plant of the present technology or the extract thereof can be used for manufacturing these various preparations.

The plants of the present technology or the extracts may be used for human or non-human animals, and may be used for therapeutic or non-therapeutic purposes.
"Non-therapeutic purpose" is a concept that does not include medical treatment, that is, treatment of the human body through treatment. An example of this is beauty practices.
"Amelioration" refers to improvement of a disease, symptom or condition; prevention or delay of worsening of a disease, symptom or condition; reversal, prevention or delay of progression of a disease or condition.
"Prevention" refers to preventing or delaying the onset of a disease or symptom in a target, or reducing the risk of developing a disease or symptom in a target.

Therefore, the plant of the present technology or its extract can be used in external skin preparations, cosmetics, pharmaceuticals, quasi-drugs, foods, food compositions and functional foods with indications for use, etc., for promoting the decomposition of melanosomes as described above. (For example, foods for specified health uses, etc.).
Furthermore, the melanosome decomposition accelerator of the present technology is useful as external preparations for skin, cosmetics, food compositions with indications for use, functional foods (for example, tablets, soft drinks, etc.), and the like.

The plant of the present technology or the extract thereof is preferably used for "cosmetics, quasi-drugs, external skin preparations" etc. for whitening or reducing spots and freckles, and is applied to the skin or comes into contact with the skin. Preferably, formulations that allow External skin preparations and cosmetics include, for example, lotions, milky lotions, creams, pack cosmetics, liquid foundations, ointments, hair nourishing agents, and the like. In addition, the dosage forms of external preparations for skin and cosmetics include aqueous systems, solubilized systems, emulsified systems (O/W type, W/O type, W/O/W type), and the like.

The content of the plant or its extract in products such as the melanosome decomposition promoter and cosmetics is preferably 0.0000001 to 0.1% by mass as dry solid content, more preferably 0.000001 to 0.1% by mass. 05% by mass, more preferably 0.00001 to 0.01% by mass.

Note that, in addition to the plant of the present technology or the extract thereof, arbitrary components may be used in combination as the melanosome decomposition accelerator and the like, if necessary. Preferred other ingredients may be pharmaceutically acceptable ingredients, such as whitening ingredients, tyrosinase inhibitors, turnover promoters, anti-inflammatory ingredients, cell activators, antioxidants, moisturizers, and UV protection. agents, solvents (water, alcohols, etc.), oils, surfactants, thickeners, powders, chelating agents, pH adjusters, emulsifiers, stabilizers, colorants, brighteners, flavoring agents, flavoring agents, excipients Examples include excipients, binders, disintegrants, lubricants, diluents, osmotic pressure regulators, fragrances, etc., and these may be blended depending on the intended formulation.
Further, the form of the melanosome decomposition accelerator and the like is not particularly limited, and may be any form such as liquid, paste, gel, solid, powder, etc.

<Search for melanosome regulating substances by fluorescent staining of melanosome-phagocytosed keratinocytes>
The screening method of the present technology uses melanosome-phagocytosing keratinocytes and performs fluorescent staining observation, thereby making it possible to obtain melanosome degradation-promoting substances or melanosome-proliferation-promoting substances.
Specifically, the present technology is a method of screening for a substance that promotes melanosome degradation or increase, which has the following characteristics (a) to (c).
(a) Adding a test substance to melanosome-poor keratinocytes and culturing;
(b) Performing antibody treatment for fluorescence observation of melanosomes in melanosome-poor keratinocytes added with the test substance, and fluorescence observation of the melanosomes;
(c) Compare the fluorescence intensity of melanosomes with the addition of the test substance to the fluorescence intensity of melanosomes without the addition of the test substance. If it decreases, the test substance is determined to be a substance that promotes melanosome degradation; if it increases, the test substance is determined to be a substance that promotes melanosome degradation. It should be judged as an increase promoting substance.
In addition, in the screening method of the present technology, a commercially available medium (culture solution) may be used as the medium (culture solution) used. Further, the culture may be performed in a CO ₂ incubator at 37°C. In this technique, unless otherwise mentioned, known methods can be used for culture medium, culture, etc.

"(a) Melanosome-poor keratinocytes" in the screening method of the present technology can be obtained by adding extracted melanosomes to cultured keratinocytes (preferably human epidermal keratinocytes) and then culturing them. Note that it is also possible to use "melanosome-phagocytic keratinocytes" obtained by known methods.
As the "cultured keratinocytes", commercially available human cultured keratinocytes may be used. Alternatively, cultured keratinocytes may be obtained by culturing using a known method. The origin of keratinocytes is not particularly limited, but human, mouse, rat, and pig origins are preferred, and human origin is more preferred.
The "extracted melanosome" can be obtained by a known method.
As an example, normal human epidermal melanocytes are cultured, and after culturing, the melanocytes are peeled from the container using a proteolytic enzyme, and the cells are collected by centrifugation. Extracted melanosomes can be obtained by adding a surfactant solution to the collected melanocytes.
The "culture after addition" is preferably about 6 hours to 3 days.
Regarding the addition ratio of extracted melanosomes to the cultured keratinocytes, it is preferable to add extracted melanosomes equivalent to 0.1 to 10×10 ⁵ melanocytes per 10 ⁴ keratinocytes/well.

In "(a) Adding and culturing the test substance" in the screening method of the present technology, the test substance adjusted to various concentrations is added to the medium of melanosome-poor keratinocytes and cultured for 12 to 48 hours.
Before adding the medium containing the test substance, it is preferable to remove the medium after culturing and then wash with a buffer such as phosphate buffered saline (PBS). In this operation, since the keratinocytes are attached to the container, unnecessary components such as melanosomes that have not been phagocytosed by the keratinocytes can be removed.
After culturing in a test substance-added medium, it is preferable to remove the medium and then wash with a buffer such as phosphate buffered saline (PBS). In this operation, since the melanosome-phagocytosed keratinocytes are attached to the container, components unnecessary for staining, such as components derived from the medium, can be removed. Furthermore, when the cells are peeled off, operations such as centrifugation are required to collect the cells, but in this technique, the cells remain attached, so this operation is not necessary.

"Melanosome-phagocytosing keratinocytes" are usually used after being peeled off because they are attached to a container, but with this technology, it is possible to perform keratinocytes without peeling.
Incidentally, as an example of a method for detaching from a container, the medium is removed after culturing, and then washed with a buffer solution such as phosphate buffered saline (PBS), and then a proteolytic enzyme is applied to the melanosome-phagocytic keratinocytes that are adhered to the container. After adding (for example, trypsin degrading enzyme), the cells are incubated at 37° C. for 5 minutes, centrifuged, and the precipitated cells are washed with a medium to finally obtain melanosome-phagocytosed keratinocytes.
When "melanosome-phagocytosing keratinocytes" are detached from the container, it is generally done before adding the test substance to adjust the quantity, or after the test substance is added and cultured for the next measuring device. By not performing this peeling step, efficiency can be improved by simplifying the working process, cell loss during detection can be reduced, and detection accuracy can be improved.

In "(b) Antibody treatment for fluorescence observation of melanosomes in melanosome-poor keratinocytes added to the test substance" in the screening method of the present technology, it is desirable to perform cell fixation treatment and permeabilization treatment as pretreatments.
The cell fixation treatment may be carried out by any known cell fixation method. For example, fix the cells in the container with 4% paraformaldehyde (room temperature, about 10 to 30 degrees Celsius, 10 to 20 minutes), and fix with methanol as appropriate (refrigerate (about 1 to 5 degrees Celsius), 20 to 40 minutes). .
As the permeation treatment, a known permeation treatment that can increase the permeability of a fluorescent staining substance can be used. Examples of surfactants that increase permeability include octylphenol poly(ethylene glycol ether), polysorbate 20, saponin, and digitonin. The surfactant is preferably a nonionic surfactant, of which octylphenol poly(ethylene glycol ether) is more preferred.

"(b) Antibody treatment for fluorescence observation" in the present technology can be performed according to known antibodies for fluorescence observation and their protocols. Examples include primary antibodies derived from mice or rabbits, and secondary antibodies labeled with FITC, Alexa Fluor ^(R) 488, Alexa Fluor ^(R) 568 dyes, and the like.

"(b) Fluorescence observation of melanosomes" in the present technology can be performed using a general fluorescence observation microscope device.
By using a fluorescence observation device, it is possible to reduce the loss of detected cells caused by peeling off the melanosome-poor keratinocytes from the container, and it is possible to prevent a decrease in measurement accuracy due to loss.
This technology allows for detailed fluorescence observation of multiple melanosomes scattered within a single cell, rather than just one count of the entire cell at the time of detection, making it possible to further improve measurement accuracy. Moreover, this technology can also confirm the behavior (shrinkage or increase) of melanosomes.
By using the software of the fluorescence observation device, it is possible to further improve the detection accuracy of the fluorescence intensity of the entire field of view without inappropriately measuring the fluorescence intensity due to halation.

In "(c)" of the present technology, promotion of melanosome degradation or increase is determined by comparing the presence or absence of addition of a test substance under the same culture conditions. It is desirable to perform fluorescence observation of each melanosome after adding the test substance and blank to the medium and culturing for a certain period of time.
Specifically, the fluorescence intensity of melanosomes with the addition of the test substance is compared with the fluorescence intensity of melanosomes without the addition of the test substance, and if it decreases, the test substance is determined to be a melanosome degradation promoting substance, and if it increases, the test substance is determined to be a substance that promotes melanosome degradation. is judged to be a substance that promotes melanosome increase.
As a judgment criterion, for example, when the fluorescence intensity without the addition of the test substance is taken as 100%, if it is 99% or less, it is judged to be a melanosome decomposition promoting substance, and if it is 101% or more, it is judged to be a melanosome increase promoting substance.
As the fluorescence intensity decreases from 75% or less to 50% or less, it can be judged that the substance has a stronger effect on promoting melanosome decomposition.
As the fluorescence intensity increases from 125% or more to 150% or more, it can be judged that the substance has a stronger effect on promoting melanosome increase.
Note that "melanosomes without addition of a test substance" are treated in the same manner as in the above "step (a)" except that no test substance was added.
In addition, a substance determined to be a melanosome decomposition promoting substance has a melanosome decomposition promoting effect, and can be used for applications related to this.
In addition, substances determined to be melanosome increase-promoting substances have a melanosome increase-promoting effect, and can also be used for uses related to this.

Hereinafter, the present invention (this technology) will be explained in more detail by giving Examples, Test Examples, etc., but the present invention (this technology) is not limited to these Examples, etc. in any way.

<Production Example 1: Preparation of honeysuckle plant extract>
10 g of honeysuckle (Lonicera Japonica Thunberg) flower buds (herbal medicine: Goldenrod) belonging to the Lonicera family, Lonicera genus, were immersed in 500 mL of water, and then extracted with hot water at a temperature of 80° C. to 90° C. for 2 hours. Thereafter, impurities were removed by filtration, and the mixture was freeze-dried to obtain a powder of goldenrod extract (water extract of honeysuckle plant).

<Test Example 1: Melanosome degradation acceleration test>
[Extraction of melanosomes]
Normal human epidermal melanocytes (manufactured by Kurabo Industries, Ltd.) were seeded in a T-75 flask in Medium 254 (manufactured by GIBCO) containing Human Melanocyte Growth Supplement (manufactured by GIBCO), and cultured in a CO ₂ incubator at 37°C. . Melanocytes were peeled off from the flask with trypsin, and the cells were collected by centrifugation. A surfactant solution was added to the collected melanocytes to extract melanosomes. After centrifuging to collect a supernatant containing melanosomes, the supernatant was further centrifuged to collect melanosomes.

<Melanosome degradation acceleration test>
(1) Pre-culture of keratinocytes Primary human epidermal keratinocytes HPEKs (manufactured by CELLnTEC) were seeded in a T-75 flask in CnT-PR medium (manufactured by CELLnTEC) and cultured in a CO ₂ incubator at 37°C.
(2) Test
A glass plate (Collagen I-coated cover glass 12 mm type, manufactured by IWAKI) was placed in a 24-well plate, and keratinocytes were seeded at 2×10 ⁴ cells/well. After culturing the keratinocytes for 3 days, melanosomes equivalent to 4×10 ⁵ melanocytes were added, and the cells were further cultured for 24 hours to allow the keratinocytes to phagocytose the melanosomes. The medium was removed, and the 24-well plate was washed three times with phosphate buffered saline (PBS) to remove unphagocytosed melanosomes present outside the cells.
The goldenrod extract of Production Example 1 was added to the keratinocytes that had phagocytosed the melanosomes to a final concentration (100 μg/1 mL of medium). Water was used as a solvent during concentration adjustment. After addition, culture was performed for 24 hours, and this was designated as a "test substance addition (with)" test. In the "test substance addition (with)" test, (3) cell staining and (4) image analysis, which will be described later, were performed.
For the "Test substance addition (without)" test, culture was performed in the same manner as the "Test substance addition (with)" test, except that "Kinginka extract" was changed to "solvent: water", and then (3) cell staining. and (4) image analysis was performed.
In the "before test substance addition" test, (3) cell staining and (4) image analysis were performed without adding the test substance after washing with PBS.

(3) Cell staining The medium was removed and the cells were washed three times with PBS. Fix cells in a 24-well plate with 4% paraformaldehyde. Furthermore, the cells in the 24-well plate were permeabilized with 0.3% Triton X-100 (trade name: nonionic surfactant/octylphenol poly(ethylene glycol ether) _n )/PBS. Monoclonal Mouse Anti-Human Melanosome Clone HMB45 (manufactured by DAKO) was used as the primary antibody and Alexa Fluor 568 goat anti-mouse IgG (H+L) (Thermo Fisher Scientific).
(4) Image analysis
Images of the antibody-treated cells adhering to the 24-well plate were acquired using a fluorescence observation microscope, and the fluorescence intensity was analyzed as RGB brightness values using Image J (manufactured by the National Institutes of Health).

Figure 1 shows photographs taken using a fluorescence observation microscope for each test. Furthermore, Table 1 shows the respective fluorescence intensities when cultured with the addition of water and goldenrod extract. These fluorescence intensities are based on the fluorescence intensity before addition of the test substance as 100%. Note that the numerical values in parentheses indicate values based on water as a standard of 1.00.
From this result, it was confirmed that the goldenrod extract promoted the decomposition of melanosomes within keratinocytes.

The following is a formulation example of a cosmetic or external skin preparation containing the plant of the present technology, the extract thereof, or the melanosome decomposition promoter of the present technology. Note that the total amount in each example is 100% by mass.

[Example 1: Lotion]
(Manufacturing method)
A. Mix and dissolve the following components (5) to (9).
B. Mix and dissolve the following components (1) to (4) and (10).
C. A was added to B and mixed to obtain a lotion.

(Ingredients) (%)
1 Glycerin 5.0
2 1,3-butylene glycol 5.0
3 Lactic acid 0.05
4 Sodium lactate 0.1
5 Polyoxyethylene monooleate (20E.O.) Sorbitan 1.2
6 Ethanol 8.0
7 Kingfisher extract of Production Example 1 0.0005
8 Methyl paraoxybenzoate 0.1
9 Fragrance 0.05
10 Purified water remaining amount

[Example 2: Lotion]
(Manufacturing method)
A. Mix and dissolve the following components (1) to (9).
B. Mix and dissolve the following components (10) to (12).
C. B was added to A and mixed to obtain a lotion.

(Ingredients) (%)
1 Glycerin 5.0
2 1,3-butylene glycol 10.0
3 Sodium monohydrogen phosphate 0.1
4 Sodium dihydrogen phosphate 0.1
5 Purified water Remaining amount 6 Goldfish extract of Production Example 1 0.0005
7 Sodium hyaluronate 0.01
8 Honey 0.01
9 Collagen 0.1
10 Polyoxyethylene hydrogenated castor oil (60E.O.) 0.2
11 Methyl paraoxybenzoate 0.1
12 Fragrance 0.02

[Example 3: Beauty serum]
(Manufacturing method)
A. Mix and dissolve the following components (1) to (10).
B. Mix and dissolve the following components (11) to (17).
C. B was added to A and mixed to obtain a beauty serum.

(Ingredients) (%)
1 Dipropylene glycol 5.0
2 1,3-butylene glycol 8.0
3 Alkyl acid/alkyl methacrylate copolymer (*1) 0.16
4 Xanthan gum 0.2
5 Purified water remaining amount 6 2% sodium hydroxide solution 2.0
7 Kingfish extract of Production Example 1 0.001
8 Sodium hyaluronate 0.01
9 Hydrolyzed elastin 0.01
10 Hydrolyzed collagen 0.1
11 PEG-50 isostearate hydrogenated castor oil 0.3
12 Isostearic acid 0.2
13 Refined jojoba oil 0.2
14 Astaxanthin 0.001
15 Methyl paraoxybenzoate 0.1
16 Ethanol 2.0
17 Fragrance 0.02
(*1) CARBOPOL1382 (manufactured by LUBRIZOL)

[Example 4: Oil-in-water emulsion]
(Manufacturing method)
A. Components 1 to 5 are uniformly dissolved and mixed at 70°C.
B. Components 6 to 14 are uniformly dissolved and mixed at 80°C.
C. Add B to A and emulsify at 70°C.
D. After adding and mixing components 15 to 17 to C, the mixture was cooled to 40°C to obtain an oil-in-water emulsion.

(Ingredients) (%)
1 1,3-butylene glycol 5.0
2 Dibutylene glycol 5.0
3 Purified water Remaining amount 4 Kingfisher extract of Production Example 1 0.002
5 Sodium hydroxide 2% solution 0.2
6 N-Myristoyl-L-glutamic acid 0.2
7 Polyoxyethylene hydrogenated castor oil (*2) 0.2
8 Macadamia nut oil fatty acid phytosteryl 1.0
9 Dimethylpolysiloxane (6CS) 3.0
10 N-stearoylphytosphingosine 0.1
11 Tocopherol acetate 0.01
12 Stearyl alcohol 0.5
13 Pentanediol
0.1
14 Carbomer 0.15
15 (Sodium acrylate/Sodium acryloyldimethyltaurine)
Copolymer (*3)
0.1
16 Ethanol 5.0
17 Fragrance 0.1
(*2) HCO-10 (manufactured by Nippon Surfactant Industries)
(*3) SIMULGEL EG (manufactured by SEPIC)

[Example 5: Oil-in-water emulsion]
(Manufacturing method)
A. Components 1 to 5 are uniformly dissolved and mixed at 70°C.
B. Components 6 to 14 are uniformly dissolved and mixed at 80°C.
C. Add B to A and emulsify at 70°C.
D. After adding and mixing components 15 to 17 to C, the mixture was cooled to 40°C to obtain an oil-in-water emulsion.

(Ingredients) (%)
1 1,3-butylene glycol 5.0
2 Dipropylene glycol 5.0
3 Purified water Remaining amount 4 Kingfisher extract of Production Example 1 0.002
5 Sodium hydroxide 2% solution 0.2
6 N-Myristoyl-L-glutamic acid 0.2
7 Polyoxyethylene hydrogenated castor oil (*2) 0.2
8 Macadamia nut oil fatty acid phytosteryl 1.0
9 Dimethylpolysiloxane (6CS) 3.0
10 N-stearoylphytosphingosine 0.1
11 Tocopherol acetate 0.01
12 Stearyl alcohol 0.5
13 Pentanediol 0.1
14 Carbomer 0.15
15 (Sodium acrylate/Sodium acryloyldimethyltaurine)
Copolymer (*3)
0.1
16 Ethanol 5.0
17 Fragrance 0.1

The present technology can also have the following configuration.
[1] A melanosome decomposition accelerator or whitening cosmetic containing a Caprifoliaceae honeysuckle (Lonicera japonica Thunberg) plant or its extract as an active ingredient.
[2] The melanosome decomposition accelerator or whitening cosmetic according to [1] above, wherein the plant part is a flower bud.
[3] A melanosome decomposition accelerator, a whitening cosmetic, or a composition for improving existing spots and freckles, which contains a goldfish or its extract as an active ingredient.
[4] The melanosome decomposition promoter, whitening cosmetic, or already produced melanosome decomposition promoter according to any one of [1] to [3] above, wherein the melanosome decomposition promotion is the decomposition promotion of melanosomes present in keratinocytes. A composition for improving spots and freckles.
[5] The melanosome decomposition accelerator, whitening cosmetic, or already used as described in any one of [1] to [4] above, wherein the plant extract is extracted with water, alcohol, or hydrous alcohol. A composition for improving dark spots and freckles. It is preferably extracted with water.
[6] It is preferable that the alcohol is a monohydric alcohol and/or a dihydric alcohol. It is preferable that the alcohol has 1 to 4 carbon atoms. It is preferable that the alcohol is one or more selected from methanol, ethanol, propylene glycol (1,2-propanediol), and 1,3-butanediol. It is preferable that the concentration of the alcohol is 20% by volume or more.

Claims (3)

It contains a hot water extract of the flower buds of the honeysuckle (Lonicera japonica Thunberg) plant as an active ingredient , and the hot water extraction is a hot water extraction at 80 to 90 °C for 30 minutes to 3 hours . A melanosome decomposition promoter that is a composition for improving dark spots and freckles. The melanosome decomposition accelerator according to claim 1, wherein the melanosome decomposition accelerator is a cosmetic or external skin preparation for improving spots and freckles that have already formed. The melanosome decomposition promoter according to claim 1 or 2, wherein the melanosome decomposition promoting agent promotes the decomposition of melanosomes present in keratinocytes.