JP7115778B2 - Melanin production inhibitory composition - Google Patents

Description

Application of Article 30, Paragraph 2 of the Patent Act February 10, 2020 (Monday) 2019 Graduation Experiment Abstracts, pp. 3 and 15 (Shikoku University Junior College Division, Department of Human Health, Department of Food and Nutrition) Ordinance February 10, 2019 (Monday) 2019 graduation experiment presentation, poster number 3 (Shikoku University 30th Anniversary Hall N209 multipurpose room)

TECHNICAL FIELD The present invention relates to a melanogenesis-suppressing composition.

As for citrus fruits, in addition to those suitable for eating raw, such as mandarin oranges, there are lemons, yuzu, and sudachi, which have a strong acidity and are not suitable for eating raw, but have a good aroma, so their juice and peels are used as seasonings. be.

Awa Suzuka is a new variety of fragrant citrus that was developed by Tokushima Prefecture by crossing Sudachi and Yuzu and was registered in 2017 (registration number 26249). Awa Suzuka has almost no seeds, is rich in fruit juice, and has a unique aroma intermediate between that of Sudachi and Yuzu. Its sugar content and citric acid content are lower than those of Sudachi, but higher than that of Yuzu. , and has a higher shelf life and storability than sudachi and yuzu (Non-Patent Documents 1 and 2).

On the other hand, Patent Document 1 describes that as a result of adding extracts of immature yuzu, hassaku, grapefruit, and banpeiyu fruits to cells, melanin synthesis inhibitory action was confirmed.

JP 2017-214349 A

Tokushima Prefectural Agriculture, Forestry and Fisheries Technical Support Center News, August 2015, No.3, p6 Tokushima Prefectural Agriculture, Forestry and Fisheries Technical Support Center Research Report, November 2015, No.2, p9-12

The inventors of the present invention, as a result of extensive independent studies, found that yuzu and sudachi pericarp extracts sometimes exhibit cytotoxicity when added to cells.

The present invention has been made in view of the above problems, and an object of the present invention is to provide a melanogenesis-suppressing composition that is excellent in safety and effectively suppresses melanin production in cells.

A melanin production-suppressing composition according to an embodiment of the present invention for solving the above problems contains a pericarp extract of Suzuka Awa. INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a melanogenesis-suppressing composition that is excellent in safety and effectively suppresses melanogenesis in cells.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a melanogenesis-suppressing composition that is excellent in safety and effectively suppresses melanogenesis in cells.

FIG. 2 is an explanatory diagram showing the results of evaluating the melanin production inhibitory effect in Example 1 according to one embodiment of the present invention. FIG. 2 is an explanatory diagram showing the results of evaluating the tyrosinase activity inhibitory effect in Example 1 according to one embodiment of the present invention. FIG. 4 is an explanatory diagram showing the results of evaluating the melanin production inhibitory effect in Example 2 according to one embodiment of the present invention. FIG. 4 is an explanatory diagram showing the results of evaluating the tyrosinase activity inhibitory effect in Example 2 according to one embodiment of the present invention. FIG. 4 is an explanatory diagram showing the results of evaluation of cell viability in Example 3 according to one embodiment of the present invention.

A composition for suppressing melanin production (hereinafter referred to as "the present composition") according to one embodiment of the present invention will be described below. Note that the present invention is not limited to this embodiment.

The present composition is a melanin production-suppressing composition containing a pericarp extract of Suzuka Awa. That is, the inventors of the present invention, as a result of extensive studies, found that, as demonstrated in the examples below, the pericarp extract of Awa Suzuka, which was produced by crossing yuzu and sudachi, unexpectedly Compared to both yuzu peel extract and sudachi pericarp extract, it has significantly lower cytotoxicity, is therefore superior in safety, and has been independently found to effectively suppress melanin production in cells. I have perfected my invention.

The pericarp extract contained in the present composition is a composition obtained by extracting the pericarp of Suzuka Awa using an extraction solvent. The pericarp of Awa Suzuka to be extracted is not particularly limited as long as the effect of the present invention can be obtained, and it may be an immature green pericarp of Awa Suzuka or a mature yellow pericarp. It may be the after-ripe orange peel. Moreover, the pericarp of Awa Suzuka may be dried or non-dried, but the dried pericarp is preferably used.

Like other citrus fruits, the fruit of Awa Suzuka contains a pericarp and a sac (consisting of a sand sac and a sac that envelops it). For the preparation of the product, the pericarp separated from the funnel (for example, the pericarp dried after being separated from the funnel) is preferably used. That is, it is preferable that the pericarp of Awa Suzuka to be extracted contains, for example, exocarp (flavedo) and does not contain fungus.

The method for extracting the pericarp of Awa Suzuka is not particularly limited as long as the pericarp-derived components can be extracted from the pericarp using an extraction solvent and the effects of the present invention can be obtained. For example, a liquid or a supercritical fluid. An extraction method using an extraction solvent (eg, liquid solvent extraction or supercritical fluid extraction) is preferably used.

The extraction temperature is not particularly limited as long as the effects of the present invention can be obtained, but for example, it is preferably higher than 0 ° C. and 100 ° C. or lower, more preferably 1 ° C. or higher and 70 ° C. or lower. Particularly preferably, the temperature is 1°C or higher and 40°C or lower.

The extraction time is not particularly limited as long as the effects of the present invention can be obtained. For example, it is preferably 1 hour or more, more preferably 12 hours or more, and particularly 24 hours or more. preferable. The extraction time is, for example, preferably 12 months or less, more preferably 1 month or less, and particularly preferably 1 week or less. The extraction time may be specified by arbitrarily combining any of the above lower limits and any of the above upper limits.

The amount of the extraction solvent used for extraction is not particularly limited as long as the effects of the present invention can be obtained. . Also, the amount of the extraction solvent used for extraction is preferably 500 mL or less, more preferably 100 mL or less, and particularly preferably 20 mL or less per 1 g of the peel. The amount of extraction solvent used for extraction may be specified by any combination of any of the above lower limits and any of the above upper limits.

The extraction solvent used for extracting the pericarp of Awa Suzuka is not particularly limited as long as the effect of the present invention can be obtained, but for example, an organic solvent and/or a supercritical fluid are preferably used. The organic solvent is not particularly limited as long as it contains an organic solvent and the effects of the present invention can be obtained.

The organic solvent is not particularly limited as long as the effect of the present invention can be obtained. methyl acetate, ethyl acetate), alkanes (eg, hexane), and cycloalkanes (eg, cyclohexane), preferably one or more selected from the group consisting of alcohols.

Alcohols are not particularly limited as long as the effects of the present invention can be obtained, but for example, lower monohydric alcohols and/or polyhydric alcohols are preferable. The lower monohydric alcohol is not particularly limited as long as the effect of the present invention can be obtained. more preferably one or more selected from the group consisting of ethanol, 1-propanol, and isopropanol, and particularly preferably ethanol. The polyhydric alcohol is not particularly limited as long as the effect of the present invention can be obtained, but is selected from the group consisting of, for example, ethylene glycol, 1,3-butylene glycol, propylene glycol, isoprene glycol, glycerin, and diglycerin. is preferably one or more.

The organic solvent may consist of only an organic solvent, but is preferably a mixed solvent of an organic solvent and water. When the organic solvent is a mixed solvent of the organic solvent and water, the organic solvent is, for example, 20% by volume or more and less than 100% by volume of the organic solvent and more than 0% by volume and 80% by volume or less of water. It may contain 30% by volume or more and 95% by volume or less of an organic solvent and 5% by volume or more and 70% by volume or less of water, and 40% by volume or more and 95% by volume or less of It may contain an organic solvent and 5% by volume or more and 60% by volume or less of water.

Furthermore, the organic solvent preferably contains, for example, 50% by volume or more and 95% by volume or less of an organic solvent and 5% by volume or more and 50% by volume or less of water, and 60% by volume or more and 95% by volume or less. and an organic solvent of 5% by volume or more and 40% by volume or less of water. More preferably, it contains 70% by volume or more and 90% by volume or less of an organic solvent and 10% by volume or more and 30% by volume or less of water.

The supercritical fluid as the extraction solvent is not particularly limited as long as the effect of the present invention can be obtained, but for example, carbon dioxide is preferably used.

A pericarp extract of Awa Suzuka is obtained by the above-described extraction of Awa Suzuka pericarp. The pericarp extract contains a pericarp-derived component of Suzuka Awa. More specifically, the pericarp extract contains a pericarp-derived fat-soluble component of Suzuka Awa. The pericarp extract containing the fat-soluble component derived from the pericarp of Awa Suzuka may be, for example, an organic solvent extract and/or a supercritical extract of pericarp of Suzuka Awa. In addition, in the preparation of the pericarp extract, the pericarp extract that is finally obtained as a result of removing the residue of the pericarp that is the target of extraction by filtration or the like (for example, the pericarp extract used as a raw material in the production of the present composition product) may not contain the pericarp.

The pericarp extract may be used as a raw material of the present composition as it is after being prepared by extraction, or may be used as a raw material of the present composition after being subjected to a treatment such as filtration, concentration, dilution, fractionation or drying. may be used as

That is, for example, the extraction solvent is first removed from the pericarp extract prepared by extraction, and a solid (for example, dry powder) pericarp extract is prepared by drying, and then the solid pericarp extract is prepared. It may be used as a raw material for the present composition as it is or as a liquid pericarp composition after being dissolved in a solvent.

The present composition contains the Awa Suzuka pericarp extract prepared as described above as an active ingredient for suppressing melanin production. That is, the present composition contains the pericarp extract of Suzuka Awa in an amount within a range that enhances the melanin production inhibitory effect compared to the case where the pericarp extract is not contained.

The content of the pericarp extract in the present composition is not particularly limited as long as it is within the range in which the effects of the present invention can be obtained. 0005% by weight or more, preferably 0.001% by weight or more, more preferably 0.005% by weight or more, and particularly preferably 0.01% by weight or more.

The upper limit of the content of the pericarp extract in terms of solid content in the present composition is not particularly limited as long as it is within the range in which the effects of the present invention can be obtained. It may be 15% by weight or less, may be 10% by weight or less, may be 5% by weight or less, may be 1% by weight or less, or may be 0.1% by weight or less. , 0.05% by weight or less, or 0.03% by weight or less. The content of the pericarp extract in terms of solid content in the present composition may be specified by arbitrarily combining any of the above lower limits and any of the above upper limits.

The form of the present composition is not particularly limited as long as the effect of the present invention can be obtained, and may be liquid (composition having fluidity) or solid (composition having no fluidity). It can be.

The present composition may be, for example, a composition for external application, and specifically, it is preferably a composition for external application to the skin. That is, the present composition may be an external preparation that is applied to the skin. In this case, the composition may be, for example, an emulsion, cream, lotion, ointment, gel, foam, powder, spray, patch, pack, or mask.

The composition may also be, for example, a composition for oral ingestion. That is, the present composition may be a functional food or drink such as a so-called supplement. In this case, the composition may be, for example, a liquid, syrup, suspension, emulsion, powder, fine granules, granules, tablets, pills, capsules, or jelly. Moreover, the present composition may be, for example, a cosmetic, a quasi-drug, or a pharmaceutical.

The present composition may contain other ingredients in addition to the Awa Suzuka pericarp extract. Other ingredients are not particularly limited as long as they are ingredients that can be used in ordinary cosmetics, external preparations, quasi-drugs, pharmaceuticals, and the like.

Specifically, other components include, for example, moisturizers, antioxidants, stabilizers, oily components, emulsifiers, excipients, disintegrants, lubricants, binders, UV absorbers, solvents, surfactants. , alcohols, water-soluble polymers, disinfectants, antibacterial agents, vitamins, pH adjusters, viscosity adjusters, preservatives, fragrances, and pigments. In addition, the composition may further contain known whitening ingredients (eg, arbutin, kojic acid, ascorbic acid and derivatives thereof).

This composition effectively suppresses the production of melanin in living cells by containing the pericarp extract of Suzuka Awa. That is, as demonstrated in the examples below, even if the pericarp extract of Awa Suzuka is brought into contact with the cells, the overproduction of melanin in the cells is induced in the absence of the contact. , can effectively suppress the production of melanin in the cells.

Therefore, by applying the present composition to a living body, the production of melanin in the cells of the living body can be suppressed and, for example, spots and freckles can be effectively prevented. Specifically, the present composition can be used, for example, as a whitening agent or as an agent for improving hyperpigmentation (eg, chloasma, senile pigmentation, post-inflammatory pigmentation).

In this respect, a fat-soluble ingredient derived from the pericarp is particularly effective as a melanin production-suppressing ingredient derived from the pericarp of Awa Suzuka. Therefore, for example, when the present composition is applied to the skin as a composition for external use on the skin, the fat-soluble component is difficult to wash off with sweat and the like, so the present composition effectively acts on the skin cells to The generation can be effectively suppressed.

In addition, since the pericarp of Awa Suzuka has less bitterness than the conventional aromatic citrus, the present composition can be easily used as a functional food for whitening and improving hyperpigmentation. In addition, as the pericarp of Awa Suzuka, immature, mature, and after-ripened ones can be used, so that the production of the present composition is less likely to be restricted by the harvest time and storage period of Awa Suzuka.

Next, specific examples according to this embodiment will be described.

[Preparation of pericarp extract] Green peel of immature Awa Suzuka (green peel), yellow peel of mature Awa Suzuka (yellow peel), and orange peel of ripe Awa Suzuka (orange peel) were dried at 50° C. for 2 hours to prepare dried green peel, yellow peel, and dried orange peel of Suzuka Awa.

An extract of each dried pericarp was then prepared. An ethanol solution containing 80% by volume ethanol and 20% by volume water was used as an extraction solvent. Specifically, 40 mL of an ethanol solution was added to 5.0 g of dry pericarp, and the pericarp was pulverized using a homogenizer.

After that, extraction was performed by leaving the ethanol solution containing the pulverized pericarp at 4° C. for 24 hours. After the extraction, centrifugation was performed at 3500 rpm for 10 minutes to precipitate the residue of the pericarp, and about 40 mL of the supernatant was obtained as a pericarp extract. Thus, a green peel extract, a yellow peel extract, and an orange peel extract of Awa Suzuka were obtained from the dried green peel, the dried yellow peel, and the dried orange peel of Suzuka Awa, respectively.

In addition, when 10 mL of yellow pericarp extract (5.0 g of dried yellow pericarp extracted with 40 mL of ethanol solution and centrifuged is the supernatant) is dried and the weight of the remaining solid content is measured, it is 0.38 g. rice field. That is, the solid content of the yellow pericarp extract was 0.038 g/mL (=0.38 g/10 mL) (about 3.8% by weight).

[Pericarp extract cell addition test] Mouse B16 melanoma cells were seeded in each well of a commercially available 24-well plate (20,000 cells/well) and cultured overnight at 37°C in a 5% CO ₂ /95% air atmosphere. .

On the other hand, by adding 1 part by volume of the pericarp extract obtained as described above to 199 parts by volume of cell culture medium, the pericarp extract containing the pericarp extract diluted 200 times by volume. Supplemented medium was prepared.

Then, 0.5 mL of the pericarp extract-added medium was added to each well in which the cells were being cultured, and cultured at 37° C. for 30 minutes in a 5% CO ₂ /95% air atmosphere. In addition, the content of the yellow pericarp extract in terms of solid content in the yellow pericarp extract-added medium containing the yellow pericarp extract diluted 200 times by volume is 0.00019 g / mL (= 0.038 g / mL ÷ 200) (about 0.019% by weight).

Next, α-MSH (Melanocyte-Stimulating Hormone) was dissolved in water to prepare an α-MSH solution, and the α-MSH solution was added to each well in an amount to give a final concentration of 1 μM, followed by 5% CO ₂ / It was cultured for an additional 72 hours at 37° C. in a 95% air atmosphere. Note that α-MSH is a factor that activates melanogenesis in melanocytes. Each well was then evaluated for protein content, melanin content and tyrosinase activity as described below.

[Protein Content] After culturing the cells for 72 hours as described above, the protein content in each well was measured. That is, first, each well was washed once with PBS (0.5 mL/well), and then 0.15 mL of 0.1 M sodium phosphate buffer (1% TritonX-100, protease inhibitor, pH 6.8) was added to each well. The cells were collected by scraping with a scraper.

The recovered suspension was then centrifuged at 14000 rpm for 10 minutes at 4°C. Then, 4 μL of the supernatant after centrifugation was added to 0.2 mL of Bradford Dye Reagent in a commercially available kit (Takara Bradford Protein Assay Kit), reacted at room temperature for 5 minutes, and then measured absorbance at a wavelength of 595 nm.

[Melanin Content] After culturing the cells for 72 hours as described above, the melanin content in each well was measured. That is, after each well was washed once with PBS (0.5 mL/well), 0.15 mL of 1N sodium hydroxide was added to each well, and the cells were collected by scraping with a scraper.

Next, the collected suspension was reacted at 100° C. for 1 hour to extract melanin, and then centrifuged at 14000 rpm for 10 minutes. Then, the absorbance at a wavelength of 405 nm of the supernatant liquid after centrifugation was measured. Furthermore, for each well, the absorbance (wavelength 405 nm) obtained by measuring the melanin content is divided by the absorbance (wavelength 595 nm) obtained by measuring the protein content, and the protein content in each well It was used as the melanin content per mass.

[Tyrosinase activity] After culturing the cells for 72 hours as described above, the tyrosinase activity in each well was measured. That is, first, each well was washed once with PBS (0.5 mL/well), and then 0.15 mL of 0.1 M sodium phosphate buffer (1% TritonX-100, protease inhibitor, pH 6.8) was added to each well. The cells were collected by scraping with a scraper.

The recovered suspension was then centrifuged at 14000 rpm for 10 minutes at 4°C. Then, the supernatant after centrifugation was used as an intracellular tyrosinase-containing liquid, and 2.5 mM L-DOPA was added as a substrate to this, reacted at 37° C. for 30 minutes, and absorbance at a wavelength of 475 nm was measured. Furthermore, for each well, the absorbance (wavelength 475 nm) obtained in the tyrosinase activity measurement is divided by the absorbance (wavelength 595 nm) obtained in the protein content measurement. It was used as the tyrosinase activity per unit.

[Results] Fig. 1 shows the results of evaluating the melanin content per protein content. In the horizontal axis of FIG. 1, “α-MSH + green peel”, “α-MSH + yellow peel” and “α-MSH + orange peel” are the green peel extract and yellow peel extract of Suzuka Awa, respectively, as described above. and orange peel extract were added to the cells, and α-MSH was further added (both n=3).

On the other hand, in the horizontal axis of FIG. 1, "control" is a group (n = 3) in which an extraction solvent (ethanol solution) was added instead of the pericarp extract and α-MSH was not added, "green pericarp", "Yellow peel" and "orange peel" are groups in which green peel extract, yellow peel extract and orange peel extract were added, respectively, and α-MSH was not added (both n = 3), "α- MSH” indicates a group (n=3) to which an extraction solvent (ethanol solution) was added in place of the pericarp extract and α-MSH was further added.

The vertical axis of FIG. 1 indicates the relative value of the melanin content per protein content in each group when the melanin content per protein content in the “control” is set to “1”.

As shown in FIG. 1, in "green peel", "yellow peel" and "orange peel", the melanin content significantly decreased compared to the "control". That is, regardless of the degree of maturity of Awa Suzuka, the addition of the Awa Suzuka pericarp extract significantly inhibited the production of melanin in cells.

In addition, in "α-MSH", the melanin content was significantly increased compared to "control". That is, in the group to which α-MSH was added without previously adding the pericarp extract of Awa Suzuka, overproduction of melanin occurred in the cells.

On the other hand, the melanin content in "α-MSH + green peel", "α-MSH + yellow peel" and "α-MSH + orange peel" was significantly reduced compared to "α-MSH", compared to "control". was below the same level.

That is, even if the pericarp extract of Awa Suzuka is added in advance and then α-MSH is added (that is, even in an environment that induces overproduction of melanin), Awa Regardless of the maturity of Suzuka, overproduction of melanin in cells was remarkably suppressed.

FIG. 2 shows the results of evaluating the tyrosinase activity per protein content. The horizontal axis in FIG. 2 is the same as in FIG. The vertical axis in FIG. 2 shows the relative value of tyrosinase activity per protein content in each group, with the tyrosinase activity per protein content in the "control" being set to "1".

As shown in Fig. 2, tyrosinase activity decreased in "green peel" and "yellow peel" compared to "control". That is, tyrosinase activity in cells was suppressed by adding the pericarp extract of Awa Suzuka, regardless of the degree of maturity of Suzuka Awa.

In addition, tyrosinase activity was significantly increased in "α-MSH" compared to "control". That is, in the group to which α-MSH was added without previously adding the pericarp extract of Awa Suzuka, tyrosinase was excessively activated in the cells.

In contrast, the tyrosinase activity in "α-MSH+green peel" and "α-MSH+yellow peel" was significantly reduced compared to "α-MSH". That is, by adding the pericarp extract of Awa Suzuka in advance, excessive activation of tyrosinase in cells is remarkable, regardless of the maturity of Awa Suzuka, even when α-MSH is added afterwards. suppressed by

[Preparation of pericarp extract] A pericarp extract (yellow pericarp extract) of mature Awa Suzuka was prepared in the same manner as in Example 1 above.

[Fraction of Pericarp Extract] First, the solvent was evaporated from 500 μL of the pericarp extract prepared as described above using an evaporator. Then, 500 μL of water was added to dissolve the water-soluble components. Further, 500 μL of ethyl acetate was added and stirred using a vortex. After that, centrifugation was performed at 15000 rpm for 5 minutes to form an ethyl acetate layer and an aqueous layer separated from each other.

Then, 400 μL of the ethyl acetate layer (upper layer) was collected, dried and solidified, and the obtained solid content was dissolved in 200 μL of ethanol to obtain about 200 μL of fat-soluble fraction. On the other hand, 400 μL of the aqueous layer (lower layer) was collected, filter-sterilized, and treated with an evaporator for 30 minutes to obtain a water-soluble fraction of about 200 μL.

[Pericarp extract cell addition test] The fat-soluble fraction was added to mouse B16 melanoma cells in the same manner as in Example 1 above, except that a fat-soluble fraction or a water-soluble fraction was used instead of the pericarp extract. Minutes or water-soluble fractions were added and protein content, melanin content and tyrosinase activity were assessed.

[Results] Fig. 3 shows the results of evaluating the melanin content per protein content. In the horizontal axis of FIG. 3, “α-MSH + fat-soluble fraction” and “α-MSH + water-soluble fraction” are the fat-soluble fraction and water-soluble fraction of the yellow pericarp extract of Awa Suzuka, respectively. The results of the group to which α-MSH was added (both n=3) are shown.

On the other hand, in the horizontal axis of FIG. 3, the “control” is a group in which an extraction solvent (80% by volume ethanol solution) is added instead of the fat-soluble fraction and the water-soluble fraction, and α-MSH is not added (n = 3), "fat-soluble fraction" and "water-soluble fraction" are groups to which the fat-soluble fraction and the water-soluble fraction were added, respectively, and α-MSH was not added (both n = 3), "α-MSH" indicates a group (n=3) to which an extraction solvent (80% by volume ethanol solution) was added in place of the fat-soluble fraction and water-soluble fraction, and α-MSH was further added.

The vertical axis of FIG. 3 shows the relative value of the melanin content per protein content in each group, when the melanin content per protein content in the “control” is set to “1”.

As shown in FIG. 3, the melanin content in the "fat-soluble fraction" decreased to about 60% of the "control". In addition, the melanin content in the "water-soluble fraction" decreased to about 70% of that in the "control". That is, the fat-soluble fraction of the pericarp extract of Suzuka Awa reduced the melanin content more than the water-soluble fraction of the pericarp extract.

In addition, in "α-MSH", the melanin content was significantly increased compared to "control". That is, in the group to which α-MSH was added without previously adding the pericarp extract of Awa Suzuka, overproduction of melanin occurred in the cells.

On the other hand, the melanin content in "α-MSH + fat-soluble fraction" was significantly reduced compared to "α-MSH" and was comparable to "control". That is, even if the fat-soluble fraction of the pericarp extract of Awa Suzuka is added in advance, even if α-MSH is added afterwards (that is, the environment is such that overproduction of melanin is induced. ), the overproduction of melanin in the cells was remarkably suppressed.

On the other hand, the melanin content in “α-MSH+water-soluble fraction” was comparable to that in “α-MSH”. That is, among the pericarp-derived components contained in the pericarp extract of Awa Suzuka, the components contained in the fat-soluble fraction (pericarp-derived fat-soluble components) were thought to effectively suppress melanin production. .

FIG. 4 shows the results of evaluating the tyrosinase activity per protein content. The horizontal axis in FIG. 4 is the same as in FIG. The vertical axis in FIG. 4 shows the relative value of tyrosinase activity per protein content in each group, with the tyrosinase activity per protein content in the "control" being set to "1".

As shown in FIG. 4, the tyrosinase activity in the "fat-soluble fraction" decreased to about 20% of the "control". Also, the tyrosinase activity in the "water-soluble fraction" decreased to about 50% of the "control". That is, the fat-soluble fraction of the pericarp extract of Awa Suzuka reduced the tyrosinase activity more than the water-soluble fraction of the pericarp extract.

In addition, tyrosinase activity was significantly increased in "α-MSH" compared to "control". That is, in the group to which α-MSH was added without previously adding the pericarp extract of Awa Suzuka, tyrosinase was excessively activated in the cells.

In contrast, the tyrosinase activity in "α-MSH+lipid-soluble fraction" was markedly reduced compared to "α-MSH". That is, by preliminarily adding the fat-soluble fraction of the pericarp extract of Suzuka Awa, excessive activation of tyrosinase in cells was remarkably suppressed even when α-MSH was subsequently added.

On the other hand, the tyrosinase activity in "α-MSH+water-soluble fraction" was comparable to that in "α-MSH". That is, among the pericarp-derived components contained in the pericarp extract of Awa Suzuka, the components contained in the fat-soluble fraction (pericarp-derived fat-soluble components) were thought to effectively suppress tyrosinase activity. .

[Preparation of pericarp extract] The dried pericarp of Awa Suzuka (yellow pericarp), the pericarp of mature Sudachi, and the pericarp of mature Yuzu are each dried at 50 ° C. for 2 hours to obtain the dried pericarp of Awa Suzuka, Dried peels of sudachi and dried peels of yuzu were prepared.

Next, in the same manner as in Example 1 above, the dried peel of Awa Suzuka, the dried yellow peel of Citrus sudachi, and the dried peel of Yuzu are extracted with an ethanol solution, and the pericarp extract of Suzuka Awa and the pericarp of Ciudad Citrus are extracted. and a yuzu pericarp extract.

[Pericarp Extract Cell Addition Test] Mouse B16 melanoma cells were seeded in each well of a commercially available 96-well plate (5000 cells/well) and cultured overnight at 37° C. in a 5% CO ₂ /95% air atmosphere. .

On the other hand, by adding the pericarp extract obtained as described above to the medium for cell culture, the volume ratio is 100 times, 200 times, 300 times, 400 times, 500 times, 600 times, 700 times, Alternatively, a pericarp extract-added medium containing the pericarp extract diluted 800 times was prepared. Then, 0.1 mL of the pericarp extract-added medium was added to each well in which the cells were being cultured, and the cells were cultured at 37° C. for 72 hours in a 5% CO ₂ /95% air atmosphere (n=3 for each group).

[Cell viability] After culturing for 72 hours as described above, the cell viability in each well was evaluated by MTT assay. That is, MTT (3-(4,5-Dimethylthiazol-2yl)-2,5-Diphenyltetrazolium bromide) was added to the medium in the well in which the cells were being cultured so that the final concentration was 0.5 mg/mL. , and cultured at 37° C. for 2 hours in a 5% CO ₂ /95% air atmosphere.

The medium was then removed from the wells, DMSO was added, and the cells were further cultured at 37° C. in a 5% CO ₂ /95% air atmosphere for 1 hour. Cells and DMSO were then collected from the wells and the absorbance of the suspension was measured at a wavelength of 570 nm.

[Results] Fig. 5 shows the results of evaluating the cell viability. In FIG. 5, the black circle indicates the group to which the pericarp extract of "Awa Suzuka" was added, the black triangle indicates the group to which the pericarp extract of "Sudachi" was added, and the black square indicates " The results of the group to which the peel extract of "Yuzu" was added, and the open circles show the results of the group ("control") to which the extraction solvent (80% by volume ethanol solution) was added instead of the peel extract. (Average value of each point n=3).

In FIG. 5, the horizontal axis indicates the dilution ratio (volume ratio) of the pericarp extract or extraction solvent added to the medium, and the vertical axis indicates the extraction solvent diluted 800 times by volume and added to the medium. It shows the relative cell viability (%) of the other groups when the absorbance at a wavelength of 570 nm in the "Control" group is defined as 100% cell viability.

That is, for example, the cell viability in “× 200” of “Awa Suzuka” is the wavelength 570 nm of the group using the pericarp extract-added medium containing the pericarp extract of Awa Suzuka diluted 200 times by volume. was divided by the absorbance at a wavelength of 570 nm in the group using the extraction solvent-added medium containing the extraction solvent diluted 800 times by volume, and then multiplied by "100".

As shown in FIG. 5, the pericarp extract of "Sudachi" has a cell survival rate of about 10% or less when the dilution rate is 700 times or less, and even when diluted 800 times and added, the cell survival rate is about 10% or less. The rate was as low as 40%. That is, it was confirmed that the pericarp extract of "Sudachi" exhibits significant cytotoxicity when diluted at a rate of 800 times or less.

In addition, when the peel extract of "yuzu" was diluted 500 times or more and added, the cell survival rate was equivalent to that of "control" and "Awa Suzuka", but when the dilution rate was 400 times or less showed a marked decrease in cell viability. That is, it was confirmed that the peel extract of "yuzu" exhibits significant cytotoxicity when diluted at a rate of 400 times or less.

On the other hand, when the pericarp extract of ``Awa Suzuka'' was diluted 100 times or less, the cell viability decreased remarkably like ``Sudachi'' and ``Yuzu''. Cell viability was comparable to the 'control' when added as That is, it was confirmed that the pericarp extract of "Awa Suzuka" does not exhibit cytotoxicity when diluted at least 200-fold.

In this way, although ``Awa Suzuka'' was produced by crossbreeding ``Sudachi'' and ``Yuzu'', the pericarp extract of ``Awa Suzuka'' is surprisingly similar to that of ``Sudachi''. It was confirmed that the cytotoxicity was remarkably lower than that of both the yuzu peel extract and the yuzu peel extract.

Claims (1)

A composition for suppressing melanin production, comprising a pericarp extract of Suzuka Awa , wherein the content of the pericarp extract in terms of solid content is 0.001% by weight or more and 0.03% by weight or less .

Images