JP2021017404A - Melanogenesis inhibitor, skin cosmetics and skin external preparation - Google Patents

Abstract

To provide a safe melanogenesis inhibitor that can suppress melanogenesis, and skin cosmetics and skin external preparations including the melanogenesis inhibitor.SOLUTION: A melanogenesis inhibitor is provided that contains extract from immature olive fruits as an active ingredient.SELECTED DRAWING: Figure 2

Description

The present invention relates to a melanin production inhibitor containing an extract extracted from olive fruits, a skin cosmetic containing the melanin production inhibitor, and a skin external preparation containing the melanin production inhibitor.

Accumulation of melanin in epidermal keratinocytes is one of the causes of pigmentation such as age spots (senile pigment spots) due to aging and sunburn. This melanin is produced by oxidizing tyrosine in melanocytes by the stimulation of ultraviolet rays applied to the skin, and is accumulated in epidermal keratinocytes.

The accumulated melanin is excreted as dirt along with the keratin by the turnover of the epidermis, but it is chronically exposed to ultraviolet rays without UV protection, and the skin turnover cycle becomes longer due to aging. When this happens, the excretion of melanin becomes dull, and melanin remains in the epidermal keratinocytes, resulting in brown spots and the like. For this reason, many melanin production inhibitors that suppress the production of melanin have been proposed as substances for preventing the formation of spots and the like.

For example, Patent Document 1 discloses a melanin production inhibitor containing hydroquinone and ascorbic acid. Since hydroquinone contained in this melanin production inhibitor has a function of suppressing melanin production, it is said that melanin is produced in epidermal keratinocytes and can prevent brown spots and the like. ing.

However, since hydroquinone, which can strongly suppress the production of melanin, is known to have cytotoxicity, there is a problem from the viewpoint of safety. Therefore, it is desired to develop a component that is safe and can suppress the production of melanin.

Japanese Patent No. 2006-249000

In view of the above-mentioned problems, an object of the present invention is to provide a melanin production inhibitor that is safe and capable of suppressing melanin production, a skin cosmetic containing the melanin production inhibitor, and an external preparation for skin.

The present invention is characterized by being a melanin production inhibitor containing an extract extracted from immature olive fruits as an active ingredient.
The present invention is also characterized by being a skin cosmetic and an external preparation for skin containing the above-mentioned melanin production inhibitor.

The above-mentioned extracts derived from immature olive fruits include not only olive fruits but also extracts extracted from olive fruits containing olive seeds. The extract also includes an extract extracted from the pomace remaining after the olive oil is extracted from the olive fruit.

The above-mentioned immature olive fruits refer to olive peels in a green or yellowish green state, and generally refer to those that can be harvested from the end of August to the beginning of October in the Northern Hemisphere. In the olive fruit maturity index indicating the maturity of olive fruits (see FIG. 1), it refers to an olive fruit having a color score of 0 to 3, and more preferably an olive fruit having a color score of 0 to 2.

The method for obtaining the extract from the immature olive fruit is not particularly limited as long as it is a method for extracting the components derived from the olive fruit, and any solvent or means may be used. For example, a method of immersing an immature olive fruit or a crushed product of an immature olive fruit, a squeezed residue remaining after separating olive oil from an immature olive fruit, etc. in an appropriate extraction solvent, or an unexplored method. It can be obtained by a method of leaving or stirring an extraction solvent in which mature olive fruits and the like are immersed within a range of the boiling point of the solvent from room temperature. Examples of the method for obtaining the extract from the immature olive fruit include a method of extracting with water or a 20-70% ethanol aqueous solution and filtering. The temperature of the solvent is preferably 20 ° C. or higher and 80 ° C. or lower, and more preferably 20 ° C. or higher and 40 ° C. or lower.

When the present invention is a skin cosmetic or an external preparation for skin, the solvent used to obtain an extract from immature olive fruits is preferably a solvent capable of obtaining a product harmless to the human body. For example, it is more preferable to mix water and ethanol in an appropriate ratio.

The properties of the external preparation for skin or cosmetic for skin may be liquid, gel, cream, semi-solid, solid, stick, powder, etc., and may be milky lotion, cream, lotion, beauty essence, etc. It can be used as a skin cosmetic such as a pack, a washing pigment, and a makeup cosmetic.

In addition, as long as the above extract suppresses the production of melanin, the skin external preparation or the skin cosmetic includes a main agent, an auxiliary agent or other components used in the production of a normal skin external preparation or a skin cosmetic, for example. , Astringent, bactericidal / antibacterial agent, preservative, metal ion sequestering agent, pH adjuster, chelating agent, refreshing agent, emulsifier, foaming agent, thickener, deodorant / deodorant, ultraviolet absorber, moisturizer, Add cell activators, anti-inflammatory / anti-allergic agents, whitening agents, enzymes, hormones, oils and fats, waxes, hydrocarbons, fatty acids, alcohols, esters, surfactants, fragrances, purified water, etc. Can be done.

In addition, as long as the above extract suppresses the production of melanin, the external preparation for skin includes various components usually used for external preparation for skin, such as moisturizers, oily substances, surfactants, thickeners, and neutralizers. , Preservatives, antioxidants, pigments, fragrances, UV absorbers, cell activators, other medicinal ingredients and the like may be included.

According to the present invention, the production of melanin can be safely suppressed.
In detail, it was found that the active ingredient capable of suppressing the production of melanin is an extract derived from olive fruits that has been used and ingested by human beings for many years, so that it has little effect even when used on the human body for a long period of time. It is safe and safe.

The inventor has also found that by extracting an extract from an immature olive fruit, melanin production can be effectively suppressed as compared with an extract extracted from a mature olive fruit. As a result, melanin can suppress the accumulation of melanin in epidermal keratinocytes, and can effectively prevent the formation of spots and the like.

An aspect of the present invention is characterized in that the extract is extracted from the residue remaining after squeezing olive oil from the immature olive fruit.
According to the present invention, since the active ingredient of the melanin production inhibitor can be extracted from the squeezed residue (residue) of the olive oil that was previously discarded as waste, the residue remaining after squeezing the olive oil from the olive fruit is effective. It can be utilized, the amount of industrial waste can be reduced, and it can contribute to society.

The above-mentioned extract is an olive fruit-derived extract obtained by extracting from the immature olive fruit residue with a solvent or the like, and the residue is immersed in an appropriate extraction solvent. Examples thereof include a method of recovering the solvent later, a method of immersing the residue in a solvent within the boiling point range of the solvent from room temperature and stirring, and then recovering the solvent. The solvent is preferably water or a 20 to 70% ethanol aqueous solution, and the temperature is preferably 20 ° C. or higher and 80 ° C. or lower, and more preferably 20 ° C. or higher and 40 ° C. or lower.

Further, as an aspect of the present invention, the active ingredient is characterized in that it inhibits the activity of tyrosinase.
According to the present invention, the activity of tyrosinase, which oxidizes tyrosine to produce melanin precursors such as dopaquinone, can be inhibited, so that the production of melanin in melanocytes can be suppressed and melanin can be prevented from being delivered to keratinocytes. This makes it possible to prevent melanin, which causes spots and the like, from accumulating in keratinocytes.

Further, as an aspect of the present invention, the active ingredient inhibits the biosynthesis of tyrosinase.
According to the present invention, the biosynthesis of tyrosinase, which produces dopaquinone, which is a melanin precursor, can be inhibited, so that the production of melanin in melanocytes can be suppressed. Therefore, it is possible to prevent melanin, which causes spots and the like, from accumulating in keratinocytes.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a safe melanin production inhibitor capable of suppressing melanin production, a skin cosmetic containing the melanin production inhibitor, and an external preparation for skin.

Explanatory drawing of olive fruit ripeness index. A bar graph showing the results of a melanin production suppression evaluation test using an extract derived from olive fruit. A bar graph showing the results of a tyrosinase activity inhibition test using an extract derived from olive fruit. Explanatory drawing which shows the change of the expression level of tyrosinase by the extract derived from olive fruit. Explanatory drawing which shows the change of the expression level of tyrosinase by the extract derived from olive fruit.

The melanin production inhibitor according to the present embodiment contains an extract extracted from immature olive fruit as an active ingredient. More specifically, an extract extracted from the squeezed residue (residue) of olive fruit obtained by squeezing olive oil from olives harvested in an immature state is used.

In this embodiment, the residue which is the squeezed residue of olive oil squeezed from the immature olive fruit is used, but it does not necessarily have to be the squeezed residue of the olive fruit, and the immature olive fruit itself or the unripe olive fruit itself. The mature olive fruit may be crushed, or the olive fruit may be kneaded into a paste, and the state before squeezing the olive oil may be used.

However, it has been previously considered that an active ingredient capable of suppressing the production of melanin can be obtained by extracting from the squeezed residue (residue) of olive fruit obtained by squeezing olive oil and using the extract. In addition, by using the residue from which olive oil has been removed, the active ingredient that suppresses the production of melanin can be extracted efficiently and in high concentration, and the squeezed residue of olive fruits, which was previously a waste, is effective. It can be utilized.

Here, the immature olive fruit refers to an olive fruit whose skin is green or yellowish green, and generally refers to an olive fruit that can be harvested from the end of August to the beginning of October in the Northern Hemisphere.
Specifically, in the olive fruit maturity index indicating the maturity of olive fruits (see FIG. 1), olive fruits having a color score of 0 to 2 are used. In the present embodiment, olive fruits having a color score of 0 to 2 are harvested to obtain an extract. However, even if olive fruits having a color score of 3 are used, the effect is weakened. A similar effect can be obtained.

The extract extracted from the immature olive fruit is extracted by the following procedure. First, the squeezed residue (residue) obtained by squeezing olive oil from immature olive fruits is collected and immersed in a 50% ethanol aqueous solution at room temperature for 7 days. Then, using a vacuum solid-liquid separator (evaporator), the squeezed residue separated from the solvent and biological water from the ethanol aqueous solution containing the squeezed residue was collected as a solid and dried to obtain a powder (dry residue). This dry residue was prepared to a predetermined concentration using a 50% aqueous ethanol solution.

In the extraction method of extracting the extract from the olive fruit, the squeezed residue obtained by squeezing olive oil is immersed in an aqueous ethanol solution, and then the solid matter separated from the solvent or the like is dried, but this method is not always used. It is not necessary to extract, and the residue left after squeezing olive oil from the olive fruit may be dried into a powder. The extract may be extracted from the state before the olive oil is squeezed, instead of the squeezed residue obtained by squeezing the olive oil.

The solvent used for extraction does not have to be limited to ethanol as long as it can extract the active ingredient derived from olive fruits, and any solvent that is usually used can be used. For example, alcohols such as water, methanol, ethanol and propylene glycol, organic solvents such as chloroform, dichloroethane, carbon tetrachloride, acetone, ethyl acetate and hexane, etc., can be used alone or in combination.

It was found that the extract derived from the immature olive fruit thus obtained suppressed the production of melanin.
Further, when the extract extracted from the immature olive fruit or the extract extracted from the mature olive fruit extracted from the mature olive fruit was mixed with a liquid containing tyrosinase and the tyrosinase activity was compared, the activity of tyrosinase was increased. I was able to confirm that there was a difference. This was found to inhibit the activity of tyrosinase, which produces the melanin precursor dopaquinone.

Further, with respect to a medium containing an extract derived from an immature olive fruit extracted from an immature olive fruit, a medium containing a mature olive extract extracted from a mature olive fruit, or a medium containing nothing. By culturing B16F10 melanoma cells and comparing the expression levels of tyrosinase, it was found that the cells have an inhibitory effect on the expression of tyrosinase.

In this way, the extract derived from immature olive fruits suppresses the accumulation of melanin in epidermal keratinocytes by suppressing the production of melanin, and effectively prevents the formation of spots and the like. can do. In addition, since this effect can exert a whitening effect and an effect of preventing skin aging, it can be used, for example, as a skin cosmetic or an external preparation for skin.

The properties of the external preparation for skin or cosmetic for skin may be liquid, gel, cream, semi-solid, solid, stick, powder, etc., and may be milky lotion, cream, lotion, etc. It can be used as a skin cosmetic such as a beauty liquid, a facial mask, a washing pigment, and a makeup cosmetic.

In addition, the skin external preparation or skin cosmetic according to the present embodiment is used in the production of a normal skin external preparation or skin cosmetic as long as the effect of suppressing the production of melanin contained in the extract is not impaired. Main agent, auxiliary agent or other ingredients, such as astringent, bactericidal / antibacterial agent, preservative, metal ion sequestering agent, pH adjuster, chelating agent, refreshing agent, emulsifier, foaming agent, thickener, deodorant / Deodorants, UV absorbers, moisturizers, cell activators, anti-inflammatory / anti-allergic agents, whitening agents, enzymes, hormones, fats and oils, waxes, hydrocarbons, fatty acids, alcohols, esters, surfactants , Perfume, purified water, etc. can be blended.

Furthermore, the external preparation for skin may be any kind as long as it does not interfere with the effect of suppressing the production of melanin contained in the extract, and various components usually used for external preparation for skin, such as moisturizers and oily substances. , Surfactants, thickeners, neutralizers, preservatives, antioxidants, pigments, fragrances, UV absorbers, cell activators, other medicinal ingredients and the like.

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

<How to select immature olive fruits>
Olive fruits were harvested when they were not yet ripe and selected according to their maturity.

More specifically, olive fruits harvested between late August and early October in the Northern Hemisphere were sorted and selected according to their maturity. Here, the olive fruit ripeness index shown in FIG. 1 was used as a criterion for determining the ripeness of the olive fruit. Specifically, among the harvested olive fruits, those having a color score of 0 to 2 in the olive fruit maturity index were selected as immature olive fruits in the present embodiment.

Here, an olive fruit maturity index used for sorting according to the maturity of olive fruits will be described.
The olive fruit ripeness index is an index for selecting the ripeness of olive fruits by the color of the fruit skin and flesh of the olive fruits, and is used to keep the quality of the olive oil produced constant and to guide the future. Has been done. Specifically, by recording the maturity distribution of harvested olive fruits based on this index and recording the characteristics of olive oil made from these olive fruits, the maturity distribution of olive fruits and olives It is related to the quality of oil.

As shown in FIG. 1, this olive fruit maturity index classifies the color of olive fruit and the color of flesh into eight stages (color score 0 to 7) according to the maturity. The higher the color score, the higher the maturity.
Specifically, as shown in FIG. 1, a color score of 0 refers to a state in which the olive fruit skin is deep green, and a color score 1 is a state in which the olive fruit skin is yellow or yellowish green. Color score 2 indicates that the skin of the olive fruit is yellowish green, and about half of the whole is reddish spots or purple.

Color score 3 indicates that approximately half of all olive fruits are red or bluish purple, and color score 4 indicates that the olive fruit skin is pale bluish purple to black and the flesh is white or greenish white. Color score 5 means that the olive fruit skin is bluish purple or black, and about half or less of the flesh is changed to purple, and color score 6 is that the olive fruit skin is bluish purple or black. More than half of the flesh is purple and the center is changing to purple, and the color score 7 is that the olive fruit skin is dark purple or black, and the flesh is almost purple to the center. Refers to the state.

<How to select mature olive fruits>
Similarly, for the mature olive fruits used as comparative experiments, among the olive fruits harvested between the end of October and the beginning of December in the Northern Hemisphere, those having a color score of 4 to 7 in the olive fruit maturity index are compared. It was selected as the target mature olive fruit.

<Extraction method of olive fruit-derived extract>
The sorted immature or mature olive fruits were washed with water, coarsely crushed, and kneaded into a paste. Next, the olive oil and the squeezed residue are separated from the pasted olive fruit by centrifugation, and the squeezed residue of the separated olive fruit is dried and then crushed into a 10-fold volume 50% aqueous ethanol solution. , Soaked at room temperature for 7 days.

Then, the solvent was removed from the filtered extract using an evaporator (manufactured by Nippon Buch Co., Ltd.), and the obtained solid substance was further dried into a powder. The dry powder thus obtained was prepared to 100 mg / ml using a 50% aqueous ethanol solution, and used as an extract derived from the squeezed residue of olive fruits in the following experiments.

Test Example 1: Melanin production suppression test
A method for measuring the melanin production inhibitory effect of an extract extracted from olive fruit pomace will be described.

<Culture method>
B16F10 melanoma cells were seeded 0.8 × 10 ⁴ cells / well on a 96-well plate supplemented with 5% FBS (fetal bovine serum) (S1820, manufactured by Biogest) / DMEM medium (5915, manufactured by Nissui Pharmaceutical Co., Ltd.). , Extracts extracted from squeezed dregs of immature or mature olive fruits (prepared to be 62.5, 125 μg / ml, 250, 500 μg / ml, respectively) or β-arbutin (0) after culturing for 6 hours. .14 mg / ml: A0522, manufactured by Tokyo Kasei Kogyo Co., Ltd.) was added to the medium containing 1 mM Theophylline (5% FBS / DMEM), and the cells were cultured for 48 hours.

After culturing for 48 hours, the medium was exchanged again with the extract extracted from the squeezed residue of immature or mature olive fruits or 1 mM Theophylline-containing medium (5% FBS / DMEM) supplemented with β-arbutin, and cultured for 24 hours.

<Quantification of melanin amount>
After culturing in 1 mM Theophylline-containing medium (5% FBS / DMEM) supplemented with β-arbutin or an extract extracted from the pomace of immature or mature olive fruit for 24 hours, it was washed twice with HBSS (+) buffer. 4 μM Hoechst33342 (H1399, manufactured by Thermo Fisher Scientific Co., Ltd.) in HBSS (+) was added, incubated for 30 minutes, and DNA was stained.

After that, the cells were washed twice again with HBSS (+), fluorescence was measured at 320 nm for excitation light (Excitation, Ex) and 465 nm for fluorescence (emission, Em), and the number of DNAs per well was calculated. Further, 50 μl / well of 10% DMSO / NaOH (4M) was added, cells of all wells were disrupted using a handy Sony kata, and the absorbance at 405 nm was measured.

The absorbance at 405 nm measured in this way was compared with the calibration curve prepared using synthetic melanin, the amount of melanin in each well was quantified, and the ratio of the amount of melanin to the fluorescence intensity of DNA was calculated. The result is shown in FIG.

The amount of melanin synthesized was represented by the amount of melanin / the fluorescence intensity of Hoechst33342 (μg melanin / Fluorescence intensity). ( ^* P <0.05, ^** p <0.01, ^*** p <0.001).
Here, the fluorescence intensity of Hoechst33342 was calculated as the ratio of the fluorescence intensity at 320 nm of excitation light (Excitation) to the fluorescence intensity at 465 nm of fluorescence (emission, Em).

FIG. 2 is a bar graph showing the amount of melanin produced with respect to the concentration of the extract extracted from the pomace of immature or mature olive fruits added to the medium. The vertical axis shows the amount of melanin, and the horizontal axis shows the concentration of the extract extracted from the pomace of immature or mature olive fruits.

As described above, the amount of melanin is calculated by dividing the value calculated from the calibration curve prepared using synthetic melanin and the absorbance at 405 nm by the fluorescence intensity (Ex320 / Em465) in each well. The amount of melanin in the medium to which the extract extracted from the pomace of immature or mature olive fruits was not added was set to 100.0.

According to FIG. 2, it can be seen that the amount of melanin produced decreases as the concentration of the extract increases in the medium to which the extract extracted from the pomace of immature or mature olive fruit is added. In addition, the medium to which the extract extracted from the squeezed residue of the immature olive fruit was added suppressed the production of melanin more than the medium to which the extract extracted from the squeezed residue of the mature olive fruit was added. Do you get it.

For example, when the concentration of the extract extracted from the squeezed residue of immature or mature olive fruit is 0.5 mg / ml, the extract extracted from the squeezed residue of mature olive fruit (hereinafter referred to as the extract derived from mature olive fruit). In the medium to which the above) was added, the amount of melanin was 24.2 μgmelanin / Fluorescenseincentity, whereas the extract extracted from the squeezed residue of the immature olive fruit (hereinafter referred to as the extract derived from the immature olive fruit) In the added medium, the amount of melanin was 0.9 μg melanin / Fluorescencentency, and it was found that the extract derived from immature olive fruit could suppress the production of melanin more than the extract derived from mature olive fruit.

It was compared with β-arbutin, which inhibits the activity of tyrosinase, which acts on melanin synthesis. As a result, the medium supplemented with the extract derived from immature or mature olive fruits at substantially the same concentration (0.13 mg / ml) as β-arbutin (0.14 mg / ml) suppressed the production of melanin. It turned out that there was.
From the above, it was found that the extract extracted from the pomace of immature olive fruits has a function as a melanin production inhibitor.

Test Example 2: Tyrosinase activity inhibition experiment
The experiment of inhibiting the activity of tyrosinase by the extract extracted from the pomace of olive fruit is described.
<Recovery of crude enzyme solution stock solution>
After culturing 10% B16F10 melanoma cells in FBS / DMEM for 72 hours, add 6 ml of PBS (-) on ice and wash, then add 600 μl of Assaybuffer containing 0.1% Triton, and use a cell scraper. The cells were collected in an Eppen tube. The Eppen tube was centrifuged (10000 G, 4 ° C., 5 min), and the supernatant was collected. This was used as a crude enzyme solution stock solution in the following experiments.

<Tyrosinase activity inhibition experiment>
Next, 50 μl of Assaybuffer, 50 μl of crude enzyme solution stock solution diluted 5-fold with Assaybuffer containing 0.1% Triton, and 100 μl of 5.0 mM L-dopa (14211-81, manufactured by Nacalai Tesque, Inc.). It was mixed and the absorbance at 550 nm was measured. Then, after incubating at 37 ° C. for 2 hours, the absorbance at 550 nm was measured again, and the crude enzyme solution stock solution was diluted so that the average value of the absorbance was 0.3. The crude enzyme solution stock solution diluted in this way was used as a tyrosinase enzyme solution.

Then, 100 μl of 5.0 mM L-dopa was added to the 96-well plate, and the extracts were extracted from the pomace of immature or mature olive fruits (62.5, 125, 250, 500 μg / ml, respectively). ), 50 μl of Assaybuffer mixed with 0.1% Triton-containing Assaybuffer (referred to as tyrosinase enzyme solution (-)), agitated with TwinMIXERTM-282 (manufactured by AS ONE Corporation) for about 110 seconds, and 550 nm. The absorbance in was measured. Then, it was incubated at 37 ° C. for 2 hours, and the absorbance measurement (550 nm) was performed again. A mixed solution of 5 wells was prepared for each extract concentration, and the absorbance was measured.

Similarly, 100 μl of 5.0 mM L-dopa is added to a 96-well plate to obtain extracts extracted from the pomace of immature or mature olive fruits (62.5, 125, 250, 500 μg / ml, respectively). The mixture was mixed with 50 μl of tyrosinase enzyme solution (referred to as tyrosinase enzyme solution (+)) and agitated with TwinMIXERTM-282 for about 110 seconds, and the absorbance at 550 nm was measured (Abs (0 h)). To do). Then, the mixture was incubated at 37 ° C. for 2 hours, and the absorbance was measured again (550 nm) (referred to as Abs (2h)). A mixed solution of 5 wells was prepared for each extract concentration, and the absorbance was measured.

For comparison, a 96-well plate was mixed with 100 μl of 5.0 mM L-dopa, 50 μl of 300 μg / ml β-arbutin, and 50 μl of tyrosinase enzyme solution (referred to as tyrosinase enzyme solution (+)). The agitation was carried out with TwinMIXERTM-282 for about 110 seconds, and the absorbance at 550 nm was measured (referred to as Abs (0h)). Then, the mixture was incubated at 37 ° C. for 2 hours, and the absorbance was measured again (550 nm) (referred to as Abs (2h)). As a comparison target, a mixed solution was prepared with 5 wells, and the absorbance was measured.

In addition, as a control (ctl), 100 μl of 5.0 mM L-dopa was added, and extracts extracted from the pomace of immature or mature olive fruits (62.5, 125, 250, 500 μg / ml, respectively). 50 μl of () and 50 μl of Assaybuffer were mixed, and agitation was performed with TwinMIXERTM-282 for about 110 seconds to measure the absorbance at 550 nm. Then, it was incubated at 37 ° C. for 2 hours, and the absorbance measurement (550 nm) was performed again. Similarly, a mixed solution is prepared with 5 wells, and the absorbance is measured.

The inhibition rate of tyrosinase activity is calculated from the difference in absorbance before and after the incubation thus obtained. The result is shown in FIG.
More specifically, the inhibition rate of tyrosinase activity is the absorbance (Abs (2h)) measured after incubating at 37 ° C. for 2 hours in each well containing the tyrosinase enzyme solution (+) and the tyrosinase enzyme solution (−). ) To the absorbance measured before incubating (Abs (0h)), and the difference is divided by the difference between Abs (2h) and Abs (0h) in the control (ctl) to obtain a percentage. Calculated.

In addition, FIG. 3A shows the tyrosinase activity inhibition rate with respect to the concentration of the extract derived from mature or immature olive fruit in the tyrosinase enzyme solution (-), and FIG. 3B shows the tyrosinase enzyme solution (+). ) Indicates the rate of inhibition of tyrosinase activity with respect to the concentration of the extract derived from mature or immature olive fruit.

First, the tyrosinase activity inhibition rate in the tyrosinase enzyme solution (-) will be described.
As shown in FIG. 3A, the inhibitory activity of tyrosinase is 90% or more with respect to both the extracts derived from mature or immature olive fruits. From this, it can be seen that melanin is not synthesized in the well containing the extract derived from the mature or immature olive fruit. It is considered that this is because the well containing the tyrosinase enzyme solution (-) does not contain tyrosinase that produces melanin from L-dopa.

Next, the tyrosinase activity inhibition rate in the tyrosinase enzyme solution (+) will be described.
As shown in FIG. 3 (b), among the wells containing the tyrosinase enzyme solution (+), the wells to which the extract derived from mature olive fruit is added hardly inhibits the activity of tyrosinase. It turned out. That is, it is considered that melanin is produced from L-dopa by the activity of tyrosinase.

On the other hand, among the wells containing the tyrosinase enzyme solution (+), the wells to which the extract derived from the immature olive fruit was added were found to inhibit the activity of tyrosinase (inhibition rate). 10-35%). That is, it is considered that melanin synthesis is suppressed.

From the above, it was found that the extract derived from immature olive fruit has an inhibitory effect on the activity of tyrosinase, which is not found in the extract derived from mature olive fruit. Therefore, by using the extract derived from immature olive fruit in skin cosmetics, external preparations for skin, etc., the activity of tyrosinase in melanocytes can be inhibited and the production of melanin can be suppressed, which has a whitening effect.

Test Example 3: Method for measuring the expression level of tyrosinase
A method for measuring the expression level of tyrosinase by an extract of olive fruit pomace will be described.

<Recovery of all proteins>
B16F10 melanoma cells were seeded using 10% FBS / DMEM, cultured for 24 hours, and then prepared to extracts derived from immature or mature olive fruits (0.0 (corresponding to ctl) and 250,500 μg / ml, respectively). ) Was replaced with 1 mM Theophylline / 10% FBS / DMEM medium, and the cells were further cultured for 24 hours. After 24 hours, the cells were washed once with PBS (−), 200 μl of Assaybuffer containing 0.1% Triton was added, and cells were collected in an Eppen tube using a cell scraper. Then, the cells were disrupted using a Handy Sony Kata, and then centrifuged (4 ° C., 12000 G, 10 min) to collect the supernatant. After diluting this supernatant 5-fold with PBS (−), the amount of protein was determined using BCAproteinAssayKit.

<Western blotting method>
Then, the amount of tyrosinase in the cultured B16F10 melanoma cells was measured by Western blotting.

More specifically, all proteins of B16F10 melanoma cells were separated by electrophoresis on SDS polyacrylamide gel, then blotting on a nitrocellulose membrane, and then the membrane was immersed in 1% BSAinPBS (-) at room temperature for 1 hour. It was shaken and blocked. Then, the cells were washed with 0.05% PBS-T, and the primary antibody was immersed in Anti-Tyrosinase Polyclonal Antibody, BiotinConjugated (bs-0819R-Biotin) (Bioss) (1: 250) overnight. Then, after washing with 0.05% PBS-T, immersion shaking was performed in the secondary antibody Streptavidin, HRPConjugate (DY998, manufactured by R & DSYSTEMS) (1: 2000) for 1 hour.

Then, after washing with 0.05% PBS-T, the band was detected using Ezblue, and an image was taken (FIG. 4). The captured image was analyzed using imageJ. In the analysis by imageJ, the ratio of the band concentration of tyrosinase to the control β-actin is calculated. The analysis result is shown in FIG.

Regarding β-actin, which is a control, the primary antibody was Beta-ActinRabbitmAb (lotNo.8, manufactured by CellSignalingTechnology) (1: 10000), and the secondary antibody was Goatanti-RabbitIgG (H + L), SensoryAnisite, H.R. (1: 20000) was used.

As shown in FIGS. 4 and 5, in B16F10 melanoma cells cultured with the extract derived from mature olive fruit added, compared with the case where the extract derived from mature olive fruit was not added (corresponding to ctl). Although the expression level of tyrosinase was decreased, there was no change due to the difference in the concentration of the extract derived from mature olive fruits.

On the other hand, in B16F10 melanoma cells cultured with the addition of an extract derived from immature olive fruit, when the extract derived from immature olive fruit was not added (ctl (concentration of extract derived from mature olive fruit). Corresponds to 0.0 μg / ml)), the expression level of tyrosinase was decreased. In addition, the higher the concentration of the extract derived from the immature olive fruit, the more the expression level of tyrosinase tended to decrease significantly.
From this, it was found that the extract extracted from the pomace of immature olive fruits has an effect of inhibiting the biosynthesis of tyrosinase.

From this, the extract derived from olive fruit has the effect of suppressing the expression of tyrosinase that contributes to the production of melanin in melanocytes, and the extract derived from immature olive fruit is extracted from mature olive fruit. It was found that the expression of tyrosinase can be suppressed more than that of the product.

Claims (6)

A melanin production inhibitor containing an extract derived from immature olive fruits as an active ingredient. The melanin production inhibitor according to claim 1, wherein the extract is extracted from a residue remaining after squeezing olive oil from the immature olive fruit. The melanin production inhibitor according to claim 1 or 2, wherein the active ingredient inhibits the activity of tyrosinase. The melanin production inhibitor according to any one of claims 1 to 3, wherein the active ingredient inhibits the biosynthesis of tyrosinase. A skin cosmetic containing the melanin production inhibitor according to any one of claims 1 to 4. An external preparation for skin containing the melanin production inhibitor according to any one of claims 1 to 4.