JP7399730B2 - Keratinocyte PAR-2 expression inhibitor and melanocyte dendrite formation inhibitor - Google Patents

Description

The present invention suppresses melanin uptake by suppressing the formation of dendrites in melanocytes and further suppressing the expression of protease activated receptor-2 (hereinafter referred to as PAR-2) in keratinocytes. The present invention relates to an invention that prevents and improves pigmentation in the skin.

The skin on the face and hands has more opportunities to be exposed to ultraviolet rays than other parts of the body, resulting in sunburn, which is temporary pigmentation caused by ultraviolet rays, and local pigmentation that occurs with age, such as senile pigmentation spots. stains are likely to occur. Pathological images of these pigmented areas show excessive melanin deposition throughout the epidermis, which begins with the production of melanin in melanocytes, transport of melanin to the tips of dendrites, release to the outside of the cells, and melanin taken up by keratinocytes. This is thought to be caused by an imbalance in the process by which the skin ultimately becomes dirt and is excreted from the skin.

The mainstream method for suppressing melanin production in melanocytes is to inhibit the activity of tyrosinase, which is the key enzyme for melanin production, and various whitening active ingredients such as kojic acid and arbutin have been developed. On the other hand, some tyrosinase activity-inhibiting components have a mechanism of action that inhibits tyrosinase activity by binding to tyrosinase instead of the substrate tyrosine. It is known to induce cell damage and cause vitiligo vulgaris (Non-Patent Document 1). Therefore, ingredients that have a tyrosinase activity inhibitory effect may have this disorder, and while tyrosinase activity inhibition can be expected to have a high melanin production suppressing effect, safety concerns remain.

In recent years, efforts have been made to develop ingredients that focus on the transfer of melanin from melanocytes to keratinocytes. The delivery process of melanin can be broadly classified into two processes: release from melanocytes and uptake by keratinocytes. It is known that PAR-2 expressed in keratinocytes controls melanin uptake in keratinocytes, and Patent Document 1 describes that an extract of Safflower of the family Asteraceae, genus Safflower, has an inhibitory effect on melanin uptake, and the mechanism thereof is explained. It has been reported that it inhibits PAR-2 activity (Patent Document 1)

On the other hand, in melanocytes, melanin produced within the cells is transported to the tips of dendrites and delivered to keratinocytes. Recently, it has been reported that the number of dendrites of melanocytes in blemished areas is significantly higher than in non-spotted areas, and this also suggests that reducing the number of dendrites per melanocyte is an effective way to improve blemishes. It is assumed that this is an important mechanism (Non-patent Document 2)

Therefore, ingredients that have the effect of suppressing dendrite formation, which suppresses the transport and release of melanin from melanocytes, and the effect of suppressing melanin uptake by keratinocytes, have an excellent preventive effect against excessive melanin deposition inside the epidermis. Furthermore, by suppressing the release and uptake of melanin, melanin is excreted as plaque, which is thought to lead to improvement of pigmentation. However, there are few reports of such components.

Niihime is a new citrus fruit that was accidentally discovered in Atashika-cho, Kumano City, and is said to be not an ancient Japanese wild species, but a hybrid variety that is the natural hybridization of tachibana and Unshu mandarin (Non-patent Document 3). The origin, Satsuma mandarin peel extract, is known to have a whitening effect, and one of its mechanisms of action is reported to have a high tyrosinase activity inhibition effect (Patent Document 2). From this, it is inferred that Nihime pericarp extract also has a high tyrosinase activity inhibiting effect.

On the other hand, Patent Document 3 discloses that hesperidin, a flavonoid that is abundantly contained in citrus fruits, has a tyrosinase activity inhibiting effect, and that hesperidin is abundantly contained in immature fruit peels. It has been shown to be effective as a whitening ingredient.

It has also been reported that the immature pericarp of Niihime contains a large amount of hesperidin (Non-Patent Document 3). Therefore, the use of Niihime's immature peel extract can be expected to have the same whitening effect as the conventionally used Tachibana and Satsuma mandarin extracts, but as mentioned above, ingredients that inhibit tyrosinase activity are not common. As the risk of causing vitiligo cannot be completely ruled out, safety concerns remain.

On the other hand, it is known that the amount of flavonoids such as hesperidin decreases as citrus fruit matures, and similar results have been shown for Niihime (Non-Patent Document 3). Therefore, it is assumed that the use of ripe fruit peel extracts can reduce the risk of vitiligo due to the inhibitory effect on tyrosinase activity, but on the other hand, a high whitening effect cannot be expected due to the loss of the inhibitory effect on tyrosinase activity. The problem arises that it disappears.

JP2017-100960 JP2006-089452 JP2005-132793

New Dermatology Seminar Shosuke Ito 2017 Volume 127 Issue 2 p. 155-160 Japanese Society of Photodermatology 1st Academic Conference Abstracts P5 General Presentation Three-dimensional structural analysis of melanocytes and keratinocytes in senile pigment spots Miyake, Y. Characteristics of flavonoids in niihime fruit-a new sour citrus fruit. Food Science and Technology Research, 12(3), 186-193, 2006

Under these circumstances, the inventors measured the inhibitory effect on tyrosinase activity in the peels of fully ripened Tachibana, Satsuma mandarin, and Niihime, and found that the tyrosinase activity was relatively high in the peel extract of Tachibana and Satsuma mandarin, which is thought to be the origin. Although the inhibitory effect remained, it was discovered that the inhibitory effect of Niihime peel extract on tyrosinase activity was much lower than that of Tachibana and Satsuma mandarin peel extracts (Figure 1).

As mentioned above, the whitening effect of the ripe Nihime peel extract, which has a low tyrosinase activity inhibition effect, was completely unexpected, but as a result of intensive study by the inventors, it was found that the ripe Nihime peel extract has melanocyte dendrites. The present invention was completed by discovering an inhibitory effect on cell formation and an inhibitory effect on PAR-2 expression in keratinocytes.

The object of the present invention is to find a component that suppresses both the transport and release of melanin in melanocytes and the uptake of melanin in keratinocytes without strongly suppressing tyrosinase activity in melanocytes.

The above problem was solved by using a ripe Niihime pericarp extract.

According to the present invention, ripe Niihime pericarp extract suppresses melanin transport and release by suppressing dendrite formation in melanocytes, and further suppresses melanin uptake by suppressing PAR-2 expression in keratinocytes. By doing so, it is possible to inhibit the transfer of melanin between melanocytes and keratinocytes and improve pigmentation such as sunburn, senile pigment spots, and inflammatory pigment spots. In addition, the ripe Niihime pericarp extract does not strongly inhibit tyrosinase activity, so it is safe for the skin.

Graph showing the tyrosinase activity inhibitory effect of ripe orange peel extract, ripe unshiu orange peel extract, and ripe Niihime peel extract Photo showing the inhibitory effect of ripe Niihime pericarp extract on dendrite formation on melanocytes Photo showing the suppressive effect of ripe Niihime pericarp extract on keratinocytes on PAR-2 gene expression

The ripe Nihime pericarp extract of the present invention can be extracted from the pericarp of Citrus tachibana/reticulata. Here, fully ripe Niihime pericarp refers to the state in which the blue part of the Nihime pericarp has disappeared and the entire pericarp has turned yellow. Niihime fruit is usually harvested from early October to early November, and the skin of the fruit is blue in appearance. After that, the ripening of the fruit progresses, and the pericarp of fruits picked after mid-December is in a fully ripe state.For example, the pericarp of Niihime, which is picked from that period until the spring of the following year, remains as a mature pericarp. In addition to this, there is no problem in using the pericarp obtained by harvesting the fruit in its green state and then leaving it in its natural state or artificially ripening it using ethylene gas or the like. The preparation of the extract mentioned here is not particularly limited, and for example, it may be added to water, various suitable organic solvents, or mixed solvents thereof, and extracted at low temperature and/or under heating. can be used.

The amount of each agent of the ripe Nihime pericarp extract relative to the entire composition is not particularly limited as long as it is an effective amount, but it is preferably 0.0001 to 1% by mass in terms of dry mass, more preferably 0.001% by mass. ~0.1% by mass.

Examples of extraction solvents include, but are not limited to, water; lower monohydric alcohols such as methyl alcohol and ethyl alcohol; liquid polyhydric alcohols such as glycerin, propylene glycol, and 1,3-butylene glycol; and ketones such as acetone and methyl ethyl ketone. One or more of the following can be used: alkyl esters such as ethyl acetate; hydrocarbons such as benzene and hexane; ethers such as diethyl ether; and halogenated alkanes such as dichloromethane and chloroform. Among these, one or a mixed solvent of two or more of water, ethyl alcohol, and 1,3-butylene glycol (1,3 BG, hereinafter referred to as BG) is particularly suitable.

The ripe Niihime pericarp extract obtained under the above conditions may be used as an extracted solution, but if necessary, it may be further processed such as filtration, concentrated, and powdered before being used as appropriate. be able to.

Examples of the preparation and efficacy tests of the ripe Niihime pericarp extract in the present invention will be shown below, but it goes without saying that the present invention is not limited to the examples described here.

<Sample preparation>
Niihime ripe fruit was provided by the Kumano City Furusato Promotion Corporation (picked in February). The pericarp was removed from the ripe Niihime fruit, dried, and ground in a mill mixer. 50% EtOH (10 times the original weight) was added thereto, and the mixture was extracted at 60°C for 3 hours. This is referred to as a ripe Niihime pericarp extract. As a comparative example, a ripe orange peel extract and a ripe unshu orange peel, which are considered to be the origin of Niihime, were used. "Ripe" in this case is synonymous with [0019].
In addition, in the next measurement test of tyrosinase activity, the above extract was dried and redissolved in the same amount of 50% BG. Solid content was approximately 4 mg/mL.

<Measurement of tyrosinase activity>
Using a 96-well plate, 10 μL of various extracts and 10 μL of tyrosinase (2500 units/mL, derived from mushroom, manufactured by Sigma) were mixed and incubated at 37° C. for 10 minutes. Thereafter, 75 μL of McIlvaine's buffer, 75 μL of L-tyrosine, and 75 μL of distilled water were added in this order, and the mixture was allowed to react at 37° C. for 20 minutes. After the reaction was completed, the absorbance at 475 nm was measured using a plate reader (infinite F200Pro, TECAN) (A value). For control, 50% BG was used instead of each extract. As a blank, the same amount of distilled water was used instead of L-tyrosine (B value). Tyrosinase activity inhibition rate was determined using the following formula.

From the graph shown in Figure 1, ripe orange peel extract and ripe unshiu orange peel extract have a high tyrosinase activity inhibitory effect, but ripe Niihime peel extract has a higher tyrosinase activity inhibitory effect than ripe orange peel extract. It was shown that the amount was significantly lower than that of the ripe Satsuma mandarin peel extract.

<Melanocyte dendrite formation suppression test>
Melanocytes derived from moderate donors were purchased from Thermo Fisher Scientific and used. Melanocytes (passage 2) were suspended in Medium 254 containing growth factors to a concentration of 2.0 x 10 ⁴ cells/mL, and 2 mL each was seeded in a 6-well plate. The cells were incubated at 37° C. under 5% CO ₂ for 24 hours, followed by medium exchange and addition of samples (Nihime pericarp extract and the solvent of the extract as a control). The sample was added to the medium at a concentration of 0.5%. Samples were added and the medium was exchanged once every 3 or 4 days, and 11 days after the cells were seeded, the dendrites were observed using a microscope (BZ-700X, Keyence) and photographs were taken.

From FIG. 2, it was confirmed that the number of dendrites per cell was clearly suppressed in melanocytes treated with the ripe Niihime pericarp extract compared to untreated melanocytes.

<PAR-2 gene expression confirmation test in keratinocytes>
Keratinocytes derived from newborn babies were purchased from Kurabo Industries and used. In this example, gene expression was used as an indicator as a method for confirming PAR-2 expression, but protein expression may also be used as an indicator. Keratinocytes (Passage 3) were suspended in Humedia KG-2 containing growth factors to a concentration of 5×10 ⁴ cells/mL, and seeded in 500 μL portions onto a 24-well plate. The cells were incubated at 37° C. under 5% CO ₂ for 24 hours, followed by medium exchange and addition of samples (Nihime pericarp extract and the solvent of the extract as a control). The sample was added to the medium at a concentration of 0.5%. Furthermore, after 24 hours of incubation, total RNA was extracted. Total RNA was extracted using the JanaBioscience Nucleic Acid Extraction Kit according to the attached protocol. cDNA was synthesized by reverse transcription using PrimeScript RT Reagent kit (TAKARA). Using the obtained cDNA as a template, the expression levels of PAR-2 and GAPDH (glyceraldehyde triphosphate dehydrogenase: used as a housekeeping gene) were measured using real-time PCR (7500 RealTime PCR system, Applied Biosystems).

The primers are sense primer for PAR-2 (5'-GCTCTCCTTTGCCGAAGTGT-3'), antisense primer (5'-TTTGAGGTGAGGGATACTTGCA-3'), sense primer for GAPDH (5'-CCACATCGCTCAGACACCAT-3'), antisense primer ( 5'-TGACCAGGCGCCCAATA-3') was used. SYBR SELECT MASTER MIX (Thermo Fisher Scientific) was used for the PCR reaction, and gene expression analysis was performed using the comparative Ct method.

From Figure 3, it was confirmed that the expression level of the PAR-2 gene in keratinocytes was suppressed by the treatment with the ripe Niihime pericarp extract compared to the control (when the gene expression level of the control was set to 100, the ripe Niihime pericarp extract items decreased to 68).

A composition according to the present invention was prepared below. Furthermore, all Niihime used were fully ripe.
<Formulation example 1> Lotion (mass%)
Niihime peel extract (solid content) 0.02
Polyoxyethylene sorbitan monolaurate (20E.0.) 1.5
1,3-butylene glycol 5.0
Glycerin 3.0
Preservatives/antioxidants Appropriate amount Fragrance Appropriate amount Purified water Remainder Total 100.0

<Formulation example 2> Emulsion (mass%)
Niihime peel extract (solid content) 0.01
Squalane 8.0
Vaseline 2.0
Beeswax 0.5
Sorbitan sesquioleate 0.8
Polyoxyethylene oleyl ether (20E.0.) 1.2
Carboxyvinyl polymer 0.2
Propylene glycol 0.5
Potassium hydroxide 0.1
Ethanol 7.0
Preservatives/antioxidants Appropriate amount Fragrance Appropriate amount Purified water Remainder Total 100.0

<Formulation example 3> Cream (mass%)
Niihime peel extract (solid content) 0.001
Beeswax 2.0
Stearyl alcohol 5.0
Stearic acid 8.0
Squalane 10.0
Self-emulsifying glyceryl monostearate 3.0
Polyoxyethylene cetyl ether (20E.0.) 1.0
Propylene glycol 5.0
Potassium hydroxide 0.3
Preservatives/antioxidants Appropriate amount Fragrance Appropriate amount Purified water Remainder Total 100.0

<Formulation example 4> Cream foundation (mass%)
Niihime peel extract (solid content) 0.05
Talc 5.0
Sericite 8.0
Titanium oxide 5.0
Color pigment Appropriate amount Polyglyceryl monoisostearate 3.0
Polyoxyethylene hydrogenated castor oil 1.5
Isotridecyl isononanoate 10.0
1,3-butylene glycol 5.0
Antioxidant Appropriate amount Preservative Appropriate amount Purified water Balance Total 100.0

<Formulation example 5> Sunscreen cosmetic (mass%)
Niihime peel extract (solid content) 0.0002
Titanium oxide 10.0
Zinc oxide 10.0
PEG-9 polydimethylsiloxyethyl dimethicone 1.5
Lauryl PEG-9 polydimethylsiloxyethyl dimethicone 1.5
Cyclopentasiloxane 20.0
Dimethicone 10.0
(Dimethicone/vinyl dimethicone) crosspolymer 0.5
Cetyl dimethicone 0.25
Glycyrrhetinic acid ester 0.05
Methylgluceth-20 1.0
1,3-butylene glycol 10.0
Sodium chloride Appropriate amount Antioxidant Appropriate amount Preservative Appropriate amount Purified water Balance Total 100.0

In order to confirm its usefulness for pigmentation, formulation examples 1, 2, and 3 containing ripe Niihime pericarp extract were used to test four female panelists in their 30s and 40s who are concerned about the visibility of pigmentation on their cheeks. We conducted a usage test for this. In the test, a preparation containing ripe Niihime peel extract was applied to one face, and a preparation (control) in which the ripe Niihime peel extract was removed and replaced with water was applied to the other face for one month after washing the face in the morning and evening. did. Before and after the test, photographs of the facial and cheek areas were taken using VISIA (registered trademark) Evolution (Canfield Scientific), and the average value of the stain score was calculated by image analysis. The rate of change in stain score was calculated by dividing the average value after use (B) of the group to which each formulation was applied by the average value before use (A).

From Table 1, it was confirmed that the pigmentation improvement effect was achieved by using the ripe Niihime pericarp extract.

Industrial availability

According to the present invention, ripe Niihime pericarp extract suppresses dendrite formation in melanocytes, thereby suppressing melanin transport and release, and further suppresses melanin uptake in keratinocytes, thereby promoting melanin formation between melanocytes and keratinocytes. By inhibiting the delivery of melanin, it is possible to reduce the amount of melanin in the epidermis and improve pigmentation such as sunburn, senile pigment spots, and inflammatory pigment spots. Furthermore, since the ripe Nihime pericarp extract does not strongly inhibit tyrosinase activity, it is safer for the skin than conventional whitening cosmetics that contain ingredients that inhibit tyrosinase activity.

Claims (3)

Melanocyte dendrite formation inhibitor containing ripe Niihime pericarp extract . A pigmentation improving agent containing ripe Niihime pericarp extract . A whitening cosmetic containing ripe Niihime fruit peel extract .