JP5053519B2 - Plant trypsin inhibitor - Google Patents

Description

The present invention relates to a serine protease activity inhibitor. In particular, the present invention relates to a serine protease activity inhibitor having an excellent antagonistic action against the activity of serine protease related to trypsin.

By activating protease activator receptor-2 (PAR-2) on the cell membrane of keratinocytes by serine protease, incorporation of melanosomes into keratinocytes is activated. The serine protease activity inhibitor of the present invention inhibits the activation of PAR-2 on the keratinocyte cell membrane, thereby suppressing the incorporation of melanosomes into keratinocytes and preventing the diffusion of melanin into the stratum corneum. By preventing the diffusion of melanin, the skin color is not darkened. Furthermore, a cosmetic for whitening having a strong whitening effect is provided by reducing the production amount of melanin itself in combination with an ascorbic acid derivative that is a material that directly inhibits melanin production in melanocytes. Serine protease is a protease having a serine residue in the active site, and representative ones include trypsin, chymotrypsin, thrombin, plasmin, and elastase.

Conventionally, whitening cosmetics have been screened for ingredients that have the action of inhibiting melanin production or reducing the produced melanin pigment in order to prevent or improve skin darkening due to ultraviolet rays and skin pigmentation such as spots and freckles. Has been formulated. For example, ascorbic acid, cysteine, and derivatives thereof, placental extracts, extracts from plants and algae are used.

However, ascorbic acid, cysteine, and derivatives thereof are susceptible to redox reactions and are unstable. In addition, when an effective amount of a placenta extract, an extract from a plant, or an algae is added, an unpleasant odor or color is imparted to the whitening cosmetics, and the whitening effect is not sufficient, resulting in many problems.

Recently, attempts have been made to suppress skin pigmentation by suppressing melanosome phagocytosis by keratinocytes as described in JP-T-2001-502360. Melanin is produced in melanosomes among melanocytes, which are phagocytosed by keratinocytes. This causes melanin to be distributed throughout the epidermis. By using a trypsin inhibitor, the melanosome phagocytosis by keratinocytes is suppressed and the diffusion of melanin is prevented.

However, even with this method, the trypsin inhibitor does not directly act on melanin production, so that it does not exhibit a sufficient effect as a whitening cosmetic.
JP-T-2001-502360

The problem of the present invention is that there is no safety problem such as skin irritation and skin sensitization, a strong skin melanin production inhibitory action, and a high whitening effect that inhibits the dispersion of produced melanin to keratinocytes The provision of cosmetics having

In the present invention, by solving the above problems, having a strong skin melanin production inhibitory effect, and inhibiting the dispersion of the produced melanin to keratinocytes, skin irritation and skin sensitization This led to the development of whitening cosmetics that are highly effective and have no safety problems.

That is, the present invention is a lily family Amadokora, leek genus, leguminous clara genus, kudzu genus, soybean genus, fuji genus, foenum glaecum genus, cucurbitaceae pumpkin genus, nigauri genus, loofah genus, aceae genus buckwheat, sericidae The present invention provides a whitening cosmetic composition containing a serine protease inhibitor and a melanin production inhibitor containing one or two or more compounds selected from the genus Falcon and Brassicaceae Alba.

The cosmetic of the present invention does not have safety problems such as skin irritation and skin sensitization, and has a high whitening effect by blending a melanin production inhibitor and a serine protease inhibitor.

Hereinafter, the present invention will be described in detail. Examples of the plant belonging to the genus Amidokolo, which exhibits serine protease inhibitory action for use in the present invention, are Amadochoro (Polygonatum odoratum (Mill.) Druce var.
pluriflorum (Miq.) Ohwi), P. humile Fisch., Naruko Yuri (P.
falcatum A. Gray) is a leek plant.
tuberosum R _OTLER ), but the leguminous Clara genus is Clara (Sophora flavescens Aiton), Enju (S. japonica L.) Isofuji (S.
tomentosa L.), as a genus of genus Kudzu (Pueraria)
lobata (Willd.) ohwi), soybeans (Glycine max Merr.), peas (G.soja Siebold et Zucc.) as genus plants, Nodafuji (Wistaria Floribunda (Willd.) DC.) , Yamafuji (W.brachybotrys Siebold et Zucc.), Foenum Graekum genus Koroha (Trigonella foenum-graecum L.), Cucurbita moschata (Duchesne) Poir. Var. Meloniformis (Cucurbita moschata (Duchesne) Poir. Carriere) Makino), pumpkin (C. pepo L.), but as a plant of the genus Nigauri, Momordica charantia L. (aka Nigauri), and as a plant of the genus Locha (Luffa aegyptiaca Mill.) Buckwheat (Fagopyrum esculentum Moench) and (F.dibotrys (D.Don) H.Hara) are the common buckwheat plants, Feniculum vulgare Mill. Brush (Sinapis alba L.) is available.

Moreover, the said plant can be used as an extract extracted under various low temperature and / or heating using various suitable organic solvents.

  The extraction solvent is not particularly limited. For example, water; lower monohydric alcohols such as methyl alcohol and ethyl alcohol; liquid polyhydric alcohols such as glycerin, propylene glycol, and 1,3-butylene glycol; acetone, methyl ethyl ketone, and the like. One type or two or more types of ketones; alkyl esters such as ethyl acetate; hydrocarbons such as benzene and hexane; ethers such as diethyl ether; halogenated alkanes such as dichloromethane and chloroform can be used. In particular, water, ethyl alcohol, or a mixed solvent of one or more of 1,3-butylene glycol is particularly suitable.

Melanin production inhibitors used in the present invention are ellagic acid and its derivatives and their salts, endothelin antagonists, ascorbic acid and its derivatives and their salts, glutathione and their derivatives and their salts, cysteine and its derivatives and their salts Commercially available reagents can be used for resorcin and its derivatives and their salts, hydroquinone and its derivatives and their salts. Various solvents from plants containing a large amount of these compounds, such as water; lower monohydric alcohols such as methyl alcohol and ethyl alcohol; liquid polyhydric alcohols such as glycerin, propylene glycol and 1,3-butylene glycol; acetone Extraction using 1 type or 2 types or more of alkyl esters such as ethyl acetate; hydrocarbons such as benzene and hexane; ethers such as diethyl ether; halogenated alkanes such as dichloromethane and chloroform; It can be used after purification. Furthermore, the above compound can be prepared by chemical synthesis.

Also, grabrizine, glabrene, liquiritin, isoliquiritin and licorice extract, placenta extract, carotenoids and animal and plant extracts containing these, irasen extract, sunflower seed extract, ibukitoranoo extract, age extract , Ogon extract, Ononis extract, Seaweed extract, Gokahi extract, Plant extract containing linoleic acid, Plant extract containing linolenic acid, Saisin extract, Hawthorn extract, Shirayuri extract, Peonies extract , Sempukuka extract, Sakuhakuhi extract, tea extract, Toki extract, sandalwood extract, beech extract, grape seed extract, hop extract, mica squid extract, saxifrage extract, Yokuinin extract are particularly limited Not at low temperature, for example using various suitable organic solvents It is extracted under heating from. Examples of the extraction solvent include water; lower monohydric alcohols such as methyl alcohol and ethyl alcohol; liquid polyhydric alcohols such as glycerin, propylene glycol, and 1,3-butylene glycol; ketones such as acetone and methyl ethyl ketone; and ethyl acetate. One or more of alkyl esters; hydrocarbons such as benzene and hexane; ethers such as diethyl ether; and halogenated alkanes such as dichloromethane and chloroform can be used. In particular, one, two or more mixed solvents of water, ethyl alcohol, and 1,3-butylene glycol are particularly suitable.

Lentiaceae Amadokolo, Amadokolo, Nymphaea lily, Leek genus leek, Lariaceae Clara genus Clara, Enju, Isofuji, Kudzu genus kudzu, Soybean genus soybean , Wild bean, nodafuji of the genus Fuji, Yamafuji, Koroha of the genus Forenum graekum, pumpkin of the cucurbitaceae pumpkin, pumpkin of the sun 0.0001-10.0 wt% is preferable, and the range of 0.01-1.0 wt% is most suitable for the blending amount of the plant extract of Fennel spp.

Ellagic acid and its derivatives and their salts, endothelin antagonists, ascorbic acid and its derivatives and their salts, glutathione and their derivatives and their salts, cysteine and its derivatives and their salts, resorcin and their use as melanin production inhibitors Derivatives thereof and salts thereof, hydroquinone and derivatives thereof, and salts thereof, glabrizine, glabrene, liquiritin, isoliquiritin and licorice extract containing them, placenta extract, carotenoids and animal and plant extracts containing these, irasen extract , Sunflower seed extract, Ibukitorano extract, Ages extract, Ogon extract, Ononis extract, Seaweed extract, Gokahi extract, Plant extract containing linoleic acid, Plant extract containing linolenic acid, Saishi Extract, hawthorn extract, white lily extract, peony extract, sempukuka extract, Sakuhakuhi extract, tea extract, tea extract, juniper extract, beech extract, grape seed extract, hop extract, squid extract In the case of extraction at room temperature, using a 1 to 1000-fold amount, especially 10 to 100-fold amount of a solvent by weight of a finely pulverized product after drying each material, Is preferably carried out at 0 ° C. or higher, particularly 20 ° C. to 40 ° C. for 1 hour or longer, particularly 3 to 7 days. Moreover, you may heat-extract at 60-100 degreeC for 1 hour.

Each of the above-mentioned extracts obtained under the above conditions may be used as an extracted solution. However, if necessary, it is necessary to appropriately use a concentrated and powdered product after filtration or the like. I can do it.

The blending amount of the melanin production inhibitor in the cosmetic of the present invention is preferably 0.0001 to 20.0% by weight, particularly 0.01 to 10.0% by weight in terms of the evaporated and dried content. .

In addition to the above essential components, the cosmetics of the present invention are aqueous components, oily components, plant extracts, animal extracts, powders, excipients, surfactants, oils, alcohols, pH adjusters, preservatives, antioxidants. It is prepared by mixing agents and thickeners, sweeteners, pigments, fragrances, and the like as necessary and blending appropriately. The dosage form of the cosmetic of the present invention is not particularly limited, and can be various dosage forms such as lotion, milky lotion, cream, pack, powder, spray, ointment, dispersion, and cleaning agent.

Examples of serine protease inhibitors according to the present invention, in particular, trypsin inhibitors, and whitening effects by melanin production inhibitors will be described below, and examples of prescriptions applied to cosmetics using the materials will be described. It goes without saying that the present invention is not limited to the embodiment described.

The plant extracts for measuring the trypsin inhibitory effect include the roots of Narcolili as a plant of the genus Amadocora, the seeds of leek as the plant of the genus Leek, the seeds of Clara as the plant of the genus Clariaceae, the flower of the genus Kuzu, Soybean seeds as plants, Nodafuji's ears as genus Fuji, Koroha seeds as Forenum glacum genus plants, Pumpkin seeds as cucurbitaceae genus pumpkins, Nigauri fruit as genus genus plants, Loofah seeds as seedlings, Tade Dry buckwheat seeds as genus buckwheat plants, fennel seeds as celebratory genus plants, and white seeds as cruciferous alba genus plants, pulverize them, leave them in 50% ethanol aqueous solution at room temperature for 1 week, and extract the extract . The extracted extract was prepared by measuring the evaporation residue to a concentration of 2%. Furthermore, an equal amount of the precipitation reagent described in 0023 was added to the plant extract prepared to a concentration of 2% to remove proteins to obtain a plant extract of 1% concentration.

(Measurement of trypsin inhibitory activity)
(Reagent)
Phosphate buffer solution: 0.1M-NaH ₂ PO ₄ and 0.1M-Na ₂ HPO ₄ are mixed at 5:25 to prepare PH7.4.
Substrate solution: 0.6 g of Hammerstein Cazein to 80 ml of 0.05M-Na ₂ HPO ₄
In addition, it is dissolved by heating in a boiling water bath. After cooling, adjust PH to 7.4.
Trypsin solution: 1: 250 Trypsin (DIFCO) dissolved in phosphate buffer to a concentration of 10 μg / ml
To do.
Precipitation reagent: Trichloroacetic acid, Na acetate, and acetic acid solution, 0.11M, 0.22M, 0.33M, respectively
Prepare the solution to a concentration.
Sample solution: Prepare by adding water or ethanol to a predetermined concentration.
(Measurement)
Add 0.1 ml of sample solution to 0.9 ml of casein substrate solution and mix. In addition, trypsin solution 2ml
Add and stir and leave at 37 ° C for 10 minutes. Add 3 ml of precipitation reagent, stir well and let stand at room temperature for 15 minutes. Thereafter, the mixture is centrifuged at 3.000 rpm for 15 minutes, and the supernatant is taken and the absorbance at 280 nm is measured.
(Measurement of inhibition rate)
The inhibition rate is calculated by Equation 1.
Control (C): Trypsin solution is added after the precipitation reagent.
Control (T): Distilled water was used instead of the sample.
Test section (S): A sample obtained by performing the above operation using each sample.

[Formula 1]

Table 1 shows the trypsin inhibition rates of various plant extracts. As a positive control substance, leupeptin, which is widely used in the biochemical field as a trypsin inhibitor, was used and compared with various plant extracts.

From Table 1, leupeptin tested as a positive control substance showed a trypsin inhibition rate of 65.0% at 0.1% concentration. In addition, the plant extract used for this test prepared the extract which precipitated the protein with the precipitation reagent containing a trichloroacetic acid, and used it for the test. This is because many conventional trypsin inhibitors derived from plants are known to be proteins. However, when a plant extract is applied to the skin, proteins have a large molecular weight and are hardly absorbed through the skin. In this experiment, considering this point, we searched for trypsin inhibitors that are not proteins. As a result, from Table 1, all plant extracts showed high inhibitory activity. Among them, extracts of soybean, fenugreek, loofah, and buckwheat show inhibition of 50% or more at 0.5% concentration, and soybean and loofah extract have almost the same inhibitory activity as leupeptin as a positive control substance at 0.1% concentration. I found out.

Next, although the example of the formulation example of the cosmetics which mix | blended various components of this invention is shown, this invention is not limited to this.

(1) Cosmetic cream
(weight%)
a) Beeswax
2.0
b) Stearyl alcohol
5.0
c) Stearic acid
8.0
d) Squalane
10.0
e) Self-emulsifying glyceryl monostearate
3.0
f) Polyoxyethylene cetyl ether (20E.O.)
1.0
g) Jojoba oil 10.0
h) Loofah extract
0.5
i) Ascorbic acid glucoside 2.0
j) 1,3-butylene glycol
5.0
k) Potassium hydroxide
0.3
l) Preservatives and antioxidants
Appropriate amount
m) Purified water
The remaining preparation methods a) to f) are dissolved by heating and kept at 80 ° C. Heat up to H) to m), keep at 80 ° C., add to a) to f), emulsify, and cool to 40 ° C. with stirring. Then g) is added and stirred to dissolve uniformly.

(2) Latex
(weight%)
a) Beeswax
0.5
b) Petrolatum
2.0
c) Squalane
8.0
d) Sorbitan sesquioleate
0.8
e) Polyoxyethylene oleyl ether (20E.O.) 1.2
f) Scrap extract
1.0
g) Arbutin
2.0
h) 1,3-butylene glycol
7.0
i) Carboxyvinyl polymer
0.2
j) Potassium hydroxide
0.1
k) Purified water
The rest
l) Preservatives and antioxidants
Appropriate amount
m) ethanol
7.0
The process up to production methods a) to e) are dissolved by heating and kept at 80 ° C. Heat up to h) to l), keep at 80 ° C., add to a) to e), emulsify, and cool to 50 ° C. with stirring. Add f), g), m) at 50 ° C., stir to 40 ° C. and cool.

(3) Lotion
(weight%)
a) Buckwheat extract
0.1
b) Magnesium ascorbate phosphate
1.0
c) Glycerin
5.0
d) Polyoxyethylene sorbitan monolaurate (20E.O.) 1.0
e) ethanol
6.0
f) Perfume appropriate amount
g) Preservatives and antioxidants
Appropriate amount
h) Purified water
The remaining preparation methods a) and b) are mixed uniformly. c) to h) are mixed and dissolved uniformly. Mix two agents when using.

(4) Lotion
(weight%)
a) Fenugreek extract 0.05
b) Glutathione 0.1
c) Glycerin

5.0
d) Polyoxyethylene sorbitan monolaurate (20E.O.)
1.0
e) ethanol
6.0
f) Perfume appropriate amount
g) Preservatives and antioxidants
Appropriate amount
h) Purified water
Mix the remaining preparation methods a) to b). c) to h) are mixed and dissolved uniformly. Mix two agents when using.

(5) Face wash
(weight%)
a) Soybean extract
0.5
b) Ascorbic acid glucoside
2.0
c) Peonies extract 0.5
d) Talc
The rest
e) Cellulose
20.0
f) Potassium myristate 30.0
g) Sodium lauryl phosphate
10.0
h) Fragrance
Appropriate amount
i) Preservatives
Appropriate amount of production method a) to i) are mixed and stirred and dispersed well to make uniform.

[Effect confirmation test]
(1) As a test subject for human effect by application, 20 females aged 25 to 55 years old use the products of the present invention and comparative products twice a day (morning and night) for 2.5 consecutive months. The state of the application site was compared before and after the test to investigate the improvement effect. In this study, cosmetics using shown in 0029 as a test, cosmetics excluding loofah extract from cosmetics shown in 0029 in the control product 1, the cosmetics shown in 0029 in the control product 2 A cosmetic material from which ascorbic acid glucoside was removed was prepared, and the effects of its use were investigated. Then, while using the cosmetics containing the active ingredient of the present invention every day, skin smears and whitening effects were tabulated before and after application for 2 months, and the effects were confirmed. The results are shown in Table-2.

As is clear from Table 2, the test consisting of a melanin production inhibitor and a serine protease inhibitor compared to a control product 1 consisting only of a conventional melanin production inhibitor and a control product 2 consisting only of a serine protease inhibitor. The product scored a total of 80 points. On the other hand, the control product 1 had 53 points and the control product 2 had a total score of 52 points, and the high whitening effect of the product of the present invention was recognized.

The present invention has no safety problems such as skin irritation and skin sensitization, and is widely expected to be applied to pharmaceuticals and cosmetics having a high whitening effect by providing a melanin production inhibitor and a serine protease inhibitor. it can.

Claims (1)

Lilyaceae Amadokora plants are Amagonokoro (Polygonatum odoratum (Mill.) Druce var. Pluriflorum (Miq.) Ohwi), Japanese apricots (P. humile Fisch.), Allium plants are leek (Allium tuberosum ROTLER), Leguminosae Are Clara (Sophora flavescens Aiton), Enju (S. japonica L.), Isofuji (S. tomentosa)
L.), Kudzu plant is Kudzu (Pueraria lobata (Willd.) Ohwi), Fuji genus is Nodafuji (Wistaria Floribunda (Willd.) DC.), Yamafuji (W.brachybotrys Siebold et Zucc.), Forenum Graekum genus Fenugreek (Trigonella foenum-graecum L.), Cucurbita moschata (Duchesne) Poir. Var. Meloniformis (Carriere) Makino, E. paposa (C. pepo L.), Bitter moth (Momordicacharantia L) .) (Also known as bitter gourd), the genus buckwheat is composed of an extract from one or more deproteinized species selected from buckwheat (Fagopyrum esculentum Moench) and (F.dibotrys (D.Don) H.Hara) Serine protease inhibitor.