JP2002308753A - Elastase activity inhibitor, estrogenic activator and anti- aging agent for skin containing the same inhibitor or the same activator - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an anti-aging agent for the skin, capable of preventing aging of the skin by recovering and maintaining tenseness and resilience of skin by action inhibiting activity of elastase modifying and decomposing elastin which is one of substance imparting tenseness and resilience to skin or by estrogenic activity promoting synthesis of collagen which is one of a substance imparting tenseness and resilience to skin. SOLUTION: This elastase activity inhibitor is obtained by formulating an extract of Lactuca indica plant with a solvent. The extract has excellent elastase activity-inhibiting action and estrogenic activity and prevents aging of skin by recovering and maintaining tenseness and resilience of skin.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an elastase activity inhibitor and an estrogen-like active agent effective for preventing aging of human skin, and to a composition containing these. Furthermore, the present invention relates to an anti-aging agent which can be used in the fields of cosmetics, quasi-drugs and the like containing an elastase activity inhibitor or an estrogen-like active agent or both agents.

[0002]

2. Description of the Related Art According to recent studies on skin aging, changes in the tissue level occurring in the skin where external aging is observed include reduction of collagen and elastin in the skin dermis, hyaluronic acid and the like. Reduced mucopolysaccharides and damage to epidermal cells. As the cause of skin aging that progresses in parallel, aging is generally the most important, but dryness, oxidation, sunlight and the like can also be mentioned as direct factors related to skin aging.

[0003] Among the above-mentioned skin aging phenomena, the reduction of elastin, which gives a particularly remarkable result, is caused by ultraviolet rays or by aging. Elastin is a coil-shaped hard protein that is entangled with fibrous collagen, and has the property of returning to its original state after stretching like an elastic body, giving the skin firmness and elasticity in cooperation with collagen. Is what it is. Ultraviolet rays activate the action of elastase, a protease that specifically acts on this elastin, so that the skin exposed to a large amount of ultraviolet rays accelerates the decomposition of elastin, and as a result, the skin becomes firmer and more elastic. Losing (Fragrance Journal, Vol. 25,
No. 4). The action of elastase is activated by aging, with similar results. Therefore, if the action of elastase can be inhibited, skin aging can be prevented.

[0004] In addition, a decrease in the amount of estrogen secreted with aging leads to a decrease in the amount of collagen and hyaluronic acid synthesized in the skin, resulting in a loss of elasticity of the skin and causing a skin without moisture and firmness ( Fragrance Journal, extra edition, No. 16). Therefore, a compound having an estrogenic activity is expected to have an effect of improving the synthesis of collagen and hyaluronic acid in the skin and preventing aging of the skin.

[0005]

SUMMARY OF THE INVENTION An object of the present invention is to provide a plant extract having a high elastase activity inhibitory activity, a plant extract that suppresses elastin denaturation and destruction caused by elastase, or an estrogen-like activity, such as collagen and An elastase activity inhibitor or estrogenic activator comprising a plant extract that enhances the synthesis of hyaluronic acid,
And an anti-skin aging agent containing them.

[0006]

Means for Solving the Problems In view of the above problems, the present inventors evaluated elastase inhibitory activity and estrogen-like activity of a variety of plant extracts, and as a result, asteraceous plant lactouka indica ( Scientific name: Lactuc
a indica ) have a strong elastase activity inhibitory effect and an estrogenic activity.

That is, the present invention provides an elastase activity inhibitor and an estrogen-like active agent containing an extract of Lactuca indica (scientific name: Lactuca indica ), and cosmetics and quasi-drugs containing these. And the like.

[0008]

Embodiments of the present invention will be described below in detail. Lactuka indica according to the present invention (scientific name: Lact
uca indica ) is widely distributed from East Asia to Southeast Asia and India. The Lactuca indica extract used in the present invention is obtained by extracting from a part or the whole of a plant such as a leaf, stem, flower, rhizome or the like of the plant. Preferably, it may be obtained by extracting from one or both of the leaves and stems. Generally, after drying or after cutting a live plant as it is, it is used.

[0009] The extraction solvent used is 2 to 20 times the extraction solvent per dry matter of the present plant or fresh plant. As an extraction solvent, generally, water, lower 1
Polyhydric alcohols (methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, etc.), liquid polyhydric alcohols (1,3-butylene glycol, propylene glycol, etc.), lower alkyl esters (ethyl acetate, etc.) ), Hydrocarbons (benzene, hexane, pentane), ketones (acetone, methyl ethyl ketone, etc.), and ethers (diethyl ether, tetrahydrofuran, dipropyl ether, acetonitrile, etc.). These solvents may be used alone or as a mixture of two or more. Preferably, one or more of water or a water-soluble solvent (eg, methanol, ethanol, propylene glycol, etc.) is used. The extraction method is not particularly limited, but the extraction is performed at normal temperature or under heating. As the method, there are ordinary extraction, Soxhlet extraction and the like. The extraction time is not limited, but is preferably several hours to two weeks.

[0010] As described above, the extract according to the present invention is usually obtained as a liquid extract. The extract may be used as it is, or a processed product obtained by performing various treatments may be used. Such a processed product is also included in the extract according to the present invention. As such, for example, a concentrated liquid obtained by concentrating an extract under normal pressure or reduced pressure, a solid obtained by evaporating the solvent in the concentrated liquid to dryness, a solid obtained by crystallizing an active ingredient from the concentrated liquid, followed by filtration and drying. Objects and the like. The amount of the Lactuca indica extract in the composition of the present invention per dry matter is not particularly limited, but is 0.002 to 20.0% by weight, preferably 0.1 to 20.0% by weight based on the total amount.
It is desirably from 0.01 to 10.0% by weight.

[0011] The extract of lactoca indica in the present invention has an excellent elastase activity inhibitory effect on human skin and an estrogenic activity. Therefore, an anti-aging agent containing this extract is effective in reducing skin aging. Prevent and maintain youthful and healthy skin condition. The anti-aging agent of the present invention can be widely used for cosmetics, quasi-drugs and the like, and particularly preferably can be widely used for cosmetics. The cosmetics to which the present invention is applied are not particularly limited, such as the dosage form. For example, lotions, emulsions, creams, foundations, packs, facial cleansers, shampoos, rinses, hair tonics and the like can be mentioned. These cosmetics include:
Various components commonly used in cosmetics, that is, aqueous components,
Oily components, powder components, alcohols, esters, surfactants, humectants, whitening agents, antioxidants, ultraviolet absorbers,
Components such as a thickener, a coloring agent, a fragrance, an antioxidant, a pH adjuster, a chelating agent, and a preservative can be blended.

[0012]

EXAMPLES Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited to these Examples. Prior to the examples, preparation examples of the plant extract of the present invention, test methods for elastase activity inhibition and estrogenic activity of the plant extract, and the results thereof will be described.

[Extraction Example 1] A 5-fold amount of 50% ethanol was added to 100 g of stems and leaves of Lactuca indica (scientific name: Lactuca indica ), and immersion extraction was performed at room temperature for 196 hours. After the treatment, the residue was filtered off, and the obtained extract was concentrated and freeze-dried to obtain Lactuca indica extract 1
2.5 g were obtained. Dissolve this extract in DMSO 2%
The following test was performed by appropriately diluting this solution to adjust the concentration.

[Test Example 1] Test on elastase activity inhibition In order to examine the elastase activity inhibition ability of the lactoca indica extract obtained in [Extraction Example 1], the elastase activity inhibition measurement method used in the present invention was as follows. It is as follows. 0.1M HEPE as reaction buffer
S, 0.5 M NaCl (pH 7.4) was used. MeOSuc-Ala-Ala-Pro-Val as elastase substrate
-pNA (L-1335 BACHEM) is dissolved in DMSO to a concentration of 80 mM, dispensed in 20 μl aliquots, and stored frozen (−
80 ° C). At the time of measurement, the reaction buffer was diluted to 8 mM before use. Elastase was prepared using human-derived saliva (SE-563 Cosmo Bio Inc.) at 200 μg /
The resulting solution was dissolved in a reaction buffer so as to obtain a volume of 10 ml, dispensed in 10 μl portions, and stored frozen (−80 ° C.). At the time of measurement, the reaction buffer was diluted to 5 μg / ml before use. 9
To a 6-well plate (Corning No. 25860), 25 μl of each 8 mM elastase substrate was dispensed,
Further, 50 μl of the inhibitor (sample diluted appropriately and adjusted in concentration) was added. Next, 25 μl of 5 μg / ml elastase was added on ice, and the mixture was immediately incubated at 37 ° C. for 20 minutes. Thereafter, the absorbance was measured at 415 nm. However, the inhibition rate was determined by the following formula.

[0015]

## EQU1 ## Inhibition rate (%) = 100− (presence of inhibitor / no inhibitor) × 100

FIG. 1 shows the result. Further, as a reference example, a test similar to that described above was performed for fetal calf serum (Moregate), which is an in vivo substance showing elastase activity inhibition, and compared. In the following, examples of various formulations and formulations of the anti-aging agent according to the present invention are described.

[Test Example 2] Test on estrogen-like activity (1) Acquisition of stable expression strain for evaluation of human estrogen β receptor action Reporter plasmid pGL2-UAS5-bG-luc (Fig.
2) and human estrogen β receptor (ERβ) expression plasmid p
CI-neo-Gal-ERb (FIG. 3) was introduced to prepare a stable expression strain to be used in the ERβ action evaluation system. First, pGL2-UAS5-bG-l
uc and pCI-neo-Gal-ERb for Qagen Plasm
It was prepared using id Maxi Kit (Qiagen, Cat. No. 1263). CV-1 cells were cultured in Dulbecco's modified Eagle's medium (DM
EM; Nissui Pharmaceutical), fetal bovine serum (Intergen, Catalog N)
o. 1020-90) to a final concentration of 10%, and further use a medium (hereinafter referred to as a growth medium) containing 50 U / ml final concentration of penicillin G and 50 μg / ml streptomycin sulfate. The cells were cultured at 37 ° C. in the presence of 5% CO ₂ , and 3 × 10 ⁶ cells were seeded on a Petri dish having a diameter of about 10 cm. The next day, the cells were treated with pGL2-U by the calcium phosphate method or lipofection method.
AS5-bG-luc and pCI-neo-Gal-ERb were introduced. Calcium Phosphate Transfection System
(GIBCOBRL, Cat.No.18306-019) and according to the attached manual, 18 μg / dish of pGL2-UAS5-b
G-luc and 2 μg / dish of pCI-neo-Gal-ERb were added.
The next day, the cells were washed with PBS (Phospahte Buffered Saline), and then added with 15% glycerol in HBS (HEPES Buffered Salin).
The cells were immersed in e) for 1 minute, washed twice with PBS, subjected to glycerol shock, and returned to the growth medium. The conditions for the lipofection method were Lipofectamine (GIBCO BRL,
Cat.No. 26300-61)
3 μg / dish of pGL2-UAS5-bG-luc and 375 ng / dish of pCI-neo-Gal-ERb were added, treated for 16 hours, and then returned to the growth medium. Two days after introduction of the plasmid, the cells were detached from the petri dish by trypsin treatment and collected,
G418 at a final concentration of 750 μg / ml (CalBiochem, Cat.No.345810)
Was transferred to a Petri dish containing a growth medium supplemented with, and the medium was cultured for about one month while replacing the medium with a new medium (a growth medium supplemented with G418 at a final concentration of 750 μg / ml) every 3 to 4 days. When the cells formed colonies of 1 mm to several mm, the growth medium containing G418 at a final concentration of 750 μg / ml was added.
The colony was transferred to a 4-well plate and further cultured. Once the cells have grown to fill almost the entire surface of the well, trypsinize the cells, detach them, transfer 4/5 cells evenly to 2 wells of a new 24-well plate for measurement, and separate the remaining 1/5 cells. Was transferred to a new 24-well plate, and the culture was continued as a master plate. The cells for measurement were cultured until the cells occupied the entire surface of the well, and one of the two wells contained 17β-estradiol (E2; Sigma) dissolved in DMSO.
Cat.No.E8875) at a final concentration of 10 nM (DMSO has a final concentration of 0.
1%) phenol red-free DMEM
(GIBCOBRL, Cat.No.11054-620) -10% activated charcoal / dextran-treated fetal calf serum (HyClone, Cat.No.SH30068.0)
3) Replace the medium (hereinafter referred to as the measurement medium) with the measurement medium supplemented with DMSO so that the final concentration becomes 0.1%, culture the cells at 37 ° C. for 24 hours, and wash the cells with PBS. , Passi
ve Lysis Buffer (Promega, Dual-Luciferase Ass
ay System Cat.No.E1910)
The mixture was shaken at room temperature for 30 minutes. 20 μl of cell lysate for 96
Transfer the well plate and add 100 μl substrate solution (Promega, Ca
t.No.E1483) is automatically dispensed and the luminometer CT-9000D
(Dia-iatron) and the luciferase activity was measured. E
Cells showing the luciferase activity of the 2-added system more than twice as high as the E2-free system were selected. The selected cells were further recloned by the dilution limit method, and E2
Cells ERβ13-3 which showed a high induction factor by addition and a high luciferase activity were selected and used for activity evaluation.

(2) S. of Lactuca indica extract
Evaluation of trogen-like activity Lactuca indica extract obtained in [Extraction Example 1] above
The estrogenic activity of the product is determined by the human estrogen obtained in (1) above.
Stable expression strain ERβ13-3 for evaluation of toestrogen β receptor action
Were examined by a reporter gene assay using a chromosome.
ERβ13-3 growth medium supplemented with 750μg / ml G418
5% CO at 37 ° C using _TwoAfter culturing in the presence, about 1.0 x 10^Five
Cells / well were seeded on a 24-well plate. next day,
Measurement medium to which each sample (final concentration of DMSO is 0.1%) was added
The cells were washed at 37 ° C for 24 hours, and the cells were washed with PBS.
Thereafter, a Repoter Lysis Buffer (Promega, Cat.No.E397)
1) Add 100 μl` per well and shake at room temperature for 30 minutes
did. Transfer 25 μl of cell lysate to a 96-well plate,
μl of the substrate solution is automatically dispensed, and the luminometer CT-9000D is used.
The luciferase activity was measured. Estrogen-like activity
The negative control (0.1% DMSO) was 0%, 10 μM E2
The activity was shown as a relative activity when the activity at the time of addition was 100% (FIG. 4).

[Example 1] Cream At the mixing amount described below, the component A was heated and mixed at 70 ° C.
To keep. Next, the component B is heated and mixed and kept at 70 ° C. The component B was mixed with the component A so as to be uniform, and then the component C was injected and mixed, and then cooled to 30 ° C. with stirring to obtain a cream.

Ingredients Ingredients Amount (wt%) (A) Squalane 10.0 Olive oil 10.0 Solid paraffin 5.0 Cetanol 4.0 Sorbitan monostearate 2.0 Polyoxyethylene sorbitan monostearate 2.0 (B ) Lactuca indica extract (Extraction Example 1) 0.05 (C) Glycerin 5.0 Methyl parapen 0.1 Perfume Appropriate amount Purified water Balance

Comparative Example 1 Cream Except for removing the component (B) lactoca indica extract in Example 1, the cream was prepared in the same manner as in Example 1 and used for a use effect test.

Example 2 Emulsion Emulsion A was heated and mixed at 70 ° C.
To keep. Next, the component B is heated and mixed and kept at 70 ° C. The component B was mixed with the component A so as to be uniform, then the component C was injected, mixed and stirred, and then cooled to 30 ° C. with stirring to obtain an emulsion.

Ingredients Ingredients Amount (wt%) (A) 1,3-butylene glycol 5.0 ethyl alcohol 5.0 Lactuca indica extract (Extraction Example 1) 5.0 Appropriate amount of purified water (B) Squalane 5. 0 Vaseline 2.0 Beeswax 0.5 Sorbitan sesquioleate 0.8 Polyoxyethylene oleyl ether (20E.O.) 1.2 Preservative proper amount Perfume proper amount (C) Carboxyvinyl polymer (1% aqueous solution) 20.0 Potassium hydroxide 0.1 Purified water Remaining

Comparative Example 2 Emulsion The emulsion was prepared in the same manner as in Example 2 except that the component (A) Lactuca indica extract was omitted, and used in a test for use effect.

Test Example 3 Use Effect Test The use tests shown below were performed on the cream and emulsion cosmetics prepared in Examples 1 and 2 and Comparative Examples 1 and 2, respectively. Table 1 shows the results. The use test was performed on 40 healthy adult women aged 28 to 55 years, which were randomly selected, as subjects, and applied each cosmetic to the skin after face washing for 4 weeks every day. The improvement effect on wrinkles and fine wrinkles after use and the effect on firmness and tarmi were examined. The condition of the skin was visually observed and evaluated based on the following evaluation criteria. (Evaluation criteria) A: Effective B: Somewhat effective C: No change D: Invalid

[0026]

[Table 1]

From the results shown in Table 1, when the cosmetic containing the extract of Lactuca indica prepared in Examples 1 and 2 was used, Comparative Examples 1, 2, and 3 not containing the extract of Lactuca indica were used. Wrinkles, wrinkles and skin tension
A good result was obtained, which was improved in terms of tarmi. From these results, it is recognized that blending Lactuca indica extract as an active ingredient is a very useful formulation.

[0028]

As described above, the degeneration and destruction of elastin, which is an elastic fiber, is suppressed by blending the lactuka indica extract of the present invention, which has excellent elastase activity inhibition and estrogenic activity, as an active ingredient. ,
An anti-aging agent is provided which promotes the synthesis of collagen and hyaluronic acid, can maintain elastic, wrinkle-free and skin-free skin, and can prevent skin aging.

[Brief description of the drawings]

FIG. 1 shows the inhibition of elastase activity of Lactuca indica extract in comparison with fetal calf serum.

FIG. 2 shows the reporter plasmid pGL2-UAS5-bG
Show the map for -luc. UAS5 indicates that five GAL4 binding region UASs are linked, bG indicates a rabbit β globin promoter, luc indicates a luciferase gene, and Amp indicates an ampicillin resistance gene.

FIG. 3 shows a map of a chimeric receptor expression plasmid pCI-neo-Gal-ERb in the Gal4 DNA binding region-hERβ ligand binding region. CMV IE Enhancer / Promoter is Cytome
garovirus immediate-early enhancer and promoter, Gal4
Indicates a Gal4 DNA binding region, ERb (LBD) indicates a hERβ ligand binding region, Neo indicates a neomycin resistance gene, and Amp indicates an ampicillin resistance gene.

FIG. 4 shows the results of a reporter assay using ERβ13-3, a stable expression strain for evaluating estrogen β receptor action, showing that estrogen receptor β of 10 nM E2, 100 μg / ml lactuka indica extract, 0.1% DMSO was used. FIG. 4 is a graph showing the results of measuring the transcription promoting ability via, and comparing estrogenic activities.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 43/00 111 A61P 43/00 111 F-term (Reference) 4C083 AA082 AA111 AA112 AA122 AB032 AC012 AC022 AC072 AC102 AC122 AC182 AC442 AC482 AD092 BB51 CC05 DD31 EE12 4C088 AB26 AC01 BA08 BA10 BA37 NA14 ZA89 ZC11 ZC20

Claims (6)

[Claims] [1] Lactuca indic
a) An elastase activity inhibitor, which comprises a solvent extract of a plant. 2. A composition comprising the elastase activity inhibitor according to claim 1. 3. An anti-skin aging agent comprising the elastase activity inhibitor according to claim 1. 4. Lactuca indica (scientific name: Lactuca
indica) An estrogenic activator characterized by containing a solvent extract of a plant. 5. A composition comprising the estrogenic activator according to claim 4. 6. An anti-skin aging agent comprising the estrogen-like active agent according to claim 4.