JP5306605B2 - Topical skin preparation - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an external preparation for skin, a functional agent and a functional oral composition comprising an ingredient derived from a natural product having excellent anti-aging actions, anti-oxidizing actions, bleaching actions, humectant actions, anti-inflammatory actions and slimming actions. <P>SOLUTION: The external preparation for skin includes an extract from a fruit and/or a leaf of Actinidia arguta of the genus Actinidia of the family Actinidiaceae. The functional agent includes the extract from the fruit and/or the leaf of the Actinidia arguta of the genus Actinidia of the family Actinidiaceae as an active ingredient. The functional oral composition includes the extract from the fruit and/or the leaf of the Actinidia arguta of the genus Actinidia of the family Actinidiaceae. <P>COPYRIGHT: (C)2008,JPO&amp;INPIT

Description

  The present invention relates to a skin external preparation, a functional agent, and a functional oral composition containing a naturally derived ingredient as an active ingredient.

  Conventionally, in the field of topical skin preparations, various anti-aging agents, whitening agents, antioxidants, and neutral fat accumulation inhibitors using natural ingredients have been studied. However, the effect is not sufficient, and the development of better components has been demanded.

  Lipid metabolism consisting of a moisturizer (Patent Documents 1 and 2) using a sap (Sarnasi sap) that emerges from the cut end when the vine of Salna pear is cut, and an extract of kiwifruit peel. A cosmetic composition (Patent Document 5) containing an improvement composition (Patent Document 3), a tyrosinase activity inhibitor (Patent Document 4) using an extract of a stem of a striped pear, and a steamed distilled water of Sarunasi is known. .

JP 2006-249101 A Japanese Patent Laid-Open No. 2004-2458 JP 2006-117596 A JP 2006-69954 A JP 2001-220344 A

  As described above, the use of the matabidae plant has been studied as a naturally-derived component, but it has not been possible to utilize a sufficient physiologically active component by any method, and development of various skin external preparations and the like having more excellent actions. Is expected.

  The present invention relates to the development of naturally-derived ingredients having excellent anti-aging action, antioxidant action, whitening action, moisturizing action, anti-inflammatory action, and slimming action, as well as a skin external preparation and functional agent having these effects, It is another object of the present invention to provide a functional oral composition.

As a result of studying various components derived from nature, the present inventors have found that a fruit or leaf extract of Actinidia arguta has a particularly excellent action, and further studies have been made. Over time, the present invention has been completed.
That is, this invention relates to the skin external preparation containing the extract of the fruit and / or leaf of the scorpion family Matatabi genus Sarnasi.
Another aspect of the present invention relates to a functional agent comprising, as an active ingredient, a fruit and / or leaf extract of the genus Matatabidae Sarnusi.
Yet another aspect of the present invention relates to a functional oral composition characterized in that it comprises an extract of fruit and / or leaf of the genus Matatabidae.

  According to the present invention, an excellent anti-aging effect, antioxidant effect, whitening action, moisturizing action, anti-inflammatory action, and slimming action can be obtained by including the fruit and / or leaf extract of the genus Matabiaceae genus Sarnashi. It is possible to provide an external preparation for skin and an oral composition to be exerted. Furthermore, the functional agent which shows the outstanding effect can be provided by using the matabidae department | strip genus Sarunasi or its extract as an active ingredient.

Matatabidae is a deciduous vine that grows naturally in the mountains.
In the present invention, an extract obtained from the fruit and / or leaf of this Sarnashi (hereinafter also referred to as “Sarnashi extract” or “extract”) is used. Here, a fruit is both a pulp and a skin, and may only be described as "fruit". The resulting extract contains an extremely large number of components that cannot be analyzed, and it is presumed that these effects act comprehensively to obtain the effects of the present invention.

  For the preparation of the pear extract, fruits and leaves may be used as they are, but considering the extraction efficiency, it is preferable to perform extraction after processing such as chopping, drying, and pulverization. Extraction can be performed using an arbitrary solvent described later, and two or more extracts extracted using different solvents may be mixed and used.

Examples of the extraction method include a method of extracting by immersing in an arbitrary extraction solvent for a predetermined time in a cooled or heated state at room temperature, and a pressing method of obtaining an extract by pressing from raw fruits and leaves. These arbitrary methods can be extracted alone or in combination of two or more. Alternatively, extraction can be performed using a supercritical fluid or a subcritical fluid. In order to increase the extraction efficiency, stirring or homogenization in an extraction solvent may be performed.
Among these, extraction by a solvent extraction method is preferable in order to efficiently extract the physiologically active component of Sarnashi. The solvent extraction method is a method in which the extraction time and the extraction temperature can be easily adjusted. For example, even when a component that easily undergoes heat denaturation is included, the solvent extraction method can be appropriately applied. In addition, by performing extraction by a solvent extraction method, for example, a physiologically active ingredient of a kind of Sarnashi that cannot be extracted by a steam distillation method that mainly extracts water-soluble volatile components can be used.

The extraction temperature is suitably about 5 ° C. to the boiling point of the extraction solvent. The extraction time varies depending on the type of extraction solvent and the extraction temperature, but is suitably about 1 hour to 14 days.
The ratio of the fruits and / or leaves to the solvent during the extraction is not particularly limited, but is preferably 0.5 to 1000 times the solvent with respect to the fruit 1 and the like, and particularly 0 in terms of extraction operation and efficiency. 0.5 to 100 mass times is preferable.

  As an extraction solvent, in addition to water, lower (carbon number 1 to 3) alcohols such as methanol, ethanol, propanol, and isopropanol; polyvalent (2 to 2,3-butylene glycol, propylene glycol, dipropylene glycol, glycerin, etc. Trivalent) alcohols; ethers such as ethyl ether and propyl ether; esters such as butyl acetate and ethyl acetate; and solvents such as ketones such as acetone and ethyl methyl ketone can be used. These may be used alone or in combination of any two or more. Saline, phosphate buffer, phosphate buffered saline, and the like may be used. Furthermore, you may use 1 type, or 2 or more types of supercritical liquids and subcritical liquids, such as water, a carbon dioxide, ethylene, propylene, ethanol, methanol, ammonia. Among these, extraction with water, lower alcohol, or polyhydric alcohol is preferable for efficiently extracting the physiologically active component of Sarnashi, and in particular, water, ethanol, 1,3-butylene glycol, dipropylene glycol, It is preferable to use one or more selected from the group consisting of glycerin.

  The extract can be used as it is, but it can be left for a certain period of time and aged, or the concentrated and dried product can be used again by dissolving it in water or a polar solvent. Alternatively, it may be used after performing purification treatment such as decolorization, deodorization, and desalting, and fractionation treatment by column chromatography or the like within a range not impairing these physiological functions. The obtained extract and its treated product and fraction can be lyophilized after each treatment and fractionation and dissolved in a solvent before use. It can also be used by encapsulating in vesicles such as liposomes or microcapsules.

Sarnashi extract has excellent anti-aging, antioxidant, whitening, moisturizing, anti-inflammatory, and slimming effects. Oral compositions can be provided.
Here, the external preparation for skin means all external compositions for external use on the skin or hair, such as cosmetics, quasi-drugs, and external medicines. The functional oral composition also means all compositions that are taken orally regardless of the type of pharmaceuticals, foods, beverages and the like. The function of the oral composition is preferably one or more of anti-aging, antioxidant, whitening, moisturizing, anti-inflammatory, and slimming.

The dosage form of the external preparation for skin is arbitrary, and can be provided, for example, as a solubilizing system such as lotion, a dispersion system such as calamine lotion, or an emulsifying system such as cream or emulsion. Further, it can be provided in various dosage forms such as an aerosol form, an ointment, and a poultice filled with a propellant.
Specifically, various cosmetics such as emulsions, creams, lotions, lotions, packs, cosmetic liquids, cleaning agents, makeup cosmetics, etc .; liquids, ointments, powders, granules, aerosols, patch agents, poultices, etc. Various forms of quasi-drugs and topical drugs can be exemplified.
The form of the functional oral composition is also arbitrary and is not particularly limited. Specifically, general foods including beverages; health foods (supplements) such as tablets, capsules, granules, powders or functional foods; oral drugs such as tablets, capsules, granules, powders, syrups, extracts Etc. can be exemplified.

  For example, by blending salnashi extract into cosmetics, topical pharmaceuticals, quasi drugs, etc., it exhibits excellent effects in preventing and improving various skin symptoms such as wrinkles, tarmi, reduced skin elasticity, spots and dullness. For example, it can be used as an anti-aging (anti-aging improvement) skin external preparation, an antioxidant skin external preparation, a whitening skin external preparation, a moisturizing skin external preparation, and the like. Furthermore, it can also be used as a slimming skin external preparation or an anti-inflammatory skin external preparation.

For external preparations for skin or functional oral compositions, in addition to the extract of sarnashi, it is usually used for preparations such as skin cosmetics, cosmetics for hair, quasi-drugs, and pharmaceuticals, depending on the use and necessity. Optional ingredients are included. Such components include water, oils (oil-based ingredients), moisturizers, powders, pigments, emulsifiers, solubilizers, gelling agents, cleaning agents, UV absorbers, thickeners, pH adjusters, surface activity. Agents, chelating agents, drugs (medicinal components), fragrances, resins, antibacterial and antifungal agents, pH adjusters, antioxidants, alcohols and the like. In addition, other anti-aging agents, whitening agents, antioxidants, anti-inflammatory agents, or other plants or extracts thereof can be used as long as the effects of the present invention are not impaired.
Also in the case of oral compositions such as foods and drinks, there is no particular limitation in combination with various components that are usually used for oral use.

  The blending amount of the pear extract in the external preparation for skin or functional oral composition can be adjusted depending on the type and purpose of use of the external preparation or oral preparation. As a general blending amount, 0.0001 to 10% by mass in terms of solid content is preferable, more preferably 0.001 to 5% by mass, based on the total amount from the viewpoints of effects and stability. Preferably it is 0.01-5 mass%, More preferably, it is 0.1-5 mass%.

Next, the functional agent which uses a sarnashi extract as an active ingredient is demonstrated.
This functional agent is a preparation that exhibits the specific functions of the extract of Sarunashi, and is preferably an anti-aging agent, antioxidant, whitening agent, moisturizer, anti-inflammatory agent, or slimming agent (inhibition of neutral fat accumulation) Agent or adiponectin producing agent).
Each of these preparations is not limited at all in terms of its form and the presence or absence of other ingredients, as long as it contains the sarnashi extract as an active ingredient. As for the form, any form such as liquid, paste, gel, solid, powder, etc. can be selected according to its use and the like, vehicle (excipient), solvent, Other general additives (antioxidants, colorants, dispersants, etc.) can optionally be included.
Each of these preparations can be blended with the above-mentioned external preparation for skin or oral composition to provide various forms of compositions, and can also be used as it is for external use or oral use.

  An anti-aging agent comprising a Sarnashi extract as an active ingredient has an excellent cell activation effect, collagen production promoting action, and ATP production promoting action, and exhibits an excellent effect in preventing and improving aging symptoms.

  Antioxidants containing the Sarnashi extract as an active ingredient have an excellent lipid peroxide resistance action, and exhibit a protective action against cell damage caused by lipid peroxide. In addition, it has an excellent free radical scavenging effect and superoxide anion scavenging effect, prevents skin photoaging and the like, and exhibits an excellent antioxidant action.

  A whitening agent containing a sarnashi extract as an active ingredient has an excellent inhibitory effect on melanin production and an inhibitory effect on tyrosinase activity, and prevents and improves pigmentation, blemishes, freckles, etc., and exhibits an excellent whitening effect.

  A moisturizing agent containing an extract of Sarnashi as an active ingredient provides an excellent moisturizing effect on the skin and hair, etc., and also has an excellent effect on improving skin symptoms such as rough skin, fine lines, dullness, and the like. Can improve the transparency.

  An anti-inflammatory agent containing a Sarnasashi extract as an active ingredient has an excellent hyaluronidase inhibitory effect and an arginase activity promoting action, and suppresses skin inflammation and exhibits an excellent anti-inflammatory action.

  The slimming agent (neutral fat accumulation inhibitor or adiponectin producing agent) containing the extract of sarunasi as an active ingredient has excellent neutral fat accumulation inhibiting effect and adiponectin production promoting action (or production action), and excellent slimming action Demonstrate.

This adiponectin is a metabolic syndrome (built-in fat syndrome) ameliorating factor and has an effect of reducing visceral fat. More specifically, it is a substance that is activated in the liver and skeletal muscle to improve the burning of fatty acids and the uptake of sugar. When diabetic, the blood concentration of adiponectin decreases, and when the concentration of adiponectin increases, the inulin sensitivity that lowers the blood sugar level increases, or enters the blood vessels and suppresses cellular phenomena that promote arteriosclerosis such as inflammation. I know. More recently, it has been reported that adiponectin has a potent anticancer effect.
Therefore, this adiponectin producing agent can be used as an arteriosclerosis preventive agent, an anticancer agent and the like.
As described above, the sarnashi extract having an adiponectin production promoting action is useful as an excellent health food material.

  In the following, preparation examples of the extract of saruna pear, tests for evaluating each action, and formulation examples of external preparations for skin and oral compositions will be described in more detail, but the technical scope of the present invention is limited by these. Is not to be done.

<Extraction method 1: Ethanol extract>
Each dried pulverized product of Sarnashi fruit and leaves was dispersed in 50 mass% aqueous ethanol solution or 75 mass% ethanol aqueous solution 20 times the amount of dry pulverized substance, and extracted at room temperature for 3 hours with stirring. After extraction, insolubles were removed by filtration, concentrated under reduced pressure, and lyophilized to obtain ethanol extracts of fruits and leaves.

<Extraction method 2: hot water extract>
Each dried pulverized product of Sarnashi fruit and leaves was dispersed in purified water 20 times the amount of dry pulverized substance, and extracted by autoclave (120 ° C., 20 minutes). After extraction, insolubles were removed by filtration, concentrated under reduced pressure, and freeze-dried to obtain hot water extracts of fruits and leaves.
In the table below, * and ** indicate a significance probability of less than 5% (P <0.05), and a significance probability of less than 1% (P <0.01) with respect to the significance P value in the t-test. It is represented by **.

1. Human dermal fibroblast activation (anti-aging)
Normal human dermal fibroblasts manufactured by Kurashiki Boseki Co., Ltd. were seeded in a 96-well microplate so that there were 2.0 × 10 ⁴ cells per well. As the seeding medium, Dulbecco's modified Eagle medium (DMEM) to which 1% by mass of fetal bovine serum (FBS) was added was used. After culturing for 24 hours, the medium was replaced with a sample culture solution adjusted to each sample concentration shown in Table 1 with 1% by mass FBS-added DMEM medium, and further cultured for 48 hours.
After removing the supernatant, the medium was replaced with a medium containing 400 μg / ml of 3- (4,5-dimethyl-2-thiazolyl) -2,5-diphenyltetrazolium bromide (MTT reagent) and cultured for about 2 hours. Thereafter, formazan produced by the opening of the tetrazolium ring was extracted with 2-propanol, and the absorbance at 550 nm was measured with a microplate reader. At the same time, the absorbance at 650 nm was measured as turbidity, and the cell activation effect was evaluated by the difference between the two measured values. In the evaluation, in addition to the sample culture solution, 1% by mass FBS-added DMEM medium was used as a negative control, and 5% by mass FBS-added DMEM medium was used as a positive control.
The obtained results are shown in Table 1 as relative values when the cell activation effect in the negative control is taken as 100.

2. Human epidermal keratinocyte activation (anti-aging)
Human epidermal cell line HaCaT was seeded in a 96-well microplate so as to be 2.0 × 10 ⁴ cells per well. The seeding medium used was Dulbecco's modified Eagle medium (DMEM) supplemented with 5% by weight fetal bovine serum (FBS). After 24 hours, the sample culture solution was adjusted to the concentration of each sample shown in Table 2 with 5% by mass FBS-added DMEM medium, and further cultured for 24 hours.
Next, the MTT reagent was adjusted with a medium to a concentration of 100 μg / ml, added to the cells from which the supernatant was removed, and cultured for about 2 hours. Finally, formazan produced by 2-propanol was extracted, and the absorbance at 550 nm was measured with a microplate reader. At the same time, the absorbance at 650 nm was measured as turbidity, and the cell activation effect was evaluated using the difference between the two measured values.
The evaluation results are shown in Table 2 by relative values when the cell activation effect in the control with no sample added is taken as 100.

3. Human dermal fibroblast type I collagen production promoting action (anti-aging effect)
Normal human dermal fibroblasts were seeded in a 96-well microplate at 2.0 × 10 ⁴ cells per well. The seeding medium used was Dulbecco's modified Eagle medium (DMEM) supplemented with 5% by weight fetal bovine serum (FBS). After culturing for 24 hours, the sample culture solution was adjusted to the sample concentration shown in Table 3 with a DMEM medium supplemented with 0.5% by mass FBS, and further cultured for 24 hours.
The ELISA method was used to quantify type I collagen secreted into the culture supernatant, and finally 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) against labeled peroxidase. ) And hydrogen peroxide were added and reacted, and the absorbance at 405 nm was measured with a microplate reader. In the evaluation, in addition to the sample culture solution, 0.5% by mass FBS-added DMEM medium as a negative control and 0.5% by mass FBS containing 50 μM L-ascorbic acid phosphate magnesium salt (VCPMg) as a positive control are added. DMEM medium was used.
The amount of protein was measured by BCA Protein Assay Kit manufactured by PIERCE, and the amount of type I collagen produced per unit protein was determined.
The obtained results are shown in Table 3 by relative values when the type I collagen production amount per unit protein amount in the negative control is 100.

4). Human dermal fibroblast ATP production promoting action (anti-aging effect)
Normal human dermal fibroblasts were seeded in a 48-well microplate at 4.0 × 10 ⁴ cells per well. As the seeding medium, Dulbecco's modified Eagle medium (DMEM) to which 1% by mass of fetal bovine serum (FBS) was added was used. After culturing for 24 hours, the sample culture solution was adjusted to the sample concentration shown in Table 4 with 1% by mass FBS-added DMEM medium, and further cultured for 24 hours.
The cell supernatant was discarded and washed, and the cells were sonicated to elute the ATP in the cells. At that time, ATP-degrading enzyme in cells is also eluted, so that an ATP-degrading enzyme inhibitor (Cellstein Hoechst 33342 manufactured by Wako Pure Chemical Industries, Ltd .; Cellstein Hoechst 33342) is used as a buffer for ultrasonic treatment. ) Has been added.
The prepared cell lysate was dispensed into a test tube, luciferase and a luciferin reagent were added, and chemiluminescence intensity was measured using a compact Lumi VS501 from Yamato Scientific Co., Ltd.
The obtained results are shown in Table 4 as relative values when the value in the control with no sample added is 100.

5. DPPH radical scavenging action (antioxidant action)
Using 50% by mass ethanol, the sample solution was adjusted so as to have each sample concentration shown in Table 5, and 100 μl was added to each 96-well microplate. Thereto, 100 μl of 0.2 mM 1,1-diphenyl-2-picrylhydrazyl (DPPH) ethanol solution was added, mixed well, and allowed to stand in the dark at room temperature for 10 minutes or 24 hours. Finally, the absorbance at 516 nm derived from the DPPH radical was measured.
When the absorbance when the sample was not added was (A) and the absorbance when the sample was added was (B), the DPPH radical elimination rate was determined from the following equation.
Radical scavenging rate = {1- (B) / (A)} × 100
Table 5 shows the results after 10 minutes of standing time, and Table 6 shows the results after 24 hours.

The company standards (reference figures) for antioxidant activity are as follows.
Radical scavenging rate 75-100%: strength of action ◎ (with strong antioxidant effect)
Radical scavenging rate 50-74%: strength of action ○ (with antioxidant effect)
Radical scavenging rate 25-49%: strength of action Δ (has weak antioxidant effect)
Radical scavenging rate 0-24%: strength of action x (no antioxidant action)

6). Human epidermal melanocyte tyrosinase activity inhibitory action (whitening action)
Normal human epidermal melanocytes manufactured by Kurashiki Boseki Co., Ltd. were seeded in a 96-well microplate so as to be 3.0 × 10 ⁴ cells per well. As a seeding medium, Medium 254S manufactured by Kurashiki Boseki Co., Ltd. was used. After culturing for 24 hours, the medium was replaced with a sample culture medium adjusted to each sample concentration shown in Table 7 using Medium 254S, and further cultured for 48 hours. Next, the cells were completely lysed by exchanging with 75 μl of a phosphate buffer containing 1% by weight Triton-X, and 50 μl of this was used as a crude enzyme solution. To the crude enzyme solution, 50 μl of 0.05% by mass L-dopa-containing phosphate buffer as a substrate was added and allowed to stand at 37 ° C. for 2 hours. The absorbance at 405 nm immediately after the addition of the substrate and at the end of the reaction was measured with a microplate reader, and each measured value was introduced into the following equation to determine the amount of produced dopamelanin.
Dopamelanin production =
{(405 nm value after reaction−405 nm value before reaction) −2.166} /5.238
Moreover, the amount of protein was measured by BCA Protein Assay Kit made by PIERCE, and the amount of dopamelanin produced per unit protein was determined.
The obtained results are shown in Table 7 as relative values when the value in the control with no sample added is 100.

7). Human skin keratinocyte type IV collagen production promoting action (anti-aging effect)
Human skin keratinocytes were seeded in a 96-well microplate at 2.0 × 10 ⁴ cells per well. The seeding medium used was Dulbecco's modified Eagle medium (DMEM) supplemented with 5% by weight fetal bovine serum (FBS). After culturing for 24 hours, the culture medium was replaced with a sample culture solution adjusted to each sample concentration shown in Table 8 using a DMEM medium supplemented with 5% by mass FBS, and further cultured for 5 days.
For the quantification of type IV collagen secreted in the culture supernatant, avidinized horseradish peroxidase was added using a sandwich ELISA method using a monoclonal antibody against IV collagen (recognition site: α2 chain) and a biotinylated polyclonal antibody. The color was developed with 3,3 ′, 5,5′-tetramethylbenzidine, and the absorbance at 650 nm was measured with a microplate reader.
The amount of protein was measured by BCA Protein Assay Kit manufactured by PIERCE, and the amount of type IV collagen produced per unit protein was determined.
The obtained results are shown in Table 8 as relative values when the type IV collagen production amount per unit protein amount in the control with no sample added is defined as 100.

8). Human epidermal keratinocyte arginase activity promoting action (anti-inflammatory and moisturizing action)
Human epidermal keratinocytes HaCaT were seeded in a 96-well microplate so as to be 2.0 × 10 ⁴ cells per well. The seeding medium used was Dulbecco's modified Eagle medium (DMEM) supplemented with 5% by weight fetal bovine serum (FBS). After culturing for 24 hours, the culture medium was replaced with a sample culture solution adjusted to each sample concentration shown in Table 9 with 5% by mass FBS-added DMEM medium (differentiation induction medium) containing 1.2 mM CaCl ₂ , and further cultured for 9 days. The medium was exchanged once every 3 days.
Urea nitrogen B-Test Wako (manufactured by Wako Pure Chemical Industries, Ltd.) was used for quantification of urea secreted into the culture supernatant. Arginase hydrolyzes arginine to produce ornithine and urea. Urea is decomposed into ammonia by urease, and ammonia reacts with salicylic acid and hypochlorous acid in the presence of sodium pentacyanonitrosyl iron (III) dihydrate (nitroprusid sodium) to produce indophenol. Based on this principle, the absorbance at 570 nm derived from indophenol was measured with a microplate reader under alkaline conditions, the urea concentration was determined, and the arginase activity was quantified. The amount of protein was measured by BCA Protein Assay Kit manufactured by PIERCE, and the arginase activity per unit protein amount was determined.
The obtained results are shown in Table 9 by relative values when the arginase activity per unit protein amount in the control with no sample added is defined as 100.

9. Hyaluronidase inhibitory action (anti-inflammatory and moisturizing action)
Commercially available hyaluronic acid potassium salt (derived from human umbilical cord) was dissolved in 0.1 M phosphate buffer (pH 7.0) to a concentration of 0.9 mg / ml to obtain a substrate solution. A commercially available hyaluronidase (derived from bovine testis) was dissolved in 0.1 M phosphate buffer (pH 7.0) so as to be 5,300 units / ml to obtain an enzyme solution. The enzyme solution was prepared at the time of use.
A sample solution (0.1 ml) prepared with a buffer solution at each sample concentration shown in Table 10 and an enzyme solution (0.03 ml) were placed in a test tube and reacted at 37 ° C. for 20 minutes. Next, 0.06 ml of activator was added and reacted at 37 ° C. for 20 minutes. Further, 0.15 ml of the substrate solution was added and reacted at 37 ° C. for 1 hour. The reaction was stopped by adding 0.06 ml of 0.4N NaOH aqueous solution, immediately cooled on ice, 0.06 ml of borate buffer (pH 9.1) was added and boiled for 3 minutes. did. After 2.0 ml of p-DABA (p-dimethylaminobenzaldehyde) solution was added and reacted at 37 ° C. for 20 minutes, the reaction solution was transferred from each test tube to a 96-well microplate and 585 nm using a microplate reader. The absorbance at was measured. As a control, a sample-free buffer solution was used. When the activity of hyaluronidase is inhibited, the degradation product N-acetylglucosamine (GlcNAc) is decreased, and the absorbance by p-DABA is lowered. Therefore, the hyaluronidase inhibitory action is defined by the following equation.
Inhibition rate (%)
= (Control absorbance-sample absorbance) / control absorbance x 100
The results are shown in Table 10.

10. SOD-like activity evaluation (Evaluation of superoxide anion scavenging ability)
To 75 μl of the HANK ′S (+) solution containing 0.25 mM WST-1 and 1 mM Hypoxanthine, 25 μl of the sample solution prepared to the respective sample concentrations shown in Table 11 with the HANK ′S (+) solution was added. Further, 25 μl (0.0075 Units) of xanthine oxidase was added and reacted at 37 ° C. for 15 minutes, and then the absorbance at 450 nm was measured. The superoxide anion elimination rate is defined by the following equation, where (A) is the absorbance when only the HANK'S (+) solution is added instead of the sample solution, and (B) is the absorbance when the sample solution is added. Is done.
Erase rate (%) = {1- (B) / (A)} × 100
The obtained results are shown in Table 11.

11. Neutral fat accumulation inhibitory action (slimming action) using human preadipocytes
Subcutaneous fat-derived normal human preadipocytes Cryo • HPRAD-SQ (Sanko Junyaku Co., Ltd.) were seeded on a 96-well microplate so that the number was 1.0 × 10 ⁴ per well. PGM medium (containing 10% by mass fetal bovine serum (FBS), 2 mM L-glutamine, 100 units / mL penicillin, 100 μg / mL streptomycin) was used as a seeding medium. Immediately before the cells are saturated, the medium is replaced with a PGM differentiation medium (10 μg / mL insulin, 1 μM dexamethasone, 200 μM indomethacin, 500 μM isobutylmethylxanthine) added with a sample having a concentration shown in Table 12. Differentiation induction was performed. After initiation of differentiation induction, the cells were cultured for 10 to 14 days until the control group matured and many lipid droplets accumulated in the cells. After the cells were collected, the cells were fixed using a 10% neutral buffered formaldehyde solution. After washing with PBS (−), 0.5 w / v% oil red O solution was added and incubated at 37 ° C. for 2 hours. After washing with PBS (−), methanol was added to extract the dye. After extraction, the absorbance at 550 nm was measured with a microplate reader. At the same time, the absorbance at 650 nm was measured as turbidity, and the amount of accumulated triglyceride was measured using the difference between the two measured values.
The measurement results are shown in Table 12 in terms of relative values with the neutral fat accumulation amount in the control using the medium without the sample as 100.

12 Adiponectin production promoting action using human preadipocytes (metabolic syndrome improving action, anticancer action)
Subcutaneous fat-derived normal human preadipocytes Cryo HPRAD-SQ (Sanko Junyaku Co., Ltd.) were seeded on a 96-well microplate so that the number was 5.0 × 10 ³ per well. PGM medium (containing 10% by mass fetal bovine serum (FBS), 2 mM L-glutamine, 100 units / mL penicillin, 100 μg / mL streptomycin) was used as a seeding medium. After culturing for 2 days, the medium was replaced with a PGM differentiation medium (10 μg / mL insulin, 1 μM dexamethasone, 200 μM indomethacin, 500 μM isobutylmethylxanthine) supplemented with samples having the concentrations shown in Table 13 to induce differentiation into adipocytes. Went. After initiation of differentiation induction, the cells were cultured for 10 to 14 days until the control group matured and many lipid droplets accumulated in the cells. Thereafter, the culture supernatant was recovered, and the amount of adiponectin in the cell supernatant was quantified using a human adiponectin / Acrp30 immunoassay manufactured by R & D Systems.
The measurement results are shown in Table 13 as relative values with the amount of adiponectin production in the control with no sample added as 100.

13. Lipid peroxide resistance (antioxidant) using human epidermal keratinocytes
Human epidermal cell line HaCaT was seeded in a 96-well microplate so as to be 2.0 × 10 ⁴ cells per well. As the seeding medium, Dulbecco's modified Eagle medium (DMEM) to which 10% by mass of fetal bovine serum (FBS) was added was used. After 24 hours, the culture medium was replaced with a sample culture solution adjusted to each sample concentration shown in Table 14 with 10% by mass FBS-added DMEM medium, and further cultured for 24 hours.
It replaced | exchanged for the Hanks (+) solution which added arbitrary concentration t-butyl hydroperoxide, and culture | cultivated for 2 hours. Furthermore, it replaced | exchanged for PBS (-) containing 150 microgram / ml neutral red, and culture | cultivated at 37 degreeC for 2 hours. Next, it was replaced with a 50% by mass ethanol aqueous solution containing 1% by mass acetic acid, neutral red incorporated into the cells was extracted, and the absorbance of the extract was measured at 540 nm.
The obtained results are shown in Table 14 by relative values when the cell viability of the control without addition of t-butyl hydroperoxide is defined as 100.

From the above results, the extract of Sarunashi has a significant anti-aging effect (cell activation action, collagen production promotion action, ATP production promotion action), antioxidant action (DPPH radical scavenging action, SOD-like activity, lipid peroxide) at low concentrations. Tolerance effect), whitening effect (tyrosinase activity inhibitory action), anti-inflammatory / moisturizing action (arginase activity promoting action, hyaluronidase inhibiting action), slimming action (neutral fat accumulation inhibiting action), and adiponectin production promoting action It became.
Then, the formulation example of the skin external preparation and oral composition which mix | blended the pear extract obtained by each said preparation method is shown.
[Example 1] Emulsion (1) Squalane 10.0 (mass%)
(2) Methylphenylpolysiloxane 4.0
(3) Hydrogenated palm kernel oil 0.5
(4) Hydrogenated soybean phospholipid 0.1
(5) Polystearic acid polyoxyethylene sorbitan (20E.O.)
1.3
(6) Sorbitan monostearate 1.0
(7) Glycerin 4.0
(8) Methyl paraoxybenzoate 0.1
(9) Carboxyvinyl polymer 0.15
(10) The balance to be purified water 100 (11) Arginine (1% by mass aqueous solution) 20.0
(12) Ethanol extract 3.0
Production method: The oil phase components (1) to (6) are heated and dissolved at 80 ° C. On the other hand, the aqueous phase components (7) to (10) are dissolved by heating at 80 ° C. The oil phase component is added to this while stirring and uniformly emulsified with a homogenizer. After emulsification, start cooling and add (11) and (12) sequentially and mix uniformly.

[Example 2] Lotion (1) Ethanol 15.0 (mass%)
(2) Polyoxyethylene (40E.O.) hydrogenated castor oil 0.3
(3) Fragrance 0.1
(4) Purified water 100 (5) Citric acid 0.02
(6) Sodium citrate 0.1
(7) Glycerin 1.0
(8) Hydroxyethyl cellulose 0.1
(9) Hot water extract 5.0
Production method: (2) and (3) are dissolved in (1). After dissolution, (4) to (8) are sequentially added, and then sufficiently stirred, (9) is added and mixed uniformly.

[Example 3] Cream (1) Squalane 10.0 (mass%)
(2) Stearic acid 2.0
(3) Hydrogenated palm kernel oil 0.5
(4) Hydrogenated soybean phospholipid 0.1
(5) Cetanol 3.6
(6) Lipophilic glyceryl monostearate 2.0
(7) Glycerin 10.0
(8) Methyl paraoxybenzoate 0.1
(9) Arginine (20 mass% aqueous solution) 15.0
(10) Remainder 100 (11) Carboxyvinyl polymer (1% by weight aqueous solution) 15.0
(12) Ethanol extract 5.0
Production method: The oil phase components (1) to (6) are heated and dissolved at 80 ° C. On the other hand, the aqueous phase components (7) to (10) are dissolved by heating at 80 ° C. The oil phase component is added to this while stirring and uniformly emulsified with a homogenizer. After the emulsification is completed, add (11), start cooling, add (12) at 40 ° C., and mix uniformly.

[Example 4] Cosmetic liquid (1) The balance (mass%) of purified water 100
(2) Glycerin 10.0
(3) Sucrose fatty acid ester 1.3
(4) Carboxyvinyl polymer (1% by weight aqueous solution) 17.5
(5) Sodium alginate (1% by weight aqueous solution) 15.0
(6) Polyglyceryl monolaurate 1.0
(7) Macadamia nut oil fatty acid phytosteryl 3.0
(8) N-lauroyl-L-glutamate di (phytosteryl-2-octyldodecyl)
2.0
(9) Hardened palm oil 2.0
(10) Squalane (from olive) 1.0
(11) Behenyl alcohol 0.75
(12) Beeswax 1.0
(13) Jojoba oil 1.0
(14) 1,3-butylene glycol 10.0
(15) L-arginine (10% by mass aqueous solution) 2.0
(16) Hot water extract 5.0
Production method: The aqueous phase components (1) to (6) are mixed and dissolved by heating at 75 ° C. On the other hand, the oil phase components (7) to (13) are mixed and dissolved by heating at 75 ° C. Next, the oil phase component is added to the aqueous phase component and preliminary emulsification is performed, followed by uniform emulsification with a homomixer. Cooling is started after completion of emulsification, and (14) and (15) are added at 50 ° C. Cool further to 40 ° C, add (16) and mix evenly.

[Example 5] Aqueous gel (1) Carboxyvinyl polymer 0.5 (mass%)
(2) The balance made into purified water 100 (3) Sodium hydroxide (10 mass% aqueous solution) 0.5
(4) Ethanol 10.0
(5) Methyl paraoxybenzoate 0.1
(6) Fragrance 0.1
(7) Hot water extract 3.0
(8) Polyoxyethylene (60E.O.) hydrogenated castor oil 1.0
Manufacturing method: (1) is added to (2), and after stirring uniformly, (3) is added. After stirring uniformly, (5) previously dissolved in (4) is added. After stirring uniformly, the previously mixed (6) to (8) are added and stirred and mixed uniformly.

[Example 6] Cleansing fee (1) Squalane 81.0 (mass%)
(2) Polyoxyethylene glyceryl isostearate 15.0
(3) The remainder made into purified water 100 (4) Ethanol extract 4.0
Manufacturing method: (1) and (2) are uniformly dissolved. (3) and (4) are sequentially added to this and mixed uniformly.

[Example 7] Face-wash foam (1) Stearic acid 16.0 (mass%)
(2) Myristic acid 16.0
(3) Lipophilic glyceryl monostearate 2.0
(4) Glycerin 25.0
(5) Sodium hydroxide 7.5
(6) Palm oil fatty acid amidopropyl betaine 1.0
(7) The balance made into purified water 100 (8) Hot water extract 5.0
Production method: The oil phase components (1) to (4) are heated and dissolved at 80 ° C. On the other hand, the aqueous phase components (5) to (7) are heated and dissolved at 80 ° C., and mixed and stirred uniformly with the oil phase components. Cooling is started, and (8) is added at 40 ° C. and mixed uniformly.

[Example 8] Make-up base cream (1) Squalane 10.2 (% by mass)
(2) Cetanol 2.0
(3) Glycerin tri-2-ethylhexanoate 2.5
(4) Lipophilic glyceryl monostearate 1.0
(5) Propylene glycol 11.0
(6) Sucrose fatty acid ester 1.3
(7) The balance which makes 100 purified water (8) Titanium oxide 1.0
(9) Bengala 0.1
(10) Yellow iron oxide 0.4
(11) Fragrance 0.1
(12) Hot water extract 3.0
Production method: The oil phase components (1) to (4) are mixed and dissolved by heating at 75 ° C. On the other hand, the aqueous phase components (5) to (7) are mixed and dissolved by heating at 75 ° C., and the pigments (8) to (10) are added thereto and dispersed uniformly with a homomixer. The oil phase component is added to the water phase component and emulsified with a homomixer. Cooling is started after the emulsification is completed, and the components (11) and (12) are added at 40 ° C. and mixed uniformly.

[Example 9] Emulsion foundation (1) Methylpolysiloxane 2.0 (mass%)
(2) Squalane 5.0
(3) Octyldodecyl myristate 5.0
(4) Cetanol 1.0
(5) Polyoxyethylene (20E.O.) sorbitan monostearate
1.3
(6) Sorbitan monostearate 0.7
(7) 1,3-butylene glycol 8.0
(8) Xanthan gum 0.1
(9) Methyl paraoxybenzoate 0.1
(10) The balance of purified water 100 (11) Titanium oxide 9.0
(12) Talc 7.4
(13) Bengala 0.5
(14) Yellow iron oxide 1.1
(15) Black iron oxide 0.1
(16) Fragrance 0.1
(17) Ethanol extract 4.0
Production method: The oil phase components (1) to (6) are mixed and dissolved by heating at 75 ° C. On the other hand, the aqueous phase components (7) to (10) are mixed and dissolved by heating at 75 ° C., and the pigments (11) to (15) are added thereto and uniformly dispersed with a homomixer. Add oil phase ingredients and emulsify. Cooling is started after the emulsification is completed, and components (16) and (17) are sequentially added at 40 ° C. and mixed uniformly.

[Example 10] Water-in-oil emollient cream (1) Liquid paraffin 34.0 (mass%)
(2) Microcrystalline wax 2.0
(3) Vaseline 5.0
(4) Diglycerin oleate 5.0
(5) Sodium chloride 1.3
(6) Potassium chloride 0.1
(7) Propylene glycol 3.0
(8) 1,3-butylene glycol 5.0
(9) Methyl paraoxybenzoate 0.1
(10) Ethanol extract 3.0
(11) The balance of 100 purified water (12) Fragrance 0.1
Production method: Dissolve (5) and (6) in a part of (11) to 50 ° C., and gradually add to (4) heated to 50 ° C. with stirring. After mixing this, it disperse | distributes uniformly to (1)-(3) heated and melt | dissolved at 70 degreeC. (7) to (10) are added to the remainder of (11) heated and dissolved at 70 ° C. while stirring, and emulsified with a homomixer. Cooling is started after completion of emulsification, and (12) is added at 40 ° C. and mixed uniformly.

[Example 11] Pack (1) Balance (mass%) of purified water 100
(2) Polyvinyl alcohol 12.0
(3) Ethanol 17.0
(4) Glycerin 9.0
(5) Polyethylene glycol (average molecular weight 1000) 2.0
(6) Hot water extract 5.0
(7) Fragrance 0.1
Production method: (2) and (3) are mixed, heated to 80 ° C, and then dissolved in (1) heated to 80 ° C. After uniformly dissolving, add (4) and (5), and start cooling while stirring. Cool to 40 ° C, add (6) and (7) and mix uniformly.

[Example 12] Bath agent (1) Fragrance 0.3 (% by mass)
(2) Ethanol extract 3.0
(3) Sodium bicarbonate 50.0
(4) Sodium sulfate 46.7
Production method: (1) to (4) are mixed uniformly.

[Example 13] Hair wax (1) Stearic acid 3.0 (mass%)
(2) Microcrystalline wax 2.0
(3) Cetyl alcohol 3.0
(4) Highly polymerized methylpolysiloxane 2.0
(5) Methylpolysiloxane 5.0
(6) Poly (oxyethylene / oxypropylene) methylpolysiloxane copolymer
1.0
(7) Methyl paraoxybenzoate 0.1
(8) 1,3-butylene glycol 7.5
(9) Arginine 0.7
(10) Purified water 100 (11) Hot water extract 4.0
(12) Fragrance 0.1
Production method: The oil phase components (1) to (6) are mixed and heated and dissolved at 75 ° C. On the other hand, the aqueous phase components (7) to (10) are dissolved by heating at 75 ° C., the oil phase component is added, and the mixture is emulsified with a homomixer. Cooling is started after the emulsification is completed, and the components (11) and (12) are added at 40 ° C. and mixed uniformly.

[Example 14] Hair artic (1) Ethanol 50.0 (mass%)
(2) Remainder 100 (3) Ethanol extract 3.0
(4) Fragrance 0.1
Production method: Components (1) to (4) are mixed and homogenized.

Example 15 Tablet (1) Corn Starch 44.0 (mass%)
(2) Crystalline cellulose 40.0
(3) Carboxymethylcellulose calcium 5.0
(4) Silicic anhydride 0.5
(5) Magnesium stearate 0.5
(6) Ethanol extract 10.0
Production method: (1) to (6) are uniformly mixed, and compression-molded with a tableting machine to obtain one tablet of 200 mg.

Example 16 Powder (1) Magnesium aluminate silicate 95.3 (mass%)
(2) Carboxymethylcellulose calcium 4.5
(3) Ethanol extract 0.2
Production method: After the powders (1) to (3) are mixed, they are pulverized by a pulverizer and uniformly dispersed.

[Example 17] Candy (1) Sucrose 60.0 (mass%)
(2) Minamata 39.5
(3) Hot water extract 0.4
(4) Fragrance 0.1
Manufacturing method: (1) and (2) are heated, mixed and homogenized, then cooled, components (3) and (4) are added at 70 ° C., and the mixture is homogenized and then molded.

[Example 18] Drink (1) Aminoethylsulfonic acid 1000 mg
(2) Thiamine nitrate 10mg
(3) Riboflavin sodium phosphate 5mg
(4) Pyridoxine hydrochloride 10mg
(5) Anhydrous caffeine 50mg
(6) Citric acid 250mg
(7) D-sorbitol solution 8g
(8) Hot water extract 10mg
(9) Fragrance Trace amount (10) Purified water Mass production method to make the whole 100 mL: (1) to (9) are sequentially added to (10) and homogenized.

Claims (1)

A skin external preparation for moisturizing, comprising an extract from one or two kinds of solvents selected from water and ethanol, of the leaves of the genus Matatabidae.