JP2010150207A - Moisturizer, anti-aging agent, antioxidant, neutral fat-accumulation inhibitor, skin whitening agent, anti-inflammatory agent, immunostimulator, skin care preparation, oral medication - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a moisturizer, an anti-aging agent, an antioxidant, a neutral fat-accumulation inhibitor, a skin whitening agent, an anti-inflammation agent, and an immunostimulator, which are capable of widely applying in fields of skin care preparations, and oral medications, by finding an active constituent having excellent cell stimulating action, skin whitening action, anti-oxidation action and the like. <P>SOLUTION: An extract of Ficus erecta Thunb., a genus Ficus in the family Moraceae, is used as a moisturizer, an anti-aging agent, an antioxidant, a neutral fat accumulation inhibitor, a skin whitening agent, or an anti-inflammation agent. Prevention or amelioration of various skin symptoms such as wrinkles, sagging, tense skin, blotches, and somberness, or inhibition of neutral fat accumulation is exerted by blending the obtained moisturizer, the anti-aging agent, the antioxidant, the neutral fat accumulation inhibitor, the skin whitening agent, the anti-inflammation agent, or the immunostimulator in a composition of a skin care preparation, an oral medication or the like. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a humectant, an anti-aging agent, an antioxidant, a neutral fat accumulation inhibitor, a whitening agent, an anti-inflammatory agent, and an immunostimulant. More specifically, it contains a moisturizer, a cell activator, an antioxidant, a neutral fat accumulation inhibitor, a whitening agent and an anti-inflammatory agent containing an extract of Inubiwa (Ficus erecta Thumb.), And an extract of Inubiwa. The present invention relates to an external preparation for skin and an oral preparation.

  Wrinkles, tarmi, pigmentation / color change, skin elasticity decline, skin surface morphology disorder, and other factors causing deterioration of skin symptoms include, for example, wrinkles and tarmi, a decrease in dermal fibroblast function due to drying, aging, etc. In addition, there are important factors such as reduction or degeneration of the dermal matrix such as collagen and elastin, and oxidative damage caused by external stress such as ultraviolet rays. In addition, pigmentation and color change, including skin darkness, are partly unknown, but are caused by hormonal abnormalities and melanin production by the stimulation of ultraviolet rays of sunlight. Among them, spots and freckles are melanin. It is believed that abnormal pigmentation is the cause.

  In order to solve such a problem, various methods have been studied conventionally. For example, as a cell activator, the essence of the genus Passifloraceae (see Patent Document 1) and the like, as the antioxidant, an extract of eucommia (see Patent Document 2), and the like, and as a melanin production inhibitor, a Symbidium genus Plant extracts (see Patent Document 3) and the like are known.

JP 2002-131945 A Japanese Patent Laid-Open No. 10-17436 JP 2002-33336 A

  Naturally-derived components are known to have various pharmacological and cosmetic effects, and so far many plants and fungi have been widely applied to fields such as external preparations for skin and oral use. However, there are many naturally-derived ingredients whose effects are not yet known, and the development of effective ingredients having excellent moisturizing action, anti-aging action, antioxidant action, whitening action, etc. was expected. . The present invention was made to find such an active ingredient, and is a moisturizer, anti-aging agent, antioxidant, neutral fat accumulation inhibitor, which can be widely applied to skin external preparations and oral preparations, The object is to provide a whitening agent, an anti-inflammatory agent, an immunostimulant, and a skin external preparation or oral preparation containing these.

  In order to find a moisturizer that can be widely applied to external preparations for skin, oral preparations, and the like, the present inventors have conducted intensive research on various naturally-derived substances. As a result, they found excellent moisturizing action, anti-aging action, antioxidant action, neutral fat accumulation-inhibiting action, whitening action, anti-inflammatory action, and immunostimulatory action for the extract of Inubiwa, and completed the present invention. That is, the present invention contains a moisturizer, an anti-aging agent, an antioxidant, a neutral fat accumulation inhibitor, a whitening agent, an anti-inflammatory agent and an immunostimulant, and an extract of Inubiwa, which contains an extract of Inubiwa. An external preparation for skin and an oral preparation are provided.

  According to the present invention, it is possible to provide a moisturizer, anti-aging agent, antioxidant, neutral fat accumulation inhibitor, whitening agent, anti-inflammatory agent and immunostimulant having excellent effects. Moreover, the skin external preparation and oral preparation which contain these as an active ingredient can be provided.

  Inubiwa (Ficus erecta Thunb.) Used in the present invention is a deciduous small Takagi of the genus Asteraceae, and is distributed in Honshu, Shikoku, Kyushu, Okinawa and Taiwan west of the Kanto region.

  As the Inubiwa in the present invention, the raw material or the dry pulverized one can be used as it is, but it is preferable to use an extract extracted with a solvent or the like. At the time of extraction, it may be used as it is, but considering the extraction efficiency, it is desirable to perform the extraction after performing processing such as shredding, drying, and pulverization. The extraction can be performed by a general method such as a method of immersing in an extraction solvent or an extraction method using a supercritical fluid or the like. The extraction temperature is preferably 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 preferably about 1 hour to 14 days.

  Examples of the extraction solvent include water, lower alcohols such as methanol, ethanol, propanol, and isopropanol, polyhydric alcohols such as 1,3-butylene glycol, propylene glycol, dipropylene glycol, and glycerin, ethyl ether, propyl ether, and the like. Solvents such as ethers, esters such as butyl acetate and ethyl acetate, and ketones such as acetone and ethyl methyl ketone can be used, and one or more of these can be selected and used. Further, physiological saline, phosphate buffer, phosphate buffered saline, or the like may be used. Furthermore, you may use 1 type, or 2 or more types of supercritical fluids and subcritical fluids, such as water, a carbon dioxide, ethylene, propylene, ethanol, methanol, ammonia. It is also possible to extract under pressure using an autoclave or the like.

  The obtained extract can be used as it is, but the concentrated and dried product can be dissolved again in water or a solvent, or purified such as decolorization, deodorization, and desalting as long as these physiological functions are not impaired. You may use after performing a process or performing the fractionation process by column chromatography. For storage, it can be freeze-dried after purification and dissolved in a solvent when used.

  When using the extract of Inubiwa, the use part is not particularly limited, and any part such as the whole, flowers, leaves, stems, branches, roots, seeds, bark, sap, pericarp, and fruits may be used. Absent. From the viewpoint of availability and effectiveness, it is more preferable to use leaves, fruits and branches.

  Inubiwa extract has excellent moisturizing, anti-aging, antioxidant, neutral fat accumulation-inhibiting, whitening, anti-inflammatory, and immunostimulatory, moisturizing, anti-aging, and antioxidants It can be used as a neutral fat accumulation inhibitor, whitening agent, anti-inflammatory agent, and immunostimulant. In addition, moisturizers, anti-aging agents, antioxidants, neutral fat accumulation inhibitors, whitening agents, anti-inflammatory agents, and immunostimulants, which contain Inuviwa extract as an active ingredient, are not only applied to the skin, but also to the hair. It can also be used for oral administration and can be applied to pharmaceuticals, quasi-drugs, cosmetics or oral preparations.

  A moisturizing agent containing an extract of Inubiwa exhibits an excellent moisturizing action on the skin and hair, and has a particularly high moisturizing effect on the skin.

  An anti-aging agent containing an extract of Inubiwa exhibits an excellent cell activating effect on various cells, particularly an excellent effect on dermal fibroblasts and human epidermal keratinocytes. It is useful as a fibroblast activator and a human epidermal keratinocyte activator.

  An antioxidant containing an extract of Inubiwa exhibits an excellent antioxidant action and is useful as an antioxidant.

A neutral fat accumulation inhibitor containing an extract of Inubiwa exhibits an excellent neutral fat accumulation inhibitory effect and is useful as a neutral fat accumulation inhibitor. Known diseases caused by excessive accumulation of neutral fat include hyperlipidemia, arteriosclerosis, fatty liver and the like. It can be expected to be effective not only in preventing and improving the disease, but also in preventing and improving such diseases. A slimming effect can also be expected.
A whitening agent containing an extract of Inubiwa exhibits an excellent whitening effect, but particularly exhibits an excellent effect on a melanin production promoting action and is useful as a whitening agent.

  A whitening agent containing an extract of Inubiwa exhibits an excellent whitening effect, but particularly exhibits an excellent effect on a melanin production promoting action and is useful as a whitening agent.

  The anti-inflammatory agent containing the extract of Inubiwa exhibits an excellent anti-inflammatory action and is useful as an anti-inflammatory agent.

  The immunostimulant containing the extract of Inubiwa exhibits an excellent immunostimulatory action and is useful as an immunostimulant.

  In addition, by adding Inuviwa extract to an external preparation for skin, prevention and improvement of skin symptoms such as wrinkles, tarmi, skin firmness, spots, kusumi, dryness, fine wrinkles, and parts such as the abdomen, thighs, face, etc. Skin external preparation that exhibits an excellent effect in the prevention and improvement of general obesity, skin external preparation for moisturizing, skin external preparation for anti-aging improvement, skin external preparation for whitening, or skin for inhibiting neutral fat accumulation It can also be used as an external preparation. Furthermore, the extract of Inubiwa can also be used for pharmaceuticals, quasi-drugs, oral preparations for the purpose of beauty, health maintenance or nutritional supplementation.

  The amount of Inubiwa extract extracted into the topical skin preparation or oral preparation can be adjusted depending on the type of skin external preparation or oral preparation, purpose of use, etc. , 0.0001 to 50.0 mass% is preferable with respect to the total amount, and more preferably 0.001 to 25.0 mass%.

  In the case where the extract of Inubiwa is added to the external preparation for skin, the dosage form is arbitrary, and for example, it can be provided as a solubilizing system such as lotion, an emulsifying system such as cream or emulsion, or a dispersing system such as calamine lotion. Furthermore, it can also be provided in various dosage forms such as aerosols, ointments, powders and granules filled with a propellant.

  Oil preparations, moisturizers, and powders, which are usually added to pharmaceuticals, quasi-drugs, skin cosmetics, hair cosmetics, and detergents, etc., as needed Other components such as body, pigment, emulsifier, solubilizer, cleaning agent, ultraviolet absorber, thickener, drug, fragrance, resin, antibacterial and fungicidal agent, alcohols and the like can be appropriately blended. In addition, other moisturizers, cell activators, antioxidants, slimming agents, anti-aging agents, whitening agents, and anti-inflammatory agents can be used in combination as long as the effects of the present invention are not impaired.

  When the extract of Inubiwa is blended into the oral preparation, its form is not particularly limited, but liquid forms such as liquid preparations, solid preparations such as granules, tablets, capsules, glazes, jelly, gummi, gum, etc. And can be used as pharmaceuticals, quasi drugs, oral supplements for nutritional supplements, oral preparations for health, and the like. In doing so, other additives such as excipients, binders, extenders, disintegrants, surfactants, lubricants, dispersants, buffers, preservatives, coating agents, preservatives, flavoring agents, flavoring agents. Coloring agents, plasticizers and the like can be added. In addition, other moisturizers, cell activators, antioxidants, slimming agents, anti-aging agents, whitening agents, and anti-inflammatory agents can be used in combination as long as the effects of the present invention are not impaired.

  In the following, production examples of Inubiwa extract, tests for evaluating each action, formulation examples of external preparations for skin and oral preparations will be described in more detail, but the technical scope of the present invention is not limited thereby. It is not something.

  [Production Example 1] 100 g of dried ground powder of Inubiwa leaf was dispersed in 2.0 kg of purified water and extracted by heating at 120 ° C. for 20 minutes using an autoclave. The extract supernatant was filtered and lyophilized to obtain extract 1.

  [Production Example 2] 100 g of dried ground crumbs of Inubiwa leaves were dispersed in 2.0 kg of 50 vol% ethanol aqueous solution and extracted at room temperature for 2 hours with stirring. The extract supernatant was filtered off, concentrated under reduced pressure, and lyophilized to obtain extract 2.

  [Production Example 3] 100 g of dried ground crumbs of Inubiwa fruit were dispersed in 2.0 kg of purified water and extracted by heating at 120 ° C. for 20 minutes using an autoclave. The extract supernatant was filtered and lyophilized to obtain extract 3.

  [Production Example 4] 100 g of dried ground crumbs of Inubiwa fruit was dispersed in 2.0 kg of 50 vol% ethanol aqueous solution and extracted at room temperature for 2 hours with stirring. The extract supernatant was filtered off, concentrated under reduced pressure, and then lyophilized to obtain extract 4.

  [Production Example 5] 100 g of a dry pulverized product of Inubiwa branch was dispersed in 2.0 kg of purified water, and extracted by heating at 120 ° C. for 20 minutes using an autoclave. The extract supernatant was filtered and lyophilized to obtain extract 5.

  [Production Example 6] 100 g of a dry pulverized product of Inubiwa branch was dispersed in 2.0 kg of 50 vol% ethanol aqueous solution and extracted at room temperature for 2 hours with stirring. The extract supernatant was filtered off, concentrated under reduced pressure, and then lyophilized to obtain extract 6.

  The effectiveness evaluation test of the extract of Inubiwa was conducted using the above production example.

[Test Example 1] Human dermal fibroblast hyaluronic acid production promoting action (moisturizing effect)
Evaluation was performed using Extract 3 (Inubiwa (real) hot water extract) as a sample.

Normal human dermal fibroblasts were seeded in a 96-well microplate at 2.0 × 10 ⁴ cells per well. Dulbecco's modified Eagle medium (DMEM) supplemented with 5% by weight fetal bovine serum (FBS) was used as the seeding medium. After 24 hours, the medium was replaced with 0.5% by mass FBS-added DMEM to which the extract 3 was added so as to have the concentration shown in Table 1, and further cultured for 3 days. For the quantification of hyaluronic acid secreted into the culture supernatant, an indirect ELISA method using proteoglycan was used, and finally 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid against labeled peroxidase was used. ) After diammonium salt (ABTS) and hydrogen peroxide were added and reacted, the absorbance at 405 nm was measured with a microplate reader. The amount of protein in each well was measured using BCA Protein Assay Kit manufactured by PIERCE, and the amount of hyaluronic acid produced per unit amount of protein was determined. The evaluation results are shown in Table 1 as relative values with the amount of hyaluronic acid produced per unit protein in the control with no sample added as 100.
Regarding the effectiveness evaluation test result, the significance probability of less than 5% (P <0.05) is represented by * and the significance probability of less than 1% (P <0.01) is represented by ** with respect to the significance probability P value in the t-test.

  As is clear from Table 1, Extract 3 showed a significant human dermal fibroblast hyaluronic acid production promoting effect. From this, it was clarified that the extract of Inubiwa has an excellent moisturizing action because it has an effect of promoting hyaluronic acid production of human dermal fibroblasts.

[Test Example 2] Human dermal fibroblast activation effect (anti-aging effect)
Evaluation was performed using Extract 5 (Inubiwa (branch) hot water extract) as a sample.

Normal human dermal fibroblasts were seeded in a 96-well microplate at 2.0 × 10 ⁴ cells per well. Dulbecco's modified Eagle medium (DMEM) supplemented with 1% by weight fetal bovine serum (FBS) was used as the seeding medium. After 24 hours, the concentration was changed to DMEM supplemented with 1% mass FBS to which the extract 5 was added so as to have the concentration shown in Table 2, and further cultured for 48 hours. Next, the MTT reagent adjusted with the culture medium so that it might become 400 micrograms / mL was added to the cell except the supernatant, and it culture | cultivated for about 2 hours. Finally, formazan produced in 2-propanol was extracted and the absorbance at 550 nm was measured. 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. In the evaluation, in addition to the sample culture solution, 1% FBS-added DMEM was used as a control, and 5% by mass FBS-added DMEM was used as a positive control. The evaluation results are shown in Table 2 as relative values with the cell activation effect in the control with no sample added being taken as 100.

  As is clear from Table 2, Extract 5 showed a significant human dermal fibroblast activation effect. From this, it was clarified that the extract of Inubiwa has a human dermal fibroblast activation effect and has an excellent anti-aging effect.

[Test Example 3] Human dermal fibroblast type I collagen production promoting action (anti-aging effect)
Evaluation was performed using Extract 4 (Inubiwa (fruit) 50% volume ethanol extract) as a sample.

Human epidermal keratinocytes HaCaT were seeded in a 96-well microplate so as to be 2.0 × 10 ⁴ cells per well. Dulbecco's modified Eagle medium (DMEM) supplemented with 5% by weight fetal bovine serum (FBS) was used as the seeding medium. After 24 hours, it was replaced with 0.5% by mass FBS-added DMEM to which the extract 4 was added so as to have the concentration shown in Table 3, and further cultured for 24 hours. For quantification of type I collagen secreted into the culture supernatant, an ELISA method was used, and labeled 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) and excess After adding hydrogen oxide and reacting, absorbance at 405 nm was measured. The amount of protein in each well was measured by BCA Protein Assay Kit manufactured by PIERCE, and the amount of type I collagen produced per unit protein was determined. The evaluation results are shown in Table 3 as relative values when the amount of type I collagen produced per unit protein in the control with no sample added is defined as 100.

  As apparent from Table 3, Extract 4 showed a significant human dermal fibroblast type I collagen production promoting effect. From this, it became clear that the extract of Inubiwa has an excellent anti-aging effect.

[Test Example 4] Human dermal fibroblast ATP production promoting action (anti-aging effect)
Evaluation was performed using Extract 2 (Inubiwa (leaf) 50% volume ethanol extract) as a sample.

Normal human dermal fibroblasts were seeded in a 48-well microplate at 4.0 × 10 ⁴ per well. Dulbecco's modified Eagle medium (DMEM) supplemented with 1% by weight fetal bovine serum (FBS) was used as the seeding medium. After 24 hours, the medium was replaced with 1% FBS-added DMEM to which the extract 2 was added so that the concentrations shown in Table 4 were obtained, and further cultured for 24 hours. The cell supernatant was removed and washed, and the cells were sonicated to elute the ATP in the cells. At that time, an ATP-degrading enzyme inhibitor (Cellstein Hoechst 33342) was added to prevent elution of ATP-degrading enzyme in the cells. ATP determination kit manufactured by Molecular Probes was used for quantification of ATP. The cell lysate was dispensed into a test tube, luciferase and a luciferin reagent were added, and chemiluminescence was measured. The evaluation results are shown in Table 4 as relative values with the ATP production ability in the control with no sample added as 100.

  As is apparent from Table 4, Extract 2 showed a significant human dermal fibroblast ATP production promoting effect. From this, it became clear that the extract of Inubiwa has an excellent anti-aging effect.

[Test Example 5] Human epidermal keratinocyte activation action (anti-aging effect)
Evaluation was performed using Extract 4 (Inubiwa (fruit) 50% volume ethanol extract) as a sample.

Human epidermal keratinocytes HaCaT were seeded in a 96-well microplate so as to be 2.0 × 10 ⁴ cells per well. Dulbecco's modified Eagle medium (DMEM) supplemented with 5% by weight fetal bovine serum (FBS) was used as the seeding medium. After 24 hours, it was replaced with 5% by mass FBS-added DMEM to which the extract 4 was added so as to have the concentration shown in Table 5, and further cultured for 24 hours. Next, the MTT reagent adjusted with the culture medium so that it might be set to 100 microgram / mL was added to the cell except the supernatant, and it culture | cultivated for about 2 hours. Finally, formazan produced in 2-propanol was extracted and the absorbance at 550 nm was measured. 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 3 as relative values when the cell activation effect in the control with no sample added is taken as 100.

  As is clear from Table 5, Extract 4 showed a significant human epidermal keratinocyte activation effect. From this, the extract of Inubiwa was found to have a human keratinocyte activating effect and to have an excellent anti-aging effect.

[Test Example 6] DPPH radical scavenging action (antioxidant action)
Evaluation was performed using Extract 3 (Inubiwa (real) hot water extract) as a sample.

Add 100 μL of 0.2 mM 1,1-diphenyl-2-picrylhydrazyl (DPPH) ethanol solution to 100 μL of the sample solution to which the extract 3 is added to 50% by mass ethanol to the concentration shown in Table 6. After mixing well, the mixture was allowed to stand in the dark at room temperature for 24 hours, and the absorbance at 516 nm derived from the DPPH radical was measured. The DPPH radical elimination rate is defined by equation (1), where (A) is the absorbance when no sample is added and (B) is the absorbance when the sample is added.
Erase rate = {1- (B) / (A)} × 100 Formula (1)
The evaluation results are shown in Table 6.

  As is clear from Table 6, Extract 3 showed a significant DPPH radical scavenging action. From this, it was clarified that the extract of Inubiwa has a DPPH radical scavenging action and has an excellent antioxidant action.

[Test Example 7] SOD-like activity (antioxidant effect)
Evaluation was performed using Extract 5 (Inubiwa (branch) hot water extract) as a sample.

To 75 μL of the HANK ′S (+) solution containing 0.25 mM WST-1 and 1 mM Hypoxanthine, 25 μL of the sample solution in which the extract 5 was added to the concentration shown in Table 7 was added to HANK ′S (+). Further, 25 μL (0.0075Units) of Xanthine Oxidase was added, and after reacting at 37 ° C. for 15 minutes, the absorbance at 450 nm was measured. When the absorbance when no sample is added is (A) and the absorbance when the sample is added is (B), the superoxide anion elimination rate is defined by equation (2).
Erase rate (%) = {1- (B) / (A)} × 100 Formula (2)
Table 7 shows the evaluation results.

  As is clear from Table 5, extract 5 showed a significant SOD-like activity. From this, it was revealed that the extract of Inubiwa has an SOD-like activity and has an excellent antioxidant effect.

[Test Example 8] Neutral fat accumulation inhibitory action As a sample, the extract 3 (Inubiwa (actual) hot water extract) was used for evaluation.

Subcutaneous fat-derived normal human preadipocytes Cryo HPRAD-SQ were seeded in a 96-well microplate so as to be 5.0 × 10 ³ cells per well. PGM medium (10% FBS, 2 mM L-glutamine, 100 units / mL Penicillin, containing 100 μg / mL Streptomycine) was used as the seeding medium. After culturing for 48 hours, extract 3 was added to the PGM differentiation medium adjusted to the concentration shown in Table 8 (containing 10 μg / mL insulin, 1 μM Dexamethasone, 200 μM Indomethacin, 500 μM Isobutylmethylxanthine), and replaced with adipocytes 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 harvesting the cells, the cells were fixed using a 10% neutral buffered formaldehyde solution. After washing with PBS (−), 0.5 mass / volume% oil red O solution was added and cultured at 37 ° C. for 2 hours. After washing with PBS (−), methanol was added to extract the dye, and the absorbance at 550 nm was measured. At the same time, the absorbance at 650 nm was measured as turbidity, and the neutral fat accumulation inhibitory action was evaluated using the difference between the two measured values. The evaluation results are shown in Table 8 as relative values when the neutral fat accumulation amount in the control with no sample added is defined as 100.

  As is clear from Table 8, the extract 3 showed a significant effect of inhibiting neutral fat accumulation in human preadipocytes. From this, it became clear that the extract of Inubiwa has an excellent neutral fat accumulation inhibitory action.

[Test Example 9] Human epidermal melanocyte tyrosinase activity inhibitory action (whitening action)
Evaluation was performed using Extract 4 (Inubiwa (leaves) hot water extract) as a sample.

Normal human epidermal melanocytes were seeded in a 96-well microplate so as to be 3.0 × 10 ⁴ cells per well. Medium 254S was used as the seeding medium. After 24 hours, the extract 3 was added and replaced with Medium 254S adjusted to the concentration shown in Table 9, and further cultured for 48 hours. Next, it was replaced with 75 μL of a phosphate buffer containing 1% by weight Triton-X to completely lyse the cells, and 50 μL was used as a crude enzyme solution. To the crude enzyme solution, 50 μL of a 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 was measured immediately after the addition of the substrate and at the end of the reaction, and the amount of dopamelanin produced was determined by introducing the difference between the two measured values into the following equation (Equation 3).
Amount of dopamelanin produced = {(405 nm value after reaction−405 nm value before reaction) −2.166} /5.238 (formula 3)
In addition, the amount of protein in each well was measured with BCA Protein Assay Kit manufactured by PIERCE, and the amount of dopamelanin produced per unit protein amount was determined. The evaluation results are compared with the amount of dopamelanin produced per unit protein amount in the control with no sample added, and are shown in Table 9.

[Test Example 10] Melanin production inhibitory action (whitening action)
Evaluation was performed using Extract 4 (Inubiwa (leaves) hot water extract) as a sample.

  B16 mouse melanoma cells (B16F0 cells) were seeded in a 90 mm dish so that there were 18000 cells per dish. Dulbecco's modified Eagle medium (DMEM) supplemented with 5% by weight fetal bovine serum (FBS) was used as the seeding medium. After 24 hours, the extract 4 was added and replaced with 5% mass FBS-added DMEM adjusted to the concentration shown in Table 10, and further cultured for 5 days. After completion of the culture, the cells were detached by trypsin treatment, transferred to a 1.5 mL microtube, and centrifuged to obtain a cell precipitate. The resulting precipitate was visually determined for its blackening status based on the following determination table. In the evaluation, 5% by mass FBS-added DMEM was used as the negative control, and 5% by mass FBS-added DMEM containing 50 mM sodium lactate was used as the positive control. These visual determination results were determined as determination 5 and determination 1, and used as an index for sample determination. As shown in Table 10, the visual judgment was evaluated in five stages. At the same time, a tissue solubilizer (trade name Solvable) was added to the precipitate, boiled, returned to room temperature, and the absorbance at 500 nm was measured with a spectrophotometer (HITACHI spectrophotometer U-3010) to determine the total amount of melanin. Asked. The evaluation results are shown in Table 11.

  As can be seen from Tables 9 and 11, Extract 4 was found to significantly suppress melanin production in pigment cells. Moreover, it became clear from Table 11 that the melanin production in a pigment cell was suppressed remarkably. From these results, it was clarified that the extract of Inubiwa has an excellent inhibitory effect on melanin production and has a high whitening effect.

[Test Example 11] Phospholipase A ₂ (PLA ₂ ) inhibitory action (anti-inflammatory action)
Evaluation was performed using Extract 6 (Inubiwa (branch) 50% ethanol extract) as a sample.

Phospholipase A ₂ (PLA ₂ ) adjusted to 60 ng / mL, a sample prepared by adding extract 6 to a concentration shown in Table 12, and DTNB (5,5-dithio) adjusted to 10 mM 10 μL each of -bis- (2-nitrobenzoic acid)) was mixed and allowed to stand at room temperature for 10 minutes. Further, 50 μL of 1.66 mM Diheptanoyl Thio-PC was added as a substrate, reacted at room temperature for 45 minutes, and the absorbance at 414 nm was measured. In addition, the absorbance at the time of buffer addition was measured instead of the PLA2 solution, and the difference between the two measured values was determined. When the value of the control with no sample added is (A) and the value at the time of sample addition is (B), the PLA ₂ inhibitory action is defined by the following formula (Formula 4).
Inhibition rate (%) = {1− (B) / (A)} × 100 (Formula 4)
The evaluation results are shown in Table 12.

As is apparent from Table 12, the extract of Inubiwa was found to have a Phospholipase A ₂ (PLA ₂ ) inhibitory action. From this, it was revealed that Phospholipase A ₂ (PLA ₂ ) inhibitory action was found in the extract of Inubiwa and had an excellent anti-inflammatory action.

[Test Example 12] Cell activation using human acute monocyte leukemia cell line (THP-1) (immunostimulation)
Evaluation was performed using Extract 2 (Inubiwa (leaf) 50 vol% ethanol extract) as a sample.

Human acute monocytic leukemia cell line (THP-1) was seeded in a 96-well microplate so as to be 5.0 × 10 ⁴ cells per well. As a seeding medium, Roswell Park Memorial Institute medium (RPMI) supplemented with 1% by mass of fetal bovine serum (FBS) was used. After 24 hours, Phorbol 12-Myristate 13-Acetate (PMA) was added to the cell culture solution to 20 ng / mL. After further 24 hours, the extract 2 was added and replaced with a 1% by mass FBS-added RPMI medium prepared to have the concentration shown in Table 13, and cultured for 48 hours. Next, RPMI medium supplemented with 1% by mass FBS supplemented with 1/10 amount of living cell count measuring reagent SF (Dojindo Laboratories) was added to the cells from which the supernatant had been removed, and cultured for about 2 hours. After mixing, the absorbance at 450 nm was measured. 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 13 as relative values when the cell activation effect in the control with no sample added is taken as 100.

  As is apparent from Table 13, the extract of Inubiwa showed a cell activation effect on human acute monocyte leukemia cells, and it was revealed that it has an excellent immune activation effect.

  The formulation example which implemented this invention is shown.

[Formulation 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) Polyoxyethylene monostearate
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) Purified water 53.85
(11) Arginine (1% by weight aqueous solution) 20.0
(12) Extract 1 (Inubiwa (leaves) hot water 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, cooling is started, and (11) and (12) are sequentially added and mixed uniformly.

[Prescription Example 2] Lotion (1) Ethanol 15.0 (mass%)
(2) Polyoxyethylene (40E.O.) hydrogenated castor oil 0.3
(3) Fragrance 0.1
(4) Purified water 78.38
(5) Citric acid 0.02
(6) Sodium citrate 0.1
(7) Glycerin 1.0
(8) Hydroxyethyl cellulose 0.1
(9) Extract 2 (Inubiwa (leaves) 50 vol% ethanol 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.

[Prescription 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) Purified water 40.7
(11) Carboxyvinyl polymer (1% by weight aqueous solution) 15.0
(12) Extract 3 (Inubiwa (real) 50 vol% ethanol extract) 1.0
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. (11) is added after completion | finish of emulsification, cooling is started, (12) is added at 40 degreeC, and it mixes uniformly.

[Formulation Example 4] Cosmetic liquid (1) Purified water 27.45 (mass%)
(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-glutamic acid
Di (phytosteryl-2-octyldodecyl) 2.0
(9) Hardened palm oil 2.0
(10) Squalane (derived 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) Extract 1 (Inubiwa (leaves) 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 (14) 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 (15) is added at 50 ° C. Cool further to 40 ° C., add (16) and mix evenly.

[Formulation Example 5] Aqueous gel (1) Carboxyvinyl polymer 0.5 (mass%)
(2) Purified water 86.7
(3) Sodium hydroxide (10% by mass aqueous solution) 0.5
(4) Ethanol 10.0
(5) Methyl paraoxybenzoate 0.1
(6) Fragrance 0.1
(7) Extract 2 (Inubiwa (leaf) 50 vol% ethanol extract) 2.0
(8) Polyoxyethylene (60E.O.) hydrogenated castor oil 0.1
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.

[Formulation Example 6] Cleansing Fee (1) Squalane 50.5 (mass%)
(2) 2-ethylhexane sancetyl 23.0
(3) Octyl palmitate 10.5
(4) Polyoxyethylene glyceryl isostearate 15.0
(5) Purified water 0.5
(6) Extract 2 (50% by volume ethanol extract of Inubiwa (leaf)) 0.5
Production method: The oil phase components (1) to (3) are uniformly dissolved. (4) to (6) are sequentially added to this and mixed uniformly.

[Formulation Example 7] Face-wash foam (1) Stearic acid 16.0 (mass%)
(2) Myristic acid 16.0
(3) Lipophilic glyceryl monostearate 2.0
(4) Glycerin 20.0
(5) Sodium hydroxide 7.5
(6) Palm oil fatty acid amidopropyl betaine 1.0
(7) Purified water 36.5
(8) Extract 2 (Inubiwa (leaf) 50 vol% ethanol extract) 1.0
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.

[Prescription Example 8] Make-up base cream (1) Squalane 10.0 (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) Purified water 69.4
(8) Titanium oxide 1.0
(9) Bengala 0.1
(10) Yellow iron oxide 0.4
(11) Fragrance 0.1
(12) Extract 3 (Inubiwa (real) 50 vol% ethanol extract) 1.2
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.

[Prescription 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) Purified water 57.4
(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) Extract 1 (Inubiwa (leaves) hot water extract) 1.2
Production method: The oil phase components (1) to (6) are mixed and dissolved by heating at 75 ° C. On the other hand, the water phase components (7) to (10) are mixed, dissolved by heating at 75 ° C., the pigments (11) to (15) are added thereto, and the mixture is 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.

[Formulation Example 10] Water-in-oil emollient cream (1) Liquid paraffin 30.0 (% by 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) Extract 2 (Inubiwa (leaf) 50 vol% ethanol extract) 1.0
(11) Purified water 47.4
(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.

[Prescription Example 11] Pack (1) Purified water 58.9 (mass%)
(2) Polyvinyl alcohol 12.0
(3) Ethanol 17.0
(4) Glycerin 5.0
(5) Polyethylene glycol (average molecular weight 1000) 2.0
(6) Extract 3 (Inubiwa (fruit) 50 vol% ethanol 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.

[Prescription Example 12] Bath agent (1) Fragrance 0.3 (% by mass)
(2) Extract 3 (Inubiwa (real) 50 vol% ethanol extract) 1.0
(3) Sodium bicarbonate 50.0
(4) Sodium sulfate 48.7
Production method: (1) to (4) are mixed uniformly.

[Prescription Example 15] Oral fluid (1) Extract 2 (Inubiwa (leaf) 50 vol% ethanol extract) 8.0 (mass%)
(2) Erythritol 1.0
(3) Citric acid 0.1
(4) Stevia 0.01
(5) Purified water 90.89
Production method: (1) to (5) are mixed uniformly.

[Prescription Example 16] Granule (1) Extract 1 (Inbiwa (leaf) hot water extract) 0.2 (parts by mass)
(2) Lactose 0.65
(3) Corn starch 0.15
Production method: (1) to (3) were passed and mixed, granulated with a granulator, dried and sized to obtain granules having a total amount of 1500 mg.

Claims (9)

A humectant containing an extract of Ficus erecta Thumb. An anti-aging agent comprising an extract of Ficus erecta Thumb. An antioxidant containing an extract of Ficus erecta Thunb. A neutral fat accumulation inhibitor containing an extract of Ficus erecta Thumb. A whitening agent containing an extract of Ficus erecta Thunb. An anti-inflammatory agent comprising an extract of Ficus erecta Thumb. An immunostimulant containing an extract of Ficus erecta Thumb. An external preparation for skin containing an extract of Ficus erecta Thumb. An oral preparation containing an extract of Ficus erecta Thumb.