JP5388555B2 - Moisturizer, anti-aging agent, antioxidant, slimming agent, whitening agent, anti-inflammatory agent, immunostimulant, external preparation for skin, functional oral composition - Google Patents

Description

  The present invention relates to a moisturizer, an anti-aging agent, an antioxidant, a slimming agent, a whitening agent, an anti-inflammatory agent, an immunostimulant, an external preparation for skin, a functional oral composition containing a plant of Woodwardia or an extract thereof. Related to things.

  Conventionally, in order to provide a moisturizing ingredient that prevents the skin from drying and moisturizes the skin, various active ingredients have been studied. Moreover, as factors of skin symptoms such as aging, diseases, stress, wrinkles due to ultraviolet rays, blemishes, reduced skin elasticity, dryness, cellular function decline, melanin production and pigmentation due to ultraviolet rays, decrease and degeneration of dermal matrix components, ultraviolet rays Oxidative damage of cells due to the above. In order to prevent and ameliorate such skin symptoms, various active ingredients have been searched and formulated. In particular, naturally-derived components are known to have various pharmacological and cosmetic effects, and the application of extracts such as plants and fungi to skin external preparations and oral compositions has been studied so far.

  For example, in order to obtain an extract of a plant belonging to the genus Halibut (see Patent Document 1) as a moisturizing agent excellent in skin moisturizing effect and safety, and to obtain a skin external preparation having an anti-aging and improving action on the skin, The essence of Ponkan as a component having an activation or growth promoting action (see Patent Document 2), an extract of plants belonging to the genus Genus ginseng (see Patent Document 3), a chlorella extract (see Patent Document 4), and a loquat extract (see Patent Document 5) ) Etc. are disclosed. As an antioxidant, an extract of a plant belonging to the genus Sarogaceae (see Patent Document 6) or the like is used, and as an ingredient that suppresses fat accumulation in the living body, an extract of a plant belonging to the family Schipokeaceae (see Patent Document 7) or the like is whitened. As an agent, an extract of white crane reishi (see Patent Document 8) and the like, a plant extract having an anti-inflammatory action, a dried product of soba, cacao and musk or a hot water extract thereof (see Patent Document 9), A solvent extract of Cannaceae plants (see Patent Document 10) and the like, and leek seeds and / or extracts thereof (see Patent Document 11) and the like are already known as immunostimulants.

JP 2007-77072 A JP 2001-131045 A JP 2000-178198 A JP 11-335293 A Japanese Patent Publication No. 5-17206 Japanese Patent Laid-Open No. 10-182413 JP 2005-60345 A JP 2003-89630 A JP 2003-201246 A JP-A-4-270224 JP 2008-31122 A

  Thus, various naturally-derived components have been applied so far. However, there are many naturally derived ingredients whose effects are not yet known. Excellent moisturizing effect, anti-aging effect, antioxidant effect, slimming effect, whitening effect, anti-inflammatory effect, immunostimulatory effect The development of an active ingredient having a potential has been expected.

  As a result of studying various components derived from nature, the present inventors, as a result, have excellent moisturizing effect, anti-aging effect, anti-oxidation effect, etc. The present inventors have found that a slimming effect, a whitening effect, an anti-inflammatory effect, and an immunostimulatory effect exist, and have further studied and completed the present invention.

  That is, the present invention relates to a moisturizer, an anti-aging agent, an antioxidant, a slimming agent, a whitening agent, an anti-inflammatory agent, an immunostimulant, and an external preparation for skin containing at least one plant selected from the plant belonging to the genus Comotida The present invention relates to an agent and a functional oral composition.

  According to the present invention, a moisturizer, an anti-aging agent, an antioxidant, a slimming agent, a whitening agent having excellent effects can be obtained by blending one or two or more kinds of plants selected from the plant belonging to the genus Cocomitida or an extract thereof. An agent, an anti-inflammatory agent, an immunostimulant, an external preparation for skin, and a functional oral composition can be provided.

  The plant belonging to the genus Kochicida used in the present invention is a perennial plant belonging to the family Blencaceae, distributed in Honshu, Shikoku, and Kyushu, south of Miyagi Prefecture and west of Toyama Prefecture, and is ornamental in Europe and the United States. The Komochishida genus Aiookaguma (Woodwardia × intermedia H.Christ), eye Komochishida (Woodwardia orientalis Sw. × Woodwardia orientalis Sw. Var. Formosana Rosenst.), IS Komochishida (Woodwardia × izuensis Sa.Kurata), Ookaguma (Woodwardia japonica ( L. f.) J. Sm.), Woodwardia harlandii Hook. Bearwardia orientalis Sw. Var. Formana Rosenst., Woodwarda japonica (L. fil. EWo wa. .

  The present invention is not particularly limited as long as it is a plant belonging to the genus Comotidida, but as a suitable one from the viewpoint of the effect of the present invention, there is exemplified bee brown bear (alias: Thaiwan Komotida) (Woodwardia orientalis Sw. Var. Formosa Rosenst.).

  When using these plants belonging to the genus Komotichida, there are no particular restrictions on the site of use, and any part of the spore body or gametophyte may be used. Use roots, whole leaves, leaf blades, petiole, etc. Preferably, all leaves including leaf blades, petiole and the like are good.

  In the extraction, the plant may be used as it is, but considering the extraction efficiency, it is preferable to perform the extraction after performing processing such as shredding, drying, and pulverization.

  The extraction can be performed by immersing in an arbitrary extraction solvent for a predetermined time. The extraction solvent may be heated as necessary. Alternatively, extraction can be performed using a supercritical fluid or a subcritical fluid. In order to increase extraction efficiency, stirring or homogenization in an extraction solvent may be performed. 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.

  As an extraction solvent, in addition to water, lower alcohols such as methanol, ethanol, propanol and isopropanol; polyhydric alcohols such as 1,3-butylene glycol, propylene glycol, dipropylene glycol and glycerin; ethers such as ethyl ether and propyl ether Solvents such as esters such as butyl acetate and ethyl acetate; ketones such as acetone and ethyl methyl ketone can be used, and water and ethanol are preferable. 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, one kind or two or more kinds of supercritical liquids and subcritical liquids such as water, carbon dioxide, ethylene, propylene, ethanol, methanol, and ammonia may be used, and carbon dioxide is preferred.

  Extracts of the above-mentioned solvents of the genus Cocomicida can be used as they are, but they may be left to stand for a certain period of time and matured, or the concentrated and dried solids are dissolved again in water or a polar solvent. Can also be used. 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 above-mentioned extract of the genus Cocomitida, its treated product and fractionated product can be freeze-dried 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.

  The plant belonging to the genus Comotidida or its extract has excellent moisturizing effect, anti-aging effect, antioxidant effect, slimming effect, whitening effect, anti-inflammatory effect, immunostimulatory effect, moisturizing agent, anti-aging agent, antioxidant agent, It can be used as a slimming agent, whitening agent, anti-inflammatory agent, immunostimulant, skin external preparation, and functional oral composition.

  A moisturizing agent containing a plant belonging to the genus Komotichida or its extract as an active ingredient has an excellent hyaluronic acid production promoting action of human dermal fibroblasts and an arginase activity promoting action of human epidermal keratinocytes, and exhibits an excellent moisturizing effect. To do.

  An anti-aging agent comprising a plant belonging to the genus Cocomicida or an extract thereof as an active ingredient is an excellent human dermal fibroblast activation effect, a human dermal fibroblast cell activation effect, and a human dermal fibroblast type I collagen production promoting effect. Has the effect of promoting ATP production of human dermal fibroblasts, activating human epidermal keratinocytes, and promoting collagen production of human epidermal keratinocytes type IV, and is excellent in preventing and improving aging symptoms To do.

  Antioxidants containing Komotichida plants or their extracts as active ingredients have excellent lipid peroxide resistance, radical scavenging action, and superoxide anion scavenging action, and exhibit excellent antioxidant effects.

  A slimming agent comprising a plant belonging to the genus Komotichida or an extract thereof as an active ingredient has an excellent action of inhibiting neutral fat accumulation of human preadipocytes and exhibits an excellent slimming effect.

  A whitening agent containing a plant belonging to the genus Komotichida or an extract thereof as an active ingredient has an excellent inhibitory effect on tyrosinase activity of human epidermal melanocytes and an inhibitory effect on melanin production of B16 mouse melanoma cells, and prevents pigmentation, spots, freckles, etc. Improve and show excellent whitening effect.

  An inflammatory agent comprising a Cocomitida plant or an extract thereof as an active ingredient has an excellent hyaluronidase inhibitory action and exhibits an excellent anti-inflammatory effect.

  An immunostimulant comprising a Comotidida plant or an extract thereof as an active ingredient has a cell activation effect using a human acute monocyte leukemia cell line and exhibits an excellent immunostimulatory effect.

  An external preparation for skin containing a plant belonging to the genus Komotichida or an extract thereof exhibits an excellent moisturizing effect, anti-aging effect, antioxidant effect, slimming effect, whitening effect, anti-inflammatory effect, immunostimulatory effect, and the like.

  A functional oral composition containing a plant belonging to the genus Commonis or its extract exhibits an excellent moisturizing effect, anti-aging effect, antioxidant effect, slimming effect, whitening effect, anti-inflammatory effect, immunostimulatory effect, and the like.

  Each of these agents is not limited at all in terms of its form and presence / absence of other components as long as it contains a Cocomitida plant or an extract thereof as an active ingredient. As for the form, any form such as liquid, paste, gel, solid or powder can be selected according to its use and the like, vehicle (excipient), solvent, Or other general additives (an antioxidant, a coloring agent, a dispersing agent etc.) can be included arbitrarily.

  Here, the external preparation for skin means all external compositions for external use on the skin or hair, such as cosmetics, quasi-drugs or external pharmaceuticals. The functional oral composition also means all compositions that are taken orally, regardless of the type of medicine, food, beverage or the like.

  The dosage form of the external preparation for skin is arbitrary, and can be provided, for example, as a solubilization system such as lotion, a dispersion system such as calamine lotion, or an emulsification system such as cream or emulsion. Furthermore, it can also be provided in various dosage forms such as aerosol form, ointment or poultice filled with propellant.

  Specifically, various cosmetics such as emulsions, creams, lotions, lotions, packs, cosmetic liquids, cleaning agents or makeup cosmetics; liquids, ointments, powders, granules, aerosols, patches or poultices Various forms of cosmetics, quasi-drugs, or external medicines 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 or extracts Etc. can be exemplified.

  For topical skin preparations or functional oral compositions, in addition to the plants belonging to the genus Cocomicidae or extracts thereof, pharmaceuticals, quasi-drugs, skin cosmetics, cosmetics for hair, cleaning agents, etc. Optional ingredients usually formulated in water, such as water, oily ingredients, moisturizers, powders, pigments, emulsifiers, solubilizers, gelling agents, cleaning agents, UV absorbers, anti-inflammatory agents, thickeners, pH adjustment Agents, chelating agents, drugs (medicinal ingredients), fragrances, resins, fungicides, fungicides, antioxidants, alcohols, and the like can be appropriately blended. Furthermore, within the range that does not impair the effects of the present invention, other moisturizers, anti-aging agents, antioxidants, slimming agents, whitening agents, anti-inflammatory agents, immunostimulants or plants other than the genus Cocomitida or extracts thereof Can be used in combination.

  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 amount of the common genus plant or its extract to the topical skin preparation or functional oral composition can be adjusted depending on the type, purpose, etc. In terms of conversion, it is preferably 0.0001 to 10.0% by mass, more preferably 0.001 to 5.0% by mass, still more preferably 0.01 to 5% by mass, and still more preferably 0.00. 1 to 5% by mass.

  Examples of preparations of the plant extract of the genus Komotichida, test methods for evaluating moisturizing effect, anti-aging effect, antioxidant effect, slimming effect, whitening effect, anti-inflammatory effect and immunostimulatory effect, topical skin preparation or functional food However, the technical scope of the present invention is not limited in any way by these.

[Extract 1]
To 100 g of dry ground pulverized product of honey bee bear, 2.0 kg of 50 mass% ethanol aqueous solution was added and extracted at room temperature for 2 hours with stirring. The extract was collected by filtration, concentrated under reduced pressure, and lyophilized to obtain extract 1.

[Extract 2]
To 100 g of dry ground pulverized leaves of bee bear, 2.0 kg of 50 mass% ethanol aqueous solution was added and extracted at room temperature for 2 hours with stirring. The extract was collected by filtration, concentrated under reduced pressure, and lyophilized to obtain extract 2.

[Extract 3]
100 g of purified water was added to 5 g of dry ground pulverized product of bee brown bear and extracted at 120 ° C. for 20 minutes. The extract was collected by filtration and lyophilized to obtain Extract 3.

[Extract 4]
100 g of purified water was added to 5 g of dried ground pulverized leaves of bee bears and extracted at 120 ° C. for 20 minutes. The extract was collected by filtration and freeze-dried to obtain extract 4.

[Extract 5]
The new leaves of bee bear bear are dried, crushed and filled into cells. Extract 2 was obtained by feeding liquefied carbon dioxide into the cell under the conditions of 25 Mpa, 40 ° C., and 5 mL / min for 2 hours.

[Extract 6]
The old leaves of bee bears are dried, crushed and filled into cells. Extract 2 was obtained by feeding liquefied carbon dioxide into the cell under the conditions of 25 Mpa, 40 ° C. and 5 mL / min for 2 hours.

  The new leaves used here are red leaves, and the old leaves are green leaves. Each effect was evaluated using the said extract. Note that * and ** described in each evaluation result indicate that the significance probability P value in the t-test is less than 5% (P <0.05), and less than 1% significance (P <0.01). ) Is represented by **.

<Moisturizing effect (Evaluation of human dermal fibroblast hyaluronic acid production promoting effect)>
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 culture medium was replaced with a sample culture solution to which the extract 3 was added so as to have each concentration shown in Table 1 in a DMEM medium supplemented with 0.5% by mass FBS, 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. ) 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 with a BCA protein assay kit manufactured by PIERCE, and the amount of hyaluronic acid produced per unit protein amount 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.

As is clear from the results in Table 1, the bee brown bear (new leaf) hot water extract (Extract 3) showed a significant moisturizing effect.

<Moisturizing effect (Evaluation of human epidermal keratinocyte arginase activity promoting effect)>
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, the sample culture medium was replaced with a 5% FBS-added DMEM medium (differentiation induction medium) containing 1.2 mM CaCl ₂ with each extract 5 added to each concentration shown in Table 2, and further cultured for 9 days. The medium was exchanged once every 3 days. Urea nitrogen B-Test Wako (Wako Pure Chemical Industries) 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 (sodium nitroprusside) to produce indophenol. Absorbance at 570 nm derived from indophenol was measured under alkaline conditions, the urea concentration was determined, and arginase activity was quantified. The protein amount in each well was measured with a BCA protein assay kit manufactured by PIERCE, and the arginase activity per unit protein amount was determined.
The evaluation results are shown in Table 2 as relative values when the arginase activity per unit protein amount in the control with no sample added is defined as 100.

As is clear from the results in Table 2, the bee brown bear (old leaf) supercritical extract (Extract 5) showed a significant moisturizing effect.

As described above, the bee brown bear extract has a hyaluronic acid production promoting action and an arginase activity promoting action, and exhibits an excellent moisturizing effect.

<Anti-aging effect (evaluation of human dermal fibroblast activation)>
Normal human dermal fibroblasts manufactured by Kurabo Industries Co., Ltd. (Kurashiki Boseki Co., Ltd.) were 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 1% by mass of fetal bovine serum (FBS) was added was used. After culturing for 24 hours, the medium was exchanged with 1% by mass FBS-added DMEM medium to which extract 1 was added so as to have each concentration shown in Table 3, 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) 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 medium containing the sample, 1% by mass FBS-added DMEM medium was used as a control.
The evaluation results are shown in Table 3 as relative values when the cell activation effect in the control is 100.

As is clear from the results in Table 3, a significant cell activation effect was observed in the medium supplemented with 50% ethanol extract (extract 1) of bee brown bear (new leaf).

<Anti-aging effect (Evaluation of human dermal fibroblast type I collagen production promoting effect)>
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 a culture solution to which the extract 1 was added so that each concentration shown in Table 4 was obtained in a DMEM medium supplemented with 0.5% by mass FBS, and further cultured for 24 hours. The ELISA method was used for the quantification of type I collagen secreted into the culture supernatant. Finally, 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt ( ABTS) and hydrogen peroxide were added and reacted, and then the absorbance at 405 nm was measured. The amount of protein in each well was measured with a BCA protein assay kit manufactured by PIERCE, and the amount of type I collagen produced per unit protein amount was determined.
The evaluation results are shown in Table 4 as relative values when the type I collagen production amount per unit protein amount in the control with no sample added is defined as 100.

  As is clear from the results in Table 4, the bee brown bear (new leaf) 50% ethanol extract (Extract 1) showed a significant collagen production promoting effect.

<Anti-aging effect (Evaluation of human dermal fibroblast ATP production promoting action)>
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 a culture solution to which the extract 2 was added so as to have each concentration shown in Table 5 with 1% FBS-added DMEM, 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 5 as relative values with the ATP producing ability in the control with no sample added as 100.

  As is clear from the results in Table 5, the bee brown bear (old leaf) 50% ethanol extract (extract 2) showed a significant ATP production promoting effect.

<Anti-aging effect (evaluation of human epidermal keratinocyte activation)>
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, the medium was replaced with a culture solution in which the extract 4 was added to each concentration shown in Table 6 in a 5% by mass FBS-added DMEM medium, 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 6 as relative values when the cell activation effect in the control with no sample added is taken as 100.

  As is clear from the results in Table 6, the bee brown bear (old leaf) hot water extract (extract 4) showed a significant cell activation effect.

<Anti-aging effect (Evaluation of human epidermal keratinocyte type IV collagen production promoting action)>
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, the culture medium was replaced with a culture medium in which the extract 1 was added to each concentration shown in Table 7 in a DMEM medium supplemented with 0.5% by mass FBS, and further cultured for 5 days. For the quantification of type IV collagen secreted into the culture supernatant, a sandwich ELISA method using a monoclonal antibody against type IV collagen (recognition site: α2 chain) and a biotinylated polyclonal antibody was used. Finally, avidinized horseradish peroxidase was used. 3,3 ′, 5,5′-tetramethylbenzidine was added and reacted, and then the absorbance at 650 nm was measured. The amount of protein in each well was measured with a BCA protein assay kit manufactured by PIERCE, and the amount of type IV collagen produced per unit protein was determined.
The evaluation results are shown in Table 7 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.

  As is clear from the results in Table 7, the bee brown bear (new leaf) 50% ethanol extract (Extract 1) showed a significant collagen production promoting effect.

  As described above, the bee brown bear extract has a cell activating action, a collagen production promoting action, and an ATP production promoting action, and exhibits an excellent anti-aging effect.

<Antioxidant effect (Evaluation of resistance to lipid peroxidation of human epidermal keratinocytes)>
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 10% by weight fetal bovine serum (FBS) was used as the seeding medium. After 24 hours, the medium was replaced with a culture solution to which the extract 4 was added so as to have each concentration shown in Table 8 in a DMEM medium supplemented with 10% mass FBS, and further cultured for 24 hours. The solution was replaced with a HANK'S (+) solution to which an arbitrary concentration of t-butyl hydroperoxide was added, and cultured 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% aqueous ethanol solution containing 1% acetic acid, neutral red incorporated into the cells was extracted, and the absorbance at 540 nm of the extract was measured.
The evaluation results are shown in Table 8 as relative values when the cell viability in the control without addition of t-butyl hydroperoxide is defined as 100.

  As is clear from the results of Table 8, the bee brown bear (old leaf) hot water extract (Extract 4) was found to have a significant lipid peroxide resistance effect.

<Antioxidant effect (DPPH radical scavenging action)>
Extract 6 was adjusted to the concentration shown in Table 9 using 50% by mass ethanol to obtain a sample solution, and 100 μL was added to each 96-well microplate. Thereto was added 100 μL of 0.2 mM 1,1-diphenyl-2-picrylhydride radical (DPPH) ethanol solution, mixed well, and allowed to stand at room temperature in the dark for 24 hours. Thereafter, the absorbance at 516 nm derived from the DPPH radical was measured. When the absorbance of the control when the sample was not added was (A) and the absorbance when the sample was added was (B), the DPPH radical elimination rate was calculated by introducing the formula (1). Table 9 shows the measurement results.
Formula (1): Radical scavenging rate = {1- (B) / (A)} × 100 (%)

  As is clear from the results in Table 9, the bee brown bear (old leaf) supercritical extract (Extract 6) showed a significant DPPH radical scavenging effect.

<Antioxidant effect (superoxide anion scavenging action)>
To 75 μL of Hanks (+) solution containing 0.25 mM WST-1 and 1 mM hypoxanthine, 25 μL of a sample obtained by diluting extract 4 with Hanks (+) solution to a concentration shown in Table 10 was added, and xanthine oxidase 25 μL (0.0075 units) was added, and after reacting at 37 ° C. for 15 minutes, the absorbance at 450 nm was measured. When the absorbance of the control to which the sample was not added was (A) and the absorbance when the sample was added was (B), the value of formula (2) was defined as the superoxide anion elimination rate. The evaluation results are shown in Table 10.
Formula (2): Erase rate = {1- (B) / (A)} × 100 (%)

  As is clear from the results in Table 10, the bee brown bear (old leaf) hot water extract (extract 10) showed a significant superoxide anion scavenging effect.

  As described above, the bee brown bear extract has a lipid peroxide resistance action, a DPPH radical scavenging action, and a superoxide anion scavenging action, and exhibits an excellent antioxidant effect.

<Slimming effect (Evaluation of neutral fat accumulation inhibitory action of human preadipocytes)>
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 (containing 10% FBS, 2 mM L-glutamine, 100 units / mL penicillin, 100 μg / mL streptomycin) was used as the seeding medium. After culturing for 48 hours, the medium was exchanged with a PGM differentiation medium (10 μg / mL insulin, 1 μM dexamethasone, 200 μM indomethacin, 500 μM isobutylmethylxanthine) supplemented with extract 1 so as to have the concentration shown in Table 11. 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 11 as relative values when the neutral fat accumulation amount in the control with no sample added is taken as 100.

  As is clear from the results in Table 11, the bee brown bear (new leaf) ethanol extract (Extract 1) has a significant triglyceride accumulation effect and exhibits an excellent slimming effect.

<Whitening effect (inhibition of human epidermal melanocyte tyrosinase activity)>
Normal human epidermal melanocytes manufactured by Kurabo Industries Co., Ltd. were seeded in a 96-well microplate so as to be 3.0 × 10 ⁴ cells per well. As a seeding medium, Medium154S manufactured by Kurabo Industries Co., Ltd. was used. After 24 hours, the medium was replaced with Medium 154S to which the extract 4 was added so as to have each concentration shown in Table 12, and the culture was further continued for 48 hours. Next, the cells were completely lysed by exchanging with 75 μL of 1% by weight Triton-X-containing phosphate buffer, and 50 μL of the cells was used as a crude enzyme solution. To the crude enzyme solution, 50 μL of 0.05% by mass L-dopa-containing phosphate buffer serving 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 with a microplate reader, and the amount of dopamelanin produced was determined by introducing each measured value into Equation (3).
Formula (3): Amount of produced dopamelanin = {(405 nm value after reaction−405 nm value before reaction)} − 2.166 / 5.238
In addition, the amount of protein in each well was measured with a BCA protein assay kit manufactured by PIERCE, and the amount of dopamelanin produced per unit protein was determined. Tyrosinase activity inhibitory action was evaluated from the relative value when the control value was 100. The results are shown in Table 12.

  As is clear from the results in Table 12, the bee brown bear (old leaf) hot water extract (Extract 4) was found to have a significant tyrosinase activity inhibitory effect.

<Whitening effect (evaluation of B16 mouse melanoma cell melanin production inhibitory effect)>
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 medium was replaced with a culture solution to which the extract 2 was added to a concentration shown in Table 13 in a DMEM medium supplemented with 5% mass FBS, and further cultured for 5 days. After completion of the culture, cells were peeled off by trypsin treatment, transferred to a 1.5 mL microtube, and centrifuged to obtain a cell precipitate. The obtained precipitate was visually determined based on the following determination table. In the evaluation, a 5% mass FBS-added DMEM medium containing 5% mass FBS was used as a negative control, and a 5% mass FBS-added DMEM medium containing 50 mM sodium lactate was used as a positive control. These naked eye determination results were determined to be determination 5 and determination 1, and used as an index for sample determination. The naked eye evaluation was evaluated in five stages as shown below. At the same time, the 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 13.
Judgment and Standard Judgment Criterion 1 Same as positive control (almost white)
2 Slightly darker than the positive control (light brown)
3 Between positive control and negative control (brown)
4 Slightly less blackening than the negative control (blackish brown) 5 Same level as the negative control (almost black)

  As is clear from the results in Table 13, the bee brown bear (old leaf) 50% ethanol extract (Extract 2) was found to have a significant melanin production inhibitory effect.

  The bee brown bear extract has a tyrosinase inhibitory activity and a melanin production inhibitory action, and exhibits an excellent whitening effect.

<Anti-inflammatory effect (evaluation of hyaluronidase inhibitory action)>
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. 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. 0.1 mL of the solution to which the extract 2 was added and 0.03 mL of the enzyme solution so as to have the concentrations shown in Table 14 in a buffer solution were placed in a test tube and reacted at 37 ° C. for 20 minutes. Next, 0.06 mL of an 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. 0.06 mL of 0.4 N NaOH was added, and ice-cooled immediately after the reaction was stopped. Boric acid buffer (pH 9.1) was added in an amount of 0.06 mL, boiled for 3 minutes, and further ice-cooled. After 2.0 mL of p-DABA solution was added and reacted at 37 ° C. for 20 minutes, the reaction solution was transferred to a 96-well microplate, and the absorbance at 585 nm 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) decreases, and the absorbance by p-DABA decreases. Hyaluronidase inhibitory action is defined by the following equation.
Inhibition rate (%) = (control absorbance−sample absorbance) / control absorbance × 100
The evaluation results are shown in Table 14.

  As is clear from Table 14, a bee brown bear (old leaf) 50% ethanol extract (Extract 2) has a significant hyaluronidase inhibitory effect and exhibits an excellent anti-inflammatory effect.

<Immune activation effect (cell activation using human acute monocyte leukemia cell line)>
A human acute monocyte leukemia cell line (THP-1) was seeded on a 96-well microplate so that the number of human acute monocytic leukemia cell line (THP-1) was 5.0 × 10 ⁴ per well. As a seeding medium, Rpswell Park Memorial Institute medium (RPMI) supplemented with 1% by mass of FBS was used. After 24 hours, phorbol 12-myristate 13-acetate (PMA) was added to the cell culture to 20 ng / mL. Further, after 24 hours, the culture medium was added with the extract 1 added to 1% by mass FBS-added RPMI medium so that each concentration shown in Table 15 was obtained, and cultured for 48 hours. Next, RPMI medium supplemented with 1% by mass FBS to which 1/10 amount of living cell count reagent SF (Dojindo Laboratories) was added was added to the cells from which the supernatant had been removed, and cultured for 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 15 as relative values when the cell activation effect in the control with no sample added is taken as 100.

  As can be seen from Table 15, in the medium supplemented with bee brown bear (new leaf) 50% ethanol extract (Extract 1), a significant human acute monocytic leukemia cell line (immune cell) activation effect was observed, which is excellent. Demonstrate the immunostimulatory effect.

  Then, the formulation example of the skin external preparation and the functional oral composition which mix | blended the Comotochida genus plant extract obtained by said each preparation method is shown.

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) The balance to be purified water 100 (11) Arginine (1% by mass aqueous solution) 20.0
(12) Extract 1 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 cooling, add (11) and (12) sequentially at 40 ° C. and mix uniformly.

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) Extract 2 1.0
Production method: (2) and (3) are dissolved in (1). Further, after sequentially adding (4) to (8), the mixture is sufficiently stirred, and (9) is added and mixed uniformly.

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) Extract 3 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. Add (11), stir, cool and add (12) at 40 ° C. and mix uniformly.

Essence (1) Purified water 100 balance (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 (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 4 3.0
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. After cooling, add (15) at 50 ° C and (16) at 40 ° C and mix uniformly.

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) Extract 1 0.5
(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.

Cleansing fee (1) Squalane 81.0 (mass%)
(2) Polyoxyethylene glyceryl isostearate 15.0
(3) Remainder to be purified water 100 (4) Extract 6 4.0
Manufacturing method: (1) and (2) are uniformly dissolved. (3) and (4) are sequentially added to this and mixed uniformly.

Facial cleansing 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 which is made into purified water 100 (8) Extract 2 0.1
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. After cooling, add (8) at 40 ° C. and mix uniformly.

Makeup base cream (1) Squalane 10.2 (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) Extract 3 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. After cooling, add components (11) and (12) at 40 ° C. and mix uniformly.

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) Extract 4 0.5
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. After cooling, components (16) and (17) are added sequentially at 40 ° C. and mixed uniformly.

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) The balance of 100 purified water (11) Fragrance 0.1
(12) Extract 5 3.0
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 (9) are added to the remainder of (10) heated and dissolved at 70 ° C. with stirring, and emulsified with a homomixer. After cooling, add (11) and extract 5 at 40 ° C. and mix uniformly.

Pack (1) Remainder 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) Extract 1 1.0
(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 evenly dissolving, add (4) and (5) and cool with stirring. Add (6) and (7) at 40 ° C. and mix uniformly.

Bath agent (1) Fragrance 0.3 (mass%)
(2) Extract 2 3.0
(3) Sodium bicarbonate 50.0
(4) Sodium sulfate 46.7
Production method: (1) to (4) are mixed uniformly.

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) The balance of 100 purified water (11) Extract 3 2.0
(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. After cooling, add components (11) and (12) at 40 ° C. and mix uniformly.

Hair artic (1) Ethanol 50.0 (mass%)
(2) The balance of purified water 100 (3) Extract 4 3.0
(4) Fragrance 0.1
Production method: Components (1) to (4) are mixed and homogenized.

Tablet (1) Corn starch 44.0 (mass%)
(2) Crystalline cellulose 100 (3) Carboxymethylcellulose calcium 5.0
(4) Silicic anhydride 0.5
(5) Magnesium stearate 0.5
(6) Extract 5 5.0
Production method: (1) to (6) are uniformly mixed, and compression-molded with a tableting machine to obtain one tablet of 200 mg.

Powder (1) Magnesium aluminate silicate 95.3 (% by mass)
(2) Carboxymethylcellulose calcium 100 The remainder (3) Extract 6 4.0
Production method: After the powders (1) to (3) are mixed, they are pulverized by a pulverizer and uniformly dispersed.

Candy (1) Sucrose 60.0 (mass%)
(2) Remaining as Minamata 100 (3) Extract 1 5.0
(4) Fragrance Appropriate amount Manufacturing method: (1) and (2) are heated, mixed and homogenized, cooled, components (3) and (4) are added at 70 ° C., mixed and homogenized, and then molded.

Drink (1) Aminoethylsulfonic acid 1000mg
(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 8mg
(8) Extract 2 1000mg
(9) Fragrance Trace amount (10) Purified water 100 mL of the remaining manufacturing method: (1) to (9) are sequentially added to (10) and homogenized.

  The external preparation for skin shown in Examples 16 to 29 was a composition having a moisturizing effect, an anti-aging effect, an antioxidant effect, a slimming effect, a whitening effect, an anti-inflammatory effect, and an immunostimulatory effect. The functional oral compositions shown in Examples 30 to 33 were compositions having a moisturizing effect, an anti-aging effect, an antioxidant effect, a slimming effect, a whitening effect, an anti-inflammatory effect, and an immunostimulatory effect.

Claims (1)

  A moisturizer comprising bee brown bear or an extract thereof as an active ingredient.