CN107970251A - Dermatologic preparation composition containing ginsenoside Mc - Google Patents

Dermatologic preparation composition containing ginsenoside Mc Download PDF

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Publication number
CN107970251A
CN107970251A CN201711171123.1A CN201711171123A CN107970251A CN 107970251 A CN107970251 A CN 107970251A CN 201711171123 A CN201711171123 A CN 201711171123A CN 107970251 A CN107970251 A CN 107970251A
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ginsenoside
skin
effect
composition
formulation example
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CN107970251B (en
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金东泫
柳权烈
李沃澯
廉明勋
曺濬喆
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Amorepacific Corp
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Amorepacific Corp
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/12—Carboxylic acids; Salts or anhydrides thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/14—Esters of carboxylic acids, e.g. fatty acid monoglycerides, medium-chain triglycerides, parabens or PEG fatty acid esters
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/63—Steroids; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0014—Skin, i.e. galenical aspects of topical compositions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/10—Anti-acne agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/02—Local antiseptics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/10—Washing or bathing preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q5/00—Preparations for care of the hair
    • A61Q5/02—Preparations for cleaning the hair
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q5/00—Preparations for care of the hair
    • A61Q5/006—Antidandruff preparations

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Abstract

The present invention relates to a kind of composition for containing ginsenoside Mc as active ingredient, the composition can not only provide anti-aging, improvement wrinkle of skin, whitening, moisturizing improvement, but also it is capable of providing anti-inflammatory, improves acne and skin problem, and effect, convergence skin and the pore refining effect of allergic symptom, and be capable of providing skin color improvement, promote hair growth, improve white hair, anti-dandruff and anti-corrosion effect.

Description

Dermatologic preparation composition containing ginsenoside Mc
The application is Application No. 201480029499.7, the applying date to be on April 24th, 2014, entitled " contain The divisional application of the Chinese patent application of the Dermatologic preparation composition of ginsenoside Mc ".This application claims April 24 in 2013 The priority for the 10-2013-0045117 korean patent applications that day submits in Korean Intellectual Property Office.
Technical field
The present invention relates to a kind of composition, the composition contains ginsenoside Mc (ginsenoside Mc), so that not Only be capable of providing it is anti-aging, improve wrinkle of skin, whitening, moisturizing improvement, but also be capable of providing anti-inflammatory, improve acne and Skin problem, and the effect of allergic symptom, convergence skin and pore refining effect, and it is capable of providing skin color improvement effect Fruit, promote hair growth, improve white hair, anti-dandruff and anti-corrosion effect.
Background technology
First defensive barrier of the skin as human body, have so that intracorporeal organ from temperature and humidity change and it is ultraviolet The stimulations of external environment condition such as line, public hazards material and protect the function of intracorporeal organ.With advancing age, skin will be due to a variety of Inherent, external factor and change.That is, in terms of the inherence, since the various hormone secretions for adjusting metabolism are reduced, And the function and cytoactive of immunocyte reduce, therefore the biology of the immune protein of needed by human body and organism constitutive protein Synthesis is reduced.For in terms of external, due to the destruction of ozone layer, the ultraviolet content that earth's surface is reached from sunray increases Add, and as the in-depth of environmental pollution, free radical and active oxygen increase, not only cause the thickness reduction of skin, wrinkle to increase, Elastic force reduces, and makes skin complexion dimmed, and problem (trouble) often occurs for skin, can also increase mole and freckle and old age Spot, and cause the various changes such as complexion is deteriorated, the colour of skin is dimmed.
In order to prevent these due in skin and external factor and occur skin condition change, and maintain health skin Skin state, people make great efforts by the way that the physiological activator obtained from existing various animals, plant, microorganism etc. is added always Enforcement of going forward side by side is added in cosmetics to be used for improving skin condition.
Ginsenoside Mc be the natural goods form contained in ginseng ginsenoside (Rb1, Rg1, Rd, Rb2, Rc, Rf) by What digestive ferment and intestinal microbial were decomposed and generated.It has been reported that the active anticancer of ginsenoside Mc is strong, and Korean granted is special The method for using ginsenoside Mc as anticancer preparation is recorded in profit the 164266th, but is not reported ginsenoside Mc is as such as overall skin condition improvement of the composition of active ingredient, promotion hair growth and improves white hair, anti-head The overall effect as skin preparations for extenal use such as scurf and anti-corrosion.
The content of the invention
Technical problems to be solved
In this regard, the inventors discovered that the ginsenoside Mc contained in ginseng can not only provide anti-aging, improvement skin wrinkle Line, whitening and moisturizing improvement, but also be capable of providing anti-inflammatory, improve the effect of acne, skin problem and allergic symptom, and Skin color improvement is capable of providing, promotes hair growth, improve white hair, anti-dandruff and anti-corrosion effect, so as to complete The present invention.
Therefore, it is an object of the present invention to provide a kind of Dermatologic preparation composition, the composition to contain ginsenoside Mc, so as to show that the integrality of skin improves and promote hair growth, improvement white hair, anti-dandruff and anti-corrosion effect Fruit.
Technical solution
To achieve these goals, present invention offer is a kind of contains ginsenoside Mc as active ingredient for anti-aging Dermatologic preparation composition.
In addition, the present invention provides a kind of external preparation for skin for being used to improve wrinkle for containing ginsenoside Mc as active ingredient Agent composition.
In addition, the present invention provides a kind of skin preparations for extenal use group for moisturizing for containing ginsenoside Mc as active ingredient Compound.
In addition, the present invention provides a kind of external preparation for skin for being used to improve acne for containing ginsenoside Mc as active ingredient Agent composition.
In addition, the present invention provides a kind of skin for being used to improve color and the colour of skin for containing ginsenoside Mc as active ingredient Skin preparation composition for external use.
In addition, the present invention provides a kind of external preparation for skin for pore refining for containing ginsenoside Mc as active ingredient Agent composition.
In addition, the present invention provides a kind of skin for being used to improve allergic skin for containing ginsenoside Mc as active ingredient Skin preparation composition for external use.
In addition, the present invention provides a kind of skin preparations for extenal use group for anti-inflammatory for containing ginsenoside Mc as active ingredient Compound.
In addition, the present invention provides a kind of skin preparations for extenal use group for whitening for containing ginsenoside Mc as active ingredient Compound.
In addition, the present invention provides a kind of ginsenoside Mc that contains is used for trichogenous skin as active ingredient Preparation composition for external use.
In addition, the present invention provides a kind of external preparation for skin for being used to prevent white hair for containing ginsenoside Mc as active ingredient Agent composition.
In addition, the present invention provides a kind of external preparation for skin for anti-dandruff for containing ginsenoside Mc as active ingredient Agent composition.
In addition, the present invention provides a kind of natural antiseptic agent composition by ginsenoside Mc.
Beneficial effect
The composition of the present invention contains ginsenoside Mc, so that anti-aging, improvement wrinkle of skin, U.S. can not only be provided In vain, moisturizing improvement, but also it is capable of providing anti-inflammatory, improvement acne and the effect of skin problem and allergic symptom, convergence Skin and pore refining effect, and be capable of providing skin color improvement, promote hair growth, improve white hair, anti-dandruff And anti-corrosion effect.
Embodiment
Dermatologic preparation composition according to the present invention contains ginsenoside Mc as active ingredient.
The ginsenoside Mc used in the present invention has the structure of following chemical formula 1：
[chemical formula 1]
The ginsenoside Mc of the present invention can be extracted from plant, can also be synthesized simultaneously by method as known in the art Use, the ginsenoside Mc of mercantile-type sale can also be used.In addition, ginsenoside Mc can be obtained from ginseng extract. At this moment the species of the ginseng used is not particularly limited, and can use water ginseng, red ginseng, white ginseng, Tai Ji ginseng and tail ginseng etc..Also, The ginseng extract not only comprising extracted as ginseng, decoct carry obtained from leachate, also include to the part or whole of leachate Concentrate obtained from body is concentrated again, or impregnating, decoction, the tablet dried the concentrate again and preparedExtracts are flowed, and the chemical substance of main efficacy results is played included in ginseng, but also include plant sheet Body, and the extract of the whole parts of ginseng such as stem, root, leaf, flower and fruit can be used, it is not limited to a certain specific part Extract.In addition, the method for ginsenoside Mc is extracted from ginseng extract can use known method.
Specifically, the ginsenoside Mc can be made by method as known in the art using water or organic solvent After standby ginseng extract, therefrom it is isolated.The organic solvent used in the present invention can be selected from ethanol, methanol, butanol, Ether, ethyl acetate, chloroform and the mixed solvent of these organic solvents and water, preferably using 80% ethanol.At this moment, Extracting temperature Preferably 10~80 DEG C, and can extract 3~24 it is small when.If beyond the Extracting temperature and the scope of extraction time, Extraction efficiency can reduce, or the change of component can occur.
Composition according to the present invention, in terms of composition total weight, preferably comprises the ginsenoside of 0.001 to 50 weight % Mc.If this is because the content of the ginsenoside Mc were less than 0.001 weight %, the effect of being brought by the component and effect Fruit is faint, if it exceeds 50 weight %, then can be there are on cutaneous safety or formulation the problem of.
The composition of the present invention can be used as anti-aging Dermatologic preparation composition, it is improving skin Effect in terms of elastic force and improvement wrinkle protrudes.
The composition of the present invention can be used as the Dermatologic preparation composition for moisturizing, it can strengthen skin Barrier function, and it is capable of the differentiation of induced skin keratinocyte.Therefore, can effectively serve as preventing or improving because of epidermis Not xerodermia, allergic dermatitis, the skin preparations for extenal use for contacting atopic dermatitis or psoriasis etc. completely caused by differentiation Composition.
The composition of the present invention can be used as improving the Dermatologic preparation composition of acne, its antibacterial effect It is excellent, it is especially excellent to the antibacterial effect of acne pathogenic bacteria, and antiphlogistic effects are provided.
The composition of the present invention can be used as improving the Dermatologic preparation composition of color and the colour of skin, by it When being used in skin, by expanding capillary, and promote blood circulation nutritional ingredient smoothly is fed to skin, and suppress Skin aging, therefore, the effect for improving color and the colour of skin are remarkable.
The composition of the present invention can be as pore refining, adjusting sebum and the skin preparations for extenal use for improving skin problem Composition uses, and when being used in skin, suppresses the sebum of excessive secretion, and by promoting elimination and the collagen of active oxygen The synthesis of albumen carrys out pore refining, and due to the reduction of inflammatory Cytokines Expression, the effect for suppressing skin problem is remarkable.
The composition of the present invention can be used as improving the composition of allergic skin, it not only passes through suppression Induce the work of the protein decomposition enzyme active acceptor -2 (Proteinase-Activated Receptor-2, PAR-2) of itch Property, so as to provide excellent antipruritic effect, but also can be by reducing the secretion of interleukin (Interleukin-8, IL8) To provide anti-inflammatory effect.Therefore, the ginsenoside Mc of the invention, which can be used as, is used for stabilized susceptibility, irritation or mistake Quick property skin and scalp, and it is appropriate for improving or alleviating the active ingredient of Dermatologic preparation composition of thermal sensation and inflammation Ground uses.
The composition of the present invention can be used as the composition for whitening, its work by hindering tyrosinase Property, suppress the generation of melanin, so as to provide excellent whitening effect.
The composition of the present invention can be used as and be used for trichogenous Dermatologic preparation composition, it promotes From hair cycle stand-down to the conversion in anagen hair cycle, so as to provide the growth including promoting hair, and promote new The generation of hair, and make the effect of existing hair health growth, also provides and prevents and suppress hair to show from what scalp came off As or hair is scattered or the effect of state that attenuates.
The composition of the present invention can be used as preventing the Dermatologic preparation composition of white hair, it passes through raising The expression of transcription factor (MITF) in melanocyte carrys out activation of melanocyte, and promotes the synthesis of melanin, so as to provide The induction of advance preventing white hair, and promote to induce the effect of dark hair.
The composition of the present invention can be used as preventing the Dermatologic preparation composition of dandruff, it is by having Effect discharge is accumulated in the toxin on hair and scalp to purify scalp, and by suppressing propagation and the growth of dandruff bacterium and can Prevent scalp inflammation reaction, also, since the anti-oxidation efficacy of generation and the effect of inhibitory activity oxygen protrudes, therefore, it is possible to carry For calming and strengthening scalp, and strengthen the effect of intrinsic phylactic power defensive power.
The composition of the present invention can be used as natural antiseptic agent composition, since it is natural component, anti-corrosion It is also harmless while effect is remarkable.
The composition of the invention can the agent containing acceptable carrier or base material on cosmeceutical or Dermatology Type.It can be provided as the whole formulations for being adapted to locally use, for example, can be provided as solution, gel, solid, paste The anhydrous product of shape In water phase disperse oil phase and obtain lotion, suspension, microemulsion, Microcapsules, subparticle ball or the form of ionic (liposome) and non-ionic folliculus dispersant, or frost, toner, wash Agent, powder, ointment, spray or the form for hiding flaw rod.And it is possible in the form of foam (foam) or further include pushing away for compression Form into the aerosol combination of agent uses.These compositions can be prepared according to method commonly used in the art.
Especially, Dermatologic preparation composition of the invention is used to prevent dandruff, is for hair growth or white for preventing During hair, can as the composition for scalp and hair and formulation, formulation are not particularly limited, such as can be turned to formulation Hair oil, trichotrophy toner, scalp care agent, hair-care agent, shampoo, hair conditioner, hair lotion or hair of scalp Dual-purpose care agent etc..
In addition, composition can include fatty material, organic solvent, lytic agent, concentrating agents, gelling according to the present invention Agent, softening agent, antioxidant, suspending agent, stabilizer, foaming agent (foaming agent), aromatic, surfactant, water, Ionic or nonionic emulsifier, filler, chelating agent, complexing agent, preservative agent, vitamin, blocking agent, wetting agent, essential oil, Dyestuff, pigment, hydrophily or lipophile activating agent, lipid folliculus are usually used in any other component of cosmetics etc. in cosmetics Common adjuvant in or Dermatology field.The adjuvant is with usually used in cosmeceutical or Dermatology field Amount import.
In addition, the composition of the present invention, in order to increase skin improvement effects, can contain skin absorption enhancement material.
Embodiment
Hereinafter, the structure and effect of the present invention will be further illustrated by test example and formulation example.However, these are tested Example and formulation example only to assist in understand the present invention and illustratively purpose is come what is provided, scope of the invention and scope are not It is defined in following examples.
The preparation of [reference example 1] ginsenoside Mc
In order to which the ginsenoside Mc that the effect of present composition is tested and used is studied purchased from peace rich (AMBO) Institute.
[test example 1] elastase activity suppresses the measure of effect
Ability is hindered for the elastase activity of ginsenoside Mc, with Epigallo-catechin gallate (EGCG) (EGCG) it is compared and measures.The elastoser and matrix used is commercially public purchased from U.S.'s Sigma-Aldrich Take charge of the elastoser of (Cat.No.E0127).
Elastase activity inhibition is tested with following test methods.
In 96 orifice plates, 10mg/L Tri(Hydroxymethyl) Amino Methane Hydrochlorides (Tris-HCL) buffer solution is being modulated into (pH8.0) in, 20 μ g/mL elastoser-type III solution of the ginsenoside Mc and 50 μ L of 200 μ L are mixed.By 250 μM EGCG is used as positive controls, and has used distilled water as the non-process group of negative control group.Afterwards, add N- succinyl-alanine-the Ala-Alas-of the 0.4514mg/mL modulated with the buffer solution of 100 μ L are to nitro acyl Aniline (N-SUCCINYL-ALA-ALA-ALA-p-NITROANILIDE), and reacted 15 minutes at 25 DEG C.Reaction terminates Afterwards, absorbance (cooperative effect 2, the microplate reader (BioTek) (VT, the U.S.) under 415nm wavelength are measured.With identical method come real Apply blank test and be corrected.
The computational methods of elastase activity inhibition the results are shown in table 1 below as shown in following mathematical expressions 1 In.
Mathematical expression 1
Elastase activity obstruction rate (%)=1- (C-D)/(A-B) × 100
A：In no addition substances, and when adding enzyme, the absorbance under 415nm wavelength
B：In no addition substances, enzyme, the absorbance under 415nm wavelength
C：When adding substances, enzyme, the absorbance under 415nm wavelength
D：Addition substances, without add enzyme when, the absorbance under 415nm wavelength
Table 1
Compound Inhibition level (%)
Non-process group 0
EGCG 65
Ginsenoside Mc 75
As shown in above-mentioned table 1, inhibition levels of the ginsenoside Mc to elastase activity is shown, than being known as bullet Property proteinase activity inhibitor EGCG it is significantly excellent, thus it is confirmed that the present invention ginsenoside Mc elastin laminin enzyme activity Property inhibition is excellent.
[test example 2] clostridiopetidase A (MMP-1) hinders ability
Obstruction ability is generated for the clostridiopetidase A of the ginsenoside Mc of the present invention, is measured compared with retinoic acid.
Equipped with DMEM (the Dulbecco's Modified Eagle's Media) trainings containing 2.5% fetal bovine serum In the 96 hole plate incubators (96-well microtiter plate) for supporting base, added with the amount of 5,000 cells/wells (well) Human fibroblasts, and in 5%CO2, culture is carried out in 37 DEG C of incubators untill 70~80% degree are grown to.With 10 μ g/ After when the ginsenoside Mc or small retinoic acid treatments 24 of ml concentration, cell culture fluid is taken.
Using available commercially as clostridiopetidase A sensing equipment (Amersham Phamarcia companies of the U.S., Catalog#： RPN2610), the clostridiopetidase A generation degree for the cell culture fluid taken is measured.First, once clostridiopetidase A antibody is smeared uniform 96- hole plates (96-well plate) in add the cell culture fluid taken, and it is anti-to implement in thermostat Ag-Ab Answer 3 it is small when.3 it is small when after, the secondary collagen antibody for being combined with colour developing group is added in 96- hole plates, and secondary response again 15 minutes.After 15 minutes, 3,3', 5, the 5'- tetramethyl benzidines (3,3', 5,5'- as colour developing evocating substance are added Tetramethylbenzidine, Sigma), and colour developing 15 minutes is induced at room temperature, add 1M sulfuric acid again and stop showing Colour response, then the color of reaction solution is in yellow, and according to the carry out degree of reaction, the degree of the yellow shown is different.
Using absorptiometer, measure is in the absorbance of the 96- hole plates of yellow under 405nm, and according to following mathematical expressions 2 The synthesis degree of clostridiopetidase A is calculated, and the results are shown in table 2 below.At this moment, the group of compositions-treated will never be used In the reaction absorbance of cell culture fluid taken as a control group.
Mathematical expression 2
Absorbance × 100 of absorbance/control group of collagenase expression degree (%)=material processing groups of cells
Table 2
Compound Expression degree (%)
Non-process group 100
Retinoic acid 75
Ginsenoside Mc 73
As shown in above-mentioned table 2, it is known that the collagenase expression degree of ginsenoside Mc is with being known as collagenase expression The retinoic acid of inhibitor is compared, its collagenase expression hinders effect level similar.
By the above results, it can confirm that the ginsenoside Mc of the present invention has and hinder matrix metalloproteinase (MMP-1) Effect.
[formulation example 1 and compare formulation example 1]
According to the composition of Table 3 below, nourishing cream (unit is prepared by conventional method：Weight %).
Table 3
[test example 3] skin elasticity improves effect confirmation
In order to confirm the effect to the raising of the skin elasticity of people, using the formulation example 1 and the formulation for comparing formulation example 1, And following evaluation is made.
For the healthy women of 20 30 to 40 years old age brackets, it is divided into two groups with every group 10, by formulation example 1 and compares agent After the nourishing cream of 1 two groups of type example is applied to face 12 weeks with frequency 1 time a day, skin elasticity tester is utilized (Cutometer SEM575, C+K Electronics Co., Ltd.s (C+K Electronic Co.), Germany) measure skin elasticity.It is tied Fruit is represented in table 4 below.The end value of table 4 is remembered with the Δ R8 values of Cutometer (Cutometer SEM 575) Carry, wherein R8 values represent the property of skin viscoplasticity (viscoelasticity).
Table 4
Experimental products Skin elasticity effect
Formulation example 1 0.44
Compare formulation example 1 0.10
As shown in above-mentioned table 4, the formulation example 1 of the ginsenoside Mc containing the present invention, the group of formulation example 1 compared with smearing Compare, its skin elasticity improves more.
Therefore, it can confirm that the composition of the ginsenoside Mc containing the present invention is highly effective to skin elasticity raising.
[test example 4] improves the confirmation of wrinkle of skin effect
In order to confirm that the composition of the present invention to the effect improving wrinkles of people, make use of the formulation example 1 and compare formulation Example 1.
For the effect improving wrinkles for confirming the formulation example 1 He comparing formulation example 1, following evaluation has been made.For 20 The healthy women of 40 years old age bracket of name, is divided into two groups with every group 10, by formulation example 1 and compares the nutrition of 1 two groups of formulation example After frost is applied to face 12 weeks with frequency 1 time a day, replica (replica) is taken out using silicon, and use skinanalysis apparatus The state of (visiometer, SV600, Courage+Khazaka electronic GmbH, Germany) measure wrinkle, and carry out Graphical analysis.Its result is represented in table 5 below.The result of table 5 below is represented in each parameter (parameter) after smearing 12 weeks Subtract the average value after the parameter value before smearing.
Table 5
As shown in above-mentioned table 5, it is known that the preparation composition for external use of formulation example 1 is to improving the effect of wrinkle of skin very It is excellent.
[test example 5] tyrosinase hinders effect
Tyrosinase is the extraction from mushroom class (Mushroom), has used the tyrosinase of Sigma.First, It will be dissolved in as the tyrosine of matrix in distilled water and the solution of 0.3mg/ml be made, and by the solution with every pipe 1.0ml Amount be added in test tube after, wherein add 1.0ml potassium-phosphate buffer solution (0.1mol concentration, pH6.8) and The distilled water of 0.7ml.
Sample liquid is prepared to mix ginsenoside Mc with 10ppm in the ethanol solution of the present invention, by the above-mentioned of 0.2ml Sample liquid is added in reaction solution, is then reacted 10 minutes in 37 DEG C of thermostats.At this moment, 0.2ml solvents will only be added and carrys out generation For adding the group of each sample liquid as a control group, and used using ascorbic acid as positive controls.In the reaction solution The tyrosinase solution of 2500 units/ml of 0.1ml is separately added into, and is reacted 10 minutes in 37 DEG C of thermostats again.Will dress The test tube for having the reaction solution, which is put into frozen water, makes its rapid cooling, so as to stop reacting, and with photoelectricity spectrum analysis instrument, measure Absorbance (synergy 2, microplate reader (VT, the U.S.), and the results are shown in table 6 below under 475nm wavelength.Under State mathematical expression 3 and hinder effect to calculate each tyrosinase.
Mathematical expression 3
Tyrosinase obstruction rate (%)=100- (the reaction absorbance of reaction absorbance/control group of substances × 100)
Table 6
Substances Tyrosinase obstruction rate (%)
Control group (does not add) 0
Ascorbic acid 52
Ginsenoside Mc 63
It is recognised that ginsenoside Mc is to the inhibiting rate ratio of tyrosinase according to the present invention from the result of above-mentioned table 6 Ascorbic acid as known tyrosinase inhibitor is much higher, therefore whitening effect is very excellent.
[test example 6] generates inhibition using the melanin of B16/F10 melanoma cells
The sample of ginsenoside Mc and kojic acid will be contained as substances using the amount of 0.001 weight % respectively, and with one Determine concentration to be added in the nutrient solution of B16/F10 melanoma cells (bank of Korea Cell system), and cultivate 3 days after, remove training Nutrient solution, is then cleaned with phosphate buffer (PBS), and with after 1N NaOH dissolving cells, absorbance (association is measured under 405nm With effect 2, microplate reader (VT, the U.S.).To be not added with the cells of substances as a control group, and with the melanin in control group Content is compared, and measures the degree that each substances hinder melanin generation.Melanin generation suppression is calculated according to mathematical expression 4 Rate processed, and the results are shown in table 7.
Mathematical expression 4
Melanin generating suppression (%)=100- (absorbance × 100 of absorbance/control group of substances)
Table 7
Substances Melanin generating suppression (%)
Control group (does not add) 0
Kojic acid 53
Ginsenoside Mc 66
It is recognised that the inhibiting rate that ginsenoside Mc generates melanin according to the present invention from the result of above-mentioned table 7 It is more much higher than the kojic acid as known melanin generation Inhibitors, therefore whitening effect is very excellent.
[test example 7] skin moisture-keeping power increases effect measuring
In order to measure the effect that ginsenoside Mc produces the increase of skin moisture-keeping power, it make use of the formulation example 1 and compare Formulation example 1, and made following evaluation.
For the adult men and women for being categorized as dry skin of 20 40 to 50 years old age brackets, it is divided into two groups with every group 10, Formulation example 1 and the nourishing cream for comparing 1 two groups of formulation example are applied to face 4 weeks with frequency 2 times a day.Smearing start before, After smearing 1 week, after 2 weeks, after 4 weeks when, and stop smear pass through 2 weeks (altogether by 6 weeks) after, in constant temperature, constant humidity condition Under (24 DEG C, relative humidity 40%), moisture of skin tester (Corneometer CM825, C+K Electronics Co., Ltd. (C+ is used K Electronic Co.), Germany) measure moisture of skin amount.Its result is represented in table 8 below.Table 8 the result is that with experiment The value for the moisture of skin tester for starting to measure before is as benchmark, by the increase part of the measured value after processing a period of time Result expressed as a percentage.
Table 8
From the result of the table 8 it has been confirmed that when smearing compares formulation example 1, untill 4 weeks by smearing, show About 30% moisture increment rate is shown, but stops moisture of skin amount after smearing and reduces, on the contrary, smearing the agent containing ginsenoside Mc During type example 1, also major part shows more than 30% moisture of skin increment rate after stopping smearing.It is possible thereby to know containing ginseng The skin moisture-keeping power excellent effect of the composition of the invention of saponin(e Mc.
[test example 8] Keratinocyte differentiation facilitation effect measures
It is as follows, in order to understand ginsenoside Mc Human Keratinocytes differentiation facilitation effect, using absorbance come Measure hornification coating (Cornified Envelope, the CE) amount generated during Keratinocyte differentiation.
First, the keratinocyte of the people cultivated after being separated from the epidermis of baby and once is put into culture and burns In bottle, after it is attached to bottom, with after the ginsenoside Mc processing of the concentration of 5ppm in nutrient solution, culture 5 days until Untill cell grows to the 70~80% of bottom area.At this time, low calcium (0.03mM) treatment group and high calcium (1.2mM) are handled into component Zuo Wei not negative control group and positive controls.Then the cell of above-mentioned culture is obtained, after being washed with phosphate buffered saline (PBS), Add the Soviet Union of two sulphur containing 2% lauryl sodium sulfate (sodium dodecyl sulfate, SDS) and 20mM concentration of 1ml The 10mM concentration of sugar alcohol (Dithiothreitol, DTT) Tri(Hydroxymethyl) Amino Methane Hydrochloride buffer solution (Tris-HCl, PH7.4), and (sonication) is ultrasonically treated, (boiling) is boiled, centrifuges, then precipitation is suspended in 1ml In PBS, and measure absorbance (synergy 2, microplate reader (VT, the U.S.) under 340nm.In addition, take out a part of described super Solution after sonication, measures protein content, as the benchmark of evaluation cell differentiation.And it the results are shown in In table 9 below.
Table 9
Substances Differentiation capability (%) in keratinocyte
Low calcium (0.03mM) solution (negative control group) 100
High calcium (1.2mM) solution (positive controls) 210
Ginsenoside Mc 285
As shown in Table 9 above, it can confirm that the differentiation facilitation effect of keratinocyte is excellent when being handled with ginsenoside Mc It is different.
[test example 9] skin barrier function recovery effects measure
It is real in order to measure the effect that ginsenoside Mc recovers to produce to the skin barrier function impaired due to skin injury Following experiments are applied.For the upper arm of 10 adult men and women, the method that (Tape Stripping) is peeled off with adhesive tape, damages skin Barrier, smears the formulation example 2 prepared according to the composition of table 10 below and compares 2 two groups of formulation example, use Vapometer respectively (dolphin (Delfin), Finland), measures the recovery extent of 1 percutaneous moisture loss amount (TWEL) daily, measures seven days, and carry out Compare.Here comparison formulation example 2 is the carrier (vehicle) of negative control group.Tested that the results are shown in table 1 below 1 In.Table 11 the result is that the difference of the before processing before barrier injury and after barrier injury is compared as 100% benchmark As a result.
Table 10
Food ingredient Formulation example 2 Compare formulation example 2
Purified Water 69 70
Propane diols 30 30
Ginsenoside Mc 1 -
Table 11
It was found from from above-mentioned table 11, it can confirm that when being handled using the comparison formulation example 2 for not containing ginsenoside Mc, with The passage of time, percutaneous moisture loss amount gradually increases, on the contrary, when being handled using the formulation example 2 containing ginsenoside Mc, Percutaneous moisture loss amount recovers normal with speed quickly, and barrier injury is restored.
[test example 10] color improvement
It is how general using laser in order to evaluate cosmetic composition according to the present invention to promoting the effect that skin blood circulates Strangle blood flow imaging instrument (Laser Doppler Perfusion Imager, LDPI；Periscan PIM II, lark prestige (Perimed) (Stockholm, Sweden)), measure blood circulation degree in skin.Known LDPI is the blood measured in skin The instrument of circulation, and it is now widely used instrument, is a kind of blood that can not only measure in capillary of skin Speed and amount, and the very delicate of the flowing in parteriole and veinlet can be measured.
In thermostatic constant wet chamber, after being washed one's face with perfumed soap, adapt to 30 minutes, and initial value is determined using LDPI.First, It is determined with the blood flow at initial stage below the forehead of 30 ice-cold to usually trick LDPI women.Then, experiment pair is made As using 1 week formulation example 1 and comparing blood flow being measured after formulation example 1, then by the blood flow of measure and the initial stage The result (skin blood flow change) that measured value is compared is represented in table 1 below 2.
Table 12
It has been confirmed that cosmetic composition is not with containing ginsenoside Mc according to the present invention from the result of above-mentioned table 12 Comparison formulation example 1 compare so that skin blood flow dramatically increases, and is promoted by this blood circulation so that complexion obtains Improve.This finally shows that the cosmetic composition according to the present invention containing ginsenoside Mc can be to effectively transmitting skin-nourishing Component, and suppress simultaneously delay skin aging and contribute.
[test example 11] colour of skin improvement
In order to understand the formulation example 1 and compare the colour of skin improvement of formulation example 1,30 test objects are made to use respectively After (in 1 times/day of evening, smear 1 week altogether), Facial Stage DM-3 (jasmine spy (Moritex), Japan) instrument evaluation is utilized The colour of skin improves degree.For colour of skin improvement rate, become using the lightness and color measured value of skin, and the lightness of skin and color Change value is judged, and the results are shown in table 1 below 3.Lightness and the higher expression colour of skin of color change value are changed It is kind.
Table 13
It has been confirmed that not containing the comparison formulation example 1 of ginsenoside Mc according to the present invention from the result of table 13, do not show Show that the significant colour of skin improves effect, on the contrary, using containing formulation examples 1 of the ginsenoside Mc as active ingredient, after use The colour of skin is greatly improved compared with before use.
[test example 12] pore refining effect
1. the effect of the pore refining of the biosynthesis by promoting collagen
By the facilitation effect and Peritoneal fibrosis of biosynthesis of the ginsenoside Mc according to the present invention to collagen β (TGF-β) is compared and is determined.First, by fibroblast with every hole 105The amount of a cell is inoculated in 24 holes (well) in, and culture is carried out untill growing to 90% or so.It is small with serum-free cell culture medium (DMEM) culture 24 When after, respectively using the ginsenoside Mc of the invention and the TGF-β that are dissolved in serum free medium of 10ng/ml at Reason, and in CO2When culture 24 is small in incubator.Their supernatant is taken out, and utilizes procollagen type (I) Enzyme-linked Immunosorbent Assay Measure (ELISA) kit (procollagen type (I) (procollagen type (I)；#MK101, TAKARA (Shiga, Japan) Whether to observe the increase and decrease of procollagen (procollagen), and it the results are shown in table 1 below 4.Non-process group is set to 100 and contrasted for the synthesis capability of collagen.
Table 14
Substances Collagen synthesis ability (%)
Non-process group 100
TGF-β 183.5
Ginsenoside Mc 195.7
It has been confirmed that ginsenoside Mc according to the present invention and positive controls TGF-β phase from the result of above-mentioned table 14 Than showing higher levels of excellent collagen synthesis ability.Therefore, it can confirm that ginsenoside Mc according to the present invention The pore to broaden is shunk by increasing the collagen growing amount on pore periphery.
2. pore refining effect
In order to understand formulation example 1 and compare the pore refining effect of formulation example 1, evaluated as follows.Select 20 pores The wide men and women's test object of size, is divided into two groups by every group 10, and is smearing formulation example 1 on the face daily according to group and comparing The nourishing cream of formulation example 1, is smeared 4 weeks altogether.Judge the effect of pore refining in the following way.Before shooting experiment and after 4 weeks Photo, and allow expert by visually being evaluated.Its result represents the (opinion rating in table 1 below 5：0- is not received completely Contracting；5- shrinks very much).
Table 15
Substances Opinion rating
Formulation example 1 4
Compare formulation example 1 0
It was found from from the result of above-mentioned table 15, compare effect of the formulation example 1 without pore refining, however, formulation example 1 is shown The pore refining effect that can with the naked eye confirm, so as to understand that the ginsenoside Mc of the present invention is excellent to the effect of pore refining size It is different.
[test example 13] sebum secretion inhibition
1. the effect of the suppression skin hypersecretion by suppressing 5α-reductase activity
In order to confirm 5α-reductase activity suppression effect, determined in HEK293-5 α R2 cells [14C] testosterone changes into [14C] dihydrotestosterone (DHT：Dihydrotestosterone ratio).P3x FLAG-CMV-5 α are transfected on HEK293 cells After R2, and by per hole 2.5 × 105The amount of a cell is inoculated in 24 orifice plates, and cultivated (Park et al., 2003, JDS.Vol.31, pp.191-98).Change within second day the new culture medium added with zymolyte and inhibitor into.By 0.05 μ Ci [14C] testosterone (kit (Amersham Pharmacia biotech), Britain) be used as culture substrate.
In order to confirm 5α-reductase activity suppression degree, ginsenoside Mc is added, and at 37 DEG C, 5%CO2In incubator Cultivate 2 it is small when.At this time, the group for not adding ginsenoside Mc is used as negative control group, Finasteride will be added (finasteride) group is used as positive controls.Recycle culture medium afterwards, and with 800 μ l ethyl acetate extraction steroids it Afterwards, the organic solvent layer on top is separated, and after drying, is dissolved to remaining residue, then with 50 μ l ethyl acetate, and in silicon On plastic film silica gel 60F254 (Silica plastic sheet kieselgel60F254), ethylacetate-hexane is used (1:1) it is unfolded as solvent.
By plastics sample after being dried in air, shower system has been used in order to measure the amount of isotopeDry sheet plastic and x-ray film are together added to bath boxIn, Measure stays in the isotopic mass of the testosterone and dihydrotestosterone on film after 1 week, then according to following mathematical expressions 5 and 6, calculates respectively Conversion ratio and obstruction rate, and the results are shown in table 1 below 6.
Mathematical expression 5
Radiant/total radiant × 100 in conversion ratio (%)=DHT regions
Mathematical expression 6
Conversion ratio × 100 of inhibiting rate (%)=[conversion ratios of conversion ratio-substances of control group]/control group
Table 16
Substances Conversion ratio (%) Obstruction rate (%)
Negative control group 48.0 -
Positive controls 27.6 42.5
Ginsenoside Mc 14.8 67
It has been confirmed that ginsenoside Mc can effectively inhibit the activity of 5α-reductase from above-mentioned 16 result of table, so that Block testosterone to be converted into dihydrotestosterone, and show compared with the Finasteride of known suppression 5α-reductase activity more Add excellent inhibition.The 5α-reductase makes testosterone be converted into dihydrotestosterone, thus with intracytoplasmic receptor protein knot Close and enter in core, so as to activate sebocyte cell and promote to break up, so that the sebum excessive secretion in sebaceous glands.Cause This, it is thus identified that ginsenoside Mc suppresses the excessive secretion of sebum by effectively suppressing the activity of 5α-reductase.
2. sebum secretion inhibition
In order to understand the formulation example 1 and compare the sebum secretion inhibition of formulation example 1, following evaluation has been carried out.Choosing Go out 30 men and women's test objects thought more than sebum secretion, smear formulation example 1 daily in the appointed part of skin on the face and compare The nourishing cream of formulation example 1, is smeared 4 weeks altogether.Judgement for sebum minimizing effect, by using sebum tester (Sebumeter SM810, C+K Electronics Co., Ltd.s (C+K Electronic Co.), Germany) measure passes through 2 weeks and 4 respectively Average sebum slip (%) after week, and the results are shown in table 1 below 7.
Table 17
It was found from from the result of above-mentioned table 17, it is of the invention contain ginsenoside Mc as active ingredient formulation example 1 with Comparison formulation example 1 without it is compared, and can effectively inhibit the sebum excessively secreted.
[formulation example 3 and compare formulation example 3~4]
Prepare formulation example 3 according to the component and content (weight %) shown in table 1 below 8 and compare formulation example 3~4, It is described as follows.Formulation example 3 is the material of mixing ginsenoside Mc, compares formulation example 3 and contains acne to improve completely without bag The material of the active ingredient of skin, standard substance of the comparative example 4 as the benchmark for antimicrbial power, which contains is used as Cuo more The erythromycin (erythromycin) that sore therapeutic agent uses.
Formulation example 3 and compare formulation example 3~4 preparation method it is as follows.The component of the A items of table 1 below 8 is completely dissolved, And in other dissolving tanks, the component of B is completely dissolved, B are added in A afterwards, makes its mixing solubilized.And Wherein, the component of C is added with the mixed proportion according to described in table 18, and after mixing, is filtered, so as to prepare This composition.
Table 18
[test example 14] tests the antimicrbial power of acne bacterium
Using each cosmetic composition according to the formulation example 3 and the composition preparation for comparing formulation example 3~4, to conduct Propionibacterium acnes (the ATCC6919 of acne pathogenic strain：Culture medium-brain heart infusion broth culture medium (BHI broth))) into Row antimicrbial power is tested.
It is as follows to the antimicrbial power test method of acne bacterium.
(1) preparation of test bacteria liquid
Using using propionibacterium acnes be inoculated in brain heart infusion broth culture medium carry out Anaerobic culturel nutrient solution as Test bacteria liquid.
(2) preparation of dilute solution
The addition 0.15ml in the brain heart infusion broth culture medium (pH6.8) or LB broth bouillons (pH4.5) of 15ml The test bacteria liquid, and use using the mixed liquor mixed as dilute solution.
(3) preparation of sample
By formulation example 3 and the cosmetic composition stoste prepared in formulation example 3~4 will be compared, made directly as sample With.
(4) antimicrbial power is tested
1) sample is added in No. 1 row of cell culture tube (96well plate) in 96 holes so that matched with initial concentration, And adding dilute solution makes total amount be 200 μ l.
2) by the mixed liquor of No. 1 row after mixing, take out the mixed liquor of 100 μ l and be added in No. 2 rows, and mix equal After even, the mixed liquor of 100 μ l is taken out again and is added in No. 3 rows.Secondary dilution (double is carried out in this way dilution)。
3) after when quiescent culture 24 is small at 32 DEG C and when 48 is small, judge whether bacterium rises in value with suspension degree, and will The Cmin for not having the propagation of bacterium is set to minimum inhibitory concentration (Minimum Inhibitory Concentration, MIC) Value.It is difficult to judge whether bacterium rises in value if mixed liquor is opaque, confirms by using micro- sem observation.
The antimicrbial power result of the test of acne bacterium is represented in table 1 below 9.For minimum inhibitory concentration, formulation is converted into In the concentration of active ingredient that contains and mark.
Table 19
Project pH Propionibacterium acnes
Formulation example 3 5.7 >52ppm
Compare formulation example 3 5.7 Maximum concentration (does not have antimicrbial power)
Compare formulation example 4 5.7 >100ppm
In the result of table 19, in minimum inhibitory concentration, ppm concentration is smaller, it is believed that the material is to acne bacterium The effective material of antimicrbial power, during using formulation example 3, with using the comparison formulation as the erythromycin of known acne therapeutic agent Example 4 is compared, and the ppm concentration shown is significantly low, whereby it was confirmed that the composition containing ginsenoside Mc has test organisms More excellent antimicrbial power.
[test example 15] lipid synthesis (Lipogenesis) suppresses experiment
Using as the 3T3-L1 cells of the fibroblast of mouse (fibroblast cell line), with 1 × 105Carefully Born of the same parents/hole, which is attached at, fills the DMEM (Dulbecco ' s containing 10% fetal bovine serum (fetal Bovine Serum, FBS) Modified eagle ' s medium, GIBCO BRL, live technology company) culture medium 6 well culture plate (culture Plate in).After 2 days, new DMEM (containing 10% FBS) culture medium is re-replaced, and cultivate 2 days.Then, with containing There are 1 μ g/ml insulin (insulin), 0.5mM isobutyl methylxanthines (IBMX) and 0.25 μm of dexamethasone (dexamethasone) DMEM (containing 10% FBS), induction is carried out to the cell of the culture, and with 50 μM Ginsenoside Mc and caffeine processing, then, after 2 days, re-replace into the DMEM comprising insulin, and cultivate 5 days.5 After it, normal incubation medium (DMEM, contains 10% FBS) is re-replaced into, and the cell is observed and is cultivated to institute State cell and change lipoblast from form.
In order to evaluate the effect of ginsenoside Mc is to suppressing Fat Accumulation in adipocyte, complete what is broken up using described 3T3-L1 adipocytes, implement the Sudan three and dye (S4136, Sigma-Aldrich).At normal temperatures, in phosphate buffer In, after 4% paraformaldehyde (pH7.2) fixed fat cell, washed with phosphate buffer, then, with the Sudan three into Photo is shot after row dyeing, and by being visually compared.It will be not added with substances or compare material and culture medium is used only Group use as a control group, for other comparative groups, handled with 50 μM of caffeines.Fat Accumulation inhibition level is to pass through The degree of dyeing is divided into +++, ++ ,+,-, so that grade is assigned, and it is at this time, closer +++, represent that dye levels are bigger.It is tied Fruit is represented in table 2 below 0.
Table 20
As shown in above-mentioned table 20, the ginsenoside Mc used in the present invention can confirm that, not only accumulation in adipocyte Fat mass it is few, and compared with as the caffeine of known lipid synthesis inhibiting substances, also with excellent lipid synthesis Inhibition.Therefore, sebum is reduced by suppressing lipid synthesis, so as to suppress the generation of acne.
[test example 16] acne improves and sebum secretion reduces and stimulate the experiment whetheing there is
Using 30 people with acne as subjects, it is divided into three groups with every group 10, and to the examination corresponding to each group Object is tested using with the formulation example 3 and comparing formulation example 3~4 cosmetic composition for preparing 1 month.Improve for acne Standard, is set as 1 point to 5 points, and mark 1 divides for " not having ", and 3 points are " common ", and 5 points are " very good ".For experimental result, with The average mark of 10 is marked in table 2 below 1.
Period is eliminated for acne, the number of days to recognize elimination is used as benchmark, for acne recurrence, to have after 1 month Benchmark is used as without recurrence.Reduction for sebum secretion, is set as 1 point to 5 points, and it is " not having " to be marked as 1 point, and 3 points are " general It is logical ", 5 points are " very good ".For experimental result, marked with the average mark of 10 in table 2 below 1.With (showing stimulates instead The number answered)/(overall test number) judge the presence or absence of skin irritatin.
Table 21
As shown in above-mentioned table 21, it is known that the formulation example 3 compared with of formulation example 3 is compared, acne does not recur, and whole Acne, which is improved, on body has excellent effect.In addition, during using comparison formulation example 4 containing antimicrbial power standard substance, although Stimulation when showing acne improvement, but using the material to skin is strong, therefore is not suitable for long-time service, however, Composition according to the present invention does not stimulate but, thus it is shown that also being adapted for using for a long time.
[test example 17] inflammation improvement-interleukin 8 (IL-8) generation inhibition
Before one day tested, by Keratoderma epithelial cell (Normal human skin keratinocyte, NHEK, buys place：Lonza), with 5 × 104Cells/well, is inoculated in 96 orifice plates, then at 37 DEG C, 5%CO2Incubator (incubator) when culture 24 is small in.24 it is small when after, clean cell 2 times with phosphate buffer, and be replaced with serum-free angling Cell basal culture medium (serum free keratinocyte basement media (KBM)).In each hole, use respectively The ginsenoside Mc processing of 5ppm, 25ppm and 50ppm concentration, and react 30 minutes after, use staphylococcus aureus respectively Peptide glycan (PGSA) (10 μ g/ml), aureus peptide glycan (50 μ g/ml) and aureus peptide glycan (50 μ G/ml)+lipopolysaccharides (1 μ g/ml) processing.Wherein, aureus peptide glycan (PGSA：peptidoglycan from S.aureus it is the cell of Gram-positive (+) bacterium) as the peptide glycan (peptidoglycan) extracted from staphylococcus The main constituents of wall, and the cell membrane component of known bacterium can induce inflammation.According to the report, especially staphylococcus, allergy Property dermatitis patients 90% or so because the bacterium produce superinfection.Known lipopolysaccharides (LPS：Lypopolysaccaride it is) leather The main constituents of the cell membrane of Lan Shi feminine gender (-) bacterium, and be the main reason for inflammation induces.
At 37 DEG C, 5%CO2After when culture 24 is small in incubator, takes out nutrient solution and carry out to interleukin 8 The enzyme-linked immunosorbent assay (ELISA) of (Interleukin-8, IL-8), and the results are shown in table 2 below 2.For ELISA, make use of the experimental method of preparation company (BD science).
Table 22
Classification The secretion (pg/ml) of interleukin 8
Without processing control group (control) 935.12
PGSA(10μg/ml) 4812.60
PGSA(50μg/ml) 5895.08
PGSA(50μg/ml)+LPS(1μg/ml) 6814.91
Ginsenoside Mc (5ppm) 1573.32
Ginsenoside Mc (25ppm) 1203.54
Ginsenoside Mc (50ppm) 1001.23
From above-mentioned table 22 it has been confirmed that ginsenoside Mc can be substantially reduced and suppress because of PGSA and lipopolysaccharides LPS and The secretion of increased interleukin 8.As a result it will be appreciated that the present invention Dermatologic preparation composition can substantially reduce because The secretion of PGSA and LPS and increased interleukin 8, so as to provide excellent anti-inflammatory effect.
[test example 18] itch alleviates evaluation
Before one day tested, by keratinocyte (cell line title：HaCaT, buys place：US mode culture Thing preservation institute (ATCC)), with 4 × 104Cells/well, is inoculated in 96 orifice plates, then at 37 DEG C, 5%CO224 are cultivated in incubator Hour.24 it is small when after, it is clear with balanced salt solution (Hanks ' Balanced Salt solution, HBSS) buffer solution of hanks After washing (washing) 96 orifice plate 2 times, by reaction buffer (2 μM of Fluo-4-AM, 20% pluronic acid (pluronic Acid), 2.5mM probenecid (probenecid)) it is put into cell.At 37 DEG C, 5%CO2Reacted 30 minutes in incubator, and After reacting 30 minutes at normal temperatures, with HBSS buffer solution for cleaning 2 times, and (weighed with 0.05%, 0.1%, 0.5% and 1.0% concentration Amount %) ginsenoside Mc handle cell.
Reaction after ten minutes, is carried out with the trypsase (trypsin) of 2U/ml or 5 μM of PAR-2 active peptides (SLIGKV) Processing, and measure intracellular Ca2+Concentration changes 80 seconds.For intracellular Ca2+The measure of concentration change, make use of microplate reader 3 (FlexStation3：Molecular device (Molecular Device), the U.S.).With the trypsase of ginsenoside Mc and 2U/ml (trypsin) or after 5 μM of PAR-2 active peptides (SLIGKV) processing, the measure bending (flex) of 80 seconds, and obtain After the minimum value of value and the difference of maximum, by the value with using 2U/ml trypsase or 5 μM of PAR-2 active peptides (SLIGKV) difference of minimum value and maximum when handling is compared, and intracellular inhibiting rate (%) table is poured in calcium ion Show in table 2 below 3.
Table 23
It is recognised that the calcium ion caused by trypsase or PAR-2 active peptides (SLIGKV) is to thin from above-mentioned table 23 Intracellular pours in, and is reduced with the processing of ginsenoside Mc, and can confirm that with the concentration for improving ginsenoside Mc, calcium Ion is substantially reduced to intracellular pouring in.
Therefore, the Dermatologic preparation composition of the ginsenoside Mc containing the present invention, by effectively suppressing to induce itch PAR-2 activity, so as to provide excellent antipruritic effect.
[formulation example 4 and compare formulation example 5]
Shampoo is prepared with the composition of table 2 below 4.Specifically, surfactant and glycol distearate are added It is added in Purified Water, and after being heated to 80 DEG C and making its uniform dissolution, is cooled to 40 DEG C gradually under agitation, also, described After active ingredient according to the present invention and preservative, viscosity modifier, spices and hair conditioner are added in mixture and is mixed, It is cooled to room temperature under agitation, so as to prepare shampoo.
Table 24
Component Formulation example 4 Compare formulation example 5
Ammonium lauryl sulfate 10 10
Polyoxyethylene lauryl base ammonium sulfate 5 5
Cocoamidopropyl betaine 2 2
Glycol distearate 1.5 1.5
Cocomonoethanolamide 0.8 0.8
Ginsenoside Mc 5.0 -
Polyquaternium-10 0.2 0.2
Blueness 1 0.0002 0.0002
Yellow 4 0.0001 0.0001
Methyl p-hydroxybenzoate 0.1 0.1
Spices 0.8 0.8
Citric acid 0.1 0.1
Dimethicone 1.0 1.0
Water To 100 To 100
[test example 19] dandruff minimizing effect is tested
The more male of 19 to 35 years old of 24 dandruffs is selected, is divided into two groups with every group 12, and divide with the following methods Not using formulation example 4 and after comparing the shampoo 1 month of formulation example 5, dandruff slip is measured.
Before on-test, hair usually is cleaned with conventional shampoo, the dandruff for then accumulating two days after collection hair washing, And by the weight of the dandruff of collection with respectively with formulation example 4 and compared with formulation example 5 shampoo washed every two days a hair and The weight for the dandruff for accumulating two days after the test is compared and evaluates.At this time, by the dandruff vacuum suck of accumulation Device is directly gathered from scalp, and obtains dandruff slip according to following mathematical expressions 7, and the results are shown in following tables In 25.
Mathematical expression 7
Dandruff slip (%)=(dandruff weight of the dandruff weight (mg) after-one month before on-test (mg)) the dandruff weight (mg) × 100 before/on-test
Table 25
It is recognised that the formulation example 4 containing ginsenoside Mc shows that excellent dandruff prevents from imitating from above-mentioned table 26 Fruit.
[test example 20] pruritus of scalp prevents the experiment of effect
Selected 24 than the more serious men and women of 25 years old to 45 years old for feeling itching of the scalp, are divided into two groups with every group 12, And, pass through following metewands pair with each formulation example 4 of frequency usage once three days and the shampoo for comparing formulation example 5 after two weeks Pruritus of scalp prevents effect from being evaluated, and the results are shown in table 2 below 6.
[metewand]
It is -5 points very excellent
Excellent -4 points
Generally -3 points
Bad -2 points
It is -1 point very bad
Table 26
Classification Formulation example 4 Compare formulation example 5
The removal effect of pruritus of scalp 4.2 2.3
It is recognised that the formulation example 4 containing ginsenoside Mc prevents from showing to pruritus of scalp from above-mentioned table 26 More excellent effect.
[test example 21] potassium ion channel activity increases effect assessment
Minoxidil as alopeciaing therapeutic agent is known as potential mitochondria K ~+Channel Opener (KATP Channel opener), it is the representative drugs used in the treatment of androgenetic alopecia.In order to evaluate this minoxidil Mechanism, used following test method(s)s.The test method(s) is the prevention K in the fibroblast using composition scalp coriumATPIt is logical The orinase (SIGMA AIDRICH, T0891) in road is handled, so as to suppress the propagation of cell, and is again turned on potassium Ion channel and recover cell Proliferation.
In order to evaluate the K as this compositionATPThe function of channel opener, present invention uses as fibroblast Mouse embryonic fibroblasts system (Mouse embryonic fibroblast the cell line, NIH3T3 of system：).This cell It is for 3T3 schemes (protocol), to separated into fiber from NIH Swiss mouse embryos (Swiss mouse embryo) Cell line carries out cell line obtained from nature immortalization.The cell line, containing 10%FBS DMEM (Gibco BRL, Gaithersburg, MD, the U.S.) in, maintaining 5%CO2, when culture 24 is small in 37 DEG C of incubator.NIH3T3 is inoculated in 96 holes In plate, and when culture 24 is small in 37 DEG C of incubator after, handled with the orinase of 2.5mM, and at 10 minutes Afterwards, serve as 10 μM of minoxidil of positive controls and the ginsenoside Mc of 2.5ppm, 5ppm and 10ppm concentration into Row processing, and after drug-treated when 48 is small after, use WST-1 kits (Roche (Roche)) measure cell Proliferation energy Power.As a result represent in table 2 below 7.
Table 27
Classification Ability of cell proliferation (%)
Without processing control group (control) 100
Minoxidil 132
Ginsenoside Mc (2.5ppm) 115
Ginsenoside Mc (5ppm) 123
Ginsenoside Mc (10ppm) 130
From above-mentioned table 27 it is recognised that when being handled with ginsenoside Mc, fibroblastic propagation is restored, and Ability of cell proliferation increases dependent on the concentration of the ginsenoside Mc of processing, and can confirm that with 10ppm ginsenosides Mc During processing, cell Proliferation returns to level when being handled with minoxidil.
The melanin generation facilitation effect experiment of [test example 22] ginsenoside Mc
The penicillin of the fetal bovine serum of addition 5%, 100IU in serum-free cell freezing media (RPMI culture mediums) In the culture medium of G and 0.2 μM of terephthalic acid (TPA) (TPA), by melanocyte, with 50,000 cells/wells, 24 orifice plates are inoculated in In.Second day, to the cell of inoculation, by the use of at the ginsenoside Mc as substances of the ultimate density of 10ppm or 50ppm Reason.Using by the use of the group of 0.1% DMSO processing as negative control group, by with 100 μM of isobutyl methylxanthine (IBMX) place The group of reason cultivates above-mentioned each group three days as positive controls at a temperature of 37 DEG C.After culture, phosphate buffer is used (PBS) cleaning orifice, and after adding the 1N NaOH of 100 μ l in every hole, dissolve the melanin in cell.Utilize tablet culture Analyzer (microplate reader), measures absorbance (cooperative effect 2, the enzyme mark of dissolved melanin under 405nm Instrument (VT, the U.S.).Result of the melanin generation facilitation effect of ginsenoside Mc compared with control group is represented following In table 28.
Table 28
Sample B16 cell amount (%)
DMSO (0.1%) 100
IBMX(100μM) 120
Ginsenoside Mc (10ppm) 112
Ginsenoside Mc (50ppm) 121
It is recognised that ginsenoside Mc promotes the B16 cell of melanocyte, so as to increase black from above-mentioned table 28 The generation of element, therefore can show that excellent melanin generation facilitation effect.
Promotion transcription factors (MITF) and tyrosinase expression of [test example 23] the ginsenoside Mc in melanocyte Effect
Using 501mel cell lines, with 500,000 cells/wells, it is inoculated in 6 orifice plates, and in each hole, with 0.1% Dimethyl sulfoxide (DMSO) (DMSO) processing conduct negative control group, by the use of 100 μM of IBMX processing as positive controls, and By the use of the life saponin(e Mc of 10ppm handle as experimental group, and cultivated at a temperature of 37 DEG C 24 it is small when, 48 it is small when and 72 it is small when Afterwards, protein is obtained.For the protein so obtained, western blot (Western is carried out using MITF and tyrosinase Blot) method is tested.Protein Extraction and western blot are implemented by the usually used standard method of those skilled in the art. After implementing western blot, the negative control group in its result is set to 100, and represented compared with the value in table 2 below 9 In.
Table 29
As shown in above-mentioned table 29, it can confirm that ginsenoside Mc improves MITF and tyrosinase protein in melanocyte The expression of matter.
The antimicrbial power evaluation of [test example 24] ginsenoside Mc
In order to evaluate the antimicrbial power of ginsenoside Mc, antibacterial experiment is implemented.Specific test method is as described below.
Staphylococcus aureus (Staphylococcus aureus), the Escherichia coli used in experiment (Escherichia coli) and pseudomonas aeruginosa (Pseudomonas aeruginosa) are in trypticase soya broth Culture in culture medium (Tryptic Soy Broth)；Candida albicans (Candida albicans) and aspergillus niger (Aspergillus niger) is the culture in Sabouraud dextrose broth bouillon (Sabouraud Dextrose Broth). It regard the dilution that nutrient solution is diluted in each culture medium with 1/100 (albicans strain is using 1/10) as test organisms Liquid uses.For aspergillus niger, 2 × 10 will be prepared into8The spore suspension of cfu/ml is as test bacteria liquid.
The mixed liquor that the test bacteria liquid that 0.15ml is added in each culture medium of 15ml is mixed is as dilute solution To use.
In No. 1 row of 96 orifice plate, the ginsenoside Mc of 10ppm is separately added into the amount of every 16 μ l of hole, and be separately added into The dilute solution of 184 μ l.The dilute solution of 100 μ l is separately added into other holes.The mixed liquor of No. 1 row is uniformly mixed Afterwards, the mixed liquor for taking out 100 μ l is added in No. 2 rows, and after mixing, the mixed liquor for taking out 100 μ l again is added to No. 3 In row.Doubling dilution has been carried out in this way.
Staphylococcus aureus, Escherichia coli and pseudomonas aeruginosa are cultivated in 32 DEG C of thermostat；Candida albicans Bacterium and aspergillus niger are cultivated in 25 DEG C of thermostat.
48 it is small when after, whether with suspensibility and microscope to confirm the increment of bacterium, so as to determine minimum inhibitory concentration (MIC) Value, and the results are shown in Table 3 below 0.
Table 30
As shown in above-mentioned table 30, it can confirm that ginsenoside Mc shows antimicrbial power to a variety of bacterial strains, and pass through this It can predict that ginsenoside Mc can work in composition as natural antiseptic agent or antiseptic a bit.

Claims (6)

1. the cosmetic combinations of moisturizing skin are being prepared as the Dermatologic preparation composition of active ingredient comprising ginsenoside Mc Application in thing.
2. applications of the ginsenoside Mc as unique active ingredient in the cosmetic composition for preparing moisturizing skin.
3. the change of reinforcing skin barrier function is being prepared as the Dermatologic preparation composition of active ingredient comprising ginsenoside Mc Application in cosmetic compositions.
4. ginsenoside Mc answering in the cosmetic composition for strengthening skin barrier function is prepared as unique active ingredient With.
5. induced skin keratinocyte is being prepared as the Dermatologic preparation composition of active ingredient comprising ginsenoside Mc Application in the cosmetic composition of differentiation.
6. ginsenoside Mc is preparing the cosmetic combinations of induced skin Keratinocyte differentiation as unique active ingredient Application in thing.
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