CN105310923A - Preparation method of cosmetic composition and extract of tissue-cultured raoulia australis shoot - Google Patents
Preparation method of cosmetic composition and extract of tissue-cultured raoulia australis shoot Download PDFInfo
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- CN105310923A CN105310923A CN201510232464.XA CN201510232464A CN105310923A CN 105310923 A CN105310923 A CN 105310923A CN 201510232464 A CN201510232464 A CN 201510232464A CN 105310923 A CN105310923 A CN 105310923A
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- 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
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
- A61K8/9783—Angiosperms [Magnoliophyta]
- A61K8/9789—Magnoliopsida [dicotyledons]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q1/00—Make-up preparations; Body powders; Preparations for removing make-up
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- 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
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- 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
- A61Q19/02—Preparations for care of the skin for chemically bleaching or whitening the skin
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- 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
- A61Q19/08—Anti-ageing preparations
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
- A61K2800/805—Corresponding aspects not provided for by any of codes A61K2800/81 - A61K2800/95
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Abstract
The invention relates to a cosmetic composition comprising an extract of tissue-cultured raoulia australis shoot and a preparation method thereof. The tissue-cultured raoulia australis shoot extract has good oxidation resisting, anti-inflammatory and whitening effects, so that the extract can be usefully utilized to the cosmetic composition with the purposes of skin protection and skin improvement.
Description
Technical field
The present invention relates to a kind of cosmetic composition and manufacture method thereof, be specifically related to the cosmetic composition comprising Australia La Woer chrysanthemum plumelet extract of tissue culture and the manufacture method thereof of antioxidation, antiinflammatory and whitening effect excellence.
Background technology
Skin has the function of protection health from outside stimulus.This outside stimulus and environment, physics, chemical factor and physiological internal factor not only reduce skin function, compared with normal skin, scytitis and the pigmentation disease as nevus and spot and so on are caused to outside stimulus, also reduces skin function and become the reason of skin aging.
Therefore present cosmetics exploitation is in and constantly finds cutaneous safety excellence, it is that skin affects from various factors to protect, antiinflammatory, antioxidation and whitening effect etc. can improve and protect the stage of the cosmetics new material of skin.
Forming to protect skin, beautify clean and in the composition of the cosmetics of exploitation; representational have solubilizing agent, emulsifying agent, antiseptic, spice, UV blockers, synthesis viscosifier etc., but these compositions are generally considered to the induced factor of scytitis or acne etc.In addition, the sebum generated in body, fatty acid, protein etc. in antiperspirant and cosmetic composition can react with the various bacterium that exist in skin and bring out inflammation.
Active oxygen in human body is generated by the reaction of various enzyme, plays a very important role to the biosynthesis, immunologic function, drug metabolism etc. of biological active substances.If but do not maintain constancy in body, when generating excessive, there is damage to human body on the contrary.Such as, free radical and active oxygen make body material's oxidations or rotten such as Cell membrane lipids, protein, nucleic acid, reduce the function of Skin Cell, thus become the main cause of skin problem, inflammation, skin aging.
Determine a variety of because of have of human body skin color, wherein important have whether contain the factors such as pigment inside and outside the distribution of melanocyte (melanocyte), skin thickness, blood vessel and human body.Especially most important factor be generated by the various enzyme effect such as tryrosinase in the melanocyte in human body be called as melanic black pigment.Melanin pigment is easily formed by the skin pigmentation disorder such as nevus, freckle, pigmentation that produced by the external environmental factor of ultraviolet radiation and so on calmness symptom etc.In order to treat or to alleviate excessive melanin pigment calm, various material and cosmetics or medicine with the use of.But dosage form and stability problem etc. during insufficient, safety issue, cosmetic ingredients to skin due to whitening effect, its use is restricted.
The effort of the skin of beauty of keeping fit while preventing these skin phenomena is also in continuation.The research direction improved skin is recently that exploitation has no side effect and human body is affine, independently can to feel at ease the material utilizing plant extract that uses with body constitution.
Usually, the functionalization cosmetic comprising natural plant extracts can be used in all plants grown in tropical rainy climate that plant can grow, subfrigid zone, climate of frigid zone.Plant adapts to respective weather, with the best biomechanism growth under this condition.Therefore, even kindred plant, according to the different parts of sunshine amount, temperature, precipitation etc. and the weather conditions such as soil aciditiy, fertile degree of the latitude grown according to it and Various Seasonal, plant, the kind of the effective ingredient of 2 metabolites of inside plants and so on is different from content.Moreover, be difficult to overcome supply be difficult to meet manufacture, the shortcomings such as processing charges is many.In order to improve these all difficulties, utilize the cosmetic material development effectiveness of vegetable culture technique remarkable recently.
On the other hand, wild herb Australia La Woer chrysanthemum (Raouliaaustralis) grows wild of Compositae is in New Zealand and Australia.Australia La Woer chrysanthemum is with mat form growth on the humus soil of easy draining, and because tolerance to cold is strong, cobble, the rock surface of main wild growth in alpine belt, use as cover plant during Landscape always.The material of the terpene series that the La Woer acid (Raoulicacid) that Australia La Woer chrysanthemum contains is made up of 25 carbon, research shows that it has the proliferation inhibiting effect to the virus causing nasal sinusitis, otitis media, chronic bronchitis, aseptic meningitis, acute pancreatitis, myocarditis etc.
Australia La Woer chrysanthemum with these effects should not be cultivated due to geographical position and climate change Korea S is domestic.Although along with active ingredient is familiar with, its increase in demand, find various effect by research, the scientific experiments result of effect of the known Australia La Woer chrysanthemum plumelet to tissue culture is little.In addition, also without any the report of result of study being applied as cosmetic composition.
Summary of the invention
The object of the invention is to; a kind of cosmetic composition and manufacture method thereof are provided, comprise antioxidation, antiinflammatory and whitening effect in described cosmetic composition excellent and there is skin improve and the extract in Australia La Woer chrysanthemum plumelet (tissueculturedRaouliaaustralisshoots) of tissue culture of skin care effect.
Object of the present invention is not limited to the above-mentioned object mentioned, other objects NM can be understood clearly from following record.
According to cosmetic composition of the present invention, it is characterized in that, Australia La Woer chrysanthemum plumelet (tissue-culturedRaouliaaustralisshoots) extract containing tissue culture is effective ingredient.
Here, relative to described cosmetic composition gross weight, preferably comprise Australia La Woer chrysanthemum plumelet extract of described tissue culture with the scope of 0.1 ~ 10 % by weight.
Especially, Australia La Woer chrysanthemum plumelet extract of described tissue culture can with being selected from pure water, Australia La Woer chrysanthemum plumelet of more than one the solvent extraction tissue culture in the alcohol of C1 ~ C4 and the polyhydric alcohol of C1 ~ C5 and obtaining.
Further, the dosage form be selected from skin cream, toner/smoothing toner, cosmetic water, astringent lotion, emulsion, milky lotion, nutritional emulsions, massage cream, nourishing cream, hand cream, vanishing cream, sun screen, dual-purpose muffin, mascara, elite, nutrition elite, facial film, soap, clean skin foam, clean skin dew, cleansing cream, body lotion and bath gel can be had according to cosmetic composition of the present invention.
In addition, according to the manufacture method of Australia La Woer chrysanthemum plumelet extract of tissue culture of the present invention, can comprise: Australia La Woer chrysanthemum plumelet of tissue culture is formed the step of dipping solution by (a) being selected from dipping 3 ~ 30 days in more than one the Extraction solvent in pure water, the alcohol of C1 ~ C4 and the polyhydric alcohol of C1 ~ C5; And (b) filters described dipping solution and obtains the step of Australia La Woer chrysanthemum plumelet extract of tissue culture.
Here, described Extraction solvent can use any one in the mixed solvent of mixed solvent, ethanol and the propylene glycol being selected from ethanol, ethanol and 1,3 butylene glycol and the mixed solvent of ethanol and pentanediol.
In addition, relative to Australia La Woer chrysanthemum plumelet weight of described tissue culture, described Extraction solvent preferably has the weight ratio of 10 ~ 20 times.
Cosmetic composition according to the present invention comprises Australia La Woer chrysanthemum plumelet extract of tissue culture, not Diazolidinyl Urea, has excellent antioxidation, antiinflammatory and whitening effect, and skin is improved and skin care very effective.Australia La Woer chrysanthemum plumelet (tissueculturedRaouliaaustralisshoots) extract protection skin of tissue culture is not by various outside stimulus; skin slip is moistened, and demonstrates the effect to the highly significant that skin function improves.
Detailed description of the invention
The present invention can carry out numerous variations, and can have various embodiments, exemplifies specific embodiment, and is described in detail to this by detailed description.But this is not limit the invention to specific embodiment, be interpreted as being included in thought of the present invention and technical scope comprise all changes, equipollent is to substitute.
Below, the cosmetic composition comprising Australia La Woer chrysanthemum plumelet (tissueculturedRaouliaaustralisshoots) extract of tissue culture according to the present invention is described.
The present inventor is evaluated as center with effect effect of Australia La Woer chrysanthemum plumelet (tissueculturedRaouliaaustralisshoots) extract to tissue culture, continue utilizing the application possibility as cosmetics of vegetable culture technique to be studied, and then complete the present invention.Its result; the present inventor specify that Australia La Woer chrysanthemum plumelet (tissueculturedRaouliaaustralisshoots) extract protection skin of tissue culture is not by various outside stimulus; skin slip is moistened, and skin function is improved to the effect with highly significant.
Relative to the gross weight of compositions, preferably comprise the extract of Australia La Woer chrysanthemum plumelet of tissue culture with the scope of 0.1 ~ 10 % by weight according to cosmetic composition of the present invention.Meet described scope and just can show helpfulness effect, namely protect skin not by various outside stimulus, skin slip is moistened, and demonstrate the effect contributing to skin function and improve.In addition, consider the intrinsic abnormal smells from the patient, color etc. of the extract of final products, be preferably included as described scope.
The extract of Australia La Woer chrysanthemum plumelet of described tissue culture preferably uses and is selected from Australia La Woer chrysanthemum plumelet of more than one the solvent in pure water, the alcohol of C1 ~ C4 and the polyhydric alcohol of C1 ~ C5 to tissue culture and extracts.
The product of cosmetic composition of the present invention can be added, such as there are various skin cream, toner/smoothing toner, cosmetic water, astringent lotion, emulsion, milky lotion, nutritional emulsions, massage cream, nourishing cream, hand cream, vanishing cream, sun screen, dual-purpose muffin, mascara, elite, nutrition elite, facial film, soap, clean skin foam, clean skin dew, cleansing cream, body lotion and bath gel etc., but are not limited thereto.When cosmetic composition of the present invention is manufactured into the dosage form of described form, can contain the formulation required of this dosage form and suitable various substrate and additive.In addition, the kind of these compositions can easily be selected by those skilled in the art with amount.
Below, the manufacture method of Australia La Woer chrysanthemum plumelet extract of tissue culture of the present invention is described.
The present invention includes: Australia La Woer chrysanthemum plumelet of tissue culture is formed the step of dipping solution by (a) being selected from dipping 3 ~ 30 days in more than one the Extraction solvent in pure water, the alcohol of C1 ~ C4 and the polyhydric alcohol of C1 ~ C5; And (b) filters described dipping solution and obtains the step of Australia La Woer chrysanthemum plumelet extract of tissue culture.
Australia La Woer chrysanthemum plumelet of the above-mentioned tissue culture used in described (a) step is preferably drying regime.And the dip time of described (a) step is preferably 10 ~ 20 days.
Relative to the weight of Australia La Woer chrysanthemum plumelet of the tissue culture of described drying, described Extraction solvent preferably uses with the weight ratio of 10 ~ 20 times (w/w).
The alcohol of described C1 ~ C4 can be methanol, ethanol, propanol, butanols etc., is particularly preferably ethanol (Ethylalcohol).In addition, the polyhydric alcohol of described C1 ~ C5 is preferably selected from more than one in alkylene glycol, 1,3 butylene glycol, propylene glycol and pentanediol.
In addition, when described Extraction solvent is the mixed solvent of described alcohol and alkylene glycol, its mixing ratio (w/w) is preferably 6:4 ~ 9:1 (that is, relative to the gross weight of whole mixed solvents of alcohol and alkylene glycol, the content of alcohol is preferably 60 ~ 90 % by weight).The content of the mixed solvent of described alcohol and alkylene glycol, when described scope, has only extracting the more effective polarity of required effective ingredient.Here, the concentration of the alcohol used in the mixed solvent of described alcohol and alkylene glycol is preferably 60% ~ 100%.
In addition, described Extraction solvent also can be the mixed solvent of described pure water and described alkylene glycol, its mixing ratio is preferably 3:7 ~ 1:9 (that is, relative to the gross weight of whole mixed solvents of pure water and alkylene glycol, the content of alkylene glycol is preferably 70 ~ 90 % by weight).
Described Extraction solvent is preferably ethanol when being single solvent.And, the mixed solvent of ethanol and alkylene glycol can be used when Extraction solvent is mixed solvent, preferred use ethanol and 1, the mixed solvent of 3-butanediol, ethanol and propylene glycol or ethanol and pentanediol, more preferably the mixed solvent of ethanol and 1,3 butylene glycol or the mixed solvent of ethanol and pentanediol can be used.
In addition, described (b) step preferably comprises the step of removing Extraction solvent further.
The invention provides Australia La Woer chrysanthemum plumelet extract comprising the tissue culture obtained by described manufacturing process is the cosmetic composition of effective ingredient.Here, effect of Australia La Woer chrysanthemum plumelet extract according to tissue culture of the present invention is confirmed by following step, wherein, comprise: with Australia La Woer chrysanthemum plumelet extract of described tissue culture, human fibroblasts is processed, investigate the step to skin irritant cytotoxic effect; With Australia La Woer chrysanthemum plumelet extract of described tissue culture, human fibroblasts is processed, investigate the step of the cytoprotective effect to outside stimulus; By carrying out the generation rejection ability test of the suppression of lipoxygenase (Lpoxgenase) activity and nitrogen oxide to Australia La Woer chrysanthemum plumelet extract of described tissue culture, the step of investigation antiphlogistic effects; By (SUPEROXIDEANION) elimination activity and free radical (HYDROXYRADICAL) the elimination activity ability of superoxide anion, the step of investigation antioxidant effect; Investigation tryrosinase suppresses the skin-whitening brought to improve the step of effect.
And cosmetic composition according to the present invention is not limited to illustrative dosage form below, can adopt the dosage form known by the art.Cosmetic composition of the present invention can coordinate other compositions usually coordinated in cosmetics in the scope not damaging object of the present invention and effect in described extract.
Enumerate below and carry out more specific description according to Production Example of the present invention, test example and dosage form example, but these are just in order to illustrate the present invention, scope of the present invention is not limited thereto.
[Production Example 1 ~ Production Example 4 and compare Production Example 1 ~ compare Production Example 4]: the manufacture of Australia La Woer chrysanthemum plumelet extract of tissue culture
Australia La Woer chrysanthemum plumelet of tissue culture and natural Australia La Woer chrysanthemum are washed respectively, with gauze except after anhydrating, dry and chopping.Here, " Australia La Woer chrysanthemum plumelet of tissue culture " that use in this Production Example is that the most advanced and sophisticated horticulture technique developmental research center learned by loyal Beijing University provides, it is aseptically utilized by Australia La Woer chrysanthemum seed from New Zealand's import plant tissue culture technique to induce plumelet, with Solution culture method.
Australia La Woer chrysanthemum plumelet of ready tissue culture and Australia La Woer chrysanthemum are flooded 2 weeks in the mixed solvent existed with the content recorded in table 1, carries out ripening.Thereafter filter with filter paper (5C, 185mm that Japanese ToyoroshiKaisha, Ltd. sell), ethanol evaporation, obtain Australia La Woer chrysanthemum plumelet extract and Australia La Woer chrysanthemum extract of the tissue culture of filtrate shape.
[table 1]
(b1:70% ethanol/b2:1,3-butanediol/b3: propylene glycol)
The cytotoxic assay of Australia La Woer chrysanthemum plumelet extract of [test example 1] tissue culture
By to Production Example 1 and the cell toxicity test comparing Production Example 1, have rated safety.
[cell culture (Cellculture)]
By the CCD-1064sk human fibroblasts bought from ATCC company in 10cm Tissue Culture Dish, to be added with 10% hyclone (FBS), Yi Sikefu Media modified (Iscove'smodifiedDulbeco'smedium:IMDM) 10ml of 1% Pen .-Strep (penicillin-streptomycin) is that culture medium is cultivated, and makes at 37 DEG C, 5%CO
2cell fusion (confluency) 70% ~ 80% in incubator.Culture medium changes weekly 2 times, after the cell reaching fusion uses pancreas enzyme-EDTA (trypsin-EDTA) carry out pancreatin process (trypsinization), maintains with Secondary Culture.By the human fibroblasts of described cultivation respectively with 1 × 10
4cells/well is divided the upper cultivation of 96 orifice plates (96-multiwellplate) 24 hours.Replace the culture medium of cultured cells with 2%FBS culture medium, with concentration for the treatment of respectively with Australia La Woer chrysanthemum plumelet extract of the tissue culture of culture medium dilution Production Example 1 with after Australia La Woer chrysanthemum extract comparing Production Example 1, at CO
2cultivate 48 hours in incubator.
[test example 1-1]: the mensuration (MTT detects (assay)) of cytotoxic effect
The MTT being dissolved in PBS (phosphate buffered saline (PBS) (phosphatebufferedsaline)) is preserved (stock) solution (5mg/ml) and adds dilution 10 times in new culture medium, its solution is added respectively 100 μ l in the hole being placed with end cultured cells, 37 DEG C of reactions 4 hours.Because the dehydrogenase in the mitochondrion of cell lived after reaction and yellow water-soluble substrate MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazoles
bromide)] be reduced to non-water-soluble MTT-first
(MTT-formazan) crystallization, detects the survival rate that its amount just can measure cell.Removing culture supernatant, adds the MTT-first that DMSO200 μ l comes to generate in dissolved cell in each hole
after crystal, measure can make first to absorb spectrophotometer (UVmax, MolecularDevice, USA)
the maximum O.D value of test optical filter (Testfilter) 540nm wavelength of absorbance and the O.D value of basic filter (Referencefilter) 630nm wavelength, absorbance (list of references TheEMBOJournal (2002) 21,2407-2417 is outer most) is obtained with its difference.Cytotoxicity illustrates with the percentage ratio of absorbance.
※ cells survival rate (%)=(sample pretreating group absorbance/non-processor group absorbance) × 100
Based on original (Raw) data, calculate the meansigma methods of the absorbance of non-processor group and the absorbance of sample pretreating group, calculate standard deviation.
[table 2]
[test example 1-2]: the mensuration (dimethyl diaminophenazine chloride detects (NeutralRedassay)) of cytotoxic effect
In each hole of cultivating the cell terminated, add dimethyl diaminophenazine chloride (NeutralRed, Sigma-Aldrich, USA) 50 μ g/ml respectively, react 3 hours.Dimethyl diaminophenazine chloride is concentrated in the cell of surviving completely or the Cytolysosome not having damage.After reaction, with 1.0% formalin/1.0% potassium chloride fixed cell, then add 1.0% acetic acid/50% alcoholic solution, extract dimethyl diaminophenazine chloride.The dimethyl diaminophenazine chloride extracted is utilized and absorbs each levels of cytotoxic value that spectrophotometer (UVmax, MolecularDevice, USA) is determined at 540 ~ 630nm, and be shown in following table 3.Cytotoxicity illustrates with the percentage ratio of absorbance, implements 3 times with 3 batches (lot).
※ cells survival rate (%)=(sample pretreating group absorbance/non-processor group absorbance) × 100
[table 3]
Based on original (Raw) data, calculate the meansigma methods of the absorbance of non-processor group and the absorbance of sample pretreating group, calculate standard deviation.
As shown in table 2 and table 3, the MTT of manufacture grain 1 of the present invention
50, NR
50value is more than 1%, and known is safe raw material with comparing compared with Production Example 1.
[test example 2]: the cytoprotective effect of Australia La Woer chrysanthemum extract of tissue culture measures
Carry out testing with SDS (sodium lauryl sulphate (Sodiumdodecylsulfate)) the Cytotoxic cytoprotective effect comparing Production Example 1 Production Example 1.
Be added with 10% hyclone (FBS), 1% Pen .-Strep IMDM at 37 DEG C, 5%CO
2human fibroblasts is cultivated in incubator.By the human fibroblasts of cultivation with 1 × 10
4cells/well is divided respectively in 96 orifice plates, cultivates 24 hours.The culture medium of cultured cells is replaced with 2%FBS culture medium, by SDS (Sodiumdodecylsulfate) 0.002% pretreatment after 1 hour, dilute Production Example 1 with culture medium respectively with respective concentration for the treatment of and compare Production Example 1, after parallel processing, at CO
2cultivate 24 hours in incubator.The MTT that 10 times of dilutions are dissolved in PBS (phosphatebufferedsaline) in new culture medium preserves solution (5mg/ml), its solution is added respectively each 100 μ l in the respective hole of terminating cultured cells, 37 DEG C of reactions 4 hours.Because the dehydrogenase in the mitochondrion of cell lived after reaction and yellow water-soluble substrate MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide] is reduced to non-water-soluble MTT-first
(MTT-formazan) crystallization, detects the survival rate that its amount just can measure cell.Removing culture supernatant, adds the MTT-first that DMSO200 μ l comes to generate in dissolved cell in each hole
after crystal, measure can make first to absorb spectrophotometer (UVmax, MolecularDevice, USA)
the maximum O.D value of test optical filter 540nm wavelength of absorbance and the O.D value of basic filter 630nm wavelength, obtain absorbance with its difference.(list of references TheEMBOJournal (2002) 21,2407-2417 is outer most).Cytoprotective effect illustrates with the percentage ratio of absorbance.
Cells survival rate (%)=(sample pretreating group absorbance/non-processor group absorbance) × 100
Based on original (Raw) data, calculate the meansigma methods of the absorbance of non-processor group and the absorbance of sample pretreating group, calculate standard deviation.
[table 4]
As shown in table 4, known, for outside stimulus, the cytoprotective effect of Production Example 1 of the present invention with compare compared with Production Example 1, more can promote cytoprotective effect.
[test example 3]: the mensuration of the antiphlogistic effects of Australia La Woer chrysanthemum plumelet extract of tissue culture
Measure Production Example 1 and carry out antiphlogistic effects experiment with comparing Production Example 1 pair of lipoxygenase activity and nitric oxide growing amount.
Inflammatory reaction be human body in order to maintain constancy, the biophylaxis of outside stimulus is reacted.Generate and stimulate the approach of the enzyme of inflammation caused with lipoxygenase activity approach and cyclooxygenase (cycloxygenase) active approach for representative.The hydroperoxidation (hydroperoxidation) of lipoxygenase catalysis unsaturated fatty acid, generate lipid peroxide hydrogen (fattyhydroperoxides), the catabolites such as these material regeneration epoxide (epoxide), hydroxyl, ketone, aldehyde.Lipoxygenase generates the number of chemical medium (chemicalmediator) relevant to inflammatory reaction or allergy etc. in organism from by linolenic acid (linolenicacid) biosynthetic arachidonic acid (arachidonicacid).Therefore, the material of lipoxygenase is hindered to show the result of the generation of the chemical mediator (chemicalmediators) suppressing relevant to various inflammatory reaction or allergy etc.Although the nitric oxide (NO) generated by the activation of the enzyme etc. taking inflammatory reaction as medium plays an important role in immunoreation, too much nitric oxide can cause inflammation reaction, brings exception to immune system.
[test example 3-1]: the mensuration of the generation rejection ability of nitrogen oxide
The generation degree of nitrogen oxide (NO) is with the method (GreenLC of Green etc., WagnerDA, GlogowskiJ, etal.1982.Analysisofnitrate, nitrite, and [15N] nitrateinbiologicalfluids.AnalBiochem126:131-138.) measure the amount generating the nitrate generated in the culture medium of index as nitrogen oxide (NO), analyze.Namely, process sample, cultivate after 30 minutes, add LPS (lipopolysaccharide, 1 μ g/mL), cultivate 24 hours and obtain Raw264.7 macrophage-conditioned media, 1% sulfanilamide (being dissolved in the sulfanilamide (sulphanilamide) in 5% phosphoric acid (phosphoricacid)) of 50 μ L and the 0.1% naphthylenediamine dihydrochloride (naphthylenediaminedihydrochloride) of 50 μ L is added in 100 μ L medium supernatants of this Raw264.7 macrophage-conditioned media, at room temperature reaction after 10 minutes, absorbance is measured at 540nm.Matched group for described anti-inflammatory activity effect test have employed the experimental group of individual processing LPS.
※ NO suppression ratio (%)=(the NO amount of the NO amount/matched group of NO amount-each extract of matched group) × 100
Calculating mean value based on original (Raw) data, obtains standard deviation.
[table 5]
The generation rejection ability of the NO occurred in the oxidative stress that LPS causes as described in the result shown in table 5, Production Example 1 with compare compared with Production Example 1, the NO demonstrating about 1.5 ~ 2 times generates inhibition.
[test example 3-2]: the activity measuring lipoxygenase (Lipoxygenase) suppresses
Implement the active suppression test as the lipoxygenase of the representational enzyme of the inflammation forming factors to outside stimulus.Sample to be tested 0.1ml and lipoxygenase (Sigma-aldrich, USA) 0.9ml is mixed, after carrying out whirlpool concussion (voltexing), 25 DEG C of reactions 10 minutes in 1mM linolenic acid (Sigma-aldrich, USA) 1ml.Then add 20% trichloroacetic acid (trichloroaceticacid) 0.5ml and 0.6% thiobarbituricacidα-(thiobarbituricacid) 1ml, react in the hot water after 10 minutes, merceration 2-3 minute in frozen water, add butanols (butanol) 2ml, whirlpool is shaken after more than 20 seconds, with 3500rpm centrifugalize 5 minutes, O.D value is measured at 513nm with UV spectrophotometer (spectrophotometer), the maximum inhibition of lipoxygenase is calculated, shown in table 6 according to following mathematical expression 2.Determination test divides 3 batches (batch) to implement 3 times, calculates the meansigma methods of absorbance, obtain standard deviation based on original (Raw) data.
[table 6]
As shown in table 6, when Australia La Woer chrysanthemum plumelet extract of the tissue culture of Production Example 1 compares with the matched group of non-processor, rely on control of the concentration lipoxygenase, can confirm and compare compared with Production Example 1, outstanding to the active inhibition of the enzyme caused inflammation.
The antioxidant effect of Australia La Woer chrysanthemum plumelet extract of [test example 4] tissue culture measures
Implement Production Example 1 to test with the antioxidant effect comparing Production Example 1.
The anti-hydroxyl radical free radical activity (%) that [test example 4-1] DPPH (1,1-diphenyl-2-picryl hydrazine (1,1-diphenyl-2picrylhydrazyl)) free radical causes measures
The reaction that active oxygen causes almost major part is radical reaction, comprises Auto-oxidation reaction process.The effect blocking Auto-oxidation reaction is responsible for by antioxidant in vivo.Reducing power as electron donation is stronger, more can become powerful antioxidant.The reagent measuring the reducing power of antioxidant has 1,1-hexichol-2-picryl hydrazine (1,1-diphenyl-2-picrylhydrazyl:DPPH) free radical.The free radical form of the rock-steady structure of DPPH in compound centered by nitrogen exists.Show absorption maximum at 517nm, the absorption being reduced rear 517nm disappears.DPPH (D9132, SIGMA) is dissolved in the mixed solvent of 6:4 (v/v) of ethanol and distilled water, adjusts described DPPH solution and make its absorbance at 513nm be about 0.6.Add DPPH solution 2ml respectively according in the sample 1ml of concentration dilution, mix.Place about after 1 hour under room temperature, use and absorb the absorbance that spectrophotometer (UVmax, MolecularDevice, USA) measures 513nm, the results are shown in table 7.
[table 7]
As shown in table 7, Production Example 1 shows excellent antioxidant effect.
[test example 4-2]: the anti-hydroxyl radical free radical activity (%) that ABTS free radical causes measures
The method of Miller [Milleretal. (1993)] is utilized to measure the total antioxidant capacity of extract.With the concentration of 7mM by 2,2'-azine group-bis-(3-ethyl benzo thiazole phenanthroline-6-sulphonic acid ester) (2,2'-Azino-bis (3-ethylbenzothiazoline-6-sulfonate)) be dissolved in distilled water, be that the mode of 2.45mM is added potassium peroxydisulfate (Potassiumpersulfate) and made ABTS cation (cation) free radical wherein with ultimate density.By ABTS and potassium peroxydisulfate mixed in equal amounts, room temperature was placed on dark place after 12 hours, and this solution of dilution in ethanol (ethanol), being adjusted to its absorbance at 734nm is 0.7.Mixed plant extract sample 10 μ l, ABTS solution (Solution) 190 μ l, distilled water 50 μ l, absorbance is measured at spectrophotometer (spectrophotometer) 734nm after 6 minutes, obtain absorbance difference with percentage ratio (%), the results are shown in table 8.
Elimination factor (%)=(B-A)/B × 100
(B: the absorbance of the non-interpolation group of extract, A: the absorbance of extract interpolation group)
[table 8]
As shown in table 8, compare comparatively Production Example 1 according to Australia La Woer chrysanthemum extract (Production Example 1) of tissue culture of the present invention and demonstrate better antioxidant effect.
[test example 5]: the tyrosinase activity of Australia La Woer chrysanthemum plumelet extract of tissue culture and B16 cell inhibition measure.
[test example 5-1] tyrosinase activity hinders test
In order to investigate Production Example 1 and compare the whitening active of Production Example 1, carry out tyrosinase activity and hindered test.In order to test, Mushroom Tyrosinase (mushroomtyrosinase is dissolved with phosphate buffer (pH6.5), T-3824,1530U/mg, Sigma) to the enzyme liquid of 1000U/ml as tryrosinase, TYR (L-tyrosine, 45160-0410, Junseichemicalco.Ltd) is dissolved to 1.5mM as matrix liquid with phosphate buffer (pH6.5).
With buffer with variable concentrations dilution Production Example 1 and compare Production Example 1, prepare detection liquid.Add the enzyme liquid 10 μ l detecting liquid 170 μ l, tryrosinase, place 10 minutes at 37 DEG C.After wherein adding matrix liquid 20 μ l, after 10 minutes, place 5 minutes in ice immediately 37 DEG C of reactions, then utilize and absorb the absorbance that spectrophotometer (UVmax, MolecularDevice, USA) measures wavelength 490nm.The absorbance of described mensuration is substituted in following mathematical expression, calculate the activity obstruction rate of tryrosinase, be shown in table 9.The liquid that alternative detection liquid is put into phosphate buffer (pH6.5) and manufactured is as blank assay liquid, and the liquid that alternative matrix liquid is put into phosphate buffer (pH6.5) and manufactured is as opaquing fluid.
< mathematical expression >
Tyrosinase activity obstruction rate (%)=100-(A-A')/(B-B') × 100
A: detect the reacted absorbance of liquid
B: the reacted absorbance of blank assay liquid
A': the opaquing fluid detecting liquid
B': the opaquing fluid of blank assay liquid
[table 9]
[test example 4-2]: the B16 cell according to Australia La Woer chrysanthemum plumelet extract concentrations of tissue culture suppresses determination experiment
To Production Example 1 and compare Production Example 1, utilize B-16 melanocyte (Melanomacell: Korea Cell strain bank), measure whitening effect.Assay method be by derive from black Mus B-16 melanocyte with the DMEM containing 10%FBS (GIBCO., USA) with 2 × 10
4the concentration of cells/well respectively adds 2ml to 6 hole tissue culture dishes, at 5%CO
2, cultivate 24 hours under 37 DEG C of conditions.Culture medium is removed after cultivation, with containing 10%FBS, 2uMa-MSH (Sigma., USA, melanophorin (MelanocytestimulatingHormone)) and 2mM theophylline (Sigma., USA, theophylline) after DMEM replaces, after adding Australia La Woer chrysanthemum plumelet extract of the tissue culture of suitably diluting with same medium respectively, at 5%CO
2, be cultured to cell under 37 DEG C of conditions at the bottom of hole, reach about more than 80%.Remove culture medium after cultivation, then with PBS (Sigma., USA, PhosphatebufferedSaline) washing, after pancreatin process, reclaim cell.The cell reclaimed removes supernatant with 5000rpm centrifugalize after 10 minutes.The cell of removing supernatant in 60 DEG C of thermostats after dry 24 hours, adds the melanin that 1NNaOH comes in dissolved cell.Utilize and absorb spectrophotometer (Uvmax, MolecularDevice, USA) measures dissolving melanic absorbance at wavelength 490nm.
< mathematical expression >
Melanin generates obstruction rate (%)=(1-A/A') × 100
(A: test group absorbance, A': matched group absorbance)
[table 10]
As shown in table 9 and table 10, the activity of Production Example 1 concentration dependent ground restraint of tyrosinase, and compared with Production Example 1, there is more excellent intracellular melanin generation inhibition frequently.
[embodiment 1 ~ embodiment 2] manufactures the elite of Australia La Woer chrysanthemum plumelet extract containing tissue culture
The elite comprising Production Example 1 has been manufactured according to the composition of following table 11.
[table 11]
In table 11, fully dispersion, moistening ingredients A, form uniform gel (Gel) shape, adds B and neutralize.Add composition C in composition (A+B), uniform stirring adds components D after dissolving at room temperature, stirs and makes it be uniformly distributed, contain in container and complete commercialization.
[embodiment 3 ~ 4]: manufacture the soft and moist astringent (skin) comprising Australia La Woer chrysanthemum plumelet extract of tissue culture
The embodiment comprising soft and moist astringent (skin) in the cosmetic composition of Production Example 1 is as shown in table 12 below.
[table 12]
[embodiment 5 ~ 6]: manufacture the frost comprising Australia La Woer chrysanthemum plumelet extract of tissue culture and comprise the prescription of frost in the cosmetic composition of Production Example 1 such as shown in following table 13.
[table 13]
Although preferred embodiments of the present invention have been disclosed for illustrative now, those skilled in the art can realize the form of being out of shape in the scope not exceeding intrinsic propesties of the present invention.Therefore, here the embodiments of the invention illustrated are not the viewpoints limited, but consider from the viewpoint illustrated, scope of the present invention is embodied in right, instead of in described explanation, the present invention should be interpreted as and comprise all with the discrepancy in its equivalents.
Claims (7)
1. a cosmetic composition, is characterized in that, comprises Australia La Woer chrysanthemum (Raouliaaustralis) plumelet extract of tissue culture.
2. cosmetic composition according to claim 1, is characterized in that, relative to described composition total weight, the scope with 0.1 ~ 10 % by weight comprises Australia La Woer chrysanthemum plumelet extract of described tissue culture.
3. cosmetic composition according to claim 1, it is characterized in that, Australia La Woer chrysanthemum plumelet extract of described tissue culture is that use is selected from Australia La Woer chrysanthemum plumelet of more than one the solvent extraction tissue culture in pure water, the alcohol of C1 ~ C4 and the polyhydric alcohol of C1 ~ C5.
4. cosmetic composition according to claim 1, it is characterized in that, described cosmetic composition has the dosage form be selected from skin cream, toner/smoothing toner, cosmetic water, astringent lotion, emulsion, milky lotion, nutritional emulsions, massage cream, nourishing cream, hand cream, vanishing cream, sun screen, dual-purpose muffin, mascara, elite, nutrition elite, facial film, soap, clean skin foam, clean skin dew, cleansing cream, body lotion and bath gel.
5. a manufacture method for Australia La Woer chrysanthemum plumelet extract of tissue culture, is characterized in that, comprising:
A Australia La Woer chrysanthemum plumelet of tissue culture is formed the step of dipping solution by () being selected from dipping 3 ~ 30 days in more than one the Extraction solvent in pure water, the alcohol of C1 ~ C4 and the polyhydric alcohol of C1 ~ C5; And
B () filters described stain solution and obtains the step of Australia La Woer chrysanthemum plumelet extract of tissue culture.
6. the manufacture method of Australia La Woer chrysanthemum plumelet extract of tissue culture according to claim 5, it is characterized in that, described Extraction solvent is be selected from any one in the mixed solvent of ethanol, ethanol and 1,3 butylene glycol, ethanol and the mixed solvent of propylene glycol and the mixed solvent of ethanol and pentanediol.
7. the manufacture method of Australia La Woer chrysanthemum plumelet extract of tissue culture according to claim 5, is characterized in that, relative to the weight of Australia La Woer chrysanthemum plumelet of described tissue culture, described Extraction solvent has the weight ratio of 10 ~ 20 times.
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| KR10-2014-0083018 | 2014-07-03 | ||
| KR1020140083018A KR101618180B1 (en) | 2014-07-03 | 2014-07-03 | Cosmetic composition comprising an extract of the tissue-cultured raoulia australis shoot |
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| CN105310923B CN105310923B (en) | 2018-12-25 |
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| CN110382055A (en) * | 2017-01-09 | 2019-10-25 | 普兰特先进技术公司 | It revolves fruit mosquito grass roots and extracts preparation process and the utilization to cosmetics |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20020043840A (en) * | 2000-12-04 | 2002-06-12 | 서경배 | Method for stabilization of drug and decrease of skin-stimulation in the skin absorption having effect of removing wrinkle and protection wrinkle formation |
| EP2436757A2 (en) * | 2009-05-26 | 2012-04-04 | Unhwa Corporation | Plant stem cell derived from cambium of family asteraceae and method for the isolated culturing thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20020043840A (en) * | 2000-12-04 | 2002-06-12 | 서경배 | Method for stabilization of drug and decrease of skin-stimulation in the skin absorption having effect of removing wrinkle and protection wrinkle formation |
| EP2436757A2 (en) * | 2009-05-26 | 2012-04-04 | Unhwa Corporation | Plant stem cell derived from cambium of family asteraceae and method for the isolated culturing thereof |
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| H.J. CHOI ET AL: "Antiviral activity of raoulic acid from Raoulia australis against Picornaviruses", 《PHYTOMEDICINE》 * |
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| CN110382055A (en) * | 2017-01-09 | 2019-10-25 | 普兰特先进技术公司 | It revolves fruit mosquito grass roots and extracts preparation process and the utilization to cosmetics |
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| KR101618180B1 (en) | 2016-05-04 |
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