KR101618180B1 - Cosmetic composition comprising an extract of the tissue-cultured raoulia australis shoot - Google Patents

Abstract

The present invention relates to a cosmetic composition comprising a tissue cultured Raoulia australis shoot extract and a method of preparing the same, wherein the Raoulia asterrice extract obtained by the tissue culture of the present invention has antioxidative, It is excellent in whitening effect and can be usefully used in a cosmetic composition for skin protection and skin improvement.

Description

TECHNICAL FIELD The present invention relates to a cosmetic composition comprising a tissue-cultured Raoulia australis shoot extract,

The present invention relates to a cosmetic composition and a method for producing the same, and more particularly, to a cosmetic composition comprising a Laoliaostrillus sinensis extract having excellent antioxidant, anti-inflammatory and whitening effect and a method for producing the same.

Skin has the function of protecting the body from external stimuli. These external stimuli, that is, the environmental, physical and chemical factors as well as the physiological internal factors, cause the skin function to deteriorate, causing skin irritation, pigmentation diseases such as stain and dullness to external stimuli more than normal skin, Which causes skin aging.

Therefore, the current development of cosmetics is under continuous development to find new cosmetic materials that improve and protect skin, such as anti-inflammation, antioxidant and whitening, which are excellent in skin safety and can protect skin from various factors.

Among the ingredients that make up the cosmetics that are developed to protect the skin and purify the beautification are typically solubilizers, emulsifiers, preservatives, fragrances, sunscreens, and synthetic thickeners. . In addition, sebum, sweat, and fatty acids and proteins of cosmetic ingredients, which are produced in the body, may cause inflammation by reaction with various bacteria present in the skin.

Active oxygen in vivo is produced by various enzyme reactions and plays a very important role in the biosynthesis of physiologically active substances, immune function, and metabolism of drugs. However, if the homeostasis of the body is not maintained and overproduced, it may lead to injury in vivo. For example, free radicals and reactive oxygen species are major causes of skin troubles, inflammation, and skin aging by oxidizing or altering biomaterials such as cell membrane lipids, proteins, and nucleic acids to lower the function of skin cells.

Several factors are involved in the determination of human skin color, among which melanocytes, skin thickness, distribution of blood vessels and presence or absence of pigment in and out of the body are important. In particular, the most important factor is melanin, a melanin pigment produced by the action of various enzymes such as tyrosinase in melanocytes in the human body. Melanin pigment is well formed by the symptoms of skin pigmentation abnormalities such as spots, freckles, and pigmentation from external environmental factors such as UV exposure. In order to treat or alleviate excessive melanin pigmentation, various substances are used in cosmetics or pharmaceuticals. However, its use is limited due to insufficient whitening effect, safety to skin, formulation and stability problems in formulating in cosmetics.

Efforts are continuing to maintain healthy and beautiful skin while preventing such skin phenomenon. Recently, research efforts for skin improvement have been actively studied to develop materials using plant extracts that are free of side effects, are human-friendly, and can be safely used regardless of their constitution.

In general, functional cosmetics containing natural plant extracts can utilize any plant that grows in a cold climate, from a tropical rainforest climate, a condition in which plants can grow. Plants are adapted to their respective climate and grow through optimal biomechanics under those conditions. Therefore, the types and contents of effective substances such as secondary metabolites in the plant may be different depending on the sunshine, temperature, precipitation, etc. depending on the latitude in which the same species of plants grow, climate conditions such as acidity and fertility of the soil, have. In addition, it is difficult to overcome the disadvantages such as difficulty in satisfying the supply according to manufacturing and high processing cost. In order to improve these various difficulties, the development of cosmetic raw materials using plant culture technology is becoming more prominent.

On the other hand, Raoulia australis , a wild plant of the Asteraceae family, is native to New Zealand and Australia. Raulia australis grows in a matt form on a humid soil that is easily drained and has a low cold resistance and is mainly grown on the surface of highland gravel and rock, and has been used as a gypsy plant in garden landscaping. Raoulic acid contained in Raoulia australis is a 25-carbon terpene-based substance that inhibits the proliferation of viruses that cause sinusitis, otitis media, chronic bronchitis, aseptic meningitis, venous pancreatitis, myocarditis, etc. Respectively.

Raulia asterrisis, which has such an effect, is not cultivated locally due to its geographical location and climate change. Despite the increase in demand due to the known active ingredients, various effects have been revealed through research, but the scientific test results reported on the efficacy of cultured Raoulia asteris shoots are insufficient. No research results have been reported for application as a cosmetic ingredient.

The present invention provides a cosmetic composition comprising an extract of tissue cultured Raoulia australis shoots which is excellent in antioxidant, anti-inflammatory and whitening effect and further has a skin improving effect and a skin protecting effect, and a preparation method thereof .

The objects of the present invention are not limited to the above-mentioned objects, and other objects not mentioned can be clearly understood from the following description.

The cosmetic composition according to the present invention is characterized in that the tissue-cultured Raoulia < RTI ID = 0.0 > australis shoot s) extract as an active ingredient.

Here, it is preferable that the above-mentioned tissue-cultured Raoulia athrusts extract is contained in an amount of 0.1 wt% or more and 10 wt% or less based on the total weight of the cosmetic composition.

In particular, the Raoulia asteris shoot extract obtained by the above-mentioned tissue culture can be obtained by culturing the cultured Raouliastrosis shoots in one or more solvents selected from the group consisting of purified water, C1 to C4 alcohols, and C1 to C5 polyhydric alcohols . ≪ / RTI >

In addition, the cosmetic composition according to the present invention can be used as a skin lotion, a skin softener, a skin toner, an astringent, a lotion, a milk lotion, a nutrition lotion, a massage cream, a nutrition cream, a hand cream, a foundation, a makeup base, a twin cake, a mascara, Nutritional essence, pack, soap, cleansing foam, cleansing lotion, cleansing cream, body lotion and body cleanser.

In addition, the method for producing Raoulia australis shoot extract according to the present invention comprises: (a) cultivating a laurolactospora shoot obtained by cultivating a tissue, which comprises purified water, a C1 to C4 alcohol, and a C1 to C5 polyhydric alcohol At least one extraction solvent selected from the group consisting of: < RTI ID = 0.0 > 1, < / RTI > 3 to 30 days to form an immersion solution; And (b) filtering the immersed solution to obtain a culture of Raoulia asteris shoot extract.

The extraction solvent may be selected from the group consisting of ethanol; A mixed solvent of ethanol and 1,3-butylene glycol; A mixed solvent of ethanol and propylene glycol; And a mixed solvent of ethanol and pentylene glycol can be used.

Furthermore, it is preferable that the extraction solvent has a weight ratio of 10 times or more to 20 times or less of the weight of the cultured Raouliastrosis shoot.

The cosmetic composition according to the present invention is free from skin irritation, including antioxidant, anti-inflammatory and whitening effects, including Raoulia asteris shoot extract obtained by tissue culture, and is further effective for skin improvement and skin protection. The tissue cultured Raoulia australis shoots extract protects the skin from various external stimuli to provide a smooth and shiny skin, and furthermore, has an excellent effect in improving skin function.

While the present invention has been described in connection with certain embodiments, it is obvious that the invention is not limited to the disclosed embodiments, but, on the contrary, is intended to cover various modifications and similarities. It should be understood, however, that the invention is not intended to be limited to the particular embodiments, but includes all modifications, equivalents, and alternatives falling within the spirit and scope of the invention.

Hereinafter, the tissue cultured Raoulia < RTI ID = 0.0 > australis shoots) extract.

The inventors of the present invention have continued the study on applicability of cosmetics using plant culture technology, focusing on the evaluation of efficacy of tissue cultured Raoulia australis shoots obtained by tissue culture and completed the present invention. As a result, the inventors of the present invention have found that tissue cultured Raoulia australis shoots extract protects the skin from various external stimuli to make the skin smooth and radiant, and has a very good effect on skin function improvement.

The cosmetic composition according to the present invention preferably contains the extract of Raoulia asteris shoot at a concentration of 0.1 wt% or more and 10 wt% or less in terms of the total weight of the composition. When the above-mentioned range is satisfied, there is a significant effect, that is, it protects the skin from various external stimuli to make the skin soft and shiny, and helps to improve the skin function. Also, when considering the inherent odor and color of the extract in the final product, it is advantageous to include it in the range as described above.

The above-mentioned tissue-cultured Raoulia asteris shoot extract may be obtained by culturing the tissue-cultured Raouliastrosis shoots with at least one solvent selected from the group consisting of purified water, C1 to C4 alcohols, and C1 to C5 alkylene glycols It is preferable to use it for extraction.

Examples of products to which the cosmetic composition of the present invention can be added include various skin lotions, skin softeners, skin toners, astringents, lotions, milk lotions, nutrition lotions, massage creams, nutritional creams, hand creams, But are not limited to, base, twin cakes, mascara, essence, nutritional essence, pack, soap, cleansing foam, cleansing lotion, cleansing cream, body lotion and body cleanser. When the cosmetic composition of the present invention is prepared into a formulation of the above-mentioned form, it may contain various bases and additives necessary for the formulation of the formulation. The kinds and amounts of these components can be easily selected by those skilled in the art.

Hereinafter, a method of producing the Rauliostrosis shoot extract of the present invention in tissue culture will be described.

The present invention relates to a method for culturing a plant, comprising (a) culturing the cultured Raouliastrosis shoots in one or more extraction solvents selected from the group consisting of purified water, C1 to C4 alcohols, and C1 to C5 polyhydric alcohols for 3 to 30 days Depositing to form an immersion solution; And (b) filtering the immersion solution to obtain an extract of Raoulia asteris shoots cultured in a tissue culture.

The lauliaostrosis shoots cultured in the step (a) are preferably dried. Further, the deposition time in the step (a) is preferably 10 days to 20 days.

The extraction solvent is preferably used at a weight ratio (w / w) of 10 to 20 times as much as the weight of the dried cultured Raouliastrosis shoot.

The C1 to C4 alcohols may be methyl alcohol, ethyl alcohol, propyl alcohol, butyl alcohol and the like, preferably ethanol (ethyl alcohol). The C 1 to C 5 dicarcohol is preferably at least one selected from the group consisting of alkylene glycol, 1,3-butylene glycol, propylene glycol and pentylene glycol.

When the extraction solvent is used for the mixing of the alcohol and the alkylene glycol, the mixing ratio (w / w) thereof is preferably 6: 4 to 9: 1 (that is, the total mixing solvent of the alcohol and the alkylene glycol It is preferable that the content of alcohol is in the range of 60 wt% or more and 90 wt% or less. When the content of the mixed solvent of the alcohol and the alkylene glycol is within the above-mentioned range, it has a more efficient polarity for extracting only the desired affinity. Here, the concentration of the alcohol used in the mixed solvent of the alcohol and the alkylene glycol is preferably 60% to 100%.

In addition, the extraction solvent may be a mixture of the purified water and the alkylene glycol, and the mixing ratio thereof is preferably 3: 7 to 1: 9 (i.e., the total weight of the purified water and the total mixed solvent of the alkylene glycol , It is preferable that the weight of the alkylene glycol is 70% by weight or more and 90% by weight or less.

It is preferable that the extraction solvent is ethanol for a single use daily. A mixed solvent of an alcohol and an alkylene glycol may be used in the case of mixing daily, and a mixed solvent of ethanol, 1,3-butylene glycol, ethanol and propylene glycol or ethanol and pentylene glycol may be used. More preferably, a mixed solvent of ethanol and 1,3-butylene glycol or a mixed solvent of ethanol and pentylene glycol can be used.

In addition, the step (b) preferably further comprises a step of removing the extraction solvent.

The present invention provides a cosmetic composition comprising as an active ingredient a Rauliostrosis shoot extract obtained by the above-mentioned tissue culture and obtained by the above-described manufacturing process. Herein, the above-mentioned tissue culture-treated Rauliostrosis shoot extract is treated with human fibroblasts to examine the cytotoxic effect on skin irritation, and the above-described tissue culture-treated Raoulia asteris shoot extract is treated with human fibroblasts A step of examining the cytoprotective effect on the external stimulus, a step of examining the anti-inflammatory effect by inhibiting the Lpoxgenase activity and the inhibitory effect on the nitric oxide production of the Raoulia asteris shoot extract obtained from the tissue culture, A step of examining the antioxidative effect of the SUPEROXIDE ANION scavenging activity and the HYDROXY RADICAL scavenging activity and the skin whitening improvement effect by the inhibition of tyrosinase, The efficacy of the extract of Laulia austr.

Furthermore, the cosmetic composition according to the present invention is not limited to the formulations exemplified below, and can be a formulation known in the art. The cosmetic composition of the present invention may be compounded with other components, which are usually added to cosmetics, in addition to the above-mentioned extract within a range that does not impair the objects and effects of the present invention.

Hereinafter, production examples, test examples and formulation examples according to the present invention will be described in more detail, but they are for the purpose of illustrating the present invention and are not intended to limit the scope of the present invention.

[ Production Example 1  To Production Example 4 , And Comparative Preparation Example 1  To Comparative Production Example 4 ]: Tissue-cultured Raoulia  Australs Shinshu  Preparation of extract

The tissue-cultured Raoulia asteris shoots and the natural laurel australis were washed with water, gently drained and then dried and sieved. Herein, " Tissue-cultivated Raoulia australis shoots " used in the present preparations is derived from Laoliostratic seeds imported from New Zealand by inducing shoots using plant tissue culture technology under aseptic conditions and culturing them in a nutrient solution , Which was supplied by the Center for Advanced Horticultural Technology Development, Chungbuk National University

The prepared tissue-cultured Raoulia asteris sinensis and Raoulia australis were aged by immersion for 2 weeks in a mixed solvent present in the contents shown in Table 1. Thereafter, the mixture was filtered through a filter paper (5C, 185 mm, available from Toyoroshi Kaisha, Ltd., Japan), and the ethanol was evaporated to obtain a filtrate, which was then subjected to tissue culture to obtain a Raoulia asteris shoot extract and a Raoulia asteris extract.

[ Test Example 1 ]: Tissue-cultured Raoulia Australs Shinshu  Cytotoxicity measurement of extract

The safety was evaluated through cytotoxicity tests for Preparation Example 1 and Comparative Preparation Example 1.

[Cell culture]

CCD-1064sk human fibroblasts purchased from ATCC were cultured in Iscove's modified Dulbeco's medium (IMDM) supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin ), The cells were cultured in a 10-cm cell culture dish to a confluency of 70% to 80% in a 5% CO ₂ incubator at 37 ° C in a 10 ml medium. The medium was changed twice a week, and the cells that reached confluence were trypsinized using trypsin-EDTA, and then subcultured. Human fibroblasts cultured in the above were divided into 96-well plates (96-multiwell plate) at 1 × 10 ⁴ cells / well and cultured for 24 hours. The cultured cells were cultured in a medium supplemented with 2% FBS, and the cultured Raoulia asteris shoot extract, Preparation Example 1, and Comparative Preparation Example 1, which is a Raoulia asteris extract, And incubated in a CO ₂ incubator for 48 hours.

[Test Example 1-1]: Measurement of cytotoxic effect (MTT assay)

MTT stock solution (5 mg / ml) dissolved in PBS (phosphate buffered saline) was diluted 10-fold in a fresh medium, and 100 μl of this solution was added to each well of the cells at 37 ° C For 4 hours. After the reaction, the living cells were detected by the dehydrogenase present in the mitochondria, and the yellow water-soluble substrate MTT [3- (4,5-dimethylthiazol-2-yl) -2,5- diphenyltetrazolium- (MTT-formazan) crystals. Therefore, the cell viability can be measured by measuring the amount thereof. After removing the culture supernatant, 200 μl of DMSO was added to each well to dissolve the MTT-positive crystals produced in the cells, and then the absorbance of the test solution was measured with an absorption spectrophotometer (UVmax, Molecular Device, USA) The OD value at a wavelength of 540 nm as a test filter and the OD value at a wavelength of 630 nm as a reference filter were measured to obtain the absorbance of the difference value (refer to The EMBO Journal (2002) 21, 2407 -2417 et al.). Cytotoxicity was expressed as a percentage of absorbance.

* Cell survival rate (%) = (absorbance of the sample treated group / absorbance of the untreated group) x 100

The standard deviation was calculated by calculating the average of the absorbance of the untreated group and the absorbance of the sample group on the basis of the raw data.

[Test Example 1-2]: Measurement of cytotoxic effect (Neutral Red assay)

To each well of the cultured cells, 50 μg / ml of Neutral Red (Sigma-Aldrich, USA) was added and reacted for 3 hours. Neutral red is enriched in lysosomes of fully live or undamaged cells. After the reaction, the cells were fixed with 1.0% formalin / 1.0% potassium chloride, and then 1.0% acetic acid / 50% ethanol solution was added to extract the neural red. The cytotoxicity of 590-630 nm was measured by absorbance spectrophotometer (UVmax, Molecular Device, USA), and the cytotoxicity was expressed as a percentage of the absorbance. (lot) three times.

* Cell survival rate (%) = (absorbance of the sample treated group / absorbance of the untreated group) x 100

The standard deviation was calculated by calculating the average of the absorbance of the untreated group and the absorbance of the sample group on the basis of the raw data.

As shown in Table 2 and Table 3, the MTT ₅₀ , NR ₅₀ Value is not less than 1%, which is a safe raw material as compared to Comparative Production Example 1. [

[ Test Example 2 ]: Tissue-cultured Raoulia Australs  Measurement of cell protection effect of extract

A cytoprotective effect test for SDS (sodium dodecyl sulfate) cytotoxicity against Preparation Example 1 and Comparative Preparation Example 1 was carried out.

Human fibroblasts were cultured in IMDM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37 ° C in a 5% CO ₂ incubator. Cultured human fibroblasts were each divided into 96-well plate at 1 × 10 ⁴ cells / well and cultured for 24 hours. The medium of the cultured cells was replaced with 2% FBS medium, and 0.002% of SDS (sodium dodecyl sulfate) was pretreated for 1 hour. Then, Production Example 1 and Comparative Production Example 1 were diluted in the medium in each treatment concentration, Lt; RTI ID = 0.0 > CO ₂ < / RTI > incubator. To the cultured cells, MTT storage solution (5 mg / ml) dissolved in PBS (phosphate buffered saline) was diluted 10-fold in a new medium, and 100 μl of this solution was added to each well of the cells. Lt; / RTI > After the reaction, the living cells were detected by the dehydrogenase present in the mitochondria, and the yellow water-soluble substrate MTT [3- (4,5-dimethylthiazol-2-yl) -2,5- diphenyltetrazolium- And the cell viability can be measured by measuring the amount thereof. After removing the culture supernatant, 200 μl of DMSO was added to each well to dissolve the MTT-positive crystals produced in the cells, and then the absorbance of the test solution was measured with an absorption spectrophotometer (UVmax, Molecular Device, USA) The OD value at a wavelength of 540 nm, which is a reference filter, and the OD value at a wavelength of 630 nm, which are reference filters, were measured, and the absorbance of the difference value was obtained (References The EMBO Journal (2002) 21, 2407-2417 et al.). The cytoprotective effect was expressed as a percentage of the absorbance.

* Cell survival rate (%) = (absorbance of the sample treated group / absorbance of the untreated group) x 100

The standard deviation was calculated by calculating the average of the absorbance of the untreated group and the absorbance of the sample group on the basis of the raw data.

As shown in Table 4, it can be seen that the cytoprotective effect of Preparation Example 1 of the present invention promotes the cytoprotective effect against external stimuli,

[ Test Example  3]: Tissue-cultured Raoulia Australs Shinshu  Measurement of anti-inflammatory effect of extract

The anti-inflammatory effect test was carried out by measuring the activity of lipoxygenase and the production of nitric oxide in Production Example 1 and Comparative Production Example 1.

The inflammatory response is the biological response to external stimuli to maintain homeostasis in our bodies. The pathway of the enzyme that produces inflammation by stimulation is the Ripoxygenase activity pathway and the Cycloxygenase activity pathway. Lipoxygenases catalyze the hydroperoxidation of unsaturated fatty acids to produce fatty hydroperoxides, which in turn produce decomposition products such as epoxide, hydroxyl, ketone, aldehyde, and the like. Lipoxygenase produces a variety of chemical mediators from arachidonic acid biosynthesized from linolenic acid, such as in vivo inflammation reactions and allergies. Therefore, the substance inhibiting the lipoxygenase inhibits the production of chemical mediators involved in various inflammatory reactions and allergies. Nitric oxide (NO), which is produced by activation of enzymes that mediate inflammatory reactions, plays a pivotal role in the immune response, but overexpressed nitric oxide causes an inflammatory reaction and an abnormal immune system.

[Test Example 3-1]: Measurement of nitric oxide production inhibition ability

The degree of nitric oxide (NO) production was measured by the method of Green et al. (Green LC, Wagner DA, Glogowski J, et al. 1982. Analysis of nitrate, nitrite, and [15N] nitrate in biological fluids Anal Biochem 126: 131-138. ) Was used to measure the amount of nitrate produced in the medium, which is an indicator of nitric oxide (NO) production. After incubation for 30 min, LPS (lipopolysaccharide, 1 μg / mL) was added to the culture supernatant of Raw264.7 macrophage cultured for 24 hours, and 50 μL of 1% sulfinilamide (5% phosphoric acid And 50 μL of 0.1% naphthylenediamine dihydrochloride were added. After reacting at room temperature for 10 minutes, the absorbance was measured at 540 nm. The control group for the anti-inflammatory effect test was an experimental group treated with LPS alone.

* NO inhibition rate (%) = (NO level of control group - NO level of each extract / NO level of control group) × 100

The average was calculated based on the raw data and the standard deviation was calculated.

As shown in Table 5, the inhibitory effect of NO production on oxidative stress caused by LPS was about 1.5 to 2 times that of Comparative Preparation Example 1, as shown in Table 5 above.

[Test Example 3-2]: Lipoxygenase activity inhibition assay

Inhibition of the activity of lipoxygenase, which is a representative enzyme of inflammation for external stimuli, was performed. 0.1 ml of a sample to be measured and 0.9 ml of a lipoxygenase (Sigma-aldrich, USA) were mixed in 1 ml of 1 mM linolenic acid (Sigma-aldrich, USA) and subjected to voltexing at 25 ° C for 10 minutes. After that, 0.5 ml of 20% trichloroacetic acid and 1 ml of 0.6% thiobarbituric acid were added, and the mixture was reacted with boiling water for 10 minutes. Then, the mixture was ice-cooled for 2-3 minutes on ice water, ) Was vortexed for 20 seconds or more and then centrifuged at 3500 rpm for 5 minutes. The OD value was measured at 513 nm using a UV spectrophotometer, and the inhibition rate of the lipoxygenase activity was calculated according to the following equation (2) The results are shown in Table 6 below. The measurement was carried out three times in three batches, and the standard deviation was calculated by calculating the average of the absorbance based on the raw data.

As shown in Table 6, the Rauliostrosis shoot extract of Tissue Culture Example 1 showed a concentration-dependent inhibition of the lipoxygenase as compared with the untreated control group, and the activity of the enzyme producing inflammation It was confirmed that the activity inhibition effect was excellent.

[ Test Example  4] Tissue-cultured Raoulia Australs Shinshu  Determination of antioxidative effect of extract

Antioxidative effect tests were carried out for Preparation Example 1 and Comparative Production Example 1.

[Test Example 4-1] Measurement of anti-hydroxyl radical activity (%) by DPPH (1,1-diphenyl-2-picrylhydrazyl)

The reaction with active oxygen is almost a radical reaction and involves an autoxidation process. Antioxidants are responsible for blocking the autoxidation reaction in vivo. As the ability to give electrons, the greater the reducing power, the stronger the antioxidant. A reagent for measuring the reducing power of an antioxidant is 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical. DPPH is present as a radical in the stabilized structure of the nitrogen center in the compound. The maximum absorption at 517 nm is shown, and the absorption disappears at 517 nm. DPPH (D9132, SIGMA) was dissolved in a mixed solvent of 6: 4 (v / v) of methanol and distilled water, and the DPPH solution was adjusted to have an absorbance of about 0.6 at 513 nm. 2 ml of the DPPH solution was added to 1 ml of the diluted sample, and mixed. After standing at room temperature for about 1 hour, the absorbance was measured at 513 nm using an absorption spectrophotometer (UVmax, Molecular Device, USA). The results are shown in Table 7 below.

As shown in Table 7, Production Example 1 showed excellent antioxidative effect.

[Test Example 4-2] Measurement of anti-hydroxyl radical activity (%) by ABTS radicals

The total antioxidant capacity of the extract was measured by Miller et al. (1993) method. 2-ethyl-benzothiazoline-6-sulfonate was dissolved in distilled water at a concentration of 7 mM and potassium persulfate was added thereto to a final concentration of 2.45 mM to obtain an ABTS cation cation radical. The same amount of ABTS and potassium persulfate was mixed and allowed to stand in the dark at room temperature for 12 hours. The solution was diluted with ethanol and adjusted to an absorbance of 0.7 at 734 nm. The absorbance difference was determined as a percentage (%) by measuring the absorbance at 734 nm by a spectrophotometer 6 minutes after mixing 10 시 of the plant extract sample, 190 AB of ABTS solution (Solution) and 50 증 of distilled water, Respectively.

Erase rate (%) = (B-A) / B x 100

(B: absorbance of the group of the non-extractant group, and A: absorbance of the group of the extractant group)

As shown in Table 8, the Laoliaostratus extract (Preparation Example 1) obtained by tissue culture according to the present invention showed an antioxidative effect superior to Comparative Preparation Example 1.

[ Test Example  5]: Tissue culture Raoulia Australs Shinshu  Extract Tyrosinase  Measurement of inhibition of activity and melanin synthesis

[Test Example 5-1] Tyrosinase Inhibition Test

Production Example 1 and Comparative Production Example 1 To investigate whitening activity, a tyrosinase inhibiting activity test was conducted. For the experiment, the tyrosinase enzyme solution was prepared by dissolving mushroom tyrosinase (T-3824, 1530 U / mg, Sigma) in phosphate buffer (pH 6.5) so as to have a concentration of 1000 U / - tyrosine (L-tyrosine, 45160-0410, Junsei chemical co., Ltd.) was dissolved in phosphate buffer (pH 6.5) so as to be 1.5 mM.

Preparation Example 1 and Comparative Preparation Example 1 were diluted in the buffer solution by concentration to prepare a sample solution. 170 μl of the test solution and 10 μl of Tyrosinase enzyme solution were placed and left at 37 ° C for 10 minutes. After incubation at 37 ° C for 10 minutes, 20 μl of the substrate solution was added, and the mixture was allowed to stand in ice for 5 minutes. Absorbance was measured at an absorbance spectrophotometer (UVmax, Molecular Device, USA) at a wavelength of 490 nm. The absorbance measured above was substituted into the following equation to calculate the inhibitory activity of tyrosinase, and it is shown in Table 9 below. The solution prepared by adding phosphate buffer (pH 6.5) instead of the sample solution was used as a blank test solution, and a phosphate buffer solution (pH 6.5) was added in place of the substrate solution.

≪ Equation &

[Test Example 4-2]: Measurement of inhibition of melanin synthesis according to the concentration of Rauliaostrissis shoot extract obtained by tissue culture

The whitening effect was measured using B-16 melanoma cells (Korean Cell Line Bank) for Production Example 1 and Comparative Production Example 1. Measuring method, derived from the black mice B-16 melanoma cells with 10% FBS is added in 2㎖ to 2 x 10 ⁴ cells / well density in 6-well tissue culture with DMEM containing (GIBCO., USA) and the plate 5% CO ₂ , 37 캜 for 24 hours. After cultivation, the medium was removed and replaced with DMEM containing 10% FBS, 2 μM α-MSH (Sigma, USA, Melanocyte stimulating hormone) and 2 mM theophylline (Sigma, USA, theophylline) Stratified shoot extract was diluted with the same medium and then added to each well. Then, the cells were cultured at a temperature of 5% CO ₂ and 37 ° C until the cells were about 80% or more at the bottom of the wells. After the culture, the medium was removed and washed with PBS (Sigma, USA, Phosphate buffered saline) and treated with trypsin to recover the cells. The recovered cells were centrifuged at 5000 rpm for 10 minutes and the supernatant was removed. Cells from which the supernatant was removed were dried in a thermostat at 60 ° C for 24 hours, and 1N NaOH was added to dissolve the melanin in the cells. Absorbance of dissolved melanin was measured at a wavelength of 490 nm using an absorption spectrophotometer (Uvmax, Molecular Device, USA).

≪ Equation &

Melanin formation inhibition rate (%) = (1-A / A ') x 100

(A: test group absorbance, A ': control group absorbance)

As shown in Tables 9 and 10, Production Example 1 inhibited tyrosinase activity in a concentration-dependent manner, and was superior to Comparative Preparation Example 1 in inhibiting intracellular melanin production.

[ Example 1  To Example 2 ] Tissue cultured Raoulia Australs Shinshu  Preparation of Essences Containing Extracts

Essences containing Preparation Example 1 were prepared according to the composition of Table 11 below.

In Table 11, component A was sufficiently dispersed and wetted to be a homogeneous gel phase, and component B was added to neutralize it. The component C was added to the component (A + B) and homogeneously stirred to be solubilized. Then, the component D was added at room temperature and stirred so as to be uniformly distributed.

[ Example  3 to 4]: Tissue culture Raoulia Australs Shinshu  Flexible longevity including extract ( skin )

Examples of the softening longevity (skin) in the cosmetic composition containing Preparation Example 1 are shown in Table 12 below.

[ Example  5 to 6]: Tissue-cultured Raoulia Australs Shinchosha extract  Manufacture of cream containing

The formulation of cream in cosmetic composition containing Preparation Example 1 is shown in Table 13 below.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the invention. It is therefore to be understood that the embodiments of the invention described herein are to be considered in all respects as illustrative and not restrictive, and the scope of the invention is indicated by the appended claims rather than by the foregoing description, Should be interpreted as being included in.

Claims (7)

And a tissue-cultured Raoulia australis shoots extract,
Characterized in that the Raoulia asteris shoot extract obtained by the above-mentioned tissue culture is contained in an amount of not less than 0.1% by weight and not more than 10% by weight based on the total weight of the composition comprising the above-mentioned tissue-cultivated Rauliostratus shoot extract. Cosmetic composition.
delete The method according to claim 1,
The lauliaostrosis shoot extract obtained by the above-mentioned tissue culture is obtained by culturing the tissue-cultured Raouliastrosis shoots using at least one solvent selected from the group consisting of purified water, C1 to C4 alcohols, and C1 to C5 polyhydric alcohols And extracting the cosmetic composition.
The method according to claim 1,
Makeup Base, Twin Cake, Mascara, Essence, Nutrition Essence, Pack, Soap, Cleansing Foam, Lotion, Milk Lotion, Nourishing Lotion, Massage Cream, Nourishing Cream, Hand Cream, Foundation, Makeup Base, Twin Cake, Skin Toner, A cleansing lotion, a cleansing cream, a body lotion and a body cleanser.
delete delete delete