KR101093863B1 - A cosmetic composition comprising an extract of the tissue cultured morinda citirifolia callus extracts and a preparation method of the extract - Google Patents

Abstract

The present invention relates to a cosmetic composition comprising a tissue cultured noni cultured cell (Tissue cultured Morinda citirifolia callus) extract, and a method of manufacturing the same, which is useful for a cosmetic composition for preventing skin aging, improving skin light, and protecting skin against skin damage. Can be used.

Cosmetics, Noni, Tissue Culture, Antioxidant, Cell Protection, Whitening, Wrinkles

Description

A cosmetic composition comprising a tissue cultured noni cultured cell extract and a method for producing the tissue cultured noni cultured cell extract {A COSMETIC COMPOSITION COMPRISING AN EXTRACT OF THE TISSUE CULTURED MORINDA CITIRIFOLIA CALLUS Extra

The present invention relates to a cosmetic composition comprising a tissue cultured Morinda citirifolia callus extract excellent in cell protection, anti-oxidation, anti-wrinkle and whitening effects, and a method for preparing the tissue cultured noni culture cell extract.

Skin protects the body from external stimuli. These external stimuli, that is, environmental, physical and chemical stimuli, deteriorate skin function and cause skin aging. There are two main causes of skin aging: endogenous aging and exogenous aging. Endogenous aging is an action of genetic factors involved in skin cells in accordance with the biological clock as the body ages, and aging progresses as skin regeneration rate and collagen synthesis become slower and slower. Exogenous aging is when aging is caused by external factors such as skin exposure, pollution, free radicals and other stresses. Exogenous aging, unlike endogenous aging, thickens the skin and causes a lot of pigmentation.

As the aging progresses, the dermal tissue of the skin is weakened and the degree of skin cell binding is significantly reduced. In addition, the amount of collagen and elastin involved in the elasticity of the skin is drastically reduced, the shape of the face gradually changes, and the dryness of the skin becomes very severe, resulting in rough skin and deep wrinkles. In addition, as metabolism decreases, blood is not supplied properly to the skin, making the skin look dull. Efforts have been made to maintain healthy and beautiful skin while preventing such skin phenomenon. Recently, research efforts to improve skin have been actively studied for developing materials using plant extracts that have no side effects, are friendly to human beings, and can be used safely regardless of constitution.

In general, functional cosmetics containing natural plant extracts can use any plant that grows in cold, cold climates from the rainforest climate, which is the condition under which plants can grow. Plants adapt to each climate and grow under the optimal biomechanisms under those conditions. Therefore, even the same species of plants may have different types and contents of active ingredients such as secondary metabolites in plants by climatic conditions such as sunshine, temperature, precipitation, soil acidity, fertility, soil, fertility, etc. have. In addition, it is difficult to meet the disadvantages, such as difficult to meet the supply according to the manufacturing, high processing costs. In order to improve these various difficulties, the development of cosmetic raw materials used by plant culture technology is emerging.

On the other hand, Noni ( Morinda Citrifolia ), an evergreen shrub of the horned pod , has been widely used as a traditional medicine in Polynesia, the South Pacific Islands, Taiti, Hawaii, Malaysia, and China for 2000 years, and is known to have a wide range of effects on diseases. have. Also, in Korea, it is recorded as Hae-gaeok and Paeokcheon in the agreement and has been used since ancient times. Noni is a tree that grows deeply rooted in volcanic soil, ranging from small bushes to trees about 9 meters high. It blooms beautiful and fragrant white flowers all year round. When the fruit ripens, it has a unique smell and taste, and the fruit contains a significant amount of fiber, which resembles potatoes about 10cm long. Noni is a traditional medicine that has been used by Polynesians for many years as a folk remedy, and all parts of the tree, including fruit, leaves, roots, stems, and seeds, have been used. The efficacy of these noni has been found in many studies. Noni is an excellent antioxidant that strengthens the immune system and helps remove harmful oxygen from the body. It purifies blood, heals damaged areas, and enhances cellular function. In addition, it has been proven to have more than 100 medical benefits, including stabilizing blood pressure, lowering cholesterol and stabilizing the mind and body. However, the reported scientific experiments on the efficacy of tissue cultured Noni callus are insufficient. In addition, there have been no reports on the application of cosmetic ingredients. Tissue cultured Morinda citirifolia callus (tissue cultured Morinda citirifolia callus) used in this experiment means that the callus is derived from the leaves of Noni by using plant tissue culture technology and cultured in nutrient solution.

Accordingly, the present inventors have completed the present invention by continuing to study the applicability as a cosmetic using plant culture technology, mainly on the evaluation of the efficacy effect of tissue cultured Morinda citirifolia callus extract.

An object of the present invention is to provide a cosmetic composition comprising a tissue cultured Morinda citirifolia callus extract having a cytoprotective , antioxidant, anti-wrinkle and whitening effect and a method for producing the same.

The present invention provides a cosmetic composition comprising a tissue cultured noni cultured cells (tissue cultured Morinda citirifolia callus) extract.

In addition, the present invention a) tissue cultured noni cultured cells (Tissue cultured Morinda citirifolia callus) 3 to 30 in at least one extraction solvent selected from the group consisting of purified water, C1 to C4 alcohol and C1 to C5 alkylene glycol. Depositing for days to form an immersion solution; And b) provides a method for producing a tissue cultured noni cultured cell (tissue cultured Morinda citirifolia callus) extract comprising the step of obtaining the noni cultured cell cultured by filtration of the deposition solution.

Since the cosmetic composition of the present invention uses a natural extract, it is free from skin irritation, and has a cytoprotective effect, antioxidant, anti-wrinkle and whitening effect, and thus is very effective in preventing skin aging, skin improvement and protection.

Hereinafter, the present invention describes a cosmetic composition comprising an extract of tissue cultured Morinda citirifolia callus.

The cosmetic composition of the present invention contains natural tissue cultured noni cultured cell culture (Tissue cultured Morinda citirifolia callus) extract, protects the skin from various external stimuli, makes the skin soft and shiny, and helps improve skin function.

Here, tissue cultured Morinda citirifolia callus (tissue cultured Morinda citirifolia callus) used in the present invention was used that was provided by Chungbuk National University Advanced Horticultural Technology Development Research Center.

The cosmetic composition of the present invention preferably comprises 0.1 to 10.0 wt% of tissue cultured Morinda citirifolia callus extract based on the total weight of the composition. If the above-mentioned range is satisfied, a significant effect, that is, protects the skin from various external stimuli, makes the skin soft and shiny, and helps to improve skin function. In addition, when considering the intrinsic smell, color, etc. of the extract in the final product is advantageously included in the above range.

The tissue cultured noni cultured cells (Tissue cultured Morinda citirifolia callus) extract is a tissue cultured noni cultured cells using at least one solvent selected from the group consisting of purified water, C1 to C4 alcohol and C1 to C5 alkylene glycol. It is preferable to extract by.

As a product to which the cosmetic composition of the present invention can be added, for example, various skin lotions, skin softeners, skin toners, astringents, lotions, milk lotions, nutrition lotions, massage creams, nutrition creams, hand creams, foundations, Makeup base, twin cake, mascara, essence, nutrition essence, pack, soap, cleansing foam, cleansing lotion, cleansing cream, body lotion and body cleanser, but is not limited thereto. When the cosmetic composition of the present invention is prepared in a formulation of the form described above, it may contain various bases and additives necessary and appropriate for the formulation of the formulation. In addition, the type and amount of these components can be easily selected by those skilled in the art.

Hereinafter, a method of preparing a tissue cultured Noni cultured cell (Tissue cultured Morinda citirifolia callus) extract of the present invention.

In addition, the present invention a) tissue cultured noni cultured cells (Tissue cultured Morinda citirifolia callus) 3 ~ in one or more extraction solvents selected from the group consisting of purified water, C1 to C4 alcohol and C1 to C5 alkylene glycol. Depositing for 30 days to form an immersion solution; And b) filtering the immersion solution to obtain a tissue cultured Morinda citirifolia callus extract.

In step a), the tissue cultured noni cultured cells (tissue cultured Morinda citirifolia callus) is preferably in a dried state. The deposition time is preferably 10 to 20 days.

The extractant is preferably used in an amount of 10 to 20 times (w / w) based on the weight of the dried tissue cultured Noni culture cells (tissue cultured Morinda citirifolia callus).

The alcohol of C1 to C4 is preferably ethanol, and the alkylene glycol of C1 to C4 is preferably one or more selected from the group consisting of 1,3-butylene glycol, propylene glycol and pentylene glycol.

In addition, when the extraction solvent is a mixed solvent of the alcohol and alkylene glycol, it is preferable that their mixing ratio (w / w) is 6: 4 to 9: 1. In the case of the mixed solvent of the alcohol and the alkylene glycol, when the content is in the above-described range, it has a more efficient polarity to extract only the desired useful components. Here, the concentration of the alcohol used in the mixed solvent of the alcohol and alkylene glycol is preferably from 60 to 100%.

In addition, the extraction solvent may be a mixed solvent of the purified water and the alkylene glycol, the mixing ratio thereof is preferably 3: 7 to 1: 9.

When the extraction solvent is a single solvent, preferably ethanol, in the case of a mixed solvent, a mixed solvent of alcohol and alkylene glycol, preferably ethanol and 1,3-butylene glycol, ethanol and propylene glycol or ethanol and pen It is a mixed solvent of styrene glycol, More preferably, it is a mixed solvent of ethanol and 1, 3- butylene glycol or ethanol and pentylene glycol.

The step b) preferably further comprises the step of removing the extraction solvent.

The tissue cultured Noni cultured Morinda citirifolia callus extract of the present invention is treated with the tissue cultured Noni cultured Morinda citirifolia callus extract on human fibroblasts to investigate the cytotoxic effect on skin irritation. In the step, the tissue cultured noni cultured cells (Tissue cultured Morinda citirifolia callus) extract to the human fibroblasts to investigate the cytoprotective effect on external stimulation, the tissue cultured noni cultured cells (tissue cultured Morinda citirifolia callus) The extracts are treated with human fibroblasts to investigate the anti-aging effect of collagen biosynthesis and the skin whitening improvement effect of tyrosinase inhibition. Can be.

Hereinafter, the present invention will be described in more detail with reference to Preparation Examples, Test Examples, Examples, and the like. However, the production examples, test examples, examples, and the like described below are for illustrating the present invention, and the present invention is not limited thereto and may be variously modified and changed.

Preparation Example 1 to Preparation Example 4, Comparative Preparation Example 1 to Comparative Preparation Example 4: Tissue cultured noni cultured cells (tissue cultured Morinda citirifolia  callus) Preparation of extract

Tissue cultured noni cultured cells (Tissue cultured Morinda citirifolia callus) and Noni (Morinda citrifolia ) were each washed with water to remove the water with gauze, and then dried and prepared. The tissue cultured Noni culture cells (tissue cultured Morinda citirifolia callus) and Noni (Morinda citrifolia ) were prepared by immersing for 2 weeks in a mixed solvent present in the content shown in Table 1 to the content shown in Table 1. Thereafter, the resultant was filtered on filter paper (5C, 185 mm commercially available from Toyoroshi Kaisha, Ltd., Japan), and ethanol was evaporated to obtain tissue cultured Morinda citirifolia callus extract and Noni (Morinda citrifolia ) extract in the filtrate state. .

Noni cultured cells in dried tissue culture (g) Dried Noni (g) Extraction solvent (g) Type (mixing ratio) content Preparation Example 1 20 - b-1: b-2 = 7: 3 980 Preparation Example 2 20 - b-1: b-2 = 6: 4 980 Preparation Example 3 20 - b-1: b-3 = 7: 3 980 Production Example 4 20 - b-1: b-3 = 6: 4 980 Comparative Production Example 1 - 20 b-1: b-2 = 7: 3 980 Comparative Production Example 2 - 20 b-1: b-2 = 6: 4 980 Comparative Production Example 3 - 20 b-1: b-3 = 7: 3 980 Comparative Production Example 4 - 20 b-1: b-3 = 6: 4 980

b-1: 70% ethanol b-2: 1,3-butylene glycol

b-3: propylene glycol

Test Example 1: tissue cultured noni tissue cells Morinda citirifolia  callus) measurement of cytotoxicity of extracts

Safety was evaluated through the cytotoxicity test for Preparation Example 1 and Comparative Preparation Example 1.

Cell culture

CCD-1064sk human fibroblasts purchased from ATCC were used with Iscove's modified Dulbeco's medium with 10% fetal calf serum (FBS) and 1% penicillin-streptomycin. ) Were cultured in a 10 cm cell culture dish with 10 ml of medium at 37 ° C. in a 5% CO ₂ incubator so that the cells were 70-80% confluent. The medium was changed twice a week, and the cells reached the confluence were maintained by passage culture after trypsinization using trypsin-EDTA. Human fibroblasts cultured above were divided into 96 × multiwell plates at 1 × 10 ⁴ cells / well and incubated for 24 hours. In the culture medium by replacing the medium of the cultured cells in 2% FBS medium and the tissue culture by say cell culture (tissue cultured Morinda citirifolia callus) extract of Preparation Example 1 and noni (Morinda citrifolia) extract of Comparative Production Example 1 Each concentration After dilution treatment, the cells were incubated in a CO ₂ incubator for 48 hours.

Test Example 1-1: Measurement of Cytotoxic Effect (MTT assay)

To the cultured cells, MTT stock solution (5 mg / ml) dissolved in PBS (phosphate buffered saline) was diluted 10-fold in fresh medium, and 100 µl of each solution was added to each well of the cell at 37 ° C. The reaction was carried out for 4 hours. After the reaction, the living cells were dehydrated by the dehydrogenase present in the mitochondria and the yellow water-soluble substrate MTT [3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium-bromide] was insoluble in water. Since it is reduced to (MTT-formazan) crystals, the amount of this can be measured to measure cell viability. Remove the culture supernatant and add 200 μl of DMSO to each well to dissolve the MTT-formenic crystals produced in the cells, and then use the absorption spectrophotometer (UVmax, Molecular Device, USA) to filter the maximum absorbance of the formazin. The absorbance of the difference value was obtained by measuring the OD value at a wavelength of 540 nm as a test filter and a 630 nm wavelength as a reference filter (The EMBO Journal (2002) 21, 2407). -2417 and many more). Cytotoxicity is expressed as a percentage of absorbance.

※ Cell survival rate (%) = (Sample absorbance / untreated group absorbance) × 100

The standard deviation was obtained by calculating the average of the absorbance of the untreated group and the absorbance of the sample treated group based on raw data.

MTT assay OD Mean ± SD
(% of control) Sample concentration (W / W%) Control group One 0.5 0.1 0.05 0.01 Preparation Example 1:
Tissue Cultured Noni Culture Cells 0.404 ± 0.09
(100.0) 0.383 ± 0.013
(94.80) 0.405 ± 0.011
(100.25) 0.428 ± 0.008
(105.94) 0.436 ± 0.014
(107.92) 0.445 ± 0.010
(110.15) Comparative Production Example 1:
Noni 0.404 ± 0.09
(100.0) 0.371 ± 0.017
(91.83) 0.408 ± 0.012
(100.99) 0.424 ± 0.013
(104.95) 0.430 ± 0.009
(106.44) 0.441 ± 0.011
(109.16)

Test Example 1-2: Measurement of Cytotoxic Effects (Neutral Red assay)

Neutral Red (Neutral Red, Sigma-Aldrich, USA) was added to the cells after the culture was incubated for 3 hours. Neutral Red is concentrated in the lysosomes of completely living or intact cells. After the reaction, cells were fixed with 1.0% formalin / 1.0% potassium chloride and then neutral red was extracted by adding 1.0% acetic acid / 50% ethanol solution. The extracted neutral red was measured by absorbance spectrophotometer (UVmax, Molecular Device, USA) to measure the cytotoxicity value of each concentration of 540-630 nm. Table 2 shows the cytotoxicity expressed as a percentage of absorbance and 3 lots. (lot) was performed three times.

※ Cell survival rate (%) = (Sample absorbance / untreated group absorbance) × 100

NR assay OD Mean ± SD
(% of control) Sample concentration (W / W%) Control group One 0.5 0.1 0.05 0.01 Preparation Example 1:
Tissue Cultured Noni Culture Cells 0.229 ± 0.004
(100.0) 0.223 ± 0.011
(97.38) 0.228 ± 0.008
(99.56) 0.238 ± 0.012
(103.93) 0.249 ± 0.007
(108.73) 0.248 ± 0.018
(108.30) Comparative Production Example 1:
Noni 0.229 ± 0.004
(100.0) 0.226 ± 0.014
(98.69) 0.230 ± 0.010
(100.44) 0.241 ± 0.012
(105.24) 0.247 ± 0.015
(107.86) 0.248 ± 0.009
(108.30)

The standard deviation was obtained by calculating the average of the absorbance of the untreated group and the absorbance of the sample treated group based on raw data.

As shown in Table 2 and Table 3, MTT ₅₀ , NR ₅₀ value of Preparation Example 1 of the present invention is 1% or more, it can be seen that the raw material is safe compared to Comparative Preparation Example 1.

Test Example 2: Tissue Cultured Tissue Cultured Cells Morinda citirifolia  callus) Measurement of cytoprotective effect of extracts

Cytoprotective effect experiments were performed on SDS (Sodium dodecyl sulfate) cytotoxicity against Preparation Example 1 and Comparative Preparation Example 1.

Human fibroblasts were cultured in 37 ° C., 5% CO ₂ incubator with IMDM with 10% fetal calf serum (FBS) and 1% penicillin-streptomycin. Cultured human fibroblasts were dispensed at 1 × 10 ⁴ cells / well in 96-multiwell plates and incubated for 24 hours. The medium of cultured cells was replaced with 2% FBS medium, and 0.002% of SDS (Sodium dodecyl sulfate) was pretreated for 1 hour, followed by dilution of Preparation Example 1 and Comparative Preparation Example 1 in the medium by treatment concentration for 24 hours. Incubated in a CO ₂ incubator. In cultured cells, MTT stock solution (5 mg / ml) dissolved in PBS (phosphate buffered saline) was diluted 10-fold in fresh medium, and 100 µl of each solution was added to each well of the cell, followed by 4 hours at 37 ° C. Reacted. After the reaction, the living cells were dehydrated by the dehydrogenase present in the mitochondria, and the yellow water-soluble substrate MTT [3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium-bromide] was insoluble in water. Since it is reduced to crystals, the amount of this can be measured to determine cell viability. Remove the culture supernatant and add 200 μl of DMSO to each well to dissolve the MTT-formenic crystals produced in the cells, and then use the absorption spectrophotometer (UVmax, Molecular Device, USA) to filter the maximum absorbance of the formazin. The OD value at a wavelength of 540 nm and the OD value at a wavelength of 630 nm as a reference filter were measured to determine the absorbance of the difference (Ref. EMBO Journal (2002) 21, 2407-2417 et al.). The cytoprotective effect is expressed as a percentage of absorbance.

※ Cell survival rate (%) = (Sample absorbance / untreated group absorbance) × 100

The standard deviation was obtained by calculating the average of the absorbance of the untreated group and the absorbance of the sample treated group based on raw data.

MTT assay OD Mean ± SD
(% of control) Sample concentration (W / W%) Control group SDS Only SDS + 0.01 SDS + 0.05 SDS + 0.1 SDS + 0.5 Preparation Example 1:
Tissue Cultured Noni Culture Cells 0.421 ± 0.011
(100.0) 0.217 ± 0.013
(51.54) 0.242 ± 0.009
(57.48) 0.299 ± 0.013
(71.02) 0.328 ± 0.017
(77.91) 0.342 ± 0.010
(81.24) Comparative Production Example 1:
Noni 0.421 ± 0.011
(100.0) 0.217 ± 0.013
(51.54) 0.246 ± 0.011
(58.43) 0.283 ± 0.015
(67.22) 0.307 ± 0.012
(72.92) 0.318 ± 0.007
(75.53)

As shown in Table 4, it can be seen that the cell protective effect of Preparation Example 1 of the present invention promotes the cell protective effect against external stimulation.

Test Example 3 Tissue Cultured Tissue Cultured Cells Morinda citirifolia  antioxidative effect of extract

Antioxidant effect test was carried out for Preparation Example 1 and Comparative Preparation Example 1.

<Measurement of anti-hydroxyl radical activity (%) by DPPH (1,1-diphenyl-2 picrylhydrazyl) radicals>

Reactions by free radicals are almost always radical reactions, including the process of automatic oxidation. Antioxidants are responsible for blocking the automatic oxidation in vivo. The greater the reducing power as an electron-giving ability, the stronger the antioxidant. A reagent that measures the reducing power of antioxidants is the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical. DPPH exists as a radical of a stabilized structure of the nitrogen center in the compound. The maximum absorption at 517 nm is shown and the reduction is absent at 517 nm. DPPH (D9132, SIGMA) was dissolved in a 6: 4 (v / v) mixed solvent 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 DPPH solution was added to 1 ml of the diluted sample and mixed. After standing at room temperature for about 1 hour, absorbance at 513 nm was measured using an absorption spectrophotometer (UVmax, Molecular Device, USA) and the results are shown in Table 5 below.

DPPH assay OD Mean ± SD
(% of control) Sample concentration (W / W%) Control group One 0.5 0.25 0.1 0.05 Preparation Example 1:
Tissue Cultured Noni Culture Cells 0.866 ± 0.006
(0.00) 0.083 ± 0.011
(90.42) 0.120 ± 0.006
(86.14) 0.284 ± 0.008
(67.21) 0.483 ± 0.011
(44.23) 0.697 ± 0.010
(19.52) Comparative Production Example 1:
Noni 0.866 ± 0.006
(0.00) 0.098 ± 0.009
(88.68) 0.198 ± 0.011
(77.14) 0.378 ± 0.010
(56.35) 0.631 ± 0.015
(27.14) 0.811 ± 0.006
(6.35)

As shown in Table 5, Preparation Example 1 showed an excellent antioxidant effect.

Test Example 4: tissue cultured noni tissue cells Morinda citirifolia  measurement of tyrosinase activity and inhibitory effect of melanin synthesis

Test Example 4-1. Tyrosinase Activity Inhibition Test

Preparation Example 1 and Comparative Preparation Example 1 Tyrosinase activity inhibition test was conducted to investigate the whitening activity. For the experiment, tyrosinase enzyme solution was prepared by dissolving mushroom tyrosinase (mushroom tyrosinase, T-3824, 1530U / mg, Sigma) in phosphate buffer (pH 6.5) to 1000U / ml, and the substrate solution was L. -Tyrosine (L-tyrosine, 45160-0410, Junsei chemical co. Ltd) was prepared by dissolving in phosphate buffer (pH 6.5) to 1.5 mM.

Preparation Example 1 and Comparative Preparation Example 1 were prepared by diluting each sample in a buffer at different concentrations. 170 μl of sample solution and 10 μl of tyrosinase enzyme solution were added thereto, and the mixture was left at 37 ° C. for 10 minutes. 20 μl of substrate solution was added thereto, followed by reaction for 10 minutes at 37 ° C., and then left for 5 minutes in ice, and then absorbance was measured at wavelength 490 nm using an absorption spectrophotometer (UVmax, Molecular Device, USA). Substituting the absorbance measured in the above Equation 1 to calculate the tyrosinase activity inhibition rate is shown in Table 6 below. A solution prepared by adding phosphate buffer (pH 6.5) instead of the sample solution was used as a blank test solution, and a solution prepared by adding phosphate buffer (pH 6.5) instead of the substrate solution was used as a calibration solution.

&Quot; (1) &quot;

Tyrosinase activity inhibition rate (%) = 100-(A-A ') / (B-B') X 100

A: absorbance after the liquid reaction

B: absorbance after reaction of blank test solution

A ': Correction liquid of the test liquid

B ': Correction amount of blank

Tyrosinase
Activity inhibition measurement OD Mean ± SD
(% of control) Sample concentration (W / W%) Control group 5 2.5 One 0.5 Preparation Example 1:
Tissue Cultured Noni Culture Cells 0.247 ± 0.011
(0.00) 0.035 ± 0.09
(85.83) 0.074 ± 0.012
(70.04) 0.125 ± 0.005
(49.39) 0.197 ± 0.009
(20.24) Comparative Production Example 1:
Noni 0.247 ± 0.011
(0.00) 0.046 ± 0.011
(81.38) 0.087 ± 0.013
(64.78) 0.149 ± 0.008
(39.68) 0.218 ± 0.013
(11.74)

Test Example 4-2: Measurement of melanin synthesis inhibition according to the concentration of tissue cultured Morinda citirifolia callus extract

For Preparation Example 1 and Comparative Preparation Example 1, the whitening effect was measured using B-16 melanoma cells (prepared Yonsei University Wonju Medical College). As a measuring method, B-16 melanoma cells derived from black mice were added to a 6 well tissue culture dish at 2 × 10 ⁴ cells / well concentration with DMEM containing 10% FBS (GIBCO., USA). 5% CO ₂ , and incubated for 24 hours under 37 ℃ conditions. After incubation, the medium was removed, and then replaced with DMEM containing 10% FBS, 2uM α-MSH (Sigma., USA, Melanocyte stimulating Hormone) and 2 mM theophylline (Sigma., USA, theophylline), and then cultured wild ginseng root muscle extract Were diluted in the same medium and added, respectively, and then cultured under 5% CO ₂ , 37 ° C. until the cells were at least about 80% at the bottom of the well. After incubation, the medium was removed, 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 for 24 hours at 60 ° C. incubator and lysed melanin by adding 1N NaOH. The dissolved melanin was measured for absorbance at wavelength 490 nm using an absorption spectrophotometer (Uvmax, Molecular Device, USA).

&Quot; (2) &quot;
Melanin synthesis inhibition rate (%) = (1- A / A ') X 100

A: melanin concentration A ': number of cells

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Melanin Synthesis Inhibition OD Mean ± SD
(% of control) Sample concentration (W / W%) Control group 0.25 0.1 0.05 0.01 Preparation Example 1:
Tissue Cultured Noni Culture Cells 0.238 ± 0.010
(0.00) 0.046 ± 0.008
(80.67) 0.089 ± 0.0015
(62.61) 0.161 ± 0.012
(32.35) 0.219 ± 0.009
(7.98) Comparative Production Example 1:
Noni 0.238 ± 0.010
(0.00) 0.073 ± 0.012
(69.33) 0.112 ± 0.018
(52.94) 0.196 ± 0.007
(17.65) 0.225 ± 0.012
(5.46)

As shown in Table 6 and Table 7, Preparation Example 1 inhibited tyrosinase activity in a concentration-dependent manner, and was excellent in inhibiting melanin production in the cell.

Test Example 5: tissue cultured noni tissue cells Morinda citirifolia  measurement of collagen biosynthesis of extract

One of the causes of skin wrinkles is a deficiency of collagen, or collagen. Collagen is a major protein that makes up the skin dermis and plays a role in maintaining skin structure and elasticity. Collagen is known to show a decrease in production with increasing age and to increase the decomposition to induce depression of the dermal dermal layer to produce wrinkles of the skin.

<Measurement of collagen biosynthesis amount>

In order to investigate the anti-wrinkle effect of the cosmetics according to the present invention, a collagen biosynthesis promoting test (Sircol ^™ Soluble Collagen Assay Kit) using fibroblasts was performed.

Cultured human fibroblasts were dispensed in a medium containing 5% FBS to be 1 × 10 ⁵ cells / well in a 12 well cell culture dish and incubated for 24 hours, followed by tissue culture in a medium containing 2% FBS. And Comparative Preparation Example 1 were diluted for each treatment concentration, treated to cells, and cultured for 48 hours, and used in the following experiment. In order to confirm that the synthesis of collagen was actually increased in the fibroblasts, Biocolor's Sircol ^TM soluble collagen assay kit (Soluble Collagen Assay kit) was purchased, and the amount of collagen was measured according to the experimental method of the product manual. Sircol ^TM soluble collagen assay kit is a quantitative method that can be identified by combining collagen and staining reagent secreted into the culture medium during cell culture. Specifically, 1 ml of a dyeing reagent (a solution containing Sirius red reagent in picric acid) was added to 100 µl of the culture supernatant and allowed to react for 30 minutes (concentration of the culture solution or the culture supernatant during the test depending on the amount of collagen production). Up to 200 μl can be reacted with 1 ml of the dyeing reagent.) Sirius red is an anionic pigment that specifically binds to collagen. After reacting for 30 minutes, the reaction solution was centrifuged at 12,000 × g for 10 minutes to precipitate the collagen-pigment conjugate. 1.0 ml of alkaline reagent was added to the precipitate and dissolved for 5 minutes at room temperature. Absorbance of the solution was measured at 540 nm using an absorbance spectrophotometer (UVmax, Molecular Device, USA), and is shown in Table 8 below. The effect of promoting collagen biosynthesis was evaluated by comparing the absorbance of the test group.

sample OD Mean ± SD
(% of control) Sample concentration (W / W%) Control group 0.01 0.05 0.1 0.5 Preparation Example 1:
Tissue Cultured Noni Culture Cells 0.424 ± 0.032
(100) 0.451 ± 0.018
(106.37) 0.489 ± 0.021
(115.33) 0.568 ± 0.023
(133.96) 0.554 ± 0.016
(131.13) Comparative Production Example 1:
Noni 0.424 ± 0.032
(100) 0.463 ± 0.015
(109.20) 0.481 ± 0.019
(113.68) 0.529 ± 0.031
(124.76) 0.546 ± 0.022
(128.77)

As shown in Table 8, Preparation Example 1 confirmed that the collagen biosynthesis effect is excellent.

Example 1 Noni Culture Cells Tissue Cultured Morinda citirifolia  callus) Preparation of essence containing extract

An essence comprising Preparation Example 1 was prepared according to the composition of Table 9 below.

Ingredient Name Content (% by weight) A 1,3-butylene glycol 1.0 glycerin 1.0 Carbomer 0.2 Glyceryl Methacrylate 0.35 Parabens 0.20 Purified water Balance B Potassium hydroxide 0.06 C PEG-6-hydrogenated castor oil 1.0 Spices 0.5 D Preparation Example 1 5.0 system 100.00

A was sufficiently dispersed and wetted to form a uniform gel, and neutralized by adding B. C was added to (A + B), uniformly stirred and solubilized, and then D was added at room temperature, stirred to distribute uniformly, and put into a container to produce a product.

Examples 2 to 4: tissue cultured noni tissue cells Morinda citirifolia  callus) Preparation of Softener (Skin) Containing Extracts

Examples of the flexible longevity (skin) in the cosmetic composition containing Preparation Example 1 are shown in Table 10 below.

ingredient Example 2 Example 3 Example 4 Content (% by weight) Content (% by weight) Content (% by weight) Article 1 10.00 5.00 2.00 Butylene glycol 2.00 2.00 2.00 glycerin 3.00 3.00 3.00 Oleyl alcohol 2.00 2.00 2.00 Polysorbate 20 1.00 1.00 1.00 ethanol 6.00 6.00 6.00 antiseptic 0.5 0.5 0.5 incense 0.5 0.5 0.5 Purified water Balance Balance Balance system 100.00 100.00 100.00

Examples 5 to 7: Tissue cultured tissue cultured Morinda citirifolia  callus) Preparation of nutritional milk (milk lotion) containing extract

Formulation example of nutrient emulsion (milk lotion) in the cosmetic composition containing Preparation Example 1 is shown in Table 11.

ingredient Example 5 Example 6 Example 7 Content (% by weight) Content (% by weight) Content (% by weight) Preparation Example 1 10.00 5.00 2.00 Butylene glycol 4.00 4.00 4.00 glycerin 3.00 3.00 3.00 Oleyl alcohol 1.00 1.00 1.00 Polysorbate20 1.00 1.00 1.00 antiseptic 0.5 0.5 0.5 incense 0.5 0.5 0.5 Purified water Balance Balance Balance system 100.00 100.00 100.00

Examples 8 to 10 tissue cultured noni tissue cells Morinda citirifolia  callus) Preparation of cream containing extract

Formulation example of the cream in the cosmetic composition containing Preparation Example 1 is shown in Table 12 below.

ingredient Example 8 Example 9 Example 10 Content (% by weight) Content (% by weight) Content (% by weight) Preparation Example 1 10.00 5.00 2.00 Butylene glycol 2.00 2.00 2.00 glycerin 4.00 4.00 4.00 Potassium hydroxide 0.10 0.10 0.10 Tocopheryl acetate 1.0 1.0 1.0 Liquid paraffin 5.0 5.0 5.0 Squalane 5.0 5.0 5.0 Macadamia Nut Oil 10.0 10.0 10.0 Polysorbate 60 1.0 1.0 1.0 Sorbitan sesquioleate 1.0 1.0 1.0 Carbomer 0.3 0.3 0.3 antiseptic 0.5 0.5 0.5 incense 0.5 0.5 0.5 Purified water Balance Balance Balance system 100.00 100.00 100.00

Claims (8)

Tissue cultured noni cultured cells (tissue cultured Morinda citirifolia callus) extract, Cosmetic composition, characterized in that used for antioxidant, skin wrinkle improvement, skin whitening or skin protection. The cosmetic composition of claim 1, comprising 0.1 to 10.0 wt% of tissue cultured Morinda citirifolia callus extract based on the total weight of the composition. The noni culture cells of tissue cultured noni culture cells (tissue cultured Morinda citirifolia callus) extract is selected from the group consisting of purified water, alcohol of C1 to C4 and alkylene glycol of C1 to C5. Cosmetic composition, characterized in that the extraction using more than one solvent. delete The method of claim 1, wherein the cosmetic composition is a skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, nutrition lotion, massage cream, nutrition cream, hand cream, foundation, makeup base, twin cake, mascara, essence, nutrition Cosmetic composition, characterized in that formulated in the form selected from essence, pack, soap, cleansing foam, cleansing lotion, cleansing cream, body lotion and body cleanser. a) tissue cultured Noni cultured Morinda citirifolia callus was immersed in at least one extractant selected from the group consisting of purified water, C1 to C4 alcohol and C1 to C5 alkylene glycol for 3 to 30 days Forming an immersion solution; And b) a method of producing a tissue cultured noni culture cell extract comprising the step of obtaining a tissue cultured Morinda citirifolia callus extract by filtration of the deposition solution. The method of claim 6, wherein the extraction solvent is ethanol; Ethanol and 1,3-butylene glycol; Ethanol and propylene glycol; And non-cultured tissue cultured Morinda citirifolia callus extract, characterized in that at least one selected from the group consisting of ethanol and pentylene glycol. The method of claim 6, wherein the step b) further comprises the step of removing the extracting solvent tissue cultured noni cultured cells (tissue cultured Morinda citirifolia callus) extract manufacturing method.