KR101146133B1 - Antiaging cosmetic composition containing extract of machilus thunbergii - Google Patents

Abstract

The present invention relates to a tohubak extract manufacturing method and a functional cosmetic composition having a function of at least one of wrinkle improvement, antioxidant and antibacterial function containing the same, more specifically, tohubak extract manufacturing method and the extract prepared by 0.01 ~ It relates to a functional cosmetic composition containing 30.0% by weight and excellent in antioxidant effect, antibacterial effect, anti-wrinkle effect, skin irritation effect.

Tofu Park Extract, Ultra High Pressure Extraction, Antioxidant Effect, Antibacterial Effect, Anti-Wrinkle Effect, Skin Stimulation Relaxation Effect, Functional Material, Cosmetic Composition

Description

Anti-aging functional cosmetic composition containing epoch extract {Antiaging cosmetic composition containing extract of machilus thunbergii}

The present invention relates to a tohubak extract manufacturing method and a functional cosmetic composition containing the same, more specifically, tohubak extract manufacturing method and 0.01 ~ 30.0% by weight of the extract prepared through this, antioxidant effect, antibacterial effect, wrinkles The anti-aging functional cosmetic composition excellent in the prevention effect and the skin irritation relief effect.

Human skin undergoes various physical and chemical changes during the aging process, and is classified into internal aging and photo-aging according to the causes. In other words, when ultraviolet rays, stress, disease state, environmental factors, wounds, and age, free radicals are activated and the condition is intensified, the antioxidant defense network existing in the living body is destroyed, and cells and tissues are damaged to promote adult disease and aging. do. More specifically, lipids, proteins, polysaccharides, nucleic acids, and the like, which are major constituents of the skin, are oxidized to destroy skin cells and tissues, thereby causing skin aging. In particular, the oxidation of protein is caused by severe damage of the skin's connective tissues such as collagen, hyaluronic acid, elastin, proteoglycan, fibronectin, severe inflammatory reactions and elasticity of the skin. This can lead to cancer and reduced immune function. Therefore, cell membranes are protected by eliminating the free radicals generated by the body's metabolic processes, irradiated by UV irradiation, or inflammatory reactions.Also, damaged cells must be regenerated by active metabolism to proliferate the cells. Can become healthy skin.

Aging involves not only free radicals but also enzymes called matrix metalloproteinases (MMPs). In vivo, the synthesis and degradation of extracellular matrix such as collagen is properly regulated, but its synthesis decreases as aging progresses. Expression of the substrate metalloproteinase (MMP), which is an enzyme that breaks down, is promoted, thereby decreasing the elasticity of the skin and forming wrinkles. Ultraviolet irradiation also activates these degrading enzymes. Therefore, there is a need for the development of a substance capable of regulating MMP expression that inhibits activation or inhibiting its activity in cells.

Until now, most of the raw materials used as materials for cosmetics simply inhibited enzyme activity. In this regard, the present inventors attempted to control the expression and activity of MMP expression induced by intracellular natural and photoaging.

Next is skin change by pigmentation. The pigments that affect skin color include melanin, melanoids, carotene, and hemoglobin, the most important of which is melanin, which affects biosynthesis. Melanin plays an important role in preventing or damaging the skin from ultraviolet rays by absorbing or scattering ultraviolet rays. There is no special maximum absorption wavelength and it absorbs light in all areas. In addition, it is excellent in removing active oxygen species, but sometimes melanin itself generates free radicals, and other substances are reduced or oxidized by catechol or quinone in melanin structure, and melanin itself shows the properties of free radicals. Pray. The overproduction of melanin is known to play an important role in forming blemishes, freckles, etc., promoting skin aging, and inducing skin cancer, and research and development for preventing melanin overproduction are being actively conducted.

Ascorbic acid (JP-A 4-9320), hydroquinone (JP-A 6-192062), kojic acid (JP-A-56-7710), arbutin (JP-A 4-93150) and some extracts inhibit tyrosinase. Although it is used as a whitening cosmetic because it has activity, the materials are used due to the disadvantages of synthetic substances and poor stability in cosmetic formulations, resulting in discoloration due to decomposition, odor occurrence, unclear effect of efficacy and effect at the biological level, and safety issues. This situation is limited.

Patent No. 531686 "Whitening Cosmetic Composition Containing Tobacco Extract" relates to extracts soluble in lower alcohols such as water, methanol, ethanol, etc., or mixtures thereof, preferably 70% methanol, which inhibit melanin synthesis. It is superior to Kojisan and is safe as a herbal extract, and is used in whitening cosmetics contained in creams, skins, lotions and packs. The patent described only the whitening function of the Tohubac extract. Thus, the present inventors found the biological efficacy in addition to the whitening function and applied it to the functional cosmetic composition.

An object of this invention is to provide the antiaging cosmetic composition which has the outstanding functionality by confirming the efficacy as a raw material of a functional cosmetic composition with respect to Tohubak extract, developing the use method, and using this raw material.

An object of the present invention is to provide an extraction method for increasing the efficacy of the Tohubak extract.

Thus, the present inventors studied the application possibility of functional cosmetic composition to the Tobacco extract, which has not been known so far, as a result of measuring the antioxidant effect, antimicrobial effect, anti-wrinkle effect, skin irritation effect, the effect is excellent anti It has been found that the efficacy as an aging functional cosmetic can be expected.

Tohubak is an evergreen broad-leaved arborescent tree of the dicotyledonous plant of the genus Amphitaceae. Jeju Island and the southern coastal areas grow relatively well under 500m above sea level. Its sound resistance is good in seedlings, but the trees are amniotic, grow well in red velvet soil and high air humidity, cold resistance is weak, but grows in fertile coastal areas, grows fast, wind resistance is strong, and it is good for pollution. Endure

Tofu foil is used as a coating material, and it is good as a roadside tree. It is an expansive tree and serves as a windbreak forest on the beach, and is suitable for landscape gardens, park trees, road trees, road gardens, green trees, and ecological parks. The root or bark is called namnampi (紅 楠 皮) medicinal. It is collected in summer and dried in the sun. Bark contains 0.48% of tannin, 12.38% of resin, 0.688% of rubber, and a large amount of mucus. The roots are n-norarmepavine, a benzyl isoquinoline alkaloid. Contains reticulin The heartwood contains lignoceric acid, quercetin, and catechol, and the wick is a raw material for mosquitoes. The mucus contained in the leaves is a complex of polysaccharides and proteins, and the components of the viscous polysaccharide are arabinose, xylose, rhamnose, galactose, glucose and gluconic acid. Leaves and twigs contain myristic acid. Tohubak's medicinal effect is to squeeze the root of the left muscles or salt to the roots and crush them. For pregliomas, fumigation with a full amount of bark and washing is performed.

The present invention is to provide a functional cosmetic composition containing the Tohubak extract. Tofu Bak extraction is usually extracted using hot water or a soluble organic solvent of ethanol, ethyl acetate, nucleic acid, diethyl ether as a solvent. It is also possible to extract using ultra high pressure, and the anti-oxidation effect, antibacterial effect, anti-wrinkle effect and irritation-releasing effect are increased when the ultra high pressure extraction is performed using hot water or soluble organic solvents of ethanol, ethyl acetate and nucleic acid diethyl ether.

Ultra-high pressure extraction refers to a technique that extracts by applying a pressure of more than 100MPa (1,000atm) physically, it is an extraction method having the same effect as extracting the material under the pressure of 10,000m in the sea. Existing extraction methods such as hot water extraction, ultrasonic extraction, and reflux extraction have low extraction yield and high energy consumption, and have disadvantages such as destruction of useful components and protein denaturation, loss of components, heat instability, and color change due to heat. , Ultra High Pressure Extraction is a useful extraction method with high extraction yield, less destruction of useful components, less color change and less odor.

The present invention provides an anti-aging functional cosmetic composition, characterized in that the content of Tohubak extract is 0.0001 to 30.0% by weight based on the total cosmetic composition. When the content of the extract is less than 0.001% by weight, there is almost no skin improvement effect, and when the content of the extract is more than 30.0% by weight, the degree of increase in the effect of increasing the content is insignificant and thus it is not economical.

In addition, the present invention, the functional cosmetic composition is a lotion, gel, water-soluble liquid, cream, ointment, essence, shampoo, hair rinse, hair treatment, hair mousse, lipstick, oil-in-water (O / W) type and water-in-oil (W) It provides a functional cosmetic composition containing the Tohubak extract, characterized in that selected from the formulation of / O) type.

In addition, the present invention is the process of adding the solvent to the Tohubak extract, the Tohubak extract;

A pressurizing step of putting a solvent containing a sample into a chamber of an ultrahigh pressure machine and applying an ultrahigh pressure of 100 MPa or more to extract the same;

A separation and purification process of separating the extract from the sample;

It relates to a Tohubak extract manufacturing method comprising a; lyophilized or concentrated to the purified extract.

In addition, in the present invention, the solvent added to the dried Tohubak sample is water, ethanol, methanol, butanol, propanol, butylene glycol, propylene glycol, chloroform, ethyl acetate, dichloromethane, hexane, petroleum ether and diethyl ether. At least one selected from the group consisting of.

In addition, the present invention may add a process of adding a crude extract material such as glass beads (silica beads) or sea sand after preparing the sample.

Functional cosmetic composition containing the Tohubak extract according to the present invention showed an antioxidant effect, antibacterial effect, anti-wrinkle effect, alleviating skin irritation.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these Examples are only illustrative description of the present invention, and the scope of the present invention is not limited to these Examples.

Example 1

60 g of the dried Tobacco sample was placed in a vacuum bag and 540 g of water was added thereto, followed by 6 g of glass beads (GHM-30, B & K Media, Korea). After the addition of the crude extract material was vacuumed using a vacuum (IS-100, Namwoong, Korea). Thereafter, a super high pressure device (TFS-2L, TOYO KOATSU CO., LTD., Japan) was used to treat a double liquid phase ultra high pressure for 1 hour under conditions of 100 MPa (1,000 bar), 25 ° C., and water (buffer). After the ultra-high pressure treatment was completed using a centrifuge (Supra 22K, Hanil, Korea) centrifuged for 20 minutes at 8,000MPa, 15 ℃ conditions to separate the soil samples and crude extract glass beads and extracts. The separated extract was filtered through a 0.8 μm, 0.45 μm filter to obtain a filtrate, and the filtrate was freeze-pretreated at -21 ° C and then -80 ° C using a freeze dryer (Bondiro, Ilshin, Korea). Lyophilization was performed for 48 hours to obtain Tobacco extract.

Example 2

60 g of the dried Tobacco sample was put in a vacuum bag and 540 g of water was added thereto, followed by vacuum treatment using a vacuum machine (IS-100, Namwoong, Korea). Thereafter, a high-pressure apparatus (TFS-2L, TOYO KOATSU CO., LTD., Japan) was used to treat a double liquid phase ultra high pressure for 1 hour under conditions of 100 MPa (1,000 bar), 25 ° C., and water (buffer). After the ultrahigh pressure was completed, centrifugal separator (Supra 22K, Hanil, Korea) was centrifuged at 8,000MPa, 15 ℃ for 20 minutes to separate the Tobacco samples and extracts. The separated extract was filtered through a 0.8 μm, 0.45 μm filter to obtain a filtrate, and the filtrate was freeze-pretreated at -21 ° C and then -80 ° C using a freeze dryer (Bondiro, Ilshin, Korea). Lyophilization was performed for 48 hours to obtain Tobacco extract.

Example 3

60 g of the dried Tobacco sample was placed in a vacuum bag, 540 g of a 70% ethanol aqueous solution was added, and 6 g of glass beads (GHM-30, B & K Media, Korea) was added to the dipped sample. After the addition of the crude extract material was vacuumed using a vacuum (IS-100, Namwoong, Korea). Thereafter, a high-pressure apparatus (TFS-2L, TOYO KOATSU CO., LTD., Japan) was used to treat a double liquid phase ultra high pressure for 1 hour under conditions of 100 MPa (1,000 bar), 25 ° C., and water (buffer). After the ultra-high pressure treatment was completed using a centrifugal separator (Supra 22K, Hanil, Korea) was centrifuged for 20 minutes at 8,000rpm, 15 ℃ condition to separate the Tobacco samples, crude extracts and extracts. The separated extract was filtered through a 0.8 μm, 0.45 μm filter to obtain a filtrate, and the filtrate was concentrated at 60 ° C., 30 MPa for 2 hours using a rotary evaporator NE-series, Eyela, Japan. After freeze-treatment at -21 ° C, the concentrate was freeze-dried at -80 ° C for 48 hours using a freeze-dryer (Bondiro, Ilshin, Korea) to obtain a Tobacco extract.

Example 4

600g of dried dried Tobacco samples were placed in a vacuum bag and 540g of 70% ethanol aqueous solution was added thereto, followed by vacuum treatment using a vacuum machine (IS-100, Namwoong, Korea). Thereafter, a super high pressure device (TFS-2L, TOYO KOATSU CO., LTD., Japan) was used to treat a double liquid phase ultra high pressure for 1 hour under conditions of 100 MPa (1,000 bar), 25 ° C., and water (buffer). After the ultra-high pressure treatment was completed using a centrifuge (Supra 22K, Hanil, Korea) centrifuged for 20 minutes at 8,000rpm, 15 ℃ condition to separate the Tobacco samples and extracts. The separated extract was filtered through a 0.8 μm, 0.45 μm filter to obtain a filtrate, and the filtrate was concentrated at 60 ° C., 30 MPa for 2 hours using a rotary evaporator NE-series, Eyela, Japan. After freeze-treatment at -21 ° C, the concentrate was freeze-dried at -80 ° C for 48 hours using a freeze-dryer (Bondiro, Ilshin, Korea) to obtain a Tobacco extract.

Comparative Example 1

60 g of dried Tobacco samples were added to a beaker, 540 g of water was added, and hydrothermal treatment was performed at 80 ° C. for 1 hour using a water tank (HB-205WP, Hanbaek, Korea). After the hot water treatment was completed, centrifugal separator (Supra 22K, Hanil, Korea) was used to centrifuge for 20 minutes at 8,000 MPa, 15 ℃ condition to separate the Tobacco samples and extracts. The separated extract was filtered through a 0.8 μm, 0.45 μm filter to obtain a filtrate, and the filtrate was freeze-pretreated at -21 ° C and then -80 ° C using a freeze dryer (Bondiro, Ilshin, Korea). Freeze-drying for 48 hours to obtain the Tobacco extract.

Comparative Example 2

60 g of dried Tobacco samples were placed in a beaker, 540 g of water was added thereto, and then ultrasonicated at 20 kHz, 750 W, and 25 ° C. for 1 hour using an ultrasonic apparatus (5510R-DTH, BARANSON, USA). After the sonication was completed, centrifugal separator (Supra 22K, Hanil, Korea) was used to centrifuge for 20 minutes at 8,000 rpm and 15 ° C. to separate the Tobacco samples and extracts. The separated extract was filtered through a 0.8 μm, 0.45 μm filter to obtain a filtrate, and the filtrate was freeze-pretreated at -21 ° C and then -80 ° C using a freeze dryer (Bondiro, Ilshin, Korea). Freeze-drying for 48 hours to obtain the Tobacco extract.

Comparative Example 3

60 g of dried Tobacco samples were placed in a round flask, and 540 g of an aqueous 70% ethanol solution was added thereto, followed by hydrothermal treatment (reflux) for 1 hour at 80 ° C. in an extractor equipped with a cooling condenser. After completion of the hot water (reflux) treatment using a centrifuge (Supra 22K, Hanil, Korea) was centrifuged for 20 minutes at 8,000rpm, 15 ℃ conditions to separate the Tobacco samples and extracts. The separated extract was filtered through a 0.8 μm, 0.45 μm filter to obtain a filtrate, and the filtrate was concentrated at 60 ° C., 30 MPa for 2 hours using a rotary evaporator NE-series, Eyela, Japan. After freeze-treatment at -21 ° C, the concentrate was freeze-dried at -80 ° C for 48 hours using a freeze dryer (Bondiro, Ilshin, Korea).

Comparative Example 4

60 g of dried Tobacco samples were placed in a beaker, 540 g of water was added, and then ultrasonicated at 20 kHz, 750 W, and 25 ° C. for 1 hour using an ultrasonic apparatus (5510R-DTH, BARANSON, USA). After the sonication was completed, centrifugal separator (Supra 22K, Hanil, Korea) was used to centrifuge for 20 minutes at 8,000 rpm and 15 ° C. to separate the Tobacco samples and extracts. The extract thus separated was filtered through a 0.8 μm, 0.45 μm filter to obtain a filtrate, and the filtrate was concentrated using a concentrator (Rotary Evaporator NE-series, Eyela, Japan) at 60 ° C., 30 MPa for 2 hours. The concentrate was freeze-dried at -21 ° C and then freeze-dried at -80 ° C for 48 hours using a lyophilizer (Bondiro, Ilshin, Korea) to obtain a Tobacco extract.

Experimental Example 1: Comparison of extraction yield

In order to confirm the yield of the Tobacco extract according to the extraction method of the present invention, the yields of the extracts of Examples 1 to 4 and Comparative Examples 1 to 4 are compared to the yield of the dried Tobacco sample, and the results are shown in Table 1. It was.

Extraction Method yield(%) Water solvent 70% ethanol solvent Ultra High Pressure Extraction (Add Crude Extraction) Example 1 12.18 Example 3 13.24 Ultra High Pressure Extraction Example 2 10.25 Example 4 11.28 Hot water (reflux) extraction Comparative Example 1 6.48 Comparative Example 3 5.04 Ultrasonic extraction Comparative Example 2 4.45 Comparative Example 4 4.08

As shown in Table 1, the yields of Examples 1 to 4 subjected to ultra-high pressure extraction were higher than those of Comparative Examples 1 to 4, which were subjected to hot water (reflux) extraction and ultrasonic extraction, at about two times higher yields in both solvent conditions. In addition, the yield increases when the crude extract such as glass beads, silica beads, or sea sand is added during extraction.

Experimental Example 2: Component Identification and Content Experiment

To determine the total phenolic content and total flavonoid content of the phenolic substances, flavonoids related to the antioxidant effect among the main components of Tohubak extract according to the present invention was carried out as follows.

Total Phenolic Content Determination

10 ml of distilled water was added to 1 ml of the extract, 2 ml of Folin-Ciocalteu phenol reagent (Sigma) was added and mixed, followed by reaction at room temperature for 5 minutes. After adding 2 ml of 20% sodium carbonate to the reaction mixture, the mixture was allowed to stand at room temperature for 1 hour, and the absorbance was measured at 680 nm using a microplate reader (UVT-06685, Thermo max, USA). In this case, gallic acid (gallic acid, Sigma, USA) was used as the indicator material, and a calibration curve for each concentration of the indicator material was made and shown in Table 2 by substituting absorbance values of each sample.

Total Flavonoid Content Determination

1.5 ml of the extract was mixed with 2% AlCl ₃ 6H ₂ O dissolved in the same amount of methanol, and then reacted at room temperature for 10 minutes, and the absorbance was measured at 367 nm using a microplate reader (UVT-06685, Thermo max, USA). It was. At this time, the catechin (catechin, Sigma, USA) was used as the indicator material, and the calibration curve for each concentration of the indicator material was made, and the absorbance values of each sample were substituted.

Extraction solvent Extraction Method sample Total phenolic content
(gallic acid equivalents) (/ of extract) Total Flavonoid Content
(catechin equivalents) (/ of extract)
water

Ultra high pressure extraction (addition of crude extract material) Example 1 294 160 Ultra High Pressure Extraction Example 2 244 131 Hot water (reflux) extraction Comparative Example 1 146 73 Ultrasonic extraction Comparative Example 2 133 70
70% ethanol aqueous solution

Ultra high pressure extraction (addition of crude extract material) Example 3 356 194 Ultra High Pressure Extraction Example 4 332 166 Hot water (reflux) extraction Comparative Example 3 134 69 Ultrasonic extraction Comparative Example 4 116 46

As a result of Table 2, Examples 1 to 4, which were subjected to ultra high pressure extraction than Comparative Examples 1 to 4, which were subjected to hydrothermal (reflux) extraction and ultrasonic extraction, showed significantly higher polyphenol content and flavonoid content in both solvent conditions.

Experimental Example 3: Measurement of Antioxidant Effect

The free radical scavenging test and the free radical scavenging test were performed to confirm the antioxidant effect in the skin aging improvement effect assay of the Tohubak extract of the present invention.

Free radical scavenging test

By using the stable absorbance of DPPH exhibiting maximum absorbance at 540 nm, the absorbance at the 540 nm wavelength decreases as the free radical DPPH is erased by the sample and becomes purple to transparent color, that is, as the free radical scavenging rate increases. Application was carried out by the following assay test method.

First, 1 mL of 0.1 mM DPPH (2,2-diphenyl-1-picryl-hydrazyl radical, Sigma) solution was diluted to an appropriate concentration in Examples 1-4 and Comparative Examples 1-4 in methanol, and 1 mL was mixed. After standing at 37 ° C. for 15 minutes, the absorbance was measured at 540 nm using a microplate reader (UVT-06685, Thermo max, USA).

In the free radical scavenging test, the control group was measured in the same manner by adding 1 ml of DPPH and 1 ml of methanol, and 1 ml of methanol and 1 ml of sample were set to obtain respective color correction values for the sample and the control group.

The free radical scavenging ratio was calculated using Equation 1 and is shown in Table 3. In Table 3, SC ₅₀ is the concentration of the sample required to remove 50% of free radicals, and the smaller the value, the higher the antioxidant activity.

% Free radical scavenging rate = 100-((absorbance of sample / absorbance of control) x 100)

Free radical scavenging test

Reduction of free radicals by measuring the change in absorbance using oxidation of nitroblue tetrazolium (NBT) by free radicals using the generation of free radicals by enzyme reaction of xanthine / xanthine oxidase (Sigma) The scavenging ability can be seen.

Add 2.4 ml of sodium carbonate (Na ₂ CO ₃ ), 0.1 ml of xanthine (Sigma), 0.1 ml of EDTA (ethylenediamine tetraacetic acid), 0.1 ml of BSA (bovine serum albumn, Sigma), 0.1 ml of NBT and 0.1 ml of sample. After mixing by using (Type 37600 Mixer, Mini mix, USA) and left at 25 ℃ for 10 minutes, 0.1 ml of xanthine oxidase was added and reacted for 20 minutes at 25 ℃, 6mM copper (CuCl ₂₎ The reaction was stopped and absorbance was measured at 540 nm using a microplate reader (UVT-06685, Thermo max, USA).

The control group in the active oxygen scavenging activity test was measured in the same way to put the third distilled water instead of the sample solution, and put the third distilled water instead of the xanthine oxidase solution to the respective color correction value for the extracted sample and the control It was set to obtain.

Using the equation (2) to calculate the active oxygen scavenging rate as shown in Table 3. In Table 3, IC ₅₀ is the sample concentration required to remove 50% of the active oxygen, and the smaller the value, the stronger the antioxidant activity.

Free radical scavenging rate (%) = 100-((absorbance of sample / absorbance of control) x 100)

Extraction solvent Extraction Method sample Free radical scavenging ratio SC ₅₀ (mg / mL) Free radical removal rate
IC ₅₀ (mg / ml)

water
Ultra High Pressure Extraction (Add Crude Extraction) Example 1 0.29 0.17 Ultra High Pressure Extraction Example 2 0.33 0.21 Hot water (reflux) extraction Comparative Example 1 0.58 0.49 Ultrasonic extraction Comparative Example 2 0.69 0.71

70% ethanol aqueous solution
Ultra High Pressure Extraction (Add Crude Extraction) Example 3 0.13 0.11 Ultra High Pressure Extraction Example 4 0.20 0.21 Hot water (reflux) extraction Comparative Example 3 0.38 0.41 Ultrasonic extraction Comparative Example 4 0.44 0.51

As a result of Table 3, Examples 1 to 4 using the ultra-high pressure extraction method than the Comparative Examples 1 to 4 using the hydrothermal (reflux) extraction and the ultrasonic extraction method showed superior antioxidant effects in both solvent conditions.

Experimental Example 4: Antibacterial Activity

As an antibacterial effect assay for the Tohubak extract of the present invention, the antimicrobial test was performed by a solid culture dilution method and a paper disk method. Experimental strains were distributed from Korea Research Institute of Bioscience and Biotechnology, and were Gram-positive bacteria ( Staphylococcus aureus KCTC 6910), Gram-negative bacteria ( Pseudomonas aeruginosa KCTC 1637), Escherichia coli ( E. Coli KCTC 1039), and Candida yeast ( Candyda). albicans KCTC 7965), four strains were used as filamentous fungi ( Aspergillus niger KCTC 6910) or more.

Agar Serial Dilution Method

Bacteria were used as tryptic soy medium, and inoculated with the bacteria, and precultured in a 37 ° C. shaking incubator for 24 hours. Potato dextrose medium was used for the cultivation of the yeast, and the medium was inoculated with the bacteria and pre-cultured for 2 days in a 25 ° C shaking incubator. Potatodextrose agar medium was used for the cultivation of filamentous fungi, and the culture was inoculated with the medium and precultured at 25 ° C. for 7 days.

For more specific antimicrobial test, inoculate bacteria in tryptic soy medium for 24 hours and pre-culture at 37 ℃ for yeast, inoculate bacteria in potato dextrose medium for 2 days for 2 days pre-culture at 25 ℃ In addition, filamentous fungi were inoculated with bacteria in potato dextrose agar medium, pre-cultivated in a 25 ° C. incubator for 7 days, and then spores of filamentous fungi formed on the surface of the medium were collected using a smeared rod and diluted in sterile saline. 2 ml of each extract diluted in appropriate concentration in 5% DMSO (Dimethylsulfoxide) saline solution for each sample and experimental strain was added to the sterilized petri dish, and the control group was diluted to the appropriate concentration in 5% saline solution. ㎖ was added to each control group, 5ml DMSO physiological saline solution was added to 2ml, sterilized in each petri dish and cooled to 48 ℃ agar medium and potato dextrose agar medium by 18ml by stirring and then left to stand Solidified.

Then, each pre-cultured test bacteria were spread on each Petri dish at a final concentration of about 1 X 10 ⁶ CFU / mL for bacteria, 1 X 10 ⁵ CFU / mL for yeast, and about 1 for filamentous fungi. Each Petri dish was plated at a bacterial concentration of X 10 ⁴ CFU / mL. Each petri dish was incubated for 24 hours at 37 ° C, yeast for 3 days at 25 ° C, and filamentous fungi for 7 days at 25 ° C incubator. The minimum sample concentration was defined as the minimum inhibitory concentration (MIC), and the results are shown in Table 4. At this time, the minimum inhibitory concentration means that the smaller the value, the higher the antimicrobial effect.

Strain
Extraction Method
Minimum inhibitory concentration (MIC, ㎍ / ㎖) Water solvent 70% ethanol solvent
S. aureus

Ultra High Pressure Extraction (Add Crude Extraction) Example 1 315 Example 3 245 Ultra High Pressure Extraction Example 2 363 Example 4 281 Hot water (reflux) extraction Comparative Example 1 497 Comparative Example 3 377 Ultrasonic extraction Comparative Example 2 534 Comparative Example 4 433
P. aeruginosa

Ultra High Pressure Extraction (Add Crude Extraction) Example 1 276 Example 3 211 Ultra High Pressure Extraction Example 2 297 Example 4 231 Hot water (reflux) extraction Comparative Example 1 439 Comparative Example 3 298 Ultrasonic extraction Comparative Example 2 513 Comparative Example 4 319
E. Coli

Ultra High Pressure Extraction (Add Crude Extraction) Example 1 122 Example 3 98 Ultra High Pressure Extraction Example 2 154 Example 4 132 Hot water (reflux) extraction Comparative Example 1 298 Comparative Example 3 169 Ultrasonic extraction Comparative Example 2 331 Comparative Example 4 197
C. albicans

Ultra High Pressure Extraction (Add Crude Extraction) Example 1 271 Example 3 231 Ultra High Pressure Extraction Example 2 298 Example 4 255 Hot water (reflux) extraction Comparative Example 1 351 Comparative Example 3 401 Ultrasonic extraction Comparative Example 2 399 Comparative Example 4 436
A. niger

Ultra High Pressure Extraction (Add Crude Extraction) Example 1 39 Example 3 31 Ultra High Pressure Extraction Example 2 49 Example 4 38 Hot water (reflux) extraction Comparative Example 1 198 Comparative Example 3 121 Ultrasonic extraction Comparative Example 2 273 Comparative Example 4 151

As shown in Table 4, Examples 1 to 4 using the ultra-high pressure extraction method compared to Comparative Examples 1 to 4 using the hot water (reflux) extraction and the ultrasonic extraction method were carried out in two solvent conditions for four kinds of bacteria, yeast and filamentous fungi. All showed superior antimicrobial effect.

 Paper Disc Method

Take one platinum equivalent of each strain cultured on a plate medium, incubate for 24 hours in 10 mL of liquid medium, activate it, inoculate 0.1 mL of the solution in 10 mL of liquid medium, and incubate for 6 hours. Inoculated to ⁷ CFU / mL and spread evenly with sterile taunt rods. Thereafter, sterilized paper discs (6 mm, Satorius, Germany) were placed on a solid flat medium, and the samples of Experimental and Comparative Examples dissolved in a solvent were absorbed to 0.05 mL / disk and cultured. Staphylococcus aureus, Pseudomonas aeruginosa, E. Coli, 37 ° C, fungi varieties Candida albicans, Aspergillus niger ) Was incubated at 27 ° C. for 24 hours and 120 hours to measure the diameter of the transparent zone around the disk. As a comparison group, methyl paraben, which is known to have strong antimicrobial activity, was used, and the results are shown in Table 5. At this time, the transparent zone around the disk is an indicator of the degree of growth inhibition for the strain, the larger the diameter means that the antimicrobial effect on the strain.

Strain
Extraction Method
Transparent zone diameter (mm) Water solvent 70% ethanol solvent
S. aureus

Ultra High Pressure Extraction (Add Crude Extraction) Example 1 10 Example 3 14 Ultra High Pressure Extraction Example 2 8 Example 4 12 Hot water (reflux) extraction Comparative Example 1 - Comparative Example 3 10 Ultrasonic extraction Comparative Example 2 - Comparative Example 4 8 Methyl paraben 11
P. aeruginosa

Ultra High Pressure Extraction (Add Crude Extraction) Example 1 9 Example 3 13 Ultra High Pressure Extraction Example 2 7 Example 4 12 Hot water (reflux) extraction Comparative Example 1 - Comparative Example 3 9 Ultrasonic extraction Comparative Example 2 - Comparative Example 4 7 Methyl paraben 12
E. Coli

Ultra High Pressure Extraction (Add Crude Extraction) Example 1 12 Example 3 20 Ultra High Pressure Extraction Example 2 10 Example 4 19 Hot water (reflux) extraction Comparative Example 1 8 Comparative Example 3 10 Ultrasonic extraction Comparative Example 2 8 Comparative Example 4 9 Methyl paraben 18
C. albicans

Ultra High Pressure Extraction (Add Crude Extraction) Example 1 10 Example 3 16 Ultra High Pressure Extraction Example 2 9 Example 4 13 Hot water (reflux) extraction Comparative Example 1 7 Comparative Example 3 9 Ultrasonic extraction Comparative Example 2 7 Comparative Example 4 8 Methyl paraben 13
A. niger

Ultra High Pressure Extraction (Add Crude Extraction) Example 1 13 Example 3 28 Ultra High Pressure Extraction Example 2 11 Example 4 25 Hot water (reflux) extraction Comparative Example 1 8 Comparative Example 3 10 Ultrasonic extraction Comparative Example 2 7 Comparative Example 4 8 Methyl paraben 24

- : no effect

As shown in Table 5, Examples 1 to 4 using the ultra-high pressure extraction method compared to Comparative Examples 1 to 4 using the hot water (reflux) extraction and the ultrasonic extraction method were carried out in two solvent conditions for four kinds of bacteria, yeast and filamentous fungi. All of them showed wider transparent zone diameters and showed superior antimicrobial effect. Especially, ultra high pressure extract using 70% ethanol aqueous solvent showed stronger antimicrobial activity against 4 kinds of bacteria, yeast and filamentous fungus than methyl paraben, which is known as a strong antimicrobial agent. It was.

Experimental Example 5: elastay activity inhibition test

In order to test the skin wrinkle inhibition and improvement effect of the Tohubak extract sample of the present invention, an elastase inhibition activity test was performed. Elastin is a component of the matrix layer in the dermis of the skin. As the skin ages, elastin decomposes, the matrix layer in the dermis collapses, and wrinkles are formed. Using this principle to measure the activity of elastin, an enzyme that degrades elastin, N-succinyl- (Ala) ₃ p-nitroaniline (N-succinyl- (Ala)) ₃ p-nitroaniline, Sigma) is a method of measuring the elastay activity by measuring the absorbance at a wavelength of 405nm the change in color generated by the decomposition of p-nitroaniline. PH 8.0, 0.267 M Trizma-HCl (Sigma), substrate solution 8.8 mM N-Succinyl- (Ala) ₃ p-nitroaniline, enzyme solution 10 μg / ml of pig pancreatic elastase (Elastase, Sigma, USA) Used as a concentration. After mixing 60 μl of buffer solution, 20 μl of substrate solution, and 100 μl of the sample solution diluted in each distilled water by concentration, 20 μl of enzyme solution was added and reacted for 15 minutes in a 25 ° C. constant temperature water bath. P-nitroaniline The amount of produced was measured at a wavelength of 405 nm using a microplate reader (UVT-06685, Thermo max, USA). At this time, the control group for measuring the elastase activity was measured in the same manner by adding the third distilled water instead of the sample, the case was set to obtain a color correction value for each of the third distilled water instead of the enzyme solution. The sample used in the test was set to PMSF (phenylmethylsulfonyl fluoride, Sigma, USA) which is a specific inhibitor of elastays to determine the epoch extract and the elastase inhibitory effect of the above Examples, Comparative Examples, Elastase inhibition rate was calculated numerically using 3 shown in Table 6. In Table 6, IC ₅₀ is the concentration of the sample required to inhibit the elastase activity by 50%, and the smaller the value, the higher the inhibition rate.

Elastase activity inhibition rate (%) = 100-((absorbance of sample / absorbance of control) x 100)

Extraction Method Elastase activity inhibition rate (IC ₅₀ , mg / ml) Water solvent 70% ethanol solvent Ultra High Pressure Extraction (Add Crude Extraction) Example 1 78.92 Example 3 2.81 Ultra High Pressure Extraction Example 2 80.11 Example 4 4.10 Hot water (reflux) extraction Comparative Example 1 More than 100 Comparative Example 3 9.24 Ultrasonic extraction Comparative Example 2 More than 100 Comparative Example 4 10.66 PMSF 0.07

As shown in Table 6, Examples 1 to 4 using the ultra-high pressure extraction method than the Comparative Examples 1 to 4 using the hot water (reflux) extraction, ultrasonic extraction method showed superior wrinkle improvement effect in both solvent conditions.

Experimental Example 6: Collagen Synthesis Effect Test

Collagen synthesis ability was measured to assay the skin wrinkle improvement effect of Tohubak extract of the present invention. Collagen is a component that forms the dermal matrix layer in the skin together with the elastin, and the skin gradually ages and components of the matrix layer in the dermis are decomposed to form wrinkles. The collagen is a component of the dermal matrix layer. By checking the synthetic effect, it is possible to confirm the effect of improving skin wrinkles.

Human dermal fibroblasts were seeded in 24-well microplates (1 × 10 ⁵ cells / well) and incubated in 37 ° C., 5% CO ₂ incubator for 24 hours. Each sample was incubated for 24 hours in DMEM medium containing no added serum. Collagen synthesis amount is carried out as follows using enzyme immunoassay.

Barries incubated for 24 hours were dispensed into 96-well microplates and coated overnight at 4 ° C. Washed three times with washing buffer (PBS-T; 0.05% Tween 20 in phosphate buffered saline) and a blocking solution (5% skin milk, Fluka) was added and blocked at 37 ° C for 1 hour. The blocking solution was discarded and washed three times with a washing buffer, and the primary antibody (rabbit anti-collagen type I, Sigma) was diluted in PBS-T, and added to 100 µl and reacted at 37 ° C for 2 hours. After washing three times with a washing buffer, the secondary antibody (anti-rabbit IgG alkaline phosphatase conjugate, Sigma) was diluted in PBS-T and added to 100μL and reacted for 2 hours at 37 ℃. After washing three times with washing buffer, 100 μl of alkaline phosphatase substrate solution (Sigma) was added and developed at 25 ° C., followed by a microplate reader (UVT-06685, Termo max, USA) at 405 nm. Absorbance was measured.

The control group for measuring the collagen synthesis effect is the reaction absorbance of the cell culture without the sample, in order to determine the collagen synthesis effect, ascorbic acid was set as a comparison group three times, and the average value was obtained.

The collagen synthesis effect was calculated numerically using Equation 4 and is shown in Table 7.

Collagen
Synthetic Effect (%)







Extraction solution
Extraction Method
sample Concentration (ug / ml) 0 One 10 100 water
Ultra High Pressure Extraction (Add Crude Extraction) Example 1 100.00 103.37 108.16 111.64 Ultra High Pressure Extraction Example 2 100.00 102.13 104.55 109.58 Hot water (reflux) extraction Comparative Example 1 100.00 94.71 98.37 101.53 Ultrasonic extraction Comparative Example 2 100.00 95.41 96.54 98.29 70% ethanol aqueous solution

Ultra High Pressure Extraction (Add Crude Extraction) Example 3 100.00 105.97 113.54 126.84 Ultra High Pressure Extraction Example 4 100.00 104.88 111.33 121.19 Hot water (reflux) extraction Comparative Example 1 100.00 101.31 102.46 106.36 Ultrasonic extraction Comparative Example 2 100.00 100.35 101.79 102.99 Ascorbic acid 100.00 108.45 123.57 131.58

As shown in Table 7, Examples 1 to 4 using the ultra-high pressure extraction method than the comparative examples 1 to 4 using the hot water (reflux) extraction, ultrasonic extraction method showed a superior wrinkle improvement effect in both solvent conditions .

Experimental Example 7: Evaluation of MMP-1 Expression Inhibition by Tohubac Extract after UV Irradiation

In order to assay MMP-1 expression by measuring the concentration of MMP-1 after UV irradiation and sample addition of the Tohubac extract of the present invention, enzyme immunoassay (ELISA) was performed as follows.

Human dermal fibroblasts are irradiated with UVA at an energy of 5 J / cm 2 using a UV chamber. UV irradiation dose and culture time were established through preliminary experiments to maximize the expression of MMP in fibroblasts. Negative controls were wrapped in tinfoil and maintained at the same time in the environment of UVA. UVA emission was measured using a UV radiometer. The cells during the UVA irradiation were intact in the previously dispensed medium and irradiated with UVA and then exchanged with the medium containing the samples for 24 hours of incubation and the medium was recovered and coated in 96-well. The primary antibody (MMP-1 (Ab-5) monoclonal antibody and MMP-2 (Ab-3) monoclonal antibody) was treated and reacted for 60 minutes at 37 ℃. After reacting the secondary antibody anti-mouse IgG (whole mouse, alkaline phosphatase conjugated) for about 60 minutes, the alkaline phosphatase substrate solution (1mg / ml ρ-nitrophenyl phosphate in diethanolamine buffer solution) was reacted at room temperature for 30 minutes and microplates. Absorbance is measured at 405 nm using a reader (UVT-06685, Termo max, USA). In this case, the control group was used without adding a sample, and in order to evaluate the inhibition of MMP-1 expression, retinol, which was known to be excellent in inhibiting MMP-1 expression, was set as a comparative group, and the results are shown in Table 8.

Extraction solvent Extraction Method sample Treatment concentration (%) MMP-1 expression inhibition rate (%)
water

Ultra High Pressure Extraction (Add Crude Extraction) Example 1 0.1 11 Ultra High Pressure Extraction Example 2 0.1 10 Hot water (reflux) extraction Comparative Example 1 0.1 5 Ultrasonic extraction Comparative Example 2 0.1 3
70% ethanol aqueous solution

Ultra High Pressure Extraction (Add Crude Extraction) Example 3 0.1 28 Ultra High Pressure Extraction Example 4 0.1 19 Hot water (reflux) extraction Comparative Example 3 0.1 13 Ultrasonic extraction Comparative Example 4 0.1 11 Retinol 0.1 18

As shown in Table 8, Examples 1 to 4 using the ultra-high pressure extraction method compared to Comparative Examples 1 to 4 using the hot water (reflux) extraction, ultrasonic extraction method is superior to both MMP-1 expression inhibition effect in both solvent conditions In particular, the ultra high pressure Tobacco extract showed more than 28% inhibition rate compared to the control group that did not process the MMP-1 expression induced by UV irradiation, which is superior to the inhibition rate of the retinol used as a control group. It was.

Experimental Example 8: Mitigation Effect of Cytotoxicity by Ultraviolet Irradiation

To confirm the cytotoxicity mitigation effect by UV irradiation of the Tohubak extract of the present invention was tested as follows.

Fibroblasts were placed in 24-well test plates 1 × 10 ⁵ and attached for 24 hours. Each well was washed once with PBS and 500 μl of PBS was added to each well. The fibroblasts were irradiated with ultraviolet rays 1 mJ / cm ² using an ultraviolet B (UVB) lamp (Model: F15T8, UV B 15W, Sankyo Dennki, Japan), and then PBS was removed and 10% FBS was added to the cell culture medium (DMEM). 1 ml) was added. After treating the Tohubak extract to be evaluated therein was incubated for 24 hours. After 24 hours, the medium was removed, and 500 μl of cell culture medium and 60 μl of MTT solution (2.5 mg / ml) were added to each well, followed by incubation in a 37 ° C. CO ₂ incubator for 2 hours. The medium was removed, and 500 μl of isopropanol-HCl (0.04N) was added thereto. After shaking for 5 minutes, the cells were lysed and 100 μl of the supernatant was transferred to a 96-well test plate, and then absorbance was measured using a microplate reader (UVT-06685, Termo max, USA). The cell survival rate (%) was measured by Equation 5 and the cytotoxicity relaxation rate by ultraviolet rays was calculated by Equation 6 and shown in Table 9.

Cell survival rate (%) = [(St-Bo) / (Bt-Bo)] X 100

Bo: 565 nm absorbance of wells that developed cell culture media only

Bt: 565 nm absorbance of the wells which developed and reacted wells without sample

St: 565 nm absorbance of the wells which reacted the sample treated wells

Reduction rate of cytotoxicity by ultraviolet ray (%) = [1- (St-Bo) / (Bt-Bo)] X 100

Bo: cell viability of wells without UV irradiation and no sample treatment

Bt: cell viability of wells that were not irradiated with UV light

St: Cell survival rate of wells treated with UV

Extraction solvent Extraction Method sample Treatment concentration (%) Cytotoxic Relaxation Rate (%)
water

Ultra High Pressure Extraction (Add Crude Extraction) Example 1 0.1 29 Ultra High Pressure Extraction Example 2 0.1 27 Hot water (reflux) extraction Comparative Example 1 0.1 8  Ultrasonic Extraction Comparative Example 2 0.1 8
70% ethanol aqueous solution
Ultra High Pressure Extraction (Add Crude Extraction) Example 3 0.1 40 Ultra High Pressure Extraction Example 4 0.1 31 Hot water (reflux) extraction Comparative Example 3 0.1 13 Ultrasonic extraction Comparative Example 4 0.1 11

As shown in Table 9, Examples 1 to 4 using the ultra-high pressure extraction method than the comparative examples 1 to 4 using the hot water (reflux) extraction, ultrasonic extraction method shows superior cytotoxicity relaxation rate in both solvent conditions It was found to effectively protect against cytotoxicity caused by UV light.

Experimental Example 9: Inhibitory Effect of Inflammatory Cytokine Expression by Ultraviolet Irradiation

To confirm the inhibitory effect of inflammatory cytokine expression by UV irradiation of the Tohubak extract of the present invention was tested as follows.

Fibroblasts isolated from human epidermal tissue (Fibroblast) were placed in a 24-well test plate each 5 x 10 ⁴ and attached for 24 hours. Each well was washed once with PBS and 500 μl of PBS was added to each well. The fibroblasts were irradiated with UV 10mJ / cm ² using an ultraviolet B (UVB) lamp (Model: F15T8, UV B 15W, Sankyo Dennki Co., Japan), after which PBS was removed and FBS was not added to the cell culture medium (DMEM). Medium) 350 μl was added. After treating the Tohubak extract to be evaluated here was incubated for 5 hours. 150 μl of the culture supernatant was taken to quantify IL-1α to determine the inhibitory effect of inflammatory cytokine expression of Tohubac extract. The amount of IL-1α was quantified using Enzyme-linked Immunosorbent Assay, and the production rate of IL-1α was calculated by Equation 7 and the results are shown in Table 10.

Inhibitory rate of inflammatory cytokine expression (%) = [1- (St-Bo) / (Bt-Bo)] X 100

Bo: IL-1α production in wells without UV irradiation

Bt: IL-1α production in wells that were not irradiated with UV light

St: IL-1α production amount of wells that were irradiated with UV light

Extraction solvent Extraction Method sample Treatment concentration (%) Inhibitory rate of inflammatory cytokine expression (%)
water

Ultra High Pressure Extraction (Add Crude Extraction) Example 1 0.1 22 Ultra High Pressure Extraction Example 2 0.1 16 Hot water (reflux) extraction Comparative Example 1 0.1 4  Ultrasonic Extraction Comparative Example 2 0.1 2
70% ethanol aqueous solution
Ultra High Pressure Extraction (Add Crude Extraction) Example 3 0.1 40 Ultra High Pressure Extraction Example 4 0.1 38 Hot water (reflux) extraction Comparative Example 3 0.1 11 Ultrasonic extraction Comparative Example 4 0.1 8

As shown in Table 10, Examples 1 to 4 using the ultra-high pressure extraction method compared to Comparative Examples 1 to 4 using the hot water (reflux) extraction, ultrasonic extraction method using a general extraction method is superior inflammatory in both solvent conditions Inhibition of cytokine expression was found to effectively protect against inflammation caused by ultraviolet rays.

Formulation Example 1 and Formulation Comparative Example 1

The ingredient composition of the nourishing cream, including the ultra-high pressure Tobacco extract according to the embodiment was prepared as shown in Table 11.

number ingredient Formulation Example 1 Formulation Comparative Example 1 One Lipophilic monostearate 2.0 2.0 2 Stearic acid 1.5 1.5 3 Cetearyl alcohol 2.2 2.2 4 Beeswax 1.0 1.0 5 Squalane 3.0 3.0 6 Hardened vegetable oil 1.0 1.0 7 Sorbitan stearate 0.6 0.6 8 Mineral oil 5.0 5.0 9 Polysorbate 60 1.5 1.5 10 Dimethicone 1.0 1.0 11 Trioctanoine 5.0 5.0 12 Betaine 3.0 3.0 13 Triethanolamine 1.0 1.0 14 glycerin 5.0 5.0 15 Sodium hyaluronate 3.0 3.0 16 Example 1 2.0 - 17 Distilled water Balance Balance 18 Preservative, fragrance, coloring a very small amount a very small amount

Of the components composed of the composition of Table 11, the aqueous components of the raw materials 12-17 are first dissolved completely by heating to 80 ° C., and then the raw materials 1 to 11 are heated to 80 ° C., added to a solution of the raw materials 12-17, and a homomixer. (Homo Mixer Mark II, Primix, Japan) was used to emulsify at 3,000 rpm for 15 minutes. Then, the raw material 18 was thrown in, stirred for 5 minutes, and cooled to room temperature.

Formulation Example 2 and Formulation Comparative Example 2

The composition of the composition of the nutrition cream including the ultra high pressure Tobacco extract according to the embodiment was prepared as shown in Table 12.

number ingredient Formulation Example 2 Formulation Comparative Example 2 One Lipophilic monostearate 2.0 2.0 2 Stearic acid 1.5 1.5 3 Cetearyl alcohol 2.2 2.2 4 Beeswax 1.0 1.0 5 Squalane 3.0 3.0 6 Hardened vegetable oil 1.0 1.0 7 Sorbitan stearate 0.6 0.6 8 Mineral oil 5.0 5.0 9 Polysorbate 60 1.5 1.5 10 Dimethicone 1.0 1.0 11 Trioctanoine 5.0 5.0 12 Betaine 3.0 3.0 13 Triethanolamine 1.0 1.0 14 glycerin 5.0 5.0 15 Sodium hyaluronate 3.0 3.0 16 Example 2 2.0 - 17 Distilled water Balance Balance 18 Preservative, fragrance, coloring a very small amount a very small amount

Among the components composed of the composition of Table 12, first, the aqueous components of the raw materials 12-17 are completely dissolved by heating to 80 ° C., and then the raw materials 1 to 11 are heated to 80 ° C., added to a solution of the raw materials 12-17, and a homomixer. (Homo Mixer Mark II, Primix, Japan) was used to emulsify at 3,000 rpm for 15 minutes. Then, the raw material 18 was thrown in, stirred for 5 minutes, and cooled to room temperature.

Formulation Example 3 and Formulation Comparative Example 3

The ingredient composition of the nourishing cream including the ultra high pressure Tobacco extract according to the embodiment was prepared as shown in Table 13.

number ingredient Formulation Example 3 Formulation Comparative Example 3 One Example 3 2.0 - 2 Lipophilic monostearate 2.0 2.0 3 Stearic acid 1.5 1.5 4 Cetearyl alcohol 2.2 2.2 5 Beeswax 1.0 1.0 6 Squalane 3.0 3.0 7 Hardened vegetable oil 1.0 1.0 8 Sorbitan stearate 0.6 0.6 9 Mineral oil 5.0 5.0 10 Polysorbate 60 1.5 1.5 11 Dimethicone 1.0 1.0 12 Trioctanoine 5.0 5.0 13 Betaine 3.0 3.0 14 Triethanolamine 1.0 1.0 15 glycerin 5.0 5.0 16 Sodium hyaluronate 3.0 3.0 17 Distilled water Balance Balance 18 Preservative, fragrance, coloring a very small amount a very small amount

Of the components composed of the composition of Table 13, first, the aqueous components of the raw materials 13-17 are completely dissolved by heating to 80 ° C, and then the raw materials 1-11 are heated to 80 ° C and added to the solution of the raw materials 13-17 and homomixer (Homo Mixer Mark II, Primix, Japan) was used to emulsify at 3,000 rpm for 15 minutes. After that, the raw material 18 was added and stirred for 5 minutes, and then cooled to room temperature.

Formulation Example 4 and Comparative Formulation Example 4

The composition of the composition of the nourishing cream, including the ultra high pressure Tobacco extract according to the embodiment, was prepared as shown in Table 14.

number ingredient Formulation Example 4 Formulation Comparative Example 4 One Example 4 2.0 - 2 Lipophilic monostearate 2.0 2.0 3 Stearic acid 1.5 1.5 4 Cetearyl alcohol 2.2 2.2 5 Beeswax 1.0 1.0 6 Squalane 3.0 3.0 7 Hardened vegetable oil 1.0 1.0 8 Sorbitan stearate 0.6 0.6 9 Mineral oil 5.0 5.0 10 Polysorbate 60 1.5 1.5 11 Dimethicone 1.0 1.0 12 Trioctanoine 5.0 5.0 13 Betaine 3.0 3.0 14 Triethanolamine 1.0 1.0 15 glycerin 5.0 5.0 16 Sodium hyaluronate 3.0 3.0 17 Distilled water Balance Balance 18 Preservative, fragrance, coloring a very small amount a very small amount

Of the components composed of the composition of Table 14, first, the aqueous components of the raw materials 13-17 are completely dissolved by heating to 80 ° C., and then the raw materials 1 to 11 are heated to 80 ° C., added to a solution of the raw materials 13-17, and a homomixer. (Homo Mixer Mark II, Primix, Japan) was used to emulsify at 3,000 rpm for 15 minutes. Then, the raw material 18 was thrown in, stirred for 5 minutes, and cooled to room temperature.

Experimental Example 10: Wrinkle improvement effect measurement

In order to measure the anti-wrinkle effect of the nourishing cream prepared in the formulation example and the formulation comparative example was measured through the following method. Forty females in their 30s or older undergoing skin aging are treated with the nourishing creams of Formulation Example 1 and Comparative Example 1 on both crow feet for 12 weeks. At intervals, the wrinkles around the eyes (replica) were measured using a skin visiometer (SV-600, C + K, Germany) by image analysis, and the depth (μm) of the wrinkles was measured.

Average value (A.U.) Formulation
Example 1 Formulation
Comparative Example 1 Formulation
Example 2 Formulation
Comparative Example 2 Formulation
Example 3 Formulation
Comparative Example 3 Formulation
Example 4 Formulation
Comparative Example 4 Before application 330 ± 31 312 ± 27 324 ± 33 320 ± 29 341 ± 13 336 ± 29 310 ± 33 325 ± 20 12 weeks after application 258 ± 17 301 ± 15 269 ± 26 311 ± 22 234 ± 21 319 ± 28 239 ± 19 315 ± 16 Wrinkle depth
Decrease (%) 21.8 3.5 17.0 2.8 31.3 5.1 23.0 3.1

As shown in the results of Table 15, it was confirmed that the nutrition cream of the formulation example to which the ultra high pressure Tobacco extract was added, compared with the nutrition cream of the comparative example of the formulation without the ultra high pressure Tobacco extract, had an excellent wrinkle improvement effect.

Experimental Example 11: HPLC Analysis

Analysis was performed using HPLC (High pressure liquid chromatography) to compare the constituents of the Tohubak extract of the present invention. Samples were prepared with appropriate concentrations in each solvent, filtered through a 0.2 μm filter, and injected into the apparatus after pretreatment. The instrument was analyzed in the UV 254-340 nm region using HPLC (Agilent 1200series, Agilent, USA) and 2% acetic acid solvent and aceto using C ₁₈ column (Zorbax XDB-C ₁₈ 4.6 x 250 mm, Agilent, USA) It was analyzed by crying method using nitrile solvent. The solvent used for the analysis was an HPLC grade reagent, and the standards were caffeic acid (Sigma, USA), (+)-catechin (Sigma, USA), (-)-epicatechin (Sigma, USA), epigallo Component analysis was performed using catechin gallate (EGCG, Sigma, USA), Didzin (Sigma, USA), and meso-dihydroguaiatinic acid (Elcom science, Korea). The analysis results are shown in FIG. 1.

Hereinafter, as an example of the formulation of the present invention, a flexible lotion (Table 16), a convergent lotion (Table 17), a nourishing lotion (Table 18), an essence (Table 19) and a pack (Table 20) are exemplified, but the functional cosmetic composition of the present invention The formulations should not be construed as limited thereto, and conventional variations of those skilled in the art are possible within the scope of the present invention.

number ingredient content(%) One glycerin 5.00 2 1,3-butylene glycol 3.00 3 PEG 1500 1.00 4 Allantoin 0.10 5 DL-Panthenol 0.30 6 EDTA-2Na 0.02 7 Benzophenone-9 0.04 8 ethanol 10.00 9 Octicdodeceth-16 0.20 10 Polysorbate 20 0.20 11 Ultra High Pressure Tobacco Extract 5.0 12 Preservative, fragrance, coloring a very small amount 13 Distilled water Balance

number ingredient content(%) One glycerin 2.00 2 1,3-butylene glycol 2.00 3 Allantoin 0.10 4 DL-Panthenol 0.30 5 EDTA-2Na 0.02 6 Benzophenone-9 0.04 7 ethanol 15.00 8 Polysorbate 20 0.20 9 Ultra High Pressure Tobacco Extract 3.0 10 Citric acid a very small amount 11 Preservative, fragrance, coloring a very small amount 12 Distilled water Balance

number ingredient content(%) One Cetearyl alcohol 1.00 2 Glyceryl Stearate 0.50 3 Polysorbate 60 1.00 4 Sorbitan sesquioleate 0.30 5 Cetyloctanoate 6.00 6 Squalane 4.00 7 Flower oil 4.00 8 Butylene Glycol 4.00 9 glycerin 4.00 10 Carbomer 0.10 11 Triethanolamine 0.10 12 Ultra High Pressure Tobacco Extract 1.00 13 Preservative, fragrance, coloring a very small amount 14 Distilled water Balance

number ingredient content(%) One glycerin 10.00 2 Betaine 5.00 3 PEG 1500 2.00 4 Allantoin 0.10 5 DL-Panthenol 0.30 6 EDTA-2Na 0.02 7 Benzophenone-9 0.04 8 Hydroxyethylcellulose 0.10 9 Carboxyvinyl Polymer 0.20 10 Triethanolamine 0.18 11 Octyldodecanol 0.30 12 Octyldodec-16 0.40 13 ethanol 6.00 14 Ultra High Pressure Tobacco Extract 5.00 15 Preservative, fragrance, coloring a very small amount 16 Distilled water Balance

number ingredient content(%) One Polyvinyl alcohol 15.00 2 Cellulose gum 0.15 3 glycerin 3.00 4 PEG 1500 2.00 5 Chicdestrin 0.15 6 DL-Panthenol 0.30 7 Allantoin 0.10 8 Glycyrisin Monoammonium 0.20 9 Nicotine amide 0.40 10 ethanol 5.00 11 PEG 40 Cured Castor Oil 0.30 12 Ultra High Pressure Tobacco Extract 1.00 13 Preservative, fragrance, coloring a very small amount 14 Distilled water Balance

Figure 1a relates to HPLC component analysis chromatogram of Tohubak ultra high pressure extract.

Figure 1b relates to HPLC component analysis chromatogram of Tohubak hydrothermal extract.

Claims (6)

delete delete Adding an extraction solvent to the soil foil sample; A pressurizing step of putting an extraction solvent containing a sample into a chamber of an ultrahigh pressure machine and applying an ultrahigh pressure of 100 MPa or more to extract the sample; A separation and purification process of separating the extract from the sample; Tofubak extract manufacturing method comprising a; lyophilized or concentrated the purified extract. The method of claim 3, The extraction solvent is characterized in that using water or ethanol. The method of claim 3, And adding a crude extract material after the step of adding the extraction solvent to the sample. The method according to claim 5, The crude extract material is characterized in that at least one selected from glass beads (glass beads), silica beads (silica beads) and sand.

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