KR101894254B1 - Cosmetic composition for improving anti-microbial and anti-wrinkling comprising extract of coptidis rhizoma and manufacturing method thereof - Google Patents

Abstract

The present invention relates to an antibacterial and wrinkle-ameliorating cosmetic composition containing a Coptis chinensis extract, and a production method thereof. To this end, the cosmetic composition contains the Coptis chinensis extract and berberine isolated from the Coptis chinensis extract and improves antibacterial and wrinkle-ameliorating functions. In addition, the cosmetic composition is composed of natural substances only, thereby enabling production of cosmetic compositions capable of normally recovering skin functions with little skin irritation.

Description

TECHNICAL FIELD The present invention relates to a cosmetic composition for antimicrobial and wrinkle-improving cosmetic compositions,

The present invention relates to a cosmetic composition for antimicrobial and wrinkle-reducing cosmetic compositions containing a Rhodiola extract and a method for producing the same. More particularly, the present invention relates to a cosmetic composition having antimicrobial and wrinkle-improving effects, including Berberine isolated from Rhodiola extract and Rhodiola, and a method for producing the same.

Skin aging can be divided into stress, disease, environmental factors, wounds, or photo aging that occurs due to internal aging and exposure to light, such as ultraviolet light, as a result of age.

Skin aging is caused by the destruction of the antioxidant defense membrane in vivo by the activated free radicals, which can result in the destruction of skin cells and tissues due to oxidation of lipids, proteins, polysaccharides and nucleic acids, which are major constituents of the skin.

In particular, when proteins are oxidized, collagen, hyaluronic acid, elastin, proteoglycan and fibronectin, which are the connective tissues of the skin, are severed, If it gets worse, DNA mutation leads to mutation, cancer induction, immune function deterioration.

Therefore, in order to prevent skin aging, it is necessary to prevent skin damage by eliminating free radicals caused by various causes. In addition, it is necessary to restore skin by regenerating and proliferating already damaged cells through active metabolism have.

In addition, skin aging can be promoted by matrix metalloproteinase (MMP), a collagenolytic enzyme. In other words, the synthesis and degradation of extracellular matrix such as collagen are controlled in vivo, but when aging proceeds, collagen synthesis is reduced and the expression of collagenase MMP is promoted, resulting in reduced skin elasticity and wrinkles .

Therefore, in order to prevent skin aging and wrinkle formation, it is necessary to regulate MMP expression induced in cells or inhibit its activity.

In addition, human skin is an organ in direct contact with the surrounding environment, and infections such as bacteria and fungi which are involved in various skin diseases are easily caused. For example, Staphylococcus aureus, which is a staphylococcus aureus causing skin irritation to skin, Staphlyococcus epidermidis, a skin-damaging agent, and Propionibacterium acnes, acnes) are present in the skin. These fungi decompose the sebum and sweat secreted on the skin to generate an odor. In addition, the degradation products stimulate the skin and cause inflammation. Propionibacterium acnes, in particular, uses lipase, a fatty acid degrading enzyme, to decompose sebum to produce free fatty acids. These free fatty acids not only stimulate the skin but also cause inflammatory lesions such as papules, pustules, and nodules, which are red rashes found in acne.

Antimicrobial compounds such as triclocarban and triclosan have been used as antimicrobial agents against such bacteria, but they are known to cause side effects such as skin burning, burning, and skin redness during long-term use.

Conventional anti-aging-related cosmetic compositions or antimicrobial cosmetic compositions use synthetic materials. However, the cosmetic composition containing such a synthetic substance may cause skin irritation when used and may cause skin troubles It is necessary to develop a cosmetic composition using natural materials.

(Patent Document 1) KR 10-2016-0122994 A1 (Patent Document 2) KR 10-2013-0027362 A1

It is an object of the present invention to provide a cosmetic composition for antibacterial and wrinkle reduction comprising a Rhodiola extract and a method for producing the same.

Another object of the present invention is to provide a cosmetic composition which has improved antimicrobial and wrinkle-reducing effects, including Berberine, which is isolated from Rhodiola extract and Rhodiola extract, and a method for producing the same.

In order to attain the above object, the cosmetic composition for antibacterial and wrinkle improvement according to one embodiment of the present invention includes a water extract of Wanghsin and a berberine, wherein the bergulin is a low molecular weight gel extract The filtrate was subjected to a primary separation using Sephadex LH-20, and the separated fractions were secondarily separated using MCI-gel CHP 20 as a porous gel column chromatography. The separated fractions were separated into YMC-gel ODS-A -HG column chromatography, followed by tertiary separation.

The Hwangryun hot-water extract is a fermented fermented hot-water extract obtained by fermenting rice with hot water.

The cosmetic composition for antibacterial and wrinkle-improving may further comprise a water extract of Yedok tree.

The extract of Yedok tree is a fermented Yedok tree hydrothermal extract fermented with rice flour.

The cosmetic composition for antibacterial and wrinkle-improving may further comprise a hill root hot water extract.

The above-mentioned hot-water extrudate is a fermented hot-water extruded product obtained by fermenting rice with hot water.

The cosmetic composition may be in the form of a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax foundation and spray Lt; / RTI >

According to another embodiment of the present invention, there is provided a method for preparing a cosmetic composition for antibacterial and wrinkle improvement comprising the steps of: 1) preparing a hydrolyzed hot water extract using dry fermentation; 2) introducing the fermented hot-water extract of step 1) into a column filled with low-molecular gel filtration Sephadex LH-20 to obtain a first fraction; 3) introducing the primary fraction of step 2) into MCI-gel CHP 20, which is a porous gel column chromatography, to obtain a second fraction; 4) separating the separated second fraction from step 3) by YMC-gel ODS-A-HG column chromatography to obtain berberine; And 5) mixing berberine in step 4) with the fermented hot-water extract of step 1).

The step 5) may further include a Yedok tree hot water extract.

5 to 10 parts by weight of a saccharide and 1 to 2 parts by weight of a strain are mixed with 100 parts by weight of rice soot, 5 to 10 parts by weight of the pulverized product may be immersed in water, and then air may be cut off at 10 to 40 DEG C, followed by extraction from the fermented yeast tree for 5 to 10 days.

The strain may be an EM (Effective Micro-Organisms) activated solution.

The step 5) may further include a Hill root root hydrothermal extract.

Wherein the step (5) further comprises a fermentation hill root hot water extract,

5 to 10 parts by weight of a saccharide and 1 to 2 parts by weight of a strain are mixed with 100 parts by weight of rice soot, and 5 to 10 parts by weight of the dry finely pulverized product is immersed, Lt; RTI ID = 0.0 > 5 C < / RTI > for 10 days.

Hereinafter, the present invention will be described in more detail.

According to one embodiment of the present invention, the cosmetic composition for antibacterial and wrinkle improvement of the present invention includes a hydrothermal extract and a berberine.

Coptis chinensis is a perennial herbaceous plant belonging to the family Ranunculaceae. It is also grown in Korea, Japan, and China for herbal medicine. It lives in the ecological land of shrubs in mountainous regions. It is thick and broad in side, has many hairs and has 4 ~ 5 roots at the end of stem. Its total length is about 10 ~ 27cm. In Oriental medicine, the roots of 5 ~ 6 year old chrysanthemum and chrysanthemum are picked up in November and dried in the sun is called chrysanthemum, has a peculiar smell, its taste is used, and the quality is true. Components include isoquinoline alkaloids such as berberin, jateorrhizine, palmatine, coptisine, magnoflorine, epiberberin, berbestine, worenine and ferulic acid.

The Rhodiola extract may be extracted with a solvent selected from the group consisting of water, an alcohol having 1 to 6 carbon atoms, and a mixed solvent thereof. The term " Rhodiola extract " means an extract obtained by extracting Rhodiola. The Rhodiola extract may be eluted with water. At this time, the extraction temperature may be an extract extracted at 10 to 100 캜, preferably at room temperature, and for an extraction period of 12 to 4 days, preferably 24 hours. The filtered extract may be a result obtained by concentration under reduced pressure with a vacuum rotary condenser. However, it is not limited to this, and it includes all of the extract, the diluted solution or the concentrate of the extract, the dried product obtained by drying the extract, or the adjusted product or the purified product thereof.

More specifically, 1) a step of washing impurities (Coptis chinensis) in purified water to remove impurities; 2) drying the washed brass of step 1) in shade at 20 to 25 ° C; 3) adding 5 to 10 times of water to the dried fermentation and repeating the reflux extraction process for 3 hours at 80 to 90 ° C for 3 to 5 times to prepare a fermented extract; 4) obtaining a supernatant through a centrifugal separation process of step 3), and removing the extract residue by vacuum filtration to obtain a filtrate; And 5) removing the solvent by using a rotary evaporator in a rotary evaporator to freeze-dry the filtrate of step 4).

Berberine is a component contained in the extract of Huangryn extract. When the extract of Huangryn is taken or applied to the skin, the effect of berberine contained in Huangryn extract may be effective. However, the extract of Huangryn contains many components other than berberine, and when using only Huangryn extract, there is a problem that it is not enough to exhibit the antibacterial and wrinkle-reducing effect of berberine.

In the present invention, berberine is separately isolated from Huangyan extract. Therefore, the antibacterial and wrinkle-reducing effect can be enhanced as compared with the case where Huangyan extract alone is used.

The berberine was firstly isolated by using a gelatin hot-water extract using MCI-gel CHP 20 as a porous gel column chromatography, and the separated fractions were subjected to secondary separation by performing YMC-gel ODS-A-HG column chromatography will be. Column chromatography was carried out by increasing the concentration of methanol from water by 10% to 100%. The fractions were divided by confirming by TLC method.

That is, the extract of HuangYuan hydrothermal extract was separated by using porous gel column chromatography Sephadex LH-20 (H ₂ O → 100% MeOH, gradient system). The fractions were separated into three fractions (CWF 1, CWF 2 and CWF 3) Respectively. CWF3 was separated into CWF3-1, CWF3-2 and CWF3-3 by performing MCI-gel CHP 20P column chromatography (H ₂ O → 10% MeOH, gradient system). CWF3-3 was subjected to YMC-gel ODS-A-HG column chromatography (55% methanol) to obtain berberine.

More specifically, Coptis chinensis is washed in purified water to remove impurities, and the washed phloem is dried in the shade at 20 to 25 ° C. The dry fermentation is repeated 5 to 10 times with water, and the reflux extraction process is repeated 3 to 5 times at 80 to 90 ° C for 3 hours to prepare a Rhodiola extract. The fermentation extract is centrifuged to obtain only supernatant, and the filtrate is obtained by vacuum filtration to remove the extracted residue. The filtrate was subjected to rotary evaporation using a vacuum concentrator to remove the solvent, followed by lyophilization to give a yellowtail extract. The freeze-dried Rhodiola extract is mixed with water and filtered using a nonwoven filter. The filtered Huanghui extract is introduced into a column packed with Sephadex LH-20 for low molecular weight gel filtration and separated into three fractions (CWF1, CWF2 and CWF3). The separated product (CWF3) is separated into CWF3-1, CWF3-2 and CWF3-3 using MCI-gel CHP 20, a porous gel column chromatography. The separated CWF3-3 was subjected to YMC-gel ODS-A-HG column chromatography (55% methanol) to obtain berberine.

As described above, through the plurality of separations, it is possible to exhibit the optimum yield of berberine at the time of obtaining berberine.

The Huangyan hot-water extract may be a fermented Huanglong hot-water extract obtained by fermenting the rice with hot rice. In the case of using the fermented yellow radish hot water extract as compared to the case of using the fermented hot water extract as it is, the toxicity of the skin is weakened and the occurrence of side effects is less and the antibacterial and wrinkle reducing effect can be further enhanced even if the content of the yellow radish extract is increased.

In this case, the fermented yellowtail hot-water extract may be prepared by fermenting the dried and pulverized fermented rice with rice flour, and then extracting from the fermented fermented rice using the above extraction method.

The cosmetic composition of the present invention may further comprise a Yedok tree hot water extract.

It is also known that yeodong is also called yodong or redbag, and Yedok tree pex is currently used as a formulary (Korean Journal of Pharmacopoeia, 1994 Japanese Pharmacopoeia Explanatory Notebook, 1996). It has been reported that it is not toxic and has no effect on general pharmacological action and fetus. There are more than 20 species such as bergenin, malloperenol and rutin. Berbenin is an active ingredient of Pseudomonas plexus. It has antitumor activity, antihyperlipidemic effect, and toxicity in acute, night and chronic toxicity tests. It is said that there is little.

However, as compared with the case where the extract of Yedoaceae is used alone as a cosmetic, in the case of using the extract of Huanghui extract as in the present invention, the antimicrobial and wrinkle-reducing effect Rise.

The extract of Yedok tree is a fermented Yedok tree hydrothermal extract fermented with rice flour. As in the case of Rhodiola extract, the extract is superior to the extract extracted directly from the jade tree at the time of preparing the extract using the fermented jade tree using the rice flour.

The cosmetic composition for antibacterial and wrinkle improvement of the present invention may further comprise a hill root hot water extract. In addition to the Hill root root hydrothermal extract, it may further comprise a Hill root root hydrothermal extract and a Soybean curd hydrothermal extract.

By further including the hot-water extract of the present invention and the hot-water extract of the cone of the present invention in the cosmetic composition of the present invention, it is possible to further enhance the antibacterial and wrinkle- It is possible to show an effect of further increasing the feeling of use.

In other words, unlike the conventional natural cosmetics, the cosmetic composition of the present invention can minimize the skin irritation and exhibit a feeling similar to that of using the synthetic ingredient, by using a natural component.

The above-mentioned heel root refers to a dried root of the pupa. The spiny grass grows in a rather humid place. The main stem is 40 to 90 cm high, with vertical lines, the root leaves are about 50 cm long, and the five small leaves are white and long. The stem leaf is opposite, odd numbered compound leaf, composed of 3 to 7 small leaves with sawtooth on the edge. The flowers bloom in June to July, and are light pink.

It is an evergreen perennial plant that grows on the surface of wet rocks or in the snow. Its rhizome extends sideways, and the leaves run sparse. Nutrient is round or oval and has a flat edge. Sporophyll with sporang is spatulate, rounded and narrowed. It is known that it has excellent effects on improvement, acupuncture, acupuncture, blood decolonization, hemostasis, decolonization, toothache, nosebleeds, bruises, eruptions, rubella, seawater and hematuria.

The above-mentioned hot-water extract of the root and the hot-water extract of the bean curd are hot-water extracts obtained from the fermented product obtained by fermenting with rice flour.

More specifically, 5 to 10 parts by weight of saccharide and 1 to 2 parts by weight of a strain are mixed with 100 parts by weight of rice soot, and 5 to 10 parts by weight of the dried natural ground product is immersed The fermentation product can be fermented for 5 to 10 days after shutting off the air at 10 to 40 ° C. And is fermented at a temperature of 30 to 37 占 폚. The above temperature is a temperature at which yeast can grow. When the temperature exceeds 40 ° C, yeast growth is inhibited. When the temperature is higher than 50 ° C, the cells are destroyed. When the temperature is lower than 10 占 폚, there is a problem that the growth of yeast stops.

For example, the above-mentioned Hansenula polymorpha and Saccharomyces cerevisiae are optimal growth conditions at 30 ° C and Pichia pastoris at 37 ° C. The fermentation time is from 24 hours to 48 hours. When the fermentation product is cultured for less than 24 hours, sufficient fermentation does not occur. Therefore, when the fermentation product is cultured for more than 48 hours, Is not preferable.

In addition, the culture is a batch culture in which the cells are initially fermented without feeding any nutrients and without removing the product after the medium is initially filled. The rice bran is characterized in that the growth of the bacterium is not inhibited by the initially mixed sugar even if the oil-rich culture method is not used.

Preferably, the cosmetic composition for antimicrobial and wrinkle improvement of the present invention comprises a hydrolyzed hydrothermal extract, wherein 5 to 10 parts by weight of berberine, 10 to 20 parts by weight of herbal extract, 10 to 20 parts by weight of herb extract, 1 to 5 parts by weight of the extract and 1 to 5 parts by weight of the hot water extract of the bean curd. Within the above range, the synergistic effect of the antibacterial and wrinkle-reducing activities can be maximized by the synergistic action depending on the mixing use of each component.

The cosmetic composition may be in the form of a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax foundation and spray Lt; / RTI >

The cosmetic composition of the present invention may be prepared in any of the formulations conventionally produced in the art and may be in the form of a solution, a suspension, a paste, a gel, a cream, a lotion, a powder, a soap, a surfactant- A powder foundation, an emulsion foundation, a wax foundation, and a spray, but the present invention is not limited thereto. More particularly, the present invention relates to a cosmetic composition which is formulated into a flexible lotion, a nutritional lotion, a nutritional cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray or a powder, (W / O) type formulation or ointment.

When the formulation of the present invention is a paste, a cream or a gel, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component. In particular, in the case of a spray, a mixture of chlorofluorohydrocarbons, Propane / butane or dimethyl ether.

When the formulation of the present invention is a solution or an emulsion, a solvent, a dissolving agent or an emulsifying agent is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, , 1,3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or fatty acid esters of sorbitan.

In the case where the formulation of the present invention is a suspension, a carrier such as water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, Cellulose, aluminum metahydroxide, bentonite, agar or tracant, etc. may be used.

When the formulation of the present invention is an interfacial active agent-containing cleansing, the carrier component is selected from aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters.

The antimicrobial and wrinkle-improving cosmetic composition containing the extract of Rhododendron rhizome of the present invention and the method of manufacturing the same can improve antibacterial and wrinkle-reducing effects, including Berberine isolated from Rhodiola extract and Rhodiola extract.

In addition, it is possible to provide a cosmetic composition which is composed of only natural ingredients in the cosmetic composition and which can reduce skin irritation and restore skin function normally.

Fig. 1 shows experimental results on the antimicrobial effect of Huanglung extract.
Fig. 2 shows the results of experiments on antimicrobial activity after applying the extract of the Japanese agar to the wallpaper.
FIG. 3 shows the results of an experiment on the antimicrobial activity against the drop-dead bacteria of Huanghui extract.
FIG. 4 shows the results of experiments on the antimicrobial effect persistence of the extract of Huanghui.
FIG. 5 shows experimental results on the antimicrobial effect after the heat-treatment of the extract.
6 shows MIC and MBC test results.
Fig. 7 shows the results of toxicity test for berberine in CD-986sk cells.
Figure 8 shows the results of ROS production for CCD-986sk cells from berberine.
Fig. 9 shows experimental results on the production of hyaluronic acid during treatment with berberine.
Fig. 10 shows the results of protein synthesis and mRNA expression of MMP-2 and MMP-9.
Figure 11 shows the results of protein synthesis and mRNA expression of TIMP-1 and TIMP-2.
Figure 12 shows the expression of inflammatory cytokine TNF-a.
FIG. 13 is a result of skin irritation test on the extract of Huanghui.
FIG. 14 shows the results of an eye mucous membrane stimulation test for the extract of Angelica keiskei.

Hereinafter, embodiments of the present invention will be described in detail so that those skilled in the art can easily carry out the present invention. The present invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

[Preparation Example: Preparation of Rhodiola extract and the like]

1. Preparation of HuangYin extract

Coptis chinensis was washed with purified water and dried in the shade at room temperature. Thereafter, the dried rhododendrons were pulverized using a pulverizer, and then water in an amount of 5 to 10 times the weight of the pulverized pulverulent pulverized product was added thereto, followed by reflux extraction at 80 to 90 ° C for 3 hours. The above reflux extraction process was repeated three times. Thereafter, only the supernatant was obtained through a centrifugation process, and the filtrate was subjected to vacuum filtration to remove the extract residue and obtain a filtrate. The filtrate was subjected to rotary evaporation using a vacuum concentrator to remove the solvent, followed by lyophilization to prepare a Chinese Rhizome extract.

2. Obtaining berberine

The freeze-dried Rhodiola extract was mixed with water and filtered using a nonwoven filter. The filtered Huanghui extract was introduced into a column packed with a low-molecular gel filtration Sephadex LH-20 and separated into three fractions (CWF1, CWF2 and CWF3). The isolated product (CWF3) was separated into CWF3-1, CWF3-2 and CWF3-3 using MCI-gel CHP 20, a porous gel column chromatography. The separated CWF3-3 was subjected to YMC-gel ODS-A-HG column chromatography (55% methanol) to obtain berberine.

3. Preparation of natural extract

The extracts of Yedok tree, Hill root extract and Kidney japonica were prepared in the same manner as in the method of producing chrysanthemum extract.

4. Preparation of Fermented Rhodiola Extract

5 to 10 parts by weight of saccharide and 1 to 2 parts by weight of a strain were mixed with 100 parts of rice skin. Thereafter, 5 to 10 parts by weight of dried pulverized pulverized product was immersed, and air was shut off at 37 DEG C, followed by fermentation for 10 days to produce a fermented fermented product. Then, the fermented Rhodiola extract was prepared using the same method as the above-described method for producing the Rhodiola extract.

5. Preparation of fermented natural extract

Fermented Yedoaceae extract, Fermented Hill root extract and Fermented Cowpea vine extract were prepared in the same manner as in the above-mentioned four fermented hot pepper extracts.

6. Preparation of cosmetic composition

The composition of the cosmetic composition was prepared by mixing the extracts of Huangyu extract, Berberine, Yukdok tree extract, Hill root extract, Cowberry vine extract, Fermented Huanghui extract, Fermented Yukdeok extract, Fermented Hill root extract and Fermented Cowberry extract as shown in Table 1 below.

A1 A2 A3 A4 A5 A6 A7 A8 A9 Experimental Example 1 100 - - - - - - - - Experimental Example 2 - 100 - - - - - - - Experimental Example 3 100 10 - - - - - - - Experimental Example 4 100 One 5 0.5 - - - - - Experimental Example 5 100 5 10 One - - - - - Experimental Example 6 100 10 20 5 - - - - - Experimental Example 7 100 15 25 10 - - - - - Experimental Example 8 100 5 10 One One - - - - Experimental Example 9 100 5 10 One 5 - - - - Experimental Example 10 - 5 - - - 100 10 One 5

[ Example : Antibacterial and wrinkle improvement effect test]

1. Experimental Method

Disk diffusion method

The antibacterial activity was measured by disc diffusion method. When _OD660 = 0.8, the liquid medium was cultured on a solid medium to have a concentration of 1 x 10 ⁶ CFU / mL. A sterilized paper disc (8 mm) was adhered onto the solid medium, and the sample was treated and sufficiently absorbed. After culturing in an incubator at 37 ° C for 24 hours, the clear zone diameter around the disc was measured to compare the antimicrobial activity of each extract.

Replica Plating Method (JIS Z 2801: 2000)

The specimens were cut into 5x5 cm squares and autoclaved. The sterilized specimens were mixed with the bacterial solution and the samples, respectively, or mixed and then smeared. The stained specimens were incubated at 37 ° C for 24 hours.

MIC and MBC experiments

Samples (500μ) in a liquid medium (500μL) so that the culture, 1x10 ⁴ CFU / mL when _.660 OD = 0.8 were mixed in 24well il. After 24 h, the cells were plated on solid medium.

Antifungal activity test

The modified agar diffusion method was used to measure antifungal activity. A mold plug made of a punching machine was placed on the medium and a paper disc containing the sample was placed. Thereafter, the cells were cultured in an incubator at 25 캜 for 3 to 7 days depending on the type of the cells.

MTT analysis

Cells were seeded in 96-well plates at a density of 5 × 10 ³ cells / well (0.18 mL). Samples were prepared at respective concentrations, 0.02 mL was added, and the cells were incubated at 37 ° C in a 5% CO ² incubator for 24 hours. After adding 0.02 mL of MTT solution (5 mg / mL), the culture solution was removed for 4 hours. Then, 0.15 mL of DMSO was added to each well and reacted at room temperature for 15 minutes. Absorbance was measured at 540 nm with an ELISA reader. Cytotoxicity measurements were expressed as the absorbance reduction rate of the sample solution addition group and the no addition group.

Measurement of reactive oxygen species between cells

Cells were incubated at 2 × 10 ⁵ cells / well in a 96-well plate and stabilized for 24 hours. After replacing with the same medium and stimulating with UVB of 20 mJ / cm ² , the sample was diluted by concentration and cultured for 48 hours. Then, the ROS Glo H ₂ O ₂ assay was performed according to the manufacturer's manual (Thermo Fisher Scientific, Waltham, USA).

HA ELISA assay

The amount of HA in the cell culture was measured using an ELISA kit (Echelon, UT, USA). The cells were adjusted to 5 × 10 ⁴ cells / mL using DMEM medium, inoculated in a 48-well plate, and cultured in a 5% CO ₂ incubator for 24 hours. The cells were stimulated with UVB at 20 mJ / cm ² and then treated with a concentration-dependent sample for 48 hours. After incubation, supernatant was taken and HA was measured.

Protein determination using Western blot

Cells were plated at 6 × 10 ⁵ cells / well in a 6-well plate, cultured for 24 hours, stimulated with UVB at 20 mJ / cm ² , and cultured for 48 hours. Cells were lysed with RIPA buffer (Pierce, IL, USA) and centrifuged (12,000 rpm, 4 ° C, 30 min) to remove cell membrane components and supernatants were obtained and quantified by Bradford assay. 20 μL of protein was electrophoresed using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred to a PVDF membrane to inhibit non-specific binding of the antibody and then transferred at 60 V for 2 hours or more. MMP-9, TNF-α, TIMP-1, TIMP-2, and β-actin were reacted with primary and secondary antibodies of MMP-2, . After reaction, the cells were reacted with Immobilon Western Chemiluminescent HRP substrate (Millipore, MA, USA) for 60 minutes and developed using a LAS 4000 analyzer (Fuji Film Life Science, Tokyo, Japan).

RNA isolation and RT-PCR

Cells were plated at 6 × 10 ⁵ cells / well and cultured for 48 hours. The medium was removed and stimulated with UVB at 20 mJ / cm < ² >, then the sample was treated with concentration and cultured for 48 hours. The total RNA was washed three times with PBS, and the cells were harvested and then separated using trizol reagent (Invitrogen, Carlsbad, CA, USA). 2 μg of total RNA and PCR primer oligonucleotide were mixed with RT- PCR was performed.

Cells were lysed with lysis buffer and centrifuged at 4 ° C and 12,000 rpm for 20 min. RT was allowed to stand at 42 ° C for 1 hour, and cDNA was prepared and incubated at 94 ° C for 5 minutes to inactivate the reverse transcriptase. The subsequent PCR conditions were repeated 30 times at 94 ° C for 30 seconds (denaturation), 50 ° C for 30 seconds (annealing) and 72 ° C for 90 seconds, The amplified cDNA was separated by electrophoresis using 1.2% agarose gel and confirmed by LAS 4000 image analyzer.

The primer sequences used in the PCR are shown in Table 2 below.

Gene Sense antisense MMP-2 GCATGGCTCGCCTACAGACT GCAGGGAGGGAGGCAGAGGA MMP-9 ATGGCCCCACTGAAGATGCT TGAACACCAACGGTGGAGGT TNF-a AAGCTTCATGGTGATGCGAC TCAAGCAGAAGAGGAAGGCA TIMP-1 ACCTAGCGGACCCGAAATAA TGGAACAGCAGGAAAAGCAC TIMP-2 CACTGGGGTTGGGAGGTAGT GCTCTCATGATGCTACCCGA GAPDH CCACCCAGTACAGCGTCAAC CATGGTGCTTCTGTCGCTCT

Patch experiment

The skin stability test, the patch test, was based on the criteria of the International Contact Dermatitis Research Group. The sample was applied to the Finn chamber (on Scanpor tape) and attached to the skin. After 48 hours, the skin reaction was confirmed.

The criteria of the test are shown in Table 3 below.

Grade Result - No reaction ± Weak positive reaction (erythema) + Moderate positive reaction (erythema) ++ Strong positive reaction (erythema, edema) +++ Strong positive reaction (erythema, edema, vesicles)

BCOP (Bovine Corneal Opacity and Permeability)

This protocol is a version established in accordance with the OECD Test Guideline 437 for use as a step-by-step test strategy for classifying substances with eye irritation or severe irritation without further testing on rabbits. In vivo It is an in vitro experiment that replaces the Draize test.

Using the corneal tissue obtained from the slaughterhouse, the permeability barrier function of the cornea by the test material and the damage of the corneal epithelial cell membrane junction were observed to evaluate the degree of stimulation of the test material. According to OECD Guideline 437, DW was used as a negative control and Ethanol was used as a positive control. Stimulation of the test substance was judged using the IVIS formula, and corrosion or irritation was judged when more than 55.1.

In extraction of small eyeballs, we extracted as soon as possible after slaughter to minimize mechanical damage and other damage to eyes. No chemical detergents were used to clean the animals' heads, or no eye drops in hot water. In order to minimize eye deterioration and bacterial contamination, it was thoroughly soaked in HBSS (Hank's ball-balanced salt) supplemented with PS (Penicillin 100,000 U / ml and Streptomycin 100,000 U / ml). In order to collect fresh cornea, the experiment should be performed on the same day as the day of slaughter in the slaughterhouse, and the eye used for the experiment should be on the same day.

The analysis and IVIS (In Vitro Irritation Score) used in the BCOP analysis are shown in Table 4 below.

IVIS UN GHS * ≤ 3 Not classified > 3, ≤ 55 No prediction can be made > 55 Category 1

(UN GHS: United Nations Globally Harmonized System)

Concentration of extract

For the progress of the experiment, the freeze-dried Rhodiola extract was mixed with purified water to prepare a concentration of 1 wt% to 10 wt%, and then the experiment was conducted.

2. Experimental results

Antibacterial evaluation

Antimicrobial activity of Staphylococcus aureus, Staphylococcus epidermidis and Propionibacterium acnes was tested as shown in Figs. 1 and 5.

Concentration (mg / disc) 100 (D) 10 (C) 5 (B) Clear zone S.aureus 20 15 11 Clear zone S. epidermidis 25 19 10 50 (D) 10 (C) 5 (B) Clear zone P.acne 17 10 a

(A: control, a: no inhibition, in mm)

Referring to FIGS. 1 and 5, it was confirmed that Staphylococcus aureus, Staphylococcus epidermidis and Acne bacteria have antibacterial effect.

Evaluation of antimicrobial activity in wallpaper

Fig. 2 shows the results of measuring the activity of the sulfuric acid by applying the extract of Rhododendrons to the wallpaper of the building material. Four cases of bacterial smear were applied to the wallpaper which was coated with sterilized wallpaper by mixing germanium smear, bacterium, According to Fig. 2, it was confirmed that the antimicrobial effect was exhibited from 24 hours after the application of the extract of Chrysanthemum morifolium extract.

Evaluation of antimicrobial activity against dropping bacteria in indoor

After collecting and culturing the indoor dropping bacteria in different places, the antimicrobial activity of Huangliao extract against the dropping bacteria in the room was evaluated.

The evaluation results are shown in Table 6 and FIG.

Concentration (mg / disc) Clear zone
(mm ) Case 100 50 10 Case1 20 17 9 Case2 18 13 8 Case3 18 11 8 Case4 19 15 9

According to the above Table 6 and FIG. 3, it was confirmed that the antibacterial effect of Rhodiola extract on the indoor falling bacteria collected at different places.

Evaluation of Antimicrobial Activity against Staphylococcus aureus Culture

The culture broth was streaked on the 1st, 7th, 14th, 21st, 30th, and 42th days of each culture at 0%, 1%, 5% and 10% .

The experimental results are shown in Fig. According to FIG. 4, no antioxidant effect and antimicrobial effect were observed at 1% concentration and no addition of Huanglung extract. At 5% concentration, antimicrobial activity and antimicrobial activity were higher than 1% concentration. At 10% concentration, no antimicrobial activity was observed 1 day after adding the extract of Rhododendron to the bacterial culture solution, but it was confirmed that the antimicrobial activity was strong after 7 days and persisted even after 42 days.

Evaluation of Antimicrobial Activity against Staphylococcus aureus Cultures (Evaluation by Temperature Variation)

FIG. 5 shows the result of evaluating the antimicrobial activity against the culture of Staphylococcus aureus according to the temperature change. Antimicrobial activity against Staphylococcus aureus after heat treatment at 0, 70 ℃, 90 ℃, 110 ℃, 130 ℃, 150 ℃ and 180 ℃ for 1 min was obtained. This means that there is no change in the antimicrobial activity of the extracts of Rhododendron japonica according to the temperature, and the Rhododendron japonica extract has heat stability.

Analysis of MIC and MBC test results

Staphylococcus aureus Staphylococcus epidermidis Propionibacterium acnes MIC (mg / ml) 0.03 0.50 0.10 MBC (mg / ml) 0.05 0.75 0.15

The MIC is the most basic indicator for determining the antimicrobial activity, which is defined as the lowest concentration of antibiotic that can inhibit microbial propagation in an in vitro sensitivity test. MBC is defined as the minimum concentration of antibiotic that reduces the amount of pathogens by at least 99.9% (ie 10 ^5-6 / mL to ≤ ≤ 10 ^2-3 / mL).

As shown in Table 7 and FIG. 6, it can be confirmed that the Huanghui extract has an excellent antibacterial effect even in a small amount.

To determine the cytotoxicity of berberine

The result of the cytotoxicity test of berberine by MTT assay is shown in Fig. According to Fig. 7, the cell survival rate was above 90% at a concentration of 25 μg / mL or less, and it was confirmed that CCD-986sk cells were not toxic. Thus, all of the following experimental results were conducted at a concentration of 25 μg / mL or less.

Result of ROS generation measurement

According to FIG. 8, the amount of ROS produced by UVB irradiation in CCD-986sk cells was increased in the group irradiated with UVB at 20 mJ / cm ² compared with the control group. After UVB irradiation, ROS production was decreased in the concentration-dependent (5, 10, 25) μg / mL concentration of berberine, especially at 25 μg / mL concentration.

Measurement results of HA generation

According to Fig. 9, when berberine was treated, it was confirmed that hyaluronic acid was increased. That is, it was confirmed that the effect of improving the wrinkles of the skin due to the increase of hyaluronic acid was increased.

Protein synthesis and mRNA expression of MMP-2 and MMP-9

As shown in FIG. 10, protein levels of MMP-2 and -9 were inhibited with increasing concentrations of berberine, and (10, 25) μg / mL of MMP-2 and 25 μg / / mL. < / RTI > In addition, mRNA expression was measured based on GAPDH expression as a house keeping gene. As the concentration increased, the expression of MMP-2 and -9 was decreased and the significance was confirmed at the concentration of 25 μg / mL.

Protein synthesis and mRNA expression of TIMP-1 and TIMP-2

11, the expression of TIMP-1, -2 was significantly decreased in the UVB group, and the expression of TIMP-1, -2 protein was increased in a concentration-dependent manner after treatment with berberine. Was increased in a concentration-dependent manner.

Expression of inflammatory cytokine TNF-α

12, expression of TNF-α and MMP-9 was increased in UVB-only treated group and that of berberine-treated group was decreased in TNF-α and MMP-9 expression, and expression of TNF-α and MMP-9 .

Analysis of patch test results

And were judged on the basis of the International Contact Dermatitis Research Group. The sample was applied to the Finn chamber (on Scanpor tape) and attached to the skin. After 48 hours, the skin reaction was confirmed.

According to Fig. 13, skin irritation test was performed on 22 male and female subjects, and no skin irritation reaction was observed in all subjects. In addition, as a result of requesting skin irritation test by the Korea Dermatological Research Institute, no stimulation was observed 30 minutes after the removal of the information, and one ± irritation was observed after 24 hours and 48 hours, and the average skin response was 0.36 Was judged to be unstimulated according to the judgment criteria.

BCOP experiment result

IVIS pro Gruppe mw stsabw NC D.I water 3.0 1.0 PC Ethanol 62.1 4.0 Susbt 1 T-1% -1.7 0.5 Susbt 2 T-10% 0.6 1.7

According to FIG. 14 and Table 8, according to the BCOP experiment of the mucosal irritation test for the bovine cornea of the extract of Rhododendron japonica extract, 1% by weight of Rhodiola extract and 10% by weight of Rhodophylla extract were all stimulated according to OECD guideline 437 It was confirmed that there is no.

Antimicrobial activity of cosmetic composition

Through the above experiment, the antimicrobial test and the toxicity test for the Huangliao extract and berberine itself proceeded. In addition, the antibacterial properties of the cosmetic compositions of Experimental Examples 1 to 10 were evaluated and the results are shown in Table 9 below.

The antimicrobial activity of the 10% by weight antler extract was evaluated as an index of 5 and the antimicrobial activity of the cosmetic compositions of Experimental Examples 1 to 10 was evaluated as an index of 1 to 10.

Experimental Example 1 Experimental Example 2 Experimental Example 3 Experimental Example 4 Experimental Example 5 Experimental Example 6 Experimental Example 7 Experimental Example 8 Experimental Example 9 Experimental Example 10 Antimicrobial activity 5 5 6 4 6 7 3 7 7 9

As shown in Table 9, it was confirmed that there was no significant difference in the case of using the extract of Rhododendron and the case of using only berberine. Further, the antimicrobial activity was excellent in the content range of the present invention, but it was confirmed that the antimicrobial activity was inferior when the content was less than or greater than the above range.

Wrinkle improvement test result for cosmetic composition

Experimental Example 1 Experimental Example 2 Experimental Example 3 Experimental Example 4 Experimental Example 5 Experimental Example 6 Experimental Example 7 Experimental Example 8 Experimental Example 9 Experimental Example 10 wrinkle improvement One 5 5 2 6 7 3 6 6 9

As shown in Table 10, it can be confirmed that the effect of improving wrinkles is insufficient when berberine is not contained separately. It was confirmed that the effect of improving wrinkles was excellent within the content range of the constituent components of the present invention.

Sensitivity to cosmetic composition

Nutrition lotions were prepared using the compositions of Examples 1 to 10. The nutritional lotion contained 3% by weight of the composition of Experimental Examples 1 to 10, 1% by weight of ascorbic acid-2-phosphate, 1% by weight of water soluble collagen (1% aqueous solution), 0.01% by weight of sodium citrate, 3% by weight of 3-butylene glycol and the remaining purified water. Twenty adult men and women were selected and applied twice a day for one week before applying the sensory evaluation.

The sensory evaluation was evaluated by an index of 1 to 10 according to taste, on the basis of stickiness, feeling and spreadability, and classified by an average index. The results are shown in Table 11.

On the other hand, the higher the number is, the higher the palatability.

Experimental Example 1 Experimental Example 2 Experimental Example 3 Experimental Example 4 Experimental Example 5 Experimental Example 6 Experimental Example 7 Experimental Example 8 Experimental Example 9 Experimental Example 10 Stickiness 3 5 5 2 6 6 3 8 8 6 Feeling 3 3 5 4 5 6 4 7 9 8 Brittle 4 3 4 3 5 6 4 9 8 7

As shown in Table 11, when the content of the cosmetic composition of the present invention was within the range, the evaluation was generally excellent, but in particular, it was confirmed that the best evaluation was obtained in Experimental Examples 8 and 9.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments, Of the right.

Claims (13)

Extract of Chrysanthemum indicum, and berberine,
The Berberine was firstly separated by using a gel filtration Sephadex LH-20 for low molecular weight, and the separated fractions were secondly separated using MCI-gel CHP 20, a porous gel column chromatography, The separated fractions were obtained by tertiary separation by performing YMC-gel ODS-A-HG column chromatography,
And further comprising a hot water extract of Yedok tree, a hot water extract of Hill root and a hot water extract of Kizuka vine,
5 to 10 parts by weight of berberine, 10 to 20 parts by weight of Yedok tree hot water extract, 1 to 5 parts by weight of hot water extract of Hill root and 1 to 5 parts by weight of hot water extract of bean curd are added to 100 parts by weight of the above-
The fermented fermented hot-water extract is a fermented fermented hot-water extract which is fermented by inoculating a strain to dry fermentation,
The hot water extract of Yedok tree is fermented Yeokdok tree hydrothermal extract fermented by using rice cakes and the hot water extract of Hill cocoon and the hot water extract of Codonthus japonica are hydrothermal extracts of fermented hemlock root fermented by fermentation using rice cakes and hot water extract of fermented cowberry vine ,
The method of fermenting rice with soot is to mix 5 to 10 parts by weight of saccharide and 1 to 2 parts by weight with respect to 100 parts by weight of rice soap, immersing 5 to 10 parts by weight of the dried natural crushed product, Wherein the fermented product is fermented for 5 to 10 days after air is shut off at 10 to 40 占 폚 to produce a fermented product.
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