KR101850601B1 - A functional cosmetic composition for anti-inflammatory and moisturizing effect containing polysaccharide of green tea produced by supercritical fluid technology and bioconversion process, and the method for preparing of the same - Google Patents

Abstract

The present invention relates to a composition for enhancing the anti-inflammatory and moisturizing function of a polysaccharide prepared through a bioconversion process and a method for preparing the green tea polysaccharide purified by a supercritical fluid technique, There is an effect of providing a method for producing a green tea polysaccharide extract having a low polysaccharide loss and a high content thereof and an excellent effect of providing a cosmetic composition having enhanced antiinflammatory and moisturizing functions produced by the above production method.

Description

TECHNICAL FIELD The present invention relates to a cosmetic composition containing a green tea polysaccharide prepared by supercritical fluid technology and bioconversion, and a cosmetic composition containing the green tea polysaccharide, process, and the method for preparing the same.

The present invention relates to a cosmetic composition having enhanced anti-inflammatory and moisturizing function through a bioconversion process and a method for preparing the same, more specifically to a green tea polysaccharide obtained by a supercritical fluid technique, The present invention also provides a functional cosmetic composition comprising a polysaccharide, a polysaccharide, and a polysaccharide.

In the cosmetics industry, there is a continuous increase in demand due to an increase in income level, an increase in interest in beauty due to population aging, and an increase in demand for youthful and resilient skin. In particular, demand for functional cosmetics Increase.

The history of skin moisturization began with the introduction of paraffin-based petrolatum, which is a typical moisturizer using this ingredient. The mechanism of action of Vaseline can be explained by the physical occlusive film on skin surface which prevents moisture loss by simply forming a paraffin film on the stratum corneum surface to prevent water evaporation. Since NMF (natural moisturizing factor) has been discovered, a new understanding of skin moisturization has become possible.

NMF is found inside the stratum corneum and consists mainly of amino acids and their metabolic byproducts. NMF loss due to lack of NMF in the stratum corneum or use of detergent causes skin problem of stratum corneum. Moisture binding ability in the stratum corneum is related to PCA content especially in NMF. In addition, NMF is an absorbing or water binding function that holds skin moisture and forms a part of moisturizing function. Unlike the water holding function of NMFs, epidermis skin hydration is important in epidermis. On the other hand, the moisturizing mechanism in Dermis is different from that of Epidermisis. HA (hyaluronan) and GAGs (glucosaminoglycans) in Dermis are typical ECMs and are closely related to dermis metabolism. Especially, it is known as the key substance of hydration mechanism, so synthesis or degradation of HA and GAG is the mechanism of action of dermis hydration, and its regulation is a fundamental clue of dermal hydration. Previously, the function and research and development of moisturizers focused mainly on the stratum corneum water holding function and the water binding activity by NMF. However, as the hydration mechanism in Epidermis is known, moisturization is controlled by various functional groups. It is known that the hydration is affected by the function of HA and GAG in Dermis, and the mechanism of action of moisturizing is found to have a more complex regulatory function.

Therefore, it is necessary to study the new moisturizing efficacy evaluation to study this complex mechanism. In addition, since the moisturizing complex action mechanism is closely related to the anti-inflammatory mechanism in skin, a new study is needed to evaluate the anti-inflammatory effect as well as the evaluation of moisturizing effectiveness.

Research on physiological activity and pharmacological effects of tea on the tea as the world's three major beverages has been mainly focused on tea catechins. Catechins have been shown to lower serum lipids, hypertension, atherosclerosis and obesity, antiplatelet activity, Antioxidant production is effective in prevention of aging, antimicrobial action, anti-cancer, anti-inflammatory action, and prevention of tooth decay. In November 2006, an external skin dermatosis drug using tea catechin was approved by the FDA.

The market for green tea such as domestic leaf tea is about 300 billion won (AP occupies 43% market share). Green tea drink market is about 200 billion won (owns Dongwon F & B market), and in Japan, green tea drink market is 3 trillion won.

As a result of studies on acidic green tea polysaccharides which have been subjected to non-enzyme / enzyme hydrolysis as a prior art of the present invention, the total polysaccharide content of the extract and the skin's efficacy mechanism thereof have been known from inflammatory skin diseases, It has not been confirmed at all, it is an acidic green tea polysaccharide, and the price is too expensive to use.

In addition, as disclosed in Korean Patent Laid-open Publication No. 10-2011-0045662 as another prior art of the present invention after hot water extraction with distilled water, the green tea leaves are extracted with hot water, filtered and ethanol precipitated to obtain an acidic green tea polysaccharide and pine mushroom extract Korean Patent No. 10-0602841 discloses an acidic polysaccharide separated by ethanol precipitation after hot water extraction of green tea and a composition for treating and treating peptic diseases containing the same, Acid green tea polysaccharides are too expensive to use. However, there are no known functional cosmetic compositions containing a green tea polysaccharide prepared by sequentially performing supercritical fluid technology and biological conversion, and methods for preparing the same.

Accordingly, it is an object of the present invention to provide a method for producing a green tea polysaccharide extract in which the inventors of the present invention sequentially add a known green tea polysaccharide prepared by supercritical fluid technology to a bioconversion process to increase total sugar content.

It is another object of the present invention to provide a cosmetic composition having enhanced anti-inflammatory and moisturizing function using the green tea polysaccharide extract prepared through the above-described method as an active ingredient.

The above object of the present invention can be achieved by a method for producing a green tea extract, comprising the steps of: a) preparing a green tea extract cake from which caffeine and caffeine have been removed using a supercritical extraction apparatus; b) extracting the green tea extract powder obtained in the above step by hot water extraction with purified water; c) adding a hydrolytic enzyme to the green tea crude polysaccharide obtained in the above step to decompose it; d) adding a low concentration of ethanol to the enzyme reaction product obtained in the enzyme decomposition step, and then centrifuging to prepare a supernatant; e) adding a secondary high concentration of ethanol to the supernatant obtained in the above step and centrifuging to obtain a precipitate; f) drying the precipitate obtained in the above step to prepare a green tea polysaccharide extract, wherein the green tea polysaccharide extract is obtained by evaluating the intercalation and moisturizing effect thereof.

The present invention provides a method for producing a green tea polysaccharide extract which minimizes the loss of polysaccharide and maximizes the total sugar content by sequentially performing a supercritical fluid extraction process and a biological conversion process, There is an excellent effect of providing a cosmetic composition which is further strengthened by anti-inflammation and moisturizing effect produced according to the production method.

Fig. 1 is a schematic diagram of a supercritical extraction process.
FIG. 2 is a diagram illustrating a process for producing a green tea polysaccharide extract. FIG.
3 is a graph showing the moisturizing effect of the green tea polysaccharide extract of the present invention.
4 is a graph showing a treatment method of SLS in a skin barrier regeneration effect experiment process.
5 is a graph showing the TEWL reduction effect of the green tea polysaccharide extract of the present invention.
FIG. 6 is a graph showing the NO reduction effect of Raw 264.7 cell activated by the green tea polysaccharide extract of the present invention.
FIG. 7 is a graph showing the effect of decreasing the expression of COX-2 in activated fibroblast when treated with green tea polysaccharide extract of the present invention.
FIG. 8 is a graph showing the effect of reducing PEG-2 release in activated fibroblast when treated with the green tea polysaccharide extract of the present invention.
9 is a graph showing skin erythema reduction upon treatment with the green tea polysaccharide extract of the present invention.

The present invention provides a method for producing a green tea extract, comprising the steps of: a) preparing a green tea extract cake having caffeine and caffeine removed using a supercritical extraction apparatus; b) extracting the green tea polysaccharide by hot water extraction of the green tea extract foil obtained in the above step (most preferably, purified water at 95 ° C for 1 hour); c) mixing and adding to the green tea crude polysaccharide obtained in the above step the most preferable four kinds of hydrolytic enzymes in an equal ratio; d) adding a low concentration of ethanol to the enzyme reaction product obtained in the enzyme decomposition step, and then centrifuging to prepare a supernatant; e) secondarily adding a high concentration of ethanol to the supernatant obtained in the above step and then centrifuging to obtain a precipitate; f) The precipitate obtained in the above step is dried to prepare the green tea polysaccharide extract of the present invention.

The green tea polysaccharide extract is characterized by enhancing or enhancing anti-inflammatory and moisturizing effects by minimizing loss of polysaccharide and maximizing total sugar content by means of sequentially performing a supercritical extraction process and a biological conversion process.

The present invention provides a cosmetic composition having anti-inflammatory and moisturizing properties and containing a green tea polysaccharide extract as an active ingredient. The cosmetic composition may be used by mixing a skin-tolerant carrier in addition to the green tea polysaccharide extract. The dermatologically acceptable carriers may be selected from the group consisting of purified water, oils, waxes, fatty acids, fatty acid alcohols, fatty acid esters, surfactants, humectants, thickening agents, antioxidants, viscosity stabilizers, , Buffers, preservatives, lower alcohols, and the like, but the present invention is not limited thereto, and the types and concentrations thereof may vary, and those skilled in the art may modify or change the scope of the present invention without departing from the spirit and scope thereof.

The cosmetic composition according to the present invention may contain a whitening agent, a moisturizing agent, an anti-inflammatory agent, an antibacterial agent, an antifungal agent, a vitamin, an ultraviolet screening agent, an antibiotic, an anti- .

Specific examples of the oil include hydrogenated vegetable oil, pharmacopoeia, cottonseed oil, olive oil, palm oil, jojoba oil, and avocado oil. Examples of the wax include wax, wax, carnauba, candelilla, montan, Paraffin, lanolin may be used.

As the fatty acid, stearic acid, linoleic acid, linolenic acid and oleic acid may be used. As the fatty acid alcohol, cetyl alcohol, octyldodecanol, oleyl alcohol, panthenol, lanolin alcohol, stearyl alcohol and hexadecanol may be used As the fatty acid ester, isopropyl myristate, isopropyl palmitate, butyl stearate and the like can be used, but not limited thereto.

Examples of the surfactant include cationic surfactants such as sodium stearate, sodium cetyl sulfate, polyoxyethylene lauryl ether phosphate, anionic surfactant such as sodium N-acyl glutamate, stearyldimethylbenzylammonium chloride and stearyltrimethylammonium chloride. Amphoteric surfactants such as glycerin monostearate, sorbitan monostearate, propylene glycol monostearate, polyoxyethylene oleyl ether, polyethylene glycol monostearate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan monopalmitate, Nonionic surfactants such as polyoxyethylene coconut fatty acid monoethaolanolide, polyoxypropylene glycol, polyoxyethylene castor oil, polyoxyethylene lanolin, and the like.

As the hygroscopic agent, glycerin, 1,3-butylene glycol, propylene glycol may be used, and as the lower alcohol, ethanol and isopropanol may be used. Examples of thickeners include sodium alginate, sodium caseinate, gelatin agar, xanthan gum, starch, cellulose ethers (e.g., hydroxyethylcellulose, methylcellulose, carboxymethylcellulose, hydroxypropylmethylcellulose), polyvinylpyrrolidone, But are not limited to, polyvinyl alcohol, polyethylene glycol, and sodium carboxymethylcellulose.

Examples of the antioxidant include butylated hydroxytoluene, butylated hydroxyanisole, propyl gallate, citric acid, and ethoxyquin; chelating agents include disodium edetate, ethanedioxy diphosphate Is available.

As the buffer, citric acid, sodium citrate, boric acid, borax, disodium hydrogen phosphate can be used. As the preservative, methyl parahydroxybenzoate, ethyl parahydroxybenzoate, dihydro acetic acid, salicylic acid , Benzoic acid can be used, but the present invention is not limited thereto.

Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples. The following examples are for illustrative purposes only and are not intended to limit the invention to the following examples and experimental examples.

Example  One. Green tea extract  Produce

As shown in FIG. 1, the green tea is firstly subjected to supercritical fluid extraction to prepare impurities-removed green tea extract foil. In the present invention, impurities are collectively referred to as wax, catechin and caffeine.

For details of the supercritical fluid extraction process, green tea is first filled in the extractor (1), and supercritical carbon dioxide heated to be suitable for extraction is supplied to the bottom of the extractor (1) through the heat exchanger (9). The supplied supercritical carbon dioxide contacts the filled green tea to extract the impurities. The supercritical carbon dioxide is discharged to the outside of the extractor, and the mixture of extracted supercritical carbon dioxide and the impurities is passed through the pressure reducing valve ② and then reduced to the pressure reducing chamber ③. In the pressure reducer 3, the extracted impurities and carbon dioxide are separated, and the separated carbon dioxide is liquefied through the heat exchanger 4 and circulated to the storage tank 5 for reuse. The impurities separated from the decompressor ③ are collected in the final impurity ⑩, and the carbon dioxide in the carbon dioxide storage tank ⑥ is supplemented with carbon dioxide supplied from the outside in addition to the carbon dioxide supplied in the circulation. The carbon dioxide stored in the storage tank 5 is pressurized through the pump 7 and supplied to the extractor through the heat exchanger 9 in a supercritical state (FIG. 1). When the supercritical fluid extraction process according to the present invention is completed, the green tea extract powder from which the impurities are removed is obtained from the extractor (1).

Green tea leaves were treated with 300 g of green tea leaves in an extractor, and the temperature was 70 ° C., the pressure was 300 bar, supercritical carbon dioxide flow rate was 150 ml / min, 80% ethanol was used as an auxiliary solvent, The fluid extraction process was performed.

Example  2. Green tea  Produce

For the preparation of green tea jodada, 4500 mL of purified water was mixed with 300 g of the green tea extract obtained in Example 1, stirred at 95 ° C for 1 hour and concentrated or dried to prepare green tea polysaccharide (FIG. 2).

Example  3. Green tea polysaccharide extract  Produce

(W / w / w / w) of the hydrolytic enzymes Pectinex Ultra SP-L, β-glucanase, Termamyl-120L and AMG-300L were mixed with the green tea crude polysaccharide prepared in Example 2 2% by weight based on the green tea group sugar was added to perform the enzyme reaction. Ethanol was added to the enzyme reaction product, which was first precipitated with solvent in a 55% (v / v) ethanol solution at a low concentration and centrifuged to obtain a supernatant. To the supernatant obtained above, a high concentration of ethanol was again added, and the mixture was further subjected to solvent precipitation in a 75% (v / v) ethanol solution, followed by secondary centrifugation, and then a precipitate was obtained. The precipitate obtained above was dried to prepare the green tea polysaccharide extract of the present invention. The green tea polysaccharide extract of the present invention was stored in a refrigerated room (4 ° C) and used as a test material in the following experiment (FIG. 2).

Experimental Example 1. Analysis of polysaccharide content of green tea polysaccharide extract of the present invention

1) Preparation of the test solution

2.5 g of the inventive green tea polysaccharide extract obtained in Example 3 was placed in a flask and dissolved in 100 mL of HPLC analysis water. The solution obtained in the above step was subjected to ultrasonic extraction for 30 minutes, and the heated extract was cooled in cold water and cooled to room temperature. A portion of the extract was filtered using a 45 μm syringe filter and used as a test solution. As a control group, commercially available acid green tea polysaccharide extracts prepared by a known method without supercritical fluid extraction process and biological conversion process according to the present invention were used.

2) HPLC analysis

The standard solution and the sample solution transferred to the vial were allowed to stand for 30 minutes or more in a sample tray set at 10 ° C, and the sample solution was injected under the following conditions. The peak areas of the chromatograms obtained by the analysis were summed and quantified by comparing them to the average value for the standard product of the same concentration.

Standard product: Pullulan series (P-5,10,20,50,100,200,400 and 800)

Column: Asahi-Pak GS-520 + GS-320 + GS-220 packed column (7.6 * 300 mm)

Flow rate: 0.5 mL / min

Injection volume: 20 μL

Detector: RI

Column temperature: 25 degrees

Mobile phase: 50 mM ammonium formate buffer (pH 5.5)

Total polysaccharide content The green tea polysaccharide extract of the present invention Control group Standard Form (average) area 2514322 1821416 5505557 content
(% By weight based on the total weight of solids) 45.7 33.1 100

As a result, the total polysaccharide content of the green tea polysaccharide extract according to the present invention increased to 45.7% of the total solids weight, which was 12.6% higher than that of the control group (Table 1).

Experimental Example 2: Moisturizing effect of the green tea polysaccharide extract of the present invention

Water holding capacity evaluation and TEWL evaluation were performed to measure the moisturizing effect and the regenerating effect of the destroyed skin barrier obtained from the green tea polysaccharide extract obtained according to the present invention. The green tea polysaccharide extract of the present invention was dissolved in DMSO (Dimethylsulfoxide) at a concentration of 100 mg / L and used in the experiment.

1) Moisturizing effect experiment

 The subject's upper arm was washed with a cleansing foam, and then dried naturally for 10 minutes after removal of sebum. Then, 100 μl of 100 mg / L green tea polysaccharide extract was treated and skin moisture was measured for 2 hours in 8 hours. As a control group, BG, which is well known for moisturizing effect, was used. The subjects were 6 males and 4 females.

As a result, as shown in FIG. 3, the water content of the untreated skin decreased with time, and it was 53% after 6 hours in the skin treated with the green tea polysaccharide extract of the present invention. This indicates that the green tea polysaccharide extract of the present invention inhibits water evaporation by forming a film inhibiting moisture evaporation in the stratum corneum of the skin.

2) Skin barrier regeneration effect (TEWL) experiment

As shown in FIG. 4, the upper side of the subject's upper arm was washed with a cleansing foam, and the skin was naturally dried for 10 minutes after removal of sebum. Then, 200 μl of 5% SLS (sodium lauryl sulfate) Damaged. After TEWL measurement of the damaged skin barrier, 100 ㎕ of green tea polysaccharide extract was treated in each part, and the degree of regeneration of the skin barrier was checked by TEWL measurement every 2 hours. The subjects had the same composition as the humectant effect measuring personnel.

As a result, as shown in FIG. 5, in the TEWL experiment, the skin treated with the green tea polysaccharide extract of the present invention reached a normal state after 6 hours, and it took 12 hours for the control olive oil and 24 hours for the untreated group.

Experimental Example 3. Anti-inflammatory effect of the green tea polysaccharide extract of the present invention

In order to measure the anti-inflammatory activity of the green tea polysaccharide extract of the present invention extracted using supercritical CO2, the amount of nitric oxide (NO) produced by LPS-induced macrophages (Raw 264.7 cells) and COX- 2, and PGE2 expression were measured. Clinically, erythema test was also performed to confirm the anti - inflammatory effect of green tea polysaccharide extract.

1) Identification of nitric oxide (NO)

The ability of green tea polysaccharide extract to inhibit nitric oxide production was measured using Raw 264.7 cell. Raw 264.7 cells were cultured in DMEM (Dulbecco's modified Eagle's medium) supplemented with penicillin (10 U / ml) and 10% FBS (Fetal bovine serum) in an incubator maintained at 37 ° C and 5% carbon dioxide. Raw 264.7 cells were plated at a density of 5 × 10 ⁴ cells / well in a 96-well plate, cultured for 24 hours, and treated with 2 μl of LPS (0.1 μg / ml). The green tea polysaccharide extract was dissolved in DMSO (dimethyl sulfoxide) at a concentration of 10, 100 and 1000 mg / L. Then, 2.5 μl of the green tea polysaccharide extract was added to each LPS-treated raw 264.7 cell and cultured at 37 ° C. in 5% carbon dioxide. After incubation for 24 hours, the supernatant was separated and 100 μl of supernatant and 100 μl of Griess reagent (1% sulfanilamide, 0.1% N-1-naphthylenediamine dihydrochloride, 5% phosphoric acid) were mixed and reacted for 10 minutes. The absorbance was measured at 540 nm using a microplate reader to measure nitric oxide production. Olive oil was used as a control.

As shown in FIG. 6, the green tea polysaccharide extract of the present invention reduced NO production in macrophages to 81.5% at a concentration of 1000 mg / L of the green tea polysaccharide extract and 36.2% higher activity than the control olive oil .

2) Identification of expression of Cyclooxigenase-2 (COX-2)

Human fibroblast cells were seeded at a density of 5 × 10 ⁴ cells / ㎖ in a 6-well plate (10 ㎖), cultured for 24 hours, and treated with 2 ㎕ of LPS (0.1 ㎍ / ㎖). The green tea polysaccharide extract was dissolved in DMSO (dimethylsulfoxide) at a concentration of 100 mg / L, added to LPS-treated fibroblast cells at 2.5 ㎕, and cultured at 37 캜 in 5% carbon dioxide. After incubation for 24 hours, fibroblast cells were washed with phosphate buffered saline (PBS) and centrifuged to obtain cells. Cells were harvested using a protein extraction solution, and protein was extracted and quantitated by the Bradford method. In each sample, 20 μg of the protein was implanted into the nitrocellulose membrane and blocked on the agitator for 90 minutes. The membrane was incubated in primary antibody (anti-COX-2) for 3 hours, then washed 3 times with 0.5% TBS, then with HRP-conjugated secondary antibody for 90 minutes. The band was measured with a chemiluminescence reagent and exposed to photographic film for 1 minute to confirm the expression of COX-2.

As shown in FIG. 7, the green tea polysaccharide extract of the present invention significantly reduced COX-2 production in fibroblast.

3) Confirmation of prostagladin-E2 (PGE2) release

PGE2 release was measured using an ELISA kit. The LPS and green tea polysaccharide extracts were treated at the same concentration as the above 1) Nitric oxide measurement method, and then cultured under the same conditions. After culturing for 24 hours, the supernatant was obtained by centrifugation, and PGE2 was quantified using an ELISA kit.

As a result, as shown in FIG. 8, it was confirmed that the expression level of PGE2, which is an inflammation inducer produced by COX-2, decreased by 35.5% at a concentration of 1000 mg / L and 71.3% at a concentration of 100 mg / L .

4 ) Identification of erythema

The effect of green tea polysaccharide extract on human skin was investigated. First, the inside of the upper arm of the subject is cleaned using a cleansing foam, and then dried naturally until the water disappears. 100 μl of 5% SLS (sodium lauryl sulfate) was applied to the dried area at 2.5 cm × 2.5 cm for 30 minutes to induce inflammation. After the inflammation was induced, erythema was measured using a colorimeter CR-14 (Minolta, Japan). 100 μl of 100 mg / L green tea polysaccharide extract and olive oil (control) were added to the inflamed area, and the erythema was measured every 2 hours to confirm the reduction of inflammation.

As a result, as shown in FIG. 9, the green tea polysaccharide extract of the present invention reduced the redness of erythema occurring in human skin to a normal state in 6 hours, higher than that of the untreated group 24 hours and olive oil 12 hours Respectively.

As described above, the green tea polysaccharide extract prepared through the supercritical extraction process and the bioconversion process of the present invention has a lower polysaccharide loss in the extraction process than the polysaccharide obtained in the conventional green tea polysaccharide extraction process, It is an extremely useful invention in the functional cosmetics industry because it has an effect of providing a cosmetic composition excellent in anti-inflammation and moisturizing effect.

Claims (5)

A method for preparing a green tea polysaccharide extract through a supercritical fluid extraction process and a biological conversion process;
(a) preparing green tea extract leaves from which caffeine and catechin have been removed using a supercritical extraction apparatus; (b) extracting the green tea extract obtained in the above step by hot water extraction with purified water to prepare green tea polysaccharide; (c) an enzymatic decomposition step of adding a hydrolytic enzyme to the green tea crude polysaccharide obtained in the step (a); (d) adding a 55% (v / v) concentration of ethanol to the enzyme reaction product obtained in the step (a) to precipitate a solvent, followed by centrifuging to prepare a supernatant; (e) secondarily adding the supernatant obtained in the above step to ethanol at a concentration of 75% (v / v) to precipitate a solvent, and then centrifuging to obtain a precipitate; (f) drying the precipitate obtained in the above step to prepare a green tea polysaccharide extract
The method according to claim 1, wherein the enzymatic degradation is carried out by mixing the hydrolytic enzymes pectinex ultra Sp-L,? -Glucanase, Termamyl-120L and AMG-300L in an equivalent ratio (w / w / w / w) By weight based on the total weight of the composition.
delete A green tea polysaccharide extract prepared by the method of claim 1 or 2 and having a total sugar content of 45.7%
The anti-inflammatory and moisturizing functional cosmetic composition containing the green tea polysaccharide extract of claim 4 as an active ingredient

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