KR20130027821A - A composition for anti-aging or wrinkle improvement of skin comprising enzyme-treated red ginseng extracts - Google Patents

Abstract

PURPOSE: A composition containing an enzyme-treated red ginseng extract is provided to promote collagen biosynthesis and to improve anti-aging and anti-wrinkling. CONSTITUTION: A composition for improving anti-aging and anti-wrinkling contains a red ginseng extract treated with Aspergillus-derived enzyme. The Aspergillus is Aspergillus niger KACC 40280, 41018, 41858, 42589, 43547, or 44333. The enzyme-treated red ginseng extract is prepared by culturing a red ginseng powder or a red ginseng extract with Aspergillus-derived enzyme.

Description

[0001] The present invention relates to a composition for preventing skin aging and skin wrinkles containing an enzyme-treated red ginseng extract,

The present invention relates to a food, pharmaceutical or cosmetic composition for improving skin beauty containing natural extracts.

The phenomenon of 'well-being', which has a great influence on modern society, includes not only the primary meaning of 'eating well and living well' but also the meaning of 'the health of the body is expressed by the beauty of skin'. This phenomenon is appearing in the field of skin beauty a lot, and the approach for skin beauty now requires a complex function and a scientific approach, instead of pursuing fragmentary efficacy (Kim Eun-young, Journal of The Korean Society of Skin Aesthetics, 2005).

Skin aging can be divided into two types. One of them is intrinsic aging, which is an unavoidable aging phenomenon as the years go by. The second is photoaging, which refers to the aging observed in the skin of the face, back of the hand, back of the neck, etc., which has been exposed to sunlight for a long time. It occurs as a result of the combination of endogenous aging and the effects of UV rays (Park Geum-ju, J Korean Soc Food) Sci Nutr, 2010).

The pores are distributed throughout the facial skin, but the nose and cheek areas can be identified with the naked eye, and the appearance of the pores varies depending on the endogenous and exogenous factors such as sex, genetics, aging, acne and chronic UV exposure. The pores are widened because the sebum is excessively secreted or skin aging begins, and as the collagen fibers and elastic fibers supporting the wall of the pores degenerate and decrease, the skin elasticity is lost and drooped. Society, 2010).

It is known that the cause of skin aging is that matrix proteins such as collagen and elastic fibers are damaged and the amount of collagen in the skin is insufficient, and elastic fibers are denatured. In addition, the synthesis of collagen is reduced through various signaling pathways and the expression of matrix metalloproteinases (MMPs), an enzyme that degrades extracellular matrix proteins, increases during skin aging. Became known. As such, collagen is recognized as a major factor related to skin aging, and the synthesis and degradation inhibition of collagen has become an important indicator in regard to skin aging and skin beauty (Kim Nam, Korea Ginseng Society, 2007).

Various studies have been conducted to improve collagen synthesis and inhibit degradation thereof to improve skin beauty. Natural products having superior skin stability are attracting interest in comparison with compounds.

Red ginseng, which contains a variety of ingredients, has long been used for medicinal purposes in oriental medicine. In modern times, it has been evaluated as the best health food in the world due to various pharmacological effects such as nourishing tonic and anticancer.

The main components of red ginseng are glycosides, red acryl, polyacetylene compounds, nitrogen-containing compounds, flavonoids, vitamins (group B), trace elements, enzymes, antioxidants, organic acids and amino acids, like white ginseng. It contains.

Red ginseng has sedative and excitatory effects on the central nervous system and acts on the circulatory system to prevent hypertension and arteriosclerosis. At the same time, hematopoietic effect (당 作用) and blood sugar levels (血糖 値) lowers, protects the liver, acts on the endocrine system indirectly effective on sexual behavior and reproductive effects, anti-inflammatory And anti-tumor effect (抗 腫 瘍 作用), it is known to have a protective effect against radiation, to protect and soften the skin.

As described in the following [Patent Document], there are many research reports and patent reports on the skin-related functions of red ginseng and red ginseng-derived saponins. Red ginseng, which has undergone many pharmacological studies in the past, is currently being actively researched, focusing on the field of skin improvement.

The effect of red ginseng on wrinkle improvement has already been reported in a paper (Kim, Sun-yoon, et al.), And dried red ginseng by steamed ginseng has been reported to be effective in improving wrinkles (Kang TH et al., J. Ethnopharmacology 123 446-451, 2009).

Korean Laid-Open Patent Publication No. 10-2011-0060001 (Published on June 6, 2011) Korean Laid-Open Patent Publication No. 10-2011-0048699 (published May 1, 2011) Korean Patent Publication No. 10-2011-063912 (published Jun. 2011) Korean Laid-open Patent Publication No. 10-2011-0063916 (published Jun 5, 2011)

Lee HJ et al., The Effect of Red Ginseng on Ultraviolet B-induced Skin Damages in Mouse, J. Ginseng Res. 2006 Vol. 30, no. 4, 194-198. Cho S et al., Red ginseng root extract mixed with Torilus fructus and Corni fructus improves facial wrinkles and increases type I procollagen synthesis in human skin: a randomized, double-blind, placebo- controlled study. J Med Food. 2009 Dec; 12 (6): 1252-9. Kim H et al., Stimulatory effect of dietary red ginseng on epidermal hydration and ceramide levels in ultraviolet-irradiated hairless mice, J Med Food. 2009 Aug; 12 (4): 746-54. Kim S et al., Compound K induces expression of hyaluronan synthase 2 gene in transformed human keratinocytes and increases hyaluronan in hairless mouse skin, Biochem Biophys Res Commun. 2004 Apr 2; 316 (2): 348-55. Kim YG et al., Effects of Red Ginseng extract on ultraviolet B-irradiated skin change in C57BL mice, Phytother Res. 2008 Nov; 22 (11): 1423-7. So SH et al., Mechanisms of Korean red ginseng and herb extracts (KTNG0345) for anti-wrinkle activity, 2008 J. Ginseng Res. Vol. 32, No. 1, 39-47. Kim NM et al., Effect of Korean red ginseng on collagen biosynthesis and MMP-I activity in human dermal fibroblast, 2007 J. Ginseng Res. Vol. 31, No. 2, 86-92. Hwang EY et al., Comparison of Phenolic Compounds Contents between White and Red ginseng and Their Inhibitory Effect on Melanin Biosynthesis, 2006 Journal of Ginseng Research, Vol. 30 No. 2, 82-87. Lee HJ et al., Photoprotective effect of red ginseng against ultraviolet radiation-induced chronic skin damage in the hairless mouse, Phytother Res. 2009 Mar; 23 (3): 399-403.

The present invention is to provide a pharmaceutical composition for preventing skin aging and wrinkle improvement.

In addition, the present invention is to provide a food composition for preventing skin aging and wrinkle improvement.

In addition, the present invention is to provide a cosmetic composition for preventing skin aging and wrinkle improvement.

In order to achieve the above object, the present inventors have searched for a variety of active substances capable of suppressing skin aging and improving skin wrinkles in natural products having excellent safety. As a result, it was surprisingly found that an enzyme obtained by treating a red ginseng extract with a specific enzyme - treated red ginseng extract exhibits excellent skin aging prevention and skin wrinkle reducing effect, and completed the present invention.

Accordingly, the present invention provides a pharmaceutical composition for preventing skin aging and improving skin wrinkles, which comprises an enzyme-treated red ginseng extract derived from Nuruk fungus.

The present invention also provides a food composition for improving skin beauty, particularly reducing pores, reducing sebum, preventing skin whitening, preventing skin aging, and improving skin wrinkles, which comprises an enzyme-treated red ginseng extract derived from Nuruk Fungus.

The present invention also provides a cosmetic composition for improving skin beauty, particularly reducing pores, reducing sebum, skin whitening, preventing skin aging and improving skin wrinkles, which comprises an enzyme-treated red ginseng extract derived from Nuruk Fungus.

The enzyme is preferably selected from the group consisting of yeast (fungus), preferably Aspergillus niger strain, more preferably Aspergillus niger KACC 40280, 41018, 41858, 42589, 43547, 44333, It is an enzyme obtained by culturing Aspergillus niger KACC 40280 strain. The enzyme may be prepared by a method comprising the steps of: (a) culturing yeast fungus with ginseng extract or ginseng powder to increase ginsenosidase production, and (b) separating the enzyme from the culture. .

Separation of the enzyme from the culture is a centrifugation step, the step of precipitating the enzyme by adding an organic solvent such as ethanol, acetone, isopropanol, etc. to the separated enzyme solution, buffering the precipitated enzyme, such as phosphate buffer, tris buffer, acetate buffer Eluting with solution to obtain a fraction, and reacting the eluted enzyme fraction with ginseng to obtain an enzyme fraction exhibiting activity.

The red ginseng may be used by washing red ginseng prepared by a conventional red ginseng manufacturing method by steaming ginseng with water, or a commercially available one. Here, the red ginseng may be selected from the group consisting of Cheonsam, Jisam and Yangsam.

The enzyme-treated red ginseng extract may be obtained by treating the red ginseng with an enzyme using a standardized processing method. The red ginseng can be obtained by red ginseng powder or a method commonly used in the art. The method may be carried out by standard enzyme processing methods (eg, processing using yeast) known in the art, and may use the method described in the Examples herein as an example.

The composition of the present invention exhibits the effect of promoting collagen biosynthesis in human dermal fibroblasts (HDF), preventing skin aging and wrinkle improvement through MMP-1 inhibition, shrinking pores, improving sebum, improving pigmentation, Efficacy in improving skin tone and at the same time showing no change in cell viability, thus showing excellent safety. In particular, the enzyme-treated red ginseng extract of the present invention exhibits improved skin aging resistance and wrinkle-reducing effect as compared with conventional red ginseng extract.

1 is a photograph showing the results of TLC in which the transformation activity of ginseng was sequentially confirmed by using an enzyme solution obtained from six Aspergillus niger strains (KACC 40280, 41018, 41858, 42589, 43547, 44333) One).
FIG. 2 is a graph of a TLC analysis of the change in the ginsenoside content and the change in polysaccharide glycolipids of the red ginseng extract of Example 2 and the red ginseng extract of Comparative Example 1. FIG.
FIG. 3 is a graph showing the HPLC results of the analysis of the change in the ginsenoside component and the change in polysaccharide glycolipids of the red ginseng extract of Example 2 and the red ginseng extract of Comparative Example 1. FIG. [(A); Red ginseng extract, (B); Enzyme-treated Red Ginseng Extract]
4 is a graph showing the results of confirming mRNA expression levels of type 1 procollagen and MMP-1 in the enzyme-treated red ginseng extract of Example 1 performed according to Test Example 2. FIG.
FIG. 5 is a graph showing the results of the cell viability test for the enzyme-treated red ginseng extract of Example 2 performed according to Test Example 2. FIG.
FIG. 6 is a photograph of a multi-facial examination camera system Janus (Aram Huvis, Korea) for the improvement of wrinkle and skin beauty through clinical examination using the enzyme-treated red ginseng extract of Example 2 performed according to Test Example 3, .
FIG. 7 is a graph showing the results of a questionnaire survey on the effect of improving the wrinkles and skin beauty through clinical examination using the ring made using the enzyme-treated red ginseng extract of Example 2 performed according to Test Example 3. FIG.
FIG. 8 is a graph showing the clinical findings of two specialists on the effect of improving the wrinkles and skin beauty in the clinic using the ring made using the enzyme-treated red ginseng extract of Example 2 performed according to Test Example 3. FIG.
9 is a graph showing the results of HLPC analysis of the methanol / acetone insoluble fraction bound to the QAE-column as a pellet obtained from the anion fraction of the enzyme-untreated red ginseng extract.
10 shows HLPC analysis results of methanol / acetone insoluble fractions bound to QAE-column pellets obtained from the enzyme-untreated red ginseng extract cation fraction.
FIG. 11 shows HLPC analysis results of the methanol / acetone insoluble fraction bound to the QAE-column as a pellet obtained from the enzyme-treated red ginseng extract anion fraction.
FIG. 12 shows HLPC analysis results of a methanol / acetone-soluble fraction bound to the QAE-column as a supernatant obtained from the anion fraction of the enzyme-untreated red ginseng extract.
FIG. 13 shows HLPC analysis results of a methanol / acetone-soluble fraction not bound to a QAE-column as a supernatant obtained from an enzyme-untreated red ginseng extract cation fraction.
FIG. 14 is a diagram showing the results of HLPC analysis of the methanol / acetone soluble fraction without binding to the QAE-column, supernatant obtained from the enzyme-treated red ginseng extract cation fraction.

The present invention provides a pharmaceutical composition for preventing skin aging and improving skin wrinkles, comprising an enzyme-treated red ginseng extract derived from Nuruk Fungi.

The enzyme is preferably selected from the group consisting of yeast (fungus), preferably Aspergillus niger strain, more preferably Aspergillus niger KACC 40280, 41018, 41858, 42589, 43547, 44333, It is an enzyme obtained by culturing Aspergillus niger KACC 40280 strain. The enzyme may be prepared by a method comprising the steps of: (a) culturing yeast fungus with ginseng extract or ginseng powder to increase ginsenosidase production, and (b) separating the enzyme from the culture. .

Separation of the enzyme from the culture is a centrifugation step, the step of precipitating the enzyme by adding an organic solvent such as ethanol, acetone, isopropanol, etc. to the separated enzyme solution, buffering the precipitated enzyme, such as phosphate buffer, tris buffer, acetate buffer Eluting with solution to obtain a fraction, and reacting the eluted enzyme fraction with ginseng to obtain an enzyme fraction exhibiting activity.

The red ginseng may be used by washing red ginseng prepared by a conventional red ginseng manufacturing method by steaming ginseng with water, or a commercially available one. Here, the red ginseng may be selected from the group consisting of Cheonsam, Jisam and Yangsam.

The enzyme-treated red ginseng extract may be obtained by treating the red ginseng with an enzyme using a standardized processing method. Such a method can be accomplished with standardized enzyme processing methods (eg, processing with yeast) known in the art, for example using the methods described in the Examples herein. For example, the pulverized red ginseng powder is reacted with the enzyme in an appropriate volume of a reaction solution, and then extracted using an appropriate extraction solvent, such as ethanol, and filtered to obtain a liquid form in which impurities are removed, or the obtained liquid form extract is obtained. Concentration and / or drying under reduced pressure in accordance with conventional methods can be obtained in the form of a powder.

In addition, the extraction process may further comprise the step of obtaining a fraction having a high content of the active ingredient, if necessary. For example, by dispersing the extract obtained by extraction with the primary extraction solvent in water, and then extracting with an appropriate secondary extraction solvent, for example, alcohol of saturated C4, the content of the active ingredient in the resulting extract It can be raised further.

The pharmaceutical composition of the present invention may include 0.01-50% by weight of the enzyme-treated red ginseng extract based on the total weight of the composition.

The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier, and oral dosage forms, external preparations, suppositories, and the like, powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc. according to conventional methods, respectively. It may be formulated in the form of sterile injectable solutions.

Such pharmaceutically acceptable carriers are those commonly used in the art, such as lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, Calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like. In addition, the pharmaceutical compositions of the present invention include diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, and other pharmaceutically acceptable additives.

When the pharmaceutical composition of the present invention is formulated as an oral solid preparation, it includes tablets, pills, powders, granules, capsules and the like, and such solid preparations include at least one excipient such as starch, calcium carbonate, sucrose or rock. Toze, gelatin, and the like, and include but are not limited to lubricants such as magnesium stearate, talc, and the like.

When the pharmaceutical composition of the present invention is formulated for oral liquid preparation, it includes a suspending agent, a liquid solution, an emulsion, a syrup, and the like, and includes, but is not limited to, diluents such as water and liquid paraffin, wetting agents, sweeteners, fragrances, preservatives, and the like.

When the pharmaceutical composition of the present invention is formulated for parenteral use, it includes a sterilized aqueous solution, a non-aqueous solvent, a suspending agent, an emulsion, a lyophilized preparation and a suppository. Non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, Vegetable oils such as oils, injectable esters such as ethyl oleate, and the like. Bases of suppositories may include, but are not limited to, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like.

The dose of the enzyme-treated red ginseng extract contained in the pharmaceutical composition of the present invention varies depending on the condition and body weight of the patient, age, degree of disease, drug form, route of administration and duration, but can be appropriately selected by those skilled in the art. For example, it can be administered or applied at a dose of 1 to 10,000 mg / kg, preferably 10 to 5000 mg / kg per day, the administration may be administered or applied once or several times a day.

The pharmaceutical composition of the present invention can be applied to mammals such as rats, mice, livestock, humans, etc. in various routes, for example orally, or intraperitoneally or intravenously, intramuscularly, subcutaneously, intrauterine dural or intracerebroventricular (Intracerebroventricular) injections. Or may be applied directly to the site where needed.

The present invention also provides a food composition for improving skin beauty, comprising an enzyme-treated red ginseng extract. In particular, the present invention provides a food composition comprising an enzyme-treated red ginseng extract for reducing pores, reducing sebum, skin whitening, preventing skin aging, and improving skin wrinkles.

The food composition of the present invention can be used as a dietary supplement. The term "health functional food" means a food manufactured and processed using raw materials or ingredients having functional properties useful for the human body according to Act No. 6767 of the Health Functional Food Act, and "functionality" refers to the structure of the human body. And ingestion for the purpose of obtaining nutrients for function or for obtaining useful effects in health uses such as physiological actions.

The food composition of the present invention may include a conventional food additives, and the suitability as the "food additives", unless otherwise specified, corresponding items according to the General Regulations and General Test Methods of the Food Additives approved by the Food and Drug Administration It is determined by the standard and the standard.

Examples of the items listed in the "Food Additive Revolution" include, for example, chemical compounds such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamon acid, natural additives such as color pigments, licorice extracts, crystalline cellulose, high color pigments, guar gum, Mixed preparations, such as a sodium L- glutamate preparation, an addition of an alkali, a preservative preparation, and a tar pigment preparation, are mentioned.

Food composition of the present invention, for the purpose of improving skin beauty, may comprise 0.01 to 50% by weight of enzyme-treated red ginseng extract based on the total weight of the composition. The enzyme-treated red ginseng extract contained in the food composition of the present invention can be obtained in the same manner as the extraction method mentioned in the production of the above pharmaceutical composition.

Food composition of the present invention can be produced and processed in the form of tablets, capsules, powders, granules, liquid, pills, beverages, tea, pouches and the like for the purpose of improving skin beauty.

For example, the health functional food in tablet form can be prepared by granulating a mixture of enzyme-treated red ginseng extract, excipient, binder, disintegrant, and other additives by a conventional method, The mixture can be directly compression molded.

In addition, the health functional food in the form of tablets may contain a mating agent and the like, if necessary, may be coated with a suitable coating agent.

The hard capsule of the capsule type health functional food can be prepared by filling a normal hard capsule with a mixture with an additive such as an enzyme-treated red ginseng extract and an excipient, or a granular product thereof or a gelatinized granule. The soft- And an excipient such as a soil extract and an excipient into a capsule base such as gelatin. The soft capsule agent may contain a plasticizer such as glycerin or sorbitol, a colorant, a preservative, and the like, as necessary.

The ring-shaped health functional food can be prepared by molding an appropriate mixture of enzyme-treated red ginseng extract, excipient, binder, disintegrant and the like. If necessary, the preparation may be mixed with white sugar or other suitable skin care agent, It may also be an objectionable substance.

The granular health functional food may be prepared by granulating an enzyme-treated red ginseng extract, an excipient, a binder, a disintegrant and the like in an appropriate manner, and may contain a flavoring agent, a mating agent and the like, if necessary. For the health functional food in the form of granules, No. 12 (1680 μm), No. 14 (1410 μm) and No. 45 (350 μm) were used for the next particle size test. Less than 5.0% and passing through the 45 can be less than 15.0% of the total amount.

The definitions of the above excipients, binders, disintegrants, lubricants, mating agents, flavoring agents, and the like are described in documents known in the art and include the same or similar functions (Korean Pharmacopoeia, College of Pharmacy, 5th ed., P. 33-48, 1989).

The present invention also provides a cosmetic composition for improving skin beauty, comprising an enzyme-treated red ginseng extract. In particular, the present invention provides a cosmetic composition for reducing pores, reducing sebum, skin whitening, preventing skin aging, and improving skin wrinkles, which comprises an enzyme-treated red ginseng extract.

The cosmetic composition of the present invention may include an enzyme-treated red ginseng extract in an amount of 0.01-50 wt% based on the total weight of the composition for the purpose of improving skin beauty. The enzyme-treated red ginseng extract contained in the cosmetic composition of the present invention can be obtained in the same manner as the extraction method mentioned in the production of the above pharmaceutical composition.

The cosmetic composition of the present invention may be prepared in any formulation commonly prepared in the cosmetic field, for example, emulsion, cream, lotion, pack, foundation, lotion, essence, hair cosmetics and the like. Specifically, the cosmetic composition of the present invention can be used as a skin lotion, a skin softener, a skin toner, an astringent, a lotion, a milk lotion, a moisturizing lotion, a nutrition lotion, a massage cream, a nutrition cream, a moisturizing cream, a hand cream, Packs, soaps, cleansing foams, cleansing lotions, cleansing creams, body lotions and body cleansers.

In addition to the enzyme-treated red ginseng extract, the cosmetic composition of the present invention may contain components commonly used in cosmetic compositions, and examples thereof include fatty substances, organic solvents, solubilizers, thickeners and gelling agents, softeners, Preservatives, water, ionic or nonionic emulsifiers, fillers, sequestering agents and chelating agents, preservatives, vitamins, blocking agents, moisturizers, essential oils, dyes, preservatives, Pigments, hydrophilic or lipophilic active agents, lipid vesicles or any other ingredients commonly used in cosmetics.

The enzyme-treated red ginseng extract according to the present invention has a specific red ginseng saponin substrate concentration, polysaccharide, glycoprotein, and glycolipid, which have been converted into relatively low molecular weight. The enzyme-treated red ginseng extract inhibits skin aging, Skin moisturizing food, medicines and cosmetics.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these Examples, Test Examples and Preparation Examples are for illustrating the present invention, and the scope of the present invention is not limited thereto.

[Manufacturing Example]

Preparation Example 1 Preparation of Enzyme Using Strain Culture Solution

Six strains of Aspergillus niger (KACC 40280, 41018, 41858, 42589, 43547, 44333) were cultured by liquid or solid culture. In the case of liquid culture, the culture was performed for 3-5 days in a medium containing beef extract 1.5g / L, peptone 2.5g / L, Magnesium Sulfate 0.05g / L, calcium chloride 0.05g / L (30-37 ℃, 180rpm) .

In the case of solid culture, the blending ratio of malt and bran is 1: 1 and 30% of the total weight of water is added to make a medium, and 5-10% of the strains pre-cultured in PDB (Potato dextrose broth) medium to the solid medium. After inoculation by inoculation, the mixture was mixed well with the medium. The culture temperature was 30-37 ℃, the moisture was maintained at 50-70% strain culture was carried out. In cultivation, ginseng extract or ginseng powder was added to increase the production of ginsenosidase. After solid culture, soak in buffer and enzyme was isolated. When the liquid culture was centrifuged at 7000 rpm for 30 minutes to obtain a crude enzyme (crude enzyme).

Enzyme protein was precipitated by adding an organic solvent to the crude enzyme solution obtained through liquid culture and solid culture. The precipitate was dissolved in a buffer solution, dialyzed, and the miscellaneous was removed to obtain a crude enzyme solution concentrate. It was loaded on an ion exchange resin and eluted with KCl or NaCl buffer. The obtained fraction was reacted with ginseng. Six kinds of enzyme solutions were obtained according to the above-mentioned six strains and the transformation activity of ginseng was sequentially confirmed using TLC (Fig. 1). As shown in FIG. 1, it was confirmed that the reactants reacted with the enzyme solution as compared to the control (major saponin) raised the points in the TLC (production of minor saponin). This indicates that the saponin composition has been converted. As a result, all six strains showed similar results, and the experiment was then performed using enzyme No. 1 obtained from KACC 40280.

[Example]

Example 1. Preparation of enzyme-treated red ginseng extract

Red ginseng provided by Ginseng Corp. was crushed into 20-40 mesh and placed in a flask or glass container. The enzyme obtained in Preparation Example 1 was dissolved in 10 volumes of water compared to the weight of red ginseng at a weight corresponding to 10-20% by weight of red ginseng, and mixed in a flask containing red ginseng. The mixture contained in the vessel was reacted at 50-60 ° C. for 24 hours. After the reaction, the supernatant and the precipitate were separated by centrifugation, and 50% EtOH was added to the precipitate at a weight ratio of 10 times, followed by extraction under reflux at 70 ° C. for 4 hours. After extraction, the mixture was filtered and concentrated to remove EtOH, and then mixed with the supernatant obtained after the initial reaction to proceed with lyophilization.

[Comparative Example]

Comparative Example 1. Preparation of red ginseng extract

The red ginseng extract was obtained in the same manner as in [Example 1] except for the enzyme treatment.

[Test Example]

<Test Example 1> Analysis of components of enzyme-treated red ginseng extract

(1) Saponin change analysis

The enzyme-treated red ginseng extract obtained in Example 1 and the red ginseng extract obtained in Comparative Example 1 were subjected to thin-layer chromatography (TLC) and high performance liquid chromatography (HPLC) after separating only saponin using a C18 sep-pak cartridge Respectively.

For the TLC analysis, the separated saponin was loaded onto a TLC plate (TLC silica gel 60F, merck), and the material was developed using mobile phase (Chloroform: Methanol: Water = 65: 35: 10) (Fig. 2).

For HPLC analysis, a PDA HPLC system instrument and Eclipse XDB-C18 column (4.6 X 250 mm, ID 5 μm) were used, and the mobile phase was mixed with water (H 2 O, 100%) and acetonitrile (CH 3 CN, 100%). It was. When the gradient elution ratio (water: acetonitrile) is 75:25, 68:32, 45:55, 40:60, 0: 100, 0: 100 and 75:25 minutes, 0-10, 10-15, 15-20, 20-25, 25-27, 27-30 and 30-40. The flow rate of the mobile phase was allowed to flow at 1.2 ml / min and the components of red ginseng were analyzed at 203 nm using a UV detector (FIG. 3).

As shown in Figures 2 and 3, the enzyme-treated red ginseng extract showed that the major saponins of red ginseng, Rb1, Rc, and Rd, were converted to relatively low molecular weight compounds such as F2 and C-K, which are trace saponins. That is, the enzyme-treated red ginseng extract of the present invention showed the changed saponin composition and content as compared with the non-enzyme-treated common red ginseng extract.

Test Example 2 Confirmation of skin aging inhibition and wrinkle improving effect

(1) Promoting the synthesis of type 1 procollagen (Type-1 Procollagen) and confirming the inhibitory effect of MMP-1

Human fibroblasts were inoculated with 3 ml of the culture solution in a 60 mm cell culture dish and inoculated with the cells at a concentration of about 1 × 10 ⁵ , followed by incubation for 24 hours at 37 ° C. and 5% CO ₂ . Thereafter, after irradiation with ultraviolet ray B at 20 mJ / cm ² , the medium was replaced with a medium containing 1 ppm of enzyme-treated red ginseng extract of Example 1 and 1 ppm of red ginseng extract of Comparative Example 1, followed by culturing for 2 days. After 2 days of culture, the cells were harvested, RNA was isolated, and mRNA expression levels of procollagen 1 and MMP-1 were confirmed by RT-PCR method. RT-PCR was carried out by applying the method of Kook Sung-ho (J Appl Physiol, 2011) and the like, primers of type 1 procollagen and MMP-1 were as shown in the table below. The results are shown in FIG.

The amount of mRNA expression of type 1 procollagen in the group treated with 10 ug / ml of the enzyme-treated red ginseng extract of Example 1 was 120% higher than that of the group treated with 10 ug / ml of the red ginseng extract of Comparative Example 1. In addition, the mRNA expression level of MMP-1 in the group treated with 10 ug / ml of the enzyme-treated red ginseng extract of Example 1 was found to be decreased by 60% or more as compared with the group treated with 10 ug / ml of the red ginseng extract of Comparative Example 1. As a result, it was confirmed that the enzyme-treated red ginseng extract of the present invention had a superior anti-wrinkle effect than the red ginseng extract not treated with the enzyme.

(2) Confirmation of skin aging inhibition effect using HDF-N-Cell

1) Cell culture

HDF-N cells, which are human skin fibroblasts, were purchased from Modern Cell & Tissue Technology (MCTT), Seoul, Korea and passaged in a laboratory. HDF-N cells were adhered to the cell culture dish and using DMEM (PAA, Canada) with 1% antibacterial-antifungal solution containing penicillin and streptomycin (PAA, Canada) and 10% FBS (PAA, Canada) Incubated. Humidity was maintained at 95% and HDF-N cells were maintained at 37 ° C. while 5% CO ₂ was continuously supplied, and the culture solution was replaced every two to three days. At the time of cell inoculation, the mixed medium and the trypan blue solution were mixed in a 1: 1 ratio to count the living cells not stained by trypan blue using a hemocytometer.

2) MTT analysis

Proliferation of HDF-N cells was measured using MTT colorimetric analysis (EZ-CyTox, Daeil-Lab Co., Korea), which can be searched in a short time, by applying a method such as Lee Hyang-bok (KSBB Journal, 2009). Cells were seeded at 1 × 10 ³ cells / well on a 96-well plate (ISS, korea) plate, and cultured in a 37 ° C. CO ₂ incubator for 24 hours. Then, the target (enzyme prepared in Example 1 and Comparative Example 1 - treated red ginseng extract and normal red ginseng extract) were treated with concentration (0.01, 0.1, 1, 10 ㎍ / ㎖) for 3 days. On the second day (48 h), the MTT (10 μl / well) reagent was added to each well and incubated for 3 hours at 37 ° C. The amount of MTT degraded to formazan was measured at 570 nm using an ELISA reader Respectively. Each treatment group was repeated three times in 3 wells. The cell proliferation effect of the samples was expressed as a percentage of the control group cultured in the DMEM solution after taking the average value of the 3 repeated experiments. The results are shown in Fig. 5A. As shown in FIG. 5A, it was confirmed that the group treated with 10 μg / ml of the enzyme-treated red ginseng extract prepared in Example 1 increased the proliferation of fibroblasts by about 10% as compared with that of normal red ginseng extract. This means that the enzyme-treated red ginseng extract of the present invention exhibits excellent wrinkle preventing effect and skin aging-inhibiting effect as compared with the red ginseng extract not treated with enzyme.

In addition to the above experiment, the HDB-N cells were irradiated with UVB under conditions of a UVB peak range of 312 nm and a UV dose of 20 mJ / cm ² , and then experiments were conducted to confirm cell proliferation effects. The test method was the same as that for confirming the cell proliferation rate. However, before the UVB irradiation, the sample was pretreated with concentrations of 0.01, 0.1, 1 and 10 μg / ml, After adding MTT (10 μl / well) reagent and culturing at 37 ° C for 3 hours, the amount of MTT degradation to formazan was measured at 570 nm using an ELISA reader.

The result is shown in Fig. 5B. It was confirmed that the group treated with 10 μg / ml of the enzyme-treated red ginseng extract prepared in Example 1 increased the proliferation of the fibroblasts by about 14.7% compared to the group treated with the enzyme-untreated red ginseng extract. This means that the enzyme-treated red ginseng extract of the present invention exhibits a wrinkle-preventing effect and a skin aging-inhibiting effect which are superior to the enzyme-treated red ginseng extract in the ultraviolet ray exposure environment.

&Lt; Test Example 3 > Confirmation of skin aging inhibition and wrinkle improving effect through human body application test

The enzyme-treated red ginseng extract obtained in Example 1 was mixed with maltodextrin in the form of a concentrate in a predetermined ratio and then spray-dried using a spray dryer. At this time, the amount of maltodextrin was made into the same amount as the solid content of the concentrated mixture. Crystal dry cellulose was added to the dried powder thus obtained, and kneaded, and the resultant was summoned to a diameter of 2.5 mm using a depilator. The summons were dried for at least 12 hours at 60 ° C. or less until the water content was 10% or less.

The clinical findings and subjects were surveyed by Janus (Aram Huvis, Korea) and two specialists on the effects of wrinkle improvement and skin care. Twenty healthy women and men aged 20 to 40 years old were asked to take a pill made with enzyme-treated red ginseng three times a day for two weeks.

After 2 weeks, the degree of skin wrinkle improvement, pore reduction, sebum reduction, skin tone improvement, and the like were evaluated through the above investigation. The skin wrinkle improvement, pore reduction, sebum reduction, and skin tone improvement of the test subjects were determined in three stages of no improvement, slight improvement, and significant improvement as compared with the pre-dose.

The results are shown in the table below and Figs. 6-8.

As shown in FIG. 6, wrinkle reduction, sebum reduction, pigmentation reduction and skin tone improvement were observed. As shown in the above table and FIGS. 7 and 8, reduction of skin wrinkles and pore size , Pigmentation, sebum, skin tone, etc., were found to be 80% positive. Thus, it was confirmed that the enzyme-treated red ginseng extract of Example 1 had a skin-beauty-improving effect.

<Test Example 4> Comparative analysis of components of enzyme-treated red ginseng extract and enzyme-untreated red ginseng extract

(1) Extraction and Anion Fraction Separation

5 g of the sample prepared in Example 1 and Comparative Example 1 was added to 20 ml of 50 mM Tris-HCl pH 7.8 and shaken well and extracted at room temperature. This was repeated three times and centrifuged (3500 rpm, 15 min) after every extraction to separate the pellet and solution. Column loaded with extract (1 × 5 cm) was combined with QAE-Sepadex A50. The column was washed with 5 times the amount of buffer (50 mM Tris-HCl pH 7.8) and the bound portion eluted with 3 times the amount of 3M ammonium acetate pH 8.0 of the filler. The polymer compound in the eluate of the QAE-column was precipitated by adding a 4-fold mixture of methanol-acetone (1: 2, -20 ° C). The pellet obtained by centrifugation (3500 rpm, 15 min) was dried in a vacuum dryer and the supernatant was evaporated to dryness.

(2) Separation of Cationic Fractions

The combined portion of the QAE-column (washing with loading buffer) is concentrated to 10x and the cationic (including neutral) biopolymer is added with a four-fold volume of methanol-acetone (1: 2, -20 ° C) mixture Precipitated. The pellet obtained by centrifugation (3500 rpm, 15 min) was dried in a vacuum dryer and the supernatant was evaporated to dryness.

(3) Measurement of molecular mass and RP-chromatography

The molecular mass of the obtained fraction component was analyzed by HPLC method on three tandem column ultrahydrogels 1000, 500 and 250 (Chromatograph Waters 2690, detector RID 486. Mobile phase 0.2 M KNO ₃ Flow rate 0.9 ml / min). Columns were calibrated with polyvinyl pyrrolidone (PVP) 100 and 40 kDa with dextrin 500, 20, 10 and 5 kDa.

The components of the fractions dissolved in methanol / acetone were performed by HPLC method on RP-column (0.46 × 25 cm Ultrasphere C18), chromatograph waters 2690, detector PDA 996 (scanerange 200-400 nm). Mobile phase A-0.1% formic acid in water; B-methanol, flow rate 1.0 ml / min.

The above results are shown in Figs.

The time [min] on the abscissa in Fig. 3 represents the diffusion time, and the voltage [mV] of the cell axis represents the content of the component.
(Ug / ml) represents the concentration of the transformed red ginseng extract, and the ordinate represents the concentration of mRNA of type 1 procollagen, MMP-1, and GAPDH. Expression level. In addition, mRNA (%) represents the value quantified based on the control group.

Claims (19)

A pharmaceutical composition for skin aging and skin wrinkle improvement comprising red ginseng extract treated with yeast mold derived enzyme. According to claim 1, wherein the yeast fungus-derived enzyme
(a) incubating yeast fungus with ginseng extract or ginseng powder to increase ginsenosidase production, and
(b) separating the enzyme from the culture
Pharmaceutical compositions prepared by the method comprising. The pharmaceutical composition of claim 2, wherein the yeast fungus is an Aspergillus niger strain. The pharmaceutical composition of claim 3, wherein the Aspergillus niger strain is an Aspergillus niger KACC 40280, 41018, 41858, 42589, 43547 or 44333 strain. The pharmaceutical composition according to claim 1, wherein the enzyme-treated red ginseng extract is obtained by incubating red ginseng powder or red ginseng extract with the yeast mold-derived enzyme. The pharmaceutical composition according to any one of claims 1 to 5, which is a powder, tablet, granule, pill, hard capsule, soft capsule or liquid formulation. A food composition for improving skin beauty comprising red ginseng extract treated with yeast mold-derived enzyme. The method of claim 7, wherein the yeast fungus-derived enzyme
(a) incubating yeast fungus with ginseng extract or ginseng powder to increase ginsenosidase production, and
(b) separating the enzyme from the culture
Food composition prepared by a method comprising. The food composition of claim 8, wherein the yeast fungus is an Aspergillus niger strain. The food composition of claim 9, wherein the Aspergillus niger strain is an Aspergillus niger KACC 40280, 41018, 41858, 42589, 43547 or 44333 strain. 8. A food composition according to claim 7 for reducing pores, reducing sebum, skin whitening, preventing skin aging or improving skin wrinkles. 8. The food composition of claim 7, wherein the enzyme-treated red ginseng extract is obtained by incubating red ginseng powder or red ginseng extract with the yeast mold-derived enzyme. The food composition according to any one of claims 7 to 12, which is a pouch, beverage or tea. Cosmetic composition for improving skin beauty, comprising yeast fungi-derived enzyme-treated red ginseng extract. The method of claim 14, wherein the yeast fungus derived enzyme
(a) incubating yeast fungus with ginseng extract or ginseng powder to increase ginsenosidase production, and
(b) separating the enzyme from the culture
Cosmetic composition prepared by a method comprising. The cosmetic composition according to claim 15, wherein the yeast fungus is an Aspergillus niger strain. The cosmetic composition of claim 16, wherein the Aspergillus niger strain is an Aspergillus niger KACC 40280, 41018, 41858, 42589, 43547 or 44333 strain. 15. A cosmetic composition according to claim 14 for reducing pores, reducing sebum, skin whitening, preventing skin aging or improving skin wrinkles. The cosmetic composition according to claim 14, wherein the enzyme-treated red ginseng extract is obtained by culturing red ginseng powder or red ginseng extract after incubation with the yeast mold-derived enzyme.

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