KR20190131765A - Method for detoxing fingerroot extracts and skin homeostasis and nontoxicity cosmetic compositions containing detoxed fingerroot extracts - Google Patents

Abstract

The present invention provides a method for removing toxicity of a finger root extract that is highly toxic to skin cells, and a non-toxic cosmetic composition containing the finger root extract whose toxicity is removed by the method, thereby maintaining homeostasis of skin cells. The finger root extract of which toxicity is removed according to the present invention prevents deterioration of skin cell function due to toxicity of panduratin and prevents skin aging and skin inflammation thereby, and is effective in providing a skin homeostatic non-toxic cosmetic composition.

Description

Methods for detoxing fingerroot extracts and skin homeostasis and nontoxicity cosmetic compositions containing detoxed fingerroot extracts}

The present invention relates to a finger root extract that helps maintain skin homeostasis, and in particular, a method of removing the toxicity of a finger root extract that is highly toxic to skin cells and a finger root extract from which the toxicity is removed by the method of removing the toxicity. It relates to a non-toxic cosmetic composition to maintain the homeostasis of the skin cells.

Fingerroot (fingerroot, Boesenbergia pandurata) is a plant of ginger family native to Southeast Asia, as well as traditional Asian dishes such as pickles, curry, and beverages, as well as tonics for childbirth, stomach pain, diarrhea, flatulence, indigestion, ulcer treatment It has been used for various purposes such as leukocyte prophylaxis and cost aid.

Fingerroot contains a flavonoid component that inhibits the infection of Helicobacter pylori bacteria, including indigestion and gastritis, and contains a cytoprotective pinotstrobin component that increases gastric mucus and reduces the area of gastric ulcer formation. It is also effective in treating peptic ulcers.

In addition, flavonoid derivatives that can inhibit the growth of cancer cells, pinosembrine, pinostrobin, ilpinetin, tadamonine, pandurata, panduratin A, and other ingredients are contained, especially pandurata The ingredient inhibits cancer cell growth, and panduratin A is known to have a prophylactic effect against prostate cancer and colorectal cancer.

Recently, according to environmentally friendly and natural-oriented trends, the active ingredients used in cosmetics are based on the usefulness of natural substances derived from plants as well as chemicals. It is used.

Accordingly, as a prior art related to finger root extract and panduratata or panduratin A, a study on panduratin A or Boesenbergia pandurata extract having an effect on atopic dermatological diseases (Korean Patent Publication 10-2012-0113201, published 2012.10.12.), Cosmetic composition for skin whitening containing panduratin derivatives (Korean Patent Publication No. 10-0829819, registered on 2008.05.08.), Containing Bosenbulzia rotunda extract Skin whitening and antioxidant composition (Korean Patent Publication No. 10-1521579, registered on May 13, 2015), novel use of panduratin A or its derivatives (Korean Patent Publication No. 10-0973221, registered on July 26, 2010) ), A novel use of panduratin derivatives or a camemperia pandurata extract containing the same (Korean Patent Publication No. 10-1088069, registered on November 23, 2011), New Calcon derivative (Japanese Patent Publication No. 10-3840536 Registered on Aug. 18, 2006) There is.

According to the recent literature, Pandoratin, a functional ingredient extracted from the roots of ginger plants, especially finger roots, is a light yellow brown powder extracted from the roots of finger roots, and is a functional ingredient of health functional foods in 2013 by the Ministry of Food and Drug Safety. It was recognized as a bioactive function grade 2

Panduratin is a light yellowish brown powder extracted from the root of the plant called finger root.It was recognized by the Ministry of Food and Drug Safety as a functional ingredient for health functional foods in 2013 (grade 2 bioactive function), and it protects skin health from skin damage caused by ultraviolet rays. It is known to contribute to reducing body fat by activating 'AMPK (AMP-activated protein kinase)', a sensor protein for maintaining and maintaining energy homeostasis in the body.

However, finger root root extract powder, panduratin, is highly toxic and should be used with caution.

In recent years, from the study of extracting and refining an active ingredient from natural products as a new material, the demand for functional cosmetics that has been extended to the range of skin improvement in the efficacy of cosmetics that have simply stayed in terms of skin protection is increasing rapidly.

Thus, in providing a functional cosmetic composition using the finger root extract, which has already been proved to be effective, panduratin is a major obstacle to skin cells, and thus can remove or alleviate the toxicity of the finger root extract. The solution is urgently needed.

KR 10-2012-0113201 A (published October 12, 2012), KR 10-0829819 B1 (registered May 8, 2008), KR 10-1521579 B1 (registered May 13, 2015), KR 10-0973221 B1 (registered July 26, 2010), KR 10-1088069 B1 (registered 23 November 2011), JP 10-3840536 B1 (registered August 18, 2006)

As the market demand for multifunctional cosmetics using natural materials increases, the inventors have developed a sensor protein 'AMPK (AMP-activated protein kinase)' for maintaining skin health and maintaining energy homeostasis. The present invention has been completed by discovering an effective method for removing toxicity that can remove and alleviate the toxicity of panduratin, the main component of finger root extract, which can contribute to reducing body fat by activating.

Therefore, an object of the present invention is to provide a method that can effectively remove the toxicity of the finger root extract.

In addition, another object of the present invention is to provide a skin homeostasis non-toxic cosmetic composition containing the finger root extract is removed toxicity.

The technical problems and objects to be solved by the present invention are not limited to the technical problems and objects mentioned above, and other technical problems and objects not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the above object, the present invention provides a method for removing the toxicity of a finger root extract, the process comprising: ethanol (Ethanol) in the finger root powder (Powder) and extracting at room temperature for 24h using a shaker; Filtering the ethanol extract, adding distilled water (Water) and then concentrating in vacuum to remove ethanol; And re-dissolving the concentrated solution from which ethanol has been removed with DMSO (dimethyl sulfoxide) and filtering with a sterile filter, storing the solution at a temperature of minus 20 degrees Celsius.

At this time, in the method of removing the toxicity of the finger root extract according to the present invention, before re-dissolving the concentrated solution from which the ethanol has been removed, chloroform is added to the concentrated solution from which the ethanol has been removed and shaken (shaking). Removing the supernatant after the back separation; And removing the lower layer solution, adding ethyl acetate and shaking to leave, then separating and collecting the lower layer solution and freeze-drying to form a powder; further dissolving the powder in DMSO Characterized in that.

In order to achieve the above object another object of the present invention provides a skin homeostasis non-toxic cosmetic composition containing a finger root extract of which toxicity is removed according to the method for removing toxicity.

At this time, in the skin homeostasis non-toxic cosmetic composition containing the finger root extract, the concentration of the finger root extract is preferably 0.1-20μg / ml.

As described above, the present invention provides a method that can effectively remove the toxicity of the finger root extract.

Accordingly, the finger root extract of which toxicity is removed according to the present invention is effective in preventing skin degradation due to toxicity of panduratin, skin aging and dermatitis caused by this, and thereby providing a skin homeostasis nontoxic cosmetic composition. .

1 to 3 is a graph showing the toxicity test results of the finger root extract for each solvent,
4 to 6 is a graph showing the results of measuring the protective effect on the UVB stimulation of each solvent root extract extract,
7 and 8 are graphs comparing the protective effect against UVB stimulation of the solvent-based finger root extract at concentrations of 0.5 and 1 μg / ml,
9 and 10 are graphs showing the results of cytotoxicity test according to the concentration of each fraction of the finger root ethanol extract,
11 and 12 are graphs showing the results of the cell protection effect test for UVB irradiation according to the concentration of each fraction of the finger root ethanol extract,
13 is a cytotoxicity test graph of the water fraction of the finger root ethanol extract
14 is a cell protective effect test graph of the water fraction of the finger root ethanol extract,
15 is a cytotoxicity test graph at a concentration of 1-20 μg / ml of the water fraction of the finger root ethanol extract,
16 is a graph evaluating the cell protective effect at the concentration of water fraction 1-20μg / ml of the finger root ethanol extract,
17 is a graph showing the change in collagen amount according to the concentration of water fraction 1-20μg / ml of the finger root ethanol extract,
18 is a graph showing the measurement results of MMP-1 production amount upon UVB irradiation according to the concentration of water fraction 1-20μg / ml of the finger root ethanol extract,
19 is a graph showing the measurement results of MMP-3 production amount during UVB irradiation according to the concentration of water fraction 1-20μg / ml of the finger root ethanol extract,
20 is a graph of fluorescence intensity measurement for measuring the generation of reactive oxygen species (Reactive Oxygen Species, ROS) according to the concentration of water fraction 1-20μg / ml of the finger root ethanol extract.

DETAILED DESCRIPTION Hereinafter, detailed descriptions of preferred embodiments of the present invention will be described with reference to the accompanying drawings. Specific details appear in the following description, which is provided to help a more general understanding of the present invention, and is not intended to limit the present invention to specific embodiments, and all changes included in the spirit and technical scope of the present invention, It should be understood to include equivalents and substitutes. In the following description of the present invention, if it is determined that a detailed description of a related known function or configuration may unnecessarily obscure the subject matter of the present invention, the detailed description thereof will be omitted.

Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art. Terms such as those defined in the commonly used dictionaries should be construed as having meanings consistent with the meanings in the context of the related art, and shall not be construed in ideal or excessively formal meanings unless expressly defined in this application. Do not.

As used in the description of the present invention, the term "extract" refers to an active ingredient isolated from natural products, that is, a material that exhibits the desired activity. The extract may be obtained by an extraction process using water, an organic solvent or a mixed solvent thereof, and includes the dry powder thereof or any form formulated using the extract. In addition, the extract also includes a fraction obtained by the extraction process.

First, in order to confirm the toxicity of the solvent-specific extracts before removing the toxicity from the finger root extract according to the present invention, and to determine the photoaging protection effect of the finger root extract in skin cells induced by UVB (Ultra Violet B) damage Fingerroot extract was prepared for various solvents as follows.

<Finger Root Extract Preparation>

Fingerroot extract used in the present invention is taken 5g out of powder-shaped finger root, and then by nucleic acid (hexane), chloroform (chloroform), ethyl acetate (ethyl acetate), ethanol (ethanol), methanol (methanol) and water 100 ml of water was added thereto and extracted at room temperature for 24 h using a shaker. After filtering using a filter (Whatman No. 2 filter paper), the solvent-specific extracts were concentrated in vacuo. The concentrate was redissolved with dimethyl sulfoxide (dimethyl sulfoxide), filtered through a 0.22 um sterile filter, and stored at −20 ° C. until analysis. The water extract was filtered and then lyophilized to redissolve the powder in 50% DMSO and then filtered through a sterile filter.

1 to 3 show the toxicity test results of the finger root extract for each solvent. AsA used for the test is labeled ascorbic acid (Vit.C) as a positive control.

1 to 3 show nucleic acids (hexane, H), chloroform (Ch), ethyl acetate (EA), ethanol (Etol, Et), methanol (methanol, Me) and water (W). As a result of treating skin cells Hs68 at concentrations of 0.1, 0.5, 1, and 5 μg / ml with each solvent, the extracts were highly toxic at 5 μg / ml except for the water extract.

Next, the experimental results of measuring the cellular protective effect of the UVB stimulation of the finger root extract of each solvent by treating at the same concentration as when measuring the cytotoxicity in order to measure the cytoprotective effect on the UVB stimulation Figures 4 to 6 Shown in As shown in Figure 4 to 6, the cell protective effect was not apparent in the remaining samples except ethanol and methanol.

Therefore, according to the purpose of the present invention to remove the toxicity of the finger root extract and maintain the activity, the following experiments were carried out with the ethanol extract, which has good activity except for methanol extract and can be used in food or cosmetics.

7 and 8 are graphs showing the results of experiments with the concentration of 0.5 and 1 μg / ml in order to prepare a cell protection effect for the UVB stimulation of each solvent root extract extract. As shown in FIG. 7 and FIG. 8, the experimental results of the cell protective effect on UVB stimulation showed the best effect in the ethanol extract, and the protective effect was significantly increased at 0.5 μg / ml rather than 1 μg / ml.

As a result of the above experiment, the finger root ethanol extract showed the highest cell protection effect against UVB. In the next step, we tried to find a fraction that lowers the toxicity and maintains the activity of the finger root ethanol extract. Therefore, the ethanol extract according to the polarity of the solvent to prepare a fraction in the order of ethanol, chloroform, ethyl acetate, water. Hereinafter, a method for preparing a fraction of the finger root extract will be described in detail.

<Preparation of Fraction of Fingerroot Extract>

100 g of powder-shaped fingerroot was taken, 1 L of ethanol was added, and extracted at room temperature for 24 h using a shaker. After filtering using a filter (Whatman No. 2 filter paper), 100 ml in 1 L was taken, and 200 ml of distilled water was added. The mixture of finger root ethanol extract and distilled water was concentrated in vacuo to remove all ethanol and transferred to a separatory funnel. A total of 2 L of chloroform was added thereto, shaken sufficiently, left to separate, and the lower layer was collected and concentrated.

After chloroform fractionation, a total of 1L of ethyl acetate was added, shaken sufficiently, left to separate, and the supernatant was collected and concentrated. Ethyl acetate was removed and the remaining water layer was lyophilized to form a powder. The concentrate and powder were both redissolved with DMSO and then filtered through a 0.22 μm sterile filter and stored at −20 ° C. until analysis.

Cytotoxicity experiments were performed on the finger root ethanol extract fractionated with ethanol, chloroform, ethyl acetate, and water as described above. Cell culture and cytotoxicity experiments were as follows.

Hs68 cells, a human dermal fibroblast line used for cytotoxicity experiments, were purchased from the American Type Culture Collection, Manassas, VA, USA. 37 ° C, 5% carbon dioxide (CO ₂ ) and 95% wet air using Dulbecco's Modified Eagle's Medium (DMEM) medium containing 10% FBS, penicillin (100 units / ml) and streptomycin (50 μg / ml) (humid air) was incubated in an incubator (Thermo Scientific, Tewksbury, MA, USA). Hs68 cells were dispensed in a 96-well plate at a concentration of 1.0Х104 cells / well, followed by 24h culture, and the medium was replaced with a medium containing no sample and no FBS. After 24 h sample treatment, 20 uL of MTT (1 mg / ml) solution was added to each well and incubated for 2 h. Finally, the supernatant was removed, and blue formazan crystals produced in viable cells were solubilized with DMSO to measure absorbance at 550 nm using a Microplate reader (BioTek, Inc., Winooski, VT, USA). In sample processing, the sample was diluted with medium so that the final concentration of DMSO was less than 0.1% (v / v).

In order to evaluate the cytotoxicity according to the concentration of each fraction of the finger root ethanol extract, hs68 cells were treated with 0.1, 0.5, 1, 5 μg / ml. As a result of treatment by concentration, as shown in Figures 9 and 10, all of the fractions except water fractions showed cytotoxicity at 5μg / ml. 9 and 10 are graphs showing the results of cytotoxicity test according to the concentration of each fraction of the finger root ethanol extract.

<UVB investigation>

Hs68 cells were inoculated at a concentration of 1.0Х104 cells / well in a 96-well plate and incubated for 24 h. After diluting the sample with medium without FBS in each well, the cells were incubated for 24 h. Afterwards, UVB (30 mJ / cm ² ) was irradiated using UVB lamps (Sankyo Denki Lamps, GL20SE, Marine, Japan) and UV intensity was monitored using UV LIGHT METER (LT Lutron, UV-340A, Taiwan). It was. All UVB irradiation was performed after a thin coating of PBS on the cells attached to the 96-well plate. After irradiation, the samples were diluted in media without FBS and treated with cells for 24 h. The control proceeded under the same conditions without UVB irradiation.

In order to evaluate the cellular protective effect of UVB irradiation on each fraction of the finger root ethanol extract, the extract for each solvent was treated with 0.1, 0.5, 1, 5 μg / ml concentration before and after UVB irradiation. As a result, as shown in Figs. 11 and 12, only the water fraction was maintained in the protective effect activity without toxicity up to 5 μg / ml.

Therefore, the water fraction of finger root ethanol extract was selected to measure the cytotoxicity and UV protective effect against UVB by concentration (1, 5, 25, 50 μg / ml). As shown in FIG. 13, the cytotoxicity did not show cytotoxicity up to 25 μg / ml, but at 50 μg / ml, the cell viability was reduced to 86%. On the other hand, in the case of the cell protective effect, as shown in Figure 14 showed a cell protective effect at 5μg / ml, 25μg / ml. Cytotoxicity test graphs and cell protective effect test graphs are shown in FIGS. 13 and 14, respectively.

As a result of the above test, the cell viability tended to decrease slightly in the water fraction of 25μg / ml of the finger root ethanol extract, so that the highest concentration was set to 20μg / ml in order to obtain an ideal result of increasing concentration-dependently during sample treatment.

The cytotoxicity of 1-20 μg / ml of the water fraction of Fingerroot ethanol extract and the cytoprotective effect against UVB did not show toxicity up to 20 μg / ml (see FIG. 15), and the cell viability decreased by UVB concentration. Increased dependently (see FIG. 16).

UVB irradiation, on the other hand, reduces the collagen production of cells. Therefore, the effect of water fraction of finger root extract on the amount of collagen in cells was measured. Experimental method is as follows.

<Measurement of MMP-1, MMP-3 and Water Soluble Collagen Production>

Measurements of MMP-1 and MMP-3, which are synthesized by UVB irradiation, were measured using enzyme-linked immunosorbents using human MMP-1 and MMP-3 ELISA kits (Merck & Co. Inc., Whitehouse Station, NJ, USA). The amount of pro-MMP-1 and MMP-3 in the medium was measured by the assay method. After UVB irradiation, soluble collagen production was measured using the SircolTM soluble collagen assay kit. The cultured cells were collected, mixed with Sirco dye reagent, and reacted. After centrifugation, cold acid-salt washing reagent was added to the precipitate and mixed. After centrifugation of the mixture, the precipitate was dissolved using an alkali reagent and the absorbance was measured at 555 nm.

As a result, UVB decreased the amount of collagen in the cells, and the concentration of collagen increased with increasing concentration of water fraction from 1μg / ml to 20μg / ml. The test results are shown in FIG. 17.

At this time, UVB irradiation increases the production of MMP-1 (Matrix MetalloProtease), an enzyme that decomposes Type 1 collagen, the protein most present in the skin, thereby promoting the decomposition of collagen. Therefore, the effect of the water fraction of the finger root extract on the production of MMP-1 was evaluated by the method described above. As a result, as shown in FIG. 18, MMP-1 production increased during UVB irradiation, and MMP-1 production decreased with increasing concentrations from 1 μg / ml to 20 μg / ml when treated with water fractions.

In addition, since MMP-3 activates MMP-1 to promote collagen degradation, MMP-3 production was measured in the same manner as MMP-1. As a result, MMP-3 production was increased during UVB irradiation, and MMP-3 production was decreased as the concentration was increased from 1 μg / ml to 20 μg / ml when the water fraction was treated (see FIG. 19).

UVB irradiation also stimulates the production of Reactive oxygen species (ROS) in cells. This abnormal increase in reactive oxygen species causes oxidative stress and induces MMPs in dermal fibroblasts to promote skin aging. Therefore, the effect of finger root extract on the ROS production of water fraction was evaluated. Experimental method is as follows.

<ROS generation measurement>

Samples were pretreated with Hs68 cells for 24 h to measure Reactive Oxygen Species (ROS) production, washed with PBS and irradiated with UVB (30 mJ / cm ² ). After UVB irradiation, the cells were treated with the samples for 30 minutes and stained with 25 uM of DCFH-DA. Fluorescence intensity corresponding to intracellular ROS generation was measured by a fluorescent spectrophotometer (Perkin-Elmer, Norwalk, CT, USA) for 2 hours at an excitation wavelength of 485 nm and an emission wavelength of 530 nm.

As a result, as shown in Figure 20, UVB irradiation increased the production of ROS in the cell, the concentration of the water fraction treatment ROS production was reduced. 20 is a graph of fluorescence intensity measurement for measuring ROS reactive oxygen generation (ROS) generation according to the concentration of water fraction 1-20μg / ml of finger root ethanol extract.

As described above, the method for removing the toxicity of the finger root extract according to the present invention effectively removes the toxicity of the finger root extract, thereby minimizing the time to stay in the skin due to toxicity and irritation, and solving the problem that caused the resistance Skin homeostasis can be safely maintained without toxicity and irritation.

In addition, the finger root extract from which the toxicity is removed according to the present invention does not exhibit toxicity up to 20 μg / ml, and increases the cell survival rate reduced by UVB in a concentration-dependent manner, and the amount of collagen in the cell by UVB irradiation, MMP-1 And by reducing the concentration-dependent MMP-3 and ROS, to prevent the degradation of the skin cells due to the toxicity of panduratin, thereby preventing skin aging and dermatitis, thereby providing a skin homeostasis non-toxic cosmetic composition.

The skin homeostasis non-toxic cosmetic composition according to the present invention may be prepared in any formulation conventionally prepared in the art, and may be used topically or systemically as commonly used in the field of dermatology by containing a dermatologically acceptable medium or base. It may also be prepared in the form of an adjuvant.

Formulations of suitable cosmetic compositions include, for example, emulsions, suspensions, microemulsions, microcapsules, microgranules or ionic (liposomes), nonionics obtained by dispersing an oil phase in a solution, gel, solid or pasty anhydrous product, aqueous phase. It may be provided in the form of a vesicle dispersant, in the form of a cream, skin, lotion, powder, ointment, spray or conceal stick. It may also be prepared in the form of a foam or in the form of an aerosol composition further containing a compressed propellant.

Products to which the cosmetic composition of the present invention may be added include, but are not limited to, mask packs, skin lotions, skin softeners, skin toners, sprays, astringents, softeners, nutrients, astringents, lotions, milk lotions, moisturizers Lotion, nutrition lotion, body cream, massage cream, nutrition cream, massage cream, moisture cream, hand cream, essence, eye cream, nutrition essence, pack, soap, shampoo, cleansing foam, cleansing lotion, cleansing cream, body lotion, body Formulations such as cleansers, treatments, essences, emulsions, press powders, loose powders, eye shadows and the like.

Meanwhile, in the detailed description of the present invention, specific embodiments have been described, but various modifications are possible without departing from the scope of the present invention. Therefore, the scope of the present invention should not be limited to the described embodiments, but should be determined not only by the scope of the following claims, but also by the equivalents of the claims.

As described above, the present invention provides a skin homeostasis non-toxic cosmetic composition containing a method of removing the toxicity of the finger root extract and the finger root extract from which the toxicity is removed, and the finger root extract of which the toxicity is removed according to the present invention is antioxidant, non-toxic. It has high activity and anti-inflammatory activity, so it is effective in the preparation of skin homeostasis products, and thus has high industrial availability.

Claims (4)

In the method of eliminating the toxicity of the finger root extract,
Ethanol (Ethanol) in the finger root powder (Powder) and extracting at room temperature for 24h using a shaker (shaker);
Filtering the ethanol extract, adding distilled water (Water) and then concentrating in vacuum to remove ethanol; And
Dissolving the concentrated ethanol removed with DMSO (dimethyl sulfoxide) and filtered with a sterile filter, the process of storing at 20 degrees Celsius; the method of removing the toxicity of the finger root extract.
The method of claim 1,
Before re-dissolving the ethanol-free concentrate in DMSO,
Chloroform in a concentrated solution from which the ethanol has been removed, followed by shaking to stand for shaking, and then removing the lower layer solution after separation; And
After removing the lower layer solution, the ethyl acetate (Ethyl Acetate) was added to shake, left to stand, separated after the separation of the lower layer solution, lyophilized to form a powder form; after further performing, the powder is re-dissolved in DMSO Toxicity removal method of the finger root extract, characterized in that.
A skin homeostasis non-toxic cosmetic composition containing a finger root extract of which toxicity is removed according to the method for removing toxicity of claim 1.
The method of claim 3,
Skin homeostasis non-toxic cosmetic composition containing a finger root extract, characterized in that the concentration of the finger root extract is 0.1-20μg / ml.

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