KR20230072193A - Cosmetic composition comprising crypsinus hastatus gametophyte extract - Google Patents

Abstract

The present invention relates to a cosmetic composition containing an extract of the whole leaf of Korancho, and by including the extract of the entire leaf of the coriander plant, it is possible to provide effects capable of enhancing skin anti-aging effects including skin moisturizing, antioxidant and skin whitening without cytotoxicity and skin side effects. can

Description

COSMETIC COMPOSITION COMPRISING CRYPSINUS HASTATUS GAMETOPHYTE EXTRACT}

The present invention relates to a cosmetic composition containing an extract of the whole leaf of Korancho.

Human skin is divided into three layers: epidermis, dermis, and subcutaneous fat, and serves to protect and support all stimuli from the external environment. In particular, the amount of water contained in human skin is about 15 to 20%, and it exists together with oil, and plays an important role in transporting various physiologically active factors necessary for the human body and controlling the evaporation of water in the body to the outside of the body. Moisture is moderately present in the stratum corneum to give the skin elasticity and flexibility. The skin is composed of four layers of stratum corneum, epidermis, dermis, and subcutaneous tissue, and cells and matrices related to skin aging are mainly present in the epidermis and dermis.

Collagen and elastin, which are components of the dermis, give strength and tension to the skin and affect skin elasticity. Collagen is synthesized by the action of fibroblasts and degraded by collagenase, and elastin is degraded by elastase.

As people age, their skin undergoes changes due to various internal or external factors. As the skin ages, imbalances in various hormones related to metabolism occur as an internal factor, and as free radicals and active oxygen caused by ultraviolet rays increase as an external factor, the thickness of the skin decreases, wrinkles form, and elasticity and moisturizing effect increase. It falls.

The increase in reactive oxygen species (ROS) generated by these factors can irreversibly destroy proteins, lipids, and DNA, which are cellular components of the human body.

The reactive oxygen species are antioxidant enzymes and tocopherols present in living organisms such as superoxide dismutase (SOD), catalase, peroxidase, and glutathione. ), vitamin C, and other non-enzymatic antioxidants are removed by the antioxidant defense system, thereby maintaining normal cellular functions of the human body, and various antioxidants are being researched and developed.

Meanwhile, human skin color is determined according to the concentration and distribution of melanin in the skin. Melanin protects skin organs under the dermis by blocking ultraviolet rays in the epidermal layer and protects the skin by absorbing biological free radicals. However, if it is present in excess, it may cause skin pigmentation such as melasma, freckles, and moles. Melanin pigment is produced from tyrosine by tyrosinase in pigment cells called melanocytes present in the basement membrane of the skin, and it is known that the production is promoted by stimuli such as ultraviolet light or inflammation. Therefore, it is possible to reduce the production of melanin by reducing external stimuli, blocking signal transmission, or inhibiting the synthesis or activity of tyrosinase, a melanin-producing enzyme. Arbutin, kojic acid, etc. have been known to have a melanin production inhibitory effect, but their utilization is not high due to a slight whitening effect, low stability, and skin irritation. need.

Therefore, in the field of cosmetics, research and development on functional cosmetics having moisturizing effects as well as antioxidation and whitening effects are being actively conducted.

Korean Patent Publication No. 10-2019-0004992 discloses a composition for skin whitening having an effect of inhibiting the activity of tyrosinase and melanin biosynthesis, but using a composition artificially synthesized, natural extracts The development of a cosmetic composition using the is incomplete.

Republic of Korea Patent Publication No. 10-2019-0004992 (2019.01.15. Publication)

An object of the present invention is to provide a cosmetic composition having no side effects by including the natural material, Crypsinus hasstatus whole leaf extract as an active ingredient.

In addition, an object of the present invention is to provide a cosmetic composition containing the extract of the whole leaf of Korancho exhibiting excellent skin moisturizing, antioxidant and skin whitening effects.

However, the problem to be solved by the present application is not limited to the above-mentioned problem, and other problems not mentioned will be clearly understood by those skilled in the art from the description below.

In order to achieve the above object, the present invention provides a cosmetic composition containing an extract of the whole leaf of Korancho ( Crypsinus hasstatus ).

An object of the cosmetic composition according to the present invention is to provide a cosmetic composition including skin moisturizing, antioxidant and skin whitening without cytotoxicity and skin side effects by including a natural material, Crypsinus hasstatus whole leaf extract.

Specifically, the cosmetic composition according to the present invention can provide a moisturizing effect by increasing the intracellular production of hyaluronic acid, which regulates the skin barrier function and maintains skin elasticity.

In addition, the cosmetic composition according to the present invention can provide an excellent antioxidant effect by increasing free radical scavenging activity, which is an oxygen compound that causes aging, arteriosclerosis, cancer, and the like.

In addition, the cosmetic composition according to the present invention can provide a skin whitening effect by inhibiting the activity of tyrosinase, an enzyme that promotes melanin production, and having DOPA oxidation inhibitory activity.

In addition, the cosmetic composition according to the present invention prevents melanin synthesis caused by α-MSH, inhibits melanin accumulation, and inhibits the expression of melanin synthase and factors involved in synthesis to promote skin whitening. efficacy can be provided.

The effects of the present invention are not limited to the above effects, and should be understood to include all effects that can be inferred from the detailed description of the present invention or the configuration of the invention described in the claims.

1 is a graph showing the results of cytotoxicity evaluation by concentration according to Examples and Comparative Examples of the present invention.
Figure 2 (a) is a graph showing the results of cytotoxicity evaluation by concentration of the whole leaf extract of Gorancho according to an embodiment of the present invention.
Figure 2 (b) is an image showing the results of MTT crystal violet staining for each concentration of the cytotoxicity evaluation of the whole leaf extract of Korancho according to an embodiment of the present invention.
3 is a graph showing the results of DPPH free radical scavenging rate evaluation by concentration according to Examples and Comparative Examples of the present invention.
Figure 4 is a graph showing the DPPH free radical scavenging rate evaluation results for each concentration of the whole leaf extract of Korancho according to an embodiment of the present invention.
5 is a graph showing the evaluation results of ABTS radical scavenging ability by concentration according to Examples and Comparative Examples of the present invention.
6 is a graph showing the ABTS radical scavenging ability evaluation results for each concentration of the whole leaf extract of Korancho according to an embodiment of the present invention.
7 is a graph showing the results of the FRAP assay of the whole leaf extract of Korancho according to an embodiment of the present invention.
8 is a graph showing the results of evaluation of the intracellular reactive oxygen species (ROS) antioxidant effect of the whole leaf extract of Korancho according to an embodiment of the present invention.
Figure 9 is a graph showing the evaluation results of tyrosinase activity inhibitory ability according to Examples and Comparative Examples of the present invention.
Figure 10 is a graph showing the results of evaluation of melanin synthesis inhibitory ability caused by α-MSH of the whole leaf extract of Korancho according to an embodiment of the present invention.
11 is a graph showing the experimental results confirming the hyaluronic acid production promoting effect of the whole leaf extract of Korancho according to an embodiment of the present invention.
In the drawings of the present invention, CE denotes an extract from the whole plant of Colomboaceae according to Comparative Example 1, PE denotes a bat orchid extract according to Comparative Example 2, and CGE denotes an entire leaf extract of Colombo japonica according to Example 1.

The present invention relates to a cosmetic composition comprising an extract of an anterior leaf of Crypsinus hasstatus and a method for preparing the same.

Hereinafter, the present invention will be described in detail.

<Cosmetic Composition Containing Korancho Whole Leaf Extract>

The fern used in the present invention ( Crypsinus hasstatus ) is a perennial plant that belongs to the fern plant Fern order Fernaceae and lives in low-lying trees, shady rock crevices, and crevices of cliffs.

In the present invention, in particular, it is characterized in that the extract obtained by extracting the whole leaf of Korancho is used.

In the present invention, Korancho, as a fern, has a propagation system through generational alternation of the anterior leaf and the sporophyte, and propagates the anterior leaf through tissue culture by culturing spores in vitro. The anterior halves may be obtained by inducing the anterior halves through aseptic germination of spores of Korancho plants and mass-cultivating them using a bioreactor. The anterior lobes of the Korancho of the present invention contain a higher concentration of physiologically active substances effective for moisturizing, anti-inflammatory, antioxidant, anti-wrinkle, whitening, etc. than wild Korancho plants.

Specifically, in the present invention, the induction of the prolophyte may be performed through a conventional method known in the art, and includes inducing the proletophage through aseptic germination in vitro from the spores of the plant. It is preferably achieved by culturing the in vitro germinated anterior lobes using a tissue culture medium. As the tissue culture medium, for example, one or more mediums included in MS (Murashige-Skoog) medium, Hyponex medium, and Knop medium may be used.

In addition, the tissue culture medium may further include a plant growth regulator. The plant growth regulator may use substances known in the art, but preferably include at least one of inorganic substances such as sucrose and gibellenline, vitamins, carbon sources, and derivatives thereof. . In addition, alkaloids, terpenoids, carotenoids, phenolic compounds, and natural extracts that act on cell differentiation and proliferation may be additionally added to the tissue culture medium.

In the present invention, "extract" refers to a preparation obtained by squeezing a herbal medicine into an appropriate leachate and concentrating the leachate by evaporation. It may be extracted through a method, and is not particularly limited.

As the extraction method, for example, shaking culture extraction, cold needle extraction, ultrasonic extraction, reflux cooling extraction, room temperature extraction, hot water extraction, ultra-high pressure extraction, incubator extraction, etc. may be used, and extraction by shaking culture extraction is preferable. do.

More preferably, the yield of the extract can be most effectively improved by mixing the whole leaf of Korancho and the solvent, stirring at 25 to 40 ° C. at a speed of 100 to 400 rpm, aging for 24 hours to 2 weeks, and then performing shaking culture extraction. there is.

In addition, the solvent may be at least one selected from the group consisting of water, lower alcohols having 1 to 4 carbon atoms, propylene glycol, glycerin, and mixed solvents thereof.

The content of the extraction solvent may be 1 to 50 times, preferably 1 to 20 times the solid weight of the whole leaf of Korancho.

Filtration is a process of removing floating solid particles from the extract, and particles may be filtered out using cotton, nylon, paper, etc., or ultrafiltration, cryofiltration, centrifugation, etc. may be used, but is not limited thereto.

The step of concentrating and then drying the filtrate includes, but is not limited to, freeze drying, vacuum drying, hot air drying, spray drying, reduced pressure drying, foam drying, high frequency drying, infrared drying, and the like. In some cases, a step of pulverizing the finally dried extract may be added.

According to one embodiment of the present invention, the Korancho whole leaf extract may be processed and used in various forms, such as an extract, a powder obtained by drying the extract, and a concentrate obtained by concentrating and filtering the extract.

According to one embodiment of the present invention, the Korancho whole leaf extract obtained by the method as described above may be included in 0.001 to 20% by weight, preferably 0.01 to 10% by weight, based on the total weight of the cosmetic composition. If the extract of the whole leaf of Korancho is less than 0.001% by weight relative to the total weight of the cosmetic composition, the function as a composition is deteriorated because the antioxidant and whitening effects cannot be sufficiently obtained, and if it exceeds 20% by weight, the efficacy is sufficient even if it does not contain more than that. is uneconomical because it can represent

The cosmetic composition of the present invention can be used in external skin ointments, creams, softening lotions, nutrient lotions, packs, essences, hair tonics, shampoos, rinses, hair conditioners, hair treatments, gels, skin lotions, skin softeners, skin toners, astringents, and lotions. , milk lotion, moisture lotion, nutrition lotion, massage cream, nutrition cream, eye cream, moisture cream, hand cream, foundation, nutrition essence, sunscreen, sunscreen cream, mist, aerosol, soap, cleansing foam, cleansing lotion, cleansing It may be provided in one or more formulations selected from the group consisting of cream, body lotion and body cleanser, but is not limited thereto.

In addition, in the cosmetic composition of each dosage form, other ingredients in addition to the whole leaf extract of Korancho may be arbitrarily selected and blended according to the formulation or purpose of use of other cosmetics, and the type and amount of ingredients may be appropriately selected by those skilled in the art. For example, it may contain a colorant (pigment), a flavoring agent (fragrance), a suspending agent, an emulsifier, a solubilizing agent, a stabilizer, an isotonic agent, a pH adjusting agent, a viscosity adjusting agent, a solvent, and the like.

When the formulation of the cosmetic composition according to the present invention is a paste, cream or gel, animal fiber, vegetable fiber, wax, paraffin, starch, tracanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc acid as a carrier component etc. can be used.

When the formulation of the cosmetic composition according to the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component, and in particular, in the case of a spray, additionally chlorofluoro propellants such as hydrocarbons, propane/butane or dimethyl ether.

When the formulation of the cosmetic composition according to the present invention is a solution or emulsion, a solvent, solvating agent or emulsifying agent is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan fatty acid ester.

When the formulation of the cosmetic composition according to the present invention is a suspension, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, an ethoxylated isostearyl alcohol, a suspending agent such as polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester , microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tracanth, and the like can be used.

When the formulation of the cosmetic composition according to the present invention is surfactant-containing cleansing, as carrier components, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosyl nates, fatty acid amide ether sulfates, alkylamidobetaines, fatty alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, linolin derivatives or ethoxylated glycerol fatty acid esters and the like can be used.

In addition, the cosmetic composition of the present invention may further include pharmaceutically or cosmetically acceptable additives.

<Moisturizing effect>

The cosmetic composition according to the present invention may act as a hyaluronic acid production promoter by including the extract of the whole leaf of Korancho as an active ingredient, and specifically, by increasing the intracellular production of hyaluronic acid that regulates the skin barrier function and maintains skin elasticity, It may have a moisturizing effect.

Furthermore, by filling the moisture in the epidermis through the moisturizing effect, the action of skin contraction occurs, so that the elasticity of sagging skin can be dramatically improved. In addition, skin wrinkles can be filled in and the skin can be contracted to show the effect of improving skin wrinkles and/or improving skin aging.

<Antioxidant effect>

The cosmetic composition according to the present invention can act as an active oxygen inhibitor by including the extract of Korancho chinensis as an active ingredient, specifically, free radicals, which are oxygen compounds that cause aging, arteriosclerosis, cancer, etc. Phosphorus DPPH (2,2-diphenyl-1-picrylhydrazyl) and ABTS (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) radical scavenging activity can be increased to exhibit excellent antioxidant effects.

In the present invention, the antioxidant means to protect the body from oxidative stress. Oxidants, which cause oxidative stress, are naturally generated in normal metabolic processes such as the process of making energy using nutrients and the immune response to destroy invading bacteria or viruses.

The free radical is an oxygen compound generated during the metabolic process of cells in the body of animals and plants and is related to causes such as aging, hardening of the arteries, cancer, etc., and the reactivity of the free radical is an oxygen molecule They range from very low ones such as Superoxide radical (O _2ㆍ ), Hydroxyl radical (OH _{ㆍ )} , Hydrogen peroxide (H ₂ O ₂ ), and Singlet oxygen (O ₂ ) to very high ones such as oxygen toxicity that is lethal to living organisms. causes cell membrane degradation, protein degradation, lipid oxidation, DNA denaturation, etc., resulting in cell dysfunction, cancer, brain diseases such as stroke, Parkinson's disease, heart disease, arteriosclerosis, inflammation, aging, and autoimmune diseases It is known to cause various diseases, etc. In particular, the accumulation of lipid peroxide, which is produced by attacking unsaturated fatty acids, which are components of biological membranes, is known to cause degradation of biological functions, aging, and adult diseases. For this purpose, DPPH (2,2-diphenyl-1-picrylhydrazyl) and ABTS (2,2'-azino-bis (3-ethylbe nzthiazoline-6-sulfonic acid)) can be used.

Therefore, the cosmetic composition of the present invention is free radical (DPPH (2,2-diphenyl-1-picrylhydrazyl) and ABTS (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) radicals) By increasing the clearance activity, as a result, it has preventive, therapeutic, and improving effects on brain diseases such as cancer, stroke, Parkinson's disease, heart disease, arteriosclerosis, inflammation, aging, autoimmune disease, aging, and adult diseases.

<Whitening effect>

The cosmetic composition according to the present invention can provide a skin whitening effect by inhibiting the activity of tyrosinase, an enzyme that promotes melanin production, and having DOPA oxidation inhibitory activity.

In addition, the cosmetic composition according to the present invention prevents melanin synthesis caused by α-MSH, inhibits melanin accumulation, and inhibits the expression of melanin synthase and factors involved in synthesis to promote skin whitening. efficacy can be provided.

In addition, the cosmetic composition according to the present invention is a protein that produces a precursor required for melanin production, Tyrosinase, TRP 1 (Tyrosinase-related protein 1), TRP 2 (Tyrosinase-related protein 2) or MITF (Microphthalmia) -associated transcription factor) is excellent in suppressing the production, so it can ultimately provide the effect of suppressing the production of melanin.

The cosmetic composition of the present invention may further include one or more uses selected from anti-aging and wrinkle improvement in addition to the anti-oxidation, skin moisturizing and skin whitening effects, and further has one or more effects selected from anti-aging and wrinkle improvement. can provide

<Method for producing the extract of the whole leaf of Korancho>

The present invention includes a method for producing an extract of the whole leaf of Korancho ( Crypsinus hasstatus ). The method for producing the whole leaf extract of Korancho of the present invention includes the steps of inducing the entire leaf body of Korancho; And it may include the step of preparing an extract from the whole leaf of Korancho.

Induction step of the coriander anterior lobes; And the information on the step of preparing the extract from the whole leaf of Korancho includes the above series of information. In addition, the whole leaf extract of Korancho prepared by the method of the present invention has at least one effect selected from antioxidant and skin whitening effects, anti-aging, skin moisturizing, and wrinkle improvement, to which all of the above are equally applicable.

Hereinafter, examples will be described in detail to explain the present invention in detail. However, the embodiments according to the present invention can be modified in many different forms, and the scope of the present invention should not be construed as being limited to the embodiments described below. The embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art.

Example: Preparation of Korancho whole leaf extract according to the present invention

100 mL of water was added to 2 g of the dried coriander plant phyllodes, and then sufficiently aged at 25 ° C at 140 rpm for 24 hours using a shaking incubator (VS-8480SRN, VISION SCIENTIFIC, Korea), and then filtered with filter paper (5C, 185 mm, Toyo toshi Kaisha, Ltd, Japan), the filtrate obtained by filtration was concentrated under reduced pressure, and a final extract was prepared using a lyophilizer.

Comparative Example 1: Preparation of Korancho whole plant extract

In the above Example, a Korancho extract was prepared in the same manner as in the above Example, except that the Korancho outpost was used instead of the Korancho whole leaf.

Comparative Example 2: Bat eggs ( Platycerium bifurcatum ) preparation of the extract

In the above Example, a Korancho extract was prepared in the same manner as in the above Example, except that the outpost part of Korancho was used instead of the entire lobe of Korancho.

<Experimental Example 1> Cytotoxicity evaluation - MTT assay

Experimental Example 1-1. Evaluation of cytotoxicity according to extracts

The cytotoxicity was evaluated for the whole leaf extract of the above example and Comparative Examples 1 and 2, the whole leaf extract of the coriander plant, the whole extract of the coriander plant, and the extract of the bat orchid.

(1) Cell line selection and cell culture

Human dermal fibroblasts (HDFs; ATCC, USA) are primary cells isolated from adult skin, and Mycoplasma, Hepatitis B, Hepatitis C, HIV-1, Bacteria, Yeast, and other fungi are not detected. did HDFs were inoculated on the bottom of a culture dish, and DMEM/F12 3:1 mixed medium containing 1% Antibiotic-Antimycotic and 10% FBS was added, and then placed in a 37°C, 5% CO ₂ incubator (HERAcell 150i, Thermo, USA). cultured in.

(2) MTT assay

Cell viability was analyzed using the principle of MTT.

Cell viability was analyzed using the principle of MTT. HDFs were inoculated into a 24-well plate at a concentration of 5 × 104 cells/well, cultured for 24 hours, and then exchanged with FBS-free medium containing diluted test substances for each concentration and cultured for 24 hours. After culturing for 24 hours, the presence or absence of changes in cell shape due to the treatment of each concentration was observed under a microscope, and then MTT solution was added to each well so that the final treatment concentration was 0.05%, and cultured for 4 hours. After removing the culture medium, 1,000 μL of DMSO was added, shaken for 10 minutes, and absorbance was measured at 540 nm (BioTek, USA).

(3) Results

All tests were repeated three times, and the results were expressed as cell viability compared to the untreated group, and statistical analysis was performed using the SPSS® Package Program (IBM, USA). Statistical significance was verified at the p<0.05 significance level by t-test of independent samples.

The cytotoxicity evaluation results according to the extracts are shown in Table 1 below, and the results are shown graphically as shown in FIG. 1.

Cell viability (%) ± S.D. density
(mg/ml) Example
(Extract of whole leaf of Korancho) Comparative Example 1
(Extract of coriander plant) Comparative Example 2
(bat egg extract) 0 101.44 ± 1.70 103.40 ± 3.00 100.17 ± 2.32 1.25 99.85 ± 1.04 98.83 ± 4.21 95.94 ± 1.43 2.5 101.17 ± 1.00 96.28 ± 4.26 95.35 ± 1.67 5 97.31 ± 1.46 93.41 ± 0.82 90.26 ± 1.34 10 92.03 ± 4.63 90.05 ± 2.76 85.02 ± 5.12 20 80.33 ± 1.44 79.21 ± 0.36 73.21 ± 1.21

Referring to Table 1 and FIG. 1, it was confirmed that the cell viability was higher at all concentrations in the case of the extract of the anterior phyllodes of Korancho compared to that of Korancho and bat eggs.

Experimental Example 1-2. Evaluation of cytotoxicity of extracts from the whole plant of Korancho for cell lines

Cytotoxicity was evaluated for the whole leaf extract of Korancho prepared in the above example.

(1) Cell line selection and cell culture

HaCaT (human keratinocyte, HaCaT, ATCC, USA), HDF (Human dermal fibroblasts, ATCC, USA) and CCD-986 (Human dermal fibroblasts, ATCC, USA) cells were used to evaluate the cell viability of the samples. HaCaT, HDF and CCD-986sk cells were grown in Dulbecco Modified Eagle Medium (DMEM, Welgene, Korea) supplemented with 10% fetal bovine serum (FBS, Gibco, USA) and 1% penicillin-streptomycin (Gibco, USA). ) was cultured in

(2) MTT assay

MTT tetrazolium is a yellow water-soluble solution that, when treated with cells, metabolizes the ring structure of tetrazolium by dehydrogenase in intact cell mitochondria, and uses the principle of reduction to blue-violet water-insoluble MTT formazan crystals. This is an experimental method mainly used to evaluate the viability of cells in a sample. Cell viability measurement according to the present invention was evaluated by measuring the absorbance of MTT formazan.

HaCaT, HDF, and CCD-986sk cells were each dispensed in a 96-well plate at 1 × 104 cells/well and cultured for 24 hours at 37°C and 5% CO ₂ conditions. Then, the extracts prepared in Examples to Comparative Examples were diluted in DMEM medium and cultured for an additional 24 hours at 37° C., 5% CO ₂ condition. After incubation, MTT solution was added to each well to a concentration of 0.5 mg/mL. After 4 hours, the formed MTT formazan was dissolved in 100 μl of DMSO (Dimethyl sulfoxide), and the absorbance at 420 nm (SpectraMax i3, Molecular devices, CA, USA) was measured.

(3) Results

All tests were repeated three times, and the results were expressed as cell viability compared to the untreated group, and statistical analysis was performed using the SPSS® Package Program (IBM, USA). Statistical significance was verified at the p<0.05 significance level by t-test of independent samples.

The cytotoxicity evaluation of the whole leaf extract of Korancho for cell lines is shown in Table 2, and the results are shown graphically as shown in FIG. 2 (a). Figure 2 (b) is an image showing the results of MTT crystal violet staining for each concentration of the cytotoxicity evaluation of the whole leaf extract of Korancho according to an embodiment of the present invention.

As a result, as shown in FIG. 2, it was confirmed that the coriander whole phyllodes extract according to the present invention did not affect cell viability.

<Experimental Example 2> Antioxidant effect evaluation

Experimental Example 2-1. DPPH free radical scavenging rate evaluation

(1) Materials and methods

In this experiment, DPPH (2,2-diphenyl-1-picrylhydrazyl, Sigma-Aldrich) and BHT (Butylated hydroxytoluene, Sigma-Aldrich) were used.

To evaluate the antioxidant activity, free radical scavenging rate using DPPH was measured. DPPH was prepared and used at a concentration of 300 μM, and the test substance was diluted in distilled water to a final concentration of 0.04%, 0.08%, 0.16%, 0.31%, 0.63%, 1.25%, 2.5%, and 5%. After adding 100 μL of each test substance and 100 μL of DPPH solution in order to a 96-well plate, reacting in an oven (JSOF-150, JSR, Korea) at 37 ° C for 30 minutes, and using a microplate reader (BioTek, USA) Absorbance was measured at 517 nm. As a solvent control group, distilled water was used instead of the test substance, and butylated hydroxytoluene (BHT) was used as a positive control group for comparison.

(2) DPPH free radical scavenging rate evaluation result

All tests were repeated three times, and statistical analysis was performed using the SPSS® Package Program (IBM, USA). Statistical significance was verified at the p<0.05 significance level by t-test of independent samples.

The DPPH free radical scavenging rate evaluation results for each concentration are shown in Tables 2 and 4 below.

3 is a graph showing the results of DPPH free radical scavenging rate evaluation by concentration according to Examples and Comparative Examples of the present invention.

Figure 4 is a graph showing the DPPH free radical scavenging rate evaluation results for each concentration of the whole leaf extract of Korancho according to an embodiment of the present invention.

DPPH scavenging activity (%) ± S.D. density
(mg/ml) Example
(Extract of whole leaf of Korancho) Comparative Example 1
(Extract of coriander plant) Comparative Example 2
(bat egg extract) 0.625 20.25 ± 1.77 18.68 ± 4.09 15.91 ± 2.98 1.25 46.75 ± 3.42 29.21 ± 1.95 31.95 ± 1.81 2.5 66.17 ± 1.02 40.11 ± 3.49 38.80 ± 3.19 5 81.51 ± 2.84 56.18 ± 0.98 50.78 ± 6.73 10 86.21 ± 1.41 64.98 ± 5.92 67.92 ± 2.98

DPPH scavenging activity (%) ± S.D. density
(mg/ml) Example
(Extract of whole leaf of Korancho) positive control
(BHT) 0.625 20.25 ± 1.77 80.42 ± 4.62 1.25 46.75 ± 3.42 81.33 ± 3.25 2.5 66.17 ± 1.02 90.32 ± 1.33 5 81.51 ± 2.84 95.65 ± 6.31 10 86.21 ± 1.41 99.34 ± 4.32

Referring to Table 2 and FIG. 3, it was confirmed that the DPPH free radical scavenging effect was excellent at all concentrations in the case of the Korancho anterior leaf extract compared to Korancho and Batran, and it was confirmed that the antioxidant capacity was remarkably excellent. In addition, referring to Table 3 and FIG. 4, it was confirmed that the DPPH free radical scavenging effect increased as the concentration of the whole leaf extract of Korancho increased (p<0.05).

Experimental Example 2-2. Evaluation of ABTS radical scavenging activity

(1) Materials and methods

In this experiment, ABTS (2,2-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid, Sigma-Aldrich, St. Louis, MO, USA) and L-Ascorbic acid (Sigma-Aldrich) were used. used

In the commonly used ABTS radical scavenging activity analysis method, such as the DPPH free radical scavenging rate analysis method, when ABTS cation radicals are generated by the reaction of ABTS and potassium persulfate, it has a unique blue-green color. A way to measure the degree.

ABTS radical scavenging ability was evaluated to evaluate antioxidant activity. 7.4 mM ABTS and 2.6 mM potassium persulfate were reacted for 12 hours in the dark to form ABTS cations, and then diluted with 1Х PBS so that the absorbance value at 730 nm was 0.70±0.02. 10 μL of each sample was added to 190 μL of the diluted ABTS solution, allowed to stand for 60 minutes, and then absorbance was measured at 730 nm. As a positive control, L-ascorbic acid was used for comparison.

(2) Evaluation result of ABTS radical scavenging ability

All tests were repeated three times, and statistical analysis was performed using the SPSS® Package Program (IBM, USA). Statistical significance was verified at the p<0.05 significance level by t-test of independent samples.

The ABTS radical scavenging ability evaluation results for each concentration are shown in Tables 4 and 5 below.

5 is a graph showing the evaluation results of ABTS radical scavenging ability by concentration according to Examples and Comparative Examples of the present invention.

6 is a graph showing the ABTS radical scavenging ability evaluation results for each concentration of the whole leaf extract of Korancho according to an embodiment of the present invention.

ABTS scavenging activity (%) ± S.D. density
(mg/ml) Example
(Extract of whole leaf of Korancho) Comparative Example 1
(Corancho extract) Comparative Example 2
(bat egg extract) 0.625 10.25 ± 2.77 12.80 ± 1.97 14.91 ± 2.98 1.25 36.75 ± 3.42 24.70 ± 2.32 20.40 ± 3.97 2.5 76.17 ± 1.02 51.95 ± 4.98 46.98 ± 0.91 5 86.51 ± 1.84 63.80 ± 3.91 56.78 ± 1.82 10 88.21 ± 0.41 70.98 ± 1.39 65.49 ± 1.35

ABTS scavenging activity (%) ± S.D. density
(mg/ml) Example
(Extract of whole leaf of Korancho) positive control
(ascorbic acid) 0.625 10.25 ± 2.77 80.42 ± 7.62 1.25 36.75 ± 3.42 86.33 ± 3.25 2.5 76.17 ± 1.02 90.32 ± 0.33 5 86.51 ± 1.84 95.65 ± 4.31 10 88.21 ± 0.41 99.34 ± 2.32

Referring to Table 4 and FIG. 5, it was confirmed that the ABTS radical scavenging effect was excellent at a concentration of 1.25 mg / ml or more in the case of Korancho anterior leaf extract compared to Korancho and Batran, and it was confirmed that the antioxidant ability was remarkably excellent.

In addition, referring to Table 5 and FIG. 6, it was confirmed that the ABTS radical scavenging effect increased as the concentration of the whole leaf extract of Korancho increased (p<0.05).

Experimental Example 2-3. FRAP assay

FRAP (Ferric reducing antioxidant power) assay is a method for verifying antioxidant activity through electron donating ability. Fe ²⁺ produced through the reaction between Fe ³⁺ acting as an oxidizing agent and antioxidant can be quantified through absorbance. way.

에서 가열해서 사용하였다. 추출물 300 ㎕에 FRAP solution 2,700 ㎕를 가한 후 암소에서 30분 방치 후 593 nm에서 흡광도를 측정하였다. The stock solution was prepared by dissolving 10 mM TPTZ solution and 20 mM FeCl3·H2O solution in 0.3 M sodium acetate buffer (pH 3.6) and 40 mM HCl, and then fresh working solution (25 mL acetate buffer + 2.5 mL TPTZ solution + 2.5 mL FeCl3 H2O solution) was prepared and immediately before use, 37

heated and used. After adding 2,700 μl of FRAP solution to 300 μl of the extract, the absorbance was measured at 593 nm after being allowed to stand for 30 minutes in the dark.

7 is a graph showing the results of the FRAP assay of the whole leaf extract of Korancho according to an embodiment of the present invention.

Referring to FIG. 7, the extract of the whole leaf of Korancho exhibited a concentration-dependent increase in reducing power, and it was confirmed that the extract of the whole leaf of Korancho was excellent in antioxidant effect.

Experimental Example 2-4: Intracellular ROS Detection

For HaCaT, HDF and CCD-986sk cells, untreated group (Veh, Vectored Exception Handling), AAPH (2,2'-azobis (2-amidinopropane) hydrochloride) only 20 mM positive control group, AAPH and Korancho anterior thallus extract 5 It was divided into an experimental group treated with mg/ml and an experimental group treated with 10 mg/ml of AAPH and Korancho whole leaf extract, and the intracellular reactive oxygen species (ROS) antioxidant effect of the Korancho whole leaf extract according to the present invention was evaluated.

The antioxidant effect was measured by a fluorometric assay using 2',7'-dichlorofluorescin diacetate (DCFH-DA), and DCFH-DA was non-fluorescent in cells. After being separated into DCFH, which is a polar molecule, it is oxidized by active oxygen in the cell and converted to fluorescent 2',7'-dichlorofluorescin (DCF), and the 2',7'-dichlorofluorescin The intensity of fluorescein (2',7'-dichlorofluorescin, DCF) was evaluated with a fluorescence microplate reader.

8 is a graph showing the results of evaluation of the intracellular ROS antioxidant effect of the whole leaf extract of Korancho according to an embodiment of the present invention.

Referring to Figure 8, it was found that the intensity of 2',7'-dichlorofluorescin (DCF) was reduced when the extract of the whole lobe of Korancho was treated, and the amount of active oxygen in the cells was reduced. Through this, it can be seen that the antioxidant effect of the whole leaf extract of Korancho according to the present invention is excellent.

<Experimental Example 3> Evaluation of whitening activity

Experimental Example 3-1: Tyrosinase activity inhibition ability

Tyrosinase acts as an oxidizing agent that oxidizes tyrosine to produce DOPA and oxidizes DOPA to produce dopachrome in the process of melanin synthesis occurring in melanosomes. Therefore, the whitening functionality can be evaluated by measuring the ability to inhibit the activity of tyrosinase.

(1) Materials and methods

The extracts prepared in Examples to Comparative Examples were prepared by concentration. After preparing 0.1M sodium phosphate buffer (pH 6.5), 1.5mM L-tyrosine and 1000U/ml tyrosinase were prepared with the solution to prepare reagents necessary for the experiment. After putting 170 μL of the extracts prepared in Examples to Comparative Examples into each well of a 96-well plate, 10 μL of Tyrosinase (1000 U/ml) was added and reacted at 37° C. for 10 minutes. After that, 20 μL of 1.5 mM L-tyrosine was added and the reaction was performed at 37° C. for 10 minutes, and the absorbance at 490 nm (SpectraMax i3, Molecular devices, CA, USA) was measured. Tyrosinase activity inhibitory ability was measured by Equation 3 below, and the results are shown in Table 6 below.

(2) Tyrosinase activity inhibition evaluation result

All tests were repeated three times, and statistical analysis was performed using the SPSS® Package Program (IBM, USA). Statistical significance was verified at the p<0.05 significance level by t-test of independent samples.

When the tyrosinase activity in the vector not treated with L-DOPA was set to 100%, the degree of tyrosinase activity in each experimental group was evaluated.

The evaluation results of Tyrosinase activity inhibition according to Examples and Comparative Examples of the present invention are shown in Table 6 below,

Figure 9 is a graph showing the evaluation results of tyrosinase activity inhibitory ability according to Examples and Comparative Examples of the present invention.

Tyrosinase Inhibition Assay (% of control) ± S.D. density
(mg/ml) untreated
(Control) L-DOPA only 5 10 Example 100 607 ± 10.60 426 ± 5.76 249 ± 8.90 Comparative Example 1 597 ± 5.11 377 ± 6.80 Comparative Example 2 548 ± 7.19 385 ± 11.20

Referring to Table 6 and FIG. 9, the examples inhibited tyrosinase activity in a concentration-dependent manner, and when the examples and comparative examples were treated at a concentration of 10 mg/ml, compared to the case where only L-DOPA was treated alone It can be confirmed that the tyrosinase activity is reduced, and it can be confirmed that there is an ability to inhibit tyrosinase activity.

However, when the Examples and Comparative Examples were treated at a concentration of 5 mg/ml, the levels of tyrosinase activity of the Korancho extract and the Batran extract of Comparative Example did not significantly decrease compared to the L-DOPA alone treatment group.

In the case of using the whole leaf extract of Korancho of Example, even when treated at a concentration of 5 mg/ml, the tyrosinase activity was significantly reduced, confirming that the tyrosinase activity inhibitory effect was superior to that of the Korancho extract and the Orchid orchid extract.

Experimental Example 3-2: Melanin synthesis inhibitory ability

The most important role in melanin synthesis is α-MSH (melanocyte stimulating hormone). The α-MSH is secreted from the middle lobe of the pituitary gland and binds to the melanocortin 1 receptor (MC1R, melanocortin 1 receptor), a membrane receptor expressed only in melanocytes, to activate adenylyl cyclase, and intracellular cAMP Signal amplification induces activation of PKA (protein kinase A), and intracellular CREB (cAMP response element binding protein) is activated to increase the expression of MITF (microphthalmia-associated transcription factor), a melanocyte-specific transcription factor. The MITF stimulates the expression of tyrosinase related protein (TRP), a melanin-producing enzyme, to regulate melanin production in melanocytes.

Therefore, the whitening effect can be evaluated by measuring the ability to inhibit melanin synthesis caused by α-MSH, which is the most important and highest signal in the skin pigmentation mechanism.

Regarding the examples of the present invention, melanin synthesis inhibitory ability was measured using B16F10 melanoma cells (Melanoma cell: Korea Cell Line Bank).

B16F10 cells were divided into 6 groups: untreated group, α-MSH treated group, arbutin treated group, kojic acid treated group, and 5 mg/ml and 10 mg/ml treated group of Korancho anterior leaf extracts to determine melanin formation. The stimulating hormone α-MSH (Sigma-Aldrich, USA) was treated with the medium and cultured for 3 to 4 days. After culturing, the cells were harvested, washed once with PBS, and 10% DMSO-added 1 N NaOH solution was left in a thermostat at 80° C. for 1 hour to dissolve melanin. Thereafter, the absorbance was measured at a wavelength of 475 nm using a microplate reader to compare and analyze the melanin pigment.

Figure 10 is a graph showing the results of evaluation of melanin synthesis inhibitory ability caused by α-MSH of the whole leaf extract of Korancho according to an embodiment of the present invention.

As a result of the experiment, as shown in FIG. 10, the treatment of the whole leaf extract of Korancho according to the present invention resulted in a decrease in melanin content, which can provide an effect of preventing melanin synthesis caused by melanin α-MSH. It means that there is, and it can be confirmed that it shows a whitening effect through inhibition of melanin production.

<Experimental Example 4> Evaluation of Moisturizing Effect - Hyaluronic Acid Promoting Effect

(One) Materials and Methods

In this experiment, HDFs were inoculated in a 24-well plate at 5Х104 cells/well, cultured for 24 hours, and then exchanged with FBS-free medium containing diluted test substances by concentration and cultured for 24 hours. After 24 hours, each culture supernatant was centrifuged at 10,000 rpm for 10 minutes, and the amount of hyaluronic acid produced in the medium with the supernatant from which debris was removed was measured at 450 nm using a Human Hyaluronic Acid (HA) ELISA Kit. The absorbance was measured at. The degree of hyaluronic acid production was evaluated by correcting the amount of total protein, and compared using retinoic acid (0.0015%) as a positive control group.

(2) Results of measurement of intracellular hyaluronic acid production

All tests were repeated three times, and statistical analysis was performed using the SPSS® Package Program (IBM, USA). Statistical significance was verified at the p<0.05 significance level by t-test of independent samples.

Table 7 and FIG. 11 are results showing the hyaluronic acid concentration produced by the treatment of the whole leaf extract of Korancho according to an embodiment of the present invention, compared with that of the Korancho extract (CE) and the Orchid orchid extract (PE).

As can be seen in Table 7 and FIG. 11 below, as the concentration of the whole leaf extract of Korancho increased, the amount of hyaluronic acid produced increased. Hyaluronic acid (HA) production was significantly increased (p<0.05).

In addition, as can be seen in Table 7 and FIG. 11 below, the total amount of hyaluronic acid (HA) produced in the whole phyllodes extract of the present Example 1 compared to the extracts of Korancho and Batran extracted by the extraction methods of Comparative Examples 1 and 2. further increased.

matter CE PE CGE density(%) HA concentration
(ng/μg) Standard Deviation HA concentration
(ng/μg) Standard Deviation HA concentration
(ng/μg) Standard Deviation 0 53.91 0.09 53.46 0.23 54.46 0.88 0.0001 54.90 0.72 53.96 0.14 56.89 2.59 0.0002 55.82 0.33 54.36 0.29 58.33 3.51 0.0004 57.08 0.82 55.09 0.15 61.58 1.40 0.0007 59.64 0.73 56.88 0.13 64.09 1.35 0.0013 61.86 0.66 56.94 0.07 66.89 0.94 0.0025 62.53 0.88 57.94 0.18 68.35 1.74 0.0015
positive control
(Retinoic acid)
69.2 2.26

As a result of the experiment, as shown in Table 7 and FIG. 11, the treatment of the whole leaf extract of Korancho according to the present invention showed a significant increase in the amount of hyaluronic acid production, which was excellent because the production of hyaluronic acid was promoted according to the treatment of the whole leaf extract of Korancho. This means that it has a moisturizing effect.

Claims (12)

A cosmetic composition comprising an extract of the whole leaf of Crypsinus hasstatus .
The method of claim 1,
A cosmetic composition comprising 0.001 to 20% by weight of the Korancho whole leaf extract relative to the total weight of the cosmetic composition.
The method of claim 1,
The cosmetic composition is a cosmetic composition characterized in that it has a skin moisturizing, antioxidant or skin whitening effect.
The method of claim 1,
The Korancho whole leaf extract is characterized in that it acts as an active oxygen inhibitor, a cosmetic composition.
The method of claim 1,
The Korancho whole leaf extract is a cosmetic composition for moisturizing, characterized in that acts as a hyaluronic acid production promoter.
The method of claim 1,
The Korancho whole leaf extract is characterized in that for inhibiting tyrosinase (tyrosinase) activity, a cosmetic composition.
The method of claim 1,
The Korancho whole leaf extract is characterized in that acting as a melanin synthesis inhibitor, a cosmetic composition.
The method of claim 3,
The cosmetic composition is characterized in that it further comprises at least one effect or use selected from anti-aging and wrinkle improvement, cosmetic composition.
The method of claim 1,
The cosmetic composition, characterized in that the Korancho whole leaf extract is extracted with shaking for 24 hours to 2 weeks at a temperature of 25 to 40 ℃ after mixing the Korancho whole leaf and a solvent.
The method of claim 9,
The shaking extraction comprises stirring at a speed of 100 to 400 rpm, cosmetic composition.
The method of claim 9,
The solvent is water, a lower alcohol having 1 to 4 carbon atoms, propylene glycol, glycerin, and a mixed solvent thereof characterized in that one selected from the group consisting of, cosmetic composition.
The method of claim 1,
The cosmetic composition is an ointment for skin external use, cream, softening lotion, nutrient lotion, pack, essence, hair tonic, shampoo, conditioner, hair conditioner, hair treatment, gel, skin lotion, skin softener, skin toner, astringent, lotion, milk Lotion, Moisture Lotion, Nutrition Lotion, Massage Cream, Nutrition Cream, Eye Cream, Moisture Cream, Hand Cream, Foundation, Nutrition Essence, Sunscreen, Sunscreen Cream, Mist, Aerosol, Soap, Cleansing Foam, Cleansing Lotion, Cleansing Cream, Characterized in that the formulation selected from the group consisting of body lotion and body cleanser, cosmetic composition.

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