KR101227701B1 - Cosmetic composition for improving skin comprising HC-6 complex extracts - Google Patents

Abstract

The present invention relates to a cosmetic composition for skin improvement, and more particularly, to a cosmetic composition for skin improvement comprising a situation mushroom extract, licorice extract, aloe vera gel and centella asiatica extract.
The composition according to the present invention is excellent in anti-aging, inflammation suppression, skin whitening or wrinkle improvement effect can be usefully used in the manufacture of cosmetics for skin improvement.

Description

Cosmetic composition for improving skin comprising HC-6 complex extracts

The present invention relates to a cosmetic composition for improving skin, and more particularly, including a composite extract of a situation mushroom extract, licorice extract, aloe vera gel and Centella asiatica extract, anti-aging, inflammation suppression, skin whitening or wrinkle The present invention relates to a cosmetic composition for improving skin having an excellent improvement effect.

Since the skin is in direct contact with the external environment and serves as an important protective film to protect the human body from physical, chemical and biological stimuli, various external environmental stresses lead to cell damage and aging. Specifically, UV rays, stress, disease conditions, environmental factors, wounds, and age can be caused by the activation of free radicals, if this condition is intensified, destroys the antioxidant defenses in vivo, damage cells and tissues To promote aging.

The skin color of a person is determined by the concentration and distribution of melanin in the skin. Melanin pigment produced by melanocytes of human skin is a phenolic polymer having a complex form of black pigment and protein, and plays an important role in blocking skin damage caused by ultraviolet rays. It is reported that the action of tyrosinase in melanocytes is the most important for melanin biosynthesis, and tyrosinase is a kind of amino acid tyrosine (DOPA), which is an intermediate of melanin polymer production. Conversion to dopaquinone plays a key role in the skin blackening process.

Skin wrinkles are known to be produced by a combination of several factors, including the skin's moisture content, collagen content, and the ability to act on the environment. Among these factors, the most influential effect on the formation of wrinkles is the expression and activity of collagenase, a collagen degrading enzyme that reduces collagen production and collagen content.

In addition, due to environmental pollution, stress, changes in the diet of the instant food or meat centers, discord in the skin immune system is causing various skin diseases such as dermatitis, skin cancer.

Recently, as the interest in skin care has increased, active development of multifunctional cosmetics that can simultaneously satisfy various skin-improving effects, such as anti-aging, whitening, wrinkle improvement, elasticity enhancement, and inflammation reduction, can be achieved at the same time. Is done.

However, since these multifunctional cosmetics are often added with a combination of various components, the physiological activity may be reduced due to the collision between various useful components, skin irritation may occur, and the cost increase may be caused by the addition of a plurality of components. have.

In addition, various attempts have been made to prepare functional cosmetics using natural extracts to minimize skin irritation and obtain useful ingredients in nature. However, these natural extracts are more expensive than most chemicals and are difficult to add at a concentration that is expected to have a definite effect, and if treated too high, skin irritation may occur.

The inventors of the present invention when applying the mushroom extract, licorice extract, aloe vera gel and centella asiatica extract in combination at the same time, the anti-aging, inflammation suppression, skin whitening of the known skin improvement efficacy of the situation mushroom extract and licorice extract Or by confirming that the wrinkle improvement effect can be significantly improved, the present invention was completed.

An object of the present invention comprises a complex extract of the situation mushroom extract, licorice extract, aloe vera gel and Centella asiatica extract to improve the skin composition for improving the anti-aging, inflammation inhibition, skin whitening or wrinkle improvement effect To provide.

In one embodiment, the present invention relates to a cosmetic composition for skin improvement for anti-aging, inflammation inhibition, skin whitening or wrinkle improvement, including a situation mushroom extract, licorice extract, aloe vera gel and Centella asiatica extract will be.

In the present invention, the situation mushroom extract, licorice extract, aloe vera gel and centella asiatica extract may be contained in a mixing ratio of 1 to 5: 1 to 5: 1 to 10: 1 to 15, preferably 1 to 3: It may be contained in a mixing ratio of 1 to 3: 1 to 6: 1 to 10.

The composition of the present invention may contain 0.01 to 30% by weight of mushroom extract, licorice extract, aloe vera gel and centella asiatica extract.

In the present invention, the situation mushroom extract, licorice extract, and centella asiatica extract are at least one selected from the group consisting of water, lower alcohol having 1 to 4 carbon atoms, acetone, ethyl acetate, butyl acetate, and 1,3 butylene glycol It may be extracted using a solvent.

The composition of the present invention is characterized by having antioxidant activity, anti-inflammatory activity and anticancer activity. In addition, the anti-inflammatory activity may be through the inhibitory action of inducible nitric oxide synthase (iNOS).

The composition of the present invention has a melanin biosynthesis inhibitory activity has a whitening action, has a collagenase inhibitory activity has a wrinkle improvement action.

In addition, the composition of the present invention comprises a high content of polyphenols, and has a convergent effect.

The composition of the present invention may further include a green tea extract, bokbunja extract and lettuce extract in addition to the above components.

The cosmetic compositions of the present invention may be in various dosage forms such as solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing cleansing, oils, powder foundations, emulsion foundations, wax foundations, and sprays. Can be.

The cosmetic composition according to the present invention, including complex extracts of situation mushrooms, licorice, aloe vera gel and centella asiatica significantly improves the antioxidant activity, anti-inflammatory activity, anti-cancer activity, pore reduction, whitening and wrinkle improvement, skin It has almost no irritation and has a comprehensive skin improvement effect.

Therefore, the cosmetic composition of the present invention can be usefully used for the development of skin improvement cosmetics for anti-aging, inflammation inhibition, skin whitening or wrinkle improvement.

1 is a result of measuring the proliferation inhibitory effect on the cancer cell line melanoma cells of HC-6 extract according to the present invention by MTT evaluation method.
2 and 3 are the results confirming the melanin biosynthesis inhibitory ability of the HC-6 extract according to the present invention.
Figure 4 is a result of measuring the iNOS protein expression level during LPS stimulation in Raw 264.7 cells to confirm the anti-inflammatory effect of the HC-6 extract according to the present invention.

The cosmetic composition for skin improvement of the present invention includes a complex extract (named 'HC-6 extract') of the situation mushroom extract, licorice extract, aloe vera gel and centella asiatica extract, anti-aging, inflammation suppression, skin It is characterized by having whitening or wrinkle improving efficacy.

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

The situation mushroom (Phellinus linteus) used in the present invention is a mud fungus of the genus Japonicum, also called woody mud mushroom. It is native to Korea, Japan, Australia, and North America. The shade is 6-12cm in diameter and 2-10cm in thickness, and has various shapes such as semicircle, flat shape, and round mountain shape. Edge is bright yellow, bottom is yellowish brown, live yellowish brown. No sack, spores light yellowish brown. These mushrooms contain various types of sugars, flavonoids, fiber, and isoflavones, and their pharmacological effects are effective in treating gynecological diseases such as anticancer effect, immune enhancing function, tumor suppression effect, uterine bleeding, and menstrual irregularities. It is known to be good. In addition, it is reported that the effect is very excellent in moisturizing and skin whitening effect, skin aging prevention function, antioxidant effect, anti-inflammatory effect, skin cell regeneration and collagen production ability when applied as a cosmetic.

Licorice (甘草, Glycyrrhiza Radix) used in the present invention is the root and root stem of the legume (Leguminosae) licorice (Glycyrrhiza glabra L.). Perennial herb, up to 30-80cm tall, basal woody, many branches. Young stems have hairs. The roots extend deep into the rectus, the laparoscopic diameter extends laterally, and new stems develop in the inferior child. The leaf is a single right upper lobe, and the leaf length is 5-20cm, the lobule is 5-17 sheets, and cilia are densely formed in a leaf disease. The flowers are 2 to 10 cm long, the color is purple, white or yellow and the flowers are 10 to 24 mm long. The root and the laparotomy are used, and the main medicinal ingredient contains 3.63 ~ 13.06% of glycyrrhizic acid, which is triterpenoid saponin, and there are other kinds of flavone compounds. As a botanical medicine, it protects the lungs and the nasal cavities to prevent the blood circulation of the face from disturbing due to the external turbidity and to remove the moisture inside and remove the skin inside and outside. It works to make you healthy.

In the present invention, aloe is a perennial plant belonging to the family Liliaceae, succulent CAM (Crassulacean Acid Metabolism) plant with fleshy leaves, has been widely used as a broad and versatile folk remedy for over 3,500 years, and more than 400 aloes have been identified. Among them, Aloe vera Linne gel is one of the most widely used natural materials for pharmaceuticals and cosmetics.

Centella Asiatica used in the present invention is a perennial herb of Apiaceae, and contains ingredients such as Asiaticoside, Asiatic-acid and Madecassic-acid. . Centella asiatica is particularly effective at regenerating the skin, strengthening immunity and enhancing vitality, resulting in various skin disorders, wound healing, tonics, and memory enhancers for It is known to be effective in mental, stress related disorders, and ulcers.

In the present invention, the situation mushroom extract, licorice extract and Centella asiatica extract may be prepared using conventional extraction solvents and extraction methods, and commercially available ones may be used. As the extraction solvent, for example, water, lower alcohol having 1 to 4 carbon atoms, acetone, ethyl acetate, butyl acetate, ether, chloroform and 1,3 butylene glycol can be used. The extraction solvent may be extracted by mixing about 2 to 40 times the weight of the dried material. The extraction temperature is not particularly limited as long as the effective activity of the useful component of each material is not removed. Preferably, the extraction temperature is about 50 to 100 ° C, more preferably 60 to 80 ° C.

In addition, the extract of the present invention includes not only the extract by the above-described extraction solvent, but also the extract that has undergone a conventional purification process. For example, by separation using an ultrafiltration membrane having a constant molecular weight cut-off value, separation by various chromatographies (made for separation by size, charge, hydrophobicity or affinity), and the like, Fractions are also included in the extract of the present invention. The extract of the present invention can be prepared in powder form by an additional process such as vacuum distillation and freeze-drying or spray-drying.

The aloe vera gel included in the composition of the present invention preferably uses a gel obtained from aloe vera alone, powdered aloe vera gel, or a mixture of aloe vera gel and powder in a suitable ratio. Can be.

When the composition of the present invention comprises a situation mushroom extract, licorice extract, aloe vera gel and centella asiatica extract at the same time, synergism occurs between the useful components of each material to prevent aging, inflammation, skin whitening or wrinkle improvement It can be effectively promoted, and when included in a certain composition ratio, it is possible to further enhance the efficacy. The composition of the present invention preferably contains the above situation mushroom extract, licorice extract, aloe vera gel and centella asiatica extract in a mixing ratio of 1 to 5: 1 to 5: 1 to 10: 1 to 15, preferably 1 ˜3: 1∼3: 1∼6: 1∼10 In such a mixing ratio, it is possible to expect an excellent enhancement effect of anti-aging, inflammation inhibition, skin whitening or wrinkle improvement.

In the present invention, the mushroom extract, licorice extract, aloe vera gel and centella asiatica extract composition may be included in an amount of 0.01 to 30% by weight, preferably 0.05 to 10% by weight, more preferably 1 to 5% by weight. %to be. If the content of the extract is too low, it is difficult to expect skin improvement effects such as anti-aging, inflammation suppression, skin whitening and wrinkle improvement, and too high content may irritate the skin.

The skin improvement composition of the present invention is characterized by having antioxidant activity, anti-inflammatory activity and anticancer activity. Specifically, the composition has antioxidant activity through electron donating ability, radical scavenging activity, and SOD-like activity, thereby preventing skin aging. In addition, the composition has an anti-inflammatory activity through the inhibitory effect of inducible nitric oxide synthase (iNOS), through which it can exhibit a skin inflammation inhibitory, atopic relieving action. In addition, the composition exhibits a cancer cell proliferation inhibitory effect is effective in improving skin cancer.

The composition for skin improvement of the present invention has melanin biosynthesis inhibitory activity through tyrosinase (Tyrosinase) inhibitory action, it can exhibit a whitening effect. In addition, the composition has a collagenase (collagenase) inhibitory activity, through which it is characterized by excellent skin regeneration, anti-wrinkle effect.

In addition, the composition of the present invention includes a high content of polyphenols to exhibit skin anti-aging, wrinkle improvement, whitening, moisturizing effect, it can also help to shrink pores and skin elasticity by showing a convergent effect.

The skin-improving composition of the present invention may be used by additionally including green tea extract, bokbunja extract and lettuce extract in addition to situation mushroom extract, licorice extract, aloe vera gel and centella asiatica extract. In addition, it can be used by additionally containing a known natural product extract or other ingredients for skin improvement within the range that does not inhibit the effective activity of the present invention.

The cosmetic composition of the present invention may include components commonly added to the cosmetic composition in addition to the situation mushroom extract, licorice extract, aloe vera gel and centella asiatica extract included as an active ingredient, for example, stabilizer, dissolution Conventional auxiliaries or carriers such as topical agents, vitamins, pigments and flavorings may be included.

Cosmetic compositions of the present invention may be prepared in any formulation conventionally prepared in the art, for example, solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing cleansing , Oils, powder foundations, emulsion foundations, wax foundations and sprays, and the like, but are not limited thereto. More specifically, flexible lotion, nourishing lotion, nourishing cream, massage cream, essence, eye cream, cleansing cream, cleansing foam, cleansing water, pack, spray, powder, pact, lip gloss, lipstick, shadow, shampoo and conditioner It can be prepared in the formulation of.

When the formulation of the present invention is a paste, cream or gel, an animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as the carrier component .

In the case where the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component. Especially, in the case of a spray, a mixture of chlorofluorohydrocarbons, propane / Propane or dimethyl ether.

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

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

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

[ Example ]

Hereinafter, the configuration and operation of the present invention will be described in more detail with reference to preferred embodiments of the present invention. However, the following examples are provided to aid understanding of the present invention, and the scope of the present invention is not limited to the following examples. Details that are not described herein will be omitted since those skilled in the art can sufficiently infer technically.

Example  1-8, Comparative example  1 to 3. Preparation of skin improvement composition

In order to prepare a composition for improving skin according to the present invention, the situation mushroom extract, licorice extract, aloe vera gel and centella asiatica extract were prepared by purchasing from OmniHub.

Each sample was mixed at the mixing composition ratio shown in Table 1 below to prepare the skin improvement compositions of Examples 1 to 8 and Comparative Examples 1-3.

Situation Mushroom Extract Licorice extract Aloe vera gel Centella asiatica extract Example 1 One One 12 5 Example 2 One One 3 17 Example 3 One One One 10 Example 4 One One One One Example 5 One One 6 One Example 6 One One One 6 Example 7 One One 3 One Example 8 One One One 3 Comparative Example 1 One - - - Comparative Example 2 - One - - Comparative Example 3 One One - -

Test Example  1.Comparison of efficacy according to sample composition ratio

Antioxidant effect, wrinkle improvement, whitening and anti-inflammatory effect according to the mixed composition ratio of the samples were measured for the skin improvement compositions prepared in Examples 1 to 8 and Comparative Examples 1 to 3.

<1-1> Antioxidant Effect Measurement

The electron donating ability was investigated to measure the antioxidant effect of the composition. Specifically, the electron donating ability (EDA) was measured by modifying Blois' method.

1 mL of 0.2 mM 1,1-diphenyl-2-picrylhydrazyl (DPPH) was added to 2 mL of each sample solution, stirred for 30 minutes, and the absorbance was measured at 517 nm. The electron donating ability was expressed as the absorbance reduction rate of the sample solution addition group and no addition group.

The DPPH radical scavenging activity (IC ₅₀ of DPPH (μg / ml)) of Examples 1 to 8 and Comparative Examples 1 to 3 was measured and shown in Table 2.

IC ₅₀ of DPPH (μg / mL) Example 1 98 Example 2 81 Example 3 64 Example 4 52 Example 5 51 Example 6 50 Example 7 50 Example 8 50 Comparative Example 1 133 Comparative Example 2 128 Comparative Example 3 109 ※ IC ₅₀ value: 50% of activity inhibits the concentration, the lower the better

As shown in Table 2, the composition of Examples 1 to 8 simultaneously containing the situation mushroom extract, licorice extract, aloe vera gel and centella asiatica extract is added to the situation mushroom extract and licorice extract alone (Comparative Example 1 ~) 2), compared with the composition containing only two kinds of mushrooms and licorice (Comparative Example 3), the antioxidant activity was found to be high overall.

In addition, when the mushroom extract, licorice extract, aloe vera gel and centella asiatica extract in a mixed ratio of 1 to 5: 1 to 5: 1 to 10: 1 to 15, the antioxidant activity enhancement effect was higher.

<1-2> Wrinkle improvement effect measurement

Collagenase (Collagenase) inhibitory activity was measured to determine the wrinkle improvement effect of the composition, which was measured according to the method of Wunsch et al. Specifically, the reaction zone was added with 4 mM CaCl ₂ in 0.1M tris-HCl buffer (pH 7.5), 0.25mL of substrate solution dissolved 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-D-Arg (0.3mg / mL) 0.15 mL of Collagenase (0.2 mg / mL) was added to a 0.1 mL mixture of the sample solution, which was allowed to stand at room temperature for 20 minutes, and then 0.5 mL of 6% citric acid was added to stop the reaction. Then, 1.5 mL of ethyl acetate was added to the mixture at 320 nm. Absorbance was measured. Collagenase inhibitory activity was shown by the absorbance reduction rate of the sample solution addition group and no addition group.

The collagenase inhibitory activity of the Examples 1 to 8 and Comparative Examples 1 to 3 were measured and shown in Table 3.

IC ₅₀ of Collagenase (μg / ml) Example 1 157 Example 2 149 Example 3 138 Example 4 129 Example 5 126 Example 6 125 Example 7 125 Example 8 124 Comparative Example 1 201 Comparative Example 2 184 Comparative Example 3 171

As shown in Table 3, the composition of Examples 1 to 8 containing the situation mushroom extract, licorice extract, aloe vera gel and centella asiatica extract at the same time is added to the situation mushroom extract and licorice extract alone (Comparative Example 1 ~ 2), compared to the composition containing only two kinds of mushrooms and licorice (Comparative Example 3), the collagenase inhibitory activity was shown as a whole, through which the composition of Examples 1 to 8 will show a high wrinkle improvement effect You can expect

In addition, when the mushroom extract, licorice extract, aloe vera gel and centella asiatica extract in a mixed ratio of 1 to 5: 1 to 5: 1 to 10: 1 to 15, collagenase inhibitory activity is further enhanced Found to be high.

<1-3> whitening effect measurement

In order to measure the whitening effect of the composition, Tyrosinase inhibitory activity was measured, which was measured according to the method of Yagi et al. Specifically, the reaction zone was prepared by adding 0.2 mL of mushroom tyrosinase (110U / mL) to a mixture of 0.2 mL of substrate solution dissolved in 10 mM L-DOPA in 0.5 mL of 0.175M sodium phosphate buffer (pH 6.8) and 0.1 mL of sample solution. The reaction was carried out for a minute to measure the DOPA chrome produced in the reaction solution at 475nm. The tyrosinase inhibitory activity was expressed by the absorbance decrease rate of the sample solution addition group and no addition group.

The tyrosinase inhibitory activity of the Examples 1 to 8 and Comparative Examples 1 to 3 were measured and shown in Table 4.

IC ₅₀ of Tyrosinase (μg / ml) Example 1 98 Example 2 87 Example 3 64 Example 4 53 Example 5 51 Example 6 51 Example 7 50 Example 8 50 Comparative Example 1 126 Comparative Example 2 110 Comparative Example 3 102

As shown in Table 4, the composition of Examples 1 to 8 containing the situation mushroom extract, licorice extract, aloe vera gel and centella asiatica extract at the same time is added to the situation mushroom extract and licorice extract alone (Comparative Example 1 ~ 2), compared to the composition containing only two kinds of mushrooms and licorice (Comparative Example 3), the tyrosinase inhibitory activity was found to be high overall, through which the composition of Examples 1 to 8 will be expected to show a high whitening effect Can be.

In addition, when it contains a mushroom extract, licorice extract, aloe vera gel and centella asiatica extract in a mixing ratio of 1 to 5: 1 to 5: 1 to 10: 1 to 15, the effect of enhancing tyrosinase inhibitory activity is higher. Appeared.

Test Example  2. Determination of efficacy according to extract concentration

Among the compositions of Examples 1 to 8, the composition of Example 8 (hereinafter, referred to as 'HC-6 extract') showing high efficacy in antioxidant effect, wrinkle improvement, whitening and anti-inflammatory effect was selected as a representative mixing ratio. According to the extract concentrations, various skin improvement effects were measured.

<2-1> Antioxidant Activity Test

<2-1-1> electron donating ability confirmation test

The electron donating ability of HC-6 extract was performed by modifying the method of Blois. Electron donating ability (EDA) was measured by modifying Blois' method. 1 mL of 0.2 mM 1,1-diphenyl-2-picrylhydrazyl (DPPH) was added to 2 mL of each sample solution, stirred for 30 minutes, and the absorbance was measured at 517 nm. The electron donating ability was expressed as the absorbance reduction rate of the sample solution addition group and no addition group.

DPPH radical scavenging activity of the HC-6 extract was measured and shown in Table 5 below. The active oxygen scavenging ability was confirmed to increase depending on the concentration of the extract and showed an excellent effect of 96% at 1000㎍ / ㎖ was confirmed that the extract of the present invention has an excellent electron donating ability.

sample Sample concentration (µg / mL) DPPH radical scavenging effect (%)
HC-6 10 3.0 ± 1.2 100 86.2 ± 0.4 1000 96.0 ± 0.9

<2-1-2> SOD-like activity verification test

SOD-like activity was measured according to Marklund's method. To 0.2 mL of each sample solution, 2.6 mL of Tris-HCl buffer (50 mL tris + 10 mM EDTA, pH 8.5) and 0.2 mL of 7.2 mM Pyrogallol were added to stop the reaction. The amount of oxidized Pyrogallol in the reaction solution was measured at 420 nm. .

Experimental results As shown in Table 6, the SOD-like activity of each concentration of the mixed extract showed a high effect of 96% in HC-6 extract 1000㎍ / ㎖ was confirmed that the antioxidant effect was excellent.

sample Sample concentration (µg / mL) SOD-like activity (%)
HC-6 10 0 ± 1.2 100 4.5 ± 0.2 1000 96.0 ± 0.2

<2-1-3> Superoxide anion radical scavenging activity

Superoxide anion radical scavenging ability was measured by nitroblue tetrazolium (NBT) reduction method. 0.1 mL of each sample solution, 0.1 mL of potassium phosphate buffer (pH 7.5), 0.6 mL xanthine (0.4 mL), and 1 mL of substrate solution containing NBT (0.24 mM) were added, and 1 mL of xanthine oxidase (0.049 U / mL) was added at 37 ° C. After reacting for 20 minutes, 1 mL of 1N HCl was added to terminate the reaction, and the absorbance was measured at 560 nm for the amount of superoxide anion radical generated in the reaction solution.

As a result, as shown in Table 7, it was confirmed that the effect increases as the concentration increases, it was confirmed that the antioxidant effect was very excellent by showing the maximum effect of 100% at 1000㎍ / ㎖.

sample The concentration of the sample (占 퐂 / ml) Radical scavenging effect (%)
HC-6 10 27.5 ± 0.6 100 81 ± 0.4 1000 100 ± 0.2

<2-2> Convergence Effect Measurement

Astringent activity was measured by Lee et al. For the convergence effect of the HC-6 extract. Specifically, using blood proteins similar to skin proteins (hemoglobin), each sample solution and hemoglobin solution were mixed 1: 1 in a centrifuge tube container, followed by shaking and mixing for 3 minutes at 1,500 rpm, followed by absorbance at 576 nm. Measured. Astrigent activity was measured by the absorbance reduction rate of the sample solution addition group and no addition group.

As a result, astrigent convergence effect as shown in Table 8 showed a high effect of 90% at 1000㎍ / ㎖, it was confirmed that the convergence effect is excellent.

sample The concentration of the sample (占 퐂 / ml) Astrigent Convergence Effect (%)
HC-6 10 12.0 ± 0.9 100 32.0 ± 0.3 1000 90.1 ± 0.1

<2-3> Wrinkle improvement effect measurement

Collagenase (Collagenase) inhibitory activity was measured for the anti-wrinkle effect of the HC-6 extract, which was tested according to Wunsch et al. In other words, 4 mM CaCl ₂ was added to 0.1 M tris-HCl buffer (pH 7.5), and 0.25 mL of the substrate solution in which 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-D-Arg (0.3 mg / mL) was dissolved and sample. 0.15 mL of Collagenase (0.2 mg / mL) was added to 0.1 mL of the solution, and the mixture was left at room temperature for 20 minutes. 0.5 mL of 6% citric acid was added to stop the reaction. Then, 1.5 mL of ethyl acetate was added to the absorbance at 320 nm. Measured. Collagenase inhibitory activity was expressed as the absorbance decrease rate of the sample solution addition group and no addition group.

As a result, as shown in Table 9, the collagenase wrinkle effect showed an effect of 82% at 1000㎍ / ㎖, it was confirmed that the wrinkle suppression effect to some extent to prevent the formation of wrinkles on the skin.

sample The concentration of the sample (占 퐂 / ml) Collagenase inhibitory effect (%)
HC-6 10 0.5 ± 0.4 100 10.0 ± 0.6 1000 82.1 ± 0.3

<2-4> whitening effect measurement

Tyrosinase (Tyrosinase) inhibitory activity was measured for the whitening effect of the HC-6 extract, and was tested according to Yagi et al. The reaction section was reacted for 2 minutes at 25 ° C by adding 0.2 mL of mushroom tyrosinase (110U / mL) to a mixture of 0.2 mL of substrate solution dissolved in 10 mM L-DOPA in 0.5 mL of 0.175 M sodium phosphate buffer (pH 6.8) and 0.1 mL of sample solution. DOPA chrome produced in the reaction solution was measured at 475 nm. The tyrosinase inhibitory activity was expressed by the absorbance decrease rate of the sample solution addition group and no addition group.

As a result, as shown in Table 10, the whitening effect through Tyrosinase inhibitory activity showed a very high effect of 99.95% at 1000㎍ / ㎖, it was confirmed that the whitening effect is excellent.

sample The concentration of the sample (占 퐂 / ml) Tyrosinase Inhibitory Effect (%)
HC-6 10 11.0 ± 0.6 100 83.0 ± 0.3 1000 99.0 ± 0.9

<2-5> Polyphenol Content Measurement

The total polyphenol content of the HC-6 extract was measured. Polyphenols are known to have anti-aging, anti-wrinkle, whitening, and moisturizing properties.

Polyphenol quantification was based on AOAC. That is, 1 mL of Folin-Ciocalteu phenol reagent reagent was added to 3 mL of a 100-fold diluted sample solution, 0.2 mL of 1N HCl was added, 1 mL of Na ₂ CO ₃ saturated solution was added thereto, mixed, and the mixture was left to stand at room temperature for 1 hour, followed by absorbance at 640 nm. After the measurement, the polyphenol content was calculated by comparing the absorbance value of the standard curve prepared with tannic acid as a standard substance.

The polyphenol content of the HC-6 extract was measured and shown in Table 11 below. The polyphenol content of HC-6 extract was much higher than the polyphenol content of Tannic acid.

sample Polyphenol Content (mg TEA / g) HC-6 complex 17.14 Tannic acid 5.94

<2-6> Anticancer Effect Measurement

In order to test the anticancer effect of the HC-6 extract, the antiproliferative effect on cancer cell line melanoma cells was measured by MTT assay, which was tested by MTT assay according to Carmichael's method. Specifically, 180 μl of melanoma cells were dispensed into 96 well plates at 2 × 10 ³ cells / well, and 20 μl of the sample was prepared by concentration, followed by incubation for 24 hours at 37 ° C. in a 5% CO ₂ incubator. The same amount of distilled water as the sample was added and incubated under the same conditions. 20 μl of MTT solution prepared at a concentration of 5 μg / ml was added thereto, followed by 4 hours of incubation. The culture medium was removed, and 150 μl of DMSO: EtOH (1: 1) was added to each well, followed by stirring for 30 minutes. ELISA reader The anticancer effect was determined by measuring the absorbance at 550 nm and measuring the growth inhibitory effect of cancer cell lines. Vitamin C was treated by concentration instead of sample for comparative experiments, and then the absorbance values were measured in the same manner. The results are shown in FIG.

The MTT screening method for checking cell viability is a method widely used as a cytotoxicity and cell proliferation screening method because many samples can be easily read. In the case of cancer cells, yellow water-soluble MTT tetrazolium is reduced to purple water-insoluble MTT formazan by the action of mitochondrial dehydrogenase during metabolism. When the absorbance of MTT formazan peaks at wavelengths near 550 nm, it reflects the concentration of metabolically active cells.

As shown in Figure 1, HC-extract was shown to inhibit the proliferation of melanoma cells.

<2-7> whitening effect measurement

For the whitening effect experiment of the HC-6 extract, melanin biosynthesis inhibition was measured, melanin biosynthesis inhibition from skin melanoma cells was measured according to Hosoi's method. The melanoma cells incubated in DMEM medium were dispensed to 1 × 10 ⁶ cells / dish in a 100mm culture dish, incubated for 24 hours, and then prepared by concentration, 2mL, and washed 48 hours later with PBS (pH 7.4). Then, the cells were detached with 0.25M Trypsin-EDTA solution, and the harvested cells were treated with 1 mL of 5% TCA per cell, centrifuged twice at 2,500 rpm, and the separated melanin was washed with PBS, followed by ether: ethanol. (1: 3) 1 mL was added, centrifuged twice and washed with ether 1 mL and dried. 1 mL of 1N NaOH was added to the dried melanin and reacted at 80 ° C. for 1 hour, and then the absorbance was measured at 405 nm. For accurate comparison, the melanin biosynthesis inhibition of vitamin C, which is known to have excellent whitening effect, was also measured. Inhibition of melanin biosynthesis was indicated by the decrease in absorbance of the sample solution addition group and no addition group.

As a result of confirming the melanin biosynthesis inhibitory activity of melanoma cells (B16F10) of HC-6 extract as shown in Figs. HC-6 extract showed more than 80% efficacy at 1000 ppm.

<2-8> Anti-inflammatory effect measurement

For the anti-inflammatory effect of the HC-6 extract, the expression level of inducible nitric oxide synthase (iNOS), an inflammation-related factor protein, was measured in immune-related cells.

Prior to the experiment, the results of confirming the toxicity of Raw 264.7 cells by HC-6 complex extract showed that the cytotoxicity of 99.5%, 98.5%, 99.2%, 78.9%, 75.9% at concentrations of 10ppm, 50ppm, 100ppm, 500ppm and 1000ppm, respectively. Showed. Since high concentration samples may cause cell denaturation, lethality, and functional impairment, they were applied to the experiment at a concentration of 100 ppm or less, which shows little cytotoxicity.

Specifically, the anti-inflammatory effect experiment was performed by dispensing Raw 264.7 cells into 2 × 10 ⁴ cells / well in a 60 mm tissue culture dish and incubating for 24 hours. After washing twice with PBS, serum excluded DMEM was added, and pretreated with LPS (1㎍ / ㎖) for 1 hour, HC-6 extract was treated by concentration (0, 10, 50, 100ppm). After 24 hours incubation, washed twice with PBS. Then, 100 μl of Lysis buffer was added to lyse the cells and centrifuged (4 ° C., 12000 rpm, 20 min) to remove the cell membrane components. The protein obtained by centrifugation was quantified by Bradford assay, 60 μl of protein was electrophoresed using 10% SDS-PAGE, and then transferred to PVDF membrance to inhibit nonspecific binding of the antibody, followed by at least 2 hours at 30-40 V. tranefer. After transfer, soak in ponceau, check band, wash twice with TBST, remove, overnight with 5% skim milk and remove background. After washing three times, attach the primary antibody (1: 100) (actin: mouse, iNos: rabit) for 1 hour, and then attach the secondary antibody (1: 1000) (actin: mouse, iNos: rabit) and ECL kit ( Amersham Pharmacia, England) was used for transfer to the film and measured. Band density was confirmed by Gel doc (Bid-rad, America). The anti-inflammatory effect test results are shown in FIG. 4, and as a result, it was confirmed that iNOS protein expression level was reduced to 50% or less at 100 ppm.

Preparation Example 1 ~ 4. For skin improvement Cosmetics  Produce

Among the compositions of Examples 1 to 8, the composition of Example 8 (hereinafter, referred to as 'HC-6 extract') showing high efficacy in antioxidant effect, wrinkle improvement, whitening and anti-inflammatory effect was selected as a representative mixing ratio. It was used at the time of manufacturing cosmetics.

Preparation Example 1 . Skin  Produce

To improve the skin using a conventional skin manufacturing method in the composition of Table 12 below.

number ingredient weight(%) One HC-6 extract 3.0 2 glycerin 1.0 3 Carboxyvinyl polymer 0.2 4 Alcohol 5.0 5 Tea Tree Oil 0.02 6 Phenoxyethanol 0.3 7 Sopolyence 0.2 8 Methyl paraoxybenzoate 0.15 9 Citric acid 0.04 10 Sodium citrate 0. 11 Spices 0.05 12 Purified water Balance system 100.0

Preparation Example 2 . Manufacture of lotions

To a lotion for improving skin using a conventional lotion manufacturing method in the composition of Table 13.

number ingredient weight(%) One HC-6 extract 3.0 2 glycerin 7.0 3 Carboxyvinyl polymer 0.6 4 Beads wax 1.8 5 Decaglyceryl Myristate 2.0 6 Squalane 6.0 7 Methyl polysiloxane 2.0 8 Bisabolol 0.1 9 Phenoxy ethanol 0.7 10 Butyl paraoxybenzoate 0.1 11 Spices 0.1 12 Purified water Balance system 100.0

Production Example 3 . Serum  Produce

To prepare a serum for skin improvement using a conventional serum manufacturing method in the composition of Table 14.

number ingredient weight(%) One HC-6 extract 3.0 2 Aminocoat 7.0 3 Carboxyvinyl polymer 0.6 4 Arbutin 1.8 5 ethanol 6.0 6 1,3-butylene glycol 2.0 7 Triethanolamine 0.1 8 Methyl paraoxybenzoate 0.1 9 Spices 0.1 10 Purified water Balance system 100.0

Production Example 4 . Manufacture of cream

To improve the cream using a conventional cream manufacturing method in the composition of Table 15 below.

number ingredient weight(%) One HC-6 extract 3.0 2 Stearic acid 0.5 3 Cetanol 1.5 4 glycerin 7.0 5 Solbitan Oliveate 0.3 6 Methylpolysiloxane 0.3 7 Phenoxyethanol 0.7 8 Beta Glucan 3.0 9 Methyl paraoxybenzoate 0.2 10 Bisabolol 0.05 11 Spices 0.1 12 Purified water Balance system 100.0

Test Example  3. human body Patch test

To determine the degree of skin irritation of the cosmetic preparations prepared in Preparation Examples 1 to 4, a skin patch test was performed. The subjects were selected to have no skin disease at the test site of 20 ~ 50 years old. Wipe the upper lip of the patch with 70% ethanol and dry it. Then, apply water to a blank using a fin chamber and add 0.02 g of the product to the upper arm for 24 hours, and remove the skin within 1 hour after removing the patch. The reaction was examined and examined again the next day (after 48 hours). The skin reaction was determined by Table 16, which is a test standard for the human patch test.

Table 17 shows the skin patch test results of the cosmetics according to Preparation Examples 1-4.

Skin reaction score - 0 no response ± One Weak positive reaction (erythema) + 2 Positive reaction (erythema) ++ 3 Strong positive reaction (erythema, edema) +++ 4 Severe positive reactions (erythema, edema, blisters)

Test substance Number of subjects who responded after 24 hours (persons) Number of subjects with response after 48 hours (persons) 0 One 2 3 4 0 One 2 3 4 Preparation Example 1 (Skin) 15 0 0 0 0 15 0 0 0 0 Preparation Example 2 (Lotion) 15 0 0 0 0 15 0 0 0 0 Preparation Example 3 (Serum) 15 0 0 0 0 15 0 0 0 0 Preparation Example 4 (Cream) 15 0 0 0 0 15 0 0 0 0

As a result of the human patch test test, the cosmetics of Preparation Examples 1 to 4 belonged to the non-irritating range and were confirmed to be safe substances for the human body.

Claims (10)

Situation mushroom extract, licorice extract, aloe vera gel and Centella asiatica extract, including the situation mushroom extract, licorice extract, aloe vera gel and centella asiatica extract 1 ~ 5: 1 ~ 5: 1 ~ 10: 1 to 15, a skin composition for improving skin anti-aging, skin whitening or wrinkles, contained in a mixing ratio.
delete The method of claim 1,
The situation mushroom extract, licorice extract, aloe vera gel and centella asiatica extract is characterized in that it is contained in a mixing ratio of 1 to 3: 1 to 3: 1 to 6: 1 to 10, cosmetic composition for skin improvement.
The method of claim 1,
The composition is characterized in that it contains 0.01 ~ 30% by weight of the complex extract of the situation mushroom extract, licorice extract, aloe vera gel and centella asiatica extract, cosmetic composition for skin improvement.
The method of claim 1,
The situation mushroom extract, licorice extract, and centella asiatica extract is one selected from the group consisting of water, lower alcohol having 1-4 carbon atoms, acetone, ethyl acetate, butyl acetate, ether, chloroform and 1,3 butylene glycol The cosmetic composition for skin improvement, characterized in that extracted using the above solvent.
The method of claim 1,
The composition is characterized in that it has an antioxidant activity, cosmetic composition for skin improvement.
delete The method of claim 1,
The composition is characterized in that it has melanin biosynthesis inhibitory activity and collagenase (collagenase) inhibitory activity, cosmetic composition for skin improvement.
The method of claim 1,
The composition is characterized in that it further comprises any one or more of green tea extract, bokbunja extract and lettuce extract, cosmetic composition for skin improvement.
The method of claim 1,
The cosmetic composition comprises a formulation selected from the group consisting of solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing cleansing, oils, powder foundations, emulsion foundations, wax foundations and sprays. It is characterized by having a cosmetic composition for skin improvement.

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