KR20190113185A - Cosmetic composition for skin whitening comprising bamboo leaf extract as an active ingredient - Google Patents

Abstract

The present invention relates to a cosmetic composition and, more specifically, to a cosmetic composition with antioxidative and skin whitening activities containing an extract of a bamboo leaf as an active ingredient. According to the present invention, the extract of a bamboo leaf (Sasa coreana Nakai) provides excellent safety and antioxidative activity and also effectively inhibits melanin generation through a tyrosinase inhibition activity and thus, the composition of the present invention including the extract as an active ingredient represents excellent skin whitening and antioxidative effects through inhibition of melanin pigments without irritating skin, thereby being useful as a functional cosmetic composition. Moreover, when an extract of Ixeris polycephala corresponding to a medicinal herb is used with the extract of a bamboo leaf (Sasa coreana Nakai) of the present invention, a synergy effect remarkably increasing melanin generation inhibition effect is provided and thus, a composition containing the extract of a bamboo leaf and an extract of Ixeris polycephala can be useful as a cosmetic material for skin whitening. In particular, the extract of a bamboo leaf (Sasa coreana Nakai) and an extract of Ixeris polycephala used as an active ingredient in the present invention are originated from a natural plant and thus the cosmetic composition of the present invention provides an advantage of being safe after a long-term use.

Description

Cosmetic composition for skin whitening comprising bamboo leaf extract as an active ingredient}

The present invention relates to a cosmetic composition, and more particularly to a cosmetic composition having an antioxidant and whitening activity comprising the bamboo leaf extract as an active ingredient.

Skin is a very important tissue that protects the human body while having direct contact with the external environment and has biochemical and physical functions. This tissue is largely divided into three parts: epidermis, dermis and subcutaneous tissue. Human skin color depends mainly on the number, size, type and distribution of melanocytes containing melanin in skin cells.

Melanosomes are produced by melanocytes and melanin, a black pigment produced in the epidermis, is produced in cells called melanocytes. Melanin absorbs ultraviolet light energy from sunlight, preventing deeper cells from being damaged by ultraviolet light. When abnormally low melanin is produced, skin lesions such as vitiligo are induced, and on the contrary, excessive synthesis of melanin by ultraviolet light not only damages the skin, but also forms spots, freckles, and further causes skin cancer.

The most important role in melanin formation is known as the tyrosinase enzyme, which produces dopaquinone, which acts on the amino acid called tyrosine in the tissues, and then through several steps of automatic oxidation Melanin, a black pigment, is produced. Therefore, the composition for whitening may be made in several ways as follows depending on which process of the step to inhibit melanin production.

First, it contains a component that can block ultraviolet rays, which is the main cause of melanin production, and the composition according to this method contains a light scattering agent or a light blocking agent to reduce DNA damage or inflammation caused by ultraviolet rays, such as glucosamine and tyrosinia. By inhibiting the synthesis of core carbohydrates necessary for me to be active, melanin production can be suppressed (acting on the whitening A stage). The second is to use substances that interfere with the function of tyrosinase involved in melanogenesis, such as enzymatic acid (kojic acid), rucinol, eric acid or arbutin (acting on the whitening B stage). Third, there is a method of stabilizing dopachrome to stop the progression to black melanin (Eu-Melanin) (acts on the whitening C stage). Fourth, while tyrosine undergoes complex oxidation and condensation reactions through dopa and dopachrome, it uses a substance such as vitamin C that has a strong antioxidant effect and can produce a whitening effect (acting on the whitening step D). Fifth, there is also a method that can remove and reduce the melanin produced in the stratum corneum using enzymes or AHA (Alpha Hydroxy Acid) (acts on the whitening step E).

In the field of conventional cosmetics, as a whitening component, for example, substances that inhibit tyrosinase enzyme activity such as kojic acid, arbutin, hydroquinone, vitamin C (L-Ascorbic acid) And derivatives thereof and various plant extracts have been used. However, the substance that inhibits the tyrosinase enzyme activity is deteriorated due to its low stability in the prescription system, or colored, or its use is limited due to the occurrence of odor, efficacy at the biological level, unclear effect, and safety problems. to be.

Therefore, the inventors of the present invention, while seeking to find a material that is stable to the human body while showing excellent whitening effect, the result of experimenting the effect of the bamboo leaf extract as a cosmetic material, the bamboo leaf extract has an antioxidant activity In addition, it was confirmed that the synthesis of melanin can be effectively inhibited by inhibiting tyrosinase activity in a concentration-dependent manner, and in particular, the use of the worm extract in combination with bamboo leaf extract effectively inhibits melanin production. The present invention was completed by confirming that it can be done.

Korean Patent Publication No. 10-2012-0054375

Therefore, it is an object of the present invention to provide a cosmetic composition that is stable to the human body and is excellent in antioxidant activity and melanin inhibitory activity, effective for skin whitening.

In order to achieve the object of the present invention as described above,

The present invention provides a cosmetic composition for skin whitening comprising the bamboo leaf extract as an active ingredient.

In one embodiment of the present invention, the bamboo type may be Sindaedae.

In one embodiment of the present invention, the extract may be extracted with one or more solvents selected from the group consisting of lower alcohols, ethyl acetate, acetone, water and hexane having 1 to 4 carbon atoms.

In one embodiment of the present invention, the cosmetic composition may further comprise a extract extract.

In one embodiment of the present invention, the cosmetic composition is a flexible lotion, gel, water-soluble liquid, milk lotion, nutrition cream, massage cream, essence, oil-in-water emulsion, water-in-oil emulsion, flake anhydrous product, solid anhydrous product , Oil dispersions in aqueous phase with globules, ionic lipid vesicles, nonionic lipid vesicles, ointments, cleansing foams, cleansing water, packs, body oils, oil-in-water makeup bases, water-in-oil makeup bases, foundations, skins It may be one formulation selected from the group consisting of cover, lipstick, lip gloss, face powder, two-way cake, eye shadow, mascara, cheek color and eyebrow pencils.

Bamboo (Sinyidae) leaf extract of the present invention can effectively inhibit melanin production through tyrosinase inhibitory activity with excellent safety, antioxidant activity, the composition of the present invention comprising it as an active ingredient without melanin irritation to the skin It can be used as a functional cosmetic composition because it can exhibit an excellent whitening effect and antioxidant effect through the pigment suppression. In addition, when using in combination with the bamboo leaf extract of the present invention corresponding to the worm plant extracts of bamboo (Sindaedae) according to the synergistic effect that the melanin production inhibitory effect is markedly increased, bamboo leaf extract and worms extract In the case of a composition comprising a can be usefully used as a cosmetic material for skin whitening. In particular, the bamboo (Sindaedae) leaf extract and beetle extract used as an active ingredient in the present invention is derived from a natural plant, the cosmetic composition of the present invention comprising the same has a safe long-term use.

1 is a schematic diagram showing an extraction process of the bamboo leaf extract of the present invention.
Figure 2 shows the high performance liquid chromatography (HPLC) chromatogram of the bamboo leaf methanol extract (SCM) and standard of the present invention (standard).
3 is a graph showing the mushroom tyrosinase inhibitory activity of the bamboo leaf methanol extract (SCM), ascorbic acid, catechin of the present invention.
Figure 4 is a graph showing melanin production inhibitory activity according to the concentration-specific treatment of the bamboo leaf methanol extract (SCM) of the present invention on α-MSH induced B16F10 cells (α-MSH: alpha-melanin stimulating hormone).
Figure 5 is a microscopic picture confirming the degree of melanogenesis according to the concentration-specific treatment of the bamboo leaf methanol extract (SCM) of the present invention to α-MSH-induced B16F10 cells (α-MSH: alpha-melanin stimulating hormone).
6 is a result of Western blot analysis of the expression levels of tyrosinase, TRP1, TRP2 protein according to the concentration-specific treatment of bamboo leaf methanol extract (SCM) of the present invention on α-MSH induced B16F10 cells.
Figure 7 is the result of measuring the cell survival rate according to the concentration-specific treatment of the bamboo leaf methanol extract (SCM) of the present invention in B16F10 cells through an MTT assay.
8 is a graph showing the melanin production inhibitory activity according to the combination treatment of medicinal plant extracts (Hirary extract, Echo extract, Beetle extract) with bamboo leaf methanol extract (SCM) of the present invention on α-MSH induced B16F10 cells .
Figure 9 is the result of measuring the cell survival rate according to the treatment of 100μg / mL medicinal plant extract of the present invention (Hairy extract, Echo extract, algae extract) to B16F10 cells through an MTT assay.

The present invention is characterized by providing a cosmetic composition comprising the bamboo leaf extract as an active ingredient.

In one embodiment of the present invention, the bamboo type may be Sindaedae.

As used herein, the term "sindae" is a type of bamboo and the scientific name is Sasa coreana Nakai. Biological classifications include plant, pizza plant door, monocotyledonous plant, flower tree, rice family, and cooking genus. Characteristic is that the leaves have hairs on the lower part and hairs on the upper part. The leaves are 5-8 pieces at the ends of the branches, long oval or ovate long oval, 3-12cm long, 6-22mm wide, pointed, subcardiac or wide basal, with no hairs on the surface, dense hairs on the back, There are fine teeth and 5-6 veins at the edge. The leaf is usually hairless and hair falls off early. The stem is 30-80cm in height and 3-8mm in diameter. The upper part is 5-20cm, the branch is dense and there are grooves in the main stem and branch. Roots have short roots, branches are split and nodes are short.

This sindae is used for ornamental purposes and leaves are edible.

However, the antioxidant and whitening activity of bamboo leaf extract has not been studied.

In the present invention, it was confirmed for the first time that the bamboo (Sinyidae) leaf extract can be used as a material of functional cosmetics with excellent anti-oxidant and whitening activity.

Bamboo leaf extract according to the present invention may be obtained by extracting and separating from nature using extraction and separation methods known in the art, the 'extract' as defined in the present invention using a suitable solvent (bamboo) Sindaedae), and includes, for example, all of the bamboo leaf crude extract, polar solvent soluble extract or non-polar solvent soluble extract.

As a suitable solvent for extracting the extract from the bamboo, any pharmaceutically acceptable organic solvent may be used, and water or an organic solvent may be used, but is not limited thereto. For example, purified water, methanol ( alcohols having 1 to 4 carbon atoms, acetone, ether, benzene, chloroform (including methanol, ethanol, propanol, isopropanol, butanol, etc.) Various solvents such as chloroform, ethyl acetate, methylene chloride, hexane and cyclohexane may be used alone or in combination.

As the extraction method, any one of hot water extraction method, cold leaching extraction method, reflux cooling extraction method, solvent extraction method, steam distillation method, ultrasonic extraction method, elution method and compression method can be used. In addition, the desired extract may further be subjected to a conventional fractionation process, it may be purified using conventional purification methods. There is no limitation to the method for producing the bamboo leaf extract of the present invention, any known method may be used.

For example, the bamboo (Sindaedae) leaf extract included in the composition of the present invention is a powder state by the additional process such as distillation under reduced pressure, freeze drying or spray drying, etc. It can be prepared as. The primary extract may be further purified using various chromatography such as silica gel column chromatography, thin layer chromatography, high performance liquid chromatography, and the like. You can also get

Therefore, in the present invention, the bamboo leaf extract is a concept that includes all the extracts, fractions and purified products obtained at each step of extraction, fractionation or purification, their dilutions, concentrates or dried products.

Looking at the method of producing a bamboo (Sinyidae) leaf extract according to an embodiment of the present invention in more detail as follows.

The dried bamboo leaves were put into water, extracted using auto clave at 120 ° C. for 90 minutes, filtered, and the filtrate was concentrated in a vacuum condenser to prepare a hot water extract. Alternatively, the dried bamboo leaves may be put in ethanol and refluxed at 80 ° C. for 6 hours, and the filtrate may be concentrated in a reduced pressure concentrator to finally prepare a spirit extract. Alternatively, the dried bamboo leaves were immersed and extracted for 7 days at room temperature in 100% methanol, the extracted solution was filtered, and the filtrate was concentrated in a vacuum condenser to prepare a methanol extract.

The composition of the present invention corresponds to a cosmetic composition comprising the bamboo leaf extract as an active ingredient.

In one embodiment of the invention, the bamboo leaf extract may be included in the cosmetic composition at a concentration of 0.001 to 10000 μg / ml.

In another embodiment of the present invention, the composition may further comprise a medicinal plant extract.

Examples of the medicinal plant extracts may include hydroxy extracts, chrysalis extracts, and algae extracts, preferably algae extracts.

`` Corylopsis coreana , Uyeki ) 'is a plant of the locust family, which is grown in Korea and grows in the mountains and mountains of Namdo, Jirisan and Suwon Gwanggyo. It grows up to 5m in height and forms a colony, and many neighbors rise up to form large clusters, and they grow together with azaleas, rhododendrons, sesame seeds, red bean pears, and gingko trees. They grow well in the sun and grow cold well. It is not worn and is resistant to dryness and grows well in dry soil. Small branches are yellowish brown with white cortex on bark. The elliptical is oval yellowish brown. The leaves are regenerated, ovate, short apex, heart bottom, 5-9cm long, 4-8cm wide, with sharp teeth, the surface is light green, the texture is good, the back is grayish white, and there is no hair. Total inflorescence is 3-4cm long, grows to 7-8cm after flowering. In March, 8-12 small flowers hang in a lantern shape. Petals are obovate, light yellow-green.

In the present specification, the term “hiri extract” means obtained by extracting from various organs or parts (eg, leaves, flowers, roots, stems, branches, shells, seeds, etc.) of the diary.

' Physalisalkekengi var. Francheti (Masters) Hort' is a perennial herb that is grown all over Korea and grows at the edge of forests less than 600m above sea level, about 60 ~ 90cm in height. In the basement there is a long root of creeping sideways. The leaves are regenerated, wild-type, sharp at the ends, and have a tooth-shaped cradle, and 2 sheets per section. Flowers come from mesenchymal and run one by one in white, pedicel is 3 ~ 4cm long, calyx is short tubular, has 5 ends with shallow tip and hairs on edge. Fruits ripen in red with a spherical axilla. Herbal medicine is used as a hut (Physalis Herba), also known as the dragon. Ingredients include Physalin A, B, and C (physalin A, B, C) (Jacobo-Herrera et al, J. Nat. Prod., 69, pp328-331, 2006), saponins, flavonoids ( flavonoid), alkaloid, and the fruit contains 0.12% carotenoid, red pigment, ascorbic acid, resin, pectin, tannin, bitter substance, carotene, alkaloid, quercetin, caffeic acid, cinnamic acid, Ferularic acid, roots contain 3-alpha-tiglooxytropane, pisalin, zeaxanthin, and seeds contain linolein-based oil (Weller P et al., J. Agric Food Chem., 51, pp7044-7049, 2003). Pharmacological action has the contraction of the uterus root and diuretic effect on the fruit.

In the present specification, the term 'cherry extract' means that it is obtained by extracting from various organs or parts (such as leaves, flowers, roots, stems, branches, shells and seeds, etc.) of the cherry tree.

' Ixeris polycephala Cassini ) 'is a kind of bitterworm belonging to the chrysanthemum family. It is a perennial weed in the mountains, fields, and fields of sub-central Korea. It grows to about 25 ~ 50cm in size and cuts the stem or leaves to give white milk. Flowers bloom in May-July at the size of about 1.5cm in yellow.

In the present specification, the term 'barberry extract' refers to an extract obtained from various organs or parts (for example, leaves, flowers, roots, stems, branches, shells, seeds, etc.) of the shell.

The medicinal plant extract of the present invention can be extracted using a suitable solvent, and any suitable pharmaceutically acceptable organic solvent may be used as the appropriate solvent, water or organic solvent may be used, but is not limited thereto. Although, for example, alcohol having 1 to 4 carbon atoms, acetone (acetone), ether, including purified water, methanol, ethanol, propanol, isopropanol, butanol, etc. solvents such as ether, benzene, chloroform, ethyl acetate, methylene chloride, hexane and cyclohexane can be used alone or in combination. have.

In the following experimental example of the present invention, the bamboo leaf extract according to the synergistic effect of the melanin production inhibitory effect is remarkably increased when used in combination with the bamboo leaf extract of the present invention Medicinal plant extract (sindaedae) In the case of the composition containing the extract of the algae and algae extract through objective experiments proved that it can be usefully used as a cosmetic material for skin whitening.

Examples of products to which the cosmetic composition of the present invention may be added include, for example, cosmetics such as astringent cosmetics, soft cosmetics, nourishing cosmetics, various creams, essences, packs, foundations, and the like, cleansing agents, face washes, soaps, treatments, and cosmetics Etc.

Specific formulations of the cosmetic composition of the present invention include flexible lotion, gel, water-soluble liquid, milk lotion, nourishing cream, massage cream, essence, oil-in-water emulsion, water-in-oil emulsion, flake anhydrous product, solid anhydrous product, globules Oil dispersions in used aqueous phase, ionic lipid vesicles, nonionic lipid vesicles, ointments, cleansing foams, cleansing water, packs, body oils, oil-in-water makeup bases, water-in-oil makeup bases, foundations, skin covers, lipsticks, Lip gloss, face powder, two-way cake, eye shadow, mascara, cheek color and eyebrow pencils.

According to a preferred embodiment of the present invention, the content of the active ingredient of the present invention is 0.00001-40% by weight based on the total weight of the composition, preferably 0.0005-40%, more preferably 0.0005-20% by weight. When the content of the active ingredient (bamboo leaf extract) is less than 0.00001% by weight, the skin whitening effect is greatly reduced, and when it exceeds 20% by weight, skin irritation may occur, and formulation problems may occur.

On the other hand, the cosmetic composition according to the present invention can be formulated by stabilizing by containing the active ingredient inside the nano-liposomes. When the active ingredient is contained inside the nanoliposome, the active ingredient is stabilized to solve problems such as precipitation formation, discoloration, and odor when formulated, and can increase the solubility and transdermal absorption of the component to maximize the efficacy expected from the extract. Can be expressed as.

Nanoliposomes in the present invention means a liposome having an average particle diameter of 10 ~ 500nm as having a conventional liposome form. According to a preferred embodiment of the present invention, the average particle diameter of the nanoliposomes is 50 ~ 300nm, more preferably 100 ~ 200nm. When the average particle diameter of the nanoliposomes exceeds 500 nm, the improvement of skin penetration and the improvement of formulation stability are very weak among the technical effects to be achieved in the present invention.

Nanoliposomes used to stabilize the extract according to the present invention may be prepared by a mixture comprising polyols, oily ingredients, surfactants, phospholipids, fatty acids and water.

The polyol used in the nanoliposome of the present invention is not particularly limited, preferably propylene glycol, dipropylene glycol, 1,3-butylene glycol, glycerin, methyl propanediol, isopropylene glycol, pentylene glycol, erythritol, xylitol , At least one member selected from the group consisting of sorbitol and mixtures thereof. The amount used is 10 to 80% by weight, preferably 30 to 70% by weight based on the total weight of the nanoliposomes.

The oil component used in the preparation of the nanoliposome of the present invention may be a variety of oils known in the art, preferably hydrocarbon-based oils such as hexadecane and paraffin oil, synthetic oils of esters, Silicone oils such as dimethicone and cyclomethicone, animal and vegetable oils such as sunflower oil, corn oil, soybean oil, avocado oil, sesame oil and fish oil, ethoxylated alkyl ether oils, propoxylated alkyl ether oils ₄₀ is a fatty alcohol, and mixtures thereof -, phytosphingosine, sphingosine, and scan non-ping the same scan pinggo cannabinoid lipid, cholesterol side-by celebrity, sitosterol cholesteryl sulfate, cholesteryl sulfate, cytokines, C _10. The amount may be 1.0 to 30.0% by weight based on the total weight of nanoliposomes, preferably 3.0 to 20.0% by weight.

Surfactants used in the preparation of the nanoliposomes of the present invention may be any known in the art. For example, anionic surfactants, cationic surfactants, amphoteric surfactants and nonionic surfactants can be used. Preferred are anionic surfactants and nonionic surfactants. Specific examples of anionic surfactants include alkylacylglutamate, alkylphosphates, alkyllactylates, dialkylphosphates and trialkylphosphates. Specific examples of nonionic surfactants include alkoxylated alkylethers, alkoxylated alkylesters, alkylpolyglycosides, polyglyceryl esters and sugar esters. Particularly preferred surfactants are polysorbates belonging to nonionic surfactants. The amount used may be 0.1 to 10% by weight based on the total weight of the nanoliposomes, preferably 0.5 to 5.0% by weight.

Phospholipids, another component used in the preparation of the nanoliposomes of the present invention, are amphoteric affinity lipids, and include natural phospholipids (eg, egg yolk lecithin or soy lecithin, sphingomyelin) and synthetic phospholipids (eg, Dipalmitoylphosphatidylcholine or hydrogenated lecithin), preferably lecithin. In particular, unsaturated lecithin or saturated lecithin derived from nature extracted from soybean or egg yolk are preferred. Typically, lecithin derived from nature has 23-95% phosphatidylcholine and 20% or less phosphadidylethanolamine. In the preparation of the nanoliposomes of the present invention, the amount of phospholipid used is 0.5 to 20.0% by weight, preferably 2.0 to 8.0% by weight, based on the total weight of the nanoliposomes.

Fatty acid to be used in nano-liposome preparation of the present invention is a higher fatty acid, preferably a C ₁₂ - ₂₂ as a saturated or unsaturated fatty acid of the alkyl chain, e.g., lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid and linoleic acid It includes. The amount may be 0.05 to 3.0% by weight based on the total weight of the nanoliposomes, preferably 0.1 to 1.0% by weight.

Water used in the preparation of the nanoliposomes of the present invention is generally deionized distilled water, the amount of which may be 5.0 to 40% by weight relative to the total weight of nanoliposomes.

The preparation of nanoliposomes can be accomplished through various methods known in the art, but most preferably, a mixture comprising the above components is applied to a high pressure homogenizer. The preparation of nanoliposomes by high pressure homogenizer can be carried out under various conditions (eg, pressure, frequency, etc.) according to the desired particle size, and preferably 1 to 5 times high pressure homogenizer under a pressure of 600 to 1200 bar. Nanoliposomes can be prepared by passing through.

The cosmetic composition of the present invention may be used alone or in duplicate, or may be used in combination with other cosmetic compositions other than the present invention. In addition, the cosmetic composition excellent in the antioxidant and whitening effect according to the present invention can be used according to a conventional method of use, the number of times of use can be varied according to the user's skin condition or taste.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are intended to illustrate the present invention more specifically, but the scope of the present invention is not limited to these examples.

< Example >

Statistical processing

All results were statistically treated after three replicates, and the mean ± standard deviation of the values obtained by the experiments. The statistical significance test between the control group and the experimental group was carried out using t-test using SPSS 23 (SPSS Inc., Chicago, IL), and was determined to be statistically significant when p <0.05 or less.

Cell culture

B16F10 cells were distributed by the Korea Cell Line Bank and Dulbecco's modified Eagle's medium (DMEM; Sigma-Aldrich, St. Louis, MO, USA) added 10% fetal bovine serum (FBS, Gibco BRL, USA) and 0.2% sodium bicarbontate. Was used as a medium, and cultured in a state of 5% CO2 at 37 ℃, the experiment was carried out by subcultured in the state where the cells were grown to about 90% of the floor area.

< Example  1>

Bamboo leaf extract manufacturer

In this study, Shinadae leaves (Sasa coreana Nakai) from Damyang-eup, Damyang-gun, Jeollanam-do, were dried for 5 hours at 60 ° C using a dry oven, and three different extraction methods were used (see FIG. 1).

As a first extraction method, 10 g of dried bamboo leaves were added to 500 mL of water, extracted using auto clave at 120 ° C. for 90 minutes, and filtered. The filtrate was concentrated with a reduced pressure concentrator to obtain a hot water extract.

As a second extraction method, 10 g of dried bamboo leaves were placed in 250 mL of 70% ethanol and refluxed at 80 ° C. for 6 hours, and the filtrate was concentrated with a reduced pressure concentrator to finally obtain a spirit extract.

As a third extraction method, 10 g of dried bamboo leaves were added to 500 mL of 100% methanol for immersion extraction at room temperature for 7 days, and the extracted solution was filtered, and the filtrate was concentrated using a vacuum concentrator to obtain an organic solvent extract.

The hydrothermal extract, ethanol extract, and methanol extract obtained through the three extraction methods were named SCW, SCE, SCM.

The extraction yields of SCW, SCE and SCM were found to be 13%, 16% and 17% based on solid content, respectively, and the yield of SCM extracted using 100% methanol was the highest.

Bamboo (Sindaedae) leaf extract of the present invention Kinds Abbreviation Bamboo Leaf Hydrothermal Extract SCW Bamboo Leaf Ethanol Extract SCE Bamboo Leaf Methanol Extract SCM

< Experimental Example  1>

Antioxidant Activity of Bamboo Leaf Extract

In this experiment, DPPH radical scavenging ability and ABTS radical scavenging ability were measured to evaluate the antioxidant activity of the bamboo leaf extract of the present invention prepared in Example 1.

DPPH free radical is a widely used method to measure the antioxidant activity of natural extracts. In this experiment, we determined the radical scavenging ability of the sample by modifying Blois's method of antioxidant activity [Blois, MS (1958) Antioxidant determinations by the use of a stable free radical. Nature 181: 1199-1200.]. 800 μL of 0.1 mM DPPH (2,2-diphenyl-1-picrylhydrazyl, Sigma, USA) dissolved in methanol and 200 μL of each sample were added to a micro centrifuge tube and voltexed to react in the dark for 15 minutes. The concentration of the remaining radicals was measured at 517 nm using a UV-Visible spectrophotometer (SCINCO, Seoul, Korea). Ascorbic acid (Sigma, USA) was used as a positive control, and the scavenging ability (IC ₅₀ ) of DPPH of the extract was expressed as the concentration necessary to reduce the absorbance of the control using only the solvent by 50%.

ABTS radical scavenging ability can measure both radical scavenging activity of hydrophilic and hydrophobic samples and has the advantage of wide application range. In this experiment, the modified method of Jeong et al. Was measured [Jeong, CH, G. Choi, J. Kim, J. Kwak, H. Heo, KH Shim, BR Cho, YI Bae, and JS Choi (2009) In vitro antioxidative activities and phenolic composition of hot water extract from different parts of Cudrania tricuspidata. J. Food Sci. Nutr. 14: 283-289.]. 7 mM ABTS solution dissolved in distilled water and 2.45 mM potassium persulfate were added in a 1: 1 ratio and left in the cow for 12 hours. Radical stock solution was diluted with PBS (pH 7.4) to have an absorbance value of 0.70 ± 0.02. 200 μL of each prepared sample was added to 800 μL of the diluted solution, and the absorbance was measured after reaction in the dark for 15 minutes. Ascorbic acid (Sigma-Aldrich, St. Louis, MO, USA) was used as a positive control, and the scavenging ability (IC ₅₀ ) of ABTS of the extract was indicated by the concentration necessary to reduce the absorbance of the control using only the solvent by 50%. .

As a result, as shown in Table 2 below, the DPPH IC ₅₀ range of each sample was found to be 604-797 μg / mL, and the AB ₅₀ IC ₅₀ range was found to be 117-166 μg / mL (Table 1). In particular, DPPH and ABTS IC ₅₀ of SCM extracted from 100% methanol of Shindaedae extract were 604 μg / mL and 117 μg / mL, respectively, and the highest antioxidant activity was found among the three extracts. In the previous study, the antioxidant activity of the gingiform, cotton rod, bamboo shoot, stalk and bamboo shoot was investigated and the IC ₅₀ range was reported as 200 ~ 1600 μg / mL. The results of this experiment showed higher antioxidant activity than the existing antioxidant activity because of different bamboo leaf types and extraction methods, and the content of polyphenols and physiological activities of bamboo leaves according to the habitat and body odor. It is also believed that may vary.

DPPH Radical and ABTS Radical Scavenging Activity of the Bamboo Leaf Extract of the Present Invention sample DPPH IC ₅₀ ^a (μg / mL) ABTS IC ₅₀ (μg / mL) SCW 608 166 SCE 797 138 SCM 604 117 Ascorbic acid 25 56 ^a IC ₅₀ Inhibitory concentration

< Experimental Example  2>

Determination of Total Polyphenol Content in Bamboo Leaf Extract

Folin-Ciocalteu reagent was mixed with distilled water 10-fold diluted with 2% Na ₂ CO ₃ (w / v) in a ratio of 1: 1: 1. After reacting for 30 minutes at room temperature, the absorbance of the reaction mixture was measured at 750 nm using a UV-Visible spectrophotometer (SCINCO, Seoul, Korea) [Choi, MH, MJ Kim, YJ Jeon, and HJ Shin (2014) Quality changes of fresh vegetable and fruit juice by various juicers. KSBB J. 29: 145-154.].

As a result, as shown in Table 3, when the polyphenol content of the SCM is based on gallic acid, the highest polyphenol content was confirmed as 42 μg / mg. In the previous studies, the total polyphenol contents of the sorghum hydrothermal extract and 80% methanol extract were 15 μg / mL and 12 μg / mL, respectively. The higher the total polyphenol content, the higher the antioxidant activity. Compared with the experimental results of this study, the total polyphenol content of SCM was found to be higher than that of previous studies.

Total Polyphenol Content of Bamboo Leaf Extract of the Present Invention sample TPC ^b (GAE ^c μg / mg) SCW 24 SCE 25 SCM 42 ^b TPC Total polyphenol content, ^c GAE Garlic acid equivalent

< Experimental Example  3>

Of bamboo leaf extract HPLC  analysis

High-performance liquid chromatography (HPLC) was used to quantify polyphenols (catechin, chlorogenic acid, caffeic acid, p-coumaric acid, ferulic acid, rutin) in extracts. Shimadzu HPLC LC-20AD pump, diode array detector (SPD-20A) and XBridge C18 column (4.6 x 150mm, 5μm) were used. The mobile phase of HPLC used A: water (0.2% (v / v) TFA) and B: methanol (0.2% (v / v) TFA). HPLC analysis conditions were 0-50 minutes, 25% -55% B, flow rate 0.6 mL / min. The column temperature was 40 ℃, the absorption wavelength was measured at 330 nm, the sample injection was injected into 20 μL and the compound was identified by comparing the retention time of the standard material.

As a result, as shown in Fig. 2, HPLC analysis showed that catechin 409 μg / g, chlorogenic acid 178 μg / g, caffeic acid 109 μg / g, p-coumaric acid 54 μg / g, ferulic acid 4 μg / g, rutin 39 It was confirmed that μg / g was contained. Catechin, chlorogenic acid, caffeic acid, and p-coumaric acid, which are found in high amounts in SCM, are known as natural antioxidants.

< Experimental Example  4>

Determination of Whitening Activity of Bamboo Leaf Extract

<4-1> Mushroom  Tyrosinase Inhibitory Activity

Tyrosinase is an enzyme that contains copper and is an enzyme that plays a crucial role in melanin production. A melanin polymer is synthesized by acting as a DOPA oxidase, which oxidizes DOPA with tyrosine hydroxylase, which oxidizes tyrosine in melanosomes to produce DOPA. In this study, SCM was treated with each concentration (25, 50, 100 ㎍ / mL) to measure the tyrosinase inhibitory activity of bamboo leaf methanol extract (SCM). Ascorbic acid and catechin, known as tyrosinase inhibitors, were used as controls.

The Macrini method [Macrini, DJ, IB Suffredini, AD Varella, RN Younes, and MT Ohara (2009) Extracts from Amazonian plants have inhibitory activity against tyrosinase: An in vitro evaluation. Brazilian J. Pharm. Sci. 45: 715-721. Experimental method is 20 μL of bamboo leaf methanol extract of the present invention prepared with 220 μL of 0.1 M sodium phosphate buffer (pH 6.5) and 25-100 μg / mL in a test tube, L-tyrosine (Sigma-Aldrich, St. Louis, MO). , USA) 20 μL of mushroom tyrosinase (Sigma-Aldrich, St. Louis, MO, USA) prepared in 1.5 mM 40 μL, 1500 unit was added and reacted at 37 ° C. for 15 minutes. After the reaction, the amount of DOPAchrome formed from 3, 4-dihydroxyphenylalanine (DOPA) by tyrosinase was measured at 490 nm.

As a result, as shown in FIG. 3, the IC ₅₀ value of SCM tyrosinase inhibition was measured to be 164 μg / mL, and the IC ₅₀ values of ascorbic acid and catechin used as positive controls were 38 μg / mL and 97 μg / mL, respectively. Appeared. These results show lower inhibitory activity than ascorbic acid and catechin used as a positive control, but can be said to be very high inhibitory activity at the natural level.

Previous studies have shown that catechins are potent tyrosinase inhibitors and are effective at low concentrations of less than 100 μM. In another study, we investigated the tyrosinase inhibitory activity of bamboo bamboo shoots and fermented extracts. It was reported that there was more than 50% inhibition in the concentration range of mL. These results show that the tyrosinase inhibitory activity is different depending on the type of bamboo and the extraction method. The reason why the high tyrosinase inhibitory activity was confirmed in the present study compared to the previous experiment was included in the SCM. Various polyphenols such as catechin and chlorogenic acid are thought to strongly inhibit tyrosinase activity.

<4-2> Melanin content measurement

In this experiment, SCM was treated at each concentration in B16F10 melanoma cells for evaluation of whitening activity, and treated with α-MSH (1 μg / mL) for 48 hours, followed by measurement of melanin production.

Melanin content was determined by Hosoi's method [Hosoi, J., E. Abe, T. Suda, and T. Kuroki (1985) Regulation of melanin synthesis of B16 mouse melanoma cells by 1α, 25-dihydroxyvitamin D3 and retinoic acid. Cancer Res. 45: 1474-1478.], B16F10 cells were dispensed into 96 wells at a concentration of 1 × 10 ⁴ cells / mL and after 24 hours, α-MSH and SCM at 1000 μg / mL concentrations (25-100 μg / mL), and after 48 hours, melanin production was confirmed and cells were washed with PBS. Cells were harvested, centrifuged at 12,000 rpm for 5 minutes, 1N NaOH was added to the resulting pellet, left at 80 ° C for 1 hour to dissolve, and the absorbance was measured at 405 nm. The melanin content was compared with the absorbance of the untreated group, and arbutin was used as the positive control group.

As a result, as shown in Figure 4, the α-MSH (1 μg / mL) treated group increased melanin content to 95.69% compared to the vehicle control, SCM treated group in the α-MSH (1 μg / mL) treated group Compared with 25, 50, 100 ㎍ / mL concentration, melanin production was reduced to 30.8%, 69.6%, and 86.4%, and albumin, a positive control group, decreased melanin production by 59.2% at 100 μg / mL concentration.

In addition, as shown in Figure 5, when the SCM was treated with each concentration (25, 50 μg / mL) by observing the morphology it was confirmed that after 48 hours melanin formation is inhibited.

<4-3> Analysis of Protein Expression Related to Melanin Formation

In this experiment, we used α-MSH in B16F10 melanoma cells to evaluate the whitening activity and treated each concentration of SCM extract to determine the expression patterns of melanogenesis-related proteins (tyrosinase, TRP-1, TRP-2). Analyzed via blots.

Primary antibodies of tyrosinase, TRP-1, TRP2 and actin and secondary antibodies such as anti-goat, anti-rabbit and antimouse were purchased from Santa Cruz Biotechnology (CA, USA). After extracting the cell lysate from the treated cells, the protein concentration was determined by Bradford assay, and 50 ㎍ of the protein was electrophoresed with 10% sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) and blotting onto the nitrocellulose membrane. Hybridization with primary antibody diluted to 1000. After washing with Membrane, it was reacted with a secondary antibody (1: 1000) to which horse radish peroxidase was attached for 1 hour, and protein expression was analyzed by using a chemiluminescence detection system (FluoChem® FC2, AlphaInnotech, USA).

As a result, as shown in FIG. 6, SCM was treated at 25, 50, and 100 μg / mL for 48 hours in a cell line induced with pigmentation with α-MSH. As a result, in tyrosinase, TRP-1, and TRP-2, It showed a high protein expression inhibitory effect and it was confirmed that the expression is suppressed in a concentration-dependent manner.

< Experimental Example  5>

Cytotoxicity Evaluation of Bamboo Leaf Extract

After dispensing B16F10 cell suspension into 6 wells at a concentration of 1 × 10 ⁴ cells / mL, 200 μL was stabilized in the incubator for 24 hours, and then the samples were treated by concentration (25, 50, 100 μg / mL) for 24 hours. Incubated for Choi's method [Choi, MH, HK Han, YJ Lee, HG Jo, and HJ Shin (2014). In vitro anti-cancer activity of hydrophobic fractions of Sparassis latifolia extract using AGS, A529, and HepG2 cell lines. J. Mushrooms 12: 304-310.], Add 2.5 μg / mL MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) solution at 100 μL per well for 1 hour. Leave in incubator. After removing the supernatant, 200 μL of DMSO was added per well, shaking for 1 minute to completely dissolve formazan, and the absorbance was measured at 540 nm using ELISA Bio-Tek instrument Inc (Winooski, VT, USA). MTT and DMSO used in this experiment were purchased from Sigma-Aldrich (St. Louis, MO, USA).

As a result, as shown in Figure 7, the cytotoxicity did not appear after 24 hours in the whole concentration tested. These results are similar to the previous studies, which did not show cytotoxicity at concentrations below 200 μg / mL when treated with bamboo blind sap. Cosmetic ingredients include chemicals, synthetic or natural products, and in some cases, used alone or in the form of a mixture. In order to secure the safety of the final product, the safety of the ingredients must be secured first. Based on the results of this experiment, the bamboo leaf extract SCM of the present invention is judged to be a relatively safe ingredient as a cosmetic raw material, and it is necessary to confirm the safety through human-derived cells and additional clinical experiments later.

< Example  2>

Medicinal Plant Extract Preparation

<2-1> diary extract preparation

Hirley purchases curry grown in Naju, Jeollanam-do, and removes impurities by rinsing three times in running water, and then dry the curry ground part (except roots as a whole) at 35 ° C. for 30 hours to completely clean the hungry. Dried. A dried sample was prepared for the dried curry ground portion. The extract obtained by adding 80% ethanol in a ratio of 1: 5 (w / v) to 1 kg of sample hirley, and refluxed three times at room temperature for 48 hours after mixing was mixed with No. 2 (Advantec TOYO, Japan). After filtration and concentrated under reduced pressure with a rotary vacuum evaporator (Heidolph VV2011, Switzerland) at 50 ℃ and lyophilized to obtain a hydroxy ethanol extract, stored at -20 ℃ was used as a sample of the experimental example.

<2-2> Chili extract preparation

Physalis used in the present invention alkekengi var. Francheti (Mast.) Hort was used to cut the ground parts (except the roots as a whole) and then wash and cut the test materials. The extract obtained by adding 80% ethanol at a ratio of 1: 5 (w / v) to 1 kg of the sample, 3 times under reflux for 48 hours after mixing at room temperature was analyzed as No. 2 (Advantec TOYO, Japan). After filtration and concentrated under reduced pressure with a rotary vacuum evaporator (Heidolph VV2011, Switzerland) at 50 ° C., lyophilized to obtain a brown chrysali ethanol extract, stored at -20 ℃ was used as a sample of the following experimental example .

<2-3> Word of mouth  Extract manufacturer

The algae were collected from the ground (except the roots), washed, and dried in the native area of Naju, Jeollanam-do. The extract obtained by adding 80% ethanol at a ratio of 1: 5 (w / v) to 1 kg of dry bark and 3 times reflux extraction at room temperature for 48 hours after mixing was obtained as No. 2 (Advantec TOYO, Japan After filtration was carried out under reduced pressure with a rotary vacuum evaporator (Heidolph VV2011, Switzerland) at 50 ° C. and freeze-dried to obtain the ethanol extract of bark, which was stored at -20 ° C. and used as a sample of the following experimental example. .

< Experimental Example  6>

Determination of Whitening Activity by Combined Treatment with Bamboo Leaf Extract and Medicinal Plant Extract

In this experiment, the whitening activity of the bamboo leaf methanol extract (SCM) of the present invention prepared in Example 1 as a medicinal plant extract was combined with each of the hydroxy extract, the chylo extract, and the algae extract to enhance the whitening activity compared to the SCM alone treatment group. We looked at whether or not.

The melanin content was measured to measure the whitening activity, and the melanin content was measured by a method similar to that of Experimental Example <4-2>. Briefly, α-MSH (1 μg / mL) was induced in B16F10 melanoma cells to induce melanin formation, wherein the bamboo leaf methanol extract (SCM) of the present invention was prepared through 25 μg / mL and Example 2 above. Melamine production was measured after 48 hours of incubation with 25 ㎍ / mL concentration of each of the extracted hyacinth extract, Echiuli extract, and beetle extract.

As a result, as shown in Table 4 and FIG. 8, the melanin production was significantly reduced compared to the bamboo leaf methanol extract (SCM) alone treatment group of the present invention in the experimental group treated with the extract of worms as a medicinal plant extract. there was. On the other hand, in the experimental group of the combination treatment of the hydroxy extract, Chorley extract showed melanin production almost similar or more increased than the bamboo leaf methanol extract (SCM) alone treatment group of the present invention.

Through the results as described above, it was confirmed that the combined use of the algae extract with bamboo leaf methanol extract (SCM) of the present invention may have a synergistic effect on the whitening activity. In particular, the bamboo leaf methanol extract (SCM) of the present invention, even at a concentration of 25 ㎍ / mL derived a whitening activity more excellent than the high concentration of 100 ㎍ / mL.

Melanin Content by Treatment Concentration (㎍ / mL) Activity (%) Standard Deviation CON 100 3.5 a-MSH 1 226 12.5 SCM 25 156 8.8 SCM 25 + Here 25 158 7.7 SCM 25 + Lee 25 165 5.2 SCM 25 + Word 25 102 7.5 Arbutin 100 136 10.8

< Experimental Example  7>

Cytotoxicity Assessment of Medicinal Plant Extracts

In this experiment, the cytotoxicity of the medicinal plant extracts of the present invention prepared by the above Example 2 (Hirary extract, Echo extract, Beetle extract) was evaluated. Cytotoxicity evaluation was measured in the same manner as in Experimental Example 5.

As a result, as shown in Table 5 and Figure 9, the medicinal plant extract of the present invention prepared by Example 2 (Hyerry extract, Echo extract, algae extract) is confirmed to have no cytotoxicity up to 100㎍ / mL It became. Therefore, the medicinal plant extract of the present invention was determined to be a relatively safe ingredient as a cosmetic raw material.

Cytotoxicity Assessment of Medicinal Plant Extracts of the Present Invention Concentration (㎍ / mL) Activity (%) Standard Deviation CON 100 3.5 Diary 25 101.4 8.2 Diary 50 104.3 8.8 Diary 100 103.9 7.7 Chorley 25 100.2 8.2 Chorley 50 102.8 8.8 Chorley 100 98.5 7.7 Beetle 25 102.2 8.2 Beetle 50 101.8 8.8 Beetle 100 105.4 2.5

So far I looked at the center of the preferred embodiment for the present invention. Those skilled in the art will appreciate that the present invention can be implemented in a modified form without departing from the essential features of the present invention. Therefore, the disclosed embodiments should be considered in descriptive sense only and not for purposes of limitation. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the scope will be construed as being included in the present invention.

SCM: Bamboo leaf methanol extract of the present invention

Claims (5)

Cosmetic composition for skin whitening comprising bamboo leaf extract as an active ingredient. The method of claim 1,
The bamboo is a cosmetic composition, characterized in that the Sindae. The method of claim 1,
The extract is a cosmetic composition, characterized in that extracted with one or more solvents selected from the group consisting of lower alcohols, ethyl acetate, acetone, water and hexane having 1 to 4 carbon atoms. The method of claim 1,
The composition is a cosmetic composition, characterized in that it further comprises a extract extract. The method according to any one of claims 1 to 4,
The cosmetic composition includes a flexible lotion, gel, water soluble liquid, milk lotion, nourishing cream, massage cream, essence, oil-in-water emulsion, water-in-oil emulsion, flake anhydrous product, solid anhydrous product, oil in aqueous phase using globules. Dispersions, ionic lipid vesicles, nonionic lipid vesicles, cleansing foams, cleansing water, packs, body oils, oil-in-water makeup bases, water-in-oil makeup bases, foundations, skin covers, lipsticks, lip gloss, face powders, two-way Cosmetic composition, characterized in that the one type of formulation selected from the group consisting of cake, eye shadow, mascara, cheek color and eyebrow pencils.

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