KR20190050627A - Fermented Extract of Ginseng Leaf for Skin Whitening and manufacturing method thereof - Google Patents

Abstract

The present invention relates to a fermented extract of ginseng leaves for skin whitening and a manufacturing method thereof. More specifically, the present invention relates to a fermented extract of ginseng leaves for skin whitening which is capable of obtaining a high yield of ginsenoside Rk1, Rk2, and Rh3, and exhibits an excellent whitening effect without cytotoxicity, a manufacturing method thereof, a method for manufacturing a fermented extract fractions of ginseng leaves thereby, and a cosmetic composition containing the same.

Description

FIELD OF THE INVENTION The present invention relates to a fermented extract of ginseng leaf for skin whitening,

The present invention relates to a ginseng leaf fermentation extract for skin whitening and a method for producing the same, and more particularly, to a ginseng leaf fermentation extract for skin whitening, which is capable of obtaining a high gain of ginsenosides Rk1, Rk2 and Rh3 and exhibits an excellent whitening effect The present invention relates to a method for producing a ginseng leaf fermented extract for skin whitening, a method for producing the same, a method for producing the extract fraction of a ginseng leaf, and a cosmetic composition containing the same.

Recently, interest in skin whitening is increasing rapidly. As the preference for skin whitening cosmetics has been extended to four seasons in summer, various whitening cosmetics such as creams, lotions, and oils have been used to protect the skin and maintain a healthy condition.

The skin color of a person is determined by the amount of melanin, carotene and hemoglobin, and melanin is the most crucial factor. Melanin is synthesized in melanocytes, which are pigment cells existing in the epidermal basal layer. Various enzymes are present in melanocytes, and they work together to oxidize a specific amino acid, which is always present in the living body, Of melanin. The melanin thus formed is transferred to the keratinocyte through the dendrites of the melanocyte to exhibit the skin color and disappear with the keratinization along with the keratinization of the epidermis.

On the other hand, melanin forms a hat-like structure around the nucleus, protects the gene from ultraviolet rays, removes free radicals, protects intracellular proteins, and absorbs or scatters ultraviolet rays from the skin. Of cells or tissues are prevented from being damaged by ultraviolet rays. However, melanin is abnormally overproduced due to external environmental changes such as exposure to excessive ultraviolet rays, air pollution, stress, or the like, and melanin is completely eliminated If pigmentation occurs, it causes blackening (blackening) of the skin, stain, and freckles.

Therefore, it is urgently required to study a substance capable of obtaining a high gain of ginsenosides Rk1, Rk2 and Rh3 that can exhibit whitening effect, and exhibiting an excellent whitening effect without cytotoxicity.

KR 10-2009-0032501 A

Disclosure of the Invention The present invention has been conceived to solve the above-mentioned problems, and it is an object of the present invention to provide a skin-whitening ginseng leaf fermentation extract for skin whitening which is capable of high gain of ginsenosides Rk1, Rk2 and Rh3, A method for producing the same, a method for producing the extract fraction of the ginseng leaves by fermentation, and a cosmetic composition containing the same.

In order to solve the above-mentioned problems, the present invention provides a method for producing ginseng leaf extract, comprising: (1) preparing a ginseng leaf extract concentrate; And (2) fermenting the ginseng leaf extract concentrate with Lactobacillus pentosus ATCC8041 to produce a ginseng leaf fermentation extract. The present invention also provides a method for producing a ginseng leaf fermentation extract for skin whitening.

According to an embodiment of the present invention, the step (1) comprises: (1) grinding the ginseng leaf; (1) -2 Mixing the pulverized ginseng leaves with an organic solvent to a concentration of 0.5-2 g / ml in the mixture, and repeating the extraction twice or five times for 6-18 hours; And (3) extracting the extracted ginseng leaf at a temperature of 50 ° C or less and concentrating the extract under reduced pressure and freeze-drying to prepare a ginseng leaf extract concentrate.

In addition, the organic solvent may be 70 to 95% methanol.

The fermentation can be carried out for 8 to 12 days at a temperature of 30 to 44 ° C and a pH of 4.5 to 7.5.

In the step (2), 0.5 to 5 parts by weight of carbon source is mixed with 100 parts by weight of (2) -1 ginseng leaf extract concentrate and sterilized; (2) -2: Lactobacillus pentosus is added to the sterilized ginseng leaf extract concentrate in an amount of 300 to 700 parts by weight based on 100 parts by weight of the ginseng leaf extract concentrate and cultured to produce a fermented product; (2) -3 removing lactobacillus pentosus from the fermentation product and sterilizing it; (2) -4 reflux extraction and precipitation of the sterilized fermentation product and filtration to prepare a filtrate; And (2) -5. The filtrate is concentrated under reduced pressure and freeze-dried at 45 ° C or below to prepare a ginseng leaf fermentation extract.

The present invention also provides a fermented ginseng leaf extract for skin whitening, which is produced by the above-described production method.

According to one embodiment of the present invention, the ginseng leaf fermentation extract may include ginsenoside Rk1, ginsenoside Rk2 and ginsenoside Rh3, The increase rate of ginsenoside Rk2 according to the following formula 2 may be 50,000% or more, and the increase rate of ginsenoside Rh3 according to the following formula 3 may be 45,000% or more.

[Relation 1]

[Relation 2]

[Relation 3]

At this time, fermented ginseng leaves and ginseng leaves before fermentation are ginseng leaves of 6-year-old ginseng.

Meanwhile, the present invention provides a cosmetic composition for skin whitening comprising the ginseng leaf fermentation extract as an active ingredient and satisfying all of the following conditions (a) to (c).

(a) When the concentration of fermented ginseng leaf extract is 35 to 65 μM, the melanin inhibition rate is 8% or more

(b) Melanin inhibition rate of fermented ginseng leaf extract at a concentration of 80 to 120 μM of 12% or more

(c) The melanin inhibition rate is 21% or more when the concentration of fermented ginseng leaf extract is 180 ~ 220 μM.

The present invention also relates to a method for preparing a ginseng leaf fermented extract, which comprises fractionating the ginseng leaf fermentation extract prepared by the above-mentioned production method to prepare a fraction of the ginseng leaf fermentation extract; And separating the active ingredient of the cosmetic composition for skin whitening from the fraction of the ginseng leaf fermentation extract.

According to an embodiment of the present invention, the fraction of the ginseng leaf fermentation extract may be ethyl acetate fraction.

The separation may be carried out by any one or more methods selected from the group consisting of silica gel column chromatography and reversed phase silica gel column chromatography.

The present invention also provides a cosmetic composition for skin whitening comprising an extract fraction of fermented ginseng leaf for skin whitening, which is separated by the above-described production method, as an active ingredient.

According to an embodiment of the present invention, the ginseng leaf fermentation extract fraction includes at least one selected from the group consisting of ginsenoside Rk1, ginsenoside Rk2 and ginsenoside Rh3, , The concentration of ginsenoside Rk1 is 25-100 μM, the concentration of ginsenoside Rk2 is 15-65 μM, and the concentration of ginsenoside Rh3 is 50-100 μM.

The ginseng leaf fermentation extract for skin whitening of the present invention, the method for producing the same, the method for producing the extract fraction of the ginseng leaf fermented by the method and the cosmetic composition containing the same can provide a high gain of ginsenosides Rk1, Rk2 and Rh3, At the same time, the whitening effect is remarkably excellent.

Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings. The present invention may be embodied in many different forms and is not limited to the embodiments described herein.

The method for manufacturing a skin-whitening ginseng leaf fermentation extract according to one embodiment of the present invention comprises the steps of: (1) preparing a ginseng leaf extract concentrate; And (2) fermenting the ginseng leaf extract concentrate with Lactobacillus pentosus ATCC8041 to prepare a ginseng leaf fermentation extract.

First, the step (1) of producing a ginseng leaf extract concentrate will be described.

The step (1) comprises: (1) pulverizing the ginseng leaves; (1) -2 mixing the ginseng leaves with the organic solvent to a concentration of 0.5-2 g / ml in the mixture, 5) repeatedly extracting ginseng leaf extract and (3) -3 extracting the ginseng leaf at a temperature of 50 ° C or lower under reduced pressure and freeze-drying to prepare a ginseng leaf extract concentrate.

The step (1) -1 is a step of pulverizing ginseng leaves, and any method that can be used in the art can be used without limitation. Preferably, the ginseng leaves are washed with water and dried, Preferably 3 to 5 cm, and then pulverized.

The step (1) -2 is a step of repeatedly extracting the mixed liquor containing the ground ginseng leaves and the organic solvent. The pulverized ginseng leaves are added to the mixture to a concentration of 0.5-2 g / ml, preferably 0.7-1.5 g / Ml, followed by repeated extraction 2 to 4 times for 6 to 18 hours at 2 to 5 times, preferably 9 to 15 hours. If the pulverized ginseng leaves are less than 0.5 g / ml in the mixed solution, the yield of ginseng leaf extract concentrate described later may be poor. In addition, if the condition of the repeated extraction is not satisfied, a concentrated ginseng leaf extract concentrate of the desired level may not be produced.

On the other hand, the organic solvent is not limited as long as it can be used as an extraction solvent. Preferably, one or more organic solvents selected from the group consisting of methanol, ethanol, fermentation ethanol and ethyl acetate can be used , And more preferably methanol. When methanol is used as the organic solvent, the organic solvent may be 70 to 95% methanol, and preferably 75 to 90% methanol. If the concentration of methanol is out of the above range, the high gain of the extracted ginsenosides Rk1, Rk2 and Rh3 may not be easy, or the whitening effect of the extract or fraction may be deteriorated.

The conditions for the vacuum concentration in the above (1) to (3) can be used without limitation as long as they can be used under normal conditions of reduced pressure, and the freeze-drying conditions in the above (1) The present invention is not particularly limited thereto.

The method may further include filtering the extracted ginseng leaves between the step (1) -2 and the step (1) -3 described below, but the present invention is not limited thereto.

The step (1) -3 is a step of producing a ginseng leaf extract concentrate, wherein the ginseng leaf extracted in step (1) -2 is concentrated under reduced pressure and freeze-dried To thereby produce a ginseng leaf extract concentrate. If the temperature of the reduced-pressure concentration exceeds 50 캜, there may arise a problem that the concentrated ginseng leaf extract concentrate can not be produced at a desired level.

Next, the step (2) of fermenting the ginseng leaf extract concentrate with Lactobacillus pentosus ATCC8041 to produce a ginseng leaf fermentation extract will be described.

The step (2) comprises the steps of (2) mixing and sterilizing a carbon source to the ginseng leaf extract concentrate, (2) -2 adding lactobacillus pentosus to the sterilized ginseng leaf extract concentrate, (2) -3 removing lactobacillus pentosus from the fermentation product and sterilizing the product, (2) -4 preparing a filtrate by reflux extraction and precipitation of the sterilized fermentation product and filtration, and (2) -5 The filtrate may be concentrated under reduced pressure at 45 ° C or below and lyophilized to prepare a ginseng leaf fermentation extract.

The step (2) -1 is a step of mixing and sterilizing a carbon source to a ginseng leaf extract concentrate. The step (2) - 1 is a step of mixing 0.5 to 5 parts by weight of carbon source, preferably 0.5 to 3 parts by weight of carbon source, per 100 parts by weight of the ginseng leaf extract concentrate And sterilized. If the carbon source is less than 0.5 part by weight with respect to 100 parts by weight of the ginseng leaf extract concentrate, it is difficult to cultivate the lactobacillus pentosus, which will be described later, so that the high gain of the ginsenosides Rk1, Rk2 and Rh3 may not be easy And when the carbon source is more than 5 parts by weight, the polysaccharide and oligosaccharide are converted and the targeted ginsenosides Rk1, Rk2 and Rh3 contents may be lowered.

The step (2) -2 is a step of adding and culturing Lactobacillus pentosus to produce a fermented product. The fermentation product is added in an amount of 300 to 700 parts by weight, preferably 400 to 600 parts by weight per 100 parts by weight of the ginseng leaf extract concentrate Lactobacillus pentosus can be added to the mixture in the weight portion and cultured to produce a fermented product. If the amount of Lactobacillus pentosus is less than 300 parts by weight based on 100 parts by weight of the ginseng leaf extract concentrate, fermentation of the ginseng leaf extract concentrate is not easy, so that the high gain of ginsenosides Rk1, Rk2 and Rh3 may not be easy If the amount is more than 700 parts by weight, the lactobacillus pentosus may not be completely removed through the sterilization step to be described later, and the remaining lactobacillus pentosus may remain. Therefore, the culture may be continued, , The content of the desired ginsenosides Rk1, Rk2 and Rh3 may be lowered.

On the other hand, the culturing can be carried out for 8 to 12 days at a temperature of 30 to 44 ° C and a pH of 4.5 to 7.5, preferably 9 to 11 days at a temperature of 32 to 42 ° C and a pH of 5 to 7 have. If the culture conditions are not satisfied, the ginsenoside Rk1, Rk2 and Rh3 may not have a high yield because the fermentation of the ginseng leaf extract concentrate is not easy.

The step (2) -3 is a step of removing lactobacillus pentosus from the fermentation product and sterilizing the product. The removal can be carried out without limitation as long as the bacteria can be removed normally, The bacteria that have been cultured through the separation can be removed. The sterilization can be carried out without limitation as long as the sterilization can be carried out under the condition that sterilization of the cultured bacteria is usually carried out. The sterilization is preferably carried out at 100 to 140 ° C and 0.7 to 2.5 atm for 10 to 50 minutes, more preferably 110 to 130 ° C And at 1 to 2 atmospheres for 15 to 45 minutes. If the sterilization condition is not satisfied, the lactobacillus pentosus may not be completely removed through the sterilization step, and the remaining lactobacillus pentosus may remain, resulting in the continued cultivation, or the ginsenosides Rk1, Rk2 And the yield may be lowered in the course of obtaining Rh3.

The step (2) -4 is a step of refluxing and precipitating the sterilized fermented product, and filtering to produce a filtrate. The reflux extraction can be used without limitation as long as it is capable of performing reflux extraction Therefore, the present invention is not limited thereto. For example, the reflux extraction may be performed by repeating extraction for 2 to 5 times for 6 to 18 hours, preferably for 2 to 4 times for 9 to 15 hours, but the present invention is not limited thereto. Meanwhile, in order to carry out the reflux extraction, the sterilized fermentation product may be mixed with an organic solvent and subjected to reflux extraction. The organic solvent may be any organic solvent that can be used without any limitation, , More preferably 70 to 85% methanol, and more preferably 75 to 85% methanol can be used. In addition, since the filtration can be performed through a conventional filtration method, the present invention is not limited thereto.

The step (2) -5 is a step of preparing the ginseng leaf fermentation extract by concentrating the filtrate under reduced pressure and freeze-drying. The filtrate is concentrated under reduced pressure at a temperature of 45 ° C. or lower, preferably 40 ° C. or lower, And then freeze-dried to prepare a ginseng leaf fermentation extract. If the temperature of the reduced-pressure concentration exceeds 45 ° C, there may arise a problem that the fermented ginseng leaf extract concentrated to the desired level can not be produced.

The present invention also provides a fermented ginseng leaf extract for skin whitening, which is produced by the above-described production method.

Wherein the ginseng leaf fermentation extract comprises ginsenoside Rk1, ginsenoside Rk2 and ginsenoside Rh3, wherein the increase rate of ginsenoside Rk1 according to the following formula 1 is 15,000% or more , The increase rate of ginsenoside Rk2 according to the following formula 2 may be 50,000% or more, preferably 51,000% or more, the increase rate of ginsenoside Rh3 according to the following formula 3 is 45,000% or more Can be more than 46,000%.

[Relation 1]

[Relation 2]

[Relation 3]

At this time, fermented ginseng leaves and ginseng leaves before fermentation are ginseng leaves of 6-year-old ginseng.

The present invention also provides a cosmetic composition for skin whitening comprising the above-described ginseng leaf fermentation extract as an active ingredient and satisfying all of the following conditions (a) to (c).

The condition (a) may be that the melanin inhibition rate is 8% or more, preferably 9% or more when the concentration of the fermented ginseng leaf extract is 35 to 65 μM, and the concentration of the fermented ginseng leaf extract is 80 to 120 μM , The melanin inhibition rate may be not less than 12%, preferably not less than 13% when the concentration of the fermented ginseng leaf extract is from 180 to 220 μM as Condition (c) have. By satisfying the above range, a high amount of melanin inhibition can be exhibited even in a small amount, and at the same time, no cytotoxicity can be exhibited.

The present invention also relates to a method for preparing a ginseng leaf fermented extract, which comprises fractionating the ginseng leaf fermentation extract prepared by the above-mentioned production method to prepare a fraction of the ginseng leaf fermentation extract; And separating the active ingredient of the cosmetic composition for skin whitening from the fraction of the ginseng leaf fermentation extract.

First, the step of preparing the fraction of the ginseng leaf fermentation extract by fractionating the ginseng leaf fermentation extract prepared by the above-mentioned production method will be explained.

This fraction was obtained by dividing and extracting the ginseng leaf fermentation extract using ethyl acetate (EtOAc) / water (H ₂ O) and then extracting the water layer by 2 to 4 times with butanol (n-BuOH) Layer, the water layer and the butanol layer were respectively concentrated under reduced pressure to obtain n-butanol fraction, H ₂ O fraction and ethyl acetate fraction.

On the other hand, by carrying out the fraction, an n-butanol fraction, an H ₂ O fraction and an ethyl acetate fraction can be obtained. By performing the separation step described below, the ginsenosides Rk1, Rk2 and Rh3 , The n-butanol fraction obtained through the fraction, the H ₂ O fraction and the ethyl acetate fraction in the ethyl acetate fraction can be used as fractions.

Next, the step of separating the active ingredient of the skin whitening cosmetic composition from the fraction of the ginseng leaf fermentation extract will be described.

The separation can be carried out by any one of ordinary separation methods, and preferably, it can be carried out by any one or more methods selected from the group consisting of silica gel column chromatography and reverse phase silica gel column chromatography, more preferably silica gel column chromatography And reversed phase silica gel column chromatography can be carried out by a series of processes.

In addition, the method may further include a step of purifying the separated separated product after the separating step, but the present invention is not limited thereto.

The present invention also provides a cosmetic composition for skin whitening comprising an extract fraction of fermented ginseng leaf for skin whitening, which is separated by the above-described production method, as an active ingredient.

Wherein the ginseng leaf fermentation extracted fraction comprises any one or more selected from the group consisting of ginsenoside Rk1, ginsenoside Rk2 and ginsenoside Rh3, wherein the ginsenoside Rk1 has a concentration of 25 The concentration of ginsenoside Rk2 is preferably in the range of 15 to 65 μM and preferably in the range of 15 to 55 μM and the concentration of ginsenoside Rh3 is preferably in the range of 50 to 100 μM and preferably in the range of 60 to 100 μM, Lt; / RTI > Since the ginsenoside Rk1, ginsenoside Rk2 and ginsenoside Rh3 are included as active ingredients to satisfy the above concentration range, they are free from cytotoxicity and have an excellent whitening effect. .

Meanwhile, the ginseng leaf fermentation extract for skin whitening according to the present invention, the process for producing the same, the process for producing the extract fraction of ginseng leaf fermented by the same, and the cosmetic composition containing the same can provide high yields of ginsenosides Rk1, Rk2 and Rh3, It is possible to exhibit an excellent effect of whitening activity without toxicity.

The present invention will now be described more specifically with reference to the following examples. However, the following examples should not be construed as limiting the scope of the present invention, and should be construed to facilitate understanding of the present invention.

[Example]

Example 1: Preparation of ginseng leaf fermented extract

(1) Preparation of Ginseng Leaf Extract Concentrate

The ginseng leaves of 6-year-old ginseng were harvested, washed and dried, and cut into 4 cm lengths and 4 cm lengths, respectively. Then, the ground ginseng leaves were mixed with 80% methanol at a concentration of 1 g / ml in the mixed solution, repeatedly extracted three times for 12 hours, and then filtered with a filter paper. Thereafter, the extracted ginseng leaves were concentrated under reduced pressure at a temperature of 45 캜 at a pressure of 45 psi for 1.5 hours and lyophilized at a temperature of -80 캜 for 24 hours to prepare a ginseng leaf extract concentrate.

(2) Preparation of ginseng leaf fermented extract

1.5 parts by weight of carbon source was mixed with 100 parts by weight of the obtained ginseng leaf extract concentrate and sterilized at a temperature of 120 ° C for 1 hour. 100 parts by weight of the ginseng leaf extract concentrate was mixed with 100 parts by weight of Lactobacillus pentosus , ATCC8041) was added in an amount of 500 parts by weight and cultured for 10 days at 37 ° C and pH 6 to prepare a fermented product. Then, the fermented product was centrifuged to remove the culture, sterilized at 120 ° C and 1.5 atm for 30 minutes, and the sterilized fermented product was mixed with 80% methanol to make 100 g / 1000 ml in the mixed solution. The mixture was refluxed three times each time, precipitated, and filtered through a filter paper to prepare a filtrate. The filtrate thus obtained was concentrated under reduced pressure at a temperature of 40 ° C and a pressure of 45 psi for 1.5 hours and lyophilized at a temperature of -80 ° C for 24 hours to prepare a ginseng leaf fermentation extract.

Examples 2 to 11 and Comparative Examples 1 to 3

The same procedure as in Example 1 was carried out except that the concentration of the organic solvent mixed with the crushed leaf blade, the type and content of the culture broth, the content of the carbon source, and the fermentation were examined. .

<Experimental Example 1>

The following Examples 1 to 11 and Comparative Examples 1 to 3 were measured / evaluated for the following items and are shown in Tables 1 and 2 below.

1. Peak area measurement of ginsenosides Rk1, Rk2 and Rh3

The ginseng leaf fermentation extracts prepared according to Examples 1 to 11 and Comparative Examples 1 to 3 were subjected to high-resolution liquid chromatography / mass spectrometry (UPLC-QTOF / MS, Waters) using ginsenosides Rk1, Rk2 and Rh3 Was measured.

2. Evaluation of melanin inhibitory efficacy

The melanin inhibitory effects of the ginseng leaf fermentation extracts prepared according to Examples 1 to 11 and Comparative Examples 1 to 3 were evaluated.

Specifically, Melan-a cells derived from C57BL / 6J mice were cultured from Dorothy C Bennett (St George's Hospital Medical School, London, UK). Cultures were incubated with 10% fetal bovine serum (FBS) 100 mg / mL of streptomycin-penicillin (penicillin-streptomycin) and 200 nM of TPA (12- ο -tetradecanoylphorbol-13- acetate) with 37 ℃ using RPMI-1640 medium containing, in the 5% CO ₂ environment Cell lines were cultured and used.

Subsequently, 1 x 10 ⁵ of Melan-a cells were added to each well of a 24-well plate and cultured overnight. Each sample was treated with a 200 μM concentration to culture for 4 days , Washed with phosphate-buffered saline and treated with 0.85N KOH in 250 L of each well. The dissolved cells were transferred to a 96-well plate and the optical density was measured at 405 nm using a microplate reader (3550, Bio-Rad). The rate of inhibition of melanin production was measured using a control (%) Compared to the control group.

division Example 1 Example 2 Example 3 Example 4 Example 5 Example 6 Example 7 Type / concentration (%) of organic solvent mixed with crushed leaf blade 80 60 75 90 97 80 80 Culture type /
Content (parts by weight) Lactobacillus pentosus / 500 Lactobacillus pentosus / 500 Lactobacillus pentosus / 500 Lactobacillus pentosus / 500 Lactobacillus pentosus / 500 Lactobacillus pentosus / 200 Lactobacillus pentosus / 400 Carbon source content (parts by weight) 1.5 1.5 1.5 1.5 1.5 1.5 1.5 Peak area of Rk1 713,667 495,372 681,401 672,139 488,130 391,506 690,166 Peak area of Rk2 125,950 103,445 121,195 122,988 106,552 84,078 124,149 Peak area of Rk3 148,669 124,856 141,259 140,884 122,540 91,210 142,982 Melanin inhibition (%) 30 19 29 28 19 19 29

division Example 8 Example 9 Example
10 Example
11 Comparative Example
1 ¹⁾ Comparative Example 2 Comparative Example 3 Type / concentration (%) of organic solvent mixed with crushed leaf blade 80 80 80 80 80 80 80 Type / content (parts by weight) Lactobacillus pentosus / 600 Lactobacillus pentosus / 800 Lactobacillus pentosus / 500 Lactobacillus pentosus / 500 - Lactobacillus Lymphosus / 500 Lactobacillus Plateau Room / 500 Carbon source content (parts by weight) 1.5 1.5 0.1 One - 1.5 1.5 Peak area of Rk1 686,548 388,072 446,186 692,198 3841 223,110 322,121 Peak area of Rk2 128,565 79,603 98,052 121,937 234.6 20,242 45,457 Peak area of Rk3 141,326 83,253 112,793 143,360 305.3 35,362 25,450 Melanin inhibition (%) 28 19 20 29 13 8 12 1) The above Comparative Example 1 represents an unfermented ginseng leaf extract.

As can be seen from Tables 1 and 2 above,

Examples 1, 3, 4, 7, 8, and 11, which satisfied all of the concentration of the organic solvent mixed with the crushed leaf blade of the present invention, the kind and content of the cultured strain, the content of the carbon source, The peak areas of ginsenosides Rk1, Rk2 and Rh3 were superior to those of Examples 2, 5, 6, 9, 10 and Comparative Examples 1 to 3, in which one of them was missing, and the melanin inhibition rate was excellent.

Production Example 1: Preparation of cosmetic composition for skin whitening

The ginseng leaf fermentation extract prepared according to Example 1 was mixed in the following amounts to prepare a skin whitening cosmetic composition.

Ginseng leaf fermentation extract 100mg / 10ml

4 ml of glycerin monostearate

4.5 ml of cetearyl alcohol

Stearic acid 3 ml

Wax 1.5 ml

Glycerin 3 ml

Squalane 8 ml

Betaine 6 ml

0.5 ml of sodium hypaloronate

Hardened vegetable oil 2 ml

Polysorbate 60 3 ml

Mix to make 100 ml of distilled water.

Production Examples 2 to 11 Comparative Production Examples 1 to 3

The ginseng leaf fermentation extract prepared in the same manner as in Preparation Example 1 except that the ginseng leaf fermentation extract prepared in Example 1 was changed to the ginseng leaf fermentation extract prepared in Examples 2 to 11 and Comparative Examples 1 to 3, And skin cosmetic compositions for skin whitening were prepared as shown in Table 4.

<Experimental Example 2>

The skin whitening cosmetic compositions prepared according to Preparation Examples 1 to 11 and Comparative Preparation Examples 1 to 3 were measured / evaluated for the following items and shown in Tables 3 to 5 below.

1. Melanin inhibition rate by concentration

The skin-whitening cosmetic compositions prepared according to Preparation Examples 1 to 11 and Comparative Preparation Examples 1 to 3 were measured for melanin inhibition rates at concentrations of 50 μM, 100 μM and 200 μM of ginseng leaf fermentation extract.

Specifically, Melan-a cells derived from C57BL / 6J mice were cultured from Dorothy C Bennett (St George's Hospital Medical School, London, UK). Cultures were incubated with 10% fetal bovine serum (FBS) 100 mg / mL of streptomycin-penicillin (penicillin-streptomycin) and 200 nM of TPA (12- ο -tetradecanoylphorbol-13- acetate) with 37 ℃ using RPMI-1640 medium containing, in the 5% CO ₂ environment Cell lines were cultured and used.

Subsequently, 1 × 10 ⁵ Melan-a cells were added to each well of a 24-well plate and cultured overnight. The concentration of the ginseng leaf fermentation extract was 50 μM, 100 μM And 200 μM, cultured for 4 days, washed with phosphate-buffered saline and treated with 250 L of 0.85N KOH in each well. The dissolved cells were transferred to a 96-well plate and the optical density was measured at 405 nm using a microplate reader (3550, Bio-Rad). The rate of inhibition of melanin production was measured using a control (%) Compared to the control group.

2. Assessment of cytotoxicity

The cytotoxicity of the skin-whitening cosmetic composition prepared according to Preparation Examples 1 to 11 and Comparative Preparation Examples 1 to 3 was evaluated when the concentration of the ginseng leaf fermentation extract was 200 μM.

Specifically, the measurement was carried out in the same manner as the melanin inhibition assay by the concentration, and the cell viability was measured as a ratio of the cell survival to the control (non-treated group) according to the following equation. .

[Mathematical Expression]

Cell viability (%) = [(compound treated cell number OD value - blank well OD value) / (control OD value - blank well OD value)] 100 (%)

division Production Example 1 Production Example 2 Production Example 3 Production Example 4 Production Example 5 Ginseng Leaf Fermented Extract Type Example 1 Example 2 Example 3 Example 4 Example 5 Melanin inhibition rate according to concentration of fermented ginseng leaf extract (%) 50 μM 12 7 11 10 7 100 μM 16 11 14 14 10 200 [mu] M 30 19 29 28 19 Cytotoxicity none none none none none

division Production Example 6 Production Example 7 Production Example 8 Production Example 9 Production Example 10 Ginseng Leaf Fermented Extract Type Example 6 Example 7 Example 8 Example 9 Example 10 Melanin inhibition rate according to concentration of fermented ginseng leaf extract (%) 50 μM 6 11 10 6 7 100 μM 9 15 15 9 9 200 [mu] M 19 28 27 18 20 Cytotoxicity none none none none none

division Production Example 11 Comparative Preparation Example 1 Comparative Production Example 2 Comparative Production Example 3 Ginseng Leaf Fermented Extract Type Example 11 Comparative Example 1 Comparative Example 2 Comparative Example 3 Melanin inhibition rate according to concentration of fermented ginseng leaf extract (%) 50 μM 11 0 0 0 100 μM 15 2 2 4 200 [mu] M 29 13 8 12 Cytotoxicity none none none none

As can be seen from Tables 3 to 5,

According to Examples 1, 3, 4, 7, 8 and 11 which satisfied all of the concentration of the organic solvent to be mixed with the crushed leaf blade of the present invention, the kind and content of the culture broth, the content of the carbon source, Production examples 1, 3, 4, 7, 8 and 11, each of which was prepared containing a leaf fermentation extract, contained ginseng leaves according to Examples 2, 5, 6, 9, 10 and Comparative Examples 1 to 3, The melanin inhibition rate by concentration was much superior to that of Production Examples 2, 5, 6, 9, 10 and Comparative Production Examples 1 to 3 including fermented extract, and no cytotoxicity was observed.

Example 12: Preparation of extract fractions of fermented leaves of ginseng leaves

The ginseng leaf fermentation extract prepared according to Example 1 was divided and extracted with ethyl acetate (EtOAc) / water (H2O), and then a water layer was obtained and divided and extracted three times with butanol (n-BuOH) The acetate layer, the water layer and the butanol layer were concentrated under reduced pressure to obtain n-butanol fraction, H ₂ O fraction and ethyl acetate fraction. Among them, the ethyl acetate fraction as a fraction of the ginseng leaf fermentation extract was analyzed by silica gel column chromatography and reversed phase silica gel column Chromatography was sequentially carried out to separate the active ingredients of the cosmetic composition for skin whitening to prepare a ginseng leaf fermented extract fraction.

<Experimental Example 3>

The following Preparative Examples and comparative Preparations were measured and / or evaluated and are shown in Table 6 below.

1. Evaluation of the yields of Rk1, Rk2 and Rh3 of fermented extract fractions of ginseng leaves

The ginsenosides Rk1, Rk2 and Rh3 of the ginseng leaf fermented extract fractions were evaluated for the ginseng leaf fermentation extract fraction prepared according to Example 12, and the yield of n - butanol fraction, H ₂ O fraction and ethyl acetate fraction.

division Example 1 Example 12 ethyl
acetate Butanol water Compound yield evaluation (%) Ginsenoside Rk1 100 525 20 15 Ginsenoside Rk2 100 681 11 5 Ginsenoside Rk3 100 720 33 10

As can be seen from Table 6 above,

It can be seen that Example 12 including the ethyl acetate fraction as the fraction of the ginseng leaf fermentation extract according to the present invention had much higher amounts of ginsenosides Rk1, Rk2 and Rh3 than Example 1, It can be seen that much greater amounts of ginsenosides Rk1, Rk2 and Rh3 were obtained than in the fraction and H2O fractions.

Thus, it can be seen that the yield of ginsenosides Rk1, Rk2 and Rh3 can be increased by carrying out the fractionation, and the amounts of ginsenosides Rk1, Rk2 And Rh3. &Lt; / RTI &gt;

Production Example 12

A skin whitening cosmetic composition containing 80 μM of ginsenoside Rk1 obtained from the ginseng leaf fermentation extract fraction prepared according to Example 12 was prepared.

Production Example 13 to Production Example 24

The skin cosmetic composition for skin whitening was prepared in the same manner as in Preparation Example 12 except that the concentration of ginsenoside Rk1, the kind and concentration of the ginsenoside derivative were changed as shown in Table 7 below.

<Experimental Example 4>

The skin-whitening cosmetic compositions prepared according to Preparation Examples 13 to 24 were measured / evaluated for the following items and are shown in Table 7 below.

1. Melanin inhibition rate measurement

The skin whitening cosmetic compositions prepared according to Preparation Examples 13 to 24 were cultured at a concentration of Rk1 of 20 μM, 40 μM, 80 μM and 120 μM at concentrations of 10 μM, 20 μM, 40 μM and 70 μM of Rk2 at concentrations of 40 μM, 60 μM, Melanin inhibition measurements were measured at 110 [mu] M.

Specifically, Melan-a cells derived from C57BL / 6J mice were cultured from Dorothy C Bennett (St George's Hospital Medical School, London, UK). Cultures were incubated with 10% fetal bovine serum (FBS) 100 mg / mL of streptomycin-penicillin (penicillin-streptomycin) and 200 nM of TPA (12- ο -tetradecanoylphorbol-13- acetate) with 37 ℃ using RPMI-1640 medium containing, in the 5% CO ₂ environment Cell lines were cultured and used.

Subsequently, 1 × 10 ⁵ Melan-a cells were plated in each well of a 24-well plate and cultured overnight. Each sample was treated with 20 μM, 40 μM, 80 μM and 120 μM of Rk1 , The cells were cultured for 4 days at a concentration of Rk2 of 10 μM, 20 μM, 40 μM and 70 μM to a concentration of 40 μM, 60 μM, 80 μM and 110 μM of Rh3, followed by washing with phosphate-buffered saline, N KOH was treated and dissolved in 250 L of each well. The dissolved cells were transferred to a 96-well plate and the optical density was measured at 405 nm using a microplate reader (3550, Bio-Rad). The rate of inhibition of melanin production was measured using a control (%) Compared to the control group.

2. Assessment of cytotoxicity

The skin whitening cosmetic compositions prepared according to Preparation Examples 13 to 24 were tested for the concentrations of Rk1, 20 μM, 40 μM, 80 μM and 120 μM, respectively, when the concentrations of Rk2 were 10 μM, 20 μM, 40 μM and 70 μM, , 80 [mu] M and 110 [mu] M, cytotoxicity was evaluated.

Specifically, the measurement was carried out in the same manner as the melanin inhibition assay by the concentration, and the cell viability was measured as a ratio of the cell survival to the control (non-treated group) according to the following equation. .

[Mathematical Expression]

Cell viability (%) = [(compound treated cell number OD value - blank well OD value) / (control OD value - blank well OD value)] 100 (%)

division Type of ginsenoside derivative Concentration (μM) Melanin inhibition (%) Cytotoxicity Production Example 13 Rk1 80 38 × Production Example 14 Rk1 20 9 × Production Example 15 Rk1 40 23 × Production Example 16 Rk1 120 41 ○ (83%) Production Example 17 Rk2 10 3 × Production Example 18 Rk2 20 16 × Production Example 19 Rk2 40 40 × Production example 20 Rk2 70 44 ○ (87%) Production Example 21 Rh3 40 8 × Production Example 22 Rh3 60 25 × Production Example 23 Rh3 80 34 × Production Example 24 Rh3 110 38 ○ (86%)

As can be seen from Table 7,

Production Examples 13, 15, 18, 19, 22, and 23 satisfying the concentration range of the ginsenoside derivative according to the present invention are superior to those of Production Examples 14, 16, 17, 20, 21, And that it can exhibit an effect of not cytotoxicity at the same time.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments, It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

Claims (13)

(1) preparing a ginseng leaf extract concentrate; And
(2) fermenting the ginseng leaf extract concentrate with Lactobacillus pentosus ATCC8041 to produce a ginseng leaf fermentation extract. The method of claim 1, wherein the step (1)
(1) -1 step of pulverizing ginseng leaves;
(1) -2 Mixing the pulverized ginseng leaves with an organic solvent to a concentration of 0.5-2 g / ml in the mixture, and repeating the extraction twice or five times for 6-18 hours; And
(1) -3 A method of producing a ginseng leaf extract for skin whitening comprising extracting ginseng leaves at a temperature of 50 ° C or lower under reduced pressure and freeze-drying to prepare a ginseng leaf extract concentrate. 3. The method of claim 2,
Wherein the organic solvent is 70 ~ 95% methanol. The method according to claim 1,
Wherein the fermentation is performed for 8 to 12 days at a temperature of 30 to 44 ° C and a pH of 4.5 to 7.5. 2. The method of claim 1, wherein step (2)
(2) mixing and sterilizing 0.5 to 5 parts by weight of carbon source per 100 parts by weight of the ginseng leaf extract concentrate;
(2) -2: Lactobacillus pentosus is added to the sterilized ginseng leaf extract concentrate in an amount of 300 to 700 parts by weight based on 100 parts by weight of the ginseng leaf extract concentrate and cultured to produce a fermented product;
(2) -3 removing lactobacillus pentosus from the fermentation product and sterilizing it;
(2) -4 reflux extraction and precipitation of the sterilized fermentation product and filtration to prepare a filtrate; And
(2) -5 A process for producing a ginseng leaf fermented extract for skin whitening, comprising the step of concentrating and freeze-drying the filtrate at 45 ° C or below under reduced pressure to prepare a ginseng leaf fermentation extract. A skin-whitening ginseng leaf fermented extract prepared by the method according to any one of claims 1 to 5. The method according to claim 6,
The ginseng leaf fermentation extract contains ginsenoside Rk1, ginsenoside Rk2 and ginsenoside Rh3,
The increase rate of ginsenoside Rk1 according to the following formula 1 is 15,000% or more,
The increase rate of ginsenoside Rk2 according to the following formula 2 is 50,000% or more,
A skin-whitening leaf extract of Ginseng leaf having an increase rate of ginsenoside Rh3 of 45,000% or more according to the following formula 3:
[Relation 1]

[Relation 2]

[Relation 3]

At this time, fermented ginseng leaves and ginseng leaves before fermentation are ginseng leaves of 6-year-old ginseng. A process for producing a ginseng leaf fermented extract according to claim 6,
A cosmetic composition for skin whitening satisfying all of the following conditions (a) to (c):
(a) When the concentration of fermented ginseng leaf extract is 35 to 65 μM, the melanin inhibition rate is 8% or more
(b) Melanin inhibition rate of fermented ginseng leaf extract at a concentration of 80 to 120 μM of 12% or more
(c) The melanin inhibition rate is 21% or more when the concentration of fermented ginseng leaf extract is 180 ~ 220 μM. Preparing a fraction of the ginseng leaf fermentation extract by fractionating the ginseng leaf fermentation extract prepared by the production method according to any one of claims 1 to 5; And
And separating the active ingredient of the skin whitening cosmetic composition from the fraction of the ginseng leaf fermentation extract. 10. The method of claim 9,
Wherein the fraction of the ginseng leaf fermentation extract is ethyl acetate fraction. 10. The method of claim 9,
Wherein the separation is performed by at least one method selected from the group consisting of silica gel column chromatography and reversed phase silica gel column chromatography. A cosmetic composition for skin whitening comprising, as an active ingredient, an extract fraction of fermented ginseng leaf for skin whitening which is separated by the production method according to claim 9. 13. The method of claim 12,
Wherein the ginseng leaf fermentation extract fraction comprises at least one selected from the group consisting of ginsenoside Rk1, ginsenoside Rk2 and ginsenoside Rh3,
Wherein the ginsenoside Rk1 has a concentration of 25 to 100 μM, the ginsenoside Rk2 has a concentration of 15 to 65 μM, and the ginsenoside Rh3 has a concentration of 50 to 100 μM.