KR20170088055A - Cosmetic composition for improving skin wrinkle containing plant extract removed chlorophyll - Google Patents
Cosmetic composition for improving skin wrinkle containing plant extract removed chlorophyll Download PDFInfo
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- KR20170088055A KR20170088055A KR1020160007923A KR20160007923A KR20170088055A KR 20170088055 A KR20170088055 A KR 20170088055A KR 1020160007923 A KR1020160007923 A KR 1020160007923A KR 20160007923 A KR20160007923 A KR 20160007923A KR 20170088055 A KR20170088055 A KR 20170088055A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
Abstract
The present invention relates to a cosmetic composition for improving wrinkles containing an extract of the plant chlorophyll is removed, as an active ingredient, specifically of the present invention the chlorophyll is removed, green tea (Green tea) leaf extract, camellia (Camellia japonica leaf extract and Polygonum cuspidatum extract exhibits significant anti-aging and anti-wrinkle effects, and is highly cytotoxic even in a cosmetic composition at a high concentration and maintains the stability of cosmetics. Therefore, it can be usefully used as a functional cosmetic raw material.
Description
The present invention has been removed or tea chlorophyll pigment (Green tea) leaf extract, camellia (Camellia japonica leaf extract and Polygonum cuspidatum ) as an active ingredient, and to a cosmetic composition for anti-aging and wrinkle improvement.
Aging is an indispensable part of life. All creatures, whether animals or plants, are born on the path of aging. Thanks to hygiene and health care, human longevity has been significantly extended over the last century. As a result, the proportion of elderly people is steadily increasing. The process of aging takes place in the whole body of the body, with changes in the physiological functions of organs, such as hypertension, chronic coronary artery disease or diabetes, as well as aging. Skin aging is also progressing with age.
Skin aging can be broadly categorized as a photoaging that appears on the face, neck, and back of a person by continuous exposure to environmental factors such as intrinsic aging and sunshine, which can be seen over time by biological processes. Both endogenous aging and photoaging are caused by the accumulation of damage to the constituents of our body caused by harmful free radicals, but the photoaging is different in that this active oxygen is produced by ultraviolet rays.
Ultraviolet rays are divided into UVA (320 ~ 400 nm), UVB (280 ~ 320 nm) and UVC (200 ~ 280 nm) according to the length of the wavelength, The wavelengths directly reaching UVA and UVB are UVA and UVB. Among them, UVA penetrates deeply into the dermis, which is the major cause of damaging fibroblasts that produce collagen that maintains skin elasticity. It activates free radicals in the skin, destroying the antioxidant defense network in vivo and damaging cells and tissues At this time, lipids, proteins, and polysaccharides, which are major constituents of the skin, are oxidized. Among them, the oxidation of proteins breaks down the connective tissues like collagen and, in severe cases, it induces cancer or triggers immunosuppression. Activation of free radicals in vivo accelerates the degradation of extracellular matrix by increasing matrix metalloprotease (MMP), an enzyme that degrades collagen by reactive oxygen species, resulting in reduced skin elasticity and wrinkles .
Therefore, extensive efforts have been made to inhibit wrinkle formation, and there have been studies on new natural substances which can replace EGCG (epigallocatechin gallate), retinoid and derivatives thereof as a typical inhibitor of collagen decomposition, . Therefore, in the past, chemical cosmetics were popular, but in recent years, due to the arrival of the era of well-being, interest in the skin has been amplified so that functional cosmetics using various plant extracts that are hypoallergenic and expected to have efficacy as cosmetics Cosmeceuticals) are being manufactured.
As a conventional patent related to a cosmetic composition using a plant extract that is hypoallergenic and exhibits wrinkle-reducing effect to replace chemical ingredients having skin irritation as described above, Korean Patent Registration No. 10-0429590 entitled " Korean Patent Registration No. 10-0670238 entitled " Cosmetic Composition for Improving Skin Wrinkles Containing a Herbal Extract and Its Preparation Method ", Korean Patent Laid-Open Publication No. 10-2007-0111025 " And Korean Patent Registration No. 10-1009766 entitled " Cosmetic Composition for Wrinkle Reduction ", which contains the extracts of Chrysomelus edulis and Aspergillus oryzae, and the like. However, there is no cosmetic composition containing a high concentration of a natural extract having a wrinkle-improving effect yet and ensuring the stability of the cosmetic composition.
Accordingly, the present inventors have made efforts to develop a cosmetic composition derived from natural products that exhibits anti-aging and skin-wrinkle effects, and is cytotoxic even when contained in a cosmetic composition at a high concentration and maintains stability of cosmetics. As a result, Green tea leaf extract, Camellia japonica leaf extract and Polygonum cuspidatum extract showed a significant wrinkle-improving effect, and at the same time, the stability of the cosmetic product was maintained even though it was contained in the cosmetic composition at a high concentration, thereby completing the present invention.
The object of the present invention is to provide a green tea leaf extract from which chlorophyll or pigment is removed, Camellia japonica leaf extract and Polygonum cuspidatum ) as an active ingredient, and an external preparation for skin.
In order to accomplish the above object, the present invention provides a green tea leaf extract, Camellia japonica leaf extract and Polygonum cuspidatum ) extract as an active ingredient.
In addition, the present invention relates to a composition comprising a green tea leaf extract, Camellia japonica leaf extract and Polygonum cuspidatum ) as an active ingredient, and to provide an external skin preparation for anti-aging and wrinkle improvement.
The present invention relates to a green tea leaf extract from which chlorophyll has been removed, a chlorella-free camellia The present invention relates to a cosmetic composition for improving anti-aging and wrinkles containing an extract of Polygonum cuspidatum , which is an effective ingredient, Even if it is contained in the cosmetic composition at a high concentration, it is not cytotoxic and the stability of the cosmetic product is maintained, so that it can be usefully used as a functional cosmetic raw material.
1 is a view showing a method of removing chlorophyll from a plant extract.
2 is a view showing a green leaf extract from which chlorophyll has been removed:
A: Chlorophyll was precipitated by reacting with fluoride water extracted from plant extracts, cow mugwort;
B: photographs of filtered or centrifuged chlorophyll deposited;
C: Photographs in which the chlorophyll was not precipitated when the chlorophyll was removed under the condition that the chlorophyll removal conditions (concentration, time, temperature) of the present invention were different; And
D: Photograph of chlorophyll precipitated through the chlorophyll removal condition of the present invention and the upper layer including the active ingredient was separated.
3 is a view showing a chlorophyll-removed camellia leaf extract:
A: Chlorophyll was precipitated by reacting with fluoride water extracted from plant extracts, cow mugwort;
B: photographs of filtered or centrifuged chlorophyll deposited;
C: Photographs in which the chlorophyll was not precipitated when the chlorophyll was removed under the condition that the chlorophyll removal conditions (concentration, time, temperature) of the present invention were different; And
D: Photograph of the chlorophyll precipitated through the chlorophyll removal condition of the present invention and the upper layer containing the active ingredient was separated.
4 is a view showing a method of removing pigment from a plant extract.
Fig. 5 is a diagram showing the callus root extract from which the pigment is removed:
A and B: represent the precipitated pigment by reacting with the fluoride water extracted from the plant extract, cow mugwort;
C: a photograph in which the pigment and the active ingredient are separated; And
D: Photograph depicting the extract of Hojokgang with pigment removed.
6 is a graph showing the effect of inhibiting collagenase activity of plant extracts from which chlorophyll or pigment is removed according to the present invention.
FIG. 7a is a graph showing the inhibitory effect of IL-6 and TNF-α on keratinocytes (Hacat cells) exposed to ultraviolet rays of a chlorophyll or pigment-free plant extract of the present invention.
FIG. 7B is a graph showing fold inhibition effect of IL-6 on the expression of chlorophyll or pigment-removed plant extract of the present invention.
FIG. 7c is a graph showing the effect of inhibiting the TNF-α expression of the chlorophyll or pigment-removed plant extract of the present invention as a fold change value. FIG.
FIG. 8 is a graph showing the inhibitory effect of the chlorophyll or pigment-removed plant extract of the present invention on the elastase activity.
FIG. 9 is a graph showing the inhibitory effect of the chlorophyll or pigment-removed plant extract of the present invention on the production of reactive oxygen species.
FIG. 10A is a graph showing the survival rate of cells treated with keratinocytes for 3 hours after chlorophyll or pigment-free plant extract of the present invention was treated. FIG.
FIG. 10B is a graph showing the survival rate of cells treated with keratinocytes for 12 hours after the chlorophyll or pigment-free plant extract of the present invention was treated.
11 is a graph showing the HPLC results of the chlorophyll or pigment-removed plant extract of the present invention.
12 is a view showing a method of manufacturing a cosmetic product using the chlorophyll or pigment-removed plant extract of the present invention.
13 is a view for confirming the stability of cosmetics.
Fig. 14 is a comparison of freeze-dried cosmetic raw materials prepared from chlorophyll-free green tea leaf extract and lyophilized green tea leaf extract without chlorophyll removed:
A: Green leaf extract with chlorophyll removed; And
B: Green tea leaf extract without chlorophyll removed.
Fig. 15 is a comparison of lyophilized cosmetic raw materials prepared with chlorophyll-free camellia leaves and lyophilized Camellia sinensis leaf extract without chlorophyll removed.
A: Camellia sinensis leaf extract with chlorophyll removed; And
B: Camellia sinensis leaf extract without chlorophyll removed.
FIG. 16 is a view comparing freeze-dried cosmetic ingredients extracted from the pigment-removed callus root extract and callus root extract without pigment removal.
A: Extract of Hojangguk extract with pigment removed; And
B: Root root extract without pigment removed.
Hereinafter, the present invention will be described in more detail.
The present invention is green tea (Green tea) leaf extract, camellia (Camellia japonica leaf extract and Polygonum cuspidatum ) extract as an active ingredient.
Green tea leaf extract, Camellia japonica ) leaves have been removed from the chlorophyll, and the Polygonum cuspidatum ) extract is the pigment is removed.
The chlorophyll or pigment-free green tea leaf extract, Camellia sinica leaf extract, and Hojokgang extract may be prepared by the following steps.
1) extracting green tea leaves, camellia leaves, or callus roots with an extraction solvent;
2) cooling the extract of step 1) and filtering;
3) Concentrating the filtrate obtained in step 2) under reduced pressure
4) one or more furoral water selected from the group consisting of furoraru water extracted from the leaves of Bombyx mori, furoral water extracted from the larval leaves of leaves, furoral water extracted from green tea leaves, furoral water extracted from camellia leaves, and furoral water extracted from cucumber mugwort After mixing with the alcohol, it is added to the reduced-pressure concentrated extract of step 3), or at least one of the fluorohydrides selected from the group consisting of the fluorohydrants is added to the reduced-pressure concentrated extract of step 3) Removing steps.
In the above method, the green tea leaves, camellia leaves or roots of step 1) can be used without limitation, such as cultivated or commercially available ones.
The extraction solvent in step 1) is preferably, but not limited to, water, alcohol or a mixture thereof. As the alcohol, C 1 to C 2 lower alcohols are preferably used. As the lower alcohol, ethanol or methanol is preferably used, but not limited thereto. As the extraction method, it is preferable to use shaking extraction or reflux extraction, but it is not limited thereto. The extraction solvent is preferably added in an amount of 2 to 10 times the amount of green tea leaves, camellia leaves, or acacia leaves, but is not limited thereto. The extraction temperature is preferably from room temperature to 100 ° C, but is not limited thereto. The extraction time is preferably 2 to 5 days, but is not limited thereto. The number of times of extraction is preferably 1 to 3 times, but is not limited thereto.
In the above method, it is preferable to use a vacuum decompression concentrator or a vacuum rotary evaporator for the decompression concentration in step 3), but it is not limited thereto.
Furor water extracted from the leaves of step 4), furoral water extracted from the large leaves of Shin, furoral water extracted from green tea leaves, furoral water extracted from camellia leaves, or furor water extracted from cucumber mugwort, Leaves, camellia leaves or mugwort are extracted with an essential oil extractor for at least 8 hours to prepare a plant liquid extract for chlorophyll removal.
In the step 4), the furor water, the furor water extracted from the largemouth leaves, the furor water extracted from the green tea leaves, the furor water extracted from the camellia leaves or the furor water extracted from the dogs are mixed with ethanol in a ratio of 9: 1 to 7: , And then 20 to 25 times of the concentrated extract of step 1) is added to dilute the concentrated extract of step 3), and then the mixture is diluted with the extract at a temperature of -40 to -60 ° C for 10 to 14 hours It is preferable to separate the precipitated chlorophyll or pigment. Also, only the follicular water extracted from the foliage leaf, the furoral water extracted from the flesh of the god, the follicular water extracted from the green tea leaves, the follicular water extracted from the camellia leaves, or the follicular water extracted from the fungus are used as the extraction solvent, The concentrated extract of step 3) is diluted, and the precipitated chlorophyll or pigment is separated by standing at -40 to -60 ° C for 10 to 14 hours under freezing conditions Do.
The green tea leaf extract is prepared by mixing the green tea leaf extract with Fuller's water selected from the group consisting of furor water, fuller's water extracted from the foxtail leaves and fuller's water extracted from the flesh of the foxtail leaves, And the camellia leaf extract is obtained by mixing Camellia sinensis leaf extract with Fuller's water selected from the group consisting of Furoraru water extracted from Dogwood mugwort, Furoraru water extracted from Camellia bark leaf and Furor water extracted from Beppermint leaf to remove chlorophyll It is preferable to prepare the camellia leaf extract. The extracts of Hojokgun include Hoerang water selected from the group consisting of Furor water extracted from Clary mugwort, Furor water extracted from the leaves of Bombyx mori, and Furor water extracted from the larval leaves By mixing the pigment No. been removed to produce a muscle extract is preferred.
It is preferable that the green tea leaf extract is added to the total cosmetic composition in an amount of 50 to 400 占 퐂 / ml, the camellia leaf extract is 30 to 120 占 퐂 / ml, and the caraway root extract is 20 to 120 占 퐂 / When it is contained in the cosmetic composition at 400 / / mL or more, it exhibits cytotoxicity. When the camellia leaf extract and the callus root extract are contained at a concentration of 120 ㎍ / mL or more, the stability of the cosmetic becomes problematic. Of the green tea leaf extract, 200 to 400 占 퐂 / mL of the green tea leaf extract, 50 to 120 占 퐂 / mL of the camelliace leaf extract and 20 to 120 占 퐂 / mL of the Japanese persimmon root extract, which are contained in the cosmetic composition.
In a specific example of the present invention, the inventors of the present invention produced green tea leaf extract, camellia leaf leaf extract and callus root extract, respectively, from which chlorophyll or pigment was removed (see FIGS. 1 to 5 and 11) The green tea leaf extracts of green tea leaf extracts were obtained by extracting chlorophyll - free green tea leaves from mixed with fuller 's water, fuller' s water extracted from bloomed leaves or fuller ' And the camellia leaf extract is prepared by mixing Camellia sinensis leaf extract with furorall water extracted from mugwort, fullerial water extracted from camellia leaves or furor water extracted from camphor leaves to produce chlorella-free camellia leaf extract , And the extracts of Hojangun extract were obtained from It was confirmed that it is preferable to prepare the extract of Phyllostachys succiniflora L. from the mixture of Fuller's water, Fuller's water extracted from the leaves of Bombyx mori, or Fuller's water extracted from the leaves of Bombyx mori, (see Tables 1 to 3).
In addition, the inventor of the present invention has found that the extract of chlorophyll or pigment-removed plant extract of the present invention inhibits collagenase activity. As a result, the extract of chlorophyll removed the collagenase (MMP-1) activity of keratinocytes (See Fig. 6).
In addition, the present inventors have confirmed that the chlorophyll or pigment-free plant extract of the present invention inhibits the inflammatory cytokine production. As a result, the present inventors have found that the plant extract obtained by removing chlorophyll from keratin cells significantly inhibits the expression of inflammatory cytokines (See FIG. 7).
The present inventors also confirmed that the chlorophyll or pigment-free plant extract of the present invention inhibited elastase activity. As a result, the chlorophyll or pigment-free plant extract of the present invention exhibited significant elastase activity in keratinocytes (Hacat cells) Inhibitory effect (see FIG. 8).
In addition, the present inventors have found that the chlorophyll or pigment-free plant extract of the present invention inhibits the production of reactive oxygen species. As a result, the chlorophyll-free green tea leaf extract, Camellia sinica leaf extract, Showed significant inhibition of the production of reactive oxygen species in keratinocytes (see FIG. 9).
The inventors of the present invention conducted a cytotoxicity test of the chlorophyll or pigment-free plant extract of the present invention and found that the chlorophyll or pigment-free plant extract of the present invention did not exhibit cytotoxicity (see FIG. 10 ).
The inventors of the present invention found that the chlorophyll or pigment-free plant extract of the present invention had an optimal content of 100 to 400 ㎍ / mL of green tea leaf extract, 50 to 120 ㎍ / mL of Camellia sinensis leaf extract, When the extract was contained in the cosmetic composition at a concentration of 20 to 120 / / mL, it was confirmed that the cosmetic stability was maintained without cytotoxicity even when contained in a cosmetic composition at a high concentration (green tea extract having cytotoxicity more than 400 / / mL, Journal of Nutrition, Vol. 40, No. 5, 2011.5, 619-624) (see FIG. 13).
Thus, chlorophyll or pigment of the present invention to remove tea (Green tea) leaf extract, camellia (Camellia japonica leaf extract and Polygonum cuspidatum extract exhibits significant anti-aging and anti-wrinkle effects, and is cytotoxic even when contained in a cosmetic composition at a high concentration and maintains the stability of cosmetics. Therefore, it can be usefully used as a functional cosmetic raw material.
The cosmetic composition may be in the form of a solution, a gel, a solid or a paste anhydrous product, an emulsion obtained by dispersing an oil phase in water, a suspension, a microemulsion, a microcapsule, a microgranule or an ionic (liposome) A cream, a skin, a lotion, a powder, an ointment, a spray, or a cone stick. It can also be prepared in the form of a foam or an aerosol composition further containing a compressed propellant.
In addition, the cosmetic composition may further contain a lipid, an organic solvent, a solubilizing agent, a thickening agent and a gelling agent, a softening agent, an antioxidant, a suspending agent, a stabilizer, a foaming agent, , A perfume, a surfactant, water, an ionic or nonionic emulsifier, a filler, a sequestering and chelating agent, a preservative, a vitamin, a barrier, a wetting agent, a essential oil, a dye, a pigment, a hydrophilic or lipophilic active agent, Or any other ingredient conventionally used in cosmetics. The cosmetic compositions of the present invention may also contain adjuvants commonly used in the cosmetics field.
In the cosmetic composition of the present invention, the extract of the present invention may be added in an amount of 1 to 15% by weight, preferably 2 to 10% by weight, to a cosmetic composition usually contained.
In addition, the present invention relates to a green tea leaf extract, Camellia japonica leaf extract and Polygonum cuspidatum ) as an active ingredient, and to provide an external skin preparation for anti-aging and wrinkle improvement.
Chlorophyll is a pigment or remove tea of the present invention (Green tea) leaf extract, camellia (Camellia japonica leaf extract and Polygonum cuspidatum extract exhibits significant anti-aging and anti-wrinkle effects, and is cytotoxic even when contained in a composition for external application for skin at a high concentration. Since the stability of the composition for external application is maintained, it can be effectively used as a skin external preparation for wrinkle improvement have.
Hereinafter, the present invention will be described in detail with reference to Examples, Experimental Examples and Preparation Examples.
However, the following Examples, Experimental Examples and Preparation Examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following Examples, Experimental Examples and Production Examples.
< Example 1> Preparation of natural extract
<1-1> Preparation of green tea leaf extract
Green tea leaf extract was prepared from a plant as a general procedure for extracting alcohol [fermented alcoholic beverages, Korea Diet Life Co., Ltd.] with a solvent.
Specifically, it was immersed in a wash dried green tea leaf (200 g) supplied from Hadong Green Tea Research Institute with 10 times the amount of the alcohol, allowed to stand at room temperature for 72 hours, and then put into a soup pack (medium pack, 25 cm X 35 cm, 8P, manufactured by T & C Eletronics Co., Ltd.). In order to volatilize the alcohol, the extract was distilled using a vacuum concentrator (CCA-1111, EYELA4) at 50 ° C for a certain period of time and then concentrated to prepare green tea leaf extract.
<1-2> Preparation of camellia leaf extract
In the case of camellia leaves purchased from Welpito, the procedure was carried out according to the general procedure for removing the foreign materials and then extracting the natural extract.
Specifically, to remove contaminants and fine dusts, well-dried camellia leaves were mixed with 500 g of dried camellia leaves in a soup pack, and washed with purified water containing water (Koyasan Millennium, High Hyatt Spring Water) And then dried. Then, the dried camellia leaves from which the pollutants were removed were pulverized with a pulverizer, and then extracted and concentrated in the same manner as in the method of extracting green tea leaves of Example <1-1> to produce Camellia sinica leaf extract.
<1-3> Hojo Chang Preparation of extract
The callus root purchased from Chunil Pharmaceutical Co. was extracted and concentrated in the same manner as in Example <1-1> of Example <1 - 1> to prepare callus root extract.
< Example 2> Production of Plant Liquid Extract for Chlorophyll Removal
(7.0 kg), camellia leaves (7.0 kg), and white mugwort (7 kg) were used for the production of chlorophyll and pigment removal plant liquid extracts. After purchasing directly from the local area, Chosun University Dental Precision Equipment & Component Regional Innovation Center extracted the essential oil extractor for more than 8 hours to prepare chlorophyll plant liquid extract (Furoal water).
<Example 3> Preparation of plant extract from which chlorophyll and pigment were removed
<3-1> Production of chlorophyll-free green tea leaf extract
The chlorophyll of the green tea leaf extract was removed using the plant liquid extract prepared in Example 2 for removing chlorophyll.
Specifically, as shown in FIG. 1, the plant liquid extract for chlorophyll removal (furor water extracted from Sugar of Artemisia) prepared in Example 2 was mixed with ethanol at a ratio of 9: 1, The extract was diluted 20 times and then allowed to stand at -50 ° C for 12 hours.
As a result, as shown in FIG. 2, the chlorophyll layer was precipitated and the active ingredient was separated on the upper layer. Thus, only the upper layer was harvested to produce chlorophyll-free green tea leaf extract (FIGS. 1 and 2). On the other hand, the green leaf extract with chlorophyll removed was completely frozen in the freezer, and then freeze-dried with a freeze dryer (FDU-1200 (EYEL4)) (Fig.
<3-2> Production of chlorophyll removed camellia leaves
A chlorella-free camellia leaf extract was prepared in the same manner as in <3-1> above (FIGS. 3 and 15).
≪ 3-3 > Hojo Chang Extract preparation
The pigment extract was removed in the same manner as in the above <3-1> to prepare a callus root extract (FIGS. 4, 5 and 15).
<Experimental Example 1> Determination of plant chlorophyll or pigments for removal of pigment from plant extracts
<1-1> Identification of Plant Follicle Water for Optimal Chlorophyll Removal from Green Tea Leaf Extract
Among the various types of chlorophyll-free plants prepared in Example 2, chlorophyll-free chlorophyll plants were identified.
Specifically, chlorophyll removal was performed using the same method as in Example <3-1>.
As a result, as shown in the following Table 1, the chlorophyll of the green tea leaf extract was significantly separated from the foliar water extracted from the fowl wormwood, the follicle water extracted from the foliage leaf and the follicle extracted from the foliage of the foliage, , And the chlorophyll was not separated from the fragrance water and the purified water extracted from the camellia leaves (Table 1).
<1-2> Identification of the most suitable chlorophyll-free plant water for camphor leaf extract
In the same manner as in Experimental Example <1-1>, the plant chlorophyll water for the removal of chlorophyll of the camellia leaves was identified.
As a result, as shown in Table 2 below, the chlorophyll of the Camellia sinica leaf extract was significantly separated from the follicular fluid extracted from the dogwood mugwort, the follicular water extracted from the Camellia sinensis leaf, Fuller's water, fuller's water and purified water extracted from the larval leaves of the ginseng were found to be unable to separate chlorophyll (Table 2).
<1-3> Hojo Chang Optimum pigment removal plant extract Water Confirm
In the same manner as in Experimental Example <1-1>, the plant chlorophyll water for the removal of chlorophyll was identified.
As a result, as shown in the following Table 3, follicle extracts from fowl wormwood, follicle extracts from fennel leaves and follicle extracts from fennel larvae significantly separated the pigments of the fennel extract, Water and purified water from the camellia leaves were not able to separate the pigments (Table 3).
< Experimental Example 2> The chlorophyll or pigment-free plant extract of the present invention Collagenase Identification of the activity inhibition effect
Retinol and adenosine, which are wrinkle improving substances, are widely used as an agent for inhibiting collagenase activity and collagenase activity. However, high concentrations of retinol and adenosine cause adverse effects on the skin. In the present invention, the expression of collagenase, Matric metalloprotinase (MMP-1), is suppressed in a high concentration extract (concentration of 100 μg / mL or more) from which chlorophyll and pigment are removed, The purpose of this study was to select materials that can help maintain the cholinesterase and to select raw materials derived from chlorophyll and pigment free natural products for the purpose of inhibiting collagenase expression.
Specifically, keratin cells (ATCC, American Type Culture Collection) were cultured in DMEM containing 15% fetal bovine serum (FBS) in a CO 2 incubator (5% CO 2 , 37 ° C ) And subcultured at intervals of 2 to 3 days. Subcultured cells were inoculated into a 60 mm culture dish at 1 × 10 6 cells and cultured for 18 hours. In addition, the cell sensitization was performed by removing the medium of the cultured cells, and then extracting various kinds of chlorophyll or pigment-free extracts (Hojangguk extract, Camellia sinensis leaf extract, Green tea leaf extract) in 2 ml of PBS buffer. 0.05% vitamin C + poly-gamma-glutamic acid (PGA). The cells were incubated in a CO 2 incubator (5% CO 2 , 37 ° C) for 1 hour. One hour later, the cells were irradiated with ultraviolet A (UVA: 360 mm) in a culture plate at 5 J / cm 2 , replaced with serum-free DMEM medium, and cultured in a CO 2 incubator for 24 hours. The medium was collected and used in an active MMP-1 ELISA experiment. Cells were collected and mRNA was extracted and used for RT-PCR experiments. The RT-PCR is performed by RT-PCR using 1 μg of extracted total RNA using Superscript first strand synthesis system (Invitrogen, CA, USA). Reaction buffer (3
As a result, as shown in FIG. 6, it was confirmed that the plant extract from which chlorophyll was removed significantly inhibited collagenase (MMP-1) activity of keratinocytes (Hacat cells). Specifically, keratinocytes were exposed to UVB to induce collagenase expression, chlorophyll-free green tea leaf extract, camellia crassifolia leaf extract, extracts of alcohol extracted from solvent by conventional methods, The extracts were treated at concentrations of 10, 30, and 100 ㎍ / mL. It was confirmed that the expression of collagenase was increased by the UVB stimulation and that the inhibitory effect of collagenase (MMP-1) on plant extracts of chlorophyll or pigment-free extract of the present invention was 70% or more (Fig. 6).
<Experimental Example 3> Inhibitory effect of chlorophyll or pigment-free plant extract of the present invention on inflammatory cytokine production
RNA isolation kit and agarose gel electrophoresis were performed to confirm the inhibition of the expression of inflammatory cytokines (TNF-α and IL-6) in plant extracts from which chlorophyll or pigment was removed from dermal cells.
Specifically, keratinocytes (immortal keratinocyte cell line, Hacat cells) cultured on a 6-well plate were irradiated with UVB at 100 mJ. Then, the extracts of green tea leaves and camellia leaves of the present invention in which the chlorophyll was removed and the pigment-free extracts of Hojokgang were removed, and the extracts of the extracts were concentrated in 10, 30 and 100 ㎍ / mL And reacted for 12 hours at 37 ° C in a cell incubator. When the reaction was completed, the RNA was isolated using an RNA isolation kit. The isolated RNA was synthesized with cDNA and RT-PCR was performed using human IL-6 and TNF-α primer. RT-PCR was carried out in the same manner as in <Experimental Example 2> except that the primers shown in the following Table 5 were used.
As a result, it was confirmed that the plant extract obtained by removing chlorophyll from keratin cells (hacat cells) showed an effect of significantly suppressing the expression of inflammatory cytokines, as shown in Fig. Specifically, keratinocytes are exposed to UVB to induce the expression of inflammatory cytokines, followed by removal of chlorophyll, followed by lyophilized green tea leaf extract, Camellia sinica leaf extract, and Chinese cabbage extract from which pigments have been removed, and freeze- The extracts were treated at concentrations of 10, 30, and 100 μg / mL. In the case of inhibition of IL-6 expression, it was confirmed that the expression was inhibited when treated with 10 ㎍ / mL of the lowest concentration of green tea leaf extract, Camellia sinensis leaf extract, and Hojokgang extract. However, Showed that the expression of IL-6 was not inhibited. Inhibition of TNF-α expression was confirmed by the inhibition of the expression by green tea leaf extract and callus root extract, while the extract of Camellia sinica leaf showed a slight inhibitory effect on expression. The extract of Paeoniae showed no inhibitory effect on TNF-α expression by UVB irradiation (FIG. 7).
<Experimental Example 4> Confirmation of the inhibitory effect of chlorophyll or pigment-removed plant extract of the present invention on elastase activity
Elastase is the only enzyme known to degrade elastin. The expression of the gene of elastin degrading enzyme is known to be caused by free radicals. A typical histological change in photoaging is elastin accumulation, which is a major component of elastin fibers. Unlike normal collagen fibers, the solar elastotic material is composed of macromolecules and severely impaired function by light. The expression of the gene of elastin degrading enzyme is markedly promoted in skin cells exposed to ultraviolet light. Inhibition of enzyme activity can fundamentally reduce skin aging. The present invention aims at the discovery of a substance capable of suppressing the activity of the elastase degrading enzyme and helping to maintain and generate elastic skin.
Specifically, an Elastase Assay Kit was used to measure the degree of elastase activation. N-Succinyl-Ala-Ala-Ala-p-nitroanilide (SANA) was used as an enzyme in the 96-well plate containing the sample. 1300 占 퐇 of the 1.015 mM substrate and 100 占 퐇 of the experimental group were added. 50 μl of pancreatic elastase was added to the solution (30 seconds mixing well). After incubation at room temperature for 10 minutes, 100 μl of 0.0375 unit / ml elastase was added and incubated for 10-30 minutes (reaction at room temperature). After 30 minutes of reaction, the absorbance at 410 nm was measured using an ELISA reader.
As a result, as shown in FIG. 8, the plant extract obtained by removing chlorophyll or pigment from keratin cells (Hacat cells) showed significant inhibitory effect on elastase activity. Specifically, we examined the inhibition of elastase activity by green leaf extract, camellia leaf leaf extract and pigment removed from chlorophyll, and extracts of alcohol extracted from solvent by alcohol extract The following results were obtained. When the chlorophyll or pigment-free extracts were treated to a concentration of 10, 30, 100 or 300 ㎍ / mL, the green tea extract and the green tea extract did not show any effect, but the extracts of Camellia sinica Activity was inhibited. From the concentration of 30 ㎍ / mL, camellia sinensis leaf extract inhibited the activity of elastase in a concentration - dependent manner and inhibited elastase activity by 30% at a concentration of 300 ㎍ / mL. In addition, the extracts showed a dose - dependent inhibition effect from a concentration of 10 ㎍ / mL to a concentration of 100 ㎍ / mL. In the case of 300 ㎍ / mL, inhibition of elastase activity by 60% or more was 100 ㎍ / mL (Fig. 8).
<Experimental Example 5> Effect of chlorophyll or pigment-removed plant extract of the present invention on the inhibition of reactive oxygen species formation
All aerobic organisms, including humans, use oxygen to promote energy metabolism and survive. These oxygen are generated as active oxygen as a byproduct after energy metabolism. When the cells are in a normal state, the antioxidant enzyme such as Superoxide Dismutase, which is an enzymatic defense system, and the antioxidant substance such as Ascorbic Acid, which is a non-enzymatic defense system, Therefore, the effect of active oxygen species is insignificant. When cells are in an abnormal state, the increase in reactive oxygen species and their continued activity disables the defense system and causes imbalance, leading to oxidation and denaturation of the protein, resulting in deterioration and deterioration of the function of the cell barrier in vivo . Accordingly, the present invention has confirmed the effect of inhibiting the production of reactive oxygen species by the chlorophyll or pigment-free plant extract of the present invention.
Specifically, 2 ', 7'-dichlorodihydrofluorescein diacetate (DCFH-DA) was used to measure the active oxygen species produced in the cells. The keratinocytes were cultured in a 96-well plate, and the chlorophyll or pigment-free green tea, camellia, cauliflower and horsetail extract of the present invention were added at a concentration of 10, 30 and 100 μl / ml. After incubation for 15 min at 37 ° C in a cell incubator, DCFH-DA was added at a concentration of 100 μM and allowed to react for 15 min at 37 ° C in a cell incubator. After the reaction, the culture medium was changed to HBSS, UVB was irradiated at 100 mJ, and the cells were incubated at 37 ° C for 15 minutes. After the reaction was completed, the cell membrane was punctured with 0.5% Triton X-100 and the fluorescence intensity was measured (Ex / Em = 480 nm / 530 nm).
As a result, as shown in FIG. 9, the extracts of green tea leaves, camellia leaves and pigments from which the chlorophyll was removed of the present invention showed a significant increase in the production of reactive oxygen species in keratinocytes Respectively. Specifically, after keratinocytes derived from humans were subjected to UVB irradiation, the effect of inhibiting the production of reactive oxygen species by each extract was confirmed. It was confirmed that the production of reactive oxygen species was increased by UVB irradiation, and all the extracts showed inhibitory effect on the production of reactive oxygen species in a concentration dependent manner, Respectively. The extracts of Hojokgang extract showed a slight effect compared to other extracts but showed a statistically significant inhibitory effect (Fig. 9).
<Experimental Example 6> Cytotoxicity test of the chlorophyll or pigment-free plant extract of the present invention
Chlorophyll-free extracts and pigment-free plant extracts were administered to keratinocytes and then cytotoxicity was confirmed.
Specifically, Trypan blue dye exclusion method was used for the cytotoxicity test. First, the chlorophyll or pigment-free extract of the present invention was treated with cells for 3 hours or 12 hours. At this time, the dye does not penetrate into the living cells, and only the dead cells penetrate the pigment and are dyed in blue. % Of living cells.
As a result, as shown in FIG. 10A, the chlorophyll or pigment-free plant extract of the present invention was treated with keratinocytes for 3 hours, and the viability of the cells was examined. As a result, it was confirmed that cytotoxicity was not exhibited .
Further, as shown in FIG. 10B, the chlorophyll or pigment-free plant extract of the present invention was treated with keratinocytes for 12 hours and the viability of the cells was examined. As a result, the chlorophyll or pigment- The plant extracts did not show cytotoxicity after 12 hours (FIG. 10).
< Experimental Example 6> The chlorophyll or pigment-free plant extract of the present invention chorophyll a and chorophyll b Confirm removal
As a control and a control group, the extracts of the present invention were prepared by extracting the extracts of Hojokgang extract, chlorophyll removed green tea leaves and control, The removal of chlorophyll was confirmed by high performance liquid chromatography (HPLC) on the extracts of Camellia sinica (Leptin Chorophyll a and chorophyll b were used as indicators.
As a result, as shown in Table 6, it was confirmed that both chlorophyll a and chlorophyll b were completely removed from the carotid root extract and green tea leaf extract. On the other hand, chlorophyll a and chlorophyll b were completely removed from Camellia sinica leaf extract and 4.5%, respectively. It was also confirmed that chlorophyll was contained in a large amount in Hojokgun extract and effectively removed by chlorophyll and dye removal experiments (Table 6).
In addition, the chromatogram for A and B in FIG. 11 shows that the chlorophyll a and chlorophyll b were effectively removed from this extract by the chlorophyll a And chlorophyll b. When the run time was 15 minutes, it was confirmed that peaks were formed at the position of 9.5 minutes and the position of 9 minutes from the indicator chlorophyll a and chlorophyll b. C and D are chromatograms that confirm the presence of large amounts of pigments and chlorophyll in the extracts of Hojokgang. In case of C, it was confirmed that Peak was formed at a position of 9 minutes when ethanol was extracted with a solvent. It was confirmed that the black pigment layer with a high molecular weight had broad peaks in the range of 2 to 4 minutes. It is confirmed through the previous announcement literature (Organized by the Organizing Agency for Growth of Establishment in 2012 - First Step Technology Development Project (Project Number: S2069583) It is confirmed that there are many substances which are difficult to use as raw materials for cosmetics. It was confirmed that the chlorophyll was removed from the green tea leaf extract by the method of the present invention. G and H were obtained by removing the camphor leaf extract and chlorophyll extracted by the conventional method The chlorophyll b is removed, but most of the components are not removed, but the chlorophyll a is removed, while the chlorophyll a is removed. And 4.5% remained.
< Experimental Example 7> In the production of cosmetics, the optimum content of the chlorophyll or pigment-free plant extract of the present invention was confirmed
Green tea leaf extract, chlorella leaf extract, and camellia leaf extract, from which the chlorophyll has been removed, were extracted and the pigment was removed to give a crude extract of 0.5% in the skins and 1% in the lotions and creams at the concentrations shown in Table 6 below. On the other hand, a cosmetic base of Cosmos Co., Ltd. was used as the cosmetic base (Fig. 12).
Then, the stability of the
As a result, as shown in Fig. 13, in the case of the cosmetic 1, it was confirmed that the color was darker as the temperature was increased except for 4 캜 when the stability was observed for one week (Fig. 13). The reason why discoloration occurs in the stability of cosmetics is that the high concentration (the concentration of raw material of Green tea leaf extract, Camellia sinensis leaf extract, And 3 for cosmetics 3, it was confirmed that the problem of discoloration occurred in
Accordingly, the present invention has confirmed that the content of green tea leaf extract, Camellia sinica leaf extract, and Hojokgang extract, which contain a large amount, does not affect the stability of cosmetics.
< Manufacturing example 1> Cosmetic composition containing the chlorophyll or pigment-free plant extract of the present invention of the present invention
<110> BAE, YONG TAE <120> Cosmetic composition for improving skin wrinkle containing plant extract removed chlorophyll <130> P2016-011 <160> 10 <170> Kopatentin 1.71 <210> 1 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> MMP-1 Forward primer <400> 1 tgacttttaa aacatagtct atgttca 27 <210> 2 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> MMP-1 Reverse primer <400> 2 tcttggattg atttgagata agtcatagc 29 <210> 3 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Beta actin Forward primer <400> 3 tggaatcctg tggcatccat gaaac 25 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Beta actin Reverse primer <400> 4 taaacgcagc tcagtaacag tccg 24 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TNF-alpha Forward primer <400> 5 cagagggaag agttccccag 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TNF-alpha Reverse primer <400> 6 ccttggtctg gtaggagacg 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> IL-6 Forward primer <400> 7 ccttggtctg gtaggagacg 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> IL-6 Reverse primer <400> 8 ccttggtctg gtaggagacg 20 <210> 9 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Beta actin Forward primer <400> 9 tggaatcctg tggcatccat gaaac 25 <210> 10 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Beta actin Reverse primer <400> 10 taaacgcagc tcagtaacag tccg 24
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| KR20200035571A (en) * | 2018-09-27 | 2020-04-06 | (주)화니핀코리아 | Cosmetic Composition For Mask Pack Comprising the Natural Complex Extract |
| KR20210050724A (en) | 2019-10-29 | 2021-05-10 | 강원대학교산학협력단 | Composition for anti-wrinkle effect comprising complex extract of Morus alba and Reynoutria japonica |
| CN113873997A (en) * | 2020-04-27 | 2021-12-31 | 自然灵感之园有限公司 | A functional cosmetic composition containing plant extract as active component and its preparation method |
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| KR101135340B1 (en) * | 2009-09-16 | 2012-04-17 | (주)아모레퍼시픽 | Anti-oxidating cosmetic composition |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| KR20200035571A (en) * | 2018-09-27 | 2020-04-06 | (주)화니핀코리아 | Cosmetic Composition For Mask Pack Comprising the Natural Complex Extract |
| KR20210050724A (en) | 2019-10-29 | 2021-05-10 | 강원대학교산학협력단 | Composition for anti-wrinkle effect comprising complex extract of Morus alba and Reynoutria japonica |
| CN113873997A (en) * | 2020-04-27 | 2021-12-31 | 自然灵感之园有限公司 | A functional cosmetic composition containing plant extract as active component and its preparation method |
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