WO2018105799A1 - Cosmetic composition containing eichhornia crassipes extract as active ingredient - Google Patents

Abstract

The present invention provides a cosmetic composition containing an Eichhornia crassipes extract as an active ingredient for fine dust adsorption.

Description

Cosmetic composition containing hyacinth extract as an active ingredient

The present invention relates to a cosmetic composition containing the water hyacinth extract as an active ingredient.

Fine dust is an air pollutant containing harmful substances such as sulfurous acid gas, nitrogen oxides, ozone, and carbon monoxide, which occurs in automobiles and factories, and refers to a material that floats in the air for a long time.

External industrial contaminants such as rapid industrialization, automotive exhaust gas, heavy metal powder and fine dust from air masses from China are contaminating people's skin and are considered as a major cause of skin aging and trouble.

Fine dust is invisible to small dust of less than 10㎛ diameter, it is known that contains toxic substances such as sulfate, nitrate and the like. These fine dusts are produced by photochemical reactions of toxic substances or heavy metals from automobile fumes and factory chimneys in the air.In case of exposure to high concentrations of fine dust environment, cough, eye irritation, skin trouble, and inflammation of lung and airway cells May cause.

In addition, fine dust weakens the metabolism of the skin and lowers sebum control function may worsen itching and dryness. Impurities in the skin may not be properly removed, causing problems, and the fat balance on the skin surface may be disrupted, resulting in dryness and itching. In addition, fine dust may promote skin aging. The absorbed fine dust stimulates the pigment cells of the skin, causing black mushrooms and wrinkles.

Fine dust can be removed to some extent by cleansing, but the finer it is, the stronger the adsorptive force, so deeply penetrates into the pores can not be completely removed by normal cleansing.

Meanwhile, in the cosmetic industry, many products using natural products have been developed to reduce skin irritation caused by various chemicals. Natural ingredients have less side effects on the skin, and as the consumer's response to cosmetics using natural ingredients has increased recently, the development value of cosmetics as a raw material for cosmetics is increasing.

Therefore, while improving skin stability, including extracts based on natural materials, the development of a cosmetic composition excellent in adsorption power of fine dust is required.

The present invention is to solve the above-mentioned problems of the prior art, an object of the present invention is to include a natural material as an active ingredient is excellent in biosafety, in particular, it provides a cosmetic composition excellent in fine dust adsorption power, including a water hyacinth extract It is.

According to one aspect of the present invention, there is provided a cosmetic composition for adsorption of fine dust comprising a water hyacinth extract as an active ingredient.

In one embodiment, the water hyacinth extract may be a fermentation extract prepared by inoculating a fermentation strain.

In one embodiment, the fermentation strain may be Lactobacillus kimchicus .

In one embodiment, the extract is water, anhydrous or hydrous lower alcohol having 1 to 4 carbon atoms, acetone, petroleum ether, ethyl acetate, butyl acetate, trichloromethane, dichloromethane, chloroform, hexane and 1,3-butylene It may be extracted with one or more solvents selected from the group consisting of glycols.

In one embodiment, the alcohol may be 65 to 75% concentration (v / v) of ethanol.

In one embodiment, the cosmetic composition is composed of flexible cosmetics, nourishing cosmetics, moisture cream, nutrition cream, massage cream, essence, ampoule, gel, eye cream, cleansing cream, cleansing foam, cleansing water, pack, spray and powder It may be formulated into one or more selected from the group.

According to another aspect of the invention, Eichhornia Provided is a cosmetic composition for antioxidant, anti-inflammatory, skin moisturizing, or anti-wrinkle comprising crassipes ) extract as an active ingredient.

According to the present invention, since the cosmetic composition includes a hyacinth extract, that is, an extract derived from a natural plant as an active ingredient, the skin improvement effect is excellent, and in particular, the wrinkle improvement and the adsorption and removal effect of fine dust may be remarkably improved. .

It is to be understood that the effects of the present invention are not limited to the above effects, and include all effects deduced from the configuration of the invention described in the detailed description or claims of the present invention.

The terminology used herein is to select general terms that are currently widely used as possible in consideration of the functions in the present invention, but may vary according to the intention or precedent of the person skilled in the art, the emergence of new technologies and the like. In addition, in certain cases, there is also a term arbitrarily selected by the applicant, in which case the meaning will be described in detail in the description of the invention. Therefore, the terms used in the present invention should be defined based on the meanings of the terms and the contents throughout the present invention, rather than the names of the simple terms.

Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art. Terms such as those defined in the commonly used dictionaries should be construed as having meanings consistent with the meanings in the context of the related art, and are not construed in ideal or excessively formal meanings unless expressly defined in this application. Do not.

The numerical range includes the numerical values defined in the range. All maximum numerical limits given throughout this specification include all lower numerical limits as if the lower numerical limits were clearly written. All minimum numerical limits given throughout this specification include all higher numerical limitations as if the higher numerical limit were clearly written. All numerical limitations given throughout this specification will include all better numerical ranges within the broader numerical range, as the narrower numerical limitations are clearly written.

Hereinafter, examples of the present invention will be described in detail, but the present invention is not limited by the following examples.

According to one aspect of the invention, there is provided a cosmetic composition comprising the hyacinth extract as an active ingredient. The cosmetic composition may include an extract of natural origin as an active ingredient, as well as excellent skin improvement effect and biosafety, and may minimize skin irritation.

The cosmetic composition is excellent in antioxidant, anti-inflammatory, skin moisturizing effect, as well as skin wrinkle improvement and fine dust adsorption due to various active ingredients contained in water hyacinth.

“ Eichhornia ” crassipes ) ”is an aquatic plant of the Aquaticaceae, which can be harvested year round in the tropical and subtropical regions, and is an attribute plant that can be harvested more than eight times a year if the water temperature is maintained at 20 degrees or higher. It has the ability to absorb and remove nitrogen and phosphorus, which are the causes of underwater eutrophication, at 1,700 kg / ha and 300 kg / ha. Since the water hyacinth contains a variety of effective ingredients beneficial to a variety of skin can be used for the purpose of improving the skin and has an excellent effect of adsorbing fine dust in the air.

The term "extract" refers to a solvent in which the active ingredient contained in the extract is transferred by contacting the solvent and the extract raw material under specific conditions. All may be included regardless. For example, extracting a component dissolved in a solvent from a natural product using water or an organic solvent, and may include both a specific component of the natural product, such as obtained by extracting only a specific component such as oil.

The mixed extract may be extracted with one or more solvents selected from the group consisting of purified water, lower alcohol having 1 to 4 carbon atoms, acetone, ethyl acetate, butyl acetate, and 1,3-butylene glycol, wherein the alcohol is 65 to 75% concentration (v / v) of ethanol.

The active ingredient contained in the raw material may be different in the extraction ratio according to the polarity of the solvent, the ethanol has excellent selectivity in the extraction of physiologically active substances of the natural raw material to achieve the optimal skin improvement effect by the ethanol extraction Can be.

Water and ethanol are different in polarity, the active ingredient extracted according to each polarity can be different, and the concentration of the ethanol can be appropriately controlled so that the optimum skin improvement effect can be implemented. At this time, if the concentration of the ethanol is more than 75% may not be achieved a proper yield, if less than 65% may not be sufficiently extracted the effective ingredient showing the skin improvement effect.

The extract is washed with water, washed with water, dried and pulverized, and extracted with conventional methods such as reflux circulation extraction, pressure extraction, and ultrasonic extraction for about 1 to 24 hours with a solvent having a volume of 8 to 12 times the weight of the raw material. And may be prepared by filtration. In addition, the extract may be obtained in a powder state by an additional process such as distillation under reduced pressure or freeze drying.

The extract may also include an extract that has undergone conventional purification. For example, the extract may be subjected to various purification methods additionally performed, such as separation using an ultrafiltration membrane having a constant molecular weight cut-off value, separation by various chromatography (manufactured for separation according to size, charge, hydrophobicity, or affinity). It may comprise a fraction obtained through.

The cosmetic composition may include a fermentation extract prepared by inoculating a fermentation strain in the water hyacinth extract.

The term "fermentation" refers to a process in which a specific microorganism uses an enzyme to decompose organic matter. Raw materials that have undergone fermentation may have a significantly improved skin improvement effect than before fermentation due to changes in the properties of the active ingredient. Metabolites contain various amino acids, organic acids, and antioxidants that are beneficial to the skin by fermentation, thereby promoting skin metabolism and imparting elasticity to the skin texture. The active ingredient particles may be reduced in size by fermentation and decompose toxins such as heavy metals, thereby reducing side effects such as skin troubles and allergies and improving absorption.

The "fermented extract" may be prepared by inoculating fermented strains after the hyacinth extract is dried naturally or using a rotary pressure reducer and a lyophilizer, diluted to a certain concentration in a specific solvent.

The fermentation strain is Lactobacillus genus (Lactobacillus sp.), Pseudomonas in coarse (Monascus sp.), Bacterial genus bifidobacteria (Bifidobacterium sp . ), Prevotella sp . , Fusobacterium sp . ), And Eubacterium sp . Strain, and may be selected from the group consisting of the strains of the genus Lactobacillus.

The strains of the genus Lactobacillus ( Lactobacillus kimchicus ), Lactobacillus pentosus ( Lactobacillus pentosus ), Lactobacillus brevis ( Lactobacillus brevis ), Lactobacillus plantarum ( Lactobacillus plantarum ), Lactobacillus casei (L) Or Lactobacillus acidophilus ( Lactobacillus acidophilus ), but may be preferably Lactobacillus kimchicus ( Lactobacillus kimchicus ).

The cosmetic composition is one or more selected from the group consisting of flexible cosmetics, nourishing cosmetics, moisture cream, nutrition cream, massage cream, essence, ampoule, gel, eye cream, cleansing cream, cleansing foam, cleansing water, pack, spray and powder Can be formulated.

The cosmetic composition may be for wrinkle improvement or fine dust adsorption, supple cosmetics, astringent cosmetics, nourishing cosmetics, lotions, nourishing cream, massage cream, essence, eye cream, cleansing cream, cleansing foam, cleansing water, pack, spray, And it may be formulated with one or more selected from the group consisting of a powder.

The cosmetic composition may be appropriately blended with other ingredients within the range of not impairing the object according to the present invention in accordance with the type or purpose of use of the formulation other than the active ingredient in each formulation.

The cosmetic composition may be prepared according to the quality or function of the final product, such as fatty substances, organic solvents, solubilizers, thickeners, gelling agents, softeners, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, surfactants, water, Commonly used in cosmetics or dermatology, such as ionic or nonionic emulsifiers, fillers, metal ion rod blockers, chelating agents, preservatives, blockers, wetting agents, essential oils, dyes, pigments, and hydrophilic or lipophilic active agents Supplement may be additionally included.

However, the adjuvant and its mixing ratio are preferably selected appropriately so as not to affect the desirable properties of the cosmetic composition according to the present invention.

Hereinafter, with reference to the accompanying drawings will be described in detail with respect to embodiments of the present invention, it is apparent that the present invention is not limited by the following examples.

Example 1 Water hyacinth extract

The water was washed with hyacinth, dried at room temperature and pulverized to obtain 100 g of coarse pulverized product. 100 g of each pulverized product was immersed in 10-fold volume of 70% concentration (v / v) ethanol and extracted under reflux for 12 hours at a temperature of 80 ° C.

The extract was filtered with 250 mesh and 0.5 μm filter paper and extracted three times in the same manner for the remaining raw materials and then cooled at room temperature. The extract was concentrated under reduced pressure at 20 ° C. and lyophilized to give a solid.

Example 2: Water hyacinth fermented extract

After diluting the water hyacinth extract of Example 1 with purified water to 5% by weight (v / v), Bifidobacterium bifiderum ( Bifidobacterium) bifidum ) was inoculated and fermented. The fermented extract was filtered with 500 mesh and 0.2 μm filter paper to obtain a hyacinth fermented extract and used as a sample of Example 2.

Example 3: Water hyacinth fermented extract

A sample of Example 3 was obtained in the same manner as in Example 2, except that Lactobacillus plantarum was used as the fermentation strain.

Example 4: Water hyacinth fermented extract

A sample of Example 4 was obtained in the same manner as in Example 2 except for using Lactobacillus kimchicus as a fermentation strain.

Experimental Example 1: Skin Safety Test

In order to verify skin safety of the extracts of Examples 1 to 4, MTT assay experiments were performed on fibroblasts (HDF), melanocytes (B16F10), and keratinocytes (HaCaT).

After the samples obtained in Examples 1 to 4 were suspended in purified water by concentration, cell viability was measured.

The extracts of Example 1 were suspended at concentrations of 0, 20, 40, 60, 80, and 100 ug / mL, and the fermentation extracts of Examples 2 to 4 were prepared at concentrations of 0.001, 0.005, 0.01, 0.05, and 0.1% ( w / w), respectively.

Cytotoxicity was performed by modifying Mosmann's method of measuring cell viability using MTT {3- (4,5-dimethylthiazol-2-yl) -2-5-diphenyltetrazolium bromide} reagent.

Fibroblasts (HDF), melanocytes (B16F10), and keratinocytes (HaCaT) were each dispensed in 96-well plates at a concentration of 1 × 10 ⁴ cells / well and incubated at 37 ° C., 5% CO ₂ for 48 hours. It was.

After removing the culture medium and incubating the sample for 48 hours in a concentration-treated medium, the medium was removed and washed repeatedly with PBS (Phosphate buffered saline).

MTT was dissolved in PBS at 5 mg / mL, 50 L was added, and incubated for 48 hours at 37 ° C. and 5% CO ₂ . 100 L of DMSO (dimethyl sulfoxide) was added per well, stirred for 10 minutes, and the absorbance was measured at 540 nm.

As a result, no cytotoxicity was confirmed at all concentrations of each sample. The results suggest that the extract is not only harmless to the human body but also has excellent human safety.

Experimental Example 2 Antioxidant Activity Test

Free radical decomposition effect

The free radical scavenging activity was measured using the free radical DPPH (1, 1-diphenyl-2-picryl hydrazyl).

4 mL methanol was added to the test tube, and the extract was added at different concentrations. 1 mL of 0.15 mM DPPH solution was added thereto to react at room temperature for 30 minutes, and the absorbance was measured at 520 nm.

Initial (Ai) and blank (Ab) without substrate and DPPH were measured, and α-tocopherol and BHT were used as positive controls. The results are shown in Table 1 below.

division	0 μg / mL	20 μg / mL	40 μg / mL
α-tocopherol	0	50.8	75.9
BHT	0	42.4	66.7
Example 1	0	39.3	61.4
Example 2	0	42.6	66.1
Example 3	0	45.2	69.7
Example 4	0	47.7	73.2

[DPPH% Clearance]

The extract was evaluated to have higher free radical scavenging activity than α-tocopherol and BHT, which are well known in the art. Therefore, the decomposition of free radicals may be promoted by the extract, and the results suggest that skin aging and skin condition improvement effect by the antioxidant activity is excellent.

Free radical species test in cells (DCF-DA assay)

After treating the extracts of Examples 1 to 4, the amount of reactive oxygen species generated in the cells stimulated with silica was compared with the negative control group to evaluate the ability to remove reactive oxygen species (ROS) (Cho et al., 1999; Kim et al., 2002).

Raw 264.7 cells were incubated in 96 well plates at a concentration of 1 × 10 ⁴ cells / well. The medium was exchanged with fresh medium and the cells were incubated for 24 hours after treatment of the extract.

Add 10.0 μL of WST-1 solution (EZ-CYTOX, DOGEN, Korea) to each well, incubate the cells for 1 hour, and measure the absorbance at a measurement wavelength of 450 nm and reference wavelength of 655 nm with an ELISA reader (Bio-Rad). It was.

The extract was diluted with dimethylsulfoxide (DMSO, Sigma-Aldrich, USA) to a maximum concentration of 20μM based on the final concentration, DMSO was used as a blank test (negative control). The experiment was repeated three times to secure the reliability.

The experiment was performed under the same cell line and the same cell culture conditions as the WST-1 assay, a cell activity measurement test. The cultured Raw 264.7 cells were washed once with a solution of Phosphate Buffered Saline (PBS; sodium chloride 137 mM, phosphate buffer 10 mM, potassium chloride 2.7 mM, all from Biopure, Canada) and then harvested Raw 264.7 cells. Suspended in 15 mL PBS solution, 15 μL of 20 mM 2 ', 7'-Dichlorofluorescein diacetate was added and incubated for 1 hour.

After centrifugation at 1200 rpm for 2 minutes, the solution was washed once with PBS solution, diluted to 1 × 10 ⁵ cells / mL, treated with a control material and extract, and incubated for 10 minutes. 50 μL of 20 mg / mL silica was incubated for 1 hour. Cell pellets obtained by centrifugation at 6000 rpm for 5 minutes were dispersed in 200 μL PBS solution and seeded in 96 well plates.

The amount of reactive oxygen species was evaluated by measuring the fluorescence at 485 nm excitation / 535 nm emission using a fluorescence microplate reader.

The extract treated in the reactive oxygen species measurement test for the sample concentration was used as the sample concentration set by measuring the cell activity.

As a negative control, DMSO was used instead of the sample material. As a positive control, ascorbic acid (Sigma-Aldrich) was diluted to 100 mg / mL in DMSO and used at a concentration of 100 μg / mL.

Based on the results, active oxygen species measurement test was performed for the same concentration, and the treated extract was used at a concentration of 50 mg / L. The experiment was repeated four times to secure the reliability. The measurement results are shown in Table 2 below.

division	0 μg / mL	20 μg / mL	40 μg / mL
Ascorbic acid	0	58.3	73.5
Example 1	0	48.2	61.5
Example 2	0	51.3	65.8
Example 3	0	53.7	67.4
Example 4	0	57.2	70.1

 [% Of control]

The extract was evaluated to equal levels of ascorbic acid and free radical scavenging activity well known in the art. That is, the results suggest that the active oxygen species removal ability of Examples 1 to 4 is excellent, and the antioxidant effect due to free radical scavenging activity is excellent.

Experimental Example 3: Anti-inflammatory Activity Test

NF-kB transcription inhibitory effect

NF-kB transcription inhibition effect was measured by NF-kB reporter assay. INOS, COX-2, IFN-β, TNF-α, etc. induced by LPS are expressed by the transcription factor NF-kB. The NF-kB is inactivated in complex with IkB in the cytoplasm. When I-kB-α is phosphorylated by I-kB-αkinase, the bound complex is decomposed and activated by NF-kB to move into the nucleus. )do. Induces expression of target gene of NF-kB and causes inflammatory action.

In addition, TRIF, which acts on inflammatory diseases such as multiple sclerosis, is a TIP-domain with an interferon-β-inducing receptor, which is a receptor that responds to the activity of TLRs (toll-like-receptors). It recognizes the specific composition of activating the immune response to the antigen. The receptor recognizes well-conserved pathogenic patterns and activates signals to stimulate the release of inflammatory cytokines. IRF-3 (interferon stimulatory gene 3) is a transcription activating factor induced by interferon α / β stimulation and binds to the interferon stimulation sequence (ISRE) in the transcriptional regulatory region of interferon-induced genes and activates its transcription.

NF-kB depending on the presence of receptor molecules (MyD88 and TRIF), such as β-galactosidase, using the PEI method in 12-well plates according to the manufacturer's protocol (Jin et al., 2009). 1 μg of plasmid containing each of Luc or TRIF was transfected into HEK293 (1 × 10 ⁶ cells / ml) cells. After culturing for 24 hours, LPS (100 ng / ml) and the extract were treated, and after 24 hours, luciferase assays were used to measure NF-kB activity and IRF-3 activity.

The luciferase assay was performed using a luciferase assay system (Promega Co., USA) reported in the manufacturer's protocol (Jung et al., 2009), and the analysis results are shown in Table 3 below.

division		NF-kB activity	IRF-3 activity
Control group (blank group)		1.00	1.00
Example 1	0 μg / mL	3.78	4.25
	20 μg / mL	2.12	2.26
	40 μg / mL	1.59	1.49
Example 2	0 μg / mL	3.72	4.23
	20 μg / mL	2.09	2.18
	40 μg / mL	1.52	1.44
Example 3	0 μg / mL	3.65	4.16
	20 μg / mL	2.05	2.16
	40 μg / mL	1.52	1.42
Example 4	0 μg / mL	3.63	4.12
	20 μg / mL	2.02	2.12
	40 μg / mL	1.37	1.35

 [Fold, Luciferase activity]

Referring to Table 3, the treatment of the extract significantly reduced the activity of NF-kB and IRF-3. The results suggest that the extract effectively inhibits the transcription of NF-kB, thereby inhibiting or alleviating inflammation and preventing and improving inflammatory diseases.

Inflammation-related Protein mRNA Quantitation

The anti-inflammatory activity of the extract was evaluated by analyzing the expression levels of TNF-α, iNOS, and COX-2.

RNA was prepared from LPS treated RAW264.7 cells. The method was prepared using Trizol Reagent (Gibco BRL) as described by Lee et al., 2008. All RNA was used after storage at -70 ℃. Semi-quantitative RT reaction was performed using MuLV reverse transcriptase.

All RNA (1 μg) was incubated with oligo-dT15 at 70 ° C. for 5 minutes and mixed with 5 × first strand buffer, 10 mM dNTPs and 0.1 M DTT. The reaction mixture was further incubated at 37 ° C. for 5 minutes and incubated for 60 minutes after the addition of MuLV reverse transcriptase (2 U). The reaction was terminated by incubating at 70 ° C. for 10 minutes. RNase H was added to remove all RNA and PCR reactions were performed (conditions: 2 μL cDNA, 4 μM 5 ′ and 3 ′ primers, 10 × buffer (10 mM Tris-HCl, pH 8.3; 50 mM KCl and 0.1). % Triton X-100), 250 μΜ dNTPs, 25 mM MgCl ₂ and 1 unit of Taq DNA polymerase (Promega, USA) The PCR reaction was denatured at 94 ° C. for 30 seconds and after annealing at 55 to 60 ° C. for 30 seconds, 72 It superposed | polymerized at 42 degreeC for 42 second, and finally superposed | polymerized at 72 degreeC for 5 minutes.

Primers of TNF-α, iNOS, and COX-2 are shown in Table 4 below.

division	Forward primer	Reverse primer
iNOS	5'-FGGAGCCTTTAGACCTCAACAGA-3 '	5'-RTGAACGAGGAGGGTGGTG-3 '
TNF-α	5'-FTGCCTATGTCTCAGCCTCTTC-3 '	5'-RGAGGCCATTTGGGAACTTCT-3 '
COX-2	5'-FCACTACATCCTGACCCACTT-3 '	5'-RATGCTCCTGCTTGAGTATGT-3 '

MRNA expression level measurement results according to the extract treatment is shown in Table 5. MRNA analysis of RAW264.7 cells resulted in increased expression of TNF-α, iNOS, and COX-2 by the addition of LPS. On the other hand, the expression of TNF-α, iNOS, and COX-2 induced by LPS decreased in a concentration-dependent manner when the extract was treated.

division		iNOS expression level	TNF-α expression level	COX-2 expression level
Control group (blank group)		1.00	1.00	1.00
Example 1	0% (w / w)	3.45	3.10	3.20
	0.01% (w / w)	2.20	1.75	2.10
	0.1% (w / w)	1.60	1.35	1.80
Example 2	0% (w / w)	3.35	3.06	3.15
	0.01% (w / w)	2.15	1.69	2.05
	0.1% (w / w)	1.52	1.30	1.67
Example 3	0% (w / w)	3.31	3.03	3.10
	0.01% (w / w)	2.05	1.64	2.01
	0.1% (w / w)	1.45	1.23	1.56
Example 4	0% (w / w)	3.25	2.95	3.01
	0.01% (w / w)	1.96	1.54	1.95
	0.1% (w / w)	1.36	1.19	1.34

 [Fold, GAPDH normalized]

Experimental Example 4 Evaluation of Skin Moisturizing Effect

The sample powders obtained in Examples 1 to 4 were suspended in purified water, diluted by concentration, and the skin moisturizing effect was evaluated.

The expression level of the HAS2 gene and AQP3 gene involved in skin moisturization in human keratinocytes (Keratinocytes) was measured at the mRNA level.

Human keratinocytes (HaCaT) were incubated at 37 ° C and 5% CO _{2 in} a 100 mm / 60.1 cm culture dish with Dulbecco's Modified Eagle's Medium (DMEM), 10% Fatal bovine serum (FBS) and 1% Antibiotic-Antimycotic. It was.

Keratinocytes were dispensed into 96 well plates at a concentration of 1 × 10 ⁶ cells / mL and incubated for 24 hours. After exchange with DMEM free medium was treated extracts of Examples 1 to 4 diluted by concentration and incubated for 24 hours.

Cells were recovered, washed with cold phosphate buffer (PBS), 1 ml of Trizol reagent [TRIzol ™ reagent, Life Technologies, Inc.] was added and total RNA extracted.

Gene expression changes were measured by qRT-PCR (quantitative real time PCR), which binds the fluorescent material to the DNA product amplified in the polymerase chain reaction (PCR) and continuously detects the fluorescent material.

In order to quantify changes in HAS2 and AQP3 gene expression, fluorescence values emitted by PCR products were measured through SYBR green I (Invitrogen). A reaction solution was prepared by mixing 0.2 μM primer, 50 mM KCl, 20 mM Tris / HCl pH 8.4, 0.8 mM dNTP, 0.5U Extaq DNA polymerase, 3 mM MgCl ₂ , and 1 × SYBR green.

Preliminary denaturation at 94 ° C for 3 minutes with Linegene K (BioER, China) followed by denaturation, annealing and polymerization at 94 ° C for 30 seconds, 58 ° C at 30 ° C A total of 40 cycles were performed for 30 seconds at 72 ° C., and the fluorescence intensity was measured after each cycle.

PCR results were verified by melting curve for each result. The threshold cycle (Ct) of each gene was normalized to the Ct of β-actin, and then analyzed by comparing the change in Ct.

The Ct value is the number of cycles when the amount of fluorescence generated by the PCR product (the number of amplified PCR products) reaches a predetermined reference value above the basic value. Primer sequences used in the experiment are shown in Table 6 below.

Gene	Forward primer	Reverse primer
HAS-2	5'-TTTCTTTATGTGACTCATCTGTCTCACCGG-3 '	5'-ATTGTTGGCTACCAGTTTATCCAAAGGG-3 '
AQP3	5′- ACCCTCATCCTGGTGATGTTTG-3 ′	5′- TCTGCTCCTTGTGCTTCACAT-3 ′

HAS2 and AQP3 mRNA expression level measurement results are shown in Table 7 below, mRNA expression level was increased concentration-dependently according to the treatment of the extract. The higher the HAS2 and AQP3 expression promoting ability can be evaluated as an excellent skin moisturizing effect.

The results suggest that the extract can enhance skin moisturization by promoting the expression of aquaporin protein and the synthesis of hyaluronic acid.

division		HAS2 expression level	AQP3 expression level
Example 1	0 μg / mL	1.00	1.00
	50 μg / mL	1.23	1.07
	100 μg / mL	1.86	1.24
Example 2	0 μg / mL	1.00	1.00
	50 μg / mL	1.47	1.16
	100 μg / mL	2.21	1.37
Example 3	0 μg / mL	1.00	1.00
	50 μg / mL	1.61	1.26
	100 μg / mL	2.43	1.48
Example 4	0 μg / mL	1.00	1.00
	50 μg / mL	1.76	1.35
	100 μg / mL	2.86	1.54

 [Fold of control]

Experimental Example 5: Collagen Biosynthesis Induction Test

The synthesis of collagen according to the growth of fibroblasts was observed.

Human dermal fibroblasts were dispensed in 96 well plates at a concentration of 1.0 × 10 ⁴ cells / mL, and cultured for 3 days at 37 ° C., 5% CO ₂ , and humidified conditions. A medium containing 10 wt% of LSGS (Low Serum Growth Supplement, manufactured by Kurashiki Boseki Co., Ltd.) in Medium 106S (manufactured by Kurashiki Boseki Co., Ltd.) was used at 100 µL per well.

The medium was exchanged with DMEM (Dulbecco's Modified Eagle's Medium, manufactured by Sigma), and incubated after treating the extracts 1 to 4 at a concentration of 50 mg / L. Purified water was added without treating the extract as a control.

After culturing the cells for 7 days, the culture medium was collected, and the concentration of type I collagen secreted in the culture medium was quantified by an enzyme-linked immunoassay (Procollagen type I c-peptide EIA Kit; manufactured by Takara Bio Co., Ltd.).

The amount of collagen in each test sample culture was calculated based on the type I collagen amount of the control group (100%), and the measurement results are shown in Table 8 below.

division	Collagen Production (%)
Example 1	171.3
Example 2	182.8
Example 3	191.5
Example 4	198.3
Control	101.2

Experimental Example 6: Expression Measurement Test of COL1A1 and MMP1

The sample powders obtained in Examples 1 to 4 were suspended in purified water, diluted by concentration, and the effect of improving wrinkles was evaluated.

Changes in the expression of COL1A1, one of the representative collagens expressed by human dermal fibroblasts, ELN, one of the elastins, and MMP1, a collagen degrading enzyme, were measured.

The extracts of Examples 1 to 4 diluted by concentration were treated to human dermal fibroblasts and cultured for 24 hours to determine the degree of collagen expression change.

Gene expression changes were measured by qRT-PCR (quantitative real time PCR), which binds the fluorescent material to the DNA product amplified in the polymerase chain reaction (PCR) and continuously detects the fluorescent material.

In order to quantify changes in COL1A1 and MMP1 gene expression, the fluorescence values emitted by PCR products were measured through SYBR green I (Invitrogen). A reaction solution was prepared by mixing 0.2 μM primer, 50 mM KCl, 20 mM Tris / HCl pH 8.4, 0.8 mM dNTP, 0.5U Extaq DNA polymerase, 3 mM MgCl ₂ , and 1 × SYBR green.

Preliminary denaturation at 94 ° C for 3 minutes with Linegene K (BioER, China) followed by denaturation, annealing and polymerization at 94 ° C for 30 seconds, 58 ° C at 30 ° C A total of 40 cycles were performed for 30 seconds at 72 ° C., and the fluorescence intensity was measured after each cycle.

PCR results were verified by melting curve for each result. The threshold cycle (Ct) of each gene was normalized to the Ct of β-actin, and then analyzed by comparing the change in Ct.

The Ct value is the number of cycles when the amount of fluorescence generated by the PCR product (the number of amplified PCR products) reaches a predetermined reference value above the basic value. Primer sequences used in the experiment are shown in Table 9 below.

Gene	Forward primer	Reverse primer
β-actin	5-GGATTCCTATGTGGGCGACGA-3	5-CGCTCGGTGAGGATCTTCATG-3
COL1A1	5-AGGGCCAAGACGAAGACATC-3	5-AGATCACGTCATCGCACAACA-3
MMP1	5-TCTGACGTTGATCCCAGAGAGCAG-3	5-CAGGGTGACACCAGTGACTGCAC-3

Expression measurement results according to the treatment of the extract is shown in Table 10 below. The expression level of COL1A1 mRNA increased in a concentration dependent manner, whereas the expression level of MMP1 mRNA decreased in a concentration dependent manner.

The higher the MMP-1 expression inhibitory ability can be evaluated that the skin wrinkle improvement and anti-aging effect is excellent, the result is that the extract can promote the production of collagen and thereby improve the skin elasticity and wrinkles bar use of skin care It can be used as

division		COL1A1 expression level	MMP1 expression level
Example 1	0% (w / w)	1.00	1.00
	0.01% (w / w)	1.82	0.79
	0.1% (w / w)	2.62	0.45
Example 2	0% (w / w)	1.00	1.00
	0.01% (w / w)	1.89	0.77
	0.1% (w / w)	2.95	0.42
Example 3	0% (w / w)	1.00	1.00
	0.01% (w / w)	2.07	0.79
	0.1% (w / w)	3.09	0.40
Example 4	0% (w / w)	1.00	1.00
	0.01% (w / w)	1.95	0.72
	0.1% (w / w)	3.16	0.37

 [Fold, β-actin normalized]

Experimental Example 7: Wrinkle improvement evaluation

The lotion formulated with extracts of Examples 1 to 4 was applied to 50 women aged 40 to 60 years every morning and evening twice a month for 1 month to evaluate the degree of wrinkle improvement.

Through wrinkle analysis, the total number of wrinkles, the length of wrinkles, and the area of wrinkles were measured and quantified, and the degree of wrinkle improvement was evaluated through image analysis. The evaluation results are shown in Table 11 below.

division		% Reduction in total wrinkles	Wrinkle Area Reduction (%)	Wrinkle Length Reduction (%)
Example 1	Week 0	0.0	0.0	0.0
	4 Weeks	20.0	8.2	8.5
	8 Weeks	22.7	11.4	15.8
	12 Weeks	24.9	19.8	23.8
Example 2	Week 0	0.0	0.0	0.0
	4 Weeks	22.5	9.5	10.3
	8 Weeks	23.8	14.2	18.9
	12 Weeks	26.2	22.6	25.8
Example 3	Week 0	0.0	0.0	0.0
	4 Weeks	23.8	10.2	12.8
	8 Weeks	25.7	15.7	21.3
	12 Weeks	28.5	24.6	27.5
Example 4	Week 0	0.0	0.0	0.0
	4 Weeks	25.9	12.4	13.6
	8 Weeks	28.8	17.8	25.3
	12 Weeks	31.2	29.5	32.4

Referring to Table 11, the cream formulated with the extracts of Examples 1 to 4 significantly reduced the total number of wrinkles, wrinkle area, wrinkle length, skin wrinkle parameters, the result is that the extract improves skin wrinkles and skin beauty It can be used as a purpose.

Experimental Example 8: Evaluation of skin texture improvement effect

The skin texture improvement effect of the cream formulated extract of Examples 1 to 4 was verified.

As a control, a cream formulated with aloe vera extract, which is known to have a moisturizing function, was used.

Forty women among 20 to 40 year olds were measured using PRIMOS light (field of view 45, flexible 3D measuring, GFMesstechnik GmbH) to measure skin prior to use of the subject and re-measure skin after using the cream. It was.

The measured value was expressed as the average of three times except the maximum value and minimum value of Ra. The higher the measured value means a lot of skin wrinkles, the results are shown in Table 12.

[Ra, (Arbitrary unit)]

division	Example 1	Example 2	Example 3	Example 4	Control
Before use	15.70	15.73	15.60	15.68	15.70
2 weeks later	14.26	13.95	13.55	12.97	14.54
4 weeks later	12.25	11.96	11.76	10.95	13.91

Referring to Table 12, the experimental group using the cream formulated with the extract of Examples 1 to 4 was evaluated that the measured value after use is significantly reduced and the skin texture improvement effect is remarkably excellent.

In particular, it was confirmed that the cream containing the hyacinth lactobacillus kimchicus fermented extract (Example 4) has the most excellent skin texture improvement effect, when the cream of the control group does not contain the hyacinth extract, the skin texture improvement effect is significantly insufficient. It was.

Experimental Example 9 Polycyclic Aromatic Hydrocarbon Removal Effect Test

The adsorption effect of polycyclic aromatic hydrocarbons (PAH), which is a large amount of harmful air in fine dust, was tested.

After removing the fur of the pig skin (1cm x 1cm) specimens were prepared and washed with PBS for 10 minutes.

600 ppm of Naphtalene, Acenaphthylene, Acenaphthene, Fluorene, Fluorene, Pyrene, Chrysene, and Benzopyrene after washing It was dipped in a solution (pH 11) for 2 hours and dried at room temperature.

Examples 1 to 4 extracts were treated with the specimens at a concentration of 50 mg / L and reacted for 3 hours, and the control group was coated with PBS and reacted for 3 hours.

After washing with PBS, the specimen was cut to a certain weight and ground with a homogenizer. The pulverized tissue was dried with anhydrous sodium sulfate (Na ₂ SO ₄ ) and then polycyclic aromatic hydrocarbons were extracted from the tissue by Soxlet extraction and Liquid-liquid extraction (Husain et al., 1997; Takatsuki et al., 1985), quantified by HPLC. Evaluation results were expressed as the ratio of the control group to the experimental group, the quantitative results are shown in Table 13.

[% of control]

division	Example 1	Example 2	Example 3	Example 4
naphthalene	7.1	5.5	4.7	3.4
Acenaphthylene	10.5	8.2	6.4	5.1
Fluorene	9.3	7.3	5.1	2.8
Fluoranthene	9.3	6.9	4.5	3.2
Pyren	3.6	2.5	1.9	1.8
Krissen	4.9	3.9	2.3	1.3
Benzopyrene	3.1	2.7	2.2	1.3

Referring to Table 13, the polycyclic aromatic hydrocarbons were significantly reduced by the treatment of the extracts of Examples 1 to 4, and the results indicate that the extract contains a large amount of fine dust and air, which may cause trouble to the skin. It can be effectively removed.

Experimental Example 10: fine dust removal effect test

The fine dust removal effect of the cosmetic composition which formulated Examples 1-4 was tested. After applying the fine dust corresponding to the PM2.5 to PM10 size to the artificial skin, it was washed using a cleansing cream formulated Examples 1 to 4.

The percentage of fine dust before and after the cleansing cream was washed and a cleansing cream containing no extracts of Examples 1 to 4 was used as a control. The evaluation results are shown in Table 14 below.

division	Fine dust removal rate (%)
Example 1	92%
Example 2	93%
Example 3	93%
Example 4	96%
Control	76%

Referring to Table 14, the fine dust was effectively removed by the cleansing cream formulated with the extracts of Examples 1 to 4, while the fine dust removal effect of the control containing no extract was relatively insufficient.

The foregoing description of the present invention is intended for illustration, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive. For example, each component described as a single type may be implemented in a distributed manner, and similarly, components described as distributed may be implemented in a combined form.

The scope of the invention is indicated by the following claims, and it should be construed that all changes or modifications derived from the meaning and scope of the claims and their equivalents are included in the scope of the invention.

Claims (7)

1. Water hyacinth ( Eichhornia) crassipes ) Cosmetic composition for adsorption of fine dust comprising the extract as an active ingredient.
2. The method of claim 1,

   The water hyacinth extract is a cosmetic composition is a fermentation extract prepared by inoculating fermentation strain.
3. The method of claim 2,

   The fermentation strain is Lactobacillus kimchicus cosmetic composition ( Lactobacillus kimchicus ).
4. The method of claim 1,

   The extract is selected from the group consisting of water, anhydrous or hydrous lower alcohols having 1 to 4 carbon atoms, acetone, petroleum ether, ethyl acetate, butyl acetate, trichloromethane, dichloromethane, chloroform, hexane and 1,3-butylene glycol Cosmetic composition extracted with one or more solvents.
5. The method of claim 4, wherein

   The alcohol is a cosmetic composition of 65 to 75% concentration (v / v) ethanol.
6. The method according to any one of claims 1 to 5,

   Cosmetics formulated with one or more selected from the group consisting of softening cream, nourishing cream, moisture cream, nourishing cream, massage cream, essence, ampoule, gel, eye cream, cleansing cream, cleansing foam, cleansing water, pack, spray and powder Composition.
7. Water hyacinth ( Eichhornia) Crassipes ) Cosmetic composition for antioxidant, anti-inflammatory, skin moisturizing, or wrinkle improvement comprising the extract as an active ingredient.