KR101917740B1 - Cosmetic compositon containing extracts of water hyacinth - Google Patents

Abstract

The invention water hyacinth (Eichhornia The present invention provides a cosmetic composition for adsorbing fine dust comprising an extract of Crassipes as an active ingredient.

Description

COSMETIC COMPOSITON CONTAINING EXTRACTS OF WATER HYACINTH <br> <br> <br> Patents - stay tuned to the technology COSMETIC COMPOSITON CONTAINING EXTRACTS OF WATER HYACINTH

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

Fine dusts are air pollutants that contain harmful substances such as sulfur dioxide, nitrogen oxides, ozone, and carbon monoxide, which are generated in automobiles and factories and mean long-term floating substances in the atmosphere.

Rapid industrialization, automobile exhaust gas, heavy metal dusts caused by air masses moved from China, and foreign pollutants such as fine dust pollute people's skin and are considered to be major cause of skin aging and trouble.

Fine dust refers to a small dust less than 10 μm in diameter that is invisible and is known to contain toxic substances such as sulfate and nitrate. These fine dusts are produced by the photochemical reaction in the air such as toxic substances or heavy metals from automobile soot and factory smokes. When exposed to a high concentration of fine dust environment, cough, eye sting, skin trouble, Lt; / RTI &gt;

In addition, fine dust may weaken the skin metabolism and deteriorate sebum control function, which may exacerbate itching and dryness. The waste matter in the skin is not properly removed, causing trouble, and the fat balance on the surface of the skin may be disrupted to cause dryness and itching. In addition, fine dust can promote skin aging. Absorbed fine dust stimulates the pigment cells of the skin, causing blotches and wrinkles.

Fine dust can be removed to some extent by cleansing, but it is stronger in adsorption force as it is finer, so it penetrates deeply into the pores and can not be completely removed by general cleansing.

Meanwhile, in the cosmetics industry, many products using natural products have been developed to reduce skin irritation caused by various chemical substances. In addition to low adverse effects on skin, natural materials have recently become increasingly appreciated as a cosmetic raw material as consumers' response to cosmetics using natural materials has increased.

Accordingly, there is a demand for development of a cosmetic composition having excellent adsorption ability of fine dusts while enhancing skin stability, including extracts based on natural materials.

The object of the present invention is to provide a cosmetic composition comprising a natural material as an active ingredient and having excellent biocompatibility and, in particular, an extract of water hyacinth, .

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

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

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

In one embodiment, the extract is selected from the group consisting of water, anhydrous or lower alcohols having 1 to 4 carbon atoms, acetone, petroleum ether, ethyl acetate, butyl acetate, trichloromethane, dichloromethane, chloroform, Glycol, &lt; / RTI &gt;

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

In one embodiment, the cosmetic composition is comprised of a softening agent, a nutritional lotion, a water cream, a nutritional cream, a massage cream, an essence, an ampoule, a gel, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, Lt; / RTI &gt; may be formulated into one or more selected from the group.

According to another aspect of the invention, water hyacinth (Eichhornia The present invention provides a cosmetic composition for antioxidant, anti-inflammation, skin moisturizing, or wrinkle improvement comprising crassipes extract as an active ingredient.

According to the present invention, the cosmetic composition of the present invention is excellent in skin-improving effect because it contains an extract of water hyacinth, that is, an extract derived from a natural plant, and in particular, the effect of improving wrinkles and adsorption and removal of fine dusts can be remarkably improved .

It should be understood that the effects of the present invention are not limited to the effects described above, but include all effects that can be deduced from the description of the invention or the composition of the invention set forth in the claims.

As used herein, the terminology used herein is intended to encompass all commonly used generic terms that may be considered while considering the functionality of the present invention, but this may vary depending upon the intent or circumstance of the skilled artisan, the emergence of new technology, and the like. Also, in certain cases, there may be a term selected arbitrarily by the applicant, in which case the meaning thereof will be described in detail in the description of the corresponding invention. Therefore, the term used in the present invention should be defined based on the meaning of the term, not on the name of a simple term, but on the entire contents of the present invention.

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 to which this invention belongs. Terms such as those defined in commonly used dictionaries are to be interpreted as having a meaning consistent with the contextual meaning of the related art and are to be interpreted as either ideal or overly formal in the sense of the present application Do not.

The numerical range includes numerical values defined in the above range. All numerical limitations of all the maximum numerical values given throughout this specification include all lower numerical limitations as the lower numerical limitations are explicitly stated. All the minimum numerical limitations given throughout this specification include all higher numerical limitations as the higher numerical limitations are explicitly stated. All numerical limitations given throughout this specification will include any better numerical range within a broader numerical range, as narrower numerical limitations are explicitly stated.

Hereinafter, embodiments of the present invention will be described in detail, but it should be apparent that the present invention is not limited by the following examples.

According to one aspect of the present invention, there is provided a cosmetic composition comprising a water hyacinth extract as an active ingredient. The cosmetic composition of the present invention contains an extract of natural origin as an active ingredient, so that it is excellent in skin improving effect and biosafety, and skin irritation can be minimized.

The cosmetic composition of the present invention is excellent in antioxidation, anti-inflammation, and skin moisturizing effect as well as skin wrinkle improvement and fine dust adsorption effect due to various active ingredients contained in water hyacinth.

The "water hyacinth (Eichhornia crassipes "is an aquatic plant with water fauna, which can be harvested all year round in tropical and subtropical areas. In Korea, it can be harvested more than 8 times a year if the water temperature is kept above 20 degrees. It has the ability to absorb and remove nitrogen and phosphorus, which are the cause of underwater eutrophication, to the level of 1,700 kg / ha and 300 kg / ha, and is also used as an underwater purification plant. Since the water hyacinth contains various active ingredients beneficial to various skin, it can be used for skin improvement and has excellent effect of adsorbing fine dust in the air.

The above-mentioned "extract" refers to a solvent in which an effective ingredient contained in an extraction raw material has been transferred by contacting a solvent and an extraction raw material under specific conditions. If a substance is separated from a natural product, the extraction method, You can include them all regardless. For example, it may include extracts of components dissolved in a solvent from natural products by using water or an organic solvent, and specific components of natural products, such as oils obtained by extracting only specific components such as oil.

The mixed extract may be extracted with at least one solvent selected from the group consisting of purified water, a lower alcohol having 1 to 4 carbon atoms, acetone, ethyl acetate, butyl acetate, and 1,3-butylene glycol, Ethanol at 75% concentration (v / v).

The extraction ratio of the active ingredient contained in the raw material may be different depending on the polarity of the solvent. Since the ethanol is excellent in selectivity for extracting a physiologically active substance from a natural raw material, the ethanol extract can realize an optimal skin improving effect .

Water and ethanol have different polarities, so that the effective ingredient to be extracted according to each polarity can be changed, and the concentration of ethanol can be appropriately controlled so that an optimal skin improving effect can be realized. At this time, if the concentration of ethanol is more than 75%, an adequate yield may not be realized. If the concentration of ethanol is less than 65%, the effective ingredient showing skin improvement effect may not be extracted sufficiently.

The extract is washed with water, dried and pulverized, and then extracted with a solvent having a volume of 8 to 12 times the weight of the raw material for about 1 to 24 hours by a conventional method such as circulation extraction, pressure extraction or ultrasonic extraction And filtering. In addition, the extract can be obtained in powder form by an additional process such as vacuum distillation or freeze-drying.

The extract may also contain an extract which has been subjected to a conventional purification process. For example, the extract may be subjected to various purification methods 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) Lt; RTI ID = 0.0 &gt; fractions. &Lt; / RTI &gt;

The cosmetic composition may include a fermentation extract prepared by inoculating a fermentation strain into the Buttechamp extract.

The term &quot; fermentation &quot; means a process in which a specific microorganism decomposes an organic substance using an enzyme. The raw material after the fermentation process can remarkably increase the skin improvement effect before the fermentation due to the change of the characteristics of the active ingredient. Metabolites contain various amino acids, organic acids, and antioxidants beneficial to skin by fermentation, which can promote skin metabolism and impart elasticity to skin texture. The active ingredient particles are small in size due to fermentation, and the toxicity such as heavy metals is decomposed, so that skin side effects such as skin troubles and allergies can be reduced and the absorption rate can be enhanced.

The &quot; fermented extract &quot; can be prepared by naturally extracting the water hyacinth extract or using a rotary vacuum concentrator and a freeze drier, diluting the extract to a specific concentration to a certain concentration, and inoculating the fermentation strain.

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

The Lactobacillus sp. Strain is selected from the group consisting of Lactobacillus kimchicus , Lactobacillus pentosus , Lactobacillus brevis , Lactobacillus plantarum , Lactobacillus casei , Or Lactobacillus acidophilus , but may be preferably Lactobacillus kimchicus .

The cosmetic composition may be at least one selected from the group consisting of softening longevity, nutritional lotion, moisture cream, nutritional 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 used for improving wrinkles or adsorbing fine dusts, and may be used for various purposes such as softening longevity, convergent lotion, nutritional lotion, lotion, nutritional cream, massage cream, essence, eye cream, cleansing cream, cleansing foam, cleansing water, And a powder.

The cosmetic composition may be appropriately blended with other ingredients within the range of not hindering the object of the present invention depending on the type of the active ingredient, the purpose of use, and the like in each formulation.

The cosmetic composition according to the present invention may contain one or more substances selected from the group consisting of a fatty substance, an organic solvent, a solubilizing agent, a thickening agent, a gelling agent, a softening agent, an antioxidant, a suspending agent, a stabilizer, a foaming agent, a fragrance, It is commonly used in cosmetics or dermatology, such as ionic or nonionic emulsifiers, fillers, metal ionic sequestrants, chelating agents, preservatives, blockers, wetting agents, essential oils, dyes, pigments and hydrophilic or lipophilic active agents &Lt; / RTI &gt;

However, it is preferable that the adjuvant and the mixing ratio thereof are appropriately selected so as not to affect the preferable properties of the cosmetic composition according to the present invention.

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

Example  1: Water hyacinth extract

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

The extract was filtered with 250 mesh and 0.5 mu m filter paper. The extract was repeatedly 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: Fermented water extract of water hyacinth

The waterweed extract of Example 1 was diluted with purified water to 5% by weight (v / v), and then the fermentation strain Bifidobacterium bifidum bifidum ) was inoculated and fermented. The fermented extract was filtered with 500 mesh and 0.2 mu m filter paper to obtain a fermented water extract of Fusaria officinalis and used as a sample of Example 2.

Example  3: Fermented water extract of water hyacinth

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

Example  4: Fermented water extract of water hyacinth

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

Experimental Example  1: Skin safety test

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

The samples obtained in Examples 1 to 4 were suspended in purified water by concentration, and 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 fermented extracts of Examples 2 to 4 were suspended at concentrations of 0.001, 0.005, 0.01, 0.05, w / w).

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

(HDF), melanocyte-forming cells (B16F10), and keratinocytes (HaCaT) were dispensed in 96-well plates at a concentration of 1 × 10 ⁴ cells / well and cultured at 37 ° C. and 5% CO ₂ for 48 hours Respectively.

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

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

As a result of measurement, cytotoxicity was not confirmed at all concentrations of each sample. The results suggest that the extract is not only harmless to human body but also has excellent human safety.

Experimental Example  2: Antioxidant activity test

Active oxygen decomposition effect

Free radical scavenging activity using DPPH (1, 1-diphenyl-2-picryl hydrazyl).

4 mL of methanol was added to the test tube and the concentration of the extract was varied. 1 mL of 0.15 mM DPPH solution was added, and the reaction was allowed to proceed at room temperature for 30 minutes. Then, the absorbance at 520 nm was measured.

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

division 0 μg / mL 20 μg / mL 40 μg / mL alpha-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 erase rate%]

The extracts were evaluated to have higher free radical scavenging activity than α-tocopherol and BHT, which are well known in the art. Therefore, the decomposition of active oxygen can be promoted by the extract, and the results suggest that the antioxidative activity is effective in preventing skin aging and improving skin condition.

Intracellular Active oxygen species  Measurement test ( DCF -DA assay)

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

Raw 264.7 cells were inoculated into 96 well plates at a concentration of 1 × 10 ⁴ cells / well. The medium was replaced with fresh medium, the extract was treated, and the cells were cultured for 24 hours.

10.0 μL of WST-1 solution (EZ-CYTOX, DOGEN, Korea) was added to each well, and the cells were incubated for 1 hour. The absorbance was measured at 450 nm and 655 nm using an ELISA reader Respectively.

The extracts were diluted with dimethylsulfoxide (DMSO, Sigma-Aldrich, USA) to a final concentration of 20 μM and DMSO was used as a blank test (negative control). The experiment was repeated 3 times to ensure reliability.

The experiment was carried out under the same cell line and the same cell culture conditions as the WST-1 assay, a cell activity assay. The cultured Raw 264.7 cells were washed once with phosphate buffered saline (PBS; sodium chloride 137 mM, phosphate buffer 10 mM, potassium chloride 2.7 mM, all from Biopure, Canada), and Raw 264.7 cells were harvested. The cells were suspended in 15 mL of PBS, and 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 cells were washed once with PBS solution, diluted to 1 × 10 ⁵ cells / mL, treated with the control substance and the extract, and cultured for 10 minutes. 20 mg / mL of silica was treated with 50 μL and incubated for 1 hour. The cell pellet obtained by centrifuging at 6000 rpm for 5 minutes was dispersed in 200 μL PBS solution and inoculated into a 96-well plate.

Fluorescence was measured at 485 nm excitation / 535 nm emission using a fluorescence microplate reader to evaluate the amount of reactive oxygen species.

The extracts treated for the measurement of reactive oxygen species to the concentration of the test solution were used as the concentration of the test solution set up through the cell activity measurement.

As a negative control, DMSO was used instead of the sample. 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 above results, the measurement of reactive oxygen species at the same concentration was carried out, and the treated extract was used at a concentration of 50 mg / L. The experiment was repeated 4 times to ensure 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

 [ROS generation inhibition rate (% of control)]

The extract was evaluated to have an equivalent level of ascorbic acid and free radical scavenging activity, which are well known in the art. That is, the above results indicate that the antioxidative effect due to the free radical scavenging activity is excellent because the active oxygen species removing ability of Examples 1 to 4 is excellent.

Experimental Example  3: Anti-inflammatory activity test

NF - kB  Transcriptional repression effect

The NF-kB reporter assay was used to measure the inhibitory effect of NF-kB transcription. INOS, COX-2, IFN-? Or TNF-? Induced by LPS are expressed by the transcription factor NF-kB. The NF-kB is inactivated in complex with IkB in the cytoplasm, and when I-kB-α is phosphorylated by I-kB-α kinase, the bound complex is degraded and activated as NF-kB, )do. Induces the expression of the target gene of NF-kB and causes inflammation.

TRIF, which acts on inflammatory diseases such as multiple sclerosis, is a receptor that responds to the activity of TLRs (toll-like-receptors) in the TIP-domain with receptors that induce interferon-beta, And activates the immune response to the antigen. The receptors recognize a well-preserved pathogenic pattern and activate the signal to stimulate the release of inflammatory cytokines. IRF-3 (interferon-stimulating factor 3) is a transcription factor induced by interferon alpha / beta stimulation and binds to the interferon-stimulated response sequence (ISRE) in the transcriptional regulatory region of the interferon inducible gene to activate its transcription.

According to the manufacturer's protocol (Jin et al., 2009), 12-well plates were coated with NF-kB according to the presence of receptor molecules such as? -Galactosidase (MyD88 and TRIF) -Luc or TRIF was transfected into HEK293 (1 x ¹⁰⁶ cells / ml) cells. After 24 hours of incubation, LPS (100 ng / ml) and the extract were treated and 24 hours later, the activity of NF-kB and IRF-3 activity were measured using Luciferase assays.

The luciferase assay was performed using a luciferase assay system (Promega Co., USA) reported in the manufacturer's protocol (Jung et al., 2009).

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, when the extract was treated, the activity of NF-kB and the activity of IRF-3 were remarkably decreased. These results suggest that the extract effectively inhibits transcription of NF-kB, thereby inhibiting or alleviating inflammation and preventing and improving inflammatory diseases.

Inflammation-related protein mRNA  Quantitative analysis

TNF-α, iNOS, and COX-2 were analyzed to evaluate the anti-inflammatory activity of the extract.

RNA was prepared from RAW264.7 cells treated with LPS. The method was prepared using Trizol Reagent (Gibco BRL) as described in Lee et al., 2008. All RNAs were stored at -70 ° C before use. Semi-quantitative RT reactions were 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 then incubated for 60 minutes after addition of MuLV reverse transcriptase (2 U). The reaction was terminated by incubating at 70 DEG C for 10 minutes. (Conditions: 2 μL cDNA, 4 μM 5 'and 3' primer, 10 × buffer (10 mM Tris-HCl, pH 8.3; 50 mM KCl and 0.1 % Triton X-100), 250 μM dNTPs, MgCl 2 25 mM , and Taq DNA polymerase 1unit (Promega, USA). PCR reactions after annealing and denaturation at 94 ℃ 30 seconds, 55 to from 60 ℃ 30 seconds, 72 Deg.] C for 42 seconds, and finally polymerized at 72 [deg.] C for 5 minutes.

The primers of TNF-a, 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 '

The results of the measurement of mRNA expression levels according to the above extract treatment are shown in Table 5 below. Analysis of RAW264.7 cell mRNA revealed that the expression of TNF-α, iNOS, and COX-2 was increased by the addition of LPS. On the other hand, the expression of TNF-a, iNOS, and COX-2 induced by the 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 skin moisturizing effect was evaluated.

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

Cultured human keratinocyte (HaCaT) under the conditions of Dulbecco's Modified Eagle's Medium (DMEM ), 10% Fatal bovine serum (FBS), 1% 37 ℃ at 100 mm / 60.1 cm culture dish along with Antibiotic-Antimycotic, 5% CO ₂ Respectively.

The keratinocytes were dispensed into 96-well plates at a concentration of 1x10 ⁶ cells / mL and cultured for 24 hours. After replacing with DMEM free medium, the extracts of Examples 1 to 4 diluted by concentration were treated and cultured for 24 hours.

Cells were harvested, washed with cold phosphate buffered saline (PBS), and 1 ml of TRIZol reagent (Life Technologies, Inc.) was added and the total RNA extracted.

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

To quantify the changes of HAS2 and AQP3 gene expression, fluorescence values of the PCR products were measured by 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, 0.5 U Extaq DNA polymerase, 3 mM MgCl ₂ and 1 × SYBR green in a PCR tube.

Denaturation, annealing, and polymerization were performed at 94 ° C for 30 seconds, at 58 ° C for 30 minutes, and then for 3 minutes at 94 ° C using Linegene K (BioER, China) Sec., 72 ° C for 30 sec., And the fluorescence intensity was measured after completion of each cycle.

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

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 certain reference value above the basal value, and the amount of gene expression can be confirmed through the Ct value. The primer sequences used in the experiments 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 '

The results of measurement of HAS2 and AQP3 mRNA expression amounts are shown in Table 7 below, and the amount of mRNA expression was increased in a concentration-dependent manner according to the treatment of the extract. The higher the ability to promote HAS2 and AQP3 expression, the better the skin moisturizing effect.

The results suggest that the extract may 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: Assay for inducing collagen biosynthesis

The synthesis of collagen following fibroblast growth was observed.

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

The medium was exchanged with DMEM (Dulbecco's Modified Eagle's Medium, Sigma), and the extracts of Examples 1 to 4 were treated at a concentration of 50 mg / L and then cultured. The control was used in which purified water was added without treating the extract.

After the cells were cultured for 7 days, the culture solution was collected and the concentration of secreted type I collagen in the culture solution was quantified by enzyme-linked immunosorbent assay (Procollagen type I c-peptide EIA Kit, Takara Bio).

The amount of collagen in each test sample culture fluid was calculated based on the amount of type I collagen in the control (100%). 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 group 101.2

Experimental Example 6: Measurement of expression 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.

The expression of COL1A1, which is one of typical collagen expressed by human dermal fibroblasts, ELN, which is one of elastic proteins, and MMP1, a collagenase, was measured.

To examine the extent of expression of collagen, the extracts of Examples 1 to 4 diluted by concentration were treated with human dermal fibroblasts and cultured for 24 hours.

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

To quantify the changes of COL1A1 and MMP1 gene expression, fluorescence values of PCR products were measured by 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, 0.5 U Extaq DNA polymerase, 3 mM MgCl ₂ and 1 × SYBR green in a PCR tube.

Denaturation, annealing, and polymerization were performed at 94 ° C for 30 seconds, at 58 ° C for 30 minutes, and then for 3 minutes at 94 ° C using Linegene K (BioER, China) Sec., 72 ° C for 30 sec., And the fluorescence intensity was measured after completion of each cycle.

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

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 certain reference value above the basal value, and the amount of gene expression can be confirmed through the Ct value. The primer sequences used in the experiments 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

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

The higher the inhibitory effect on MMP-1 expression, the better the skin wrinkle and the anti-aging effect. The above results show that the extract promotes the production of collagen and thereby improves skin elasticity and wrinkles. .

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

50 women of 40 to 60 years old were subjected to a lotion formulated with the extracts of Examples 1 to 4 twice daily for one month in the morning and evening for one month, and the degree of wrinkle improvement was evaluated.

The total number of wrinkles, wrinkle length and wrinkle area of each subject were measured by replica analysis. The degree of wrinkle improvement was evaluated by image analysis. The evaluation results are shown in Table 11 below.

division Reduction rate of total wrinkles (%) Wrinkle area reduction rate (%) Wrinkle length reduction rate (%) Example 1 0 weeks 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 0 weeks 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 0 weeks 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 0 weeks 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 creams formulated with the extracts of Examples 1 to 4 significantly reduced the total number of wrinkles, wrinkles, and wrinkles, which were parameters of skin wrinkles. The results showed that the extracts improved skin wrinkles and skin cosmetics And that the present invention can be utilized as an application of the present invention.

EXPERIMENTAL EXAMPLE 8: Evaluation of skin texture improvement effect

The effect of improving the skin texture of the cream formulated with the extracts of Examples 1 to 4 was verified.

As a control group, a known formulation of aloe vera extract known to have a moisturizing function was used.

For 40 selected women aged 20 to 40 years, the skin was measured before use of the subject using PRIMOS light (field of view 45, flexible 3D measuring, GFMesstechnik GmbH), and the skin was remeasured Respectively.

The measured values were expressed as three average values except for the maximum value and the minimum value of Ra. The higher the measured value, the more wrinkled the skin. The evaluation results are shown in Table 12 below.

[Ra, (Arbitrary unit)] division Example 1 Example 2 Example 3 Example 4 Control group Before use 15.70 15.73 15.60 15.68 15.70 after 2 weeks 14.26 13.95 13.55 12.97 14.54 After 4 weeks 12.25 11.96 11.76 10.95 13.91

As shown in Table 12, it was evaluated that the test group using the cream formulated with the extracts of Examples 1 to 4 remarkably reduced the measured value after use and remarkably improved the skin texture.

In particular, it was confirmed that the skin cream improving effect of the cream (Example 4) containing the hydrolyzed lactobacillus lactobacillus kimchicus extract was the most excellent, and when the cream of the control group containing no water extract was not used, Respectively.

Experimental Example 9: Test for removing polycyclic aromatic hydrocarbons

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

Porcine sheath (1 cm x 1 cm) with hair removed was prepared and washed with PBS for 10 minutes.

After washing, 600ppm of Naphtalene, Acenaphthylene, Acenaphthene, Fluorene, Fluoranthene, Pyrene, Chrysene, Benzopyrene (PH 11) for 2 hours and then dried at room temperature.

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

After washing with PBS, the specimens were cut to constant weight and crushed with a homogenizer. The pulverized tissue was dried with anhydrous sodium sulfate (Na ₂ SO ₄ ), and the polycyclic aromatic hydrocarbons were extracted from the tissue by Sohxlet extraction and liquid-liquid extraction (Husain et al. 1997; Takatsuki et al., 1985) and quantitated by HPLC. The evaluation results are shown as a ratio of the control group to the experimental group, and the quantitative results are shown in Table 13 below.

[% 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 Pyrene 3.6 2.5 1.9 1.8 Crissen 4.9 3.9 2.3 1.3 Benzopyran 3.1 2.7 2.2 1.3

As shown in Table 13, the polycyclic aromatic hydrocarbons were significantly reduced by the treatment of the extracts of Examples 1 to 4, and the results showed that the extract contained a large amount of fine dust and air, It can be effectively removed.

Experimental Example 10: Micro dust removal test

The effect of removing the fine dust of the cosmetic composition formulated in Examples 1 to 4 was tested. Fine dust corresponding to PM2.5 to PM10 size was applied to the artificial skin and then washed using the cleansing cream formulated in Examples 1 to 4. [

The ratio of fine dust before and after the cleansing cream was calculated 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 group 76%

Referring to Table 14, fine dusts were effectively removed by cleansing creams formulated with the extracts of Examples 1 to 4, while the effect of removing fine dust from the control group containing no extract was relatively inadequate.

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive. For example, each component described as a single entity may be distributed and implemented, and components described as being distributed may also be implemented in a combined form.

The scope of the present invention is defined by the appended claims, and all changes or modifications derived from the meaning and scope of the claims and their equivalents should be construed as being included within the scope of the present invention.

Claims (7)

A cosmetic composition for fine dust adsorption and antiinflammation comprising Eichhornia crassipes extract as an active ingredient. The method according to claim 1,
Wherein the waterworm extract is a fermented extract prepared by inoculating a fermentation strain. 3. The method of claim 2,
Wherein the fermentation strain is Lactobacillus kimchicus . The method according to claim 1,
Wherein the extract is selected from the group consisting of water, an anhydrous or lower alcohol having 1 to 4 carbon atoms, acetone, petroleum ether, ethyl acetate, butyl acetate, trichloromethane, dichloromethane, chloroform, hexane and 1,3-butylene glycol Gt; cosmetic &lt; / RTI &gt; composition. 5. The method of claim 4,
Wherein the alcohol is ethanol at a concentration of 65 to 75% (v / v). 6. The method according to any one of claims 1 to 5,
Cosmetics formulated with at least one selected from the group consisting of softening longevity, nutritional lotion, moisture cream, nutritional cream, massage cream, essence, ampoule, gel, eye cream, cleansing cream, cleansing foam, cleansing water, pack, spray and powder Composition. The method according to claim 1,
Antioxidant, skin moisturizing, or wrinkle improving.