KR20200081542A - Composition for cosmetic comprising compounds extracted from eisenia bicyclis - Google Patents

Abstract

The present invention relates to a cosmetic composition comprising an Eisenia bicyclis extract as an active ingredient. Particularly, the present invention relates to a cosmetic composition comprising, as an active ingredient, an Eisenia bicyclis extract which is effective in antioxidation, anti-inflammation, and skin wrinkle reduction by extracting and using useful ingredients from Eisenia bicyclis using ethanol or water.

Description

Cosmetic composition using Eisenia bicyclis extract as an active ingredient {COMPOSITION FOR COSMETIC COMPRISING COMPOUNDS EXTRACTED FROM EISENIA BICYCLIS}

The present invention relates to a cosmetic composition using an rhubarb extract as an active ingredient, specifically extracting useful components using rhubarb using ethanol or water and using it to extract antioxidants, antioxidants, and skin wrinkles that are effective in improving wrinkles. It relates to a cosmetic composition as an active ingredient.

As living and income levels have risen and improved, people have become increasingly interested in personal aesthetic life, away from everyday lifestyles due to consciousness (a study on the purchase form and preference of functional cosmetics and functional foods. Master's Thesis. 2010.). In particular, with the development of modern medicine, as the average lifespan increases and becomes more relaxed, there is much interest in anti-aging and skin health (Kim, SH Yong, HJ Shin, C. and Ko, SG Research of traditional herbal medicines for anti-aging, inhibition effect of wrinkle and whitening effect in the skin.Korean J. Ori.Physiol.Pathol. 2008, 22, 691-698.).

The aging of the skin can be divided into intrinsic and exogenous aging depending on the factors. Intrinsic aging is the deterioration of the skin's structure and physiological functions as it ages, and extrinsic aging is an aging phenomenon caused by accumulated external stress such as sunlight. In particular, skin is thinned by exogenous aging such as exposure to various pollutants and UV rays, wrinkles are increased, elasticity is reduced, and spots, freckles, and blotch are increased (Gilchrest BA. Skin aging and photoaging. Dermatol Nurs. 1990, 2, 79-82., Kang, KS Kim, ID Kwon, RH Heo, YY Oh, SH Kim, MA Jung, HJ Kang, HY Ha, BJ The evaluation of anti-wrinkle effects in oriental herb extract.J. Life Sci, 2007, 17, 1147-1151.). As skin aging progresses, the ability to produce structural proteins such as collagen, elastin, and hyaluronic acid, which are the constituents of the skin, decreases, and biosynthesis of type-1 collagenase increases, resulting in increased expression of matrix metalloproteinases (MMPs). , Induce decomposition of matrix proteins such as elastic fibers and fibronectin, thereby reducing skin elasticity and causing skin wrinkles (Brenneisen P, Sies H, Scharffetter-Kochannek K. “Ultraviolet-B irradiation and matrix metalloproteinases: from induction via signaling to initial events” Ann. NY Acad. Sci, 2002, 973, 31-43.).

In order to maintain and improve not only skin health, such as preventing skin aging and wrinkle formation, but also maintaining and improving overall health of the body, research on cosmetics, food, and medicines using various natural products has been actively conducted.

In Korea, three sides are composed of the sea, so the types of seaweeds inhabiting the sea area are very diverse and rich. These seaweeds have been widely used as food, fertilizer, medicinal, raw materials and fertilizer for the seaweed industry for a long time. Recently, as seaweeds contain as many functional ingredients as plants, it has been actively researched for the separation and verification and utilization of these functional ingredients (Choi, DM, Kim, DS, Lee, DS, Kim, HR, and Pyeun, JH 1995. Trace components and functional sacchsrides in seaweed-1. J. Korean Fish. Soc. 28: 49-59.).

The large brown algae (Eisenia bicyclis) is a perennial seaweed belonging to the kelp tree kelp tree and rhododendron. It forms a large colony on Korea's Ulleungdo and Dokdo Islands, and colonies are developing in Yeongdeok, Gyeongbuk, on the east coast. Effects of temperature, light intensity, and Gwangju period. Lee Min-jung. Graduate school of Gyeongsang National University. Master's thesis. 2018.). Rhubarb contains a large amount of iodine and potassium, and has been edible as a kelp substitute since ancient times due to its unique taste, and has recently been used as a raw material for alginic acid (physiological activity and application of rhubarb extract. Daeyong Kim. Graduate School of Pukyong National University. Master's Thesis. 2009.).

Research on the functionality of rhubarb has also been actively conducted, especially antibacterial (Eom SH, Lee BJ and Kim YM. 2010. Effect of yeast fermentation on the antioxidant and anti-inflammatory activity of sea tangle water extract.Korean J Fish Aquat Sci 43, 117-124.), anti-inflammatory (Jung, HA, Jin SE, Ahn BR, Lee CM and Choi JS. 2013.Anti-inflammatory activity of edible brown alga Eisenia bicyclis and its constituents fucosterol and phlorotannins in LPS-stimulated RAW264 .7 macrophages.Food Chem Toxicol 59, 199-206.), Antihyperlipidemia (Jang YH, Choi SW and Cho SH. 2008.Effect of Eisenia bicyclis and its pill on serum lipid status in rats fed high fat diet.Korean J Food Nutri 41, 5-12.), anti-diabetes (Okada Y, Ishimaru A, Suzuki R and Okuyama T. 2004.A new phloroglucinol derivative from the brown alga Eisenia bicyclis: potential for the effective treatment of diabetic complications.J Nat Prod 67 , 103-105.). In addition, rhubarb contains a large amount of physiologically active substances such as chlorotannin, and chlorotannin isolated from rhubarb has eckol and phlorofucofuroeckol-A, dieckol, 8,8'-bieckol, and has been reported to have antioxidant activity. (Okada Y, Ishimaru A, Suzuki R and Okuyama T. 2004.A new phloroglucinol derivative from the brown alga Eisenia bicyclis: potential for the effective treatment of diabetic complications.J Nat Prod 67, 103-105.).

On the other hand, Korean Patent Publication No. 10-2013-0141874 relates to an antimicrobial composition comprising an rhubarb-derived polotanin compound as an active ingredient, and the antimicrobial composition has particularly excellent antibacterial activity against MRSA (Methicillin-Resistant Staphylococcus aureus). Disclosed is an antimicrobial composition comprising a fluorotannin compound as an active ingredient.

Korean Registered Patent No. 10-0966128 discloses rhubarb enzyme extract, rhubarb powder, kelp powder, and excipients for rhubarb enzyme extract, which has the effect of treating hyperlipidemia and diabetes, produced by rhubarb powder production, extrusion molding and enzymatic hydrolysis from seaweed rhubarb. Disclosed is a rhubarb tablet manufactured by mixing and a method for manufacturing the same.

Korean Patent Publication No. 10-2011-0057586 discloses a pharmaceutical composition for preventing or treating diabetes and a health functional food composition for improving diabetes, consisting of brown algae rhubarb powder, rhubarb enzyme extract, kelp powder and excipients.

As described above, various studies on rhubarb have been conducted, and its functionality is known, but its utilization is still insufficient, and in particular, industrial applications for functional verification and cosmetics for skin improvement are very poor.

Korean Patent Publication No. 10-2013-0141874 (2013.12.27.) Korean Registered Patent No. 10-0966128 (2010.06.17.) Korean Open Patent No. 10-2011-0057586 (2011.06.01.)

The present invention has been devised to solve the problems as described above and to provide the necessary technology,

An object of the present invention is to provide a cosmetic composition using extracts and concentrates using seaweed rhubarb to provide a cosmetic composition that is effective in improving antioxidant, anti-inflammatory and skin wrinkles.

As one embodiment of the present invention for achieving the above object,

The present invention provides a cosmetic composition using rhubarb extract as an active ingredient.

At this time, the rhubarb extract is extracted by adding 800 to 1200 parts by weight of ethanol to 100 parts by weight of dried and crushed rhubarb, and then extracting for 20 to 25 hours at room temperature to prepare a rhubarb extract; Concentration step of separating and removing the solid content contained in the rhubarb extract prepared in the extraction step, and concentrated under reduced pressure at a temperature of 40 to 50 ℃ the solid content is removed to prepare a rhubarb concentrate of 10 to 20 Brix; And a freeze-drying step of preparing the rhubarb extract by freezing the rhubarb concentrate prepared in the concentration step at a temperature of -70 to -80°C and then freeze-drying it.

In addition, the rhubarb extract is extracted by adding 800 to 1200 parts by weight of water to 100 parts by weight of dried and crushed rhubarb, followed by extraction at 90 to 100°C for 2 to 3 hours to prepare a rhubarb extract; Concentration step of separating and removing the solid content contained in the rhubarb extract prepared in the extraction step, and concentrated under reduced pressure at a temperature of 60 to 70 ℃ the solid content is removed to prepare a 10 to 20 Brix rhubarb concentrate; And a freeze-drying step of preparing the rhubarb extract by freezing the rhubarb concentrate prepared in the concentration step at a temperature of -70 to -80°C and then freeze-drying it.

In the present invention, the cosmetic composition may be characterized in that it is made of any one selected from the group consisting of lotion, lotion, serum, massage cream, nutrition cream, moisture cream, hand cream, essence, nutrition essence, body lotion have.

Cosmetic composition containing rhubarb extract as an active ingredient according to an embodiment of the present invention extracts useful components using rhubarb using ethanol or water and prepares a cosmetic composition using them to improve antioxidant, anti-inflammatory and skin wrinkles According to this, it can be used as a cosmetic composition of various formulations.

1 is a graph showing the results of measuring the DPPH radical scavenging activity for rhubarb extract.
2 is a graph showing the results of measuring the ABTS radical scavenging activity for rhubarb extract.
3 is a graph showing the results of measuring SOD-like activity for rhubarb extract.
4 is a graph showing the results of measuring superoxide dismutase (SOD) enzyme activity for rhubarb extract.
5 is a graph showing the cell viability check results for rhubarb extract.
6 is a graph showing the cytotoxicity measurement results of rhubarb extract for RAW 264.7 cell.
7 is a graph showing the measurement results of Nitric oxide inhibition rate for rhubarb extract.
8 is a graph showing the measurement results of iNOS production inhibition of rhubarb extract through Western blot.
9 is a graph showing the measurement results of COX-2 production inhibition of rhubarb extract through Western blot.
10 is a graph showing the results of iNOS and COX-2 expression inhibition efficacy of rhubarb extract through real-time PCR analysis.
11 is a graph showing the cytotoxicity measurement results of rhubarb extract for HaCaT cells.
12 is a graph showing the measurement results of MMP-1 protein expression of rhubarb extract through Western blot.

Hereinafter, embodiments of the present invention will be described in detail so that those skilled in the art to which the present invention pertains may easily practice. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art. Accordingly, the embodiments of the present invention can be modified in various other forms, and the scope of the present invention is not limited to the embodiments described below.

Throughout the specification of the present invention, when a part “includes” a certain component, it means that the component may further include other components, not to exclude other components, unless specifically stated to the contrary.

The terms “about”, “substantially”, and the like used throughout the specification of the present invention are used in terms of or close to those values when manufacturing and material tolerances unique to the stated meanings are presented, and the present invention To help understand, accurate or absolute figures are used to prevent unconscionable abusers from unintentionally using the disclosed disclosure.

The present invention provides a cosmetic composition (hereinafter also referred to as'cosmetic composition') containing rhubarb extract as an active ingredient.

The cosmetic composition according to the present invention contains the rhubarb extract prepared by concentrating and lyophilizing the rhubarb extract extracted by adding ethanol or water to rhubarb as an active ingredient, and is expected to have high antioxidant, anti-inflammatory, and wrinkle-improving effects.

According to one embodiment of the present invention, the rhubarb extract contained in the cosmetic composition is added to 800 to 1200 parts by weight of ethanol to 100 parts by weight of dried and crushed rhubarb, and then extracted at room temperature for 20 to 25 hours to extract the rhubarb extract Extraction step to prepare, the concentration step of separating and removing the solid content contained in the rhubarb extract prepared in the extraction step, and concentrated under reduced pressure at a temperature of 40 to 50 ℃ the solid content is removed to prepare a 10 to 20 Brix rhubarb concentrate and It can be characterized in that the rhubarb concentrate prepared in the concentration step is freeze-dried at a temperature of -70 to -80°C and then freeze-dried to prepare a rhubarb extract.

In addition, according to one embodiment of the present invention, the rhubarb extract contained in the cosmetic composition is added to 800 to 1200 parts by weight of water with respect to 100 parts by weight of dried and crushed rhubarb, and then at 90 to 100° C. for 2 to 3 hours Extraction step to extract the rhubarb extract, the solid content contained in the rhubarb extract prepared in the extraction step is separated and removed, and concentrated extract is removed at a temperature of 60 to 70 ℃ concentrated under reduced pressure to 10 to 20 Brix rhubarb concentrate It can be characterized in that it comprises a concentration step to prepare and freeze-drying step to prepare the rhubarb concentrate prepared in the concentration step by freezing at a temperature of -70 to -80 ℃ and then freeze-drying it.

When the rhubarb extract is out of the limited range as described above, anti-oxidation, anti-inflammatory and skin wrinkle improvement effects cannot be expected due to low or dilute concentration of useful ingredients in the extract. Conversely, the concentration of the extract is too high for antioxidant, Although the anti-inflammatory and anti-wrinkle effect may have an improvement effect, there is a concern that the manufacturing cost is high in the manufacture of cosmetic products, and skin toxicity may also occur.

At this time, when using the rhubarb extract as a cosmetic composition, the cosmetic composition may be characterized in that the rhubarb extract is included in a proportion of 0.1 to 90% by weight relative to the total weight of the cosmetic composition.

This is that when the rhubarb extract is contained in a proportion of less than 0.1% by weight relative to the total weight% of the cosmetic composition, antioxidant, anti-inflammatory and skin wrinkle improvement effects may not be properly expressed, and rhubarb extract is 90% based on the total weight% of the cosmetic composition When included in a proportion exceeding weight%, there may be a problem that the manufacturing cost increases in the manufacture of cosmetic products.

In addition, the cosmetic composition according to the present invention is formulated into any one formulation selected from the group consisting of lotion, lotion, serum, massage cream, nourishing cream, moisture cream, hand cream, essence, nutrient essence, and body lotion in a conventional method Can be used.

Hereinafter, the present invention will be described in detail according to specific embodiments. The cosmetic composition prepared according to one embodiment of the present invention is a rhubarb extract yield and yield measurement experiment, nutrient analysis experiment, mineral and heavy metal measurement experiment, constituent amino acid measurement experiment, free amino acid measurement experiment, fatty acid composition experiment, antioxidant effect experiment, Anti-inflammatory effect experiment and wrinkle improvement effect experiment were conducted. The cosmetic composition according to an embodiment of the present invention can be more clearly understood by experiments described below.

Preparation of rhubarb extract (1): Preparation of ethanol extract

1. Extraction step: 1000 parts by weight of ethanol was added to 100 parts by weight of dried and crushed rhubarb, and then extracted at room temperature for 24 hours to prepare a rhubarb extract.

2. Concentration step: The solid content contained in the rhubarb extract is separated and removed using a filter (Advantec Filter paper No.2), and the extract from which the solid is removed is concentrated under reduced pressure at a temperature of 45° C. to prepare 15Brix rhubarb concentrate. .

3. Freeze-drying step: The rhubarb concentrate is frozen at a temperature of -70°C and then lyophilized to prepare a rhubarb extract.

Preparation of rhubarb extract (2): Preparation of water (hot water) extract

1. Extraction step: After adding 1000 parts by weight of water to 100 parts by weight of dried and crushed rhubarb, extract at 100°C for 2 hours and 30 minutes to prepare a rhubarb extract.

2. Concentration step: The solid content contained in the rhubarb extract is separated and removed using a filter (Advantec Filter paper No.2), and the extract from which the solid is removed is concentrated under reduced pressure at a temperature of 65° C. to prepare 15Brix rhubarb concentrate. .

3. Freeze-drying step: The rhubarb concentrate is frozen at a temperature of -70°C and then lyophilized to prepare a rhubarb extract.

Experiment for measuring the yield and yield of rhubarb extract

Yield and yield were measured for rhubarb extract (hereinafter referred to as “ethanol extract”) prepared using ethanol and rhubarb extract (hereinafter referred to as “hot water extract”) prepared using water. In the case of ethanol extract, extracts were prepared by varying the ethanol concentration to 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100%, and then the yield and yield were measured.

Experiment result

In the case of rhubarb extract by ethanol concentration, the extraction yield was highest in extracts using 50-70% ethanol. Table 1 shows the measurement results of the yield and yield for the ethanol extract and the hot water extract.

Rhubarb extract Yield (%) Yield (g) Hydrothermal 36.32 145.28 g (400 g extracted) 10% EtOH 11.37 22.73 g (200 g extraction) 20% EtOH 17.83 35.66 g (200 g extraction) 30% EtOH 21.38 42.76 g (200 g extraction) 40% EtOH 24.88 49.76 g (200 g extraction) 50% EtOH 27.90 55.79 g (200 g extraction) 60% EtOH 29.69 59.37 g (200 g extraction) 70% EtOH 25.98 51.95 g (200 g extraction) 80% EtOH 19.33 38.66 g (200 g extraction) 90% EtOH 8.36 16.71 g (200 g extraction) 100% EtOH 1.59 3.17 g (200 g extraction) *EtOH: Ethanol extract

Nutrient measurement experiment

1. Determination of crude protein

The crude protein was quantified using the Kjeldahl method according to AOAC. The collected sample was taken into a Kjeldahl decomposition tube, titrated with a 0.1N NaOH solution using a Kjeldahl (Foss, Sweden) device, and multiplied by a nitrogen coefficient (6.25) to calculate the content of crude protein.

2. Crude fat quantitative experiment

Crude fat was measured by the Soxhlet method. That is, about 5 g of the sample was placed in a cylindrical filter paper, the sample was blocked with cotton wool, placed in an extraction tube, filled with ether in water, extracted for 8 to 16 hours in a water bath at 60°C, the ether in the water was blown, and the weight of fat was measured. .

3. Quantification of inquiry

Ash content is obtained by taking a certain amount of sample in a crucible that knows the constant weight by direct painting method according to AOAC, painting it for 5 to 6 hours in a 550-600°C furnace, and cooling it for a period of time in a desiccator to obtain the constant weight to obtain the constant weight. The amount of inquiry was calculated as.

4. Carbohydrate determination

Carbohydrates content (%) was determined by subtracting water, crude protein, crude fat, ash, and content% from 100% of the total sample.

5. Quantification of free sugar

The content of the saccharide was about 5 g of the sample, precisely weighed into a 50 mL volumetric flask, dissolved in 25 mL of water, filled to 50 mL with acetonitrile, and filtered through a 0.45 μL membrane filter as a test solution.

Fructose, Glucose, Sucrose, Maltose, and Lactose (Sigma, USA) are precisely weighed with 100 g of a 100 mL measuring flask, dissolved in 50 mL of water, filled with acetonitrile to 100 mL, diluted, and used as a standard solution. Did. The sugar content in the sample was analyzed under the conditions shown in Table 2 using HPLC.

Items Conditions Instrument Waters, USA (Empower system) Column 250㎜×4.6㎜, Polyamine II (YMC Pack) Detector RI Mobile phase Water: Acetonitrile (20:70, v/v) Injection volume 20 μl Flow rate 1.0ml/min Column temp 35℃

6. Cholesterol measurement

Saponification process: About 2 g of the sample (rhubarb extract) was precisely weighed and taken into an Erlenmeyer flask. At this time, the amount of fat in the sample was adjusted to be 1 g or less. The magnetic rod was placed in an Erlenmeyer flask, and 40 mL of 95% ethanol and 50 mL of 8 mL 50% potassium hydroxide solution were added. A condenser was installed and heated with stirring using a magnetic stirring-heater to reflux for 70±10 minutes. For saponification, if a lump occurs while continuously observing the sample, the test solution is stirred by adding 50% potassium hydroxide solution while dispersing or stirring with a glass rod. When reflux was completed, the heater was turned off and 60 mL of 95% ethanol was added through the top of the condenser during stirring. After about 15 minutes, the condenser was removed from the flask, the flask was capped, cooled to room temperature, and the test solution was stabilized for 24 hours.

Extraction: 100 ml of toluene was added while stirring the test solution after saponification was completed, and the mixture was stirred for 30 seconds or more. After transferring it to a 500 mL separatory wort without washing, 110 mL 1M potassium hydroxide solution was added to the separatory strainer and shaken vigorously for 10 seconds to agitate and the separated lower layer was discarded. 40 mL 0.5 M potassium hydroxide solution was added to the separatory filter and the separatory filter was turned over. Then, the contents were slowly mixed for 10 seconds to form a vortex. The toluene layer was washed with 40 mL of distilled water slowly, washed with water, and left to stand. The separated lower layer was discarded and the washing process was repeated three or more times. At this time, the more the washing process was repeated, the more intense the agitation. The washing process was continued until the toluene layer was clear. The toluene washed with glass wool and a glass funnel filled with about 20 g of anhydrous sodium sulfate was dehydrated by flowing into an Erlenmeyer flask filled with about 2 g of anhydrous sodium sulfate. The cap was placed in an Erlenmeyer flask, stirred and mixed to stand for at least 15 minutes. 25 mL of the extracted toluene layer was taken into a 125 mL round flask with a flat bottom, concentrated under reduced pressure at 40±3°C, evaporated to dryness, and about 3 mL of acetone was added to the residue, followed by concentration under reduced pressure to complete dryness.

Derivatization: 1.0 mL of the standard solution and the test solution described above were respectively taken into a 15 mL centrifuge tube, 0.2 mL hexamethyldisilane was added to each centrifuge tube, 0.1 mL trimethylchlorosilane was added, the cap was closed, and this was stirred vigorously for 30 seconds. Mix and stand for 15 minutes. After adding 1.0 mL 5α-Cholestane internal standard solution and 10 mL of distilled water to each centrifuge tube, the cap was closed and stirred vigorously for 30 seconds, and this was centrifuged at 3000 rpm for 2 minutes. The heptane layer of the upper layer was taken as a test solution for gas chromatograph (Shimadzu GC-2010) measurement. The analysis conditions for analyzing the cholesterol level are shown in Table 3 below.

Items Conditions Instrument Shimadzu GC2010 Column HP-5(50m×0.25mm) Carrier gas N ₂ Detector FID Flow rate 2 (ml/min) Injection temp. 250℃ Column temp. 190℃ for 2min, 20℃/min(Rate), 230℃ for 3min,
40℃/min(Rate), 255℃ for 25min Detector temp. 300℃ Injection volume 1 μl

7. Inorganic and heavy metal measurement experiments

The preparation of the sample was precisely measured around 0.5 g of the sample using a wet decomposition method, and 5 mL of 65% HNO ₃ and ₁ mL of 30% H ₂ O ₂ were placed in a teflon bottle and used as a pretreatment test solution. The pre-treatment method was acid digestion using microwave digestion system (Ethos-1600, USA) at a maximum of 550 W for 30 minutes. The sample solution that had been subjected to the pre-treatment was filtered with a 0.45 μm PTFE filter and used as an analysis sample. Quantitative analysis of inorganic and heavy metals was carried out using an Inductively Coupled Plasma spectrometer (SHIMADZU ICPE 9000, Japan). All reagents and distilled water were used for mineral analysis.

Instrument Conditions Radio Frequency Power 1.20 kW Plasma Gas 10.0 L/min Auxiliary Gas 0.60 L/min Carrier Gas 0.70 L/min Wave length(nm) Na 589.592 Pb 220.353 CD 214.438

Experiment result

The nutrients (crude protein, crude fat, ash, carbohydrate, fatty acid, free sugar, cholesterol and minerals) and heavy metals measured by the above method are shown in Table 5 below. This experiment was conducted by requesting the Kyungpook Techno Park Daegu Oriental Medical Center Efficacy Verification Center.

Test Items content calorie 40.33 (Kcal/100g) salt 353.93 (mg/100g) carbohydrate 7.67 (g/100g) sugars 0.15 (g/100g) Fat 0.19 (g/100g) trans fat 0 (g/100g) Saturated fat 0.03 (g/100g) cholesterol 0 (mg/100g) protein 1.98 (g/100g) Lead (Pb) 0.05 (mg/Kg) Cadmium (Cd) 0.07 (mg/kg) Calcium (Ca) 161.96 (mg/100g) Copper (Cu) Non-detection Iron (Fe) 0.78 (mg/100g) Potassium (K) 564.55 (mg/100g) Magnesium (Mg) 85.98 (mg/100g) Phosphorus (P) 139.68 (mg/100g) Zinc (Zn) 0.43 (mg/100g)

Composition amino acid and free amino acid measurement experiment

1. Composition amino acid measurement experiment

6N HCl is added to 50 mg of lyophilized rhubarb extract and sealed. The sealed sample was hydrolyzed at 110° C. for 24 hours, then the seal was released and dried by heating at 80° C. for 24 hours. After diluting with 0.02N HCl according to the concentration of the dried sample, filtration using a 0.45um filter and injection into the amino acid automatic analyzer to measure the constituent amino acids. Analysis conditions are shown in Table 6 below.

Column Ion exchange Pump Pressure 0~19.6Mpa Flow Rate 0.05~0.99ml/min Auto sampler 200vials Injection Volume 1~100μl Reaction Unit Reaction Column(135℃) Detector Wavelength 570 nm. 440nm

2. Free amino acid measurement experiment

After homogenization by adding 5% trichloroacetic acid to 0.1 g of lyophilized rhubarb extract, centrifuge at 10,000 rpm for 10 minutes using a centrifuge to collect the supernatant. The collected supernatant was filtered using a 0.45um filter, and then injected into an automatic amino acid analyzer to measure free amino acids. Analysis conditions are shown in Table 6 above.

Experiment result

1. Composition amino acid measurement result

Table 7 shows the results of measuring the amino acid content of the rhubarb extract. As a result of measurement of the content of the constituent amino acids, it was confirmed that the contents of glutamic acid, alanine and proline were high.

Composition amino acid type Content (mg/g) aspartic acid 5.666 threonine 3.223 serine 2.810 glutamic acid 30.112 glycine 3.331 alanine 22.140 cystine 0.820 valine 3.435 methionine 1.385 isoleucine 2.417 leucine 4.538 tyrosine 2.700 phenylalanine 4.096 lysine 3.978 ammonia 3.768 histidine 1.098 arginine 2.730 proline 19.377 total amino acid 113.858 ND=Not Detected

2. Free amino acid measurement results

The results of measuring the free amino acid content for rhubarb extract are shown in Table 8 below. As a result of measuring the free amino acid content, it was confirmed that the content of proline, cystathionine, and clutamic acid was high.

Free amino acid type Content (ug/g) phosphoserine 230.846 taurine ND phospho ethanol amine 181.284 urea ND aspartic acid 392.447 threonine 124.128 serine 186.032 glutamic acid 877.961 sarcosine ND α-amino adipic acid ND glycine 64.400 alanine ND citrulline ND α-amino-n-butyric acid ND valine 202.537 cystine 55.933 methionine 25.653 cystathionine 565.690 isoleucine 153.545 leucine 108.290 tyrosine 100.030 phenylalanine 89.783 β-alanine 40.438 β-amino isobutyric acid 36.189 γ-amino-n-butyric acid 3.318 ethanol amine 59.848 hydroxylysine 15.184 ornithine 25.651 lysine 103.063 1-methylhistidine ND histidine 18.976 3-methylhistidine ND anserine ND carnosine ND arginine 340.833 hydroxy proline 5.132 proline 7369.833 total free amino acid 11.377 ND=Not Detected

Fatty acid composition measurement experiment

The sample (rhubarb extract) was homogenized prior to fat extraction. The homogenized sample was accurately weighed in an amount containing about 100-200 mg of fat, placed in a Majonnier tube, and about 100 mg of pyrogallol was added, and then 2 mL of an internal standard solution was added. Add the boiled side to the Majonnier tube and add 2 mL of ethanol to mix until the whole sample is well mixed. Add 10 mL of 8.3 M hydrochloric acid solution and mix. The stopper of the Majonnier tube was sealed with a rubber band or Teflon tape, and then decomposed for 40 minutes while stirring at an appropriate speed in a water bath at 70 to 80°C. The stirrer was mixed every 10 minutes so that the particles attached to the wall surface of the Majonnier tube were well mixed. After decomposition, ethanol was added to fill and gently mixed. 25 mL of diethyl ether was added to the decomposed product of the prepared Majonnier tube, the mixture was capped and shaken for 5 minutes to extract. The stopper was washed with an ether mixed extraction solvent, 25 mL of anhydrous petroleum ether was added, shake-extracted again for 5 minutes, and centrifuged at 600 rpm for 5 minutes. After washing the stopper with an ether mixed extraction solvent, the ether layer was separated in a 150 mL beaker, and then the ether was slowly evaporated in a 35-40° C. water bath using nitrogen to evaporate. The fat extracted with 2-3 mL chloroform and 2-3 mL diethyl ether was dissolved, transferred to a 15 mL test tube, concentrated in nitrogen in a 40° C. water bath, and 2.0 mL 7% trifluoroboranmethanol solution and 1.0 mL toluene were added. . Sealed well with a Teflon/silicon stopper, heated in an oven at 100° C. for 45 minutes, and then cooled to room temperature. After adding 5.0 mL of distilled water, 1.0 mL of hexane and about 1.0 g of anhydrous sodium sulfate, shaking and standing, the separated supernatant was taken, placed in another vial containing about 1.0 g of anhydrous sodium sulfate, dehydrated, and used as a test solution.

Experiment result

Fatty acid composition measurement results are shown in Table 9 below.

Fatty acid type Fatty acid in the sample
(g/100g) Methyl butyrate(C4:0) 0.0000 Methyl hexanoate(C6:0) 0.0000 Methyl octanoate(C8:0) 0.0000 Methyl decanoate(C10:0) 0.0000 Methyl undecanoate(C11:0) 0.0000 Methyl laurate(C12:0) 0.0000 Methyl tridecanoate(C13:0) 0.0000 Methyl myristate(C14:0) 0.0051 Myristoleic acid methyl ester (C14:1) 0.0000 Methyl pentadecanoate(C15:0) 0.0000 Cis-10-pentadecanoic acid methyl ester (C15:1) 0.0000 Methyl palmitate(C16:0) 0.0152 Methyl palmitoleate (C16:1) 0.0047 Methyl heptadecanoate(C17:0) 0.0000 Cis-10-heptadecanoic acid methyl ester (C17:1) 0.0000 Methyl stearate(C18:0) 0.0000 Trans-9-elaidic acid methyl ester(C18:1n9t) 0.0000 Cis-9-oleic acid methyl ester (C18:1n9c) 0.0090 Linolelaidic acid methyl ester(C18:2n6t) 0.0000 Methyl linoleate(C18:2n6c) 0.0033 Methyl arachidate(C20:0) 0.0000 Gamma-linolenic acid methyl ester (C18: 3n6) 0.0000 Methyl cis-11 eicosanoate (C20:1) 0.0000 Methyl linolenate(C18:3n3) 0.0044 Methyl heneicosanoate(C21:0) 0.0000 Cis-11,14-eicosadienoic acid methyl ester(C20:2) 0.0066 Methyl behenate(C22:0) 0.0000 cis-8,11,14-eicosatrienoic acid methyl ester(C20:3n6) 0.0000 Methyl erucate(C22:1n9) 0.0000 Cis-11,14,17-eicosatrenoic acid methyl ester(C20:3n3) 0.0000 Methyl cis-5,,8,11,14-eicosatetraenoic acid
methyl ester(C20:4n6) 0.0000 Methyl trcosanoate(C23:0) 0.0123 cis-13,16-docosadienoic acid methyl ester(C22:2) 0.0000 Methyl lignocerate(C24:0) 0.0000 Methyl cis-5,8,11,14,17-eicosapentaenoate(C20:5n3) 0.0046 Methyl nervonate(C24:1) 0.0000 Cis-4,7,10,13,16,19-docosahexaenoic acid
methyl ester(C22:6n3) 0.0000

Antioxidant effect experiment

1. DPPH radical scavenging ability

In the experimental group, rhubarb hot water extract and ethanol extract by content were diluted with 50% ethanol to a concentration of 1 g/10 ml. This experiment was measured according to the method of Blois in the antioxidant assay (Blois, 1958), 900 µl of 0.1 mM DPPH (2,2-diphenyl-1-picrylhydrazyl, Sigma, USA) dissolved in ethanol, and 100 µl of each experimental group was E.P. After putting into a tube and reacting for 10 minutes, it was measured at 517 nm using a UV spectrophotometer (TECAN, Mannedorf, Switzerland), ascorbic acid (Sigma, USA) was used as a positive control, and the concentration was measured at 1 mg/ml. . The antioxidant capacity of the experimental group was calculated by obtaining the absorbance of the sample-added and no-added.

2. ABTS radical scavenging ability

In the experimental group, rhubarb hot water extract and ethanol extract by content were diluted with 50% ethanol to a concentration of 1 g/10 ml. This experiment was measured by applying the ABTS+ decolorization assay, and mixed with 7.4 mM ABTS (Sigma, USA) dissolved in distilled water and 2.6 mM potassium persulphate 1:1 in 1 ml of radical stock solution left in the cow for 12-16 hours. After 50 μl of each experimental group was added and reacted for 60 minutes, absorbance was measured using a UV spectrophotometer (TECAN, Mannedorf, Switzerland). As a positive control, ascorbic acid (Sigma, USA) 1 mg/ml and Trolox (Sigma, USA) 1 mM were used, and the antioxidant capacity of the experimental group was calculated by obtaining the absorbance of the sample and no additives.

3. SOD-like activity measurement

In the experimental group, rhubarb hot water extract and ethanol extract by content were each diluted to a concentration of 0.5 g/10 mL of Tris-HCl buffer (pH 8.5). This experiment was measured according to the method of Marklund et al., and 0.2 ml of Tris-HCl buffer (pH 8.5) and 0.2 ml of 7.2 mM pyrogallol dissolved in distilled water were added to 0.2 ml of each experimental group and left at 25°C for 10 minutes, followed by 1N HCl 1 ml After the reaction was stopped, the absorbance was measured at 420 nm using a UV spectrophotometer (TECAN, Mannedorf, Switzerland). The antioxidant capacity of the experimental group was calculated by obtaining the absorbance of the sample-added and no-added.

4. Superoxide dismutase (SOD) enzyme activity

Cell experiment: Human hepatocellular liver carcinoma cell line was purchased from the Korean Cell Bank (KCLB) and cultured with 10% FBS (fetal bovine serum) and Dulbeccos modified Eaglesmedium (DMEM, Sigma, USA) added with penicillin-streptomycin After incubation with 5% CO ₂ at a temperature of 37° C., after passage on the plate, after the cells reach a certain level of confluency, the rhubarb hot water extract and ethanol extract by content are pretreated with a concentration of 100 μg/ml. tBHP (Sigma, USA) was reacted after treating 100 uM.

SOD assay: Superoxide dismutase (SOD) activity was measured at 450 nm using the Cayman SOD assay kit, and the SOD enzyme activity of the experimental group was calculated by obtaining the absorbance of the sample and no additives.

5. Confirmation of cell viability through MTT assay

Cell experiment: Human hepatocellular liver carcinoma cell line was purchased from the Korean Cell Bank (KCLB) and cultured with 10% FBS (fetal bovine serum) and Dulbeccos modified Eaglesmedium (DMEM, Sigma, USA) added with penicillin-streptomycin The cells were cultured in a state of 5% CO ₂ at a temperature of 37° C., and after passage on a plate, the extracts according to rhubarb hot water and ethanol content at a concentration of 100 μg/ml after cell passage reached a certain level of tBHP (Sigma, USA) 100 uM was treated and reacted.

MTT assay: After incubation, add MTT reagent to each well so that the final concentration is 0.5 mg/ml, incubate for 4 hours at 37°C, 5% CO ₂ incubator, discard the culture medium, treat DMSO and dissolve formazan to dissolve formazan, UV spectrophotometer (TECAN , Mannedorf, Switzerland) was measured at 570 nm.

Experiment result

1. DPPH radical scavenging ability measurement result

As a result of confirming DPPH radical scavenging ability of rhubarb extract, it showed high antioxidant activity at the rest of the contents except for rhubarb hot water extract, 10% rhubarb ethanol extract, and 20% rhubarb ethanol extract, based on 1 mg/mL ascorbic acid, a positive control. DPPH radical scavenging activity analysis results are shown in Figure 1 below.

2. ABTS radical scavenging ability measurement result

As a result of confirming ABTS scavenging of the rhubarb extract, it can be seen that both rhubarb hot water extract and rhubarb ethanol extract by ethanol content showed high antioxidant activity based on 1 mg/mL of ascorbic acid and 1 mM of Trolox, which are positive controls. The results of ABTS radical scavenging activity analysis are shown in FIG. 2 below.

3. SOD-like activity measurement result

As a result of confirming the SOD-like activity of rhubarb extract, it shows more than 50% antioxidant activity in the remaining contents except for rhubarb hot water extract and 100% rhubarb ethanol extract, and in particular, exhibits high antioxidant activity in 50% and 70-90% ethanol content extracts . SOD-like activity measurement results are shown in Figure 3 below.

4. Superoxide dismutase (SOD) enzyme activity measurement result

In the extract of rhubarb hot water extract and ethanol content, SOD enzyme activity showed similar or high activity compared to the control group.When tBHP treatment alone, the SOD enzyme activity decreased to 80.99% compared to the control group, whereas when treated with tBHP and the experimental group Although it showed a tendency to decrease compared to the single treatment group, it was found that the enzyme activity increased when compared to the tBHP alone group, and particularly, it showed high enzyme activity in hot water and 10%, 70-90% ethanol content extract. The results of measuring superoxide dismutase (SOD) enzyme activity are shown in FIG. 4 below.

5. Cell viability confirmation result through MTT assay

Compared to the control group, cytotoxicity was shown in rhubarb 60, 100% ethanol extract, and when treated with tBHP, cytotoxicity was high in 60, 100% ethanol extract. The results of confirming the cell viability through the MTT assay are shown in FIG. 5 below.

Anti-inflammatory effect experiment

1. Measurement of cytotoxicity by MTT assay

Macrophage cells (RAW 264.7) used to measure cell viability were purchased from the ameriacan type culture collection (Manassas, USA). As the cell culture medium, DMEM medium mixed at a ratio of 10% FBS and 1% penicillin/streptomycin (100 unit/mL) was used, and the cells were subcultured by adapting to a 37°C, 5% CO ₂ incubator. RAW 264.7 was dispensed into a 96-well plate at 0.18 mL so as to be 5×104 cells/well, and samples were prepared for each concentration, and 0.02 mL was added, followed by incubation at 37° C. in a 5% CO2 incubator for 24 hours. Rhubarb 70%ethanol extract is treated for 10 hours at concentrations of 10, 25, 50, 100, 250, 500, and 1000 μg/mL, and then incubated for 4 hours by adding 0.02 mL of MTT solution prepared at a concentration of 2.5 mg/mL. . Thereafter, the culture solution was removed, and 0.1 mL of DMSO (Dimethyl Sulfoxide, DUSAN) per well was reacted for 30 minutes at room temperature, and absorbance at 540 nm was measured using an ELISA reader. Cytotoxicity was measured as a decrease in absorbance between the sample solution added group and the non-added group.

2. Nitric oxide inhibition rate measurement

The amount of Nitric oxide (NO) production was measured using the griess reagent for NO present in the cell culture medium. Seed the cell line RAW 264.7 to 5 x 10 ⁴ cells/well in a 96 well plate and incubate for 24 hours in a 37°C, 5% CO ₂ incubator. Subsequently, the culture medium is exchanged with serum-free DMEM medium and treated with lipopolysaccharide (LPS) to 1 ppm/well to induce an inflammatory reaction, and each rhubarb 70% extract is 10, 25, 50, 100, 250, 500, 1000 μg After treating by /mL concentration, incubate for 24 hours in a 37°C, 5% CO ₂ incubator. Then, 100 μL of the cell culture was transferred to a new 96 well plate, and 100 μL of griess reagent was added to react for 5 min at room temperature, and absorbance was measured at 540 nm to calculate the result.

3. Measurement of iNOS and COX-2 protein expression through Western blot

To confirm iNOS and COX-2 protein expression, seed the cell line RAW 264.7 into 2 x 10 ⁵ cells/well in a 6 well plate, and incubate for 24 hours in a 37%, 5% CO ₂ incubator. Subsequently, after replacing the culture medium with serum-free DMEM medium, lipopolysaccharide (LPS) was treated to 1 ppm/well to induce an inflammatory reaction of cells, and each sample solution was treated by concentration, followed by 37°C, 5% CO ₂ incubator Incubate for 24 hours. After that, the medium was removed, washed once with PBS, and then treated with 60 μL/well of RIPA buffer, each containing 0.1% of phosphatase and protease inhibitor, to lyse the cells and using a centrifuge at -4°C, 15 min at 15,000 rpm. During centrifugation, the obtained supernatant is transferred to a new tube. Thereafter, the amount of protein is quantified by a bradford assay, separated by mass by 10% SDS-PAGE using a power supply electrophoresis device, and transferred to a PVDF membrane. The membrane is blocked using a digital reciprocating shaker device for 5% skin milk 2 hours, primary antibody 2 hours, TBST solution 10 minutes (3 repetitions), secondary antibody 1 hour, TBST solution 10 minutes (3 repetitions). After rough reaction with HRP substrate coloring reagent for 2 minutes, the band was checked using a western imaging system (CAS-400SM, Davinch-K Co., Ltd., Korea) device, and quantitative and results were calculated.

4. Real-time PCR analysis confirmed iNOS and COX-2 expression inhibition

1) Cell lysis and cDNA synthesis

After cell seeding on a 6 well plate and incubating for 24 hours, the cells are stimulated and the sample solution is treated by concentration, and then incubated for 24 hours in a 37°C, 5% CO ₂ incubator. After removing the supernatant and dispensing 1 mL in each well of Trizol lysis buffer, the cells are lysis and left at room temperature for 5 minutes. 200 μL of Chloroform per 1 mL of Trizol buffer is added and left for 5 minutes after inverting for 30 seconds. Centrifuge After centrifugation at -4℃ for 15 minutes at 15,000 rpm, 500 μL of the supernatant is transferred to the e-tube. To this, 500 μL of Iso-propanol was added, inverted slowly, left for 5 minutes, and then centrifuged again at 15,000 rpm at -4° C. for 15 minutes. After checking the RNA pellet on the bottom of the E-tube, remove the supernatant, wash the RNA pellet with 75% ethanol, and evaporate the remaining solvent to completely remove it. After dissolving RNA pellet by adding 50 μL of diethylpyrocarbonate (DEPC) water, check the ratio of 260 nm / 280 nm and proceed with the experiment.

2) Real-time PCR

SYBR Green PCR Mastet Mix (applied biosystem, UK) using cDNA and TOPreal ^TM qPCR 2X PreMIX ((Enzynomics, Daejeon, Korea), Primer, ABI step one plus (Applied biosystem, California, USA) After analyzing and denatured for 2 minutes at 95°C using Stepone Software (Applied biosystem, California, USA), the temperature cycling conditions of reacting at 95°C for 10 seconds, 60°C for 15 seconds, and 72°C for 20 seconds are repeated 40 times. One result was analyzed.

Experiment result

1. Cytotoxicity measurement result by MTT assay

As a result of measuring the cytotoxicity of RAW 264.7 cells, 70% ethanol extract of rhubarb showed a cytotoxicity of more than 20% from a concentration of 500 μg/mL compared to the control group. The results of measuring cytotoxicity through MTT assay are shown in FIG. 6 below.

2. Nitric oxide inhibition rate measurement result

As a result of measuring the amount of NO by processing the sample for each concentration, it was found that the concentration was dependently decreased. It showed an inhibitory effect of 54.9% at the highest concentration of 1,000 μg/mL. Nitric oxide inhibition rate measurement results are shown in Figure 7 below.

3. iNOS, COX-2 protein expression measurement result through Western blot

The inhibition rate of iNOS expression of 70% ethanol extract of rhubarb was about 50.9% at 250 μg/mL and about 17.3% at COX-2. The measurement results of iNOS production inhibition of rhubarb through Western blot are shown in FIG. 8, and the measurement results of COX-2 production inhibition of rhubarb through Western blot are shown in FIG. 9. The sequences of the primers used for INOS and COX-2 are shown in Table 10.

Gene Primer Sequence(5’→ 3’) INOS Forward ACA TCG ACC CGT CCA CAG TAT Reverse CAG AGG GGT AGG CTT GTC TC COX-2 Forward TCC CTA AAG GAA AAG TGG GAC Reverse GAG CGC ATT AACCTC AGG ACC

4. Result of confirming iNOS and COX-2 expression inhibition through real-time PCR analysis

The expression rate of iNOS was confirmed to see the mechanism of inhibition of NO in rhubarb, and the expression of COX-2 was measured at mRMA level to view the inhibitory pathway of PGE ₂ , and the results were shown above. The inhibitory effect of rhubarb on iNOS expression was about 74.3% at the highest concentration of 250 μL, and the inhibitory effect on COX-2 expression was about 22.8%. The results of confirming the inhibition of iNOS and COX-2 expression through real-time PCR analysis are shown in FIG. 10 below.

Wrinkle improvement effect measurement experiment

1. Measurement of cytotoxicity by MTT assay

HacaT cells were dispensed into a 96-well plate at 0.18 mL so as to be 5×10 ⁴ cells/well, samples were prepared for each concentration, and 0.02 mL was added, followed by incubation at 37° C. in a 5% CO ₂ incubator for 24 hours. The 70% ethanol extract of rhubarb is treated for 24 hours for each concentration of 5, 10, 25, 50, 100, 250, 500 μg/mL, and then 0.02 mL of MTT solution prepared at a concentration of 2.5 mg/mL is added and incubated for 4 hours. . Thereafter, the culture solution was removed, and 0.1 mL of DMSO (Dimethyl Sulfoxide, DUSAN) per well was reacted for 30 minutes at room temperature, and absorbance at 540 nm was measured using an ELISA reader. Cytotoxicity was measured as a decrease in absorbance between the sample solution added group and the non-added group.

2. Measurement of MMP-1 protein expression through Western blot

To confirm MMP-1 protein expression, seeding the cells in a 6 well plate using HaCat cells and incubating for 24 hours in a 37°C, 5% CO ₂ incubator. After that, the supernatant is removed as much as possible, and 2 mL of PBS is dispensed for each well, and then irradiated with UV-B for 1 minute to stimulate. After replacing the culture solution with serum-free DMEM medium, each sample solution was treated by concentration, and then incubated for 24 hours in a 37°C, 5% CO ₂ incubator. After that, the medium was removed, washed once with PBS, and treated with 60 μL/well of RIPA buffer containing 0.1% of phosphatase and protease inhibitor, respectively, to lyse the cells and using a centrifuge at -4°C and 15 minutes at 15,000 rpm. During centrifugation, the obtained supernatant is transferred to a new tube. Thereafter, the amount of protein is quantified by a bradford assay, separated by mass by 10% SDS-PAGE using a power supply electrophoresis device, and transferred to a PVDF membrane. The membrane is blocked using 5% skin milk 2 hours, primary antibody 2 hours, TBST solution 10 minutes (3 repetitions), secondary antibody 1 hour, and TBST solution 10 minutes (3 repetitions) using a digital reciprocating shaker device. After rough reaction with HRP substrate coloring reagent for 2 minutes, the band was checked using a western imaging system (CAS-400SM, Davinch-K Co., Ltd., Korea) device, and quantitative and results were calculated.

Experiment result

1. Cytotoxicity measurement result by MTT assay

As a result of measuring the cytotoxicity of 70% ethanol extract of rhubarb against HaCaT cells, compared to the control group, it showed cytotoxicity of 20% or more from a concentration of 50 μg/mL. Cytotoxicity measurement results by MTT assay are shown in Figure 11 below.

2. Measurement result of MMP-1 protein expression through Western blot

MMPs are secreted from many cells, including fibroblasts and keratinocytes in the skin. As a result of confirming the expression inhibitory effect of 70% ethanol extract of rhubarb on HaCaT cells (keratinocytes), the expression inhibition rate was about 50% at 25 μg/mL. Measurement results of MMP-1 protein expression through Western blot are shown in FIG. 12 below.

In conclusion, through the results of Examples 1 to 8, the cosmetic composition according to an embodiment of the present invention contains an active ingredient of a rhubarb extract prepared using ethanol or water, thereby providing antioxidant activity, anti-inflammatory, cytotoxic, and It was confirmed that there was a wrinkle improvement effect.

As described above, the present invention has been described in detail with reference to examples, but the present invention is not limited to the above embodiments, and may be modified in various ways, and having ordinary knowledge in the art within the technical spirit of the present invention. It is clear that many variations are possible by the ruler. In addition, various forms of substitutions, modifications and changes will be possible by those skilled in the art without departing from the technical spirit of the present invention described in the claims, and this also belongs to the scope of the present invention. something to do.

Claims (4)

Cosmetic composition comprising a rhubarb extract as an active ingredient. The method according to claim 1,
The rhubarb extract,
An extraction step of adding 800 to 1200 parts by weight of ethanol to 100 parts by weight of dried and crushed rhubarb, followed by extraction for 20 to 25 hours at room temperature to prepare a rhubarb extract;
Concentration step of separating and removing the solid content contained in the rhubarb extract prepared in the extraction step, and concentrated under reduced pressure at a temperature of 40 to 50 ℃ the solid content is removed to prepare a rhubarb concentrate of 10 to 20 Brix; And
A cosmetic composition comprising a; lyophilization step of preparing a rhubarb extract by freeze-drying the rhubarb concentrate prepared in the concentration step at a temperature of -70 to -80°C. The method according to claim 1,
The rhubarb extract,
An extraction step of adding 800 to 1200 parts by weight of water to 100 parts by weight of dried and crushed rhubarb, followed by hot water extraction at 90 to 100°C for 2 to 3 hours to prepare a rhubarb extract;
Concentration step of separating and removing the solid content contained in the rhubarb extract prepared in the extraction step, and concentrated under reduced pressure at a temperature of 60 to 70 ℃ the solid content is removed to prepare a 10 to 20 Brix rhubarb concentrate; And
A cosmetic composition comprising a; a freeze-drying step of preparing a rhubarb extract by freezing the rhubarb concentrate prepared in the concentration step at a temperature of -70 to -80°C and then freeze-drying it. The method according to claim 1,
The cosmetic composition is a cosmetic composition characterized in that it is prepared in any one formulation selected from the group consisting of lotion, lotion, serum, massage cream, nutrition cream, moisture cream, hand cream, essence, nutrition essence, body lotion.

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