KR20220117020A - Cosmetic composition comprising extract obtained from herbal mixture using low-temperature aging process - Google Patents

Abstract

The present invention relates to an extract obtained from an herbal medicine mixture consisting of Angelica gigantis, Cnidium officinale, Paeonia lactiflora, Rehmanniae radix, and Scutellariae radix by using a low-temperature aging process. The extract significantly increases the content of active ingredients compared to extracts obtained by using other methods, has useful cosmetic and pharmacological activities such as antibacterial, antioxidant, anti-inflammatory, and skin non-irritation effects, and thus can be usefully used as a cosmetic composition for sensitive skin including acne skin.

Description

COSMETIC COMPOSITION COMPRISING EXTRACT OBTAINED FROM HERBAL MIXTURE USING LOW-TEMPERATURE AGING PROCESS}

The present invention relates to an extract obtained from a crude drug mixture using a low-temperature aging process, and a method for preparing the same and a use thereof. More specifically, the extract of the present invention obtained through a low temperature aging process has useful cosmetic and pharmacological activities, including antibacterial, antioxidant, anti-inflammatory and skin hypoallergenic, and can be used as a cosmetic for sensitive skin including acne skin. .

As the field of cosmetics expands, consumers' interest in the safety and efficacy of ingredients contained in cosmetics is increasing. In particular, some ingredients known to be harmful to the skin, such as phenoxyethanol and parabens, are classified as medium or high-risk grade ingredients by EWC, an American environmental research group, or their toxicity continues in various media press releases. Consumers are increasingly concerned about raw materials. Accordingly, 'hypoallergenic cosmetics' that exclude not only harmful ingredients to the skin but also ingredients that are controversial are being released.

Moreover, the development of dermacosmetic products is actively carried out in the current cosmetics market, which is analyzed to be the cause of the change in consumers' perception of cosmetics. Dermocosmetic is a compound word of 'Dermatology' meaning skin science and 'cosmetic' meaning cosmetics.

The reason why the derma cosmetic product market is growing at an amazing rate every year is that as consumers' skin becomes more sensitive, more and more people are looking for derma cosmetic products developed based on the research results of skin science as a way to solve this problem. Most women with sensitive skin are interested in symptoms such as dryness and itching, and there are many cases where cosmetics cause serious skin problems. Accordingly, more and more consumers are taking active measures for sensitive skin that reacts sensitively to the external environment regardless of skin types such as dry, oily, neutral, or combination, and derma cosmetic products are in the spotlight among these consumers.

According to the '2017 Female Skincare Consumer Trend Report' published by NPD Group, a market research firm, approximately 50% of female consumers check the ingredients when purchasing cosmetics. Domestic consumers are also changing the trend of purchasing products after carefully examining the ingredients contained in cosmetics rather than purchasing products based on brand awareness or model. Accordingly, the beauty industry is releasing products emphasizing that they contain natural ingredients, which is intended to relieve consumers' psychological anxiety by including natural ingredients.

There are many natural products used in cosmetics that are pharmacologically effective for living things, and the substance can be said to be a mixture of various chemical substances. These herbal ingredients are applied not only for cosmetic decoration, but also for skin or hair protection, wool, and the like. When 'natural ingredients' such as herbal medicines with pharmacological efficacy are used as materials for cosmetics, they not only give consumers psychological stability but also provide practical help to the skin.

According to the naturalistic trend of using herbal medicines as materials for cosmetics, cosmetics using various natural materials have been developed, from vegetable raw materials including crude drugs to various fermented raw materials. became

On the other hand, low-temperature aging is a technology inspired by the wisdom of our ancestors who aged natural materials for a long time at low temperatures.

Moreover, with respect to the technology of fermenting natural materials in a low-temperature environment, when the low-temperature aging technology is used, it is possible to prevent excessive growth of microorganisms but not inhibit the growth, thereby increasing the efficacy. Therefore, the low-temperature aging technology can generate the most active ingredients from natural materials through decomposition or synthesis from microorganisms and minimize toxic substances, so that the obtained natural materials can maximize their efficacy when used as raw materials for cosmetics. have.

As a result of the present inventors' diligent efforts to develop a cosmetic composition that not only gives consumers psychological stability but also provides practical help to the skin, Angelica gigas Nakai, Cnidium officinale Makino, Paeonia lactiflora Pall, and Sukjihwang It was found that the extract obtained by applying a low-temperature aging process to a herbal mixture consisting of ( Rechmannia glutinosa Libosch) and gold ( Scutellaria baicalensis Georgi) exhibits optimal efficacy when applied to sensitive skin including acne-prone skin. Thus, the present invention was completed.

It is an object of the present invention to provide a cosmetic composition comprising an extract obtained by using a low-temperature aging process from a herbal mixture consisting of Angelica asiatica, chrysanthemum, peony, sukjihwang, and gold.

Another object of the present invention is to provide a cosmetic use of a cosmetic composition for application to sensitive skin including acne skin.

Another object of the present invention is to provide a formulation of the cosmetic composition having hypoallergenicity to the skin, for example, a facial cream, body lotion or body cream.

Other objects of the present invention will become apparent to those skilled in the art from the following description.

In order to achieve the above object, in one aspect, the present invention provides a cosmetic composition comprising an extract obtained by using a low-temperature aging process from a herbal mixture consisting of Angelica asiatica, cheongung, peony, sukjihwang and gold.

In one embodiment, the herbal mixture can be obtained by mixing angelica, cheongung, peony, sukjihwang and gold in the same ratio.

In another embodiment, the low-temperature aging process may include a low-temperature aging step of aging the crude drug mixture as a solvent at a temperature of 2°C to 6°C for 5 to 10 days. Preferably, the low-temperature aging process includes a low-temperature aging step of aging the crude drug mixture with a solvent at a temperature of 4° C. for 7 days.

In another embodiment, in the low-temperature aging step, the solvent may be selected from the group consisting of purified water, mineral water, and carbonated water. Preferably, the solvent is carbonated water.

In another embodiment, in the low-temperature aging step, the solvent may be used in an amount 10 times the weight of the crude drug mixture.

In another embodiment, the extract may be obtained by repeatedly extracting the low-temperature aged herbal medicine mixture with hot water (purified water) or 70% ethanol three times. Preferably, the extract is obtained by repeatedly extracting the low-temperature aged herbal medicine mixture with 70% ethanol three times using carbonated water as a solvent.

In another embodiment, the cosmetic composition may be used to improve acne.

In another embodiment, the cosmetic composition may be formulated as a facial cream, body lotion or body cream. In this case, the cosmetic composition may further include a milk protein component and a baobab seed component. The milk protein component is preferably a colostrum extract of a cow, more preferably a New Zealand cow, and the baobab seed component may be a baobab seed extract.

The extract obtained by using the low-temperature aging process from the herbal mixture consisting of Angelica asiatica, chrysanthemum, peony, Sukhumi hwang and gold according to the present invention significantly increases the content of active ingredients compared to extracts obtained using other methods, and exhibits antibacterial, antioxidant, and anti-inflammatory properties. And it has useful cosmetic and pharmacological activity including skin hypoallergenicity, so it can be usefully used as a cosmetic composition for sensitive skin including acne skin.

1 shows the results of confirming the electron donating ability of 11 extracts in relation to the in vitro efficacy evaluation of extracts for each solvent of Example 2.
2 shows the results of confirming the ABTS radical scavenging ability in 11 extracts in relation to the in vitro efficacy evaluation of the extracts for each solvent of Example 2.
3 shows the results of measuring the antibacterial activity of some extracts among 11 extracts in relation to the evaluation of the in vitro efficacy of the extracts for each solvent of Example 2.
4 shows the results of cell viability measurement by MTT assay for some of the 11 extracts in relation to the evaluation of the in vitro efficacy of the extracts for each solvent of Example 2.
5 shows the results of measuring the NO production inhibitory activity of some of the 11 extracts in relation to the in vitro efficacy evaluation of the extracts for each solvent of Example 2.
6 shows the results of measuring protein expression through Western blot for some of the 11 extracts in relation to the in vitro efficacy evaluation of the extracts for each solvent of Example 2.
7 shows the measurement results of mRNA expression through RT-PCR (reverse transcription PCR) for some of the 11 extracts in relation to the evaluation of the in vitro efficacy of the extracts for each solvent of Example 2.
Figure 8 shows the results of HPLC analysis for identifying the indicator substance in the extract E-3 of the present invention.
9 shows the results of the preservative test evaluation of the cosmetic formulation (body lotion) of the present invention.
10 shows the results of the preservative test evaluation of the cosmetic formulation (body cream) of the present invention.
11 shows the results of the preservative test evaluation of the cosmetic formulation (facial cream) of the present invention.

As mentioned above, the present invention is based on the discovery that an extract obtained using a low-temperature aging process from a herbal mixture consisting of Angelica asiatica, chrysanthemum, peony, Sukhumi hwang and gold exhibits optimal efficacy when applied to sensitive skin including acne-prone skin. it has been done

Accordingly, in one aspect, the present invention provides a cosmetic composition comprising an extract obtained using a low-temperature aging process from a herbal mixture consisting of Angelica asiatica, cheongung, peony, sukjihwang and gold.

Angelica gigas Nakai ( Angelica gigas Nakai) is a perennial herbaceous plant belonging to the Umbrella family. It is collected in late autumn, removed the root of the beard, waited for the moisture to evaporate slightly, and then dried while slowly fumigating with smoke. Angelica is used as medicine for gynecological diseases and has analgesic, sedative, antiarrhythmic, antithrombotic, hwahyeol, purifying, tonic, liver function protection and anticancer action.

Cnidium officinale Makino is a perennial herb belonging to the Umbrella family, and dried roots and stems collected from September to November are used as medicines. This drug has pharmacological actions such as sleep action, blood pressure lowering action, vasodilation action, and treatment of vitamin E deficiency, and is said to have excellent blood retention and analgesic action.

Peony ( Paeonia lactiflora Pall) is a dried root of red peony or a closely related plant, which is a perennial herb belonging to the Lepidoptera family. It contains peaoniflorin, the main component, benzoic acid, essential oil, fatty acid, tannin, sugar, triterpenoid, β-sitosterol, and the like.

Rechmannia glutinosa Libosch is a perennial plant belonging to the ginseng family. It is used as a laxative, hemostatic agent, hematemesis, constipation, hypersexuality, diabetes, etc., and is known to have high immunity.

Gold ( Scutellaria baicalensis Georgi ) is a perennial herb of the Lamiaceae family. Its main components are 30 kinds of flavonoid compounds, 14 kinds of amino acids, essential oil, and stigmasterol. It has antiviral, cellular immunity, It has anticancer action.

In one embodiment, the herbal mixture may be a mixture of angelica, cheongung, peony, sukjihwang and gold in the same ratio.

In another embodiment, the low-temperature aging process may include a low-temperature aging step of aging the crude drug mixture as a solvent at a temperature of 2°C to 6°C for 5 to 10 days. Preferably, the low-temperature aging process includes a low-temperature aging step of aging the crude drug mixture with a solvent at a temperature of 4° C. for 7 days.

In another embodiment, in the low-temperature aging step, the solvent may be selected from the group consisting of purified water, mineral water, and carbonated water. Preferably, the solvent is carbonated water.

As used herein, the term “purified water” refers to water purified through an ion exchange resin.

As used herein, the term “mineral water” refers to water containing minerals (minerals).

As used herein, the term “carbonated water” refers to water comprising carbonic acid.

In another embodiment, in the low-temperature aging step, the solvent may be used in an amount 10 times the weight of the crude drug mixture.

In another embodiment, the extract may be obtained by repeatedly extracting the low-temperature aged herbal medicine mixture with hot water (purified water) or 70% ethanol three times.

As such, the extract obtained using the low-temperature aging process (especially in the case of repeated extraction with 70% ethanol three times after the low-temperature aging step) has a high polyphenol content when measuring the total polyphenol content compared to the extract obtained only with hot water or 70% ethanol. (see Table 2), and showed a high scavenging rate when measuring electron donating ability and ABTS radical scavenging activity using 1-diphenyl-2-picrylhydrazyl (DPPH), respectively (see Table 3 and Table 4 below). In addition, the extract obtained by using the low-temperature aging process for antibacterial activity measurement (paper disc method), cell viability measurement (MTT assay) and anti-inflammatory measurement (in particular, after carbonated water is used as a solvent in the low-temperature aging step, purified water or 70 % ethanol) and showed a favorable effect (see Tables 5 to 9 below).

In particular, when carbonated water was used as a solvent in the low-temperature aging step and then extracted with purified water or 70% ethanol, as a result of analyzing the physiologically active substance content of the obtained extract, the content of decursin was detected the most, followed by paeoniflorin, baicalein, and ligustilice. , was confirmed to be detected in the order of luteolin. Accordingly, it was analyzed that a relatively large amount of decursin was extracted during the low-temperature aging process, and the main component of the extract was confirmed as decursin (see Table 12 and FIG. 8 below).

In addition, it can be seen that the cosmetic composition can be used for the improvement of acne from the advantageous effect shown when measuring the antibacterial activity.

On the other hand, the cosmetic composition according to the present invention is a stabilizer, solubilizer, vitamins, pigments and fragrances in addition to extracts obtained from a herbal mixture consisting of active ingredients such as Angelica asiatica, chrysanthemum, peony, sukjihwang and gold using a low-temperature aging process. adjuvants and cosmetically acceptable carriers.

In another embodiment, the cosmetic composition of the present invention may be prepared in any formulation conventionally prepared in the art, for example, a solution, suspension, emulsion, paste, gel, cream (facial cream and body cream). including), lotion (including body lotion), detergent, powder, oil, powder foundation, emulsion foundation, wax foundation, and spray, but is not limited thereto. More specifically, the cosmetic composition of the present invention can be prepared in the form of sun cream, softening lotion, astringent lotion, nourishing lotion, nourishing cream, massage cream, essence, eye cream, pack, spray, face wash, bath agent or powder. .

When the formulation is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tragacanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as a carrier component. have.

When the formulation is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component, and in particular, in the case of a spray, additional chlorofluorohydrocarbon, propane/butane or a propellant such as dimethyl ether.

When the formulation is a solution or emulsion, a solvent, solubilizer or emulsifier is used as a carrier component, for example, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3 -Butylglycol oil, glycerol fatty ester, polyethylene glycol or fatty acid ester of sorbitan can be used.

When the formulation is a suspension, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystalline cellulose , aluminum metahydroxide, bentonite, agar or tragacanth may be used.

In particular, the cosmetic composition may be formulated as a facial cream, body lotion or body cream. In this case, the cosmetic composition may further include a milk protein component and a baobab seed component. The milk protein component is preferably a colostrum extract of a cow, more preferably a New Zealand cow, and the baobab seed component may be a baobab seed extract.

Hereinafter, the present invention will be described in more detail by the following examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited thereto.

Example

<Reference example>

1. Reagents

Among the reagents used in the experiment, MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-tetrazolium bromide), grease reagent, and lipopolysaccharide (LPS) were Sigma (Sigma). , St. Louis, USA) was used. Cell culture reagents such as DMEM (Dulbecco's modified Eagle medium), fetal bovine serum (FBS), and penicillin/streptomycin were purchased from Gibco BRL (Grand Island, USA), and the primary antibody , Inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) from Cell Signaling (Boston, USA), secondary antibody mouse anti-rabbit IgG HRP (mouse anti-rabbit IgG HRP) was purchased from Santa Cruz (Santacruz, CA, USA).

2. Strain and Cell Culture

Propionibacterium acnes KCTC 3314 (P. acnes) used in the antibacterial activity measurement experiment was purchased from the Korea Research Institute of Bioscience and Biotechnology Biological Resources Center and used. P. acnes was cultured using a medium (Difco TM Reinforced Clostridial Medium) in an anaerobic environment (GasPak EZ Anaerobic Container System (BD, USA)) at 37°C.

RAW 264.7, a mouse macrophage cell line, was purchased from the Korea Cell Line Bank (Seoul, Korea) and added with 10% FBS and 25 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) at 37°C and 5% CO2. It was cultured using one DMEM (Dulbecco's modified Eagle's medium) medium.

3. Microorganisms for preservative testing, preparation of inoculum, inoculation method and evaluation criteria

Among the five bacteria used in the antiseptic test, bacteria E coli KCTC2571, Paeruginosa KCTC2513 and S aureus sub Aureus KCTC1916, yeast Candida albicans KCTC7965, and mold Aspergillus niger KCTC6317 were obtained from the Korea Research Institute of Bioscience and Biotechnology Biological Resources Center (KCTC). Received and used.

In addition, the inoculum for use in the preservative test was prepared and inoculated as follows. First, C. albicans cells cultured in potato dextrose broth/agar (PDA) medium were inoculated into 9 mL of sterilized saline 0.85% using an inoculation loop, and the dilution concentration was 1.0×10 ⁵ CFU/mL. The concentration was adjusted. A. niger cultured in PDA medium was dispersed into sterile 0.85% saline using an inoculation loop after flowing 9 mL of sterile 0.85% saline into the medium. The concentration was adjusted so that the dilution concentration of the bacteria was 1.0×10 ⁵ CFU/mL. Each cell of P. aeruginosa, S. aureus and E. coli cultured in tryptic soy broth/agar (TSA) was floated through an inoculation loop and inoculated in 9 mL of sterilized saline 0.85%. The dilution concentration of each bacteria was diluted to 1.0×10 ⁸ CFU/mL, and the concentration of the suspension was adjusted to 3.0×10 ⁸ CFU/mL based on McFarland standards. The sample prepared by the above method was added to the inoculated bacterial solution at 5.50×10 ⁵ CFU/mL. In addition, yeast and mold cultures were inoculated to 5.50×10 ⁵ CFU/mL and then stored at room temperature. For the sample inoculated with the bacteria, 1 g of each sample was collected for 0 days, 3 days, 7 days, 14 days, and 28 days of inoculation, and diluted 10-fold with sterilized physiological saline solution was prepared in the culture medium. Specific culture conditions for each strain are as follows:

In order to measure the preservative power in each medium treated with the sample, the number of viable cells measured was converted into a log reduction value to confirm the death rate. The preservative power of the sample fluid was analyzed according to the preservative standard of CTFA, which is judged to have preservative power.

<Production Example>

1. Preparation of extract of herbal mixture of the present invention using low temperature aging process

Angelica (domestic), cheongung (domestic), peony (domestic), Sukjihwang (domestic), and golden (domestic) medicinal materials purchased from Omnihub Co., Ltd. (located in Yeongcheon, Gyeongbuk) were washed with water and used after drying in the shade. A total of 50 g of the herbal mixture (hereinafter, referred to as "the herbal mixture of Preparation Example") was prepared by mixing the above medicinal substances in the same ratio.

A solvent (purified water, mineral water, or carbonated water) in an amount 10 times the amount thereof was added to the thus-prepared herbal mixture, and the mixture was aged at a low temperature at 4°C for 7 days. The low-temperature aged herbal medicine mixture was centrifuged to separate the supernatant and the precipitate.

Then, the precipitate was washed with purified water and dried. The dried precipitate was extracted with hot water or 70% ethanol. In the case of hot water extraction, 1,500 mL of distilled water (DW) was added to the dried precipitate, followed by extraction at 100° C. for 9 hours; In the case of 70% ethanol extraction, a 10-fold amount of 70% ethanol was added to the dried precipitate, followed by extraction for 24 hours. This extraction process was repeated three times.

The extract extracted with hot water or 70% ethanol was filtered, concentrated, and then freeze-dried to obtain an extract of a low-temperature aged herbal medicine mixture.

<Comparative example>

1. Preparation of extract of herbal mixture by only hot water extraction

1,500 mL of distilled water was added to the herbal mixture of Preparation Example, followed by extraction at 100° C. for 9 hours. Then, the resulting extract was filtered, concentrated, and freeze-dried to obtain an extract of the herbal mixture extracted only with hot water.

2. Preparation of extracts of herbal mixtures only by ethanol extraction

10 times the amount of 70% ethanol was added to the herbal mixture of Preparation Example, followed by extraction for 24 hours. This was repeated 3 times. Then, the resulting extract was filtered, concentrated, and freeze-dried to obtain an extract of the herbal mixture extracted with 70% ethanol.

The extracts obtained in <Preparation Example> and <Comparative Example> were classified by solvent and shown in Table 1 below.

[Table 1] Names of extracts by solvent

Example 1. Measurement of total polyphenol content of extracts by solvent

Extracts for each solvent obtained in <Preparation Examples> and <Comparative Examples>, that is, extracts A, B, C-1, C-2, C-3, D-1, D-2, D-3, E-1 , E-2 and E-3 as samples, their total polyphenol content was measured by modifying the Folin-Denis method. Polyphenol compounds present in plants are known to have various physiological activities such as antioxidants.

Specifically, each extract was diluted in distilled water, 2 ml of the diluted extract and 2 ml of 50% Folin-ciocalteu reagent (Sigma, USA) were mixed, and then left at room temperature for 3 minutes. Then, 10% Na ₂ CO₃ 2 ㎖ was added and mixed, and then left at room temperature for 1 hour. The absorbance was then measured at 700 nm. After preparing a calibration curve (y=0.0361x-0.2441, R ² =0.9991) using tannic acid as a standard material, the total polyphenol content was expressed as mg tannic acid equivalent (TAE) per 100 g.

Table 2 shows the results of measuring the total polyphenol content in 11 extracts using tannic acid as a standard material as described above.

[Table 2] Measurement of total polyphenol content

As can be seen from Table 2, extracts A and B showed polyphenol contents of 109.51±0.04 mg TAE/100 g and 115.75±0.37 mg TAE/100 g, respectively; Extracts C-1, C-2 and C-3 showed polyphenol content of 81.74±0.39 mg TAE/100 g, 97.64±0.35 mg TAE/100 g and 280.90±0.23 mg TAE/100 g, respectively; D-1, D-2 and D-3 showed polyphenol content of 76.90±0.59 mg TAE/100 g, 121.95±0.53 mg TAE/100 g and 284.35±0.45 mg TAE/100 g, respectively; E-1, E-2 and E-3 showed contents of 90.24±0.70 mg TAE/100 g, 121.58±0.34 mg TAE/100 g and 474.76±0.45 mg TAE/100 g, respectively.

From these results, it was confirmed that the polyphenol content of carbonated water E-3 (70% ethanol extract of the crude drug mixture of Preparation Example that was aged at low temperature by adding carbonated water) was the highest.

Example 2. Extraction of each solvent in vitro Efficacy evaluation

1) Measurement of antioxidant activity

A) Measurement of electron donating ability

The electron donating ability measurement experiment was performed as follows. 60 μl of 1-diphenyl-2-picrylhydrazyl (DPPH) and 120 μl of each concentration extract were added, mixed, left for 15 minutes, and then absorbance was measured at 517 nm using a microplate reader. At this time, vitamin C (VIT-C) was used as a control. The electron donating ability was expressed as the absorbance decrease rate of the group with and without the addition of the sample solution (refer to Equation (1) below).

(1)

(One)

The results of confirming the electron donating ability of the 11 extracts as described above are shown in Table 3 and FIG. 1 below.

[Table 3] Electron donating ability measurement result table

As a result of measuring the extracts for each solvent at a concentration of 1,000 μg/ml, as can be seen from Table 3 and FIG. 1, A and B exhibited 44% and 67% elimination rates, respectively; Purified water C-1, C-2 and C-3 exhibited 22%, 44% and 96% clearance, respectively; Minerals D-1, D-2 and D-3 exhibited 9%, 58% and 95% clearance, respectively; Carbonated water E-1, E-2, and E-3 exhibited 16%, 77%, and 94% removal rates, respectively.

As a result, the ethanol extracts aged at low temperature using all solvents (purified water, mineral water and carbonated water), i.e., purified water C-3, mineral D-3, and carbonated water E-3, have a removal rate of 94% or more at a concentration of 1,000 μg/ml. As it was confirmed to represent the, it has excellent antioxidant activity.

B) Measurement of ABTS radical scavenging activity

Measurement of antioxidant activity using ABTS radicals was performed as follows. 7 mM 2,2-azino-bis(3-ethyl-benthiazoline-6-sulfonic acid) and 140 mM potassium persulfate are mixed, stored at room temperature for 16 to 18 hours to form ABTS+, and then diluted with ethanol for use did. 150 μl of the sample was added to 3 ml of ABTS+ and absorbance was measured at 700 nm. At this time, Vitamin C (VIT-C) was used as a control.

ABTS radical scavenging ability was calculated according to the following formula (2):

(2)

(2)

As above, the results of confirming the ABTS radical scavenging ability in the 11 extracts are shown in Table 4 and FIG. 2 below.

[Table 4] ABTS radical scavenging activity measurement result table

As a result of measuring the extract for each solvent at a concentration of 1,000 μg/ml, as can be seen from Table 4 and FIG. 2, A and B exhibited 35% and 50% clearance, respectively; Purified water C-1, C-2 and C-3 exhibited 15%, 47% and 70% clearance, respectively; Minerals D-1, D-2 and D-3 exhibited 14%, 34% and 74% clearance, respectively; Carbonated water E-1, E-2 and E-3 exhibited 14%, 37% and 84% clearance, respectively.

As a result, it was confirmed that carbonated water E-3 exhibited the best ABTS radical scavenging rate (84%) among extracts that had undergone a low-temperature aging process using purified water, mineral water, and carbonated water.

2) Measurement of antibacterial activity (paper disc method)

Purified water C-2 and C-3, mineral water D-2 and D-3, and carbonated water E-2 and E-3 among the extracts for each solvent using Propionibacterium acnes ( P. acnes ), the causative bacteria of acne The antibacterial activity shown against was measured as follows.

First, about 1 platinum of each strain cultured on a plate medium was taken, cultured in 10 ml of liquid medium for 24 hours to activate, and then 0.1 ml of the bacterial solution was inoculated into 10 ml of liquid medium again, followed by main culture for 5 hours. This was inoculated so that the number of bacteria per plate medium was about 10 ⁷ cells, and spread evenly with a sterile cotton swab. A sterilized filter paper disc (8 mm, Tokyo, Japan) is placed on a solid plate medium, and the sample is absorbed by concentration so as to be 0.05 ml/disc, and incubated at 37°C for 24 hours in a clear zone ( The antibacterial activity was confirmed by measuring the diameter of the clear zone (mm).

The results of measuring the antibacterial activity as described above are shown in Tables 5 to 7 and FIG. 3 below.

[Table 5] Measurement of antibacterial activity (purified water low temperature aging)

[Table 6] Measurement of antibacterial activity (mineral water low temperature aging)

[Table 7] Measurement of antibacterial activity (low temperature aging in carbonated water)

As shown in Table 7 and Figure 3, it was confirmed that both carbonated water E-2 and E-3 had antibacterial activity against P. acnes , and when used at a concentration of 10 mg/ml or more, 13 mm or more and 15 mm or more, respectively. It was confirmed that an inhibitory ring was formed.

3) Measurement of cytotoxicity (MTT assay) and measurement of anti-inflammatory activity

i) cell culture

For culturing of RAW 264.7 macrophages used in this experiment, DMEM medium supplemented with 10% FBS and 1% penicillin/streptomycin (100 U/ml) was used, and the macrophages were incubated at 37° C. and 5% CO ₂ in an incubator. Adapted and subcultured.

ii) Cell viability measurement and results by MTT assay

Cell viability was measured by dispensing 100 μl of RAW 264.7 cells cultured in item i) to 1×104 cells/well in a 96-well plate, preparing samples by concentration, and adding them, 37°C, 5% CO Incubated for 24 hours in ₂ incubator. In this case, carbonated water E-2 and E-3 having high total polyphenol content and excellent antioxidant activity were used as samples. 40 μl of MTT solution prepared at a concentration of 2.5 mg/ml was added to the culture and cultured for 3 hours. Then, the culture solution was removed, and 100 μl of DMSO was added to each well and reacted at room temperature for 10 minutes. Then, absorbance was measured at 540 nm with an ELISA reader. Cell viability was expressed as a decrease in absorbance of the group with and without the addition of the sample solution.

The results of cell viability measurement by the MTT assay performed as above are shown in Table 8 and FIG. 4 .

[Table 8] Cell viability measurement result table

As a result of processing carbonated water E-2 and E-3 for Raw 264.7 cells by concentration, as can be seen from Table 8 and FIG. 4, both carbonated water E-2 and E-3 at a concentration of 500 μg/ml are 90 % or higher survival rate.

iii) Measurement of nitric oxide (NO) production inhibitory activity

The amount of NO produced from the RAW 264.7 cells cultured in item i) was measured in the form of NO ₂ present in the cell culture medium using griess reagent.

Specifically, RAW 264.7 cells were seeded in a 96-well plate to 1×10 ⁶ cells/well. 37° C., 5% CO ₂ Incubated in an incubator for 24 hours, and treated with LPS 10 μg/ml (except normal). After 2 hours, the sample solutions (carbonated water E-2 and E-3) prepared by concentration were treated and cultured for 24 hours to obtain a supernatant. Then, the same amount of griess reagent was added and reacted in a 96-well plate for 10 minutes, and absorbance at 540 nm was measured. The NO inhibitory activity was expressed as the absorbance reduction rate of the group with and without the addition of the sample solution.

The results of the NO production inhibitory activity measurement performed as above are shown in Table 9 and FIG. 5 .

[Table 9] NO production inhibition activity measurement result table

As can be seen from Table 9 and FIG. 5, when the LPS-treated group (Control) was 100%, carbonated water E-2 and E-3 reduced the NO production in a concentration-dependent manner.

iv) Measurement of protein expression by Western blot

In order to investigate the effect of carbonated water E-2 and E-3 on the protein expression levels of iNOS and COX-2, which are inflammatory mediators, an experiment was performed as follows.

The RAW 264.7 cells cultured in item i) were seeded in a 10 mm tissue culture dish and then cultured for 24 hours. After culturing for 24 hours in a medium treated with sample solutions (carbonated water E-2 and E-3) prepared by concentration, 100 μl of complete mini 1 tab was added to Radio-immunoprecipitation assay (RIPA) buffer and dissolved. Then, the supernatant obtained by centrifugation at 4 ℃ and 13,200 rpm was quantified using a BCA protein assay kit. After separation of the protein by electrophoresis on 10% SDS-PAGE, the separated protein was transferred to a polyvinylidene fluoride (PVDF) membrane. A blocking buffer (5% skim milk in TBST (tris-buffered saline and tween 20)) was prepared and blocking was performed at room temperature for 1 hour. The primary antibody was diluted and left at 4° C. overnight, and then washed 3 times with TBST at 10-minute intervals again. The secondary antibody was diluted and reacted at room temperature for 1 hour and 30 minutes, washed 3 times, and then the band was identified and quantified.

The results of the protein expression measurement performed as above are shown in FIG. 6 . In this case, β-actin, a house keeping gene, was used as a positive control.

As can be seen from FIG. 6 , the expression level of each protein was increased in the LPS-only treatment group; When treated at a concentration of 100 μg/ml, carbonated water E-3 (iNOS and COX-2 protein expression levels were higher than that of carbonated water E-2 (iNOS and COX-2 protein expression levels were 92% and 94%, respectively)) 34% and 60%, respectively) showed good inhibition of protein expression.

v) Measurement of mRNA expression through RT-PCR (reverse transcription PCR)

To investigate the effect of carbonated water E-2 and E-3 on the mRNA expression levels of iNOS and COX-2, which are inflammatory mediators, reverse transcription polymerase chain reaction (RT-PCR) was performed as follows. In this case, GAPDH, a house keeping gene, was used as a positive control.

[Table 10] Sequence of primers used for RT-PCR

The sequences of the primers used in the experiment are shown in Table 10. In the case of GAPDH and iNOS, PCR was performed under cycling conditions of 2 minutes at 96 °C, 10 seconds at 96 °C, 30 seconds at 64 °C, 1 minute at 72 °C, and 10 minutes (40 cycles) at 72 °C, and COX- In case 2, PCR was performed under cycling conditions of 2 minutes at 96°C, 10 seconds at 94°C, 30 seconds at 51°C, 1 minute at 72°C, and 10 minutes at 72°C (40 cycles). Then, after electrophoresis on 1.5% agarose gel with 0.002% ethidium bromide added at 100 V for 20 minutes, bands were identified, analyzed and quantified.

The results are shown in FIG. 7 . As can be seen from FIG. 7 , in the LPS-only treatment group, the mRNA expression levels of iNOS and COX-2 were increased; When treated at a concentration of 100 μg/ml, carbonated water E-2 (mRNA expression levels of iNOS and COX-2 were 29% and 57%, respectively) and carbonated water E-3 (mRNA expression levels of iNOS and COX-2 were 19% and 28% respectively) showed good inhibition of mRNA expression.

Example 3 Analysis of the content of physiologically active substances in carbonated water E-3

In order to analyze the components contained in the extract, first, literature research was conducted using specialized information and papers, and then analysis data of the extract components and standard materials were collected, and representative components of each herbal mixture were collected and selected (refer to the literature [ Jeong SY, Kim HM, Lee KH, Kim KY, Huang DS, Kim JH, Seong RS (2015) Quantitative analysis of marker compounds in Angelica gigas, Angelica sinensis, and Angelica acutiloba by HPLC/DAD. 511 https://doi.org/10.1248/cpb.c15-00081];H . Hong, JC An, JF de La Cruz, SG Hwang Cnidium officinale Makino extract induces apoptosis through activation of caspase-3 and p53 in human liver cancer HepG2 cells Exp Ther Med, 14 (2017), pp. 3191-3197; Wu, M. and Gu, Z. (2009) Screening of Bioactive Compounds from Moutan Cortex and Their Anti-Inflammatory Activities in Rat Synoviocytes Evidence-Based Complementary and Alternative Medicine, 6, 57-63; and L Yu, AG Wu, VKW Wong, LQ Qu, N Zhang, DL Qin, W Zeng, B Tang, HM Wang, Q Wang* and BYK Law*. The new application of UHPLC-DAD-TOF/MS in identification of inhibitors on beta-amyloid fibrillation from Scutellari a baicalensis. Front Pharmacol. 2019;10:194).

The analysis conditions were as follows. For HPLC analysis, waters ACQUITY H-Class Liquid Chromatograph is used, column is waters BEH C18, mobile phase is acetonitrile and water as solvent gradient, injection volume 10 ul at a flow rate of 1.0 ml/min, column temperature 30°C, UV absorbance was tested at 280 nm (see Table 11). As a result of creating a calibration curve using the peak area, a correlation coefficient value of 0.9998 or more was secured.

[Table 11] HPLC conditions

As a result of analyzing the extract E-3 under the above conditions, it was confirmed that the content of decursin was detected the most, followed by paeoniflorin, baicalein, ligustilice, and luteolin in order (see Table 12 and FIG. 8). As a result, as it was analyzed that a relatively large amount of decursin was extracted during the low-temperature aging process, the main indicator component of the low-temperature-aged herbal mixture extract of the present invention was confirmed as decursin.

[Table 12]

Example 4. Preparation of cosmetic formulations containing extract E-3 and confirmation of its efficacy

1) Preparation of body lotion (hereinafter referred to as “test body lotion” or simply “product”) and body cream (hereinafter referred to as “test body cream” or simply “product”) containing extract E-3

5 ingredients (mineral oil, silicone oil, PEG, color and artificial flavor) free, fragrance free, EWG green grade, extract E-3 (2% (w/w)), cow colostrum component and baobab seed Test body lotions and test body creams containing the ingredients were prepared based on the formulas shown in Tables 13 and 14 below, respectively.

[Table 13] Test Body Lotion Formula

[Table 14] Test Body Cream Formula

The pH slightly acidic and hypoallergenic test body lotion and test body cream prepared in this way are excellent in relieving skin irritation and soothing skin for sensitive skin. . In addition, these body lotions and body creams are quickly absorbed into the skin without stickiness, supplying moisture and nutrition to dry or sensitive skin, and at the same time making the skin comfortable with deep moisturizing and soothing effects. The efficacy of the test body lotion and test body cream as described above can be clearly seen from the evaluation results described below.

2) Preparation of a facial cream containing extract E-3 (hereinafter referred to as “test facial cream” or simply “product”)

5 ingredients (mineral oil, silicone oil, PEG, color and artificial flavor) free, fragrance free, EWG green grade, extract E-3 (2% (w/w)), cow colostrum component and baobab seed A test facial cream containing the ingredients was prepared based on the formula shown in Table 15 below.

[Table 15] Test Facial Cream Formula

The pH slightly acidic and hypoallergenic test facial cream prepared in this way has excellent skin irritation relief and skin soothing effects on sensitive skin. In addition, this test facial cream is quickly absorbed into the skin without stickiness, providing moisture and nutrition to dry or sensitive skin, while at the same time making the skin comfortable with deep moisturizing and soothing effects. The efficacy of the test facial cream as described above can be clearly seen from the various performance evaluation results described below.

3) Evaluation of various performance of test body lotion, test body cream and test facial cream

A) Evaluation of the Challenge Test

In order to secure the objectivity and reproducibility of the test, a preservative force test was performed. The preservative force test is to inoculate a cosmetic formulation to check whether the microorganisms are killed, and it was tested based on the CTFA test method. For the preservative test, microorganisms derived from formally collected strains of EU member states were used as test strains, and 3 types of bacteria and 2 types of fungi were used for the test strains. The strain information used in the experiment was as follows:

① Bacteria: E coli KCTC2571, P aeruginosa KCTC2513, S aureus sub Aureus KCTC1916

② Fungi : Candida albicans KCTC7965 , Aspergillus niger KCTC6317

The preservative test evaluation results of each of the formulations (test body lotion, test body cream and test facial cream) are shown in Tables 16 to 18 and FIGS. 9 to 11 below.

[Table 16] Evaluation result of preservative force test of test body lotion

[Table 17] Evaluation result of preservative power test of test body cream

[Table 18] Evaluation result of preservative power test of the test facial cream

As can be seen from Tables 16 to 18 and FIGS. 9 to 11 , in each formulation, no bacteria were detected from day 1 onwards, and no bacteria and fungi were detected from day 3 onwards.

B) Skin patch safety evaluation

In order to investigate the safety of each of the above formulations (test facial cream, test body cream and test body lotion), 30 healthy adults (average age: 37.0±9.6 years old) were used as test subjects, according to Semyung University Cosmetics Clinical Research Support Center standard The skin patch test was performed using IQ Ultra according to the operating procedure.

25 ml of each formulation was added dropwise to the IQ Ultra. The patch was attached for 24 hours, 30 minutes after patch removal (1st), 24 hours after patch removal (2nd), 48 hours after patch removal (3rd) by 2 experts The stimulation index was calculated according to the criteria of Dermatitis Research Group, ICDRG) and the degree of stimulation was determined. The results are shown in Tables 19 to 21.

[Table 19] Results of skin irritation determination of the test facial cream

[Table 20] Results of skin irritation of the test body cream

[Table 21] Results of skin irritation of the test body lotion

As can be seen from Tables 19 to 21, the test facial cream, test body cream and test body lotion used in this test were all judged to be non-irritating to the skin.

C) Skin irritation evaluation

(i) Trial Facial Cream

In order to examine the skin soothing (relieving redness) effect of each of the above formulations (test facial cream, test body cream and test body lotion), Semyung University Cosmetics Clinical Research Support Center standard operating procedure with 20 healthy adults as test subjects Accordingly, the effect of alleviating redness (skin soothing effect) induced by external stimulation (stimulation by SLS) on the forearm of the test subject was evaluated.

After the test subjects washed their forearms, they applied an appropriate amount of each formulation twice a day (morning and evening). The evaluation was made taking into account both the instrumental evaluation and the skin irritation evaluation as follows:

- Instrumental evaluation: *a value measurement using a colorimeter (Spectrophotometer CM-2600D, Minolta, Japan); and taking pictures using a digital camera (EOS 70D, Canon, Japan).

- Skin irritation evaluation: At each visit, the investigator evaluates the results of the researcher's visual evaluation and the test subject's questionnaire on the area where each formulation was used.

The evaluation results made as described above are shown in Tables 22 to 25.

[Table 22] *a value measurement value and redness relief rate (%) of the test facial cream area

[Table 23] *a value measurement value and redness relief rate (%) of unused area

[Table 24] Statistical analysis results between the two groups (test facial cream use group and non-use group)

[Table 25] Results of skin irritation evaluation of test subjects

As can be seen from Tables 22 to 24, there was a difference in the area where the test facial cream was used compared to the area where the test facial cream was not used ( p <0.05), and the redness reduction rate (%) of the area where the test facial cream was used was - after 24 hours - 9.80%, -6.09% after 48 hours, and 7.95% after 72 hours. In addition, as can be seen from Table 25, no special skin adverse reaction was observed with respect to the test facial cream in all test subjects. Therefore, it is judged that the test facial cream has a skin soothing effect to relieve redness induced by external stimuli.

(ii) test body cream

In order to examine the skin soothing (relieving redness) effect of the test body cream, 20 healthy adults were subjected to external stimulation (SLS) on the forearm of the test subject according to the standard operating procedure of Semyung University Cosmetics Clinical Research Support Center. The effect of alleviating redness (skin soothing effect) induced by stimuli) was evaluated.

After the test subjects washed their forearms, they applied an appropriate amount of each formulation twice a day (morning and evening). The evaluation was made taking into account both the instrumental evaluation and the skin irritation evaluation as follows:

- Instrumental evaluation: *a value measurement using a colorimeter (Spectrophotometer CM-2600D, Minolta, Japan); and taking pictures using a digital camera (EOS 70D, Canon, Japan).

- Skin irritation evaluation: At each visit, the investigator evaluates the results of the researcher's visual evaluation and the test subject's questionnaire on the area where each formulation was used.

The evaluation results made as described above are shown in Tables 26 to 29.

[Table 26] *a value measurement value and redness relief rate (%) of the test body cream area

[Table 27] *a value measurement value and redness relief rate (%) of unused area

[Table 28] Statistical analysis results between the two groups (test body cream use group and non-use group)

[Table 29] Results of skin irritation evaluation of test subjects

As can be seen from Tables 26 to 28, there was a difference ( p <0.05) in the area where the test body cream was used compared to the area where the test body cream was used, and the redness relief rate (%) of the area where the test body cream was used after 24 hours - 8.74%, -3.54% after 48 hours, and 10.00% after 72 hours. In addition, as can be seen from Table 29, no special skin adverse reaction was observed with respect to the test body cream in all test subjects. Therefore, it is judged that the test body cream has a skin soothing effect that relieves redness induced by external stimuli.

(iii) test body lotion

In order to examine the skin soothing (relieving redness) effect of the test body lotion, 20 healthy adults were subjected to external stimulation (SLS) on the forearm of the test subject according to the standard operating procedure of Semyung University Cosmetics Clinical Research Support Center. The effect of alleviating redness (skin soothing effect) induced by stimuli) was evaluated.

After the test subjects washed their forearms, they applied an appropriate amount of each formulation twice a day (morning and evening). The evaluation was done taking into account both the instrumental evaluation and the skin irritation evaluation as follows:

- Instrumental evaluation: *a value measurement using a colorimeter (Spectrophotometer CM-2600D, Minolta, Japan); and taking pictures using a digital camera (EOS 70D, Canon, Japan).

- Skin irritation evaluation: At each visit, the investigator evaluates the results of the researcher's visual evaluation and the test subject's questionnaire on the area where each formulation was used.

The evaluation results made as described above are shown in Tables 30 to 33.

[Table 30] *a value measurement value and redness relief rate (%) of the test body lotion area

[Table 31] *a value measurement value and redness relief rate (%) of unused area

[Table 32] Statistical analysis results between two groups (test body lotion use group and non-use group)

[Table 33] Results of skin irritation evaluation of test subjects

As can be seen from Tables 30 to 32, there was a difference ( p <0.05) in the area where the test body lotion was used compared to the area where the test body lotion was not used, and the redness relief rate (%) of the area where the test body lotion was used was - after 24 hours - 8.62%, -0.16% after 48 hours, and 8.75% after 72 hours. In addition, as can be seen from Table 33, no special skin adverse reaction was observed with respect to the test body lotion in all test subjects. Therefore, it is judged that the test body lotion has a skin soothing effect to relieve redness induced by external stimuli.

Claims (12)

A cosmetic composition comprising an extract obtained by using a low-temperature aging process from a herbal mixture consisting of Angelica asiatica, cheongung, peony, sukjihwang, and gold. The cosmetic composition according to claim 1, wherein the herbal mixture is obtained by mixing angelica, cheongung, peony, sukjihwang and gold in equal proportions. The cosmetic composition according to claim 1, wherein the low-temperature aging process includes a low-temperature aging step of aging the crude drug mixture at a temperature of 2° C. to 6° C. for 5 to 10 days by adding a solvent. The cosmetic composition according to claim 3, wherein the low-temperature aging process includes a low-temperature aging step of aging at a temperature of 4°C for 7 days by adding a solvent to the herbal mixture. The cosmetic composition according to claim 3, wherein the solvent is selected from the group consisting of purified water, mineral water and carbonated water. The cosmetic composition according to claim 5, wherein the solvent is carbonated water. The cosmetic composition according to claim 3, wherein the solvent is used in an amount 10 times the weight of the herbal mixture. The cosmetic composition according to claim 3, wherein the extract is obtained by repeatedly extracting the crude drug mixture that has undergone the low-temperature aging step with hot water or 70% ethanol three times. The cosmetic composition according to claim 6, wherein the extract is obtained by repeatedly extracting the crude drug mixture that has undergone the low-temperature aging step with 70% ethanol three times. The cosmetic composition according to claim 1, wherein the cosmetic composition is used to improve acne. According to claim 1, wherein the cosmetic composition is formulated as a facial cream, body lotion or body cream, the cosmetic composition. The cosmetic composition according to claim 11, wherein the cosmetic composition further comprises a milk protein component and a baobab seed component.

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