KR20210077365A - Cosmetic composition comprising colocasia esculenta biorenovate extract and method of preparing the same - Google Patents

Abstract

The present invention relates to an anti-inflammatory cosmetic composition obtained by bioconversion of an extract of Colocasia esculenta using a strain, and a manufacturing method of the anti-inflammatory cosmetic composition.

Description

COSMETIC COMPOSITION COMPRISING COLOCASIA ESCULENTA BIORENOVATE EXTRACT AND METHOD OF PREPARING THE SAME, COSMETIC COMPOSITION COMPRISING COLOCASIA ESCULENTA BIORENOVATE EXTRACT AND METHOD OF PREPARING THE SAME

The present invention relates to an anti-inflammatory cosmetic composition obtained by bioconversion of a taro extract into a strain, and a method for preparing the anti-inflammatory cosmetic composition.

Inflammation is a characteristic of the pathological response that occurs first to defend the body from harmful stimuli such as toxic substances, damaged tissue, and infection of pathogens. It is a complex process in which various immune cells express inflammatory mediators to respond to external infections. to be. The inflammatory response is a beneficial defense mechanism in the body that can restore the structure or function against tissue damage caused by external stimuli, but if the inflammatory response is chronic and excessive, it causes dysfunction and necrosis of cells or tissues and causes brain diseases, atopy, cancer, and cardiovascular diseases. Since it can develop into various diseases, such as, research is being actively conducted to find, control and remove factors that cause inflammation, and to elucidate mechanisms. Such an inflammatory response mainly involves macrophages, and among them, Lipopolysaccharide (LPS) present in the outer membrane of Gram-negative bacteria acts as a representative inflammation-inducing factor that stimulates macrophages. Macrophages recognize LPS bound to CD14 through a pattern recognition receptor called TLR4 (Toll-like receptor 4), and activate the MAPKs (mitogen-activated protein kinase) family and several signaling pathways. As a result, it induces NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells), iNOS (inducible nitric oxide synthase), COX-2 (cyclooxygenase-2), IL-1β (interleukin-1β), and IL It leads to the expression of pro-inflammatory cytokines such as -6 (interleukin-6) and TNF-α (tumor necrosis factor-α).

These inflammatory reactions are the main cause of skin troubles, and as substances used for anti-inflammatory purposes, nonsteroidal substances such as flufenamic acid and ibuprofen are known to be effective. However, these substances have a problem in that the amount of use thereof is limited due to safety issues on the skin, or the effect of these substances is insignificant, so that an inflammatory alleviation effect cannot be expected.

Taro ( Colocasia esculenta L. Schott) belonging to Cheonnamseong is an old yellow crop with high useful value, and it inhabits not only Korea, but also India and Indonesia. Taro is called taro, dasheen, eddoe, etc. depending on the region of production and consumption, and it is known that dextrin, sucrose, galactan, galactose, mannose, etc. are included except for water. According to Donguibogam, taro is slightly poisonous when raw, and has been used in folk remedies as a special medicine to get rid of hangover and morale by protecting blood and energy.

Recently, research and development of recycling known materials through various technologies is being conducted, and Republic of Korea Patent Registration No. 10-1523631 discloses an anti-inflammatory composition comprising a walnut flower extract, an Indian frankincense extract, an hollyhock tree extract and a ginseng extract as active ingredients. is starting However, there has been no report on the use of the taro extract as a cosmetic composition exhibiting an anti-inflammatory effect. Accordingly, there is a need to develop a raw material derived from a natural substance that does not stimulate the human body, has no toxicity and has an excellent anti-inflammatory effect, and can be directly applied to cosmetics.

US 10-1523631 B

The present invention bioconverts a taro extract through a biorenovation using a Bacillus amyloliquefaciens strain, and an anti-inflammatory cosmetic composition with excellent effects of treatment, improvement, prevention and relief of inflammation, and preparation thereof The purpose is to provide a method.

In order to achieve the above object, the present invention provides an anti-inflammatory cosmetic composition obtained by bioconverting a taro extract into a strain.

In addition, the present invention provides a method for preparing an anti-inflammatory cosmetic composition.

The anti-inflammatory cosmetic composition of the present invention has excellent effects of treating, improving, preventing and alleviating inflammation, has excellent effects of relieving inflammation even at the protein level, is harmless to the human body because there is no toxicity to macrophages, and has little skin irritation. It can be used in the fields of external preparations for skin of cosmetics, pharmaceuticals and quasi-drugs.

In addition, in the present invention, it is possible to prepare an anti-inflammatory cosmetic composition exhibiting an anti-inflammatory effect through biorenovation of a taro extract using a Bacillus amyloliquefaciens strain, and specifically, RAW through the bioconverted taro extract. 264.7 It is possible to provide an anti-inflammatory cosmetic composition that has no toxicity to cells and exhibits the effect of inhibiting the expression of proinflammatory cytokines of IL-6 and IL-1β and inhibiting the expression of iNOS and COX-2 proteins.

In addition, there is provided an anti-inflammatory cosmetic composition comprising a bioconverted taro root extract that can be used as a cosmetic material or a raw material for pharmaceuticals, and in particular can be used as a feminine cleanser due to its anti-inflammatory effect.

1 is an HPLC chromatogram of Bacillus amyloliquefaciens (NC), taro root extract (CE), and bioconverted taro root extract of the present invention.
2 is a graph showing the viability of RAW 264.7 cells according to each condition.
3 is a graph showing the nitric oxide (NO) production rate according to each condition in RAW 264.7 cells.
4 is a graph showing _{the PGE 2} production rate according to each condition in RAW 264.7 cells.
Figure 5a is a graph showing the protein level of iNOS (β-actin is a control) and iNOS protein expression rate analyzed by Western blot according to each condition in RAW 264.7 cells.
Figure 5b is a graph showing the protein level of COX-2 (β-actin is a control) and COX-2 protein expression rate analyzed by Western blot according to each condition in RAW 264.7 cells.
Figure 6a is a graph showing the TNF-α production rate according to each condition in RAW 264.7 cells.
6b is a graph showing the IL-6 production rate according to each condition in RAW 264.7 cells (*p<0.05;**p<0.01).
6c is a graph showing the IL-1β production rate according to each condition in RAW 264.7 cells (*p<0.05).

The present invention relates to an anti-inflammatory cosmetic composition comprising a bioconverted taro extract and a method for preparing the same.

The anti-inflammatory cosmetic composition of the present invention has excellent effects of treating, improving, preventing and alleviating inflammation, has excellent effects of relieving inflammation even at the protein level, is harmless to the human body because there is no toxicity to macrophages, and has little skin irritation. It can be used in the fields of external preparations for skin of cosmetics, pharmaceuticals and quasi-drugs.

< Anti-inflammatory cosmetic composition >

The anti-inflammatory cosmetic composition of the present invention includes a bioconverted taro extract obtained by bioconverting the taro extract into a strain as an active ingredient.

In the present invention, the taro extract is extracted by solvent extraction using an organic solvent, and may be extracted from one or more parts of leaves, stems and roots of taro, and the taro root extract is most preferred.

The organic solvent used for the solvent extraction may be a C1 to C5 alcohol, and ethanol is most preferably used for the solvent extraction. In addition, the concentration of the C1 to C5 alcohol used in the solvent extraction may be 60 to 100%, and it is preferable to use the C1 to C5 alcohol at a concentration of 70%.

The anti-inflammatory cosmetic composition of the present invention inhibits nitric oxide (NO) production, inhibits PGE ₂ production, inhibits iNOS expression, inhibits COX-2 expression, inhibits interleukin 6 (IL-6) production, interleukin 1β (IL-1β) ) exhibits one or more effects selected from the group consisting of inhibition of production and combinations thereof, and thus has an anti-inflammatory effect.

As used herein, the term "biorenovation" refers to a method of preparing a compound inducing structural changes such as phosphorylation or glycosylation from a substrate using a microorganism. Phosphorylation refers to binding to a substrate by replacing a phosphoric acid group (HOPO(OH)O-) with a hydrogen atom of the substrate by the action of a kinase. of glucoside is added.

In addition, in the present invention, the strain may be a strain of the genus Bacillus, may be Bacillus amyloliquefaciens, and KCTC 43033 Bacillus amyloliquefaciens is particularly preferred.

In one embodiment, the anti-inflammatory cosmetic composition may be used for cosmetics, pharmaceuticals or quasi-drugs. The anti-inflammatory composition is used for the cosmetic, pharmaceutical or quasi-drug use, and can be used for anti-inflammatory activity, prevention, treatment, and the like.

In addition, the anti-inflammatory cosmetic composition may further include a pharmaceutically or cosmetically acceptable component.

When the anti-inflammatory cosmetic composition is used in the cosmetic, components included in conventional cosmetics include purified water, oil, wax, fatty acid, fatty alcohol, fatty acid ester, surfactant, humectant, thickener ( thickening agent), antioxidant, viscosity stabilizer, chelating agent, buffer, and lower alcohol, but is not limited thereto.

In addition, the formulation of the cosmetic comprising the anti-inflammatory cosmetic composition of the present invention may be prepared in any formulation conventionally prepared in the industry, and may take the form of a solution, emulsion, viscous mixture, etc., preferably may be one type of formulation selected from the group consisting of paste, emulsion, cream, gel, powder, spray, solution, emulsion, suspension, pack, foundation, lotion, cosmetic water and cleansing formulation containing surfactant. More specifically, the cosmetic composition is a skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nourishing lotion, cream, massage cream, nourishing cream, moisture cream, hand cream, foundation, essence, nourishing essence. , pack, soap, cleansing foam, cleansing lotion, cleansing cream, body lotion, and one type of formulation selected from the group consisting of body cleanser.

In addition, when the anti-inflammatory composition of the present invention is used in the pharmaceutical, it may be used in the form of a solid, semi-solid, solution, emulsion, dispersion, micellar, liposome, and the like.

In addition, it is preferable that the cosmetic composition of the present invention is for a feminine cleanser.

As used herein, the term "feminine cleanser" refers to a dedicated cleanser used in and around a woman's vulva for the purpose of inhibiting the proliferation of harmful bacteria in the vagina, reducing odor and/or maintaining an appropriate pH in the vagina.

The composition for feminine cleanser of the present invention may contain excipients, disintegrants and/or lubricants acceptable for feminine wash. The excipients, disintegrants and lubricants included in the composition for feminine wash of the present invention are commonly used in the manufacture of feminine washes, and the excipients are prosolve, starch, microcrystalline cellulose, mannitol, lactose, low-substituted hydroxypropyl cellulose and/or may be sucralose, specifically, Prosolv (Prosolv SMCC90), starch (Starch 1500), microcrystalline cellulose (microcrystalline cellulose PH102) and/or mannitol (mannitol 160C), and the disintegrant may be cross It may be povidone, sodium starch gluconate, croscarmellose sodium, polysorbate and/or sodium lyryl sulfate, specifically, crospovidone (crospovidone CL-SF), and the lubricant may be magnesium stearate, hard It may be silicic anhydride, talc and/or glyceryl behenate, and specifically, it may be magnesium stearate, but is not limited thereto.

The composition for feminine hygiene products of the present invention is for the prevention or improvement of vaginal-related diseases, and the vaginal-related diseases may be vaginal laxity, vaginal dryness and/or vaginitis, but is not limited thereto.

According to one embodiment of the present invention, the composition may be any one formulation selected from the group consisting of liquid, gel, solid, cleaning composition, and vaginal insertion tablet.

The present invention is a composition for a feminine cleanser, wherein the formulation may be a formulation for parenteral administration, for example, a solution, a gel, a solid, a cleaning composition, a tablet for vaginal insertion, a suppository form, a cream, It may be in liquid formulations such as ointments, dressing solutions, sprays, and other coating agents, solutions, suspensions, and emulsions, and sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried formulations, suppositories, creams. , ointment, jelly, foam, cleansing agent, or vaginal insert, preferably a liquid, gel, solid, cleansing composition, or external skin preparation such as a tablet for vaginal insertion.

The formulation may be prepared by adding, for example, a solubilizing agent, an emulsifying agent, a buffer for pH adjustment, etc. to sterile water. As the non-aqueous solvent or suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and/or ethyl oleate may be used. On the other hand, the suppository formulation may be prepared by adding, for example, witepsol, macrogol, tween 61, cacao butter, laurin, glycero gelatin, etc., and a carrier for formulating the same The carrier may include a binder, a lubricant, a suspending agent, a solubilizer, a buffer, a preservative, a lubricant, an isotonic agent, a stabilizing agent, a dispersing agent, a suspending agent, a coloring agent, a flavoring agent, and the like.

The preferred dosage of the feminine wash composition of the present invention varies depending on the patient's condition and weight, the degree of disease, drug form, administration route and period, but may be appropriately selected by those skilled in the art.

The dosage of the composition for feminine hygiene products of the present invention may be appropriately selected by those skilled in the art in consideration of the administration method, the age, sex and weight of the user, and the severity of the disease. For example, the dosage of the feminine wash composition of the present invention may be 0.001 to 1000 mg/kg based on an adult. It may be administered once or several times a day, but is not limited thereto.

The composition for feminine hygiene products of the present invention may be administered together with a substance exhibiting preventive or therapeutic activity for one or more female genitourinary-related diseases.

In addition, the bioconverted taro extract may be contained in an amount of 0.1 to 20% by weight, preferably 0.1 to 5% by weight, and most preferably 0.1 to 0.5% by weight based on the total weight of the anti-inflammatory cosmetic composition, However, the present invention is not limited thereto.

< Method for manufacturing anti-inflammatory cosmetic composition >

The present invention provides a method for preparing the above-described anti-inflammatory cosmetic composition. In the method for manufacturing the anti-inflammatory cosmetic composition of the present invention, all of the contents described with respect to the anti-inflammatory cosmetic composition of the present invention can be applied, and detailed descriptions of overlapping parts are omitted, but the description is the same even if the description is omitted. can be applied.

Specifically, the present invention comprises the steps of preparing an extract from taro; and

It provides a method for preparing an anti-inflammatory cosmetic composition comprising the step of bioconverting the taro extract into a strain of the genus Bacillus.

More specifically, in one embodiment, the method for preparing an anti-inflammatory cosmetic composition,

solvent extraction of taro root with ethanol one or more times;

obtaining a taro extract by lyophilizing the solvent-extracted taro and dissolving it in ethanol; and

It may include the step of bioconverting the taro extract after pelleting the strain of the genus Bacillus.

The strain of the genus Bacillus may be amyloliquefaciens (Bacillus amyloliquefaciens), preferably, Bacillus amyloliquefaciens KCTC 43033 (Bacillus amyloliquefaciens KCTC 43033).

The step of preparing the extract from taro includes performing solvent extraction using an organic solvent at least once, preferably twice, from one or more parts of leaves, stems and roots of taro, preferably from taro root. . The organic solvent used for the solvent extraction may be a C1 to C5 alcohol, and ethanol is most preferably used for the solvent extraction. In addition, the concentration of C1 to C5 alcohol used in the solvent extraction may be 60 to 100%, it is preferable to use a C1 to C5 alcohol at a concentration of 70%, and it is more preferable to use ethanol at a concentration of 70% desirable.

In addition, the method may further include dissolving the taro extract extracted by the solvent extraction in an organic solvent after pre-freezing, and the freeze-drying is performed at -130 to -90°C, preferably at -120 to -100°C. , more preferably at -110°C. In addition, the freeze-drying is preferably performed in the above temperature range for 30 to 48 hours at a pressure of 0.3 mbar, but is not limited thereto. The organic solvent may be the same kind of solvent as that used for solvent extraction, but is not limited thereto.

In addition, the method for preparing the anti-inflammatory cosmetic composition of the present invention may further include adding a pharmaceutically or cosmetically acceptable additive.

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

Example: Preparation of bioconverted taro extract

1-1. Taro Extract Preparation

The roots of the taro (Colocasia esculenta (L.) Schott) used in this example were collected in April 2018 from Ara-dong, Jeju City, Jeju Special Self-Governing Province, and provided by Jeju Biological Resources Co. Taro (root) was extracted twice using 70% ethanol for 24 hours at room temperature, filtered through a paper filter, and then concentrated using a vacuum concentrator. After freeze-drying the concentrated extract at -110°C, it was dissolved in 70% ethanol to finally obtain a taro extract.

1-2. Microbial culture

The microorganism is a Bacillus amyloliquefaciens (KCTC 43033) strain, and it is received from a microbiology center and cultured for 20 hours according to optimal growth conditions in a nutrient broth (Beef extract 3.0 g/L, Peptone 5.0 g/L), A microbial pellet was obtained by centrifugation at 5,000 rpm for 10 minutes. The obtained pellet was washed twice with PG buffer (50 mM Phosphate buffer, 2% Glycerin), and then suspended in the same buffer to obtain a microorganism suspension.

1-3. Biorenovation of Taro Extract

After 72 hours of reaction the Taro extracts and microbe suspension previously prepared from 30 ℃, centrifuged and concentrated using a vacuum concentrator and the resulting supernatant was then lyophilized at -110 ℃ the biotransformation extract taro (Colocasia esculenta Biorenovate Extract, CEB) was obtained.

1-4. HPLC analysis of bioconverted taro extract (CEB)

For HPLC analysis, Shimadzu SpectroMonitor 3200 digital UV/Vis detector (Phenomenex 4 μm Hydro-RP 80 Å, 250×4.6 mm) was used, and the mobile phase was water containing 0.1% TFA (Trifluoroacetic acid) and acetonitrile (solvent B). ) was used. Analysis conditions were a flow rate of 1.0 mL/min, a temperature of 40°C, and 10 μl of a sample was injected and analyzed under gradient conditions for 30 minutes, and the detection wavelength was set to 254 nm for analysis.

Using the Bacillus amyloliquefaciens (KCTC 43033) strain, the taro root extract was biorenovated by biorenovation technique. After the reaction, the supernatant of the reactant was recovered through centrifugation and concentrated under reduced pressure. As a result of analysis through HPLC, a new peak was confirmed in the bioconversion extract sample when compared with the taro root extract (FIG. 1). This suggested the possibility of a peak that appeared when many compounds of taro extract were bioconverted into unknown compounds by Bacillus amyloliquefaciens by the bioconversion technique used in this paper.

Experimental Example: Evaluation of bioconverted taro extract

2-1. cell culture

RAW 264.7 cells were used as cells to confirm the anti-inflammatory effect of the bioconverted taro extract according to this example, and the RAW 264.7 cells were purchased from the Korea Cell Line Bank and mixed with 10% FBS (fetal bovine serum) and 100 units/mL. Using a culture medium of Dulbecco's Modified Eagle Medium (DMEM, Gibco, NY, USA) containing penicillin-streptomycin (penicillin-streptomycin), it was cultured at _{37° C., 5% CO 2 condition.}

2-2. Evaluation of the anti-inflammatory effect of bioconverted taro extract

All of the following Experimental Examples 1 to 5 were performed under the conditions shown in Table 1 below, and LPS was added as lipopolysaccharides to induce an inflammatory response in cells.

In Table 1 below, Control 1 is RAW 264.7 cells in a non-inflammatory state, and Control 2 is RAW 264.7 cells in an inflammation-induced state. In addition, Examples 1 to 3 were obtained by adding LPS and bioconverted taro extract (CEB) of the present invention to RAW 264.7 cells, respectively, in the amounts shown in Table 1 below, and then culturing. In addition, non-bioconverted taro extract (CE) was added to each of the contents shown in Table 1 below and cultured, and the effects of bioconversion were confirmed by using these as Comparative Examples 1 to 3.

control comparative example Example One 2 One 2 3 One 2 3 LPS (μg/mL) - One One One One One One One CEB (μg/mL) - - - - - 50 100 200 CE (μg/mL) - - 50 100 200 - - -

2-3. Statistical processing

All of the experimental examples below were repeated three times, and the results were expressed as mean ± standard deviation, and statistical analysis was performed by analysis of variance (ANOVA) to verify the significance (p <0.05, 0.01) of each treatment section. Afterwards, multiple comparisons were performed using Student's t-test.

Experimental Example 1: Cytotoxicity measurement

For cytotoxicity measurement, 8.0 × 10 ⁴ cells/mL of RAW 264.7 cells were dispensed in a 24-well plate, and incubated for 24 hours in an incubator at 37°C, 5%, CO ₂ conditions, samples (CEB and CE extracts) and LPS ( 1 μg/mL) were co-treated and cultured for 24 hours (Examples 1 to 3 and Comparative Examples 1 to 3). Then, MTT was added and reacted at 37°C for 4 hours, then the supernatant was removed, DMSO was added to dissolve the precipitate, and then transferred to a 96-well plate and absorbance was measured at 570 nm using an ELISA reader. The average absorbance for each sample group was measured, and cytotoxicity was evaluated by comparing the absorbance value of the control group, and the results are shown in FIG. 2 .

Comparative evaluation of cytotoxicity

To investigate the cytotoxicity of the CEB extract against RAW 264.7 cells, the cell viability was investigated using the MTT assay. As a result, the CEB extract CE extract showed a survival rate of about 90% at 50 μg/mL, and did not show a significant difference compared to Controls 1 and 2 at 100 and 200 μg/mL, indicating that toxicity to RAW 264.7 cells was reduced at all concentrations. It was confirmed that it was insignificant (FIG. 2).

Experimental Example 2: Measurement of NO production inhibitory activity

RAW 264.7 cells were aliquoted as 8.0 × 10 ⁴ cells/mL in a 24-well plate, and incubated at 37° C., 5% CO ₂ condition for 24 hours in an incubator. After that, samples (CEB and CE extracts) and LPS (1 μg/mL) were co-treated and incubated for 24 hours. Then, 100 μL of the supernatant and Griess reagent [1% (w/v) sulfanilamide, 0.1% (w/v) naphthylethylenediamine in 2.5% (v/v) phosphoric acid] 100 μL was mixed in an equal amount in a 96 well plate, and after dark reaction for 10 minutes, absorbance was measured at 540 nm using an ELISA reader, and the results are shown in FIG. 3 .

Comparative evaluation of NO production inhibitory activity

Nitric oxide, a free radical that causes inflammation The effect of CEB and CE extracts on the production of (NO) was investigated (FIG. 3). RAW 264.7 cells were simultaneously treated with the CEB extract according to the example or the CE extract (50, 100, 200 μg/mL) and LPS (1 μg/mL) according to the comparative example to induce the generation of NO, and then use the Griess reagent to measure the amount of ^{NO 2 -} present in the cell culture medium. As a result, compared with the LPS-only treatment group, the CEB extract reduced NO production in a concentration-dependent manner by 64% at a concentration of 50 μg/mL, 37% at a concentration of 100 μg/mL, and 22% at a concentration of 200 μg/mL appeared to do Therefore, the CEB extract is considered to be able to effectively control the induction of disease caused by inflammation.

On the other hand, the CE extract without bioconversion increased the NO production rate to about 105% at a concentration of 50 μg/mL, and showed an NO production rate of 80% or more even at concentrations of 100 and 200 μg/mL. It was confirmed that there was little effect of inhibiting the production. For the CE extract (Comparative Examples 1 to 3), which was confirmed not to sufficiently satisfy the NO production inhibitory effect, further experiments were not conducted thereafter.

Experimental Example 3: Prostaglandin E ₂ (PGE ₂ ) measurement of production inhibitory activity

RAW 264.7 cells (8.0 × 10 ⁴ cells/mL) were treated with CEB extract and LPS (1 μg/mL) at the same time and cultured for 24 hours. _{After 24 hours, the PGE 2} content of the supernatant obtained by centrifuging the culture medium (12,000 rpm, 3 min) was measured using a mouse enzyme-linked immnunosorbent assay (ELISA) kit (R&D Systems Inc., Minneapolis, MN, USA). , the results are shown in FIG. 4 .

PGE ₂ Comparative evaluation of production inhibition

PGE ₂ induces the activation of immune-related cells to cause an inflammatory response, and is known as a substance that causes edema, fever, and cancer. _{As a result of measuring the PGE 2} production inhibitory activity of the CEB extract under the same conditions as the NO experiment above, the control 2 and CEB extract were added to each treatment concentration (50, 100, 200 μg), which was induced by LPS alone. _{/mL) decreased the production of PGE 2} by 88%, 60%, and 31%, respectively (FIG. 4). As a result of the above results, it was confirmed that the effect of inhibiting the production of _{PGE 2 increased as the concentration of the extract increased.} In particular, at a concentration of 200 μg/mL, _{it was found that PGE 2} production was inhibited by 69% compared to Control 2. Therefore, it is thought that the CEB extract can expect a better anti-inflammatory effect by reducing the production of _{PGE 2} as well as suppressing NO production.

Experimental Example 4: Western blot analysis measurement

RAW 264.7 cells were ^{incubated for 24 hours at 4.0×10 5} cells/mL, then CEB extract and LPS (1 μg/mL) were co-treated and cultured for 24 hours. Afterwards, the cells were washed twice with PBS, and lysis buffer [1×RIPA (Upstate Cell Signaling Solution, NY, USA), 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM Na ₃ VO ₄ , 1 mM NaF, 1 μg/mL aprotinin, 1 μg/mL pepstatin, and 1 μg/mL leupeptin] for 1 hour, followed by centrifugation to separate the protein supernatant. Protein concentration was quantified using a BCA kit (Bio-Rad, USA). The quantified protein was electrophoresed on a 10% polyacylamide gel and transferred to a poly-vinylidene difluoride (PVDF) membrane (Milipore, Burlington, MA, USA) at 200 mA for 2 hours. The protein-transferred membrane was placed in 0.05% Tween20/Tris-buffered saline (0.05% T/TBS) containing 5% skim milk, blocked at room temperature for 1 hour, and then reacted with the primary antibody. The primary antibody reaction was iNOS antibody (1:5,000, Bio-Rad, USA), COX-2 antibody (1:1,000, Rockland Immunochemicals, Inc., USA), β-actin antibody clone AC-74 (1:10,000, Sigma, USA) at 4°C for one night, washed 3 times with 0.05% T/TBS solution, diluted 1:10,000 with secondary antibody (Jackson ImmunoResearch, USA), and reacted at room temperature for 1 hour. Washed 3 times with 0.05% T/TBS solution. Proteins were measured through an imaging densitometer (model GS-700, Bio-rad, USA) using an ECL kit (Bio-Rad, USA), and the results are shown in FIGS. 5A and 5B.

Comparative evaluation of iNOS and COX-2 expression inhibition

Macrophages are stimulated by LPS to express pro-inflammatory genes such as COX-2 and iNOS. Among them, iNOS is a nitric oxide synthase that continuously induces the production of NO to mediate the inflammatory response, and COX-2 is _{an enzyme that synthesizes PGE 2} and is known to be related to cancer. Therefore, in this experiment, RAW 264.7 cells were stimulated with LPS to confirm the signaling pathway for inhibition of inflammation by the CEB extract, and the protein expression levels of iNOS and COX-2 were observed by treating the CEB extract by concentration. As a result of the experiment, it was confirmed that the expression level was increased in the LPS-treated control group 2 compared to the control group 1, and it was confirmed that the increased iNOS protein expression level significantly decreased as the concentration increased when the CEB extract was treated (Fig. 5a). In addition, it was confirmed that the protein expression level of COX-2 was also decreased in a concentration-dependent manner by the CEB extract ( FIG. 5b ). Thus, it was confirmed that the inhibition of inflammation by the CEB extract was made through the signal transduction process of NF-κB. These results suggested that the CEB extract exhibits an anti-inflammatory effect by effectively reducing the expression of _{NO, PGE 2, iNOS, and COX-2.}

Experimental Example 5: Pro-inflammatory cytokines (IL-6, IL-1β) production inhibitory activity measurement

RAW 264.7 cells were pre-cultured in a 24 well plate at 8.0 × 10 ⁴ cells/mL at 37°C, 5% CO ₂ in an incubator for 24 hours, and then treated with CEB extract and LPS (1 μg/mL) at various concentrations at the same time. and cultured for 24 hours. Then, the amount of proinflammatory cytokine produced in the supernatant obtained by centrifugation of the culture medium (12,000 rpm, 3 min) was determined by Mouse TNF-α ELISA Kit (Invitrogen, California, USA), Mouse IL-6 ELISA Kit (BD Biosciences, California, USA). ), was measured using a Mouse IL-1β ELISA Kit (R&D Systems Inc., Minneapolis, MN, USA), and the results are shown in FIGS. 6a to 6c.

Comparative evaluation of inhibition of pro-inflammatory cytokines (IL-6, IL-1β) production

TNF-α, IL-1β, and IL-6, known as representative pro-inflammatory cytokines, are known to regulate inflammatory responses, and are known to regulate the activity, proliferation and differentiation of immune cells. In this experiment, the effect of the CEB extract on the LPS-stimulated RAW 264.7 cells on the expression of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α was investigated using an ELISA kit ( FIGS. 6a to 6c ). ). It was confirmed that the expression of IL-1β, IL-6, TNF-α in RAW264.7 cells was significantly increased by LPS, and increased by LPS by treating the CEB extract at concentrations of 50, 100 and 200 μg/mL. It was confirmed that the production amount of IL-1β and IL-6 was suppressed ( FIGS. 6b and 6c ). In particular, it was confirmed that the CEB extract decreased the secretion of IL-1β and IL-6 by 87% and 86%, respectively, at 200 μg/mL concentration compared to the LPS alone treatment group. On the other hand, it is considered that the CEB extract is not significantly involved in the TNF-α production mechanism, as the inhibitory ability of TNF-α is insufficiently observed when compared to the LPS-treated group (FIG. 6a). It is thought that the CEB extract has a superior effect on the production mechanism of IL-1β and IL-6 than TNF-α, suggesting that the expression of pro-inflammatory cytokines is suppressed by the CEB extract.

2-4. conclusion

In this experimental example, to confirm the anti-inflammatory effect of CEB extract obtained by biorenovation bioconversion technique of taro root extract, nitric oxide (NO), Prostaglandin E ₂ (PGE ₂ ) production inhibitory activity, inhibition of pro-inflammatory cytokines (IL-6, IL-1β) production, and inhibition of iNOS and COX-2 protein expression were measured. As a result, the CEB extract, in which a large number of bioconversion compounds were detected, showed no cytotoxicity against RAW264.7 cells at all concentrations, and inhibited LPS-induced NO production in a concentration-dependent manner. In addition, PGE ₂ production inhibitory activity was also inhibited in a concentration-dependent manner, and the secretion of the pro-inflammatory cytokines IL-1β and IL-6 was also excellent at 200 μg/mL concentration. Thus, it was confirmed that the bioconversion technique through the biorenovtion technique further enhances the original efficacy. Therefore, the CEB extract using the biorenovation technique is considered to be a result that suggests the possibility of being used as a cosmetic material with natural anti-inflammatory effects.

Preparation example: Preparation example by formulation of feminine wash

Preparation Example 1: Feminine cleanser containing taro root bioconversion extract (mist)

Preparation examples of feminine cleanser (mist) in the anti-inflammatory cosmetic composition containing the taro root bioconversion extract are shown in Table 2 below.

ingredient content (wt%) Bioconverted Taro Extract 0.1~0.5 Purified water 80-90 glycerin 5-10 dipropylene glycol 5-10 coco-betaine 2-7 1,2-Hexanediol 0.5~2 Butylene Glycol 0.5~1.5 sodium chloride 0.1~1 Disodium Cocoamphodiacetate 0.1~1 sodium citrate 0.1~1 Citric Acid 0.1~0.5 Bacillus/Western rapeseed extract fermented filtrate 0.1~0.5 lavender oil 0.1~0.5 Ethylhexylglycerin 0.05~0.2 Hexylene Glycol 0.05~0.2 Lactobacillus Lysate 0.01~0.05 Disodium EDIT 0.01~0.05 Polyglyceryl-10 Myristate 0.01~0.05 Polyglyceryl-10 Laurate 0.01~0.05

Preparation Example 2: Feminine cleanser (gel) containing taro root bioconversion extract

Preparation examples of feminine cleanser (gel) in the anti-inflammatory cosmetic composition containing taro root bioconversion extract are shown in Table 3 below.

ingredient content (wt%) Bioconverted Taro Extract 0.1~0.5 Purified water 80-90 glycerin 10-20 dipropylene glycol 5-10 Ammonium acryloyldimethyl taurate/vpicopolymer 0.5~2 Disodium Cocoamphodiacetate 0.5~2 Butylene Glycol 0.5~2 1,2-Hexanediol 0.5~1.5 sodium chloride 0.5~1 Citric Acid 0.5~1 coco-betaine 0.5~1 sodium citrate 0.1~0.5 Polyglyceryl-10 Laurate 0.1~0.5 Ethylhexylglycerin 0.1~0.5 Polyglyceryl-10 Myristate 0.1~0.5 xanthan gum 0.1~0.5 Hexylene Glycol 0.1~0.5 lavender oil 0.1~0.5 Bacillus/Western rapeseed extract fermented filtrate 0.1~0.5 Lactobacillus Lysate 0.01~0.05 Disodium EDIT 0.001~0.005

Preparation Example 3: Feminine cleanser containing taro root bioconversion extract (bubble)

Preparation examples of feminine cleanser (bubble) in the anti-inflammatory cosmetic composition containing taro root bioconversion extract are shown in Table 4 below.

ingredient content (wt%) Bioconverted Taro Extract 0.1~0.5 Purified water 80-90 glycerin 1 to 10 coco-betaine 1 to 10 Sodium Cocoyl Apple Amino Acid 1 to 10 sodium chloride 1 to 10 1,2-Hexanediol 1-5 Hexylene Glycol 0.1~1 Polyglyceryl-4 Caprate 0.1~1 Citric Acid 0.1~1 Ethylhexylglycerin 0.1~1 lavender oil 0.1~1 Disodium EDIT 0.1~1 Tetrasodium Glutamate Diacetate 0.1~1 Bacillus/Western rapeseed extract fermented filtrate 0.1~0.5 Lactobacillus Lysate 0.01~0.05

Claims (11)

An anti-inflammatory cosmetic composition obtained by bioconverting a taro extract into a strain.
The method according to claim 1,
The strain is an anti-inflammatory cosmetic composition that is Bacillus amyloliquefaciens (Bacillus amyloliquefaciens).
The method according to claim 1,
The taro is an anti-inflammatory cosmetic composition comprising taro root.
The method according to claim 1,
The taro extract is an anti-inflammatory cosmetic composition, characterized in that it is extracted by solvent extraction using an organic solvent.
The method according to claim 1,
The anti-inflammatory cosmetic composition inhibits nitric oxide (NO) production, inhibits PGE ₂ production, inhibits iNOS expression, inhibits COX-2 expression, inhibits interleukin 6 (IL-6) production, interleukin 1β (IL-1β) production with respect to cells An anti-inflammatory cosmetic composition, characterized in that it exhibits one or more effects selected from the group consisting of inhibition and combinations thereof.
The method according to claim 1,
The anti-inflammatory cosmetic composition is for a feminine cleanser, an anti-inflammatory cosmetic composition.
7. The method of claim 6,
The anti-inflammatory cosmetic composition is an anti-inflammatory cosmetic composition in any one formulation selected from the group consisting of liquid, gel, solid, cleaning composition, and vaginal insertion tablet.
The method according to claim 1,
The bioconverted taro extract is an anti-inflammatory cosmetic composition, characterized in that it is included in an amount of 0.1 to 20% by weight based on the total weight of the anti-inflammatory cosmetic composition.
preparing an extract from taro; and
Bioconverting the taro extract into a Bacillus amyloliquefaciens strain
A method for preparing an anti-inflammatory cosmetic composition comprising a.
10. The method of claim 9,
The step of preparing the extract from the taro is a method for preparing an anti-inflammatory cosmetic composition comprising performing solvent extraction using an organic solvent at least once.
11. The method of claim 10,
The method for preparing an anti-inflammatory cosmetic composition, further comprising dissolving the taro extract extracted by the solvent extraction in an organic solvent after freeze-drying.

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