KR102588988B1 - Extract of Phaseolus vulgaris germinated using Centella Asiatica extract - Google Patents

Abstract

The present invention relates to a Phaseolus vulgaris extract germinated with a Centella asiatica extract, which has an excellent anti-inflammatory effect and an excellent effect of increasing the expression of a skin barrier-related factor, and a cosmetic composition comprising the same.

Description

Tiger bean extract germinated with Centella Asiatica extract {Extract of Phaseolus vulgaris germinated using Centella Asiatica extract}

The present invention relates to a tiger bean extract sprouted with Centella asiatica extract, which has excellent anti-inflammatory effects and an effect of increasing the expression of skin barrier-related factors, and a cosmetic composition containing the same.

Human skin, especially the stratum corneum of the epidermis, protects the human body from various external stresses such as harsh weather or harmful environments, and also prevents the skin from drying out by blocking moisture within the human body from evaporating. In particular, among the components constituting the stratum corneum, lipid components such as ceramide are arranged in a layered structure within the stratum corneum and are known as substances that maintain a strong skin barrier by maintaining moisture in between. When the production of ceramide is reduced due to aging, the protective barrier function of the stratum corneum is reduced, making the skin prone to drying. As a result, not only does skin aging accelerate, but it also causes skin problems such as atopic dermatitis and psoriasis.

To solve this problem, various cosmetics have been proposed that have excellent anti-inflammatory effects and contain substances that increase the expression of skin barrier-related factors. For example, Republic of Korea Patent No. 10-2066384 discloses a cosmetic composition with excellent skin barrier improvement and filaggrin activation effects containing ceramide, cholesterol, fatty acid, acacia pollen extract, and unsaponifiable vegetable oil. However, conventional cosmetic compositions do not provide sufficient anti-inflammatory effects and skin barrier improvement effects.

Accordingly, there is a need for a material that exhibits low cytotoxicity and has excellent anti-inflammatory effects and an effect of increasing the expression of skin barrier-related factors, as well as cosmetics containing the same.

Republic of Korea Patent No. 10-2066384

The present invention provides a material that exhibits low cytotoxicity and has excellent anti-inflammatory effects and an effect of increasing the expression of skin barrier-related factors, and a cosmetic composition containing the same.

The present invention provides a tiger bean extract sprouted with Centella asiatica extract and a cosmetic composition containing the same.

The tiger bean extract germinated with the Centella asiatica extract according to the present invention exhibits low cytotoxicity while providing excellent anti-inflammatory effects and an effect of increasing the expression of skin barrier-related factors.

Figure 1 is a photograph of tiger beans of Preparation Example 1-4.
Figure 2 is a graph showing the HPLC measurement results for the tiger bean extract of Preparation Examples 1-4.
Figure 3 is a graph showing the results of anti-inflammatory cytotoxicity test of tiger bean extracts of Preparation Examples 1, 2, and 4.
Figure 4 is a graph showing the results of evaluating the anti-inflammatory efficacy of tiger bean extracts of Preparation Examples 1, 2, and 4.
Figure 5 is a graph showing the results of the barrier cytotoxicity test of tiger bean extracts of Preparation Examples 1, 2, and 4.
Figure 6 is a graph showing the results of evaluating the barrier efficacy of tiger bean extracts of Preparation Examples 1, 2, and 4.

Hereinafter, the present invention will be described. However, it is not limited to the following content, and each component may be variously modified or selectively mixed as needed. Accordingly, it should be understood to include all changes, equivalents, and substitutes included in the spirit and technical scope of the present invention.

<Tiger bean extract>

The present invention provides an extract of tiger bean sprouted with a Centella asiatica extract. The tiger bean extract germinated with the Centella asiatica extract according to the present invention exhibits low cytotoxicity while providing excellent anti-inflammatory effects and an effect of increasing the expression of skin barrier-related factors.

The tiger bean extract of the present invention can be prepared by extracting Centella asiatica, germinating the tiger bean with the Centella asiatica extract, and extracting the germinated tiger bean.

The extraction solvent used for extracting Centella asiatica is selected from the group consisting of water, anhydrous or hydrous lower alcohols having 1 to 4 carbon atoms, acetone, ethyl acetate, butyl acetate, dichloromethane, ethyl ether, hexane, and 1,3-butylene glycol. It may include one or more types. For example, the extraction solvent may be water or ethanol, for example, 60 to 80% (v/v) ethanol.

The extraction can be performed using an autoclave. For example, after adding an extraction solvent to Centella asiatica, extraction can be performed in a high pressure sterilizer at 110 to 130°C for 10 to 20 minutes. When extraction is performed under the above-mentioned conditions, centella asiatica components can be extracted with high efficiency. The extraction may be performed 1 to 5 times, for example, 3 times.

After the extraction, if necessary, the step of filtering and concentrating the extract may be further included. The filtration method is not particularly limited, and may be filtered using, for example, a 0.2 to 150 ㎛ filter, for example, a 0.4 to 0.5 ㎛ filter. The concentration method is not particularly limited, and for example, concentration can be performed using a reduced pressure concentrator.

Tiger beans are germinated with the centella asiatica extract prepared above. For example, tiger beans can be placed on cotton, covered with thin cotton, and cultured after sufficiently moistening the cotton with the above-mentioned Centella asiatica extract. Cultivation may be carried out for 5 to 10 days, for example one week. If necessary, centella asiatica extract can be added several times to the cotton covering the tiger bean during cultivation so that the cotton is sufficiently wetted.

The concentration of Centella asiatica extract may be 0.1% or more and less than 2%, for example, 0.7 to 1.3%. If the concentration of Centella asiatica extract is less than the above-mentioned range, the anti-inflammatory effect and the effect of increasing the expression of skin barrier-related factors may not be sufficiently exerted, and if it exceeds the above-mentioned range, tiger bean may not germinate.

The germinated tiger bean is extracted. For example, the germinated tiger bean can be dried, ground, and then extracted. For example, germinated tiger beans can be dried at 50°C for 48 hours, then crushed with a mortar and pestle, and then extracted.

The extraction solvent used for the extraction of tiger bean is from the group consisting of water, anhydrous or hydrous lower alcohol having 1 to 4 carbon atoms, acetone, ethyl acetate, butyl acetate, dichloromethane, ethyl ether, hexane and 1,3-butylene glycol. It may include one or more selected types. For example, the extraction solvent may be water or ethanol, for example, 60 to 80% (v/v) ethanol.

The mixing ratio of tiger bean and extraction solvent may be 1:1 to 30 weight ratio, for example, 1:1 to 10 weight ratio. If the mixing ratio of the extraction solvent for tiger bean is less than the above-mentioned range, the tiger bean component may not be sufficiently extracted, and if it exceeds the above-mentioned range, a lower yield is obtained compared to the amount of solvent used, which is inefficient.

The extraction may be performed by incubation at 20 to 40° C., for example, at room temperature, for 1 to 5 days, for example, 2 to 4 days. When extracted under the above-mentioned conditions, tiger bean components can be extracted with high efficiency. The extraction may be performed 1 to 5 times, for example, 3 times.

After the extraction, if necessary, steps of filtering, concentrating, and freeze-drying the extract may be further included. The filtration method is not particularly limited, and may be filtered using, for example, a 0.2 to 150 ㎛ filter, for example, a 0.4 to 0.5 ㎛ filter. The concentration method is not particularly limited, and for example, concentration can be performed using a reduced pressure concentrator.

<Cosmetic composition>

The cosmetic composition of the present invention includes the above-described tiger bean extract. Based on the total weight of the cosmetic composition, the tiger bean extract may be included in an amount of 0.001 to 20% by weight, for example, 0.005 to 1% by weight.

In addition, the cosmetic composition of the present invention may further include talc, pigment, etc., and may further include additives commonly used in the relevant technical field, such as antioxidants and preservatives.

The cosmetic composition of the present invention can be prepared in any formulation commonly prepared in the art, for example, solutions, suspensions, emulsions, pastes, gels, creams, lotions, surfactant-containing cleansers, oils, It can be manufactured in a formulation such as a spray.

Hereinafter, the present invention will be described in more detail through examples. However, the following examples are only intended to aid understanding of the present invention and do not limit the scope of the present invention to the examples in any way.

[Preparation Example 1: Preparation of tiger bean extract]

200 ml of distilled water was added to 2 g of Centella asiatica, and extraction was performed at 121°C for 15 minutes using an autoclave to prepare a 1% concentration Centella asiatica extract.

Place 3 tiger bean (average weight: 0.75 g) eggs on cotton, cover it with thin cotton, and pour 15 ml of the sterilized Centella asiatica extract prepared above on the cotton on the first day and 5 ml every day from the second day for one week. By culturing (i.e., treating with a total volume of 45 ml for 7 days), tiger bean was germinated.

The germinated tiger bean was dried at 50°C for 48 hours and then ground with a mortar and pestle. The pulverized tiger bean was extracted with 70% ethanol (w/w, 1:10) for 4 days, then concentrated and freeze-dried to prepare the tiger bean extract powder of Preparation Example 1.

[Preparation Example 2: Preparation of tiger bean extract]

Tiger bean extract powder of Preparation Example 2 was prepared in the same manner as Preparation Example 1, except that 2% concentration of Centella asiatica extract prepared using 4 g of Centella asiatica was used.

[Preparation Example 3: Preparation of tiger bean extract]

Tiger bean extract powder of Preparation Example 3 was prepared in the same manner as Preparation Example 1, except that 5% concentration of Centella asiatica extract prepared using 10 g of Centella asiatica was used.

[Preparation Example 4: Preparation of tiger bean extract]

Tiger bean extract powder of Preparation Example 4 was prepared in the same manner as Preparation Example 1, except that regular tiger bean that was not germinated with Centella asiatica extract was used.

[Experimental Example 1: Appearance observation]

The tiger bean of Preparation Example 1-4 was observed, and a photograph of its appearance is shown in 1. As shown in Figure 1, the outer skin of the tiger bean of Preparation Example 1, which was germinated with a 1% concentration of Centella asiatica extract, changed to brown, unlike the tiger bean of Preparation Example 4, which was not germinated. On the other hand, the tiger beans of Preparation Examples 2 and 3, which were germinated with 2% concentration of Centella asiatica extract and 5% concentration of Centella asiatica extract, respectively, did not germinate.

[Experimental Example 2: Ingredient Analysis]

HPLC measurement was performed using the tiger bean extract powders of Preparation Examples 1-4 under the following conditions, and the results are shown in Figure 2. As shown in Figure 2, the tiger bean extract of Preparation Example 1, which was germinated with a 1% concentration of Centella asiatica extract, contained a total of active ingredients (Peak 1, 2, 3, 4) compared to the non-germinated tiger bean extract of Preparation Example 4. It can be seen that the area increases by 1.6 times. On the other hand, the tiger bean extracts of Preparation Examples 2 and 3, respectively treated with 2% concentration of Centella asiatica extract and 5% concentration of Centella asiatica extract, contained more active ingredients (Peak 1, 2, 3, 4) than the tiger bean extract of Preparation Example 4. The total area appeared small.

<HPLC analysis conditions>

Column: Phenomenon Luna C18 250 x 4.6 mm

Sol A: 0.1% phosphoric acid in 3DW

Sol B: ACN

PDA: 210 nm, 300 nm

Injection volume: 10ul

Flow rate: 1 mL/min

Oven: 35℃

Sample: diluted to 5,000 ppm

[Experimental Example 3: Evaluation of anti-inflammatory cytotoxicity, Raw264.7 MTT assay]

To confirm the cytotoxicity of tiger bean extract, MTT assay was performed. Specifically, 100 μL of the RAW264.7 cell line was dispensed at an appropriate concentration per well in a 96 well plate and cultured for 24 hours under 37°C and 5% CO ₂ incubator conditions. Cultured cells were treated with samples at different concentrations and then cultured for 24 hours at 37°C and 5% CO ₂ incubator conditions. 3 hours before completion of culture, 10 μL of 5 mg/mL MTT was treated and cultured under the same conditions for 3 hours. After completion of incubation, the culture medium was discarded, 100 μL of DMSO was dispensed, and the mixture was stirred at room temperature for 10 minutes. Absorbance was measured at 570 nm using a microplate reader (Thermo, UV/Vis). Cell viability was calculated using the formula below, and when the cell viability was 80% or more, it was judged that there was no cytotoxicity.

Cell viability (%) = 100

As shown in Figure 3, the tiger bean extract of Preparation Example 1 germinated with Centella asiatica extract at 1% concentration and the tiger bean extract of Preparation Example 2 treated with Centella asiatica extract at 2% concentration were 50 μg/mL and 100 μg/mL, respectively. It can be confirmed that it is non-toxic at concentrations below mL.

[Experimental Example 4: Evaluation of anti-inflammatory efficacy (Raw264.7 NO assay)]

To confirm the anti-inflammatory effect of tiger bean extract, NO assay was performed. Specifically, 100 μL of the RAW264.7 cell line was dispensed at an appropriate concentration per well in a 96 well plate and cultured for 24 hours under 37°C and 5% CO ₂ incubator conditions. After treating the cultured cells with samples according to concentration, they were cultured for 30 minutes at 37°C and 5% CO ₂ incubator conditions, then treated with an appropriate amount of 1 μg/mL lipopolysaccharide (LPS), and cultured for 24 hours under the same conditions. After completion of incubation, the supernatant was transferred to a new 96 well plate, reacted with the Griess reagent system kit (Promega), and absorbance was measured at 540 nm using a microplate reader (Thermo, UV/Vis). The NO production amount was derived by comparing the trend line of the Sodium Nitrite standard (y = Ax + B) with the formula: NO production amount = (absorbance of treatment - B) / A. Since NO induces an inflammatory response, the higher the inhibitory activity is measured, the higher the anti-inflammatory efficacy can be evaluated.

As shown in Figure 4, the tiger bean extract of Preparation Example 1 germinated with Centella asiatica extract at a concentration of 1% showed excellent anti-inflammatory effect compared to the non-germinated tiger bean extract of Preparation Example 4. On the other hand, the tiger bean extract of Preparation Example 2 treated with Centella asiatica extract at a concentration of 2% showed an anti-inflammatory effect at the same level as the tiger bean extract of Preparation Example 1, but as described later in Experimental Example 6, it did not show a skin barrier effect. did.

[Experimental Example 5: Evaluation of barrier cytotoxicity, HaCat MTT assay]

To confirm the cytotoxicity of tiger bean extract, MTT assay was performed. Specifically, 200 μL of the HaCaT cell line was dispensed at an appropriate concentration per well in a 96 well plate and cultured for 24 hours under 37°C and 5% CO ₂ incubator conditions. After treating the cultured cells with samples at different concentrations, they were cultured for 24 hours at 37°C and 5% CO ₂ incubator conditions. 3 hours before completion of culture, 20 μL of 5 mg/mL MTT was treated and cultured under the same conditions for 3 hours. After completion of incubation, the culture medium was discarded, 150 μL of DMSO was dispensed, and the mixture was stirred at room temperature for 10 minutes. Absorbance was measured at 570 nm using a microplate reader (Thermo, UV/Vis). Cell viability was calculated using the formula below, and when the cell viability was 80% or more, it was judged that there was no cytotoxicity.

Cell viability (%) = 100

As shown in Figure 5, the tiger bean extract of Preparation Example 1 germinated with Centella asiatica extract at 1% concentration and the tiger bean extract of Preparation Example 2 treated with Centella asiatica extract at 2% concentration were 50 μg/mL and 25 μg/mL, respectively. It can be confirmed that it is non-toxic at concentrations below mL.

[Experimental Example 6: Barrier efficacy evaluation (HaCaT RT-qPCR)]

To confirm the skin barrier efficacy of tiger bean extract, RT-qPCR was performed. Specifically, 4 mL of HaCaT cell line was dispensed at an appropriate concentration per well in a 60 mm dish and cultured in an incubator at 37°C and 5% CO ₂ for 24 hours. After serum starvation was performed on the cultured cells for 24 hours, samples were treated according to concentration and cultured for 24 hours under 37°C and 5% CO ₂ incubator conditions. After completion of incubation, RNA was extracted by treatment with 1 mL of Trizol (Invitrogen), and RNA was condensed and washed using chloroform, 2-propanol, 75% EtOH, and a centrifuge. After drying RNA, an appropriate amount of nuclease free water was added and quantification was performed using Nano drop (ALLSHENG). After preparing each RNA to the same concentration, reverse transcription was performed using a cDNA Synthesis Kit (PhileKorea). After mixing the synthesized cDNA, Primer (LOR Oligonucleotide), SYBR green, and Nuclease free wate in an appropriate ratio, RT-qPCR was performed using Real time PCR equipment (Applied Biosystem, Quantstudio 5). In the analysis of PCR results, △△Ct values were calculated to compare gene expression. Since the factor (LOR) used in the efficacy evaluation is a factor with barrier function, the higher the mRNA expression of the sample compared to the negative control, the higher the barrier efficacy can be evaluated.

As shown in Figure 6, the tiger bean extract of Preparation Example 1 germinated with Centella asiatica extract at a concentration of 1% showed an excellent barrier effect. On the other hand, the tiger bean extract of Preparation Example 2 treated with Centella asiatica extract at a 2% concentration and the ungerminated tiger bean extract of Preparation Example 4 did not show a barrier effect.

Claims (5)

A tiger bean extract germinated with Centella asiatica extract,
Tiger bean extract wherein the concentration of the Centella asiatica extract is 0.1% or more and less than 2%. delete The tiger bean extract according to claim 1, wherein the tiger bean germination is performed by culturing for 5 to 10 days. A cosmetic composition comprising the tiger bean extract of claim 1 or 3. The cosmetic composition according to claim 4, which has excellent anti-inflammatory effects and an effect of increasing the expression of skin barrier-related factors.