KR102396816B1 - Cosmetic composition and cosmetic additive composition comprising extracted-fermented Glycyrrhizia uralensis leaf - Google Patents

Abstract

The present invention relates to a cosmetic and cosmetic additive composition comprising a fermented extract of licorice leaves, specifically, a fermentation obtained by fermenting an extract extracted from licorice leaves with a mixed solution of water and ethanol with Lactobacillus plantarum . It relates to a cosmetic composition comprising water and a cosmetic additive composition.
The cosmetic composition and cosmetic additive composition of the present invention utilize the leaves of licorice, which are mostly discarded as by-products, and by including the extract and fermented extract of the licorice leaves, they are safe for the human body, and contain ingredients beneficial to the skin such as polyphenols and glycizin. It contains a large amount and exhibits excellent nitrogen monoxide production inhibitory ability, antioxidant activity, and antibacterial activity to prevent or improve skin troubles.

Description

Cosmetic composition and cosmetic additive composition comprising extracted-fermented Glycyrrhizia uralensis leaf

The present invention relates to a cosmetic and cosmetic additive composition comprising a fermented extract of licorice leaves, specifically, a fermentation obtained by fermenting an extract extracted from licorice leaves with a mixed solution of water and ethanol with Lactobacillus plantarum . It relates to a cosmetic composition comprising water and a cosmetic additive composition.

According to the Ministry of Food and Drug Safety (MFDS) survey, as teenagers wear makeup faster, 17% of elementary school students and 64% of middle school students started applying makeup, and more than half of high school girls answered that they use color cosmetics. The problem is that children have thinner skin tissue than adults and have a higher absorption rate, so even a small amount of heavy metal is dangerous. In fact, it was found that 35% of patients who visit the hospital for skin problems are teenagers. In addition, in recent years, fine dust and yellow dust in Korea and many Southeast Asian countries have been at a very serious stage, causing various types of skin diseases such as contact dermatitis and itching in modern people including adolescents, causing serious damage to health and life. there is. Accordingly, there is a need to develop a product that can be used safely and can improve the problematic skin as described above.

On the other hand, as the saying goes, 'Licorice in the Pharmacopeia', licorice has been a medicinal herb that should not be omitted from traditional herbal medicines. According to the Korean Medical Association in 1983, licorice was the most prescribed herbal medicine. Licorice relieves the toxicity of other medicinal substances and harmonizes all medicines to make the medicinal effects appear well, and has many excellent effects. It is an important medicine that is indispensable to the extent that it is used in 70 out of 113 prescriptions even in ancient times. Although many farmers are cultivating licorice, licorice is not currently a product promoted by the government or a purchased crop, so it is exposed to difficulties in developing a market for the harvest by the growers themselves. Leaves and stems, which account for more than 50% of the total, are being thrown away. Accordingly, it is necessary to increase the usability of licorice to provide a more diverse market to farmers, and at the same time to develop products using leaves or stems, which are by-products of licorice, and to solve the problem of by-products that are thrown away.

In order to solve the above problems, the present inventor tried to develop a cosmetic that can be used safely by utilizing the leaves mostly discarded as a by-product of licorice, and can provide beneficial effects to the skin, such as skin inflammation relief and skin aging prevention.

Republic of Korea Patent Registration No. 10-1991830

Therefore, the main object of the present invention is to provide a cosmetic composition and cosmetic additive composition that can exhibit beneficial functionality while being safe for the human body by utilizing the leaf part, which is a by-product, which is mostly thrown away instead of the root part of licorice, which is generally used as a herbal medicine. .

According to one aspect of the present invention, the present invention provides a cosmetic composition and a cosmetic additive composition comprising a fermented product obtained by fermenting an extract extracted from licorice leaves with a mixed solution of water and ethanol with Lactobacillus plantarum to provide.

In the composition of the present invention, the fermented product is preferably obtained by pulverizing the extract obtained by the extraction to obtain an extract powder, adding the extract powder to water, inoculating Lactobacillus plantarum, and fermenting Do.

The cosmetic composition and cosmetic additive composition of the present invention utilize the leaves of licorice, which are mostly discarded as by-products, and by including the extract and fermented extract of the licorice leaves, they are safe for the human body, and contain ingredients beneficial to the skin such as polyphenols and glycizin. It contains a large amount and exhibits excellent nitrogen monoxide production inhibitory ability, antioxidant activity, and antibacterial activity to prevent or improve skin troubles.

1 is a graph showing the total polyphenol content of a fermented product of Lactobacillus plantarum of 80% ethanol shaking extract of licorice leaf according to an embodiment of the present invention compared with that of fermented products with other bacteria.
2 is a graph showing the viability of B16F1 cells according to the treatment of the Lactobacillus plantarum fermented product of 80% ethanol shaking extract of licorice leaf according to an embodiment of the present invention compared with the fermented product fermented with other bacteria. control (DW), control.
3 is a graph showing the nitrogen monoxide production inhibitory ability of a Lactobacillus plantarum fermented product of 80% ethanol shaking extract of licorice leaf according to an embodiment of the present invention compared to fermented products fermented with other bacteria. control (DW), control.
4 to 5 show the main phytochemical analysis HPLC results of the Lactobacillus plantarum fermented licorice leaf 80% ethanol shaking extract according to an embodiment of the present invention compared with the licorice leaf extract, the licorice root extract and before fermentation. It is a graph. 4 (A), licorice leaf 80% ethanol shaking extract; 4 (B), licorice root 80% ethanol shaking extract; Figure 5 (A) before fermentation of licorice leaf 80% ethanol shake extract; 5 (B) Lactobacillus plantarum fermented product of 80% ethanol shaking extract of licorice leaf (after fermentation). 1, glycyrrhizin; 2, liquiritin; 3, liquiritigenin.
6 and 7 are RAW264.7 cells according to the concentration (10, 100, 250, 500㎍ / ㎖) treatment of the Lactobacillus plantarum fermented 80% ethanol shaking extract of licorice leaves according to an embodiment of the present invention. It is a graph showing viability compared with licorice leaf extract, licorice root extract and before fermentation. con, control; Before fermentation of licorice leaves, before fermentation of 80% ethanol shaking extract of licorice leaves; After fermentation of licorice leaves, Lactobacillus plantarum fermented product of 80% ethanol shaking extract of licorice leaves; Leaf, Licorice Leaf 80% Ethanol Shaking Extract; Root, Licorice root 80% Ethanol Shaking Extract.
8 and 9 are licorice leaf extract and licorice root extract for each concentration (100, 250 μg/ml) of fermented Lactobacillus plantarum of 80% ethanol shaking extract of licorice leaf according to an embodiment of the present invention. And it is a graph shown compared to before fermentation. CON, control; LPS, LPS treated group (1㎍ / ㎖); Before fermentation of licorice leaves, before fermentation of 80% ethanol shaking extract of licorice leaves; After fermentation of licorice leaves, Lactobacillus plantarum fermented product of 80% ethanol shaking extract of licorice leaves; Licorice leaf, Licorice leaf 80% ethanol shaking extract; Licorice root, licorice root 80% ethanol shaking extract.
Figure 10 shows the antibacterial activity of the 80% ethanol shaking extract of licorice leaf according to an embodiment of the present invention compared to the extraction solvent, the extraction method and the licorice root extract. ①, 80% ethanol ultrasonic extract of licorice leaves; ②, licorice leaf 80% ethanol shaking extract; ③, licorice leaf hot water shaking extract; ④, 80% ethanol ultrasonic extract of licorice root; ⑤, Licorice root 80% ethanol shake extract; ⑥, licorice root hot water shaking extract; ⑦, control (DW).
Figure 11 shows the antibacterial activity of the fermented Lactobacillus plantarum of 80% ethanol shaking extract of licorice leaf according to an embodiment of the present invention compared with that before fermentation. ①, before fermentation of licorice leaf 80% ethanol shaking extract; ②, Lactobacillus plantarum fermented product of 80% ethanol shaking extract of licorice leaf (after fermentation), ③, control (DW).

The cosmetic composition and cosmetic additive composition of the present invention include a fermented product obtained by fermenting an extract extracted from licorice leaves with a mixed solution of water and ethanol with Lactobacillus plantarum .

Licorice leaf used as an extraction raw material in the present invention refers to a leaf of a medicinal plant belonging to licorice, that is, leguminosae and whose scientific name is Glycyrrhiza uralensis Fischer. It is known that there is a significant difference in component content and physiological activity from the root, which is mainly used as a herbal medicine.

In the present invention, the licorice leaves are preferably used in powder form. In addition, it is preferably freeze-dried and then powdered before use. In addition, preferably 10 to 100 mesh (mesh), more preferably 30 to 80 mesh, more preferably 40 to 60 mesh is used by pulverizing.

In the present invention, a mixed solution of water and ethanol is used as an extraction solvent. According to the present invention, a significant difference in functionality may occur depending on the difference in the extraction solvent. Accordingly, preferably 50 to 95% (v/v) of ethanol, more preferably 60 to 90% (v/v) of ethanol, more preferably 70 to 90% (v/v) of ethanol, more preferably For example, 75-85% (v/v) ethanol is used.

In the present invention, for the extraction, a method of adding licorice leaves to the solvent and performing stationary, shaking or ultrasonic extraction may be used. At this time, preferably licorice leaves are added to the solvent at a concentration of 10 to 200 g/L, more preferably at a concentration of 20 to 150 g/L, more preferably at 30 to 100 g/L, more Preferably, it is added in an amount of 40 to 60 g/L. In addition, the extraction time at this time is preferably 1 to 100 hours, more preferably 10 to 80 hours, more preferably 20 to 30 hours. In addition, the extraction temperature at this time is 0 ~ 90 ℃, more preferably 20 ~ 80 ℃, more preferably 40 ~ 70 ℃. In the case of shaking extraction, preferably 50 to 200 rpm, more preferably 100 to 150 rpm.

In the present invention, after the extraction, the extract is preferably filtered, more preferably filtered and concentrated under reduced pressure, and more preferably after filtration and concentration under reduced pressure, freeze-drying is used.

In the present invention, the licorice leaf extract obtained by the above extraction is fermented with Lactobacillus plantarum . At this time, for fermentation, preferably, a method of preparing a fermentation medium by adding the licorice leaf extract and inoculating the Lactobacillus plantarum therein is used. In this case, preferably, the licorice leaf extract has a concentration of 0.005 to 0.5 g/100 ml, more preferably 0.01 to 0.1 g/100 ml, and more preferably 0.02 to 0.08 g/100 ml. It is added to prepare a fermentation medium. In addition, at this time, the fermentation medium is preferably based on water. For example, a fermentation medium is prepared by adding the licorice leaf extract to water.

Fermentation in the present invention is preferably made at 30 ~ 40 ℃, more preferably made at 35 ~ 39 ℃. Also, preferably 1 to 7 days, more preferably 2 to 4 days. Also, preferably shaking fermentation, in this case, preferably 100 to 300 rpm, more preferably 150 to 200 rpm shaking fermentation.

The Lactobacillus plantarum used as the fermenting bacteria in the present invention is preferably Lactobacillus plantarum KCTC 3104 strain.

For fermentation in the present invention, it is preferably inoculated by adding a Lactobacillus plantarum culture to the fermentation medium. At this time, preferably, the concentration of Lactobacillus plantarum is 1x10 ³ ~ 1x10 ¹⁰ cells/100ml, more preferably 1x10 ⁴ ~ 1x10 ⁹ cells/100ml, more preferably 1x10 ⁵ ~ 1x10 ⁸ cells/100ml Inoculate as much as possible.

In the present invention, after the fermentation, the fermentation broth is preferably filtered, and more preferably, it is used after filtration and freeze-drying.

According to the present invention, smooth extraction and fermentation of licorice leaves can be achieved according to the method as described above.

In addition, according to the present invention, the fermented licorice leaf extract prepared according to the above method has excellent total polyphenol content, is safe for the human body due to low cytotoxicity, exhibits excellent nitric oxide production inhibitory ability, and phytochemicals such as glycyrrhizin It has excellent content, exhibits excellent antioxidant activity, and can exhibit excellent antibacterial activity.

In particular, the fermented licorice leaf extract of the present invention may exhibit antibacterial activity against Staphylococcus aureus and Pseudomonas aeruginosa. These bacteria can be dangerous to the human body for other reasons as well, but they can cause eye diseases or skin rashes if they are contaminated with cosmetics. Therefore, it is possible to use cosmetics more safely by preventing contamination of the above-mentioned bacteria through the method of adding the fermented licorice leaf extract of the present invention to cosmetics.

In the present invention, the cosmetic composition preferably contains the fermented licorice leaf extract at a concentration of 1 to 100 μg/ml in consideration of the cytotoxicity test result.

The cosmetic composition and cosmetic additive composition of the present invention may further include a cosmetically acceptable carrier or additive in addition to the fermented licorice leaf extract. In this case, the carrier also includes a pharmaceutically acceptable carrier. In addition, the cosmetic composition and cosmetic additive composition of the present invention may be formulated in various forms. For example, in the case of a cosmetic composition, it can be formulated into a lotion, a nourishing lotion, a nourishing cream, a massage cream, a pack, and a nourishing essence. And the cosmetic additive composition of the present invention may be the fermented licorice leaf extract itself.

The cosmetic composition and cosmetic additive composition of the present invention may further include other functional substances in addition to the fermented licorice leaf extract. In this case, the functional material may be a processed product of plant material, for example, an extract or a fermented product. In addition, the cosmetic composition and cosmetic additive composition of the present invention may include only the fermented licorice leaf extract as a plant fermented product, and may not include extracts or fermented products of other plants other than the fermented licorice leaf extract.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and therefore, the scope of the present invention is not to be construed as being limited by these examples.

[Example]

1. Preparation of fermented liquorice extract

1-1. extraction

After washing the licorice root, it was freeze-dried for 72 hours using a freeze-drying device. After separating the leaves and roots, each was crushed, and filtered through a 45 mesh net to obtain a powder. The prepared leaf powder or root powder was added to 80% (v/v) ethanol or water at 50 g/L, followed by shaking extraction or ultrasonic extraction at 120 rpm at 60° C. for 24 hours. Then, the obtained extract was filtered through a filter, concentrated under reduced pressure and freeze-dried to obtain a final extract.

1-2. fermentation

The licorice leaf or root extract was added to water to a concentration of 0.05 g/100 ml to prepare a fermentation medium, and then Lactobacillus plantarum (KCTC 3104), Lactobacillus acidophilus ( L. acidophilus ) (KCTC 3171), Lactobacillus casei ( L. casei ) (KCTC 3109) or Lactobacillus delbrueckii ( L. delbrueckii ) (KCTC 3635) Inoculated by adding the culture solution to a concentration of 1x10 ⁶ cells / 100 ㎖, Shaking fermentation was carried out for 3 days in a shaking incubator at 37°C and 180 to 190 rpm. Thereafter, the fermentation broth was filtered through a filter (0.45 μm or 0.2 μm pore size), lyophilized, and stored frozen.

2. Experiment

2-1. Measurement of the number of viable cells in the fermentation broth

On the 1st, 2nd, and 3rd days of fermentation, three fermentation broth samples (Lactobacillus plantarum fermentation broth of 80% ethanol shaking extract of licorice leaves) were prepared, and then the number of viable cells was measured by microscopic counting.

As a result, as shown in Table 1, it was found that the number of viable cells increased as the fermentation period elapsed, which means that the fermented strains grew smoothly and fermented normally.

sample dilution Number of measurements (repeat) Average Deviation Conversion (cells/ml) One 2 3 Fermentation 1 day 1.E-01 6.4 7.2 6.4 6.667 0.462 2.E+07 2 days of fermentation 1.E-01 13.4 13.2 13 13.200 0.200 3.E+07 Fermentation 3 days 1.E-02 6.6 5.8 6 6.133 0.416 2.E+08

2-2. Analysis of total polyphenol content

Total polyphenol contents were calculated by Na Hyun Lee et al. (2015). It was measured according to the method of Sample solution (fermented product suspension (DDW) (0.5 g/L) of licorice leaf 80% ethanol shaking extract) and gallic acid standard (0, 20, 40, 60, 80, 100 mg/L) 100 Add 2ml of 2% Na ₂ CO ₃ solution to μl, stir sufficiently, and leave for 3 minutes. Then, add 100 μl of 50% Folin &Ciocalteu's phenol reagents, vortex, and react in the dark for 30 minutes, and remove the blank at 750 nm. Absorbance was measured as a control. The total polyphenol content of each sample was calculated from the dryness curve obtained using gallic acid as a standard material.

As a result, it was found that the sample fermented with Lactobacillus plantarum as shown in FIG. 1 was superior to the sample before fermentation and the sample fermented with other fermenting strains. At this time, the total polyphenol content of the sample fermented with Lactobacillus plantarum was 300.94±0.01 mgGAE/g.

2-3. Safety evaluation of fermented products according to differences in fermentation strains

Cell viability was measured in B16F1 cells using the MTT method. B16F1 cells were treated with each fermented product of 80% ethanol shake extract of licorice leaf and fermented with each fermented strain at a concentration of 10 mg/ml, and cell viability was measured.

As a result, as shown in FIG. 2, the sample fermented with Lactobacillus plantarum showed higher cell viability than the sample before fermentation and the sample fermented with other fermentation strains. This means that the sample fermented with Lactobacillus plantarum is safe from cytotoxicity compared to other samples. At this time, the cell viability of the sample treated group fermented with Lactobacillus plantarum was 99%.

2-4. fermented strain according to the difference fermented Nitric oxide production deterrent evaluation

RAW 264.7 (TIB-71; ATCC, USA) macrophages used in the experiment were purchased from ATCC (American type cell collection, USA), and 10% FBS (Fetal Bovine Serum) in DMEM (Dulbecco's Modified Eagle's Medium; Gibco, USA) medium. ; Gibco, USA) and 1% Penicillin (100U/ml)/Streptocycin (100㎍/ml) were added to the culture medium at 37°C, 5% CO ₂ conditions.

RAW 264.7 cells were seeded in a 6-well plate at a concentration of 5×10 ⁵ cells/well, and the medium was removed after culturing for 24 hours. Here, fresh DMEM (10% FBS, 1% Penicillin (100U/ml)/Streptocycin (100㎍/ml)) medium and sample (fermented product of 80% ethanol shaking extract of licorice leaves) were pretreated for 1 hour and then LPS ( 1 μg/ml) and incubated at 37° C., 5% CO ₂ conditions for 24 hours. After 24 hours of sample treatment, 50 μl of the cell culture supernatant and 50 μl of Griess reagent (1% sulfanilamide + 0.1% α-naphthylamide in 2.5% phosphoric acid) were mixed and reacted for 10 minutes in 96 well plates. absorbance was measured. The value of nitric oxide (NO) contained in the cell culture supernatant was calculated by creating a standard curve with NaNO ₂ .

As a result, as shown in FIG. 3 , it was found that the fermented sample had a higher ability to inhibit the production of nitrogen monoxide than the sample before fermentation.

2-5. Phytochemical quantitative evaluation

For comparative evaluation of licorice extract and fermented product, quantitative analysis of the main component using HPLC was performed.

HPLC analysis outline analysis target Glycyrrhizic acid, liquiritin, liquiritigenin HPLC Agilent 1200 series Detector DAD (Diode Array Detector) at 254 and 276 nm calibration curve Calibration range: 10 ~ 50 ㎍/mL (no weight)

All concentrations used in the test were indicated as free bases. Based on the indicator components of licorice listed in the Korean Pharmacopoeia, glycyrrhizic acid, glycyrrhizin, liquiritin, and liquiritigenin were selected as phytochemicals to be analyzed, and the content in each extract and fermented product were evaluated and compared.

As a diluent for HPLC quantitative evaluation of major components, 50% (v/v) methanol was prepared and used.

The analyzed licorice extract and fermented product samples are shown in Table 3 below.

Sample ID Sample information GLY-L1 Licorice leaf extract before fermentation GLY-L2 Licorice leaf extract after fermentation GLY-L3 Licorice Leaf Extract GLY-R1 Licorice Root Extract

* GLY : Glycyrrhiza, L: leaves, R: root

The licorice leaf extract before fermentation is lyophilized after sterilization of 80% ethanol shaking extract of licorice leaves, preparing a medium at a concentration of 0.05 g / 100 ml, sterilization and filtering with a filter, and the licorice leaf extract after fermentation is licorice leaf It is a Lactobacillus plantarum fermented product of 80% ethanol shake extract, the licorice leaf extract is a licorice leaf 80% ethanol shake extract, and the licorice root extract is a licorice root 80% ethanol shake extract.

For HPLC quantitative evaluation, a certain amount (20-40 mg) of each powder sample is taken, ultrasonically extracted with 1 ml of methanol, a certain amount of the supernatant is taken, diluted with 50% (v/v) methanol, and centrifuged, and the supernatant 15 μl was injected into HPLC as a sample solution.

HPLC conditions are as follows.

HPLC: Agilent 1200 series with DAD (Diode Array Detector)

Detector: UV 254 and 276 nm

Column: Shseido CapCell PAK MGII C18 (4.6×150 mm, 3 μm pore size)

Column temperature: 35℃

Mobile phase: 0.1% formic acid (aqueous)/acetonitrile (A/B, v/v) as gradient elution: 0 min-25% B, 4 min-25% B, 8 min-30% B, 12 min-35% B, 16 min-45% B, 19 min-45% B, 22 min-70% B, 27 min-70% B, 28 min-40% B, 30 min-25% B

Flow rate: 0.6 ml/min

Injection volume: 15 μl

The concentration was calculated using the regression equation of the calibration curve according to the weighted least squares method using the peak area ratio and the concentration of the preparation to the standard material. Agilent Chemstation (Agilent 1200 series) was used to prepare the calibration curve and calculate the concentration.

Unit of concentration: μg/ml

Regression equation for calibration curve: y = ax + b

(y: peak area ratio, x: preparation concentration, a: slope, b: y-intercept)

Concentration (mg/g) = C × V × W ^-1 × f

C = result obtained (μg/ml)

V = extraction volume (ml)

W = sample weight (g)

f = dilution factor

Measured values were expressed as μg/ml or mg/g (converted as free base) and expressed with 2 decimal places, and peak area values were expressed with 4 significant digits.

As a result, as shown in Table 4, although the contents of glycyrrhizin and liquiritin of the licorice leaf extract are lower than that of the licorice root extract, the content of glycyrrhizin increases as the licorice leaf extract is fermented with Lactobacillus plantarum. appear.

Content (μg/mg) Glycyrrhizin (1) Liquiritin (2) Liquidity Genin (3) GLY-L1 1.09 0.91 0.52 GLY-L2 1.20 0.84 0.53 GLY-L3 0.81 0.70 0.36 GLY-R1 17.70 3.83 0.18

2-6. Antioxidant activity evaluation

In order to evaluate the antioxidant activity, DPPH, ABTS and SOD analysis were performed on the samples in Table 3 above.

DMSO 1ml was added to each sample, extracted to a concentration of 100mg/ml, and serially diluted to prepare a sample solution at a concentration of 1mg/ml.

Analysis of DPPH radical scavenging activity was performed by adding 20 μl of the sample solution and 80 μl of 0.5 mM DPPH solution in order to a 96 well plate, leaving it at room temperature for 30 minutes, and then measuring the absorbance at 517 nm by ELISA. Thereafter, the DPPH radical scavenging ability was calculated using Equation 1 below.

[Formula 1]

ABTS radical scavenging activity analysis was performed by putting 10 μl of each test solution into a well, adding 90 μl of ABTS+ solution, incubating at 37° C. for 30 minutes (in dark conditions), and measuring absorbance at 734 nm by ELISA. Thereafter, the ABTS radical scavenging ability was calculated using Equation 2 below.

[Formula 2]

For SOD radical scavenging activity analysis, 20 μl of each test solution was put into a well, 200 μl of WST solution was added, and 20 μl of dilution buffer was added to each well of blank2 and blank3, and then 20 μl of enzyme working solution was added to each well of blank1 and sample solution. After incubation at 37° C. for 20 minutes, absorbance was measured at 450 nm. In order to check the reaction time at which the absorbance value of Blank1 was 0.7≤blank1≤1.0, the absorbance value was measured in kinetic mode. Then, the SOD radical scavenging ability was calculated using Equation 3 below.

[Formula 3]

As a result, as shown in Table 5, the antioxidant activity of the licorice leaf extract was found to be very superior to that of the licorice root extract. And when comparing before and after fermentation, DPPH decreased while ABTS increased.

Sample ID Radical scavenging ability (%) DPPH ABTS SOD PC 92.55±0.12 99.71±0.65 - GLY-L1 54.44±2.93 49.99±5.18 85.53±0.56 GLY-L2 48.98±2.32 51.24±4.79 82.45±1.97 GLY-L3 70.60±4.49 57.09±5.67 97.98±1.84 GLY-R1 6.10±0.63 9.86±0.95 37.26±2.70

* PC: positive control

* Expressed as mean ± standard deviation according to 3 repeated analysis

2-7. Safety evaluation by concentration of fermented products

Table 3 above for RAW 264.7 cells using the MTT ([3-(4,5-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide], Sigma Chemical Co., St. Louis MO, USA) method Cell viability according to each concentration of the sample was measured. The cells were aliquoted in a 96-well plate at a concentration of 5×10 ³ cells/well and cultured for 24 hours, after which the medium was removed. Herein, 100 μl of fresh DMEM (10% FBS, 1% Penicillin (100 U/ml)/Streptocycin (100 μg/ml)) and samples diluted by concentration were added, respectively, and incubated for 24 hours, followed by MTT solution (5 mg/ml). ) 10 μl was added to each well and incubated at 37° C., 5% CO ₂ conditions for 4 hours. After removing the medium and adding 100 μl/well of DMSO (dimethylsulfoxide; Sigma Chemical Co., St. Louis MO, USA), the formazan generated in the cells was stirred for 15 minutes to elute, and then using an ELISA reader Absorbance was measured at 590 nm.

As a result, as in FIGS. 6 and 7 , all extracts and fermented products exhibited concentration-dependent cytotoxicity as a whole, and although the licorice leaf extract had lower safety than the licorice root extract, it was found to be improved through fermentation. In addition, in the case of fermented licorice leaf extract, it was found that up to 100 μg/ml can be safely used without cytotoxicity.

2-8. Evaluation of nitrogen monoxide production inhibitory ability by concentration of fermented products

According to the method of 2-4, the inhibition ability of nitrogen monoxide production according to the treatment for each concentration of the sample in Table 3 was compared.

As a result, as shown in FIGS. 8 and 9 , all extracts and fermented products exhibited a concentration-dependent nitric oxide production inhibitory ability as a whole.

2-9. Antibacterial activity evaluation

The antibacterial activity of licorice leaf or root extracts and fermented products was evaluated.

As strains, Staphylococcus aureus (KCTC3881), aerobic gram-positive bacteria, Escherichia coli (KCTC1039) and Pseudomonas aeruginosa (KCTC1637), aerobic gram-negative bacteria, Candia albicans (KCTC7965) and Aspergillus niger (KCTC7965) and Aspergillus, which cause candidiasis as fungi (KCTC) purchased and used.

Remove and thaw each strain stored at -80℃, S. aureus and P. aeruginosa in TSA (Trypicsoy agar), E. coli in LB (Luria-Bertani media), C. albicans in YEPD, A. niger was inoculated into PDA and cultured at 30°C for 18 to 48 hours, and subculture was used at intervals of once every 7 days to prevent the activity of the strain from dropping during the experiment.

strain deposit number badge Escherichia coli KCTC 1039 LB Staphylococcus aureus KCTC 3881 TSA Pseudomonas aeruginosa KCTC 1637 TSA Candida albicans KCTC 7965 YEPD Aspergillus niger KCTC 6910 PDA

Antibacterial activity was measured by the paper-disk method of the prepared sample, and the experimental method is as follows.

First, a single colony of each strain with good activity is taken through subculture and smeared evenly over the entire solid medium. Place the sterilized paper-disk on the smeared plate and inject 40 to 60 μl of each sample. Cultivate under optimal conditions for each strain. After incubation, check and measure the clear zone.

As a result, as shown in FIG. 10 , both licorice leaves and roots exhibited antibacterial activity against S. aureus and P. aeruginosa in 80% ethanol extract, whereas the hot water extract did not show antibacterial activity. In addition, as shown in FIG. 11 , antibacterial activity against S. aureus and P. aeruginosa was exhibited both before and after fermentation of 80% ethanol shaking extract of licorice leaves.

Claims (4)

The extract obtained by extracting from licorice leaves with a mixed solution of water and ethanol is powdered to obtain an extract powder, the extract powder is added to water, and Lactobacillus plantarum is inoculated and fermented. A cosmetic composition comprising water. delete The extract obtained by extracting from licorice leaves with a mixed solution of water and ethanol is powdered to obtain an extract powder, the extract powder is added to water, and Lactobacillus plantarum is inoculated and fermented. A cosmetic additive composition comprising water.
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