STEAMED GLYCYRRHIZA SP. EXTRACT WITH INCREASED GLYCYRRHETINIC ACID CONTENTS AND PREPARATION METHOD THEREOF

Abstract

Provided are a composition including a steamed Glycyrrhiza sp. extract with increased glycyrrhetinic acid contents as an active ingredient, a method of improving the skin, the method including the step of applying or administering the composition to an individual, a method of preparing the composition, and a Glycyrrhiza sp. extract prepared by way of the method. The present invention has excellent antioxidant, whitening, anti-inflammatory, skin revitalization, and regeneration effects, and is thereby used as a cosmetic composition, a food composition, or a quasi-drug composition that is safe for the skin while having an excellent effect of improving skin conditions.

Claims

1. A method of preparing a Glycyrrhiza sp. extract, the method comprising the steps of: steaming Glycyrrhiza sp. at 90° C. to 150° C. for 1 hour to 10 hours; and extracting the steamed Glycyrrhiza sp.

2. The method of claim 1, wherein the preparation method uses roots of the Glycyrrhiza sp.

3. The method of claim 1, further comprising the step of roasting the Glycyrrhiza sp.

4. The method of claim 3, wherein the roasting is performed at 150° C. to 230° C. for 15 minutes to 45 minutes.

5. The method of claim 1, further comprising the step of drying the steamed Glycyrrhiza sp.

6. The method of claim 1, wherein the Glycyrrhiza sp. extract includes 0.1 mg to 2 mg of glycyrrhetinic acid per g of the Glycyrrhiza sp.

7. The method of claim 1, wherein the Glycyrrhiza sp. extract has antioxidant use.

8. The method of claim 1, wherein the Glycyrrhiza sp. extract has whitening use.

9. The method of claim 1, wherein the Glycyrrhiza sp. extract has anti-inflammatory use.

10. The method of claim 1, wherein the Glycyrrhiza sp. extract has skin regeneration use.

11. A Glycyrrhiza sp. extract prepared by way of the method of claim 1.

12. A cosmetic composition comprising the Glycyrrhiza sp. extract of claim 11 as an active ingredient.

13. The composition of claim 12, wherein the composition is used for antioxidation, whitening, anti-inflammation, or skin regeneration.

14. A method for antioxidation, whitening, anti-inflammation, or skin regeneration of a subject, comprising applying the cosmetic composition of claim 12 to a target skin of the subject.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0085] FIG. 1 shows the results of comparing the glycyrrhetinic acid contents according to various heat treatment conditions.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0086] Hereinafter, the present invention will be described in more detail with reference to Examples. However, these Examples are for illustrative purposes only, and the scope of the present disclosure is not intended to be limited by these Examples.

Example 1. Preparation of Glycyrrhiza sp. Extract

[0087] To prepare Glycyrrhiza sp. extract with a high content of glycyrrhetinic acid, dry Glycyrrhiza sp. roots (Glycyrrhiza glabra) (#1) were cut to a thickness of about 0.5 cm to 1 cm, and roasted for 30 minutes at each temperature using a roaster at a constant temperature (#2-5, #7).

[0088] In addition, the cut dry Glycyrrhiza sp. roots were put in a thick sealed stainless steel container, and sealed under conditions where the roots could be moistened with a small amount of purified water such that the outside air did not pass therethrough, and high-pressure steaming was carried out according to temperature and time, and then the roots were thoroughly dried at 40° C. until completely dried (#6-10).

[0089] For evaluation of the glycyrrhetinic acid content, a standard material 18β-glycyrrhetinic acid (>97%) was purchased from Sigma-Aldrich. 50 g of the roasted or steamed Glycyrrhiza sp. was mixed with 1 L of HPLC grade methanol, extracted with sonication for 1 hour, and filtered through a PTFE filter (0.45 μm) to prepare an extract for analysis of the glycyrrhetinic acid content.

Example 2. Properties According to Heat Treatment and Treatment Time Conditions and Comparison of Glycyrrhetinic Acid Contents

Example 2-1. Comparison of Glycyrrhetinic Acid Contents According to Heat Treatment Conditions

[0090] The content (mg) of glycyrrhetinic acid in 1 g of the dry Glycyrrhiza sp. was evaluated using Shimadzu's high-performance liquid chromatography (HPLC) and diode array detector (DAD). An Agilent ZORBAX Eclipse Plus C18 reversed-phase column (4.6 mm×150 mm, particle size of 3.5 μm) was used as a stationary phase, and a combination of water and acetonitrile with a small amount of formic acid was used as a mobile phase to analyze glycyrrhetinic acid in the extract through a gradient elution method. The sample input amount was 10 μL, the flow rate was 1 mL/min, and the detection wavelength was 240 nm.

[0091] The glycyrrhetinic acid contents were compared (mg in 1 g of the dry Glycyrrhiza sp.) under various heat treatment conditions (untreated, roasted at 140° C. for 30 minutes, roasted at 170° C. for 30 minutes, roasted at 200° C. for 30 minutes, roasted at 230° C. for 30 minutes, steamed at 120° C. for 4 hours, roasted at 200° C. for 30 minutes, and steamed at 120° C. for 4 hours) (Table 1, FIG. 1).

[0092] As a result, it was confirmed that when no treatment was performed or roasting was performed at 200° C. or lower, glycyrrhetinic acid was not detected; when roasting was performed at 230° C., glycyrrhetinic acid was detected in an amount of less than 0.1 mg; when only steaming was performed at 120° C., the content of glycyrrhetinic acid was increased twice or more, as compared to the case of performing only roasting; and when roasting was performed at 200° C. and steaming was performed at 120° C., the content of glycyrrhetinic acid was increased about four times to nine times or more, as compared to the case of performing only roasting or steaming.

[0093] Further, it was confirmed that glycyrrhizin was converted into glycyrrhetinic acid under the roasting condition of 230° C., but the raw material Glycyrrhiza sp. was burnt, which was not suitable for use in extraction. In contrast, it was confirmed that the raw material was not burnt when steamed at 120° C. or under mixed conditions of roasting at 200° C. and steaming at 120° C., and the content of glycyrrhetinic acid was significantly further increased.

TABLE-US-00001 TABLE 1 Treatment conditions Glycyrrhetinic acid #1 Untreated Not detected #2 Roasted at 140° C. for 30 minutes Not detected #3 Roasted at 170° C. for 30 minutes Not detected #4 Roasted at 200° C. for 30 minutes Not detected #5 Roasted at 230° C. for 30 minutes 0.09 #6 Steamed at 120° C. for 4 hours 0.22 #7 Roasted at 200° C. for 30 minutes & 0.85 Steamed at 120° C. for 4 hours

Example 2-2. Comparison of Changes in Properties and Sensory Evaluation According to Heat Treatment Conditions

[0094] In order to examine changes in the properties and to perform sensory evaluation according to the heat treatment conditions, the color (inside of the epidermis) and smell were examined according to the heat treatment conditions. As a result, when roasting was performed at 230° C., the raw material was burnt, which was confirmed from its color and smell (Table 2).

TABLE-US-00002 TABLE 2 Treatment conditions Color Smell #1 Untreated Yellow - Own unique smell of light yellow Glycyrrhiza sp. root #2 Roasted at 140° C. Yellow - Own unique smell and for 30 minutes light yellow slightly nutty scent #3 Roasted at 170° C. Yellow - Slightly nutty scent for 30 minutes golden yellow #4 Roasted at 200° C. Light brown Nutty scent for 30 minutes #5 Roasted at 230° C. Dark brown - Distinct burnt smell for 30 minutes black #6 Steamed at 120° C. Yellow - Mixed smell of own unique for 4 hours dark brown smell and steaming smell #7 Roasted at 200° C. Dark brown - Mixed smell of nutty scent for 30 minutes & black and steaming smell Steamed at 120° C. for 4 hours

Example 2-3. Comparison of Glycyrrhetinic Acid Contents According to Heat Treatment and Treatment Time Conditions

[0095] In order to observe changes in the content of glycyrrhetinic acid according to the steaming conditions and to determine whether the steaming condition is more favorable in producing glycyrrhetinic acid than the roasting method, the conditions were fixed at 200° C. for 30 minutes, which are strong roasting conditions that do not burn the raw material, and the contents of glycyrrhetinic acid were compared in those only roasted (#4), those steamed at 105° C., 115° C., and 125° C. for 2 hours, 4 hours, and 6 hours (#8-10), and those roasted at 200° C. for 30 minutes and then steamed at 120° C. for 2 hours, 4 hours, and 6 hours (#7) (Table 3).

TABLE-US-00003 TABLE 3 30 minutes 2 hours 4 hours 6 hours Treatment conditions (Roasting) (Steaming) (Steaming) (Steaming) #4 Roasted at 200° C. Not detected for 30 minutes #8 Steamed at 105° C. 0.15 0.18 0.19 #9 Steamed at 115° C. 0.17 0.22 0.25 #10 Steamed at 125° C. 0.23 0.38 0.51 #7 Roasted at 200° C. 0.45 0.85 1.18 for 30 minutes & Steamed at 120° C.

[0096] From the above test results, it was confirmed that more glycyrrhetinic acid was generated under all steaming conditions than under roasting. It was confirmed that when steaming was performed in combination with roasting, a larger amount of glycyrrhetinic acid was generated by supporting the conversion of glycyrrhizin into glycyrrhetinic acid.

[0097] On the other hand, it was confirmed that the range of steaming temperature and time for increasing the generation of glycyrrhetinic acid was wide. Hereinbelow, considering the convenience of the steaming treatment in commercial facilities, roasting was performed at 200° C. for 30 minutes, and then steaming was performed at 120° C. for 4 hours, which are the upper conditions for generating glycyrrhetinic acid, and then antioxidant, whitening, anti-inflammatory, skin revitalization, and regeneration effects were examined.

Example 3. Antioxidant Effect by Scavenging Free Radicals

[0098] In order to examine the antioxidant effect of the Glycyrrhiza sp. extract, 10 g of the Glycyrrhiza sp., which was not treated, steamed at 120° C., or roasted at 200° C. and steamed at 120° C., was extracted with 200 mL of 50% butylene glycol and filtered to prepare each extract.

[0099] In the present disclosure, 1,1-diphenyl-2-picrylhydrazyl (DPPH), which is a relatively stable free radical, and exhibits a maximum absorption at 517 nm when it exists in a radical state, but loses its absorption ability as the radical is scavenged, was used to measure free radical scavenging capacity. DPPH was used after being dissolved in methanol at a concentration of 0.12 mM, and Trolox, which is a water-soluble analogue of vitamin E, was used as a positive control.

[0100] 100 μL of Trolox solutions (0.0125 mg/mL, 0.025 mg/mL, 0.05 mg/mL, 0.1 mg/mL, 0.2 mg/mL, 0.4 mg/mL, and 0.8 mg/mL) at each concentration were put into a 24-well plate, and 1,900 μL of DPPH solution was added to each. The plate was left for 1 hour while blocking the light at room temperature, absorbance at 517 nm was measured using an ELISA reader, and a calibration curve according to the concentration was prepared with absorbance values of the positive control at seven different concentrations. Likewise, 100 μL of the extract and 1,900 μL of DPPH solution were reacted. By substituting the absorbance of the extract into the calibration curve, the degree of the antioxidant effect shown by 1 mL of the extract was expressed as Trolox μmol equivalents per mL, and the experiment was conducted in triplicate, and then the average value was calculated (Table 4).

TABLE-US-00004 TABLE 4 Treatment conditions of raw material Trolox μmol used in preparation of extract equivalents per mL Untreated 0.12 Steamed at 120° C. for 4 hours 0.49 Roasted at 200° C. for 30 minutes & 0.80 Steamed at 120° C. for 4 hours

[0101] As a result, as shown in Table 4, it was confirmed that those treated with steaming, particularly those roasted at 200° C. and steamed at 120° C., showed the excellent antioxidant effect, as compared with those untreated.

Example 4. Whitening Effect by Inhibition of Melanin Production

[0102] In order to examine the whitening effect of the Glycyrrhiza sp. extract, 10 g of the Glycyrrhiza sp., which was not treated, steamed at 120° C., or roasted at 200° C. and steamed at 120° C., was extracted with 200 mL of 50% butylene glycol and filtered to prepare each extract.

[0103] To culture MNT-1 melanoma cells, a mixture of DMEM (Dulbecco's Modified Eagle's Medium) and FBS (Fetal Bovine Serum) was used as a basic medium, and MNT-1 melanoma cells were dispensed in a 6-well plate at a density of 1×10.sup.5 cells/mL to 2×10.sup.5 cells/mL and cultured for 24 hours. Each extract was treated at a concentration of 0.1% in the cell culture solution, and then cultured for 72 hours. As a control group, 50% butylene glycol was used. Arbutin, which is a positive control, was treated at 200 μg/mL (200 ppm). Thereafter, the cells were treated with trypsin, detached from the culture plate, centrifuged at 13,000 rpm for 1 minute, and the supernatant was removed. The remaining cells were subjected to lysis by adding 300 μL of a 0.5% Triton X-100 solution thereto. The cells were again centrifuged at 13,000 rpm for 3 minutes to separately recover the pellet and supernatant. Melanin in the precipitate was dissolved by adding 100 μL of 0.5 N sodium hydroxide solution and incubating for 12 hours, and absorbance at 450 nm was measured using an ELISA reader to measure the total amount of melanin produced. The total amount of melanin, of which production was inhibited as compared to the control group, was determined as the melanin production inhibition rate (%), and the experiment was conducted in triplicate, and then the average value was calculated (Table 5).

TABLE-US-00005 TABLE 5 Treatment conditions of raw material Melanin production used in preparation of extract inhibition rate (%) Arbutin (positive control) 19.7% Untreated 10.6% Steamed at 120° C. for 4 hours 13.1% Roasted at 200° C. for 30 minutes & 18.4% Steamed at 120° C. for 4 hours

[0104] As a result, as shown in Table 5, it was confirmed that those treated with steaming, particularly those roasted at 200° C. and steamed at 120° C., showed the excellent whitening effect, as compared with those untreated.

Example 5. Anti-Inflammatory Effect by Inhibition of NO Production

[0105] In order to examine the anti-inflammatory effect of the Glycyrrhiza sp. extract, 10 g of the Glycyrrhiza sp., which was not treated, steamed at 120° C., or roasted at 200° C. and steamed at 120° C., was extracted with 200 mL of 50% butylene glycol and filtered to prepare each extract.

[0106] To culture Raw264.7 cells, a mixture of DMEM (Dulbecco's Modified Eagle's Medium) and FBS (Fetal Bovine Serum) was used as a basic medium, and Raw264.7 cells were dispensed in a 24-well plate at a density of 1×10.sup.5 cells/mL to 2×10.sup.5 cells/mL and cultured for 24 hours. After removing the medium and starving with a serum-free medium for 12 hours, each extract was treated at a concentration of 1% in the cell culture solution. 30 minutes later, lipopolysaccharide was added at a concentration of 500 ng/mL, followed by culturing for 18 hours. As a control group, 50% butylene glycol was used. L-NMMA, which is a positive control, was treated at 100 μM. After culturing, the supernatant was taken and transferred to a 96-well plate, and GRIESS reagent was added and reacted at room temperature for 15 minutes. Absorbance at 540 nm was measured using an ELISA reader. The total amount of NO, of which production was inhibited as compared to the control group, was determined as the NO production inhibition rate (%), and the experiment was conducted in triplicate, and then the average value was calculated (Table 6).

TABLE-US-00006 TABLE 6 Treatment conditions of raw material NO production used in preparation of extract inhibition rate (%) L-NMMA (positive control) 60.9% Untreated 36.7% Steamed at 120° C. for 4 hours 42.3% Roasted at 200° C. for 30 minutes & 55.8% Steamed at 120° C. for 4 hours

[0107] As a result, as shown in Table 6, it was confirmed that those treated with steaming, particularly those roasted at 200° C. and steamed at 120° C., showed the excellent anti-inflammatory effect, as compared with those untreated.

Example 6. Skin Revitalization and Regeneration Effects by Keratinocyte (HaCaT) Proliferation

[0108] In order to examine the skin revitalization and regeneration effects of the Glycyrrhiza sp. extract, 10 g of the Glycyrrhiza sp., which was not treated, steamed at 120° C., or roasted at 200° C. and steamed at 120° C., was extracted with 200 mL of 50% butylene glycol and filtered to prepare each extract.

[0109] To culture HaCaT cells (human-derived keratinocytes), a mixture of DMEM (Dulbecco's Modified Eagle's Medium) and FBS (Fetal Bovine Serum) was used as a basic medium, and HaCaT cells were dispensed in a 96-well plate at a density of 1×10.sup.4 cells/mL to 2×10.sup.5 cells/mL and cultured for 24 hours. After removing the medium, a serum-free medium, in which the concentration of each extract was 0.5%, was treated, and then cultured for 24 hours. As a control group, 50% butylene glycol was used. A medium containing 5% FBS was used as a positive control. CCK reagent was added and reacted for 30 minutes. Absorbance at 450 nm was measured using an ELISA reader. The experiment was conducted in triplicate, and then the average value was calculated, and the skin revitalization and regeneration effects increased as compared to those of the control group were confirmed (Table 7).

TABLE-US-00007 TABLE 7 Treatment conditions of raw material Proliferation used in preparation of extract (relative %) 5% FBS (positive control) 109.2% Untreated 98.7% Steamed at 120° C. for 4 hours 106.9% Roasted at 200° C. for 30 minutes & 112.3% Steamed at 120° C. for 4 hours

[0110] As a result, as shown in Table 7, it was confirmed that those treated with steaming, particularly those roasted at 200° C. and steamed at 120° C., showed the excellent skin revitalization and regeneration effects, as compared with those untreated.

[0111] Based on the above description, it will be understood by those skilled in the art that the present disclosure may be implemented in a different specific form without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the above embodiment is not limitative, but illustrative in all aspects. The scope of the disclosure is defined by the appended claims rather than by the description preceding them, and therefore all changes and modifications that fall within metes and bounds of the claims, or equivalents of such metes and bounds are therefore intended to be embraced by the claims.