<i>Ganoderma lucidum </i>extract-preparing method using eco-friendly extraction technique, <i>Ganoderma lucidum </i>extract prepared thereby, and anti-aging cosmetic composition comprising same extract

Abstract

A method for preparing Ganoderma lucidum extract using an eco-friendly extraction technique is disclosed. A Ganoderma lucidum extract prepared thereby and a cosmetic composition containing the extract, and their uses are disclosed. The extraction technique use an aqueous cyclodextrin solution alone or in combination with ultrasonic extraction to afford a Ganoderma lucidum extract containing a high concentration of the effective ingredient triterpenoid.

Claims

1. A method for preparing a Ganoderma lucidum extract, the method comprising extracting Ganoderma lucidum with an aqueous cyclodextrin solution to form a clathrate of a triterpenoid component of the Ganoderma lucidum and a cyclodextrin of the aqueous cyclodextrin solution, wherein the Ganoderma lucidum extract contains triterpenoids of Ganoderma lucidum, and wherein the aqueous cyclodextrin solution contains the cyclodextrin in a concentration ranging from 0.01 to 30% by weight based on total volume of the aqueous cyclodextrin solution.

2. The method of claim 1, further comprising an ultrasonic treatment during the extracting step of forming the clathrate of the cyclodextrin and the triterpenoid component of the Ganoderma lucidum.

3. The method of claim 1, wherein the cyclodextrin in the aqueous cyclodextrin solution is a mixture of α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin.

4. The method of claim 1, wherein the aqueous cyclodextrin solution comprises a mixture of βcyclodextrin and γ-cyclodextrin at a weight ratio of 1:1; or a mixture of α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin at a weight ratio of 1:1 to 2:0.5 to 2.

5. The method of claim 1, wherein temperature of the extraction is 15° C. to 100° C.

6. The method of claim 1, wherein duration of the extraction is 10 minutes to 24 hours.

7. The method of claim 2, wherein temperature of the extraction during the ultrasonic treatment is 15° C. to 35° C., and duration of the extraction is 10 minutes to 120 minutes.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows the preparation process of the Ganoderma lucidum extract containing a high concentration of triterpenoids according to the present disclosure.

(2) FIG. 2 is a graph comparing the absorption of triterpenoids contained in Ganoderma lucidum extracts after preparing the Ganoderma lucidum extracts using aqueous cyclodextrin solutions according to various mixing ratios of α-CD, β-CD, and γ-CD.

DETAILED DESCRIPTION OF THE EMBODIMENTS

(3) One embodiment of the present disclosure relates to a method for preparing a Ganoderma lucidum extract containing triterpenoids using an aqueous cyclodextrin solution, comprising the step of adding Ganoderma lucidum containing the triterpenoid component to an aqueous cyclodextrin solution to form a conjugate of the cyclodextrin and the triterpenoid component of the Ganoderma lucidum.

(4) Another embodiment of the present disclosure relates to a method for preparing a Ganoderma lucidum extract containing triterpenoids, comprising the step of forming the conjugate by adding an ultrasonic treatment in the step of forming a conjugate of the cyclodextrin and the triterpenoid component of the Ganoderma lucidum.

(5) Further, the present disclosure relates to a Ganoderma lucidum extract containing triterpenoids prepared by the method for preparing an extract using an aqueous cyclodextrin solution.

(6) The method for preparing a Ganoderma lucidum extract according to the present disclosure is characterized in that the conjugate of the cyclodextrin and the triterpenoid is formed by adding Ganoderma lucidum containing the triterpenoid component to an aqueous cyclodextrin solution.

(7) In addition, the method for preparing a Ganoderma lucidum extract according to the present disclosure can further increase the formation efficiency of the conjugate by adding an aqueous cyclodextrin solution to Ganoderma lucidum and extracting the same in combination with ultrasonic treatment.

(8) In the extraction of the active ingredient of Ganoderma lucidum, the present disclosure has a feature of using an aqueous cyclodextrin solution instead of an organic solvent. Preferably, water at room temperature or hot water containing an appropriate concentration of cyclodextrin may be used.

(9) As the Ganoderma lucidum containing the triterpenoid component, Ganoderma lucidum, Ganoderma sinensis, Ganoderma atrum, Ganoderma neojaponicum, and the like may be used, but is not limited thereto, and lingzhi mushroom, commercially available at herbal shops, can be used as raw materials.

(10) The cyclodextrin used in the aqueous cyclodextrin solution according to the present disclosure may be α-cyclodextrin, β-cyclodextrin, or γ-cyclodextrin, and may be a cyclodextrin derivative or a polymer of cyclodextrin. It may also be a mixture of two or more selected from the above-listed cyclodextrins, cyclodextrin derivatives, and cyclodextrin polymers, and the concentration of the triterpenoid component contained in the Ganoderma lucidum extract may vary depending on the combination and mixing ratio thereof.

(11) As the cyclodextrin in the aqueous cyclodextrin solution, α-cyclodextrin, β-cyclodextrin, or γ-cyclodextrin may be used, and (2-Hydroxypropyl)-α-cyclodextrin (molecular weight of about 1180, CAS 128446-33-3), (2-Hydroxypropyl)-β-cyclodextrin (molecular weight of about 1400, CAS 128446-35-5), (2-Hydroxypropyl)-γ-cyclodextrin (molecular weight of about 1580, CAS 128446-34-4), methyl-β-cyclodextrin (molecular weight of about 1310, CAS 128446-36-6) or the like may be used. In addition, one or more selected from the group consisting of polymers of the cyclodextrins, polymers of the cyclodextrin derivatives, or polymer of the cyclodextrins and the cyclodextrin derivatives may be used.

(12) In the aqueous cyclodextrin solution, when α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, hydroxypropyl α-cyclodextrin, hydroxypropyl β-cyclodextrin, hydroxypropyl γ-cyclodextrin, or methyl β-cyclodextrin is used alone, it is not preferable to use α-cyclodextrin alone, but it is preferable to use β-cyclodextrin, γ-cyclodextrin, hydroxypropyl β-cyclodextrin, hydroxypropyl γ-cyclodextrin, or methyl β-cyclodextrin.

(13) Further, in the aqueous cyclodextrin solution, it is preferable to use a mixture of β-cyclodextrin and γ-cyclodextrin in a weight ratio of 1:1 rather than using α-cyclodextrin, β-cyclodextrin, or γ-cyclodextrin alone.

(14) In addition, in the aqueous cyclodextrin solution, when three types of α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin are mixed, the mixing ratio of α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin is preferably in a weight ratio of 1:1 to 2:0.5 to 2, and it is most preferable that the mixing ratio of α-cyclodextrin, β-cyclodextrin and γ-cyclodextrin is in a weight ratio of 1:1:1. However, all types of cyclodextrins mentioned above may be used, and the materials used in the preparation method of the present disclosure are not limited thereto.

(15) In the step of forming a conjugate of the cyclodextrin and the triterpenoid of the Ganoderma lucidum, the concentration of cyclodextrin in the aqueous cyclodextrin solution is preferably 0.01 to 30% (weight/volume), more preferably 0.5 to 5% (weight/volume), relative to the total volume of the aqueous solution. If the concentration of the aqueous cyclodextrin solution is less than 0.01% (weight/volume), the binding rate may be significantly reduced, which may cause in a reduction in extraction efficiency, and if the concentration exceeds 30% (weight/volume), the extraction efficiency may no longer increase and become saturated.

(16) The extraction time for forming the conjugate is preferably 10 minutes to 24 hours. If it is less than 10 minutes, the extraction efficiency may be reduced, and even if the extraction time exceeds 24 hours, the extraction efficiency may not increase in proportion to time and may become almost saturated, and thus, it is preferable not to exceed 24 hours in terms of cost. The more preferable extraction time for forming the conjugate is 30 minutes to 240 minutes. In particular, the extraction temperature for forming the conjugate is preferably 15° C. to 100° C.

(17) When an ultrasonic extraction is to be performed in combination during the formation process of the conjugate, the ultrasonic (20 kHz to 25 MHz) extraction is preferably performed at 15° C. to 35° C. for 10 minutes to 120 minutes, and most preferably at 15° C. to 35° C. for 15 minutes to 60 minutes.

(18) The present disclosure relates to a cosmetic composition containing a Ganoderma lucidum extract containing a high concentration of triterpenoids, as an active ingredient, prepared by the extraction method using the aqueous cyclodextrin solution.

(19) The cosmetic composition according to the present disclosure may contain the Ganoderma lucidum extract containing triterpenoids in an amount of 0.0001% by weight to 30% by weight, preferably 0.001% by weight to 10% by weight, based on the total weight of the composition, and most preferably 0.01% by weight to 1% by weight.

(20) The present disclosure relates to the use of the cosmetic composition containing the Ganoderma lucidum extract containing triterpenoids in reducing skin wrinkles and increasing skin elasticity, and a method for reducing skin wrinkles and a method for increasing skin elasticity using the composition.

(21) The cosmetic composition according to the present disclosure contains the triterpenoid component in a high concentration, thereby increasing anti-aging effects such as suppression of MMP-1 activity and inhibition of collagenase, and accordingly, it can be used to reduce skin wrinkles and increase skin elasticity.

(22) The cosmetic composition according to the present disclosure may be formulated into softening cosmetic water, astringent cosmetic water, nourishing cosmetic water, nourishing cream, massage cream, essence, eye cream, eye essence, cleansing cream, cleansing foam, cleansing water, facial pack, powder, body lotion, shampoo, conditioner, body wash, tooth paste or mouth wash, and used in cosmetics.

MODE FOR CARRYING OUT THE INVENTION

(23) Hereinafter, the present disclosure will be described by way of Example and Test Examples. However, these Examples and Test Examples are given for illustrative purposes only to help understanding of the present disclosure, and the scope of the present disclosure is not limited by these examples.

TEST EXAMPLE 1

Extraction Efficiency of Triterpenoids in Ganoderma lucidum Using Cyclodextrin

(24) In this test, the extraction using an aqueous cyclodextrin solution was carried out as follows: To 200 ml of an aqueous solution, which was prepared by dissolving α-cyclodextrin (α-CD), β-cyclodextrin (β-CD), hydroxypropyl beta cyclodextrin (HP-β-CD), or γ-cyclodextrin (γ-CD) at a concentration of 1% (weight/volume) in water was added 10 g of pulverized Ganoderma lucidum, and the mixture was extracted at 95° C. for 60 minutes. Thereby, extracts of Examples 1 to 4 were obtained, and the extracts were used for analysis.

(25) The extraction using water (distilled water) as a control was performed in the same order except that the process of dissolving cyclodextrin in water was omitted in the extraction process using the aqueous cyclodextrin solution. 10 g of Ganoderma lucidum was suspended in 200 ml of water, and then extracted under the same reaction conditions and time as the extraction process using the aqueous cyclodextrin solution to obtain an extract of Comparative Example 1, which was used for analysis.

(26) In addition, the aqueous cyclodextrin solution-ultrasonic extraction used in this test utilizes the extraction using the aqueous cyclodextrin solution in combination with ultrasonic extraction, and the ultrasonic extraction was carried out using an ultrasonic extractor (Branson Ultrasonics Sonifier™ S-450D) at room temperature (20° C.) at 60 KHz for 15 minutes.

(27) The obtained extracts were filtered through a 0.45 μm filter, and then used as HPLC samples and analyzed by HPLC-PDA system. HPLC analysis was carried out using Waters Alliance system (model 2965) and photodiode array (PDA, model 2996). Luna® 5 μm C18 (2) (100 Å, LC column 250×4.6 mm) was used as analytical columns, and water and acetonitrile were used as mobile phase.

(28) The results are shown in Table 1.

(29) TABLE-US-00001 TABLE 1 Aqueous Aqueous Cyclodextrin Cyclodextrin Solution * Solution Ultrasonic Assisted Extraction extraction Relative Relative to to Area Control Area Control Comparative Water 318486 100% 281655 100% Example 1 (Distilled water) Example 1 1% α-CD 290482  94% 321539 114% Aqueous solution Example 2 1% β-CD 416196 131% 466576 166% Aqueous solution Example 3 1% HP-β-CD 425589 134% 410915 146% Aqueous solution Example 4 1% γ-CD 438854 138% 456253 162% Aqueous solution

(30) As shown in Table 1, when the extraction method using the aqueous cyclodextrin solution according to the present disclosure was used, it was confirmed that the Ganoderma lucidum extract containing a high concentration of triterpenoids, which is an active ingredient of Ganoderma lucidum, was obtained. In the case of Examples 2 to 4 in which the extraction was carried out using 1% β-CD aqueous solution, 1% HP-β-CD aqueous solution, or 1% γ-CD aqueous solution, the concentration of triterpenoids (ganoderic acid, etc.) extracted from the Ganoderma lucidum was increased by about 30 to 40% compared to when using the conventional water extraction method. However, in the case of Example 1 in which the extraction was carried out using 1% α-CD aqueous solution, the concentration of triterpenoids was rather decreased compared to Comparative Example 1 using the simple water extraction.

(31) In addition, in the case of the extraction technique in which the extraction method using the aqueous cyclodextrin solution according to the present disclosure was combined with ultrasonic extraction, the concentration of triterpenoids extracted from Ganoderma lucidum was increased in all cases of 1% α-CD aqueous solution, 1% β-CD aqueous solution, 1% HP-β-CD aqueous solution, and 1% γ-CD aqueous solution (Examples 1 to 4 in combination with ultrasonic extraction) compared to the control group (Comparative Example 1 in combination with ultrasonic extraction). In particular, when the extraction using 1% β-CD aqueous solution or 1% γ-CD aqueous solution was combined with ultrasonic extraction, the extraction concentration of triterpenoids was increased by 60% or more compared to the control group, thereby showing excellent extraction efficiency.

TEST EXAMPLE 2

Extraction Efficiency of Triterpenoid in Ganoderma lucidum According to Types and Mixed Use of Cyclodextrins

(32) In this test, the extraction method using the aqueous cyclodextrin solution was performed in combination with ultrasonic extraction. As the aqueous cyclodextrin solution, an aqueous solution prepared by diluting cyclodextrin to 1% (weight/volume) in water was used, and in particular, the cyclodextrin was one selected from the group consisting of α-CD, β-CD, or γ-CD, or a mixture of two or more thereof. Thereafter, 10 g of pulverized Ganoderma lucidum was added to 200 ml of the aqueous solution, followed by ultrasonic extraction to obtain an extract, which was filtered and used for analysis. The ultrasonic extraction was performed at room temperature (20° C.) at 40 KHz for 30 minutes using an ultrasonic extractor (Branson Ultrasonics CPX952838R).

(33) The extraction using water (distilled water) as a control was performed in the same order except that the process of dissolving cyclodextrin in water was omitted in the extraction process using the aqueous cyclodextrin solution. 10 g of Ganoderma lucidum was suspended in 200 ml of water, and then extracted under the same reaction conditions and time as the extraction process using the aqueous cyclodextrin solution to obtain an extract, which was used for analysis.

(34) The extracts were allowed to react with 5% valine-acetic acid and perchloric acid and then analyzed by measuring the absorbance of triterpenoids at 550 nm using Biotek™ Synergy 2.

(35) The results are shown in Table 2 and FIG. 2.

(36) TABLE-US-00002 TABLE 2 Composition Cyclodextrin 1% Mixing ratio Absor- No. α-CD β-CD γ-CD bance  1 Comparative  0.0%  0.0%  0.0% 0.0270 Example 1  2 Example 1 100.0%  0.0%  0.0% 0.0227  3 Example 2  0.0% 100.0%  0.0% 0.0383  4 Example 4  0.0%  0.0% 100.0% 0.0383  5 Example 5  66.7%  0.0%  33.3% 0.0367  6 Example 6  50.0%  0.0%  50.0% 0.0273  7 Example 7  33.3%  0.0%  66.7% 0.0333  8 Example 8  0.0%  66.7%  33.3% 0.0350  9 Example 9  0.0%  50.0%  50.0% 0.0397 10 Example 10  0.0%  33.3%  66.7% 0.0377 11 Example 11  50.0%  25.0%  25.0% 0.0337 12 Example 12  40.0%  40.0%  20.0% 0.0387 13 Example 13  40.0%  20.0%  40.0% 0.0303 14 Example 14  33.3%  33.3%  33.3% 0.0407 15 Example 15  25.0%  50.0%  25.0% 0.0393 16 Example 16  25.0%  25.0%  50.0% 0.0380 17 Example 17  20.0%  40.0%  40.0% 0.0390

(37) As shown in Table 2 and FIG. 2, when the extraction was carried out using 1% aqueous cyclodextrin solution, it was confirmed that the absorbance was found to be high in all Examples except for using α-CD alone compared to Comparative Example 1 using the simple water extraction method. That is, in the case of Example 2 and Examples 4 to 17, the concentration of triterpenoids extracted from Ganoderma lucidum was higher than that of Comparative Example 1, and accordingly, high extraction efficiency was confirmed.

(38) In addition, in the 1% aqueous cyclodextrin solution, a higher absorbance was observed when two types of β-CD and γ-CD were mixed at a mixing ratio of 1:1 or when three types of α-CD, β-CD, and γ-CD were mixed at a predetermined ratio, compared to when α-CD, β-CD, or γ-CD was used alone, resulting in a higher extraction concentration of triterpenoids from Ganoderma lucidum. In particular, in the case of in which three types of α-CD, β-CD, and γ-CD were mixed and used, a higher absorbance was observed when the mixture of α-CD, β-CD, and γ-CD was used in a mixing ratio of 1:1 to 2:0.5 to 2 compared to when they were used alone, and the absorbance was found to be the highest when α-CD, β-CD, and γ-CD were mixed and used in a mixing ratio of 1:1:1, confirming that triterpenoids could be extracted at the highest concentration.

TEST EXAMPLE 3

MMP-1 Inhibitory Effect of Ganoderma lucidum Extracts Extracted with Aqueous Cyclodextrin Solution

(39) In order to confirm the inhibitory effect of Ganoderma lucidum extracts extracted with the aqueous cyclodextrin solution according to the present disclosure on matrix metalloprotease-1 (MMP-1) expression increased by ultraviolet rays, fibroblasts cells (HS68) were seeded at a concentration of 0.75*10.sup.5 in each well of a 12 well plate and made to be a starvation state for 24 hours, then washed with phosphate buffered saline and irradiated with UV (30 mJ). Subsequently, as a positive control, 1, 10, and 50 ppm of Ganoderma lucidum extracts extracted with 5 μM of retinoic Acid (RA) and an aqueous cyclodextrin solution were treated twice for 48 hours, and the amount of free MMP-1 in the media was measured using a kit (Amersham, RPN2610).

(40) The Ganoderma lucidum extracts were those extracted by using the 1% β-CD aqueous cyclodextrin solution in combination with the ultrasonic extraction, and the data values were corrected with the control that does not contain a test substance. The results are shown in Table 3.

(41) TABLE-US-00003 TABLE 3 Extracellular MMP-1 Concentration (% of control) Control   0% Positive control  5 μM 66.2% (Retinoic acid) Ganoderma lucidum extract 50 ppm 37.1% extracted using water (distilled water) Ganoderma lucidum  1 ppm 26.5% extracts extracted using 10 ppm 47.1% aqueous cyclodextrin 50 ppm 51.3% solution (1% β-CD aqueous solution * Ultrasonic Assisted extraction)

(42) As shown in Table 3, the Ganoderma lucidum extracts extracted using the aqueous cyclodextrin solution according to the present disclosure inhibited the expression of MMP-1 with increasing concentration. In addition, the Ganoderma lucidum extracts showed excellent MMP-1 expression inhibitory effect compared to the Ganoderma lucidum extract extracted with water without using the aqueous cyclodextrin solution.

TEST EXAMPLE 4

Collagenase Inhibitory Effect of Ganoderma lucidum Extracts Extracted with Aqueous Cyclodextrin Solution

(43) The inhibitory ability of Ganoderma lucidum extracts extracted with the aqueous cyclodextrin solution according to the present disclosure on collagenase production was measured in comparison with retinoic acid. Human fibroblasts were added at 5,000 cells/well in a 96-well microtiter plate containing Dulbecco's Modified Eagle's Media (DMEM) medium containing 2.5% fetal bovine serum and incubated in an incubator (5% CO.sub.2, 37° C.) until the growth reached 70 to 80%. Then, the Ganoderma lucidum extracts, which were extracted using the 1% β-CD aqueous cyclodextrin solution in combination with ultrasonic extraction, were treated with the cells at a concentration of 100 μg/ml for 24 hours, and then the cell culture solution was collected.

(44) Then, the degree of collagenase production in the thus-collected cell culture solution was measured using a commercially available collagenase measuring instrument (GE Healthcare Life Sciences). First, the collected cell culture solution was placed in a 96-well plate uniformly coated with primary collagenase antibodies, and the antigen-antibody reaction was performed in a constant-temperature bath for 3 hours. After 3 hours, chromophore-conjugated secondary collagen antibodies were placed in the 96-well plate and allowed to react again for 15 minutes. After 15 minutes, a coloring stimulant was added to cause color development at room temperature for 15 minutes, and when 1M sulfuric acid was added again to stop the reaction (color development), the color of the reaction solution became yellow, and the degree of yellow color varied according to the progress of the reaction.

(45) The absorbance of the yellowish 96-well plate was measured at 405 nm using an absorptiometer, and the expression level of collagenase was calculated by Calculation Equation 1 below. In particular, the reaction absorbance of the collected cell culture solution in the untreated group was taken as the absorbance of the control group.

(46) Collagenase expression level (%)=A/B×100

(47) A: Absorbance of the test substance-treated cell group

(48) B: Absorbance of control group

(49) Meanwhile, the results of measuring the collagenase expression inhibitory effect of the test substances in the human fibroblasts are shown in Table 4 below as the degree of collagenase expression, which were compared to the degree of collagenase expression of the untreated group as 100.

(50) TABLE-US-00004 TABLE 4 Degree of Collagenase Expression Concentration (%) Control (Untreated group)  100% Positive control  5 μM 75.0% (Retinoic acid) Ganoderma lucidum extract 50 ppm 83.7% extracted using water (distilled water) Ganoderma lucidum extracts  1 ppm 93.5% extracted using aqueous 10 ppm 82.7% cyclodextrin solution in 50 ppm 74.0% combination with ultrasonic extraction (1% β-CD aqueous solution * Ultrasonic Assisted extraction)

(51) As shown in Table 4, the Ganoderma lucidum extracts extracted with the aqueous cyclodextrin solution according to the present disclosure effectively inhibited collagenase expression. In particular, the Ganoderma lucidum extracts showed higher collagenase expression inhibitory effect than the Ganoderma lucidum extract extracted with water without using the aqueous cyclodextrin solution.

(52) From the results of Test Examples 1 and 2, it was confirmed that the Ganoderma lucidum extracts prepared according to the method for preparing an extract of the present disclosure contained a high concentration of the triterpenoid component. In addition, from the results of Test Examples 3 and 4, it was confirmed that the Ganoderma lucidum extracts prepared according to the present disclosure showed excellent MMP-1 and collagenase inhibitory effects. Accordingly, it can be seen that the Ganoderma lucidum extracts containing a high concentration of triterpenoids prepared according to the method for preparing an extract of the present disclosure have an anti-aging effect and thus can be used for reducing skin wrinkles and increasing skin elasticity.