METHOD FOR EXTRACTING EXTRACT OF LIQUOR PRODUCTION BY-PRODUCT AND COMPOSITION FOR WHITENING, WRINKLE IMPROVEMENT AND ANTI-AGING EFFECT COMPRISING THE EXTRACT THEREBY

Abstract

Provided are a method for extracting an extract of liquor production by-products and a composition for whitening, wrinkle improvement and anti-aging effects, which includes the extract obtained by the above method. The extract of liquor production by-products according to the above method has excellent whitening, wrinkle improvement or anti-aging effects and thus can be used as a material for cosmetics and the like.

Claims

1. A method for extracting an extract of a liquor production by-product, wherein the liquor production by-product is a by-product in production of alcoholic beverages, comprising: (1) drying the liquor production by-product; (2) crushing the by-product dried in (1); (3) fermenting the by-product crushed in (2) with any one or more strains of Weissella cibaria, Issatchenkia orientalis or Saccharomyces cerevisiae; and (4) extracting the by-product fermented in (3).

2. The method according to claim 1, wherein the liquor production by-product in (1) is a by-product remaining after production of fermented liquor.

3. The method according to claim 1, wherein, in the drying of the (1), a drying temperature ranges from 15 to 35° C., and a drying time ranges from 24 to 48 hours.

4. The method according to claim 1, wherein the by-product crushed in the crushing of (2) has a particle size of 0.1 to 3 mm.

5. The method according to claim 1, wherein an amount of the strain used in the fermentation of (3) ranges from 0.005 to 3% by weight based on a total weight including the by-product and an extraction solvent.

6. The method according to claim 1, wherein, in the fermentation of (3), the by-product is supplemented with sugar in an amount of 0.1 to 20% by weight based on a total weight of the by-product.

7. The method according to claim 1, wherein the fermentation of (3) includes fermenting at 25 to 50° C. for 24 to 72 hours.

8. The method according to claim 1, wherein the extraction of the (4) includes extracting with water or 70% (v/v) ethanol.

9. The method according to claim 1, further comprising (5) filtering the extract obtained through the extraction of (4), and then concentrating the same.

10. The method according to claim 9, wherein the filtered extract is concentrated at 45 to 65° C. to reach 5 to 60 brix°.

11. The method according to claim 9, further comprising (6) powdering the extract concentrated solution.

12. An extract of liquor production by-products, produced by the method according to claim 1.

13. A composition for whitening, wrinkle improvement or anti-aging effects, comprising the extract according to claim 12.

14. A method for manufacturing a scrub composition, comprising: (1) drying a liquor production by-product; (2) crushing the by-product dried in (1); (3) fermenting the by-product crushed in (2) with any one or more strains of Weissella cibaria, Issatchenkia orientalis or Saccharomyces cerevisiae; (4) extracting the by-product fermented in (3); and (5) pulverizing the remaining by-product after extracting in (4) to a particle size of 100 to 500 .Math.m.

15. A scrub composition manufactured by the method according to claim 14.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0047] FIG. 1 illustrates a cell culture process.

[0048] FIG. 2 illustrates an MTT assay measurement process for measuring cytotoxicity.

[0049] FIG. 3 shows results of the cytotoxicity test.

[0050] FIG. 4 illustrates a process of measuring the tyrosinase inhibitory activity.

[0051] FIG. 5 shows measurement results of the tyrosinase inhibitory activity according to the present invention.

[0052] FIG. 6 illustrates a process of measuring the inhibition of melanin production.

[0053] FIG. 7 shows measurement results of the inhibition of melanin production according to the present invention.

[0054] FIGS. 8A and 8B show evaluation results of reactive oxygen species inhibitory effects on B16F10 melanocytes and CCRF S-180II fibroblasts according to the present invention.

[0055] FIGS. 9A and 9B show wrinkle improvement effects based on the expression levels of MMP1 and MMP8 according to the present invention.

MODE FOR CARRYING OUT INVENTION

[0056] Hereinafter, the present invention will be described in detail by way of examples and experimental examples.

[0057] However, the following examples and experimental examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following examples and experimental examples.

Examples 1 to 3, and Comparative Examples 1 to 3

[0058] In an embodiment of the present invention, wine juice solids, which are by-products of wine production, were used. The by-products were dried at 15 to 35° C. for 24 to 48 hours, and the dried by-products were crushed to a particle size of 2 mm or less. Thereafter, using sucrose or glucose, sugar was supplemented in an amount of 10% by weight based on the total weight of the by-product.

[0059] Weissella cibaria strain in Example 1, Issatchenkia orientalis strain in Example 2, and Saccharomyces cerevisiae strain in Example 3, respectively, were used for treatment of the crushed by-products. Then, fermentation was performed by adding distilled water 10 times the volume of the by-products, followed by stationary culture or shaking culture at 25 to 40° C. for 24 to 72 hours.

[0060] The amount of strain used herein was 0.02% by weight based on the total weight including the by-products and water.

[0061] Comparative Example 1 is a normal group without any treatment, Comparative Example 2 is a negative control group treated with a stimulant, and Comparative Example 3 is a group treated with normal grape extract. After adding distilled water 5 times the volume of the by-product to the fermented by-products, the extraction process was performed at room temperature for 24 hours in order to yield an extract.

TABLE-US-00001 Comparative Example 1 Comparative Example 2 Comparative Example 3 Example 1 Example 2 Example 3 Untreated group Negative control group (stimulant treatment) General grape extract treatment Weissella cibaria treatment Issatchenkia orientalis treatment Saccharomyc es cerevisiae treatment

Experimental Example 1 Measurement of Cytotoxicity of Fermented Wine Extract

1-1. Cell Culture

[0062] Cells used herein are mouse melanoma B16F10 cells, which were purchased from the Korea Cell Line Bank (KCLB), and the medium used herein is DMEM (Dulbecco’s Modified Eagle Medium, gibco, USA) supplemented with 10% FBS (fetal bovine serum, gibco, USA), sodium bicarbonate (Sigma, USA) and 1% Penicilin. Specifically, the cells were cultured using the above medium in an incubator under 37° C. and 5% CO.sub.2 conditions, and the culture process is shown in FIG. 1.

1-2. Cytotoxicity Measurement

[0063] In order to confirm the cytotoxicity of a sample to cells, the strains in Examples 1 to 3 and Comparative Example 3 were used for treatment at concentrations of 400, 500 and 800 ppm, respectively. Thereafter, 70% (v/v) ethanol of 5 times the volume of the fermented wine juice solids was added, followed by extraction for 24 hours at room temperature and measurement by MTT assay. The specific method is as follows.

[0064] Mouse melanoma B16F10 cells were dispensed by 100 .Math.L in a 96-well plate using DMEM medium containing 10% FBS so as to reach 5 .sub.X 10.sup.3 cells/well, and cultured in an incubator under 37° C. and 5% CO.sub.2 conditions. After culturing for 24 hours for cell adhesion, the cells were treated with 150 .Math.L of each diluted sample in the medium supplemented with 5% FBS, followed by exchanging the medium. Then, after 3 hours, 2 nM α-MSH stimulant was supplied. After 72 hours from incubation, each well was treated with 7.5 .Math.L of 5 mg/mL MTT reagent and the medium was removed after 3 hours, followed by adding 100 .Math.L of DMSO to measure absorbance at 570/630 nm (FIG. 2). Cytotoxicity was indicated by the percentage as compared to the control, and comparison results are shown in FIG. 3.

[0065] As a result of the experiment, all of the treatments using the strains in Comparative Examples 2 and 3 and Examples 1 to 3, respectively, exhibited similar cytotoxicity to the normal group. Therefore, it is determined that treatment with the strains in Examples 1 to 3 and application of the same to a human body are not harmful.

Experimental Example 2 Evaluation of Whitening Effects Through Measurement of Tyrosinase Inhibitory Activity

[0066] In order to evaluate the whitening effects of the inventive composition, the strains in Comparative Example 3, Examples 1 to 3 and Comparative Examples 1 and 2 were used for treatment at 400 and 800 ppm, respectively, to measure tyrosinase inhibitory activity, and the specific method is as follows.

[0067] In order to evaluate the tyrosinase inhibitory activity, 100 .Math.L of mouse melanoma B16F10 cells were dispensed in a 96-well plate using a medium containing 10% FBS to reach 5 .sub.X 10.sup.3 cells per well, and cultured in an incubator at 37° C., 5% CO.sub.2 for 24 hours.

[0068] Thereafter, the cells were treated with 150 .Math.L of each sample diluted using a medium supplemented with 5% FBS, followed by exchanging the medium. Then, after 3 hours, 2 nM α-MSH stimulant was supplied, and the cells were cultured in an incubator at 37° C., 5% CO.sub.2 for 3 days. After incubation, the medium was removed and the product was treated with 25 .Math.L of lysis buffer containing 1% sodium deoxycholate, Triton X-100, and 100 mM PMSF, followed by shaking for 30 minutes.

[0069] After 30 minutes, Solution A (2% N,N-dimethyl formamide in 100 mM sodium phosphate), Solution B (5 mM L-DOPA in 100 mM sodium phosphate) and Solution C (20 mM MBTH in H2O) were mixed at a ratio of 2:1:1 to produce a mix buffer, and then 140 .Math.L of the mix buffer was added to each well to induce a reaction, followed by measurement of absorbance at 505 nm, and measurement results are shown in Table 2 below and FIG. 5.

TABLE-US-00002 Comparative Example 1 Comparative Example 2 Example 1 Example 2 Example 3 Fermented strain - (normal group) Negative control group (stimulant treatment) Weissella cibaria Issatchenkia orientalis Saccharomyces cerevisiae Treatment concentration (ppm) - 400 800 400 800 400 800 400 800 Tyrosinase activity (%) 100 576 638 609 640 281 225 468 399

[0070] From the result of the experiment, it could be confirmed that, as a result of treatment in Examples 1 to 3 of the present invention after introduction of the stimulant to B16F10 melanoma cells, the tyrosinase activity was inhibited in Examples 1 to 3. In particular, when Example 2 was used for treatment at a concentration of 800 ppm, the tyrosinase activity was inhibited and measured to about ⅓ level as compared to the negative control group in Comparative Example 2 at the same concentration, therefore, the remarkable tyrosinase inhibitory activity of the present invention could be confirmed.

Experimental Example 3 Evaluation of Whitening Effects Through Measurement of Melanin Production Inhibitory Ability

[0071] In order to investigate the melanin production inhibitory ability, mouse melanoma B16F10 cells were dispensed in a 6-well plate to reach to 1.sub.X10.sup.5 cells, and cultured in an incubator at 37° C., 5% CO.sub.2 for 24 hours. Then, after removing the culture medium, 2 mL of a medium in which the products of Examples 1 to 3 and Comparative Examples 1 to 3 were introduced at 40 ppm and 80 ppm, respectively, was added, followed by culturing for 3 hours. Thereafter, the product was treated with 2 nM α-MSH stimulant and cultured for 3 hours. This process is shown in FIG. 6.

[0072] After removing the culture medium, the product was washed with PBS, scrapped and centrifuged at 4° C. and 14,000 rpm for 20 minutes. Then, 50 .Math.L of 1 N NaOH solution was added to the pellets, followed by a reaction at 60° C. for 1 hour and measurement of absorbance at 405 nm. Measurement results are shown in FIG. 7.

[0073] As a result of the experiment, in the case of Comparative Example 2, 126% of melanin was produced compared to Comparative Example 1. Further, as compared to Comparative Example 1, 130% of melanin was produced when treated with Comparative Example 3 at 40 ppm, while 120 % of melanin was produced when treated with the same at 80 ppm. Further, Examples 1 to 3 showed a lower melanin production rate than Comparative Examples 1 to 3. In particular, when Example 2 was used for treatment at 40 ppm, the melanin production rate was higher than that of Comparative Example 1 but significantly lower than those of other comparative examples as well as examples. Further, when treated at 80 ppm, 81% of melanin was generated compared to Comparative Example 1, therefore, it was confirmed that the melanin production was significantly inhibited at a level of 46% compared to Comparative Example 2, and at a level of about 20% compared to Comparative Example 1 of the untreated group, respectively.

Experimental Example 4 Evaluation of Anti-Aging Effects

[0074] In order to evaluate the anti-aging effects of the inventive composition, active oxygen was induced with H.sub.2O.sub.2, and an experiment for the inhibitory effect to the generation of active oxygen was conducted. The specific experimental method is as follows.

[0075] B16F10 melanocytes and CCRF S-180II cells were dispensed in a 96-well plate to reach to 1×10.sup.4 cells/well, and cultured in an incubator at 37° C., 5% CO.sub.2 for 24 hours. The culture product was treated with samples diluted by concentration and cultured for 12 hours. Then, after removing the culture medium, the product was washed with PBS, treated with 10 .Math.M DCFDA solution, and cultured for 30 minutes.

[0076] Thereafter, the cells were treated with 1 mM H.sub.2O.sub.2 and cultured for 3 hours, followed by measuring a degree of intracellular ROS generation using a fluorescence analyzer (Excitation 485 nm/Emission 530 nm).

[0077] The present experiment was implemented by treatment using Comparative Examples 1 to 3 and Examples 1 to 3 at a concentration of 200, 400 ppm, or 400, 800 ppm, respectively.

[0078] As a result of examining the active oxygen inhibitory effect of the examples on melanocytes and fibroblasts, it was found that the active oxygen increased by H.sub.2O.sub.2 stimulation was significantly reduced (FIGS. 8A and 8B).

Experimental Example 5 Evaluation of Wrinkle Improvement Effects

[0079] In order to evaluate the wrinkle improvement effects of the inventive composition, Examples 1 to 3 and Comparative Examples 1 to 3 were used for treatment at a concentration of 400 ppm, respectively, to measure the expression levels of MMP1 and MMP8, thereby determining synthesis inhibitory effects. MMP1 and MMP8 are a type of matrix metalloproteinase (MMP) known to act on skin aging by decomposing collagen with collagenase, and specific experimental methods for the above description are as follows.

Rt-PCR

[0080] CCRF S-180II cells were dispensed in a 6-well plate to reach 1×10.sup.5 cells/well and cultured in an incubator at 37° C., 5% CO.sub.2. The cells were treated with samples diluted by concentration and cultured for 12 hours, followed by treatment with 1 mM H.sub.2O.sub.2 and culturing the same. After removing the culture medium and washing the product with PBS, total RNAs were isolated/extracted by treatment with 1 ml of trizol, dissolved in DEPC water, followed by measuring a concentration at 260 nm.

[0081] The extracted total RNAs were reacted with 10 mM dNTP mixture and oligo dT in a thermo cycler at 65° C. (PC-320, ASTEC, Japan) for 5 minutes. Then, a reaction mixture, in which 10×RT buffer, 20 mM MgC12, 0.1MDTT and RNaseout were introduced, was added, followed by a reaction at 42° C. for 2 minutes. Subsequently, Superscript III was added, followed by further reaction at 42° C. for 50 minutes and 70° C. for 15 minutes so as to synthesize cDNA. To this product, RNase H was added in the same amount as Superscript III in order to remove the remaining RNA, and the synthesized cDNA was stored at -20° C. and used for PCR experiments. The reaction mixture for PCR reaction was prepared by adding MQ-water, 10X PCR buffer containing 15 mM MgC12, sense-primer, antisense-primer, 2.5 mM dNTP mixture and Taq polymerase. After adding the reaction mixture to cDNA and mixing the same, it was amplified using a thermocycler. PCR products were subjected to electrophoresis on agarose gel (Mupid-2plus, Advance, Japan) and then comparative analysis using GelDoc-It imaging system (UVP, USA) and image acquisition & analysis software (UVP, USA).

(2) Western Blot

[0082] CCRF S-180II cells were dispensed in a 6-well plate to reach 1×10.sup.5 cells/well and cultured in an incubator at 37° C., 5% CO.sub.2 for 24 hours. The cells were treated with samples diluted by concentration and cultured for 12 hours, followed by treatment with 1 mM H.sub.2O.sub.2 and culturing the sample. After removing the culture medium, the product was washed with PBS and scrapped, followed by adding RIPA lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, pH 7.4, 1 mM PMSF and protease inhibitors) thereto. Cells were lysed with lysis buffer, centrifuged at 4° C. and 14,000 rpm for 20 minutes, and then the supernatant was quantified for protein by BCA (bicinchoninic acid) protein assay (pierce BCA protein assay kit, Thermo), followed by separation of the protein through SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel) electrophoresis.

[0083] The separated protein was moved to a polyvinylidene difluoride (PVDF) membrane and blocked (5% skim milk in 1×TBST) for 1 hour. Primary antibody (1:1000) was added and left overnight at 4° C., and then washed 3 times with 1×TBST for 15 minutes. Following this, secondary antibody (1:2000) was added to react at room temperature for 2 hours. By exposing fluorescence, which was expressed by a reaction with ECL solution in a darkroom, to an X-ray film, respective bands were identified and confirmed.

[0084] As a result of monitoring the effects of the above examples on the expression of MMP1 and MMP8 due to H.sub.2O.sub.2 stimulation in CCRF S-180 II cells, it was confirmed that the present examples could significantly inhibit MMP1 and MMP8 expression as compared to not only the negative control group treated with H2O2 but also the sample-untreated group (FIGS. 9A and 9B).