Composition for improving skin, containing pomegranate concentrate as active ingredient
09901536 ยท 2018-02-27
Assignee
Inventors
Cpc classification
A61K8/498
HUMAN NECESSITIES
A23L33/105
HUMAN NECESSITIES
A61K2800/85
HUMAN NECESSITIES
A23V2002/00
HUMAN NECESSITIES
A61P17/16
HUMAN NECESSITIES
International classification
A61K47/00
HUMAN NECESSITIES
C07C39/12
CHEMISTRY; METALLURGY
A61K36/00
HUMAN NECESSITIES
A23L33/105
HUMAN NECESSITIES
A61K8/97
HUMAN NECESSITIES
Abstract
The present disclosure relates to an effect of promoting hyaluronic acid synthesis of a pomegranate concentrate. A pomegranate concentrate, prepared by treating pomegranate with a starch-degrading enzyme and passing the same through a heating concentration process and having an ellagic acid content of 0.8 mg/g or higher and a polyphenol content of 8 mg/g or higher, has an effect of promoting hyaluronic acid. According to the present disclosure, hyaluronic acid synthesis can be promoted and skin moisturization can be improved by administering a food or pharmaceutical composition containing the pomegranate concentrate as an active ingredient to a subject in need of promotion of hyaluronic acid synthesis.
Claims
1. A method for promoting hyaluronic acid synthesis, which comprises administering a food or pharmaceutical composition comprising a pomegranate pulp concentrate as an active ingredient to a subject in need of promotion of hyaluronic acid synthesis, wherein the method provides a skin whitening effect to the subject, and wherein the pomegranate pulp concentrate comprises 0.8-3 mg/g of ellagic acid and 8-15 mg/g of polyphenol.
2. The method according to claim 1, wherein the subject is a human.
3. The method according to claim 1, wherein the pomegranate pulp concentrate is prepared through a process of treating a pomegranate fruit pulp with a starch-degrading enzyme and then concentrating by heating.
4. The method according to claim 3, wherein the treatment with the starch-degrading enzyme is performed at 40-65 C. within 60 minutes.
5. The method according to claim 3, wherein the concentrating by heating is performed at 40-110 C. for 3 or more times.
6. The method according to claim 5, wherein the concentrating by heating is performed by performing heating concentration at 70-100 C. and 400-850 mbar for 2 or more times and then performing heating concentration at 40-80 C. and 100-350 mbar for 1 or more times.
7. The method according to claim 5, wherein the concentrating by heating is performed by performing first heating concentration at 70-85 C. and 400-550 mbar, performing second heating concentration at 85-92 C. and 550-750 mbar, performing third heating concentration at 92-100 C. and 750-850 mbar, performing fourth heating concentration at 60-80 C. and 250-350 mbar and performing fifth heating concentration at 40-60 C. and 100-250 mbar.
8. The method according to claim 5, wherein the concentrating by heating is performed by performing first heating concentration at 55-90 C., performing second heating concentration at 105-110 C. and performing third heating concentration at 100-105 C.
9. The method according to claim 1, wherein the food composition is in the form of a fortified food, a dietary supplement, a non-alcoholic drink, a sports drink, a fruit drink, tea-or milk-based drink or a liquid food.
10. The method according to claim 1, wherein the method is effective for improving skin moisturization.
11. The method according to claim 1, wherein the method has an effect of inhibiting melanin synthesis.
12. The method according to claim 1, wherein the method has one or more effects selected from the group consisting of improvement of skin moisturization, improvement of wrinkles, improvement of skin elasticity and reduction of cornification.
Description
BRIEF DESCRIPTION OF DRAWINGS
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BEST MODE FOR CARRYING OUT INVENTION
(22) Hereinafter, the present disclosure will be described in detail through examples. However, the following examples are for illustrative purposes only and it will be apparent to those of ordinary skill in the art that the scope of the present disclosure is not limited by the examples.
EXAMPLE 1
Preparation of Pomegranate Concentrate through Heating Concentration
(23) (1) Preparation of 12NK26
(24) First, 1000 kg of Turkish pomegranate fruits were washed after removing impurities. 450 kg of pomegranate pulp was obtained by cutting the fruits, removing the pericarp and removing the seeds by compression. After filtration, the pomegranate pulp was sterilized at 100-105 C. for 60 seconds and then cooled to 48-55 C. The resulting pomegranate juice was treated with 70-100 mL of pectinase per 1000 L of the juice at 48-55 C. for 1 hour to degrade starches. Then, after adding 900 g of bentonite per 1000 L of the pomegranate juice for maintenance of turbidity and color, provision of viscosity suitable for intake, etc., the mixture was stirred at 48-55 C. for 10 minutes. Then, after vacuum filtration through 1.5-mm and 1-mm filters followed by heating concentration (sequentially, at 70-85 C. and 400-550 mbar up to 12 Brix, at 85-92 C. and 550-750 mbar up to 17 Brix, at 92-100 C. and 750-850 mbar up to 31 Brix, at 60-80 C. and 250-350 mbar up to 43 Brix, and then at 40-60 C. and 100-250 mbar up to 65 Brix), a pomegranate concentrate containing 1.8-3.0 mg/g of ellagic acid and 12.59 mg/g polyphenol (as measured by gallic acid colorimetry) was prepared by filtering through a 0.15-mm filter. The weight ratio of fruit: concentrate in the pomegranate concentrate was 10:1. This pomegranate concentrate was named as sample 12NK26.
(25) (2) Preparation of 12910910
(26) First, Iranian pomegranate fruits were washed after removing impurities. The fruits were cut and the pericarp was removed. Then, the seeds were removed by compressing at 160 bar. After sterilization at low temperature, the resulting pomegranate juice was treated with 100-150 g of pectinase per 6000 L of the juice at 48-60 C. for 1 hour to degrade starches. Then, after adding 1200-1800 g of gelatin, 6000 g of silicon dioxide and 14 kg of bentonite per 6000 L of the pomegranate juice for maintenance of turbidity and color, provision of viscosity suitable for intake, etc., the mixture was stirred at 50-60 C. for 30 minutes. Then, after vacuum filtration followed by heating concentration (sequentially, at 55-90 C. (3 minutes), 105-110 C. (90 seconds) and 100-105 C. (90 seconds)), a pomegranate concentrate containing 0.8-1.4 mg!g of ellagic acid and 11.79 mg/g polyphenol (as measured by gallic acid colorimetry) was prepared. The weight ratio of fruit concentrate in the pomegranate concentrate was 5:1. This pomegranate concentrate was named as sample 12910910.
COMPARATIVE EXAMPLE 1
Preparation of Pomegranate Juice (921217)
(27) First, 1000 kg of Turkish pomegranate fruits were washed after removing impurities. 450 kg of pomegranate pulp was obtained by cutting the fruits, removing the pericarp and removing the seeds by compression. After filtration, the pomegranate pulp was sterilized at 90-94 C. for 20 seconds and then cooled to 15-20 C. As a result, a pomegranate juice containing 0.2-0.32 mg/g of ellagic acid and 0.7-1.55 mg/g polyphenol (as measured by gallic acid colorimetry) was prepared. The weight ratio of fruit concentrate in the pomegranate concentrate was 2:1. This pomegranate juice was named as sample 921217.
EXAMPLE 2
Evaluation of Hyaluronan Synthesis-Promoting Effect of Pomegranate Concentrate
(28) 1. Test Materials
(29) Test solutions were prepared by diluting the samples 12NK26, 12910910 and 921217 to concentrations adequate for cell culturing. After conducting preliminary experiments by the WST-1 assay, skin-related effects of the test solutions were evaluated at concentrations not exhibiting cytotoxicity. A positive control group was diluted with a cell culture medium.
(30) TABLE-US-00001 TABLE 1 Reagents and materials Manufacturer Catalog No. Dulbecco's Modified Welgene LM-001-01 Eagle's Medium (DMEM) 48 well plate SPL 30048 96 well plate SPL 31096 Cell proliferation reagent WST-1 Roche 1644-807 DC protein assay kit Bio-Rad 500-0116 Sodium hydroxide (NaOH) welgene ML022-02 N-Acetyl-D-glucosamine (NAG) SIGMA A8625 Hyaluronan ELISA kit R&D system DY3614
(31) 2. Cell Line and Culturing
(32) Human keratinocytes (HaCaT cells, ATCC) were seeded onto the bottom of a culture dish and cultured at 37 C. in a 5% carbon dioxide incubator after adding Dulbecco's modified Eagle's medium (DMEM, Welgene, Korea) containing penicillin (100 IU/mL), streptomycin (10 g/mL) and 10% FBS.
(33) 3. Cytotoxicity
(34) The cytotoxicity of the test solutions for the HaCaT cells was measured. The HaCaT cells were seeded onto a 96 well plate with 510.sup.4 cells/well and then cultured for 24 hours. Then, after maintaining a starvation state for 12 hours, the test solutions and a fresh medium (without supplements) were added and the cells were cultured for 24 hours. 24 hours later, for measurement of cell survivability, 100 L of WST-1 solution diluted to 1/10 with a supplement-free medium was treated per well. After incubation for 1 hour, absorbance was measured at 450 nm.
(35) As a result, 12NK26 showed 80% or higher cell survivability at concentrations below 0.5%, 921217 showed 80% or higher cell survivability at concentrations below 1%, and 12910910 showed 80% or higher cell survivability at concentrations below 0.5%.
(36) TABLE-US-00002 TABLE 2 (n = 4) Sample Concentration % control Control 100.00 2.125 12NK26 0.05% 84.06 1.702 0.1% 95.87 5.598 0.5% 83.74 3.854 1% 44.45 1.912 921217 0.1% 85.11 6.651 0.5% 86.78 5.583 1% 81.01 4.944 5% 75.69 3.727 12910910 0.05% 87.08 2.575 0.1% 95.13 4.583 0.5% 104.46 4.368 1% 65.02 7.070
(37) 4. Increase in Hyaluronan Synthesis
(38) The skin-moisturizing effect of the samples was evaluated by measuring the degree of hyaluronan synthesis in keratinocytes by ELISA. For this, the HaCaT cells were seeded onto a plate at 510.sup.4 cells/well and cultured for 24 hours. 24 hours later, the cells were treated with the test samples diluted with the DMEM cell culture medium (without supplements) and cultured for 24 hours. The cultured cells were recovered and the quantity of hyaluronan was measured by hyaluronan ELISA (R&D Systems, DY3614). The cells adherent to the bottom were washed with PBS and lysed with 1 N NaOH to determine the total amount of proteins. Then, the quantity of hyaluronan per given quantity of proteins was calculated.
(39) Measurement of Protein Quantity
(40) The protein quantity was measured using a Bio-Rad DC protein kit according to the Lowry method.
(41) (1) The cell lysate (supernatant) is added to a 96-well plate with 5 L per well.
(42) (2) Preparation of protein standard
(43) 1.5 mg/mL bovine serum albumin (BSA, Bio-Rad, 500-0007) diluted with a cell lysis buffer to 8 different concentrations is added to the 96-well plate with 5 L per well.
(44) (3) The reagent A and the reagent S included in the kit are mixed at a ratio of 40:1 and added with 25 L per well.
(45) (4) The reagent B included in the kit is added with 200 L per well.
(46) (5) After incubation at room temperature for 15 minutes, absorbance is measured at 750 nm.
(47) (6) The total protein quantity is calculated from the BSA calibration curve.
(48) 5. Statistics
(49) All data were represented as meanSD. Statistical analysis was conducted according to the Student's t-test (significance probability p<0.005, two-sided).
(50) 6. Result
(51) The effect of the samples 12NK26, 921217 and 129109103 on hyaluronan synthesis was evaluated by ELISA. For this, a hyaluronan ELISA kit (R&D Systems, DY3614) was used. As a result, hyaluronan was measured as 179.6%, 214.4% and 305.1% at concentrations of 0.01%, 0.05% and 0.1% as compared to the control group for 12NK26. The hyaluronan synthesis increased in a concentration-dependent manner. The increase in hyaluronan synthesis was statistically significant at all the three concentrations. For 921217, hyaluronan was measured as 84.0%, 98.3% and 106.2% at concentrations of 0.1%, 0.5% and 1%, respectively, as compared to the control group and no statistical significance was observed at the three concentrations. For 12910910, hyaluronan was measured as 162.4%, 262.4% and 298.2% at concentrations of 0.01%, 0.05% and 0.1% as compared to the control group. The hyaluronan synthesis increased in a concentration-dependent manner. The increase in hyaluronan synthesis was statistically significant at all the three concentrations. For NAG used as a positive control group, 224.8% of hyaluronan was produced when treated with 20 mM as compared to the control group, showing that the hyaluronan biosynthesis by the HaCaT cells is promoted by NAG (see Table 3 and
(52) TABLE-US-00003 TABLE 3 (n = 4) Sample Concentration % control Control 100.00 20.876 NAG 20 mM 224.85 16.937 12NK26 0.01% 179.66 16.549 0.05% 214.44 13.714 0.1% 305.19 26.122 921217 0.1% 84.02 17.511 0.5% 98.32 14.297 1% 106.27 19.036 12910910 0.01% 162.43 6.761 0.05% 262.49 17.319 0.1% 298.27 22.858
EXAMPLE 3
Evaluation of Effect of Pomegranate Concentrate on Skin's Physiological Activity
(53) 1. Test Materials
(54) A test solution was prepared by diluting a pomegranate concentrate (sample 12NK26) prepared as described in Example 1 to a concentration adequate for cell culturing. After conducting preliminary experiments by the WST-1 assay, skin-related effects of the test solution were evaluated at concentrations not exhibiting cytotoxicity. A positive control group was diluted with a cell culture medium.
(55) Other test materials used in this example are as follows.
(56) TABLE-US-00004 TABLE 4 Reagents and materials Manufacturer Catalog No. Dulbecco's Modified Welgene LM-001-01 Eagle's Medium (DMEM) Fibroblast Basal Medium (FBM) Lonza CC-3131 48 well plate SPL 30048 96 well plate SPL 31096 Cell proliferation reagent WST-1 Roche 1644-807 Procollagen type I c-peptide (PIP) TAKARA MK101 DC protein assay kit Bio-Rad 500-0116 Sodium hydroxide (NaOH) welgene ML022-02 human total MMP-1 ELISA kit R&D system DY901 Transforming growth factor- PEPROTECH, INC 100-21C (TGF-) Retinoic acid SIGMA R2625 N-Acetyl-D-glucosamine (NAG) SIGMA A8625 Hyaluronan ELISA kit R&D system DY3614
(57) 2. Cell Line and Culturing
(58) For testing of wrinkle improvement, normal human primary dermal fibroblasts, neonatal (HDF-N) derived from the human neonatal foreskin were used. The cells were seeded onto the bottom of a culture dish and cultured at 37 C. in a 5% carbon dioxide incubator after adding the fibroblast basal medium (FBM, Lonza, CC-3131) containing 0.1% insulin, 0.1% rhFGF, 0.1% gentamicin and 2% FBS.
(59) For evaluation of skin-whitening effect, Melan-a cells derived from C57BL/6J (black, a/a) mouse, provided by Dr. Dorothy C Benette (St George's Hospital, London, UK), were used as an immortalized cell line (Bennett D C, Cooper P J, Hart I R. A line of non-tumorigenic mouse melanocyte, syngeneic with the B16 melanoma and requiring a tumor promoter for growth. Int J Cancer 1987; 39: 414-418). The cells were cultured at 37 C. in a 5% carbon dioxide incubator after adding the RPMI 1640 medium containing 10% fetal bovine serum, 50 U/mL penicillin, 50 g/mL streptomycin, 200 nM phorbol 12-myristate 13-acetate (PMA).
(60) 3. Cytotoxicity
(61) The cytotoxicity of the test solutions for the HDF-N cells and Melan-a cells was measured. The HDF-N cells and Melan-a cells were seeded onto a 96 well plate with 610.sup.3 cells/well and 910.sup.3 cells/well, respectively, and then cultured for 24 hours. Then, after maintaining a starvation state for 12 hours, the test solutions and a fresh medium (without supplements or FBS) were added and the cells were cultured for 24 hours. 24 hours later, for measurement of cell survivability, 100 L of WST-1 solution diluted to 1/10 with a supplement-free medium was treated per well. After incubation for 1 hour, absorbance was measured at 450 nm.
(62) As a result, the pomegranate concentrate sample showed 80% or higher cell survivability at concentrations below 0.1% for the HDF-N cells (Table 5) and 80% or higher cell survivability at concentrations below 0.05% for the Melan-a cells (Table 6).
(63) TABLE-US-00005 TABLE 5 (n = 5) Sample Concentration % control Control 100.00 6.147 Pomegranate 0.005% 93.78 5.175 concentrate 0.01% 100.42 6.882 0.05% 97.34 4.946 0.1% 94.14 5.402 0.5% 42.67 7.540
(64) TABLE-US-00006 TABLE 6 (n = 5) Sample Concentration % control Control 100.00 6.817 Pomegranate 0.005% 107.90 10.084 concentrate 0.01% 99.70 4.972 0.05% 80.08 8.846 0.1% 68.14 7.841
(65) 4. Wrinkle-Improving Effect
(66) 4-1. Increase in Procollagen
(67) In order to measure the effect of promoting procollagen synthesis of the test solution, HDF-N cells were seeded onto a 48-well plate with 110.sup.4 cells/well and then cultured for 24 hours. Then, after maintaining a starvation state for 24 hours, the cells were treated with the test solution diluted with the cell culture medium FBM (without supplements) at different concentrations and then cultured for 24 hours. The cultured cells were recovered and the quantity of procollagen was measured using the procollagen type I c-peptide (PIP) EIA kit (Takara, MK101). The cells adherent to the bottom were washed with PBS and lysed with 1 N NaOH to determine the total amount of proteins. Then, the quantity of procollagen per given quantity of proteins was calculated.
(68) As a result, a positive control group treated with 10 ng/mL TGF- showed 150.8% of procollagen production as compared to the control group. This suggests that the procollagen biosynthesis by the HDF-N cells is promoted by TGF-. Meanwhile, when the cells were treated with the sample at concentrations of 0.01%, 0.05% and 0.1%, the procollagen production was 96.3%, 53.3% and 53.0%, respectively, and no statistically significant difference from the control group was observed (Table 7).
(69) TABLE-US-00007 TABLE 7 (n = 4) Sample Concentration % control Control 100.00 8.955 TGF- 10 ng/ml 150.88 5.366 Pomegranate 0.01% 96.35 7.440 concentrate 0.05% 53.36 8.462 0.1% 53.02 4.416
(70) 4-2. Inhibition of UV-Induced MMP-1 Expression
(71) The effect of the test solution on UV-induced MMP-1 expression was evaluated in order to evaluate the wrinkle-improving effect of the sample by measuring the activity of matrix metalloproteinase (MMP-1) by immuno-ELISA assay. HDF-N cells were seeded onto a 24-well plate with 210.sup.4 cells/well and then cultured for 24 hours. 24 hours later, after discarding the medium and washing with DPBS, 200 L of DPBS was added and 5 J/cm.sup.2 of UV-A was irradiated. After treating the cells with the sample diluted to an adequate concentration, the cells were cultured for 24 hours. Then, the culture medium was taken and the quantity of MMP-1 was measured with the human total MMP-1 ELISA kit (R&D Systems, DY901). The measured quantity of MMP-1 was calibrated with the total protein quantity.
(72) As a result, the UVA-irradiated group produced 240.2% of MMP-1 as compared to the unirradiated group, which suggests that MMP-1 expression is induced in the HDF-N cells by 5 J/cm.sup.2 UVA. Meanwhile, when the HDF-N cells were treated with the pomegranate concentrate at concentrations of 0.001%, 0.005% and 0.01%, the MMP-1 production measured with the human total MMP-1 ELISA kit (R&D Systems, DY901) was 137.1%, 69.3% and 7.1%, respectively, 1 as compared to the UVA-unirradiated group. That is to say, the MMP-1 production decreased in a concentration-dependent manner and the decrease was statistically significant when compared with the UV-irradiated group. Also, retinoic acid used as a positive control group showed 171.6% of MMP-1 production as compared to the UVA-unirradiated group, which was statistically significant decrease as compared to the UV-irradiated group.
(73) TABLE-US-00008 TABLE 8 (n = 4) UV Sample Concentration % UV UVA 100.00 18.728 5 J/cm.sup.2 + 240.25 18.389 Retinoic acid 1 M 171.68 10.422 Pomegranate 0.0001% 137.12 21.034 concentrate 0.0005% 69.39 12.158 0.001% 7.11 2.665
(74) 4-3. Elastase Inhibition Assay
(75) The HDF-N fibroblasts were used to measure the effect of the test solution on elastase activity. For this, the cultured HDF-N cells were treated with 0.2 M Tris-HCl (pH 8.0) containing 0.1% Triton X-100 and lysed by sonication. After centrifuging at 3,000 rpm for 20 minutes and taking the supernatant, the activity of the enzyme was measured by quantifying it and the total protein quantity.
(76) For measurement of the elastase activity, the homogenized fibroblasts were treated with 200 g/mL elastase, 0.2 M Tris-HCl buffer and the sample at different concentrations. After adding 50 mM STANA, a substrate specific for elastase, and incubating at 37 C., absorbance was measured at 405 nm. The degree of elastase activity inhibition was compared with a control group not treated with the test solution. Phosphoramidon was used as a positive control group.
(77) When absorbance was measured at 405 nm after eating with the pomegranate concentrate at concentrations of 0.05%, 0.1% and 1%, the elastase activity was measured to be 92.3%, 89.5% and 63.6%, respectively, as compared to the control group, showing a decreasing tendency in a concentration-dependent manner. In particular, statistically significant decrease as compared to the control group was observed at all the three concentrations. For the phosphoramidon used as the positive control group, the elastase activity was measured as 74.9% as compared to the control group. The decrease was statistically significant.
(78) TABLE-US-00009 TABLE 9 (n = 3) Sample Concentration % control Control 100.00 0.819 Phosphoramidon 10 M 74.90 5.962 Pomegranate 0.05% 92.31 1.040 concentrate 0.1% 89.59 3.164 1% 63.65 3.478
(79) 5. Skin-Whitening Effect
(80) 5-1. Tyrosinase Inhibition Assay
(81) A sample solution for measuring tyrosinase activity was prepared by diluting the test solution with ethanol or a suitable solvent to an adequate concentration. After sequentially adding 220 L of 0.1 M phosphate buffer, 20 L of the sample solution and 20 L of a mushroom tyrosinase solution (1500-2000 U/mL) to a test tube, 40 L of a 1.5 mM tyrosine solution was added to the resulting solution. After incubation at 37 C. for 10-15 minutes, absorbance was measured at 490 nm using the ELISA reader. As a blank solution, 0.1 M phosphate buffer (pH 6.5) was added instead of the sample solution. The sample concentration (IC.sub.50) when the activity was inhibited by 50% was calculated using a suitable program.
(82) The pomegranate concentrate could not inhibit the tyrosinase activity at concentrations below 0.1%. In contrast, the IC.sub.50 value of kojic acid used as a positive control group was measured to be 3.634 ppm.
(83) TABLE-US-00010 TABLE 10 (n = 3) Pomegranate Sample concentrate Kojic acid IC.sub.50 3.634 ppm
(84) 5-2. Inhibition of Melanin Synthesis
(85) Melan-a cells were seeded onto a 24-well plate with 310.sup.4 cells/well. After culturing the cells in an incubator for 24 hours so that the cells adhered well to the plate, the test solution diluted with a culture medium to different concentrations were added to the respective wells. The test sample was treated once in 3 days, for a total of 6 days. Melanin in the cells was observed with an optical microscope (bright-field microscope). The cells were lysed with 1 N NaOH and then centrifuged. After separating the supernatant, the total amount of proteins was quantified by measuring absorbance at 405 nm. The melanin content was determined from the protein quantity.
(86) As a result, the melanin content was measured as 95,4%, 86.3% and 67.1% when the concentrations of the test solution were 0.005%, 0.01% and 0.05%, respectively, as compared to an untreated control group. That is to say, the melanin synthesis decreased in a concentration-dependent manner and the decrease was statistically significant. When 100 M arbutin was treated as a positive control group, the melanin synthesis was inhibited in a concentration-dependent manner.
(87) TABLE-US-00011 TABLE 11 (n = 4) Sample Concentration % control Control 100.00 4.904 Arbutin 100 M 73.54 5.397 Pomegranate 0.005% 95.49 9.277 concentrate 0.01% 86.32 1.206 0.05% 67.12 5.695
(88) 6. Statistics
(89) All data were represented as meanSD. Statistical analysis was conducted according to the Student's t-test (significance probability p<0.005, two-sided).
EXAMPLE 4
Clinical Assessment of Effect of Pomegranate Concentrate on Skin's Physiological Activity
EXAMPLE 4-1
Test Methods
(90) 1. Recruitment of Subjects
(91) Test was conducted for 62 female subjects aged between 25 and 60 years who had dry skin, crows' feet and pigmentation. The selected subjects were randomly given a drink containing a pomegranate concentrate (sample 12NK26) prepared according to the method described in Example 1 (test product) or a drink not containing a pomegranate concentrate (control product) and asked to take one pouch a day (50 mL/pouch) for 8 weeks (double-blind test). Before the intake of the test product and at 4 weeks and 8 weeks after the start of the intake, skin-improving effect was evaluated based on visual assessment, skin's water content, skin cornification, TEWL, crows' feet (replica), skin elasticity, skin color brightness (L* value), questionnaire, safety, diet and body weight (including BMI). Also, blood test was conducted before the intake of the test product and at 8 weeks after the start of the intake.
(92) The ingredients included in the drink (test product) containing the pomegranate concentrate (sample 12NK26) and the drink (control product) not containing the pomegranate concentrate and their contents are as follows.
(93) TABLE-US-00012 Ingredients Test product Control product Remarks Pomegranate 10 mL Main ingredient concentrate Purified water 40 mL 41.5 mL Fructooligosaccharide 6.5 mL Food additive Fructose 1 mL Food additive Red cabbage color 0.4 mL Food additive Citric acid 0.5 mL Food additive Pomegranate flavor 0.1 mL Food additive Total 50 mL 50 mL
(94) 2. Measurement and Evaluation
(95) The subjects were divided into a control product group and a test product group through block randomization and the face and left or right forearm were selected as test sites. For the face, measurement was made on the cheek where the corner of the eye meets the tip of the nose (water content, cornification, TEWL, elasticity and skin color), pigmented area (skin color) and Crow's feet (wrinkles). For the inner forearm, measurement was made at the part 5 cm distant from the elbow. The subjects were asked to wash faces and rest for 30 minutes in a space maintained at constant temperature (222 C.) and humidity (505%) with no air movement and direct sunlight before they participated in the visual assessment, instrumental measurement (skin's water content, cornification, transepidermal water loss, skin elasticity, skin color and crows' feet), survey, body weight (BMI) measurement, dietary investigation and safety evaluation.
(96) 3. Visual Assessment
(97) The degree of crows' feet of 2 subjects was evaluated between grades 0 and 9 according to the visual assessment standard of the Guideline for Efficacy Evaluation of Functional Cosmetics II of the Ministry of Korea Food and Drug Safety (July 2005). The wrinkles at the corner of the eye were imaged using the Visia facial imaging system (Canfield Imaging System, USA) in the FL mode.
(98) TABLE-US-00013 TABLE 12 Grade Description 0 No skin wrinkle and fine skin texture. 1 Fine skin wrinkles begin to appear. 2 Fine skin wrinkles are formed slightly. 3 There are many fine wrinkles and shallow wrinkles begin to appear. 4 Shallow wrinkles are formed slightly. 5 Shallow wrinkles are distinct but there is no deep wrinkle. 6 Shallow wrinkles begin to turn to deep wrinkles. 7 Deep wrinkles are formed slightly. 8 There are many deep wrinkles. 9 There are many very deep wrinkles.
(99) 4. Instrumental Measurement
(100) Before the intake of the product and at 4 weeks and 8 weeks after the start of the intake, skin's water content, transepidermal water loss, cornification, skin elasticity and skin color were measured on the left or right cheek of the subjects. Also, left or right crows' feet were measured.
(101) (1) Measurement of skin's water content using Corneometer CM825
(102) Corneometer CM825 (Courage+Khazaka GmbH, Germany) measures water content at 30-40 m beneath the skin's horny layer from capacitance. When current is supplied from a probe, the electrical conductivity of the skin due to the water contained in the skin is displayed in an arbitrary unit (AU). A higher value is measured as the skin's water content is larger. For accurate measurement, measurement was made 3 times for each test sites (cheek, inner forearm) and their mean values were analyzed.
(103) (2) Measurement of Transepidermal Water Loss using Vapometer
(104) Transepidermal water loss (TEWL) refers to loss of water from the skin and is closely related with the moisturizing function. Because the TEWL value increases as dryness is aggravated, the skin hydration can be evaluated by measuring TEWL. Vapometer (Delfin, Finland) measures evaporation of water per unit area with time (g/m.sup.2h) using temperature and humidity sensors. The evaporation of water at the test sites (cheek, inner forearm) at different times was measured and analyzed.
(105) (3) Measurement of Skin Cornification using D-Squame
(106) Cornification, which is another measure of skin hydration, is measured by sampling the horny substance from the test sites (cheek, inner forearm) using the D-Squame black tape. The sample was obtained by attaching the tape on the skin by applying a pressure of 225 g/cm.sup.2 for about 5 seconds using the D500 D-Squame disc applicator and then detaching the tape. The obtained horny substance sample was imaged with Charm View (Moritex, Japan) at 700 magnification and the area was analyzed using the Image-Pro Plus program.
(107) (4) Measurement of Crows' Feet (Replica) using Visioline VL-650
(108) Visioline VL650 (Courage+Khazaka GmbH, Germany) is an instrument which measures the depth, length, etc. of wrinkles in a prepared replica. After mounting the replica on a standard cartridge configured such that a special light passes therethrough, shadow images were obtained using a CCD camera and the maximum wrinkle depth (the depth of the thickest wrinkle) was analyzed.
(109) (5) Measurement of Skin Elasticity using Cutometer
(110) Skin elasticity is measured using Cutometer MPA 580 (Courage+Khazaka, Germany). The most frequently used method uses negative pressure and is highly reproducible. The value measured by the Cutometer is between 0 and 1 (mm, ratio). Measurement was made at the test site (front cheek) 3 times for each measurement and the mean values of the parameters (R0-R9) were analyzed. The elasticity parameters were interpreted as follows.
(111) TABLE-US-00014 TABLE 13 Parameters Description R0 Highest point of the first curve (skin firmness). R1 Lowest point of the first curve (ability to return to the skin's original state). R2 Gross elasticity the closer the value is to 1, the more elastic the skin is). R3 Highest point of the last curve (tiring effect). R4 Lowest point of the last curve (tiring effect). R5 Net elasticity (the closer the value is to 1, the more elastic the skin is). R6 Portion of viscoelasticity of the curve. R7 Biological elasticity of the curve. R8 Ua value of the first curve (the closer Ua and Uf are, the greater is the ability of the skin to return to its original state). R9 Tiring effect of the skin after repeated sucking in the skin. The skin is softer as R0 is higher and firmer as R0 is lower. The closer R2, R5, R7 and R8 are to 1, the more elastic the skin is. The lower R1, R3, R4, R6 and R9 are, the more elastic the skin is.
(112) (6) Measurement of Skin Color using Spectrophotometer CM-700d
(113) Spectrophotometer CM-700d measures tristimulus values by measuring spectral reflectance and calculates L*, a* and b* values of the CIELAB color system. In the L*a*b* color system, lightness is represented by L* and color and chroma are represented by a* and b*, a* and b* indicate color directions. a* indicates red direction, a* indicates green direction, b* indicates yellow direction, and b* indicates blue direction. The color becomes achromatic as the L*, a* and b* values approach 0 and shows high chromaticity in the opposite case. Measurement was made 3 times for each test site (front cheek, pigmented areas) and the skin color lightness (L* value) was averaged.
(114) 5. Blood Analysis
(115) The blood of the subjects was examined before the intake of the test product and at 8 weeks after the intake. It was found out that the test product does not affect metabolism.
(116) 6. Dietary Investigation and Body Weight Measurement
(117) In order to investigate whether the change in dietary habits of the subjects affected the test result, body weight was measured before the intake of the test product and at 4 and 8 weeks after the start of the intake and dietary investigation was conducted to check whether the dietary habits of the subjects were maintained as usual during the test period. The subjects were asked to self-report given questionnaires on diet. Diet: (none: 0, little: 1, moderate: 2, much: 3)
(118) 7. Survey
(119) The efficacy of the test product and satisfaction at the intake were evaluated before the intake of the test product and at 4 and 8 weeks after the start of the intake based on the questionnaires answered by the subjects.
(120) 8. Safety Evaluation
(121) The skin condition of the subjects was examined before the intake of the test product and at 4 and 8 weeks after the start of the intake. Also, the skin condition, systemic condition and irritation by the product were evaluated by interviewing the subjects.
(122) 9. Data Analysis
(123) Means and standard deviations (S.D) of the instrumental measurement values were calculated before the intake of the test product and at 4 and 8 weeks after the start of the intake and the difference of the measured values between the measurement times and groups was statistically analyzed. Comparison between the measurement times was conducted by the paired t-test (p<0.05) and comparison between the groups as conducted by the independent t-test (p<0.05). The SPSS 20.0 program was used for the statistical analysis and the result of survey was analyzed based on frequency.
EXAMPLE 4-2
Test Results
(124) 1. Visual Assessment
(125) The crows' feet were decreased (improved) with statistical significance at 8 weeks after the start of the intake of the product (product B) as compared to before the intake of the test product (p<0.05) (Table 14,
(126) TABLE-US-00015 TABLE 14 (n = 62) Group Week Mean S.D (grade) p-value Change (%) Product A Week 0 6.05 0.76 Week 4 6.03 0.78 0.326 .Math.0.33 Week 8 6.03 0.78 0.326 .Math.0.33 Product B Week 0 6.00 0.77 Week 4 5.97 0.79 0.161 .Math.0.50 Week 8 5.91 0.79 0.032* .Math.1.50 p-value: Paired t-test (*p < 0.05, **p < 0.01, ***p < 0.001, Significantly different as compared to before the intake of the product.) Change: (W.sub.x W0)/W0 100, calculated by mean value.
(127) 2. Skin's Water Content
(128) At 4 and 8 weeks after the start of the intake of the product, skin's water content was increased (improved) both on the face and forearm for the test group (product B) as compared to the control group (product A) with statistical significance (p<0.05) (Table 15,
(129) TABLE-US-00016 TABLE 15 (n = 62) Mean Site Week Group difference Std. Error (A.U) p-value Face 4 W A 0.04 0.44 0.000*** B 3.34 0.48 8 W A 0.86 0.41 0.000*** B 5.21 0.49 Forearm 4 W A 1.00 0.59 0.036* B 3.16 0.80 8 W A 2.32 0.64 0.013* B 4.53 0.57 p-value: Independent t-test (*p < 0.05, **p < 0.01, ***p < 0.001 There is significant difference between the two groups.)
(130) 3. Transepidermal Water Loss (TEWL)
(131) At 4 and 8 weeks after the start of the intake of the product, TEWL was decreased (improved) on the forearm for the product B (test group) as compared to the product A (control group) with statistical significance (p<0.05) (Table 16,
(132) TABLE-US-00017 TABLE 16 (n = 62) Mean Site Week Group difference Std. Error (g/m h) p-value Face 4 W Product A 0.29 0.28 0.631 Product B 0.49 0.29 8 W Product A 0.86 0.35 0.301 Product B 1.33 0.28 Forearm 4 W Product A 0.05 0.19 0.005** Product B 0.66 0.16 8 W Product A 0.39 0.22 0.018* Product B 1.04 0.16 p-value: Independent t-test (*p < 0.05, **p < 0.01, ***p < 0.001 There is significant difference between the two groups.)
(133) 4. Skin Cornification
(134) Skin cornification was decreased (improved) on the face at 4 weeks after the start of the intake of the product and on the face and forearm at 8 weeks after the start of the intake of the product as compared before the intake of the product, with statistical significance (p<0.05). At 8 weeks after the start of the intake of the product, the decrease in skin cornification on the face was 1.84% for the product A (control group) and 6.12% for the product B (test group). On the forearm, the decrease was 0.37% for the product A and 4.09% for the product B (p<0.05) (Table 17).
(135) TABLE-US-00018 TABLE 17 (n = 62) Change Site Group Week Mean S.D (pixel) p-value (%) Face Product Week 0 230357.50 23015.03 A Week 4 227988.19 23944.58 0.454 .Math.1.03 Week 8 226107.52 21733.64 0.229 .Math.1.84 Product Week 0 237015.07 24065.03 B Week 4 228910.36 24618.74 0.048* .Math.3.42 Week 8 222516.67 20785.20 0.000*** .Math.6.12 Fore- Product Week 0 249474.38 21609.59 arm A Week 4 249081.79 22120.34 0.806 .Math.0.16 Week 8 248550.90 20482.48 0.674 .Math.0.37 Product Week 0 247601.54 20889.48 B Week 4 247391.85 19860.34 0.929 .Math.0.08 Week 8 237486.39 19686.42 0.003** .Math.4.09 p-value: Paired t-test (*p < 0.05, **p < 0.01, ***p < 0.001 There is significant difference as compared to before the intake of the product.), Change: (W.sub.x W0)/W0 100, calculated by mean value.
(136) At 8 weeks after the start of the intake of the product, the decrease in skin cornification on both the face and forearm was decreased (improved) with statistical significance for the product B as compared to the product A (p<0.05) (Table 18,
(137) TABLE-US-00019 TABLE 18 (n = 62) Mean Site Week Group difference Std. Error (pixel) p-value Face 4 W Product A 2369.31 3123.71 0.262 Product B 8104.71 3928.50 8 W Product A 4249.98 3457.55 0.034* Product B 14,498.41 3225.33 Forearm 4 W Product A 392.59 1582.37 0.949 Product B 209.59 2319.13 8 W Product A 923.48 2172.94 0.022* Product B 10115.16 3182.20 p-value Independent t-test (*p < 0.05, **p < 0.01, ***p < 0.001 There is significant difference between the two groups.)
(138) 5. Crows' Feet (Replica)
(139) At 8 weeks after the start of the intake of the product B (test group), the maximum wrinkle depth was significantly decreased (improved) as compared to before the intake of the product (p<0.05) (Table 19,
(140) TABLE-US-00020 TABLE 19 Change Parameters Group Week Mean S.D p-value (%) Max Product 0 W 390.86 93.77 wrinkle A 4 W 406.59 101.01 0.400 .box-tangle-solidup.4.02 depth 8 W 381.14 87.78 0.615 .Math.2.49 (um) Product 0 W 426.16 70.69 B 4 W 420.61 104.50 0.756 .Math.1.30 8 W 391.12 59.42 0.021* .Math.8.22 p-value: Paired t-test (*p < 0.05, **p < 0.01, ***p < 0.001 There is significant difference as compared to before the intake of the product.), Change: (W.sub.x W0)/W0 100, calculated by mean value
(141) 6. Skin Elasticity
(142) At 8 weeks after the start of the intake of the product, R1 (the ability to return to original state) and R4 (the lowest point of the last curve) were decreased (improved) with statistical significance and R5 (net elasticity) and R7 (biologic elasticity) were significantly increased (improved) for the product B (test group) as compared to the product A (control group). Also, at 4 and 8 weeks after the start of the intake of the product, R2 (gross elasticity) was increased (improved) with statistical significance for the product B as compared to the product A (p<0.05) (Table 20,
(143) TABLE-US-00021 TABLE 20 (n = 62) Mean difference Parameters Week Group Std. Error (mm) p-value R0 4 W Product A 0.0043 0.0056 0.890 Product B 0.0030 0.0076 R0 8 W Product A 0.0198 0.0068 0.420 Product B 0.0271 0.0059 R1 4 W Product A 0.0034 0.0020 0.060 Product B 0.0045 0.0036 8 W Product A 0.0058 0.0032 0.002** Product B 0.0199 0.0029 R2 4 W Product A 0.0084 0.0031 0.001** Product B 0.0186 0.0069 8 W Product A 0.0010 0.0065 0.000*** Product B 0.0472 0.0071 R3 4 W Product A 0.0045 0.0059 0.888 Product B 0.0031 0.0080 8 W Product A 0.0209 0.0068 0.330 Product B 0.0299 0.0061 R4 4 W Product A 0.0103 0.0038 0.087 Product B 0.0013 0.0054 8 W Product A 0.0060 0.0046 0.009** Product B 0.0222 0.0040 R5 4 W Product A 0.0087 0.0123 0.202 Product B 0.0153 0.0138 8 W Product A 0.0141 0.0192 0.018* Product B 0.0733 0.0150 R6 4 W Product A 0.0013 0.0184 0.721 Product B 0.0082 0.0187 8 W Product A 0.0279 0.0276 0.405 Product B 0.0559 0.0194 R7 4 W Product A 0.0050 0.0052 0.064 Product B 0.0095 0.0057 8 W Product A 0.0036 0.0076 0.002** Product B 0.0357 0.0062 R8 4 W Product A 0.0010 0.0038 0.288 Product B 0.0075 0.0047 8 W Product A 0.0140 0.0044 0.243 Product B 0.0072 0.0037 R9 4 W Product A 0.0002 0.0009 0.917 Product B 0.0001 0.0010 8 W Product A 0.0011 0.0008 0.197 Product B 0.0028 0.0011 p-value: Independent t-test (*p < 0.005, **p < 0.01, ***p < 0.001 There is significant difference between the two groups.)
(144) 7. Skin Color Brightness
(145) Skin color brightness was increased (improved) with statistical significance for the product B (test group) as compared to the product A (control group) on the cheek at 4 weeks after the start of the intake of the product and on both the cheek and pigmented areas at 8 weeks after the start of the intake of the product (p<0.05) (Table 21,
(146) TABLE-US-00022 TABLE 21 (n = 62) Mean difference Site Week Group Std. Error (L*value) p-value Cheek 4 W Product A 0.05 0.06 0.025* Product B 0.21 0.04 8 W Product A 0.18 0.05 0.000*** Product B 0.55 0.07 Pigmented 4 W Product A 0.05 0.07 0.088 area Product B 0.23 0.07 8 W Product A 0.17 0.05 0.019* Product B 0.40 0.08 p-value: Independent t-test (*p < 0.05, **p < 0.01, ***p < 0.001 There is significant difference between the two groups.)
(147) 8. Blood Analysis
(148) No statistically significant difference was observed between the two groups in any parameter (p<0.05) (Table 22).
(149) TABLE-US-00023 TABLE 22 (n = 62) Items Group Mean difference Std. Error p-value T. Protein Product A 0.04 0.06 0.089 Product B 0.19 0.06 Albumin Product A 0.03 0.04 0.929 Product B 0.03 0.03 T. Bil Product A 0.03 0.08 0.437 Product B 0.04 0.04 SGOT Product A 0.10 0.63 0.130 Product B 1.31 0.67 SGPT Product A 0.40 1.11 0.144 Product B 1.66 0.86 T. Chol Product A 1.77 3.32 0.917 Product B 2.25 3.23 Triglyceride Product A 1.90 7.14 0.632 Product B 2.59 6.09 Blood sugar Product A 1.07 1.25 0.461 (fasting) Product B 2.25 1.01 BUN Product A 0.49 0.66 0.534 (blood urea Product B 0.01 0.48 nitrogen) Creatinine Product A 0.01 0.03 0.907 Product B 0.01 0.02 Hemoglobin Product A 0.29 0.10 0.694 Product B 0.24 0.09 p-value: Independent t-test (*p < 0.05, **p < 0.01, ***p < 0.001 There is significant difference between the two groups.)
(150) 9. Dietary Investigation and Body Weight Measurement
(151) There was no significant difference between the two groups in dietary investigation, body weight and BMI for the product A and the product B both before the intake of the product and at 4 and 8 weeks after the start of the intake of the product (p<0.05) (Table 23).
(152) TABLE-US-00024 TABLE 23 (n = 62) Items Group Week Mean S.D p-value Dietary A Week 0 2.01 0.05 investigation Week 4 2.00 0.04 0.786 Week 8 2.00 0.03 0.321 B Week 0 2.02 0.05 Week 4 2.00 0.04 0.200 Week 8 2.00 0.03 0.069 Body A Week 0 57.41 9.44 weight Week 4 57.60 9.60 0.232 Week 8 57.59 9.71 0.328 B Week 0 57.99 8.33 Week 4 57.75 8.30 0.111 Week 8 57.82 8.32 0.266 BMI A Week 0 22.74 3.72 Week 4 22.86 3.77 0.084 Week 8 22.82 3.74 0.354 B Week 0 23.19 2.75 Week 4 23.15 2.68 0.561 Week 8 23.13 2.71 0.367 p-value: Paired t-test (*p < 0.05, **p < 0.01, ***p < 0.001 There is significant difference as compared to before the intake of the product.)
(153) 10. Survey
(154) The satisfaction at the efficacy of the test product was evaluated based on the questionnaires answered by the subjects before the intake of the test product and at 4 and 8 weeks after the start of the intake. The result is as follows (Table 24).
(155) TABLE-US-00025 TABLE 24 (n = 62) Product A (n = 30) Product B (n = 32) Positive Satisfaction Positive Satisfaction Items Week responses (N) (%) responses (N) (%) Improvement of 4 W 6 20.00 10 31.25 skin moisturization 8 W 13 43.33 19 59.38 Improvement of 4 W 5 16.67 8 25.00 skin color 8 W 15 50.00 17 53.13 Improvement of 4 W 4 13.33 7 21.88 skin wrinkles 8 W 9 30.00 18 47.37 1. Moistness 4 W 19 63.33 17 53.13 8 W 21 70.00 25 78.13 2. Softness 4 W 21 70.00 22 68.75 8 W 23 76.67 28 87.50 3. Decreased tightening 4 W 16 53.33 21 65.63 8 W 20 66.67 22 68.75 4. Reduced cornification 4 W 18 60.00 19 59.38 8 W 16 53.33 18 56.25 5. Fine texture 4 W 13 43.33 18 56.25 8 W 18 60.00 23 71.88 6. Decreased drooping 4 W 9 30.00 17 53.13 8 W 10 33.33 18 56.25 7. Brightened tone 4 W 14 46.67 16 50.00 8 W 19 63.33 24 75.00 8. Healthy skin 4 W 17 56.67 20 62.50 8 W 21 70.00 24 75.00 9. Decreased fine 4 W 7 23.33 12 37.50 wrinkles 8 W 11 36.67 14 43.75 Grades: 0. no change, 1. insignificant, unperceived change, 2. slightly perceived improvement, 3. perceived improvement, 4. distinct improvement-Number (%) of subjects who answered positively: 2-4 Grades: 1. never, 2. no, 3, unlikely, 4. likely, 5. yes, 6. absolutely-Number (%) of subjects who answered positively: 4-6
(156) Grades: 0. no change, 1. insignificant, unperceived change, 2. slightly perceived improvement, 3. perceived improvement, 4. distinct improvement
(157) Number (%) of subjects who answered positively: 2-4
(158) Grades: 1, never, 2. no, 3. unlikely, 4. likely, 5, yes, 6. absolutely
(159) Number (%) of subjects who answered positively: 4-6
(160) 11. Safety Evaluation
(161) No abnormal response was observed in any subject in dermatological or systemic symptoms throughout the test period (Table 25).
(162) TABLE-US-00026 TABLE 25 (n = 62) Product A Product B Symptoms 4 W 8 W 4 W 8 W Skin Itchiness 0 0 0 0 Pricking pain 0 0 0 0 Ticklishness 0 0 0 0 Burning 0 0 0 0 Stinging 0 0 0 0 Stiffness 0 0 0 0 Tightness 0 0 0 0 Erythema 0 0 0 0 Edema 0 0 0 0 Cornification 0 0 0 0 Pimple 0 0 0 0 Systemic Headache 0 0 0 0 Dizziness 0 0 0 0 Fever 0 0 0 0 Lack of appetite 0 0 0 0 Sickness 0 0 0 0 Vomiting 0 0 0 0 Indigestion 0 0 0 0 Constipation 0 0 0 0 Diarrhea 0 0 0 0 Total number of subjects who 0 0 0 0 showed abnormal responses