Composition for preventing and treating muscle diseases or improving muscular function, containing platycodon grandiflorum extract

Abstract

The present invention relates to a composition for preventing and treating muscle diseases or improving muscular function, containing a Platycodon grandiflorum extract. More specifically, the present invention relates to: a pharmaceutical composition for preventing and treating muscle diseases, containing a Platycodon grandiflorum extract; a food composition for preventing muscle diseases or improving muscular function; or a cosmetic for improving muscular function. The Platycodon grandiflorum extract of the present invention increases muscle formation by inducing muscular protein synthesis and muscle cell differentiation. Therefore, the extract is a natural product so as to be used safely without side effects, thereby being usable as an active ingredient of a pharmaceutical, food, or cosmetic composition exhibiting an excellent effect in preventing and treating muscle diseases or improving muscular function.

Claims

1. A method for treating a muscle disease in a subject or improving a muscular function in a subject having the muscle disease, the method comprising administering an effective amount of a composition consisting of a Platycodon grandiflorum extract as an active ingredient to the subject in need thereof.

2. The method of claim 1, wherein the Platycodon grandiflorum extract is an extract obtained by using at least one solvent selected from the group consisting of water, an organic solvent having 1 to 6 carbon atoms, a subcritical fluid, a supercritical fluid, and a mixture thereof.

3. The method of claim 2, wherein the organic solvent having 1 to 6 carbon atoms is selected from the group consisting of alcohol having 1 to 6 carbon atoms, acetone, ether, benzene, chloroform, ethyl acetate, methylene chloride, hexane, cyclohexane, and petroleum ether.

4. The method of claim 1, wherein the muscle disease is selected from the group consisting of atony, muscular atrophy, muscular dystrophy, muscular degeneration, muscular spasticity, amyotrophic lateral sclerosis, myasthenia gravis, cachexia and sarcopenia.

5. The method of claim 1, wherein the Platycodon grandiflorum extract is obtained by extracting Platycodon grandiflorum under an ultra-high pressure of 100 MPa to 1000 MPa.

6. A method for treating a muscle disease in a subject or improving a muscular function in a subject having the muscle disease, the method comprising administering an effective amount of a composition comprising a Platycodon grandiflorum extract as an active ingredient to the subject in need thereof, wherein the Platycodon grandiflorum extract is an extract obtained by using at least one solvent selected from the group consisting of water, an organic solvent having 1 to 6 carbon atoms, a subcritical fluid, a supercritical fluid, and a mixture thereof.

7. The method of claim 6, wherein the organic solvent having 1 to 6 carbon atoms is selected from the group consisting of alcohol having 1 to 6 carbon atoms, acetone, ether, benzene, chloroform, ethyl acetate, methylene chloride, hexane, cyclohexane, and petroleum ether.

8. A method for treating a muscle disease in a subject or improving a muscular function in a subject having the muscle disease, the method comprising administering an effective amount of a composition comprising a Platycodon grandiflorum extract as an active ingredient to the subject in need thereof, wherein the Platycodon grandiflorum extract is obtained by extracting Platycodon grandiflorum under an ultra-high pressure of 100 MPa to 1000 MPa.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows the results of measuring the expression of p-mTOR protein in L6 muscle cells by using five kinds of the Platycodon grandiflorum extract (1. methanol extract, 2. ethanol extract, 3. ethyl acetate extract, 4. hexane extract and 5. hot water extract), respectively.

(2) FIG. 2 shows the results of measuring the expression of p-mTOR and p-p70S6K proteins in L6 muscle cells by using the methanol extract of Platycodon grandiflorum, respectively.

(3) FIG. 3 shows the results of measuring the mRNA expression of MyoD and myogenin, respectively, by treating the hexane extract, the ethyl acetate extract, the ethanol extract, and the hot water extract of Platycodon grandiflorum s.

(4) FIG. 4 shows the results of measuring the mRNA expression of MyoD and myogenin in L6 muscle cells by treating the methanol extract of Platycodon grandiflorum.

(5) FIG. 5 shows the results of measuring the mRNA expression of atrogin-1 and MuRF-1 in L6 muscle cells by treating the hexane extract, the ethyl acetate extract, the ethanol extract, and the hot water extract of Platycodon grandiflorum.

MODE FOR CARRYING OUT INVENTION

(6) Hereinafter, the present invention will be described in more detail with reference to Examples.

(7) However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Example 1

(8) Preparation of the Platycodon grandiflorum Extract

(9) <1-1> Preparation of the Methanol Extract of Platycodon grandiflorum

(10) The subterranean part of dried Platycodon grandiflorum was pulverized with a mixer, and 100 g of the milled Platycodon grandiflorum sample was added to 1 L of 100% methanol and extracted at room temperature for 48 hours. The extracted sample was filtered with Whatman No. 2 filter paper, and the filtered extract was concentrated using a vacuum rotary condenser to remove the solvent component, and then a methanol extract of Platycodon grandiflorum was obtained.

(11) <1-2> Preparation of the Ethanol Extract of Platycodon grandiflorum

(12) The subterranean part of Platycodon grandiflorum was pulverized with a mixer, and 100 g of the milled Platycodon grandiflorum sample was added to 1 L of 100% ethanol and extracted at room temperature for 48 hours. The extracted sample was filtered with Whatman No. 2 filter paper, and the filtered extract was concentrated with, a vacuum rotary condenser to remove the solvent components, and then an ethanol extract of Platycodon grandiflorum was obtained.

(13) <1-3> Preparation of the Ethyl Acetate Extract of Platycodon grandiflorum

(14) The subterranean part of the dried Platycodon grandiflorum was pulverized with a mixer, and 100 g of milled Platycodon grandiflorum sample was added to 1 L of 100% ethyl acetate and extracted at room temperature for 48 hours. The extracted sample was filtered with Whatman No. 2 filter paper, and the filtered extract was concentrated with a vacuum rotary condenser to remove solvent components to obtain an ethyl acetate extract of Platycodon grandiflorum.

(15) <1-4> Preparation of the Hexane Extract of Platycodon grandiflorum

(16) The subterranean part of the dried Platycodon grandiflorum was pulverized with a mixer, and 100 g of milled Platycodon grandiflorum sample was added, to 1 L of 100% hexane and extracted at room temperature for 48 hours. The extracted sample was filtered, with Whatman No. 2 filter paper, and the filtered extract was concentrated with a vacuum rotary condenser to remove solvent components, and then a hexane extract of Platycodon grandiflorum was obtained.

(17) <1-5> Preparation of the Hot-Water Extract of Platycodon grandiflorum

(18) The subterranean part of the dried Platycodon grandiflorum was pulverized with a mixer, and 100 g of the milled Platycodon grandiflorum sample was added to 1 L of water and extracted while stirring at 100 C. for 2 hours. The extracted sample was filtered with Whatman No. 2 filter paper, and the filtered extract was concentrated with a vacuum rotary condenser to remove solvent components, and then a hot-water extract of Platycodon grandiflorum was obtained.

(19) <1-6> Preparation of the Ultra-High Pressure Extract of Platycodon grandiflorum

(20) The subterranean part of the dried Platycodon grandiflorum was pulverized with a mixer, 1 g of the milled Platycodon grandiflorum sample and 76 ml of 18% ethanol were placed in a polyethylene pack and sealed, and then extracted with an ultra-high pressure extraction apparatus (Frescal MFP-7000; Mitsubishi Heavy Industries, Tokyo, Japan). Extraction pressure was 320 MPa and extraction time was 5 min. The extracted sample was filtered with Whatman No. 2 filter paper, and the filtered extract was concentrated with a vacuum rotary condenser to remove solvent components to obtain an ultra-high pressure extract of Platycodon grandiflorum.

(21) <1-7> Preparation of the Supercritical Fluid Extract of Platycodon grandiflorum

(22) The dried Platycodon grandiflorum was pulverized with a mixer, and 1 g of the milled Platycodon grandiflorum sample was filled into a sample cartridge and extracted with a supercritical fluid extraction apparatus (SFX 3560, Isco Inc., Lincoln, Nebr., USA). Supercritical fluid extraction conditions were extraction pressure 40 MPa, extraction temperature 50 C., supercritical carbon dioxide flow rate 60 mL/min, and extraction time 60 min. When the supercritical fluid extraction was completed, the supercritical fluid condition was released by lowering the pressure of the extraction device to obtain a supercritical fluid extract of Platycodon grandiflorum.

(23) <1-8> Preparation of the Subcritical Fluid Extract of Platycodon grandiflorum

(24) The dried Platycodon grandiflorum was pulverized with a mixer, and 1 g of milled. Platycodon grandiflorum sample was added to 10 mL of distilled water and extracted with a subcritical fluid extractor (DIONEX Accelerated Solvent Extractor 100, DIONEX co., USA). The subcritical fluid extraction condition was extraction pressure 2.5 MPa, the extraction temperature 150 C., and the extraction time 15 minutes. The extracted sample was filtered with Wattman No. 2 filter paper, and the filtered extract was lyophilized at 40 C. to obtain a subcritical fluid extract of Platycodon grandiflorum.

Example 2

(25) Effect of Platycodon grandiflorum Extract on Increasing the Synthesis of Muscle Proteins

(26) Muscle cell L6 myoblasts (ATCC, Manassas, Va., USA) were added to a 6-well plate with Dulbecco's modified Eagle's Media (DMEM; Hyclone) containing 10% fetal bovine serum (FBS; Hyclone, Logan, Utah, USA) at a concentration of 110.sup.5 cells/ml. When, the cell density reached about 80 to 85%, the medium in the wells was removed, and 10 g/mL of the Platycodon grandiflorum extract of Examples 1-1 to 1-5, respectively, were added to DMEM containing 2% horse serum (HS; Hyclone) for the purpose of inducing myotube differentiation. Meanwhile, a group treated with 0.01% DMSO instead of the sample extract was used as a control group. This process was carried out for 6 days in two days to differentiate and then dissolved in NP-40 buffer solution (ELPIS-Biotech, Daejeon, Republic of Korea) containing proteinase inhibitor cocktail. The cells dissolved in the buffer solution were transferred to a 1.5 ml tube and centrifuged at 13,000 rpm for 10 minutes to take only a supernatant. The supernatant was quantified using the Bradford method (Bio-Rad Laboratories Inc., Hercules, Calif., USA). The quantified proteins were boiled for 5 minutes, separated by electrophoresis on 10% SDS-PAGE, and the separated, proteins were transferred to the nitrocellulose membrane. p-mTOR primary antibodies (Cellular signaling technology, Beverly, Mass., USA) were diluted with 2.5% bovine serum albumin (BSA) at a ratio of 1:1000 and reacted with proteins transferred to the nitrocellulose membrane for 20 hours at room temperature. The nitrocellulose membrane incubated with the diluted primary antibodies was washed three times for 10 minutes using Tris-buffer Saline Tween 20 (TEST). After-washing, the anti-rabbit secondary antibodies (Bethyl Laboratories, Inc., Montgomery, Tex., USA) conjugated with horseradish peroxidase, which recognizes the primary antibodies, were diluted to 1:5000 in 2.5% BSA, followed by reacting with the nitrocellulose membrane at room temperature for 2 hours and washing three times for 10 minutes using TBST. Protein bands were developed using ECL western, blotting detection reagents (Amersham, Tokyo, Japan), while the developed protein bands were identified using G:BOX EE imaging system (Syngene, Cambridge, UK). The results are shown in FIG. 1.

(27) As shown, in FIG. 1, it was confirmed that the expression level of p-mTOR in L6 muscle cells was increased by the treatment of the Platycodon grandiflorum extract. This verifies that the Platycodon grandiflorum extract of the present invention is excellent in increasing the production of muscle in muscle cells.

Example 3

(28) Effect of the Methanol Extract of Platycodon grandiflorum on Increasing the Synthesis of Muscle Proteins

(29) Experiments were conducted in the same manner as in Example 2, except that the methanol extract of Platycodon grandiflorum of Example 1-1 was used at a concentration of 1 g/ml and 10 g/ml. The protein band was confirmed by reacting with p-mTOR primary antibodies (Cell signaling technology) and p-p70S6K antibodies (Santa Cruz Biotechnology, Santa Cruz, Calif., USA).

(30) As shown in FIG. 2, it was observed that the expression of p-mTOR and p-p70S6K was increased respectively in L6 muscle cells by treating the methanol extract of Platycodon grandiflorum. This verifies that the methanol extract of Platycodon grandiflorum of the present invention is excellent in increasing the production of muscle in muscle cells.

Example 4

(31) Effect of Platycodon grandiflorum Extract on Muscle Differentiation

(32) Muscle cells, L6 myoblasts (ATCC) were added to a 6-well plate at 210.sup.5 cells/ml together with DMEM (Hyclone) containing 10% FBS (Hyclone). When the cell density reached about 80 to 85%, the medium in the wells was removed. The cells were treated with the hexane extracts, the ethylacetate extract, the ethanol extract and the hot water extract of Platycodon grandiflorum prepared in Examples 1-2 to 1-5, respectively, dissolved in DMEM (Hyclone) containing 2% HS (Hyclone) at a concentration of 10 g/mL to induce myotube differentiation. Meanwhile, a group treated with 0.01% DMSO instead of the sample extract was used as a control group. This procedure was carried out for 6 days in two days to differentiate and total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, Calif., USA). The isolated total RNA was quantified using NanoDrop 1000 (Thermo Fisher Scientific Inc., Mass., USA). Quantified 16 L of RNA was synthesized into cDNA using Reverse Transcriptase Premix (ELPIS-Biotech) and PCR machine (Gene Amp PCR System 2700; Applied Biosystems, Mass., USA) at 42 C. for 55 minutes and 70 C. for 15 minutes. 4 L out of 16 L cDNA was amplified by PCR using the following specific primers (Bioneer, Daejeon, Republic of Korea) and PCR premix (ELPIS-Biotech). PCR was performed by repeating 30 cycles of 95 C. for 30 seconds, 60 C. for 1 minute, and 72 C. for 1 minute.

(33) TABLE-US-00001 MyoD (SEQIDNO:1) Forwardprimer:5-TTTCGACTCACCAGACCTGC-3 (SEQIDNO:2) Reverseprimer:5-CAGAGCCTGCAGACCTTCAA-3 Myogenin (SEQIDNO:3) Forwardprimer:5-TTTCGCACCTGATGGACCTG-3 (SEQIDNO:4) Reverseprimer:5-CTTTCTTGAGCCTGCGCTTC-3 -Actin: (SEQIDNO:5) Forwardprimer:5-AGCCATGTACGTAGCCATCC-3 (SEQIDNO:6) Reverseprimer:5-CTCTCAGCTGTGGTGCTGAA-3

(34) PCR amplified cDNA was separated by electrophoresis on 1.5% agarose gel and cDNA band was identified using G:BOX EF imaging system (Syngene). The results are shown in FIG. 3.

(35) As shown in FIG. 3, it was observed that the expression of MyoD and myogenin mRNA was increased in L6 muscle cells by treating the hexane extract, the ethyl acetate extract, the ethanol extract, and the hot water extract of Platycodon grandiflorum. This verifies that the extracts of Platycodon grandiflorum of the present invention are excellent in promoting muscle differentiation in muscle cells.

Example 5

(36) Effect of the Methanol Extract of Platycodon grandiflorum on Muscle Differentiation

(37) L6 myoblast (ATCC) was cultured in the same manner as in Example 4. The methanol extract of Platycodon grandiflorum prepared in Example 1-1 was dissolved in DMEM (Hyclone) containing 2% HS (Hyclone) at a concentration of 1 g/mL and 10 g/mL, followed by its treatment on said cells to induce myotube differentiation. Meanwhile, a group treated with 0.01% DMSO instead of the sample was used as a control group. RT-PCR was performed in the same manner as in Example 4, after proceeding for 6 days in 2 days to differentiate.

(38) As shown in FIG. 4, it was observed that the expression of MyoD and myogenin mRNA was increased in L6 muscle cells by treating the methanol extract of Platycodon grandiflorum. This verifies that the methanol extract of Platycodon grandiflorum of the present invention is excellent in promoting muscle differentiation in muscle cells.

Example 6

(39) Inhibitory Effect of Platycodon grandiflorum on Muscle Protein Degradation

(40) L6 myoblast (ATCC) was added to a 6-well plate at 210.sup.5 cells/ml together with DMEM (Hyclone) containing 10% FBS (Hyclone). When the cell density reached about 80% to 85%, the medium in the wells was removed and myotube differentiation was induced by treating the cells with DMEM (Hyclone) containing 2% HS (Hyclone). The medium was replaced with fresh medium every two days and the differentiation was carried out for a total of 6 days. After differentiation, 10 g/mL of the hexane extract, the ethylacetate extract, the ethanol extract and the hot water extract of Platycodon grandiflorum prepared in Examples 1-2 to 1-5 were respectively dissolved in DMEM (Hyclone) containing 50 ng/mL tumor necrosis factor alph (TNF-; PeproTech, Rocky Hills, N.J., USA), followed by their treatment on the cells. After 6 hours, total RNA was isolated using TRIzol reagent (Invitrogen). The isolated total RNA was quantified using NanoDrop 1000 (Thermo Fisher Scientific Inc.). The quantified 16 L of RNA was synthesized into cDNA using Reverse Transcriptase Premix (ELPIS-Biotech) and PCR machine (Gene Amp PCR System 2700; Applied Biosystems) at 42 C. for 55 minutes and at 70 C. for 15 minutes. The PCR was performed by repeating 30 cycles at 95 C. for 30 seconds, at 60 C. for 1 minute and at 72 C. for 1 minute with 4 l of 16 l cDNA, the following specific primers (Bioneer), and PCR premix (ELPIS-Biotech):

(41) TABLE-US-00002 Atrogin-1 (SEQIDNO:7) Forwardprimer:5-CCCTGAGTGGCATCGCCCAA-3 (SEQIDNO:8) Reverseprimer:5-AGGTCCCGCCCATCGCTCA-3 MuRF-1 (SEQIDNO:9) Forwardprimer:5-GAAATGCTATGCAGAACCTG-3 (SEQIDNO:10) Reverseprimer:5-ATTCCTGCTTGTAGATGTCG-3 -Actin: (SEQIDNO:11) Forwardprimer:5-AGCCATGTACGTAGCCATCC-3 (SEQIDNO:12) Reverseprimer:5-CTCTCAGCTGTGGTGCTGAA-3

(42) PCR amplified cDNA was separated by electrophoresis on 1.5% agarose gel and cDNA band was identified using G:BOX EF imaging system (Syngene). The results are shown in FIG. 5.

(43) As shown in FIG. 5, it was found that mRNA expression of atrogin-1 and MuRF-1 in L6 muscle cells was decreased by treating the Platycodon grandiflorum extracts. This verifies that the extracts of Platycodon grandiflorum of the present invention are excellent in inhibiting the degradation of muscle proteins in muscle cells.

Example 7

(44) Effect of the High Pressure Extracts of Platycodon grandiflorum on Muscle Formation

(45) The ultra-high pressure extract of Platycodon grandiflorum prepared in Example 1-6, the supercritical fluid extract of Platycodon grandiflorum prepared in Example 1-7, and the subcritical fluid extract of Platycodon grandiflorum prepared in Example 1-8 were respectively treated at a concentration of 20 ppm on muscle cells in the same manner as in Example 2. The p-mTOR protein band was developed using ECL Western Blotting Detection Reagents (Amersham, Tokyo, Japan) and a density of the developed protein band was measured using G:BOX EF imaging system (Syngene, Cambridge, UK). The density of the relative protein bands in the experimental groups treated with the samples was expressed as a percentage (%) based on the protein band of the control group of which density is designated as 100%. The results are shown in Table 1 below.

(46) TABLE-US-00003 TABLE 1 Effect of the high pressure extracts of Platycodon grandiflorum in enhancing the expression level of p-mTOR protein Experimental groups Relative density (%) Control group 100 Example 1-6 130 Example 1-7 127 Example 1-8 136

(47) As shown in the Table 1, it was confirmed that the ultra-high pressure extract, the supercritical fluid extract, and the subcritical fluid extract of Platycodon grandiflorum respectively enhanced the protein expression level of p-mTOR which is a major gene involved in the improvement of muscle function.

Example 8

(48) Evaluation on the Effect of Increasing Muscle Mass in Animal Models

(49) Five-week-old Wistar rats were adapted for 1 week and 100 ng/g of TNF- was supplied for 2 weeks to induce muscular atrophy. Then, groups were randomly assigned on the basis of body weight and divided into three groups of 8 rats per each group. 500 mg/kg body weight of the ethanol extract of Platycodon grandiflorum prepared in Example 1-2 and 500 mg/kg body weight of the hot-water extract of Platycodon grandiflorum prepared in Example 1-5 were suspended in 0.25% carboxymethylcellulose, respectively. Experimental groups were administrated with each suspended extracts once a day for 8 weeks at regular time. A control group was administered with TNF- suspended in the same amount of 0.25% carboxymethyl cellulose ingested by the experimental groups.

(50) After 8 weeks of administration, the muscles under the right calf were excised and weighed with microbalance (Mettler PE 160, USA). As a result, as shown in Table 2, the weight of muscle was significantly (p<0.01) increased by 21.18% and 18.23% in the group administered with the ethanol extract and the hot-water extract of Platycodon grandiflorum, respectively. These results indicate that the extracts of Platycodon grandiflorum according to the present invention are effective for increasing muscle mass.

(51) TABLE-US-00004 TABLE 2 Weight of calf muscle per treated material Average weight of calf Experimental groups muscle (mg) Control group 406 20.3 The ethanol extract Platycodon 492 31.6 grandiflorum The hot water extract Platycodon 480 28.7 grandiflorum

(52) Hereinafter, there are disclosed preparation examples for preparing a pharmaceutical composition for preventing and treating muscle diseases, a food composition for preventing muscle diseases or improving muscle function, or a cosmetic composition for improving muscle function, the compositions comprising the Platycodon grandiflorum extract of Example 1 according to the present invention as an active ingredient. However, these preparation examples are not intended to be limiting the present invention by any means, but merely to be illustrative. The pharmaceutical, food or cosmetic compositions of Preparation Examples 1 to 3 were prepared according to the conventional methods using the following components and composition ratios.

<Preparation Examples 1> Food

(53) <1-1> Preparation of Health Food

(54) 1000 mg of the Platycodon grandiflorum extract of Example 1, 70 g of vitamin A acetate, 1.0 mg of vitamin E, 0.13 mg of vitamin B1, 0.15 mg of vitamin B2, 0.5 mg of vitamin B6, 0.2 g of vitamin B12, 10 mg of vitamin C, 10 g of biotin, 1.7 mg of Nicotinic amide, 50 g of folic acid, 0.5 mg of calcium pantothenate, 1.75 mg of ferrous sulfate, 0.82 mg of zinc oxide, 25.3 mg of magnesium, carbonate, 15 mg of potassium monophosphate, 55 mg of dibasic calcium phosphate, 90 mg of potassium, citrate, 100 mg of calcium carbonate, and 24.8 mg of magnesium chloride may be mixed to prepare a health food, while the composition ratios may be modified. After mixing the above components according to general health food manufacturing methods, granules are prepared for use in the manufacture of a health food composition according to conventional methods.

(55) <1-2> Preparation of Health Drinks

(56) 1000 mg of the Platycodon grandiflorum extract of Example 1, 1000 mg of citric acid, 100 g of oligosaccharide, 2 g of plum concentrate and 1 g of taurine were added to purified water, followed by mixing to obtain a total of 900 ml of health drink according to general health drink manufacturing methods. After stirring for about 1 hour at 85 C., the resulting solution was filtered and placed in a sterilized 2 L container, sealed, sterilized and refrigerated for use in the manufacture of a health drink composition.

(57) <1-3> Chewing Gum

(58) After mixing 20 wt % of gum base, 76.9 wt % of sugar, 1 wt % of flavor, 2 wt % of water and 0.1 wt % of the Platycodon grandiflorum extract of Example 1, chewing gum was prepared according to well-known methods in the art.

(59) <1-4> Candy

(60) After mixing 60 wt % of sugar, 39.8 wt % of starch syrup, 0.1 wt % of flavor and 0.1 wt % of the Platycodon grandiflorm extract of Example 1, candy was prepared according to well-known methods in the art.

(61) <1-5> Biscuit

(62) After mixing 25.59 wt % of first grade soft flour, 22.22 wt % of first grade plain flour, 4.80 wt % of refined sugar, 0.73 wt % of salt, 0.78 wt % of glucose, 11.78 wt % of palm shortening, 1.54 wt % of ammonium, 0.17 wt % of sodium bicarbonate, 0.16 wt % of sodium sulfite, 1.45 wt % of rice powder, 0.0001 wt % of vitamin B, 0.04 wt % of milk flavor, 20.6998 wt % of water, 1.16 wt % of whole milk powder, 0.29 wt % of alternative milk powder, 0.03 wt % of calcium phosphate I, 0.29 wt % of sulfuric salt, 7.27 wt % of spray milk and 1.0 wt % of the Platycodon grandiflorum extract of Example 1, biscuit was prepared according to well-known methods in the art.

<Preparation Example 2> Therapeutics

(63) <2-1> Powder

(64) 50 mg of the Platycodon grandiflorm extract of Example 1 and 2 g of crystalline cellulose were mixed thoroughly and then, filled and sealed in a sealed package to obtain a powder preparation.

(65) <2-2> Tablets

(66) 50 mg of the Platycodon grandiflorm extract of Example 1, 400 mg of crystalline cellulose and 5 mg of magnesium stearate were mixed thoroughly and formulated into tablet according to conventional methods to obtain a tablet preparation.

(67) <2-3> Capsules

(68) 30 mg of the Platycodon grandiflorum extract of Example 1, 100 mg of whey protein, 400 mg of crystalline cellulose, and 6 mg of magnesium stearate were mixed thoroughly and filled in a gelatin capsule according to conventional methods to obtain a capsule preparation.

<Preparation Example 3> Cosmetics

(69) <3-1> Nourishing Lotion (Milk Lotion)

(70) According to conventional methods, nourishing lotion was prepared with the Platycodon grandiflorum extract of Example 1 and the following components at a ratio as shown in Table 3 below.

(71) TABLE-US-00005 TABLE 3 Composition ingredients Preparation Example 3-1 (wt %) Platycodon grandiflorum 2.0 extract Squalane 5.0 Wax 4.0 Polysorbate 60 1.5 Sorbitan sesquioleate 1.5 Liquid paraffin 0.5 Caprylic/capric triglyceride 5.0 Glycerin 3.0 Butylene glycol 3.0 Propylene glycol 3.0 Carboxyvinyl polymer 0.1 Triethanolamine 0.2 Preservatives, Coloring, q.s. Fragrance Purified water To 100

(72) <3-2> Softening Lotion (Skin Lotion)

(73) According to conventional methods, skin lotion was prepared with the Platycodon grandiflorum extract of Example 1 and the following components at a ratio as shown in Table 4 below.

(74) TABLE-US-00006 TABLE 4 Composition ingredients Preparation Example 3-2 (wt %) Platycodon grandiflorum 2.0 extract Glycerin 3.0 Butylene glycol 2.0 Propylene glycol 2.0 Carboxyvinyl polymer 0.1 PEG 12 Nonyl phenyl ether 0.2 Polysorbate 80 0.4 Ethanol 10.0 Triethanolamine 0.1 Preservatives, Coloring, q.s. Fragrance Purified water To 100

(75) <3-3> Nourishing Cream

(76) According to conventional methods, nourishing cream was prepared with the Platycodon grandiflorum extract of Example 1 and the following components at a ratio shown in Table 5 below.

(77) TABLE-US-00007 TABLE 5 Composition ingredients Preparation Example 3-3 (wt %) Platycodon grandiflorum 2.0 extract Polysorbate 60 1.5 Sorbitan sesquioleate 0.5 PEG60 Hardened castor oil 2.0 Liquid paraffin 10 Squalane 5.0 Caprylic/capric triglyceride 5.0 Glycerin 5.0 Butylene glycol 3.0 Propylene glycol 3.0 Triethanolamine 0.2 Preservatives q.s. Coloring q.s. Fragrance q.s. Purified water To 100

(78) <3-4> Massage Cream

(79) Massage cream was prepared according to conventional methods by using the Platycodon grandiflorum extract of Example 1 and the following components at a ratio as shown in Table 6 below.

(80) TABLE-US-00008 TABLE 6 Composition ingredients Preparation Example 3-4 (wt %) Platycodon grandiflorum 1.0 extract Wax 10.0 Polysorbate 60 1.5 PEG60 Hardened castor oil 2.0 Sorbitan sesquioleate 0.8 Liquid paraffin 40.0 Squalane 5.0 Caprylic/capric 4.0 triglyceride Glycerin 5.0 Butylene glycol 3.0 Propylene glycol 3.0 Triethanolamine 0.2 Preservatives, coloring, q.s. fragrance Purified water To 100

(81) <3-5> Packs

(82) A pack was prepared according to conventional methods at a composition ratio shown in Table 7 below.

(83) TABLE-US-00009 TABLE 7 Composition ingredients Preparation Example 3-5 (wt %) Platycodon grandiflorum 1.0 extract Polyvinyl alcohol 13.0 Sodium 0.2 carboxymethylcellulose Glycerin 5.0 Allantoin 0.1 Ethanol 6.0 PEG12 nonyl phenyl ether 0.3 Polysorbate 60 0.3 Preservatives, coloring, q.s. fragrance Purified water To 100

(84) <3-6> Gels

(85) A gel was prepared according to conventional methods by using the Platycodon grandiflorum extract of Example 1 and the following components at a ratio shown in Table 8 below.

(86) TABLE-US-00010 TABLE 8 Composition ingredients Preparation Example 3-6 (wt %) Platycodon grandiflorum 0.5 extract Ethylenediamine sodium 0.05 acetate Glycerin 5.0 Carboxyvinyl polymer 0.3 Ethanol 5.0 PEG60 Hardened castor oil 0.5 Triethanolamine 0.3 Preservatives, Coloring, q.s. Fragrance Purified water To 100