US 2013 0045289A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2013/0045289 A1 Cho et al. (43) Pub. Date: Feb. 21, 2013

(54) COMPOSITION FOR PROMOTING THE (30) Foreign Application Priority Data ACTIVITY OF PEROXSOME PROLIFERATOR-ACTIVATED Nov. 26, 2009 (KR) ...... 10-2009-O115O24 RECEPTOR-DELTA Publication Classification (75) Inventors: Si Young Cho, Seoul (KR); Pil Joon (51) Int. Cl. Park, Yongin-si (KR); Ji Hae Lee, A61E36/282 (2006.01) Yongin-si (KR); Dae Bang Seo, A6IP 25/28 (2006.01) Yongin-si (KR); Sang Jun Lee, A6IP 25/6 (2006.01) Seongnam-si (KR) A6IP3/00 (2006.01) (73) Assignee: AMOREPACIFIC CORPORATION (52) U.S. Cl...... 424/740 (57) ABSTRACT (21) Appl. No.: 13/511,964 Disclosed is a composition for promoting the activity of per oxisome proliferator-activated receptor-ö (PPAR-8), which (22) PCT Filed: Nov. 25, 2010 contains vulgaris extracts or Artemisia capillaris extracts as active ingredients. The composition is effective in (86) PCT NO.: PCTAKR1O/O84O4. strengthening muscles, improving endurance and memory, S371 (c)(1), and preventing and alleviating the symptoms of dementia or (2), (4) Date: May 24, 2012 Parkinson's disease. Patent Application Publication Feb. 21, 2013 Sheet 1 of 4 US 2013/0045289 A1

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COMPOSITION FOR PROMOTING THE memory. The composition may be variously used in the field ACTIVITY OF PEROXSOME of food, health-food Supplements or pharmaceuticals. PROLIFERATOR-ACTIVATED RECEPTOR-DELTA DESCRIPTION OF DRAWINGS 0009 FIG. 1 shows fluorescence data showing PPAR-8- TECHNICAL FIELD LBD binding affinity of Artemisia iwayOmogi extract in a test 0001. The present disclosure relates to a composition tube: including Artemisia extract as an active ingredient. 0010 FIG. 2 shows expression level of the reporter gene luciferase as a result of PPAR-ö activation by Artemisia iway BACKGROUND ART Omogi extract; 0002 Peroxisome proliferator-activated receptors 0011 FIG. 3 shows expression level of the target genes of (PPARS) are ligand-activated transcription factors belonging PPAR-8, CPT1 Band PDK4, in muscle cells treated with 200 to the nuclear receptor Superfamily activated by fatty acids. ug/mL Artemisia iwayOmogi extract; and The PPAR family consists of PPAR-C, PPAR-B/8 and PPAR 0012 FIG. 4 shows fatty acid oxidation in muscle cells Y, which show different ligand specificity and tissue distribu promoted as a result of treating with 200 g/mL Artemisia tion. PPAR-C. is expressed the most in the liver and promotes iwayOmogi extract. fatty acid oxidation in peroxisomes and mitochondria. PPAR-Y is expressed in adipose tissues and regulates Storage BEST MODE of fats. The ligand of PPAR-C, fibrate, is used as hypolipi 0013. A composition according to an exemplary embodi demic agent, and the ligand of PPAR-Y, thiazolidinedione, is ment of the present disclosure comprises Artemisia extract as used in the treatment of diabetes mellitus type 2. an active ingredient. The composition comprising. Artemisia 0003 PPAR-8 is expressed in muscles, brown adipose extract promotes the activity of peroxisome proliferator-acti tissues, etc. This transcription factor shows anti-obesity effect vated receptor delta (PPAR-8) and is effective in promoting since fatty acid oxidation in adipocytes is promoted in mice in muscular metabolism and/or improving memory. which it is overexpressed (Wang Y X et al., Cell, 113, pp. 0014. In the present disclosure, Artemisia (ssuk, Artemisia 159-170, 2003). An activator of PPAR-8 promotes metabo princeps) collectively refers to dicotyledonous perennial lism in skeleton muscle cells, improves insulin sensitivity, grasses belonging to the family of the order reduces adipocytes and inhibits inflammatory response by . Artemisia grows naturally in the whole area of increasing expression of such proteins as carnitine palmitoyl and has been widely used for food as Vegetable, Soup, transferase f (CPT1B) and pyruvate dehydrogenase kinase porridge, cake, etc. isozyme 4 (PDK4) (Barish G D et al., J. Clin. Invest., 116, pp. 0015. In an exemplary embodiment, the Artemisia extract 590-597, 2006, 116:590). When the ligand of PPAR-O, may be Artemisia iwayOmogi extract. Artemisia iwayOmogi is GW501516, was administered together with the AMP-acti a perennial grass belonging to the genus Artemisia of family vated protein kinase (AMPK) activator AICAR to a mouse, Asteraceae. It is also called Saengdang SSuk, aedang SSuk, the mouse showed strengthened muscles even without exer Sacheol SSuk, injincho, heat reliever, heuinsan SSuk or teolsan cise as well as anti-obesity effect (Vihang A Net al., Cell, 134, SSuk. In particular, the young leaves are called injin or heat pp. 1-11, 2008). reliever and traditionally have been used as food or folk 0004. However, activator or ligand of PPAR-8 with proven medicine owing to peculiar fragrance and medicinal effect. stability has not been found yet. The inventors of the present Injin, the young bud of sacheol SSuk which is a perennial grass disclosure have found out that Artemisia iwayOmogi extract belonging to the family Asteraceae, has been known from old contains a novel ligand capable of activating PPAR-8. times to be beneficial for the liver. Especially, it is known to be highly effective in jaundice. Injin promotes secretion of bile DISCLOSURE and clears the liver by excreting lumps, cholic acid and biliru bin in the bile. Also, it reduces blood pressure, relieves fever, Technical Problem and kills various germs including tubercle bacillus. In addi tion, it is effective in degrading lipids, dilating coronary arter 0005. An embodiment of the present disclosure is directed ies and promoting urination. to providing a composition including Artemisia extract. 0016. In an exemplary embodiment, the composition has 0006 Another embodiment of the present disclosure is the effect of regulating the expression of enzymes that pro directed to providing food, health-food Supplement and phar mote fat and Sugar metabolism in muscle cells. Specifically, maceutical compositions that include the composition the composition which comprises Artemisia princeps or Arte including Artemisia extract. misia iwayomogi extract as an active ingredient is effective in promoting the expression of proteins that promote fat and Technical Solution Sugar metabolism in muscles, as ligand activating PPAR-ö. In 0007. In one general aspect, the present disclosure pro an exemplary embodiment, the composition may be a com vides a composition including Artemisia princeps or Artemi position for strengthening muscles and/or improving endur ance. In another exemplary embodiment, the composition sia iwayOmogi extract as an active ingredient. may be a composition for preventing and/or alleviating the symptoms of dementia or Parkinson's disease. These effects Advantageous Effects may beachieved through promotion of the PPAR-8 activity. 0008. The composition according to the present disclosure 0017. In an exemplary embodiment, the composition may has the effect of promoting the activity of PPAR-8 and is comprise 1-100 wt %, specifically 1-80 wt % of the Artemisia effective in promoting muscular metabolism and improving princeps or Artemisia iwayOmogi extract based on the total US 2013/0045289 A1 Feb. 21, 2013 weight of the composition. More specifically, the Artemisia 0022. The pharmaceutical composition according to the princeps or Artemisia iwayOmogi extract may be included in present disclosure may be administered orally or parenterally, an amount of 10-60 wt % based on the total weight of the e.g. rectally, topically, transdermally, intravenously, intra composition. The above-described content of the Artemisia muscularly or Subcutaneously. princeps or Artemisia iwayOmogi extract is an effective con 0023. A pharmaceutically acceptable dosage, i.e. admin tent for activating PPAR-8. istration dosage, of the active ingredient will vary depending 0018. A process for preparing the Artemisia princeps or on the age, gender and body weight of the Subject, particular Artemisia iwayOmogi extract according to the present disclo disease or pathological condition to be treated, severity of the Sure is not particularly limited. The Artemisia princeps or disease or pathological condition, administration route and Artemisia iwayOmogi extract may be extracted using water or discretion of a diagnoser. Determination of the administration an organic solvent. The organic solvent may be selected from, dosage considering these factors is in the level of those skilled for example, a group consisting of ethanol, methanol, in the art. A general administration dosage may be 0.01-2000 butanol, ether, ethyl acetate, chloroform and a mixture of the mg/kg/day, specifically 1-100 mg/kg/day. However, this organic solvent with water. For example, the Artemisia prin administration dosage does not limit the scope of the present ceps or Artemisia iwayOmogi extract may be obtained by disclosure by any means. repeating a procedure of adding 70% ethanol (3 L) to Arte 0024. The composition according to the present disclosure misia princeps or Artemisia iwayOmogi (300g) and stirring at may be prepared into various types of composition for exter 70-80° C. for 3 hours 2 times. Thus obtained extract may be nal skin application, more specifically a cosmetic composi filtered through Whatman No. 1 filter paper and freeze dried tion. For example, it may be prepared into softening lotion, at -70° C. to obtain the Artemisia princeps or Artemisia astringent lotion, nourishing lotion, eye cream, nourishing iwayomogi extract in Solid form. cream, massage cream, cleansing cream, cleansing foam, 0019. The composition according to an exemplary cleansing water, powder, essence, pack, etc., but is not par embodiment of the present disclosure is applicable not only to ticularly limited thereto. animals but also to human. The present disclosure also pro 0025. The composition according to the present disclosure vides a food or health-food Supplement composition compris which contains Artemisia princeps or Artemisia iwayOmogi ing the above-described composition. The food or health extract as an active ingredient has the effect of promoting the food Supplement composition may be prepared, for example, activity of PPAR-8. The effect of a substance promoting the into tablet, granule, drink, caramel, diet bar, tea powder, typi activity of PPAR-O of strengthening muscles, improving cal tea bag, etc., but are not particularly limited thereto. The endurance and memory, and preventing or alleviating the compositions may be prepared by those skilled in the art symptoms of dementia or Parkinson's disease is known in the without special difficulty using the active ingredient and other art. Experimental details can be found in Korean Patent ingredients commonly used in the art considering particular Application Publication Nos. 10-2008-0050348 and 2008 preparation type or purpose of use. A synergic effect may be OO65234. achieved when the active ingredient is used in combination with other ingredients. MODE FOR INVENTION 0020. The present disclosure also provides a pharmaceu 0026. The examples and experiments will now be tical composition comprising the above-described composi described. The following examples and experiments are for tion. The pharmaceutical composition may further comprise a illustrative purposes only and not intended to limit the scope pharmaceutical adjuvant such as preservative, stabilizer, wet of this disclosure. ting agent or emulsifier, Salt for osmotic control and/or buffer or other therapeutically useful Substance, and may be formu Example 1 lated into various oral or parenteral administration forms according to methods known in the art. Preparation of Artemisia iwayOmogi Extract Artemisia iwayOmogi cultivated in 0021 Formulations for oral administration include, for Chengcheon-dong, Jecheon-si, Chungcheongbuk-do, example, tablet, pill, hard and soft capsule, liquid, Suspen Korea was purchased. After adding 70% ethanol (3 Sion, emulsion, syrup, powder, dust, fine granule, granule, L) to Artemisia iwayOmogi (300 g), the mixture was pellet, etc. These formulations may comprise a surfactant, a stirred at 70-80°C. for 3 hours. This procedure was diluent (e.g. lactose, dextrose, Sucrose, mannitol, Sorbitol, repeated 2 times and extract was obtained by cellulose or glycine) and a lubricant (e.g. silica, talc, Stearic filtering through filter paper. The filtrate was acid and its magnesium or calcium salt, or polyethylene gly concentrated under reduced pressure using a rotary col) in addition to the active ingredient. Further, the a tablet vacuum evaporator and then freeze dried to obtain may comprise a binder (e.g. magnesium aluminum silicate, dry powder (29 g). starch paste, gelatin, tragacanth, methyl cellulose, sodium carboxymethyl cellulose or polyvinylpyrrolidone) and, as Test Example 1 occasion demands, it may also comprise a pharmaceutical additive Such as a disintegrant, an absorbent, a colorant, a PPAR-8-LBD Binding Affinity of Artemisia flavor, a Sweetener, etc., e.g. starch, agar, alginic acid or its iwayOmogi Extract sodium salt. The tablet may be prepared by the common mixing, granulation or coating method. And, formulations for 0027 PPAR-8 is activated when a ligand is attached to the parenteral administration include, for example, injection, ligand binding domain (LBD) of this protein and regulates medicinal drop, ointment, lotion, gel, cream, spray, Suspen expression of other genes. Therefore, a Substance activating Sion, emulsion, Suppository or patch, but are not limited this transcription factor should be able to bind to the LBD. In thereto. order to investigate whether Artemisia iwayOmogi extract can US 2013/0045289 A1 Feb. 21, 2013

activate PPAR-8, the following experiment was performed hours and, after washing 2 times with cold saline, RNA was using the Artemisia iwayOmogi extract prepared in Example extracted using TRIZol agent (Invitrogen). Then, cDNA was 1. synthesized using the extracted lug/LL RNA, using a reverse 0028. The Artemisia iwayOmogi extract was dissolved in transcription PCR system (Promega). dimethylsulfoxide (DMSO) and PPAR-8-LBDbinding affin 0035 Expression level of genes such as CPT1 B, PDK4 ity was quantified by measuring fluorescence using an assay and GAPDH was measured using the synthesized cDNA as kit (LanthaScreen TR-FRET peroxisome proliferator recep well as a primer and a probe designed for the genes (Applied tor delta coactivator assay kit; Invitrogen, CA). Biosystems; CPT1B, Mm00487200 m 1, PDK4, 0029. The result is shown in FIG.1. As seen from FIG. 1, Mm00447181 ml, GAPDH, Mm.99999915 q1). The PCR fluorescence intensity increased in a manner dependent on the reaction and analysis were performed using the Rotor-Gene concentration of the Artemisia iwayOmogi extract. The ECso 3000 system (Corbett Research, Sidney, Australia). value for PPAR-O-LEBD ligand of the Artemisia iwayomogi 0036. The result is shown in FIG. 3. As seen from FIG. 3, extract was 7 ug/mL. it was confirmed that the Artemisia iwayOmogi extract Test Example 2 increases expression of CPT1B and PDK4 in the muscle cells when compared to control. PPAR-8 Reporter Activating Effect of Artemisia iWayOmogi Test Example 4 0030. In order to investigate whether the Artemisia iway Omogi extract actually activates PPAR-6, the following Promotion of Fatty Acid Oxidation in Muscle Cells experiment was performed using a human PPAR-8 reporter of Artemisia iwayOmogi Extract assay kit (NR1C2, PPAR-8/B) Reporter Assay kit (Indigo 0037 C2C12 muscle cells differentiated for 4 days as in Biosciences, Inc.). Test Example 3 were treated with the Artemisia iwayomogi 0031 PPAR-8 reporter cells were added to CRM-1 extract for 24 hours. The next day, the cells were washed 2 medium (Indigo BioSciences, Inc.), seeded on a 96-well plate, times with brine after removing the medium. After treating cultured for 4 hours, and then treated with the Artemisia with 1 mM L-carnitine and 3 uCi |9,10-H palmitic acid for iwayOmogi extract at concentrations of 50, 100 and 200 3 hours, excess 9,10-Hpalmitic acid was removed from the ug/mL. 1 uMGW501516 (Alexis Biochemicals, USA) well medium and the quantity of produced HO was measured known as a ligand of PPAR-6 was used as positive control, using a beta counter. As a result, it was confirmed that the cells and DMSO of /1000 volume equivalent of the medium was treated with the Artemisia iwayOmogi extract showed fatty used as negative control. After incubation at 37° C. for 20 acid oxidation increased by about 2 times as compared to the hours in a 5% CO incubator, the medium was discarded and cells treated only with DMSO (control). reaction was performed at room temperature for 10 minutes after adding Luc detection reagent. Then, the activity of 1. A method for promoting muscular metabolism compris luciferase reporter expressed by PPAR-8 was analyzed using ing administering an effective amount of Artemisia extract as a luminometer. an active ingredient to a subject in Such need, wherein the 0032. The result is shown in FIG. 2. As seen from FIG. 2, method is for promoting muscular metabolism. it was confirmed that the Artemisia iwayOmogi extract 2. The method for promoting muscular metabolism accord expresses the reporter gene more than negative control by ing to claim 1, wherein the Artemisia is Artemisia princeps or activating PPAR-8. Artemisia iwayOmogi. Test Example 3 3. The method for promoting muscular metabolism accord ing to claim 1, which regulates expression of an enzyme that Regulation of Expression of Genes Involved in Fatty promotes fat and Sugar metabolism in muscle cells. Acid and Sugar Metabolism in Muscle Cells of 4. The method for promoting muscular metabolism accord Artemisia iwayOmogi Extract ing to claim 1, which achieved through strengthening muscles 0033. In order to analyze the effect of the Artemisia iway or improving endurance. Omogi extract on the regulation of expression of genes 5. (canceled) involved in fatty acid and Sugar metabolism in muscle cells, 6. The method for promoting muscular metabolism accord the Artemisia iwayOmogi extract prepared in Example 1 was ing to claim 1, wherein the Artemisia extract is administered dissolved in DMSO and the following experiment was per in a form of a composition and the composition comprises formed. Artemisia extract in an amount of 1-80 wt % based on the total 0034. Immature C2C12 muscle cells were acquired from weight of the composition. the American Type Culture Collection (ATCC: USA) and 7. A method for improving memory comprising adminis cultured in Dulbecco's modified Eagle's medium (DMEM; tering an effective amount of Artemisia extract as an active Gibco 1210-0038) containing 10% fetal bovine serum (FBS) ingredient to a subject in Such need, wherein the method is for until 70% confluency in a 5% CO incubator while replacing improving memory. the medium every other day. The cells were induced to dif ferentiate into muscle cells by culturing in a medium contain 8. The method for improving memory according to claim 7. ing 2% horse serum (HS). After culturing for 4 days in the wherein the Artemisia is Artemisia princeps or Artemisia medium containing 2% HS, the muscle cells were treated iWayOmogi. with 200 ug/mL Artemisia iwayOmogi extract. A control 9. The method for improving memory according to claim 7. group was treated with DMSO (/1000 volume equivalent of which regulates expression of an enzyme that promotes fat the medium). The treated cells were cultured at 37° C. for 24 and Sugar metabolism in muscle cells. US 2013/0045289 A1 Feb. 21, 2013 4

10. (canceled) a composition and the composition comprises Artemisia 11. The method for improving memory according to claim extract in an amount of 1-80 wt % based on the total weight of 7, which is achieved through for preventing or alleviating the composition. dementia or Parkinson's disease. 13-15. (canceled) 12. The method for improving memory according to claim 7, wherein the Artemisia extract is administered in a form of k . . . .