US 2014.0004215A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2014/0004215 A1 BROWNELL et al. (43) Pub. Date: Jan. 2, 2014
(54) COMPOSITIONS AND METHODS FOR Publication Classification MANAGING WEIGHT (51) Int. Cl. (71) Applicants: Unigen, Inc., Cheonan-si (KR); Unigen, A 6LX36/575 (2006.01) Inc., Seattle, WA (US) A61E36/53 (2006.01) (72) Inventors: Lidia Alfaro BROWNELL, Tacoma, A61E36/85 (2006.01) WA (US); Byong-Il CHOI, A61E36/37 (2006.01) Cheongwon-gun (KR); Brandon (52) U.S. Cl. CORNELIUSEN, Tenino, WA (US); CPC ...... A61K 36/575 (2013.01); A61K 36/37 Mei-Feng HONG, Lacey, WA (US); (2013.01); A61 K36/53 (2013.01); A61 K Eu-Jin HYUN, Cheonan-si (KR); Qi 36/185 (2013.01) JIA, Olympia, WA (US); Ping JLAO, USPC ...... 424/745; 424/769 Lacey, WA (US); Hyun-Jin KIM, Asan-si (KR); Mi-Ran KIM, Cheonan-si (KR); Tae-Woo KIM, Ulsan (KR): Bo-Su LEE, Pohang-si (KR); (57) ABSTRACT Young-Chul LEE, Daejeon (KR): Jeong-Bum NAM, Cheongwon-gun (KR); Mesfin YIMAN, Kent, WA (US); The present disclosure provides Diels-Alder adducts of chal Ji-Hye Hwang, Jecheon-si (KR): cone and prenylphenyl moieties capable of modulating the Mi-Sun OH, Cheonan-si (KR) activity of cannabinoid receptors, and to oligomers of flavan 3-ol capable of modulating fat absorption and storage. Such (73) Assignees: Unigen, Inc., Cheonan-si (KR); Diels-Alder adducts of chalcone and prenylphenyl moieties UNIGEN, INC., Seattle, WA (US) or oligomers of flavan-3-ol can optionally be used in combi (21) Appl. No.: 13/904,851 nation with other weight management agents, such as anorec (22) Filed: May 29, 2013 tic agents, a lipase inhibitors, other cannabinoid receptor modulators, psychotropic agents, insulin sensitizers, stimu Related U.S. Application Data lants, or satiety agents, as well as to methods of use thereof (60) Provisional application No. 61/652,807, filed on May Such as treating or preventing weight gain or obesity, promot 29, 2012, provisional application No. 61/783,729, ing weight loss, appetite Suppression, modifying Satiety, or filed on Mar. 14, 2013. the like. Patent Application Publication Jan. 2, 2014 Sheet 1 of 3 US 2014/0004215 A1
Week O Week Week 2 Week 3 Week 4 Week 5 Week 6 Week 7 Week 8
NC ...... FD ?t X 40mg/kgOristat Yerba Mate Magnolia100mg/kg X- 500mg/kg 500mg/kgMutamba Morus Rosemary 200mgikg 500mg/kg Fig. 1 Patent Application Publication Jan. 2, 2014 Sheet 2 of 3 US 2014/0004215 A1
Week O Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Week 7 Week 8
Fig. 2 Patent Application Publication Jan. 2, 2014 Sheet 3 of 3 US 2014/0004215 A1
s
A9'709. 2->0gzgelz.108 - 182 66969. s ->6979vaz-5 G6'6092 c5 5 ->608/9 la Cd 3 699 OCZ S i. >gll 698 CN 25 7299.9A. s 9 - >99-'08g. 8 s 9A9977 2 is ->0Gv-6z. i5 E9EA9 d wa CN ->700 S le888.98 le S 8 g K S 33 Xe- w- ra o o O ne. Sueu US 2014/0004215 A1 Jan. 2, 2014
COMPOSITIONS AND METHODS FOR from a chemical library based on its inhibition of fatty acid MANAGING WEIGHT synthase, but was developed as a pancreatic triglyceride lipase inhibitor. But, orlistat has been placed on a list of drugs CROSS-REFERENCE TO RELATED having a potential signal of serious risk due to cases of liver APPLICATIONS toxicity, which has led to a change in the product labeling and 0001. This application claims the benefit under 35 U.S.C. the FDA is continuing to evaluate this issue to determine the S119(e) of U.S. Provisional Patent Application No. 61/652, need for any further regulatory action (see www.fda.gov/ 807, filed May 29, 2012, and U.S. Provisional Patent Appli Drugs/GuidanceComplianceRegulatory Information/Sur cation No. 61/783,729, filed Mar. 14, 2013, where these two veillance/Adverse DrugEffects/ucm 161063.htm). provisional applications are incorporated herein by reference 0009 From the foregoing, a need is apparent for improved in their entireties. compositions and methods for weight management. BACKGROUND BRIEF SUMMARY 0010. In brief, the present disclosure is directed to com 0002 1. Technical Field pounds and compositions useful for weight management and 0003. The present disclosure relates to compositions and related methods, including stereoisomers, pharmaceutically methods for weight management and, more particularly, to or nutraceutically acceptable salts, tautomers, glycosides and Diels-Alder adducts of chalcone and prenylphenyl moieties, prodrugs of the disclosed compounds. to oligomers of flavan-3-ol, or both, optionally in combina 0011. In certain embodiments, this disclosure provides a tion with other weight management agents, such as anorectic composition comprising a mixture of a Morus extract agents, a lipase inhibitors, cannabinoid receptor modulators, enriched for one or more Diels-Alder adducts of a chalcone psychotropic agents, insulin sensitizers, stimulants, or Satiety and a prenylphenyl moiety, a Magnolia extract, and a Yerba agents, as well as to methods of use thereof such as treating or mate extract. In further embodiments, this disclosure pro preventing weight gain or obesity, promoting weight loss, vides a composition comprising a mixture of a Morus extract appetite Suppression, modifying Satiety, or the like. enriched for Diels-Alder adducts of a chalcone and a pre 0004 2. Description of the Related Art nylphenyl moiety, a Magnolia extract, and a Mutamba 0005. Obesity is a food problem. In industrialized coun extract. In further embodiments, this disclosure provides a tries, affluence provides abundant and variable food items to composition comprising a mixture of a Morus extract the general public. Food, with the associated taste and olfac enriched for Diels-Alder adducts of a chalcone and a pre tory pleasures, is an indulgence, not just for basic Survival. As nylphenyl moiety, a Rosemary extract, and a Yerba mate a result, obesity and obesity-related health issues are increas eXtract. ing rapidly and there is a strong need for dietary Supplements 0012 Exemplary Diels-Alder adducts of a chalcone and a that help with weight control. The market size for food prenylphenyl moiety include compounds having a structure Supplements that decrease body weight is large and there are of Formula I or II: few effective products. 0006 For many years, Cannabis sativa (marijuana) has
been known to stimulate food consumption through the action of its active component, delta-9-tetrahydrocannabinol (THC), an exogenous cannabinoid. This effect prompted research into its mechanism of action. The binding sites for THC were eventually cloned and named CB1 and CB2. These receptors belong to the G-protein coupled family character ized by seven trans-membrane loop domains. Both receptors belong to Go Subclass and signal by negatively regulating cyclic AMP levels. CB1 was also shown to activate potassium channels. CB2 receptor is present in immune cells and is not involved in regulation of food consumption. CB1, the can nabinoid receptor involved in feeding behavior, is widely expressed both in brain and peripheral tissues, including adi pose tissue, skeletal muscles, liver, and gastrointestinal (GI) tract. II 0007 Most of the published CB1 receptor antagonists might be better termed “inverse agonists' as they are capable of inhibiting constitutive activity of non-occupied CB1 recep tors. The major clinical indications for this group of com pounds are obesity and substance abuse. In the past, five CB1 compounds have been tested in clinical studies. They include Rimonabant (Sanofi-Aventis, launched in 2006), MK-0364 (Merck, Phase III), Surinabant and AVE-1625 (Sanofi-Aven tis, Phase II), and SLV-319 (Solvay, Phase II). Rimonabant (marketed as AcompliaR), RimoslimTM or ZimultiR) was the first selective CB1 antagonist discovered in 1994. It was approved in 37 countries, but it has since been withdrawn from obesity treatment due to neurological side effects. 0008 Another product on the market is tetrahydrolipstatin (orlistat, sold as Alli(R) or Xenical(R). Orlistat was identified US 2014/0004215 A1 Jan. 2, 2014 or a pharmaceutically or nutraceutically acceptable salt, tau Alder adducts of a chalcone and a prenylphenyl moiety can be tomer, glycoside or stereoisomer thereof, wherein the Sub obtained or enriched from certain plants or certain plant parts, stituents are as defined herein. Such as Morus alba root bark, and can be used as a cannab 0013. In another aspect, the present disclosure provides inoid receptor (e.g., CB1, CB2) modulator. Modulation of methods for managing weight. In certain embodiments, the cannabinoid receptor activity can be helpful in managing compositions of this disclosure can be used in methods for weight or diabetes. Exemplary weight management agents treating, preventing, or managing weight gain or obesity or for use with the Diels-Alder adducts of a chalcone and a excess weight, promoting or managing weight loss, appetite prenylphenyl moiety include anorectic agents, lipase inhibi Suppression, reducing food craving, reducing eating between tors, other cannabinoid receptor modulators, psychotropic meals and in the evening hours, modifying Satiety, modifying agents, insulin sensitizers, stimulants, satiety agents, or any fat uptake or fat absorption, increasing metabolism to pro combination thereof. Furthermore, the Diels-Alder adducts mote weight loss or prevent weight gain, maintaining body of a chalcone and a prenylphenyl moiety of the present dis weight, promoting fat burn, increasing lipolysis, reducing closure, as well as compositions thereof, can be used in meth body fat or fatty tissues, increasing muscle or lean body mass, ods to treat or prevent weight associated disorders. reducing hepato-steatosis, improving fatty liver, improving 0019. In the following description, certain specific details one or more liver NASH scores, enhancing fat metabolism, are set forth in order to provide a thorough understanding of reducing the release of pro-inflammatory adipokines, various embodiments of this disclosure. However, one skilled increasing adiponectin from fat tissues, promoting a healthy in the art will understand that the invention may be practiced lipid profile (by, e.g., lowering LDL cholesterol, lowering without these details. total cholesterol, lowering triglyceride, or increasing HDL), 0020. In the present description, any concentration range, promoting glucose metabolism, reducing fasting glucose lev percentage range, ratio range, or integer range is to be under els, block absorption of carbohydrates, maintaining healthy stood to include the value of any integer within the recited glucose levels, reducing caloric intake, improving caloric range and, when appropriate, fractions thereof (such as one efficiency, reducing food intake, reducing visceral fat, reduc tenth and one hundredth of an integer), unless otherwise ing waist circumference, reducing body-to-mass index indicated. Also, any number range recited herein relating to (BMI), or any combination thereof. any physical feature, such as polymer subunits, size or thick 0014. These and other aspects of the invention will be ness, are to be understood to include any integer within the apparent upon reference to the following detailed description. recited range, unless otherwise indicated. As used herein, the terms “about” and “consisting essentially of meant.20% of BRIEF DESCRIPTION OF THE DRAWINGS the indicated range, value, or structure, unless otherwise indi cated. It should be understood that the terms “a” and “an as 0015 FIG. 1 shows agraph of mean body weights for mice used herein refer to “one or more' of the enumerated com on a high fat diet that had been treated for 8 weeks with one of ponents. The use of the alternative (e.g., “or) should be the following: Magnolia extract, Morus alba extract, understood to mean either one, both, or any combination Mutamba extract, Rosemary extract, or Yerba mate extract. thereof of the alternatives. Unless the context requires other The negative controls included mice on the high fat diet that wise, throughout the present specification and claims, the received no treatment (HFD) and mice kept on a normal diet word “comprise' and variations thereof, such as, “comprises' control (NC), while the positive control was mice on a high fat and “comprising, as well as synonymous terms like diet treated with orlistat. “include’ and "have” and variants thereof, are to be construed 0016 FIG.2 shows agraph of mean body weights for mice in an open, inclusive sense; that is, as “including, but not on a high fat diet that had been treated for 8 weeks with one of limited to. the following combination of extracts: (1) Composition 1A of 0021 Reference throughout this specification to “one Example 63 (3 components)—Magnolia (100 mg/kg): Morus embodiment' or “an embodiment’ means that a particular alba (200 mg/kg): Yerba mate (500 mg/kg); (2) Composition feature, structure or characteristic described in connection 2A (3 components)—Magnolia (100 mg/kg): Morus alba with the embodiment is included in at least one embodiment (200 mg/kg): Mutamba (500 mg/kg); (3) Composition 10 of of the present invention. Thus, the appearances of the phrases Example 68 (3 components)—Magnolia (100 mg/kg): Yerba “in one embodiment' or “in an embodiment” in various mate (500 mg/kg):Mutamba (500 mg/kg); (4) Composition places throughout this specification are not necessarily all 12 (3 components)—Morus alba (200 mg/kg): Yerba mate referring to the same embodiment. Furthermore, the particu (500 mg/kg):Mutamba (500 mg/kg); and (5) Composition 11 lar features, structures, or characteristics may be combined in of Example 73 (4 components)—Magnolia (100 mg/kg): any Suitable manner in one or more embodiments. Morus alba (200 mg/kg): Yerba mate (500 mg/kg): Mutamba 0022 “Amino” refers to the NH radical. (500 mg/kg). The negative controls included mice on the high (0023 “Cyano” refers to the CN radical. fat diet that received no treatment (HFD) and mice kept on a (0024) “Hydroxy” or “hydroxyl refers to the –OH radi normal diet (ND), while the positive control was mice on a cal. high fat diet treated with orlistat (ORI). 0025 “Imino” refers to the –NH substituent. 0017 FIG.3 shows MALDI-TOF positive ion mode mass 0026 “Nitro” refers to the NO, radical. spectrum of Mutamba fraction 84/F6 generated from 0027 “Oxo' refers to the =O substituent. Mutamba stem bark EtOH extract 84. 0028 “Thioxo’ refers to the =S substituent. 0029 “Alkyl refers to a straight or branched hydrocarbon DETAILED DESCRIPTION chain radical consisting solely of carbon and hydrogenatoms, 0018. The present disclosure provides compositions of which is saturated or unsaturated (i.e., contains one or more Diels-Alder adducts of a chalcone and a prenylphenyl moiety double or triple bonds), having from one to twelve carbon and at least one other weight management agent. The Diels atoms (C-C alkyl), or one to eight carbon atoms (C-Cs US 2014/0004215 A1 Jan. 2, 2014
alkyl) or one to six carbon atoms (C-C alkyl), and which is include, for example, cyclopropyl, cyclobutyl, cyclopentyl, attached to the rest of the molecule by a single bond, e.g., cyclohexyl, cycloheptyl, and cyclooctyl. Polycyclic radicals methyl, ethyl, n-propyl, 1-methylethyl (iso-propyl), n-butyl, include, for example, adamantyl, norbornyl, decalinyl, 7.7- n-pentyl, 1,1-dimethylethyl (t-butyl), 3-methylhexyl, 2-me dimethyl-bicyclo[2.2.1]heptanyl, and the like. Unless other thylhexyl, ethenyl, prop-1-enyl, but-1-enyl, pent-1-enyl, wise Stated specifically in the specification, a cycloalkyl penta-1,4-dienyl, ethynyl, propynyl, butynyl, pentynyl, hexy group may be optionally Substituted. nyl, and the like. Unless stated otherwise specifically in the 0037 “Cycloalkylalkyl” refers to a radical of the formula specification, an alkyl group may be optionally substituted. —RR, where R, is an alkylene chainas defined above and R. 0030) “Alkylene' or “alkylene chain” refers to a straight or is a cycloalkyl radical as defined above. Unless stated other branched divalent hydrocarbon chain linking the rest of the wise specifically in the specification, a cycloalkylalkyl group molecule to a radical group, consisting solely of carbon and may be optionally substituted. hydrogen, which is saturated or unsaturated (i.e., contains one 0038 “Fused refers to any ring structure described herein or more double or triple bonds), and having from one to which is fused to an existing ring structure in the compounds twelve carbon atoms, e.g., methylene, ethylene, propylene, of this disclosure. When the fused ring is a heterocyclyl ring n-butylene, ethenylene, propenylene, n-butenylene, propy or a heteroaryl ring, any carbon atom on the existing ring nylene, n-butynylene, and the like. The alkylene chain is structure which becomes part of the fused heterocyclyl ring or attached to the rest of the molecule through a single or double the fused heteroaryl ring may be replaced with a nitrogen bond and to the radical group through a single or double bond. atOm. The points of attachment of the alkylene chain to the rest of 0039) “Halo' or “halogen” refers to bromo, chloro, fluoro the molecule and to the radical group can be through one or iodo. carbon or any two carbons within the chain. Unless stated 0040 “Haloalkyl refers to an alkyl radical, as defined otherwise specifically in the specification, an alkylene chain above, that is Substituted by one or more halo radicals, as may be optionally substituted. defined above, e.g., trifluoromethyl, difluoromethyl, trichlo 0031 “Alkoxy” refers to a radical of the formula–OR, romethyl, 2.2.2-trifluoroethyl, 1,2-difluoroethyl, 3-bromo-2- where R is an alkyl radical as defined above containing one fluoropropyl, 1,2-dibromoethyl, and the like. Unless stated to twelve carbon atoms. Unless stated otherwise specifically otherwise specifically in the specification, a haloalkyl group in the specification, an alkoxy group may be optionally Sub may be optionally substituted. stituted. 0041) “Heterocyclyl or "heterocyclic ring” refers to a 0032 “Alkylamino” refers to a radical of the formula stable 3- to 18-membered non-aromatic ring radical which —NHR, or —NRR, where each R is, independently, an consists of two to twelve carbon atoms and from one to six alkyl radical as defined above containing one to twelve car heteroatoms selected from the group consisting of nitrogen, bonatoms. Unless stated otherwise specifically in the speci oxygen and Sulfur. Unless stated otherwise specifically in the fication, an alkylamino group may be optionally Substituted. specification, the heterocyclyl radical may be a monocyclic, 0033. “Thioalkyl refers to a radical of the formula—SR, bicyclic, tricyclic or tetracyclic ring system, which may where R is an alkyl radical as defined above containing one include fused or bridged ring systems; and the nitrogen, car to twelve carbon atoms. Unless stated otherwise specifically bon or sulfur atoms in the heterocyclyl radical may be option in the specification, a thioalkyl group may be optionally Sub ally oxidized; the nitrogen atom may be optionally quater stituted. nized; and the heterocyclyl radical may be partially or fully 0034 Aryl refers to a hydrocarbon ring system radical saturated. Examples of Such heterocyclyl radicals include comprising hydrogen, 6 to 18 carbon atoms and at least one dioxolanyl, thienyl 1.3dithianyl, decahydroisoquinolyl, aromatic ring. For purposes of this disclosure, the aryl radical imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidi may be a monocyclic, bicyclic, tricyclic or tetracyclic ring nyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, system, which may include fused or bridged ring systems. 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, Aryl radicals include aryl radicals derived from acean oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrro thrylene, acenaphthylene, acephenanthrylene, anthracene, lidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahy aZulene, benzene, chrysene, fluoranthene, fluorene, as-in drofuryl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thia dacene, S-indacene, indane, indene, naphthalene, phenalene, morpholinyl, 1-oxo-thiomorpholinyl, and 1,1-dioxo phenanthrene, pleiadene, pyrene, and triphenylene. Unless thiomorpholinyl. Unless stated otherwise specifically in the stated otherwise specifically in the specification, the term specification, Unless stated otherwise specifically in the “aryl' or the prefix "ar-' (such as in “aralkyl) is meant to specification, a heterocyclyl group may be optionally substi include aryl radicals that are optionally substituted. tuted. 0035 “Aralkyl refers to a radical of the formula - R - 0042 “N-heterocyclyl refers to a heterocyclyl radical as R, where R, is an alkylene chain as defined above and R is defined above containing at least one nitrogen and where the one or more aryl radicals as defined above, for example, point of attachment of the heterocyclyl radical to the rest of benzyl, diphenylmethyl and the like. Unless stated otherwise the molecule is through a nitrogen atom in the heterocyclyl specifically in the specification, an aralkyl group may be radical. Unless stated otherwise specifically in the specifica optionally substituted. tion, a N-heterocyclyl group may be optionally Substituted. 0036) “Cycloalkyl or “carbocyclic ring refers to a stable 0043 “Heterocyclylalkyl refers to a radical of the for non-aromatic monocyclic or polycyclic hydrocarbon radical mula—R.R. where R, is an alkylene chain as defined above consisting solely of carbon and hydrogen atoms, which may and R is a heterocyclyl radical as defined above, and if the include fused or bridged ring systems, having from three to heterocyclyl is a nitrogen-containing heterocyclyl, the het fifteen carbon atoms, or having from three to ten carbon erocyclyl may be attached to the alkyl radical at the nitrogen atoms, and which is Saturated or unsaturated and attached to atom. Unless stated otherwise specifically in the specifica the rest of the molecule by a single bond. Monocyclic radicals tion, a heterocyclylalkyl group may be optionally Substituted. US 2014/0004215 A1 Jan. 2, 2014
0044) “Heteroaryl” refers to a 5- to 14-membered ring or more hydrogen atoms are replaced with —NRR, system radical comprising hydrogen atoms, one to thirteen NRC(=O)R. NRC(=O)NRR, NRC(=O) carbonatoms, one to six heteroatoms selected from the group OR, NRSO.R., OC(=O)NRR, OR - SR consisting of nitrogen, oxygen and Sulfur, and at least one - SOR SOR OSO-R-SO.OR=NSO.R., and aromatic ring. For purposes of this disclosure, the heteroaryl - SONR.R. “Substituted also means any of the above radical may be a monocyclic, bicyclic, tricyclic or tetracyclic groups in which one or more hydrogen atoms are replaced ring system, which may include fused or bridged ring sys with C(=O)R = C(=O)CR C(=O)NRR, —CH2SO.R. —CH2SO.NRR. In the foregoing, R, and tems; and the nitrogen, carbon or Sulfur atoms in the het R, are the same or different and independently hydrogen, eroaryl radical may be optionally oxidized; the nitrogenatom alkyl, alkoxy, alkylamino, thioalkyl, aryl, aralkyl, cycloalkyl, may be optionally quaternized. Examples include azepinyl, cycloalkylalkyl, haloalkyl, heterocyclyl N-heterocyclyl, het acridinyl, benzimidazolyl, benzothiazolyl, benzindolyl, ben erocyclylalkyl, heteroaryl, N-heteroaryl or heteroarylalkyl. Zodioxolyl, benzofuranyl, benzooxazolyl, benzothiazolyl, “Substituted further means any of the above groups in which benzothiadiazolyl, benzob.14 dioxepinyl, 1,4-benzo one or more hydrogen atoms are replaced by a bond to an dioxanyl, benzonaphthofuranyl, benzoxazolyl, benzodiox amino, cyano, hydroxyl, imino, nitro, oxo, thioxo, halo, alkyl, olyl, benzodioxinyl, benzopyranyl, benzopyranonyl, benzo alkoxy, alkylamino, thioalkyl, aryl, aralkyl, cycloalkyl, furanyl, benzofuranonyl, benzothienyl (benzothiophenyl), cycloalkylalkyl, haloalkyl, heterocyclyl N-heterocyclyl, het benzotriazolyl, benzo 4.6imidazol-2-alpyridinyl, carba erocyclylalkyl, heteroaryl, N-heteroaryl or heteroarylalkyl Zolyl, cinnolinyl, dibenzofuranyl, dibenzothiophenyl, fura group. In addition, each of the foregoing Substituents may nyl, furanonyl, isothiazolyl, imidazolyl, indazolyl, indolyl, also be optionally substituted with one or more of the above indazolyl, isoindolyl, indolinyl, isoindolinyl, isoquinolyl, Substituents. indolizinyl, isoxazolyl, naphthyridinyl, oxadiazolyl, 0048 “Glycoside' refers to a molecule in which a sugar 2-oxoazepinyl, oxazolyl, oxiranyl, 1-oxidopyridinyl, 1-oxi group is bonded through its anomeric carbon to another group dopyrimidinyl, 1-oxidopyrazinyl, 1-oxidopyridazinyl, via a glycosidic bond. Exemplary Sugars include glucose, 1-phenyl-1H-pyrrolyl, phenazinyl, phenothiazinyl, phenox rhamnose, manose, galactose, arabinose, glucuronide and azinyl, phthalazinyl, pteridinyl, purinyl, pyrrolyl pyrazolyl, others. Glycosides can be linked by an O- (an O-glycoside), pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, quinazolinyl, N- (a glycosylamine), S- (a thioglycoside), or C- (a C-glyco quinoxalinyl, quinolinyl, quinuclidinyl, isoquinolinyl, tet side)glycosidic bond. Compounds of this disclosure can form rahydroquinolinyl, thiazolyl, thiadiazolyl, triazolyl, tetra glycosides at any suitable attachment point. Zolyl, triazinyl, and thiophenyl (i.e. thienyl). Unless stated 0049) “Prenyl refers to the otherwise specifically in the specification, a heteroaryl group may be optionally substituted. 0045 “N-heteroaryl” refers to a heteroaryl radical as defined above containing at least one nitrogen and where the point of attachment of the heteroaryl radical to the rest of the molecule is through a nitrogenatom in the heteroaryl radical. Unless Stated otherwise specifically in the specification, an N-heteroaryl group may be optionally substituted. radical. Prenyl includes isoprenyl, which refers to the 0046) “Heteroarylalkyl refers to a radical of the formula —R,R, where R, is an alkylene chain as defined above and R, is a heteroaryl radical as defined above. Unless stated other wise specifically in the specification, a heteroarylalkyl group may be optionally substituted. 0047. The term “substituted used herein means any of the above groups (i.e., alkyl, alkylene, alkoxy, alkylamino, thio alkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, haloalkyl, radical (cis or trans). Prenyl groups may be substituted or heterocyclyl N-heterocyclyl, heterocyclylalkyl, heteroaryl, unsubstituted. N-heteroaryl or heteroarylalkyl), wherein at least one hydro 0050) “Prenylphenyl refers to a phenyl moiety connected gen atom is replaced by a bond to a non-hydrogenatoms Such to a prenyl moiety as defined above. Prenylphenyls include as a halogen atom Such as F. Cl, Br, and I; an oxygen atom in substituted phenyls such as flavonoids and other substituted groups such as hydroxyl groups, alkoxy groups, and ester phenyls and heteroaryls, provided there is at least one prenyl group in the molecule. In the case of Substituted phenyls and groups; a Sulfur atom in groups such as thiol groups, thioalkyl heteroaryl, the prenyl moiety need not be directly attached to groups, Sulfone groups, Sulfonyl groups, and Sulfoxide the phenyl ring, but can be attached at any point in the mol groups; a nitrogen atom in groups such as amines, amides, ecule. alkylamines, dialkylamines, arylamines, alkylarylamines, 0051. “Chalcone' refers to a compound comprising the diarylamines, N-oxides, imides, and enamines; a siliconatom in groups such as trialkylsilyl groups, dialkylarylsilyl groups, following core structure: alkyldiarylsilyl groups, and triarylsilyl groups; and other het eroatoms in various other groups. “Substituted also means any of the above groups in which one or more hydrogenatoms are replaced by a higher-order bond (e.g., a double- or triple bond) to a heteroatom Such as oxygen in Oxo, carbonyl, car boxyl, and ester groups; and nitrogen in groups such as imi nes, OXimes, hydrazones, and nitriles. For example, “substituted includes any of the above groups in which one US 2014/0004215 A1 Jan. 2, 2014
Chalcones can be variously substituted at any of the above occupancy. Isotopically-labeled compounds of structure (I) carbon atoms. can generally be prepared by conventional techniques known 0052 “Prodrug is meant to indicate a compound that may to those skilled in the art or by processes analogous to those be converted under physiological conditions or by Solvolysis described in the Preparations and Examples as set out below to a biologically active compound of this disclosure. Thus, the using an appropriate isotopically-labeled reagent in place of term “prodrug” refers to a metabolic precursor of a compound the non-labeled reagent previously employed. of this disclosure that is pharmaceutically and nutraceutically 0057 The instant disclosure is also meant to encompass acceptable. A prodrug may be inactive when administered to the in vivo metabolic products of the disclosed compounds. a subject in need thereof, but is converted in vivo to an active Such products may result from, for example, the oxidation, compound of this disclosure. Prodrugs are typically rapidly reduction, hydrolysis, amidation, esterification, and the like transformed in vivo to yield the parent compound of this of the administered compound, primarily due to enzymatic disclosure, for example, by hydrolysis in blood. The prodrug processes. Accordingly, this disclosure includes compounds compound often offers advantages of Solubility, tissue com produced by a process comprising administering a compound patibility or delayed release in a mammalian organism (see, of this invention to a mammal for a period of time sufficient to Bundgard, H., Design of Prodrugs (1985), pp. 7-9, 21-24 yield a metabolic product thereof. Such products are typically (Elsevier, Amsterdam)). A discussion of prodrugs is provided identified by administering a radiolabelled compound of this in Higuchi, T., et al., A.C.S. Symposium Series, Vol. 14, and disclosure in a detectable dose to an animal. Such as rat, in Bioreversible Carriers in Drug Design, Ed. Edward B. mouse, guinea pig, monkey, or to human, allowing Sufficient Roche, American Pharmaceutical and Nutraceutical Associa time for metabolism to occur, and isolating its conversion tion and Pergamon Press, 1987. products from the urine, blood or other biological samples. 0053. The term “prodrug is also meant to include any 0058 “Stable compound” and “stable structure” are meant covalently bonded carriers, which release the active com to indicate a compound that is sufficiently robust to survive pound of this disclosure in vivo when Such prodrug is admin isolation to a useful degree of purity from a reaction mixture, istered to a mammalian Subject. Prodrugs of a compound of and formulation into an efficacious therapeutic agent. this disclosure may be prepared by modifying functional 0059 “Mammal’ includes humans and both domestic ani groups present in the compound of this disclosure in Such a mals. Such as laboratory animals or household pets (e.g., cats, way that the modifications are cleaved, either in routine dogs, Swine, cattle, sheep, goats, horses, rabbits), and non manipulation or in Vivo, to the parent compound of this dis domestic animals, such as wildlife or the like. closure. Prodrugs include compounds of this disclosure 0060) "Optional” or “optionally” means that the subse wherein a hydroxy, amino or mercapto group is bonded to any quently described element, component, event or circum group that, when the prodrug of the compound of this disclo stances may or may not occur, and that the description Sure is administered to a mammalian Subject, cleaves to form includes instances where the element, component, event or a free hydroxy, free amino or free mercapto group, respec circumstance occur and instances in which they do not. For tively. Examples of prodrugs include acetate, formate and example, “optionally substituted aryl' means that the aryl benzoate derivatives of alcohol oramide derivatives of amine radical may or may not be substituted and that the description functional groups in the compounds of this disclosure and the includes both substituted aryl radicals and aryl radicals hav like. ing no Substitution. 0054 The instant disclosure is also meant to encompass 0061 “Pharmaceutically or nutraceutically acceptable all pharmaceutically or nutraceutically acceptable com carrier, diluent or excipient' includes any adjuvant, carrier, pounds of structure (I) being isotopically-labelled by having excipient, glidant, Sweetening agent, diluent, preservative, one or more atoms replaced by an atom having a different dye? colorant, flavor enhancer, Surfactant, wetting agent, dis atomic mass or mass number. Examples of isotopes that can persing agent, Suspending agent, stabilizer, isotonic agent, be incorporated into the disclosed compounds include iso solvent, or emulsifier which has been approved by the United topes of hydrogen, carbon, nitrogen, oxygen, phosphorous, States Food and Drug Administration as being acceptable for fluorine, chlorine, and iodine, such as H, H, C, C, C, use in humans or domestic animals. 13N, 15 N, 15O, 17O, 18O, 3P. 32P 35S, 18F 36C1, 123, and 125I, 0062 “Pharmaceutically or nutraceutically acceptable respectively. These radiolabelled compounds could be useful salt' includes both acid and base addition salts. to help determine or measure the effectiveness of the com 0063 “Pharmaceutically or nutraceutically acceptable pounds, by characterizing, for example, the site or mode of acid addition salt” refers to those salts which retain the bio action, orbinding affinity to pharmacologically important site logical effectiveness and properties of the free bases, which of action. Certain isotopically-labelled compounds of struc are not biologically or otherwise undesirable, and which are ture (I), for example, those incorporating a radioactive iso formed with inorganic acids such as hydrochloric acid, tope, are useful in drug or Substrate tissue distribution studies. hydrobromic acid, Sulfuric acid, nitric acid, phosphoric acid The radioactive isotopes tritium, i.e. H, and carbon-14, i.e. and the like, and organic acids such as acetic acid, 2,2-dichlo '''C, are particularly useful for this purpose in view of their roacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic ease of incorporation and ready means of detection. acid, benzenesulfonic acid, benzoic acid, 4-acetamidoben 0055 Substitution with heavier isotopes such as deute Zoic acid, camphoric acid, camphor-10-Sulfonic acid, capric rium, i.e. H. may afford certain therapeutic advantages acid, caproic acid, caprylic acid, carbonic acid, cinnamic resulting from greater metabolic Stability, for example, acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane increased in Vivo half-life or reduced dosage requirements, 1.2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethane and hence may be preferred in some circumstances. Sulfonic acid, formic acid, fumaric acid, galactaric acid, gen 0056 Substitution with positron emitting isotopes, such as tisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, '''C, F, 'O and 'N, can be useful in Positron Emission glutamic acid, glutaric acid, 2-oxo-glutaric acid, glycero Topography (PET) studies for examining substrate receptor phosphoric acid, glycolic acid, hippuric acid, isobutyric acid, US 2014/0004215 A1 Jan. 2, 2014
lactic acid, lactobionic acid, lauric acid, maleic acid, malic generally accepted in the art includes all pharmaceutically or acid, malonic acid, mandelic acid, methanesulfonic acid, nutraceutically acceptable carriers, diluents or excipients mucic acid, naphthalene-1,5-disulfonic acid, naphthalene-2- therefor. Sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, 0067. As used herein, "enriched for refers to a plant oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, extract or other preparation having at least a two-fold up to propionic acid, pyroglutamic acid, pyruvic acid, salicylic about a 1000-fold increase of one or more active compounds acid, 4-aminosalicylic acid, sebacic acid, Stearic acid, suc as compared to the amount of one or more active compounds cinic acid, tartaric acid, thiocyanic acid, p-toluenesulfonic found in the weight of the plant material or other source acid, trifluoroacetic acid, undecylenic acid, and the like. before extraction or other preparation. In certain embodi 0064 "Pharmaceutically or nutraceutically acceptable ments, the weight of the plant material or other source before base addition salt” refers to those salts which retain the bio extraction or other preparation may be dry weight, wet logical effectiveness and properties of the free acids, which weight, or a combination thereof. are not biologically or otherwise undesirable. These salts are 0068. As used herein, “major active ingredient’ or “major prepared from addition of an inorganic base or an organic active component” refers to one or more active compounds base to the free acid. Salts derived from inorganic bases foundina plant extractor other preparation, or enriched for in include the sodium, potassium, lithium, ammonium, calcium, a plant extract or other preparation, which is capable of at magnesium, iron, Zinc, copper, manganese, aluminum salts least one biological activity. In certain embodiments, a major and the like. In certain embodiments, the inorganic salts are active ingredient of an enriched extract will be the one or ammonium, sodium, potassium, calcium, or magnesium more active compounds that were enriched in that extract. salts. Salts derived from organic bases include salts of pri Generally, one or more major active components will impart, mary, secondary, and tertiary amines, Substituted amines directly or indirectly, most (i.e., greater than 50%) of one or including naturally occurring Substituted amines, cyclic more measurable biological activities or effects as compared amines and basic ion exchange resins, such as ammonia, to other extract components. In certain embodiments, a major isopropylamine, trimethylamine, diethylamine, triethy active ingredient may be a minor component by weight per lamine, tripropylamine, diethanolamine, ethanolamine, centage of an extract (e.g., less than 50%, 25%, or 10% of the deanol, 2-dimethylaminoethanol, 2-diethylaminoethanol, components contained in an extract) but still provide most of dicyclohexylamine, lysine, arginine, histidine, procaine, the desired biological activity. Any composition of this dis hydrabamine, choline, betaine, benethamine, benzathine, closure containing a major active ingredient may also contain ethylenediamine, glucosamine, methylglucamine, theobro minor active ingredients that may or may not contribute to the mine, triethanolamine, tromethamine, purines, piperazine, pharmaceutical or nutraceutical activity of the enriched com piperidine, N-ethylpiperidine, polyamine resins and the like. position, but not to the level of major active components, and Particularly useful organic bases are isopropylamine, diethy minor active components alone may not be effective in the lamine, ethanolamine, trimethylamine, dicyclohexylamine, absence of a major active ingredient. choline and caffeine. 0069) “Effective amount” or “therapeutically effective amount refers to that amount of a compound or composition 0065 Often crystallizations produce a solvate of the com of this disclosure which, when administered to a mammal, pound of this disclosure. As used herein, the term “solvate Such as a human, is sufficient to effect treatment, including refers to an aggregate that comprises one or more molecules any one or more of: (1) treating or preventing weight gain in of a compound of this disclosure with one or more molecules a mammal; (2) promoting weight loss; (3) Suppressing appe of solvent. The solvent may be water, in which case the tite in a mammal; (4) modifying Satiety in a mammal; (5) solvate may be a hydrate. Alternatively, the solvent may bean treating or preventing obesity in a mammal; (6) modifying fat organic solvent. Thus, the compounds of the present inven uptake in a mammal; and (7) increasing metabolism to pro tion may exist as a hydrate, including a monohydrate, dihy mote weight loss or prevent weight gain in a mammal. The drate, hemihydrate, sesquihydrate, trihydrate, tetrahydrate amount of a compound or composition of this disclosure that and the like, as well as the corresponding Solvated forms. The constitutes a “therapeutically effective amount will vary compound of this disclosure may be true Solvates, while in depending on the compound, the condition being treated and other cases, the compound of this disclosure may merely its severity, the manner of administration, the duration of retain adventitious water or be a mixture of water plus some treatment, or the age of the subject to be treated, but can be adventitious solvent. determined routinely by one of ordinary skill in the art having 0066. A “pharmaceutical composition” or “nutraceutical regard to his own knowledge and to this disclosure. composition” refers to a formulation of a compound of this 0070 “Dietary supplements' as used herein are a product disclosure and a medium generally accepted in the art for the that improves, promotes, increases, manages, controls, main delivery of the biologically active compound to mammals, tains, optimizes, modifies, reduces, inhibits, or prevents a e.g., humans. For example, a pharmaceutical composition of particular condition associated with a natural state or biologi the present disclosure may be formulated or used as a stand cal process (i.e., are not used to diagnose, treat, mitigate, cure, alone composition, or as a component in a prescription drug, or prevent disease). For example, with regard to weight-re an over the counter (OTC) medicine, a botanical drug, an lated conditions, dietary Supplements may be used to promote herbal medicine, a homeopathic agent, or any other form of weight loss, manage weight gain, maintain weight, modify health care product reviewed and approved by a government Satiety, reduce caloric intake, increase muscle mass, or the agency. Exemplary nutraceutical compositions of the present like. Exemplary dietary Supplements include one or more of a disclosure may be formulated or used as a stand alone com dietary ingredient such as a vitamin, a mineral, an herb or position, or as a nutritional or bioactive component in food, a other botanical, an amino acid, or any other Substance used to functional food, a beverage, a bar, a food flavor, a medical Supplement the diet by increasing total dietary intake, or a food, a dietary Supplement, or an herbal product. A medium concentrate, metabolite, constituent, extract, or any combina US 2014/0004215 A1 Jan. 2, 2014 tion thereof. In certain embodiments, dietary Supplements are Accordingly, in certain embodiments the positions marked a special category of food and are not a drug. with an * above (and various other positions within com (0071 "Treating or “treatment” as used herein refers to the treatment of the disease or condition of interest in a pounds I and II) can each independently exist as either R or S mammal. Such as a human, having the disease or condition of isomers, and the present invention is meant to include all Such interest, and includes: (i) preventing the disease or condition possible isomers, as well as their racemic and optically pure from occurring in a mammal, in particular, when Such mam forms. Optically active (+) and (-), (R)- and (S)-, or (D)- and mal is predisposed to the condition but has not yet been (L)-isomers may be prepared using chiral synthons or chiral diagnosed as having it; (ii) inhibiting the disease or condition, reagents, or resolved using conventional techniques, for i.e., arresting its development; (iii) relieving the disease or example, chromatography and fractional crystallization. condition, i.e., causing regression of the disease or condition; or (iv) relieving the symptoms resulting from the disease or Conventional techniques for the preparation/isolation of indi condition, (e.g., relieving pain, reducing inflammation, caus vidual enantiomers include chiral synthesis from a Suitable ing weight loss) without addressing the underlying disease or optically pure precursor or resolution of the racemate (or the condition. As used herein, the terms “disease' and “condi racemate of a salt or derivative) using, for example, chiral tion” may be used interchangeably or may be different in that high pressure liquid chromatography (HPLC). When the the particular malady or condition may not have a known compounds described herein contain olefinic double bonds or causative agent (so that etiology has not yet been worked out) and it is therefore not yet recognized as a disease but only as other centres of geometric asymmetry, and unless specified an undesirable condition or syndrome, wherein a more or less otherwise, it is intended that the compounds include both E specific set of symptoms have been identified by clinicians. and Z geometric isomers. Likewise, all tautomeric forms are 0072. As used herein, “statistical significance” refers to a also intended to be included. p value of 0.050 or less when calculated using the Students t-test and indicates that it is unlikely that a particular event or result being measured has arisen by chance. 0074. A “stereoisomer refers to a compound made up of 0073. The compounds of this disclosure, or their pharma the same atoms bonded by the same bonds but having differ ceutically or nutraceutically acceptable salts may contain one ent three-dimensional structures, which are not interchange or more asymmetric centers and may thus give rise to enan able. The present invention contemplates various stereoiso tiomers, diastereomers, and other stereoisomeric forms that mers and mixtures thereof and includes “enantiomers', may be defined, in terms of absolute stereochemistry, as (R)- or (S)- or, as (D)- or (L). For example, the compounds of which refers to two stereoisomers whose molecules are non structure I or II may have chiral centers at least at the positions Superimposeable mirror images of one another. noted with * in the structures below. 0075. A “tautomer refers to a proton shift from one atom of a molecule to another atom of the same molecule. The present invention includes tautomers of any said compounds.
0076. The chemical naming protocol and structure dia grams used herein are a modified form of the I.U.P.A.C. nomenclature system, using the ACD/Name Version 9.07 software program or ChemDraw Ultra Version 11.0 software naming program (CambridgeSoft), wherein the compounds of this disclosure are named herein as derivatives of the cen tral core structure, e.g., the imidazopyridine structure. For complex chemical names employed herein, a Substituent group is named before the group to which it attaches. For example, cyclopropylethyl comprises an ethylbackbone with II cyclopropyl substituent. Except as described below, all bonds are identified in the chemical structure diagrams herein, except for some carbon atoms, which are assumed to be bonded to sufficient hydrogenatoms to complete the Valency. 0077. As noted herein, in certain embodiments, the present disclosure provides a composition comprising a Diels-Alder adduct of a chalcone and a prenylphenyl moiety, and at least one other weight management agent, wherein the Weight management agent is an anorectic agent, a lipase inhibitor, a cannabinoid receptor modulator, a psychotropic agent, an insulin sensitizer, a stimulant, or a Satiety agent, wherein the Diels-Alder adduct of a chalcone and a pre nylphenyl moiety is a compound having a structure of For mula I or II: US 2014/0004215 A1 Jan. 2, 2014
I0084) R'', R'', R' or R'' are each independently H, hydroxyl, halogen, sulfhydryl, amino, aldehyde, C-alkyl, C-12 alkoxy, C-12 alkthio, C-12 alkyamino, aryl, heteroaryl, aralkyl, alkyl carbonyl, aralkylcarbonyl or one of R''", R', R“, R's joins with another of R2, R, R or R's to form a heterocyclic or heteroaromatic ring or one of R''", R'', R' or R'joins with Y' to form an ether bond; I0085 R'', R', R'' and Rare each independently H, hydroxyl, halogen, Sulfhydryl, amino, aldehyde, C-2 alkyl, Calkoxy, Calkthio, Calkyamino, aryl, heteroaryl, aralkyl, alkyl carbonyl, aralkylcarbonyl or one of R'', R', R'', R'joins with another of R', R', R' or R' to form a heterocyclic or heteroaromatic ring: I0086 X joins with one of R", R, R or R7 to form a direct bond or X joins with Y' to form an oxo moiety; II I0087 X joins with one of R, R, R or R to form a direct bond or X joins with Y to form an oxo moiety; I0088 Y is H or Y' joins with one of R''", R'', R'' or R'' to form an ether bond or Y' joins with an adjacent aliphatic carbon to form an oxirane ring: I0089 Yi is HorY-joins with an adjacentaliphatic carbon to form an oxirane ring; and 0090 a dashed bond represents an optional double bond Such that all Valences are satisfied. (0091. In further embodiments of the foregoing, R'' and R" are each independently methyl,
or a pharmaceutically or nutraceutically acceptable salt, tau tomer, glycoside, prodrug or stereoisomer thereof, wherein: 0078 R'' andal R' are eachh 1ndependentindependently C- 2 alkylalKyl 0079 R2 andal R' are eachhindependently 1ndependent HorO R Oor R' joins with one of R, RS, R, R7, R2, R, R or R's or R,R,R,R7, R2, R,R or R', respectively, to form an ether bond; 0092. In some embodiments, R* and Rare H. In other 0080 R and Rare each independently H, hydroxyl or embodiments, Rand Rare H. OXO. I0081) R', R, R and R'' are each independently H, 0093. In yet other embodiments, the compound has the hydroxyl, halogen, Sulfhydryl, amino, aldehyde, C-2 alkyl, following structure (Ia) or (IIa): C-12 alkoxy, C-2 alkthio, C-12 alkyamino, aryl, heteroaryl, aralkyl, alkyl carbonyl, aralkylcarbonyl or one of R", R", R or R'joins with another of R", R, R or R7 to form a heterocyclic or heteroaromatic ring or one of R". R. R. or R'joins with X' to form a direct bond; I0082 R. R. R. and R' are each independently H, (Ia) hydroxyl, halogen, Sulfhydryl, amino, aldehyde, C-2 alkyl, C-12 alkoxy, C-2 alkthio, C-12 alkyamino, aryl, heteroaryl, aralkyl, alkyl carbonyl, aralkylcarbonyl or one of R. R. R. R7 joins with another of R. R. R. R7 to form a heterocyclic or heteroaromatic ring or one of R. R. R. or R'joins with X to form a direct bond; 0083 R8c, R8, R9a, R9b, R10a, Rioh, Rlla and R1 lb a each independently H, hydroxyl, halogen, Sulfhydryl, amino, aldehyde, C-12 alkyl, C-12 alkoxy, C-12 alkthio, C-12 alkyamino, aryl, heteroaryl, aralkyl, alkyl carbonyl, aralkyl carbonyl or one of R, R, R' or R''' or one of R, R, R' or R'joins with another of R, R, R or R'' or another one of R. R. R' or R'', respectively, to form a heterocyclic or heteroaromatic ring;
US 2014/0004215 A1 Jan. 2, 2014 10
-continued -continued
(IIc)
(Ie)
(IId)
(If)
(IIe)
(Ig) US 2014/0004215 A1 Jan. 2, 2014 11
-continued -continued
(Ig)
(I)
0.098 wherein R is, at each occurrence, independently H, hydroxyl or C-2 alkyl. 0099. In further embodiments of the foregoing, the com pound has one of the following structures (Ih), (Ili), (I), (Ik), (Il), (IIh), (IIi), (II), (IIk) or (IIT):
(Ih) (Ik)
(II) (II) US 2014/0004215 A1 Jan. 2, 2014 12
-continued -continued (III) (IIh)
0100. In still other embodiments, at least one R is hydroxyl, and in other embodiments at least one R is (III)
10101. In other related embodiments, at least two of R". R4b Risa Rish R6a R6b R7a R7b Risa R8b R9a R9b R10a Rioh Ria Rh R2 Rih Risa R35 R4, Rh Ris and R'' are hydroxyl. 0102. In some exemplary compositions, the compound has one of the following structures (Im). (In), (IIm) or (IIn): (II)
(Im)
(IIm) US 2014/0004215 A1 Jan. 2, 2014 13
-continued -continued
(In)
wherein R is, at each occurrence, independently H, hydroxyl or C-alkyl. For example, in some embodiments at least one R is hydroxyl, and in other embodiments at least one R is
(IIn) 2
(0.104) Instill other embodiments, X'joins with one of R". R, R or R7 to form a direct bond or Xjoins with one of R'', R', R' or R' to form a direct bond and the com pound has one of the following structures (Io) or (IIo):
(Io)
01.03 at least one of R, R, R, R, R, R, R7, R7, R8c, R8b, R9, R9, R10a, Rioh, R la, R1b, R12, R12b, R13a, R13b, R4a, R14b, Risa O R 15b is
In other embodiments, at least one of R. R. R. R. R. (IIo) R6b R7a R7b Risa R8b R9a R9b R10a R10b Rlla R1 lb R12a R2, R13, R3, R1, R1, Rl5 or R's is heteroaryl. For example, in Some embodiments, the heteroaryl is selected from: US 2014/0004215 A1 Jan. 2, 2014 14
For example, in some further embodiments, the compound has one of the following structures (Ip) or (IIp):
(Ip) (Iq)I
(IIp)
(Ir)
In still some other further embodiments, Y'joins with one of R''", R'', R'' or R'' to form an ether bond and the com- In some more specific embodiments, the compound is pound has one of the following structures (Iq) or (Ir): selected from any of the compounds provided in Table A. TABLE A
List of Exemplary Compounds
Molecular Structure Name Species Formula M.W.
Albafuran C Morus aiba C34H28Og S80.590
OH OH
HO O OH
O OH
HO O)-(O ) OH US 2014/0004215 A1 Jan. 2, 2014 15
TABLE A-continued
List of Exemplary Compounds
Molecular Structure Name Species Formula M.W.
OH Abafuran C: Morus C34H28Og S80.590 2-Epimer australis
O HO \
O HO
HO HO
OH Albanin F Morus aiba, CoHO 692.718 also from Morus australis, Morus bombycis, and Morus ihout
OH Albanin F Morus sp. CoH8O2 710.733 (Moracenin D): 12,13-Dihydro, 13-hydroxy
US 2014/0004215 A1 Jan. 2, 2014 16
TABLE A-continued
List of Exemplary Compounds
Molecular Structure Name Species Formula M.W.
Albanin G Morus aiba: C15HO 760.836 (Kuwanon H. also isol. Moracenin A.) from Morus australis, Morus bombycis, and Morus ihout
Albanin G: Morus CHOo. 744.837 2"-Deoxy mongolica (Mongolicin D)
Albanol A Morus ihout C34H26Os 562.575 (Mulberrofuran G.) US 2014/0004215 A1 Jan. 2, 2014 17
TABLE A-continued
List of Exemplary Compounds
Molecular Structure Name Species Formula M.W.
Albanol A: Morus ihout C39H34Os 630.693 3'- (3-Methyl-2- butenyl), Mulberrofuran F
Albanol B Morus aiba C3H22Os 558.543
Artonin C Artocarpus CHOo 678.734 heterophyllus (jackfruit) US 2014/0004215 A1 Jan 2, 2014 18
TABLE A-continued
List of Exemplary Compounds
Molecular Structure Name Species Formula M.W.
Artonin D Artocarpus 676.718
heterophyllus (jackfruit)
Artonin I Morus 692.718 heterophyllus
Australisin B Morus 646.692 australis US 2014/0004215 A1 Jan. 2, 2014 19
TABLE A-continued List of Exemplary Compounds
Molecular Structure Name Species Formula M.W.
OH Austalisin C.: Morus C34H28Og 2-Epimer australis
HO
OH
HO
Brosimone B BroSinopsis CoHsOo 678.734 oblongifolia (preferred genus name Brosimum)
Brosimone D BroSinopsis C45H44O11 760.836 oblongifolia (preferred genus name Brosimum) US 2014/0004215 A1 Jan. 2, 2014 20
TABLE A-continued
List of Exemplary Compounds
Molecular Structure Name Species Formula M.W.
Cathayanon A Morus CHO 708.717 cathayana
Cathayanon A: Morus CoHO2 708.717 14-Epimer cathayana
Cathayanon E Morus CoHO2 708.717 cathayana
US 2014/0004215 A1 Jan. 2, 2014 21
TABLE A-continued
List of Exemplary Compounds
Molecular Structure Name Species Formula M.W.
Chalcomoracin Morus aiba 648.708 and Morus mongolica
Chalcomoracin; Sopocea 648.708 3',5'-Diepimer matriculata
Chalcomoracin; Morus 648.708 3'-Epimer mongolica US 2014/0004215 A1 Jan. 2, 2014 22
TABLE A-continued List of Exemplary Compounds
Molecular Structure Name Species Formula M.W.
Dorstenone Dorstenia CaoH38O8 646.735 barrieri OH O
HO
O HO
HO OH OH
Guangsangon C Morus C5HoOo. 610.616 iOFOil:
Guangsangon D Morus C5HoOo. 610.616 iOFOil:
Guangsangon Morus C35H3OO11 626.615 D; 2'-Deoxy, iOFOil: US 2014/0004215 A1 Jan. 2, 2014 23
TABLE A-continued
List of Exemplary Compounds
Molecular Structure Name Species Formula M.W.
Guangsangon Morus 610.616 D; 3-Deoxy, 4'- iOFOil: hydroxy and Morus wittiorum
Guangsangon Morus 610.616 D; 2-Epimer, 3 iOFOil: deoxy, 4'- hydroxy
Guangsangon E Morus 648.708 iOFOil:
Guangsangon Morus 666.723 E; 3"-Epimer, iOFOil: 2",3"-dihydro, 3"-hydroxy US 2014/0004215 A1 Jan. 2, 2014 24
TABLE A-continued
List of Exemplary Compounds
Molecular Structure Name Species Formula M.W.
Guangsangon F Morus CoHOo 676.718 iOFOil:
O Guangsangon G Morus C5H2Oo. 608.600 iOFOil: OH
HO O
OOH OH
HO OH
OH
Guangsangon Morus C35H28O11 624.600 G; 1"-Epimer, iOFOil: 2'-hydroxy US 2014/0004215 A1 Jan. 2, 2014 25
TABLE A-continued
List of Exemplary Compounds
Molecular Structure Name Species Formula M.W.
Guangsangon Morus C35H28O11 624.600 G; 2'-Hydroxy iOFOil:
Guangsangon Morus C35H28O11 625.600 G: 5-Hydroxy wittiori in
Guangsangon H Morus CHOo 678.734 iOFOil: US 2014/0004215 A1 Jan. 2, 2014 26
TABLE A-continued List of Exemplary Compounds
Molecular Structure Name Species Formula M.W. Guangsangon J Morus C39H36Og 648.708 iOFOil:
Guangsangon L. Morus aiba C27H2Os 476.482
Isobavachromene Dorstenia CaoH3sOs 646.735 dimer Zenkeri
Kuwanol A Morus C34H28O8 S64.590 bombycis US 2014/0004215 A1 Jan 2, 2014 27
TABLE A-continued
List of Exemplary Compounds
Molecular Structure Name Species Formula M.W.
Kuwanol B Morus 562.575 bombycis
OH HO
OH
HO OH 21
HO
Kuwanol E Morus aiba (white mulberry)
Kuwanol E: Sopocea 668.739 2",3"-Dihydro, iiicifolia 3"-hydroxy
US 2014/0004215 A1 Jan. 2, 2014 28
TABLE A-continued
List of Exemplary Compounds
Molecular Structure Name Species Formula M.W.
Kuwanon J Morus aiba CoHsOo 678.734 and from Morus bombycus
and Morus nigra
Kuwanon J: Morus aiba CaoH38Og 662.735 16"-Deoxy (white mulberry) US 2014/0004215 A1 Jan. 2, 2014 29
TABLE A-continued
List of Exemplary Compounds
Molecular Structure Name Species Formula M.W.
Kuwanon J: 2- Morus aiba CaoH38Og 662.735 Deoxy (white mulberry)
Kuwanon J, Morus aiba CaoH38Og 662.735 A21"22"- (white Isomer, 2-deoxy mulberry) US 2014/0004215 A1 Jan. 2, 2014 30
TABLE A-continued
List of Exemplary Compounds
Molecular Structure Name Species Formula M.W.
Kuwanon J: Morus aiba CaoH38O8 646.735 2,16"-Dideoxy (white mulberry)
Kuwanon J: Morus CoHoOo. 680.750 2',3'-Dihydro mangolica US 2014/0004215 A1 Jan. 2, 2014 31
TABLE A-continued
List of Exemplary Compounds
Molecular Structure Name Species Formula M.W.
Kuwanon J: 1'- Morus aiba CoHsOo 678.734 Epimer and Morus bombycus
Kuwanon J: Artocarpus CaoH38Og 662.735 A21"22"- heterophyllus Isomer, 2-deoxy (jackfruit) (Artonin X.) US 2014/0004215 A1 Jan. 2, 2014 32
TABLE A-continued
List of Exemplary Compounds
Molecular Structure Name Species Formula M.W.
Kuwanon L. Morus aiba C5HoO 626.615 (white mulberry)
Kuwanon L; Morus aiba CHO 692.718 2,3-Didehydro, (white 3-(3-methyl-2- mulberry) butenyl) US 2014/0004215 A1 Jan. 2, 2014 33
TABLE A-continued
List of Exemplary Compounds
Molecular Structure Name Species Formula M.W.
Kuwanon N Morus ihout C15HO 760.836
Kuwanon O Morus ihout CHO 694.734 US 2014/0004215 A1 Jan. 2, 2014 34
TABLE A-continued List of Exemplary Compounds
Molecular Structure Name Species Formula M.W. HO OH Kuwanon P Morus ihout CHO 582.606
HO
OH O HO O
OH OH
OH
HO OH Kuwanon P; 2- Morus C34H30O8 Deoxy iOFOil:
HO
OH
OH
OH Kuwanon W Morus ihout C15H2O. 758.820 US 2014/0004215 A1 Jan. 2, 2014 35
TABLE A-continued
List of Exemplary Compounds
Molecular Structure Name Species Formula M.W.
Kuwanon X Morus ihout C34H30Og S82.606
Kuwanon X; Morus alba CHO 582.606 3'-Epimer (white mulberry)
Kuwanon Z Morus aiba C3H2Oo. 594.573 (white mulberry) US 2014/0004215 A1 Jan 2, 2014 36
TABLE A-continued
List of Exemplary Compounds
Molecular Structure Name Species Formula M.W.
Mongolicin C Morus 578.574 mongolica
Moracenin C Morus sp. 760.836
Mulberrofuran Morus C bombycis O (Moraceae)
OH
Mulberrofuran Morus aiba 632.709 E (white mulberry) (Moraceae) US 2014/0004215 A1 Jan. 2, 2014 37
TABLE A-continued
List of Exemplary Compounds
Molecular Structure Name Species Formula M.W.
Mulberrofuran I Morus C34H24Os 560.559
bombycis
OH
Mulberrofuran J Morus ihout C34H28Og S80.590
OH HO O
HO. O HO \ ()
OH
Mulberrofuran Morus OH J, 2-Epimer bombycis HO O
OH US 2014/0004215 A1 Jan. 2, 2014 38
TABLE A-continued List of Exemplary Compounds
Molecular Structure Name Species Formula M.W. Mulberrofuran Morus aiba 646.692 O
Mulberrofuran Morus aiba C3H22Og S74.542 P (white mulberry)
Mulberrofuran Morus aiba C3H2Oo. 592.558 Q (white mulberry) US 2014/0004215 A1 Jan. 2, 2014 39
TABLE A-continued
List of Exemplary Compounds
Molecular Structure Name Species Formula M.W.
Mulberrofuran Morus aiba C34H24Og 576.558 S (white mulberry)
Mulberrofuran Morus aiba C4H44Og 716.826 T (white mulberry)
US 2014/0004215 A1 Jan. 2, 2014 40
TABLE A-continued
List of Exemplary Compounds
Molecular Structure Name Species Formula M.W.
Mulberrofuran Morus C39H36Og 648.708 U insignis
Multicaulisin Morus CoHO 692.718 militicautis
HO Sanggenol G Morus C3oE34O7 694.734 cathayana
O OH
OH
O HOO
HO OH
HO OH US 2014/0004215 A1 Jan. 2, 2014 41
TABLE A-continued
List of Exemplary Compounds
Molecular Structure Name Species Formula M.W.
Sanggenol J Morus C15H4O2 776.835 cathayana
Sanggenol M Morus CHO 748.825 mongolica
Sanggenon B Morus C33H30Og 570.595
US 2014/0004215 A1 Jan. 2, 2014 42
TABLE A-continued
List of Exemplary Compounds
Molecular Structure Name Species Formula M.W.
Sanggenon B; Morus sp CoHO2 706.701 7-O-(2,4- HO Dihydroxy benzoyl) (Sanggenon S)
Sanggenon D Morus CoHO2 708.717 cathayana
Sanggenon E Morus Spp. CHO2 776.835
US 2014/0004215 A1 Jan. 2, 2014 43
TABLE A-continued
List of Exemplary Compounds
Molecular Structure Name Species Formula M.W.
Sanggenon G Morus aiba CoHsO 694.734
Sanggenon G: Morus sp. CoHoO12 712.749 14,15-Dihydro, 15-hydroxy
Sanggenon Q Morus CoHO2 708.717 mongolica US 2014/0004215 A1 Jan. 2, 2014 44
TABLE A-continued
List of Exemplary Compounds
Molecular Structure Name Species Formula M.W.
Sanggenon D; Morus CoHO2 708.717 3'-Epimer cathayana
Sanggenon D; Morus CHO 708.717 2,3,3'- cathayana Triepimer
Sorocein B Sopocea CaoH34Og 658.703 bonplandii
C On 2 OH O (2) HO 21 No N US 2014/0004215 A1 Jan. 2, 2014 45
TABLE A-continued
List of Exemplary Compounds
Molecular Structure Name Species Formula M.W.
Sorocein H Sopocea CHO2 776.835 bonplandii (Moraceae) and Morus spp.
Wittiorumin B Morus CoHO2 708.717 wittortin
Wittiorumin B; Morus CoHO 692.718 1"-Epimer, 2'- wittiorum deoxy US 2014/0004215 A1 Jan. 2, 2014 46
TABLE A-continued
List of Exemplary Compounds
Molecular Structure Name Species Formula M.W.
Wittiorumin E Morus CoHsOo 678.734 wittiorum
Wittiorumin F Morus C39H36Og 648.708 wittortin
Wittiorumin G Morus CHOo 678.734 wittiorum
US 2014/0004215 A1 Jan. 2, 2014 47
TABLE A-continued List of Exemplary Compounds
Molecular Structure Name Species Formula M.W.
Yunanensin A Morus C39H28O8 624.645 vunnanensis
(3) indicates text missing or illegible when filed
0105 Compounds in Table A can be extracted, isolated or in the Examples below. Appropriate concentrations and dos purified from the indicated plant species or certain plant parts ages can be readily determined by one skilled in the art. (e.g., from the bark, trunk, trunk bark, stem bark, root, root 0108) Administration of the compounds of this disclosure, bark, bark Surface, leaves, fruits, flowers, other plant parts, or or their pharmaceutically or nutraceutically acceptable salts, any combination thereof) or can be prepared synthetically or in pure form or in an appropriate pharmaceutical or nutraceu semi-synthetically as described in more detail below. In cer tical composition, can be carried out via any of the accepted tain embodiments, one or more compounds of Table A are modes of administration of agents for serving similar utilities. enriched for or are the major active ingredients in an extract of The pharmaceutical or nutraceutical compositions of this dis the indicated plant species, wherein the enriched extract is closure can be prepared by combining a compound of this obtained from a whole plant or certain plant parts, such as disclosure with an appropriate pharmaceutically or nutraceu leaves, bark, trunk, trunk bark, stem, stem bark, twigs, tubers, tically acceptable carrier, diluent or excipient, and may be root, root bark, bark Surface, young shoots, rhizomes, seed, formulated into preparations in Solid, semi-solid, liquid or fruit, androecium, gynoecium, calyx, stamen, petal, sepal. gaseous forms, such as tablets, capsules, powders, granules, carpel (pistil), flower, or any combination thereof. ointments, Solutions, Suppositories, injections, inhalants, 0106. It is understood that any embodiment of the com gels, microspheres, and aerosols. Typical routes of adminis pounds of structure (I) or (II), as set forth above, and any tering Such pharmaceutical or nutraceutical compositions specific substituent set forth herein for the compounds of include oral, topical, transdermal, inhalation, parenteral, Sub structure (I) or (II), as set forth above, may be independently lingual, buccal, rectal, vaginal, or intranasal. The term combined with other embodiments or substituents of com parenteral as used herein includes Subcutaneous injections, pounds of structure (I) or (II) to form embodiments of this intravenous, intramuscular, intrasternal injection or infusion disclosure not specifically set forth above. In addition, in the techniques. Pharmaceutical or nutraceutical compositions of event that a list of substituents is listed for any particular R this disclosure are formulated so as to allow the active ingre group in a particular embodiment or claim, it is understood dients contained therein to be bioavailable upon administra that each individual substituent may be deleted from the tion of the composition to a patient. Compositions that will be particular embodiment or claim and that the remaining list of administered to a subject or patient take the form of one or substituents will be considered to be within the scope of this more dosage units, where for example, a tablet may be a disclosure. single dosage unit, and a container of a compound of this 0107 For the purposes of administration, the compounds disclosure in aerosol form may hold a plurality of dosage of the present invention may be administered as a raw chemi units. Actual methods of preparing Such dosage forms are cal or may be formulated as pharmaceutical or nutraceutical known, or will be apparent, to those skilled in this art; for compositions. Pharmaceutical or nutraceutical compositions example, see Remington. The Science and Practice of Phar of the present invention comprise a compound of structure (I) macy, 20th Edition (Philadelphia College of Pharmacy and or (II) and a pharmaceutically or nutraceutically acceptable Science, 2000). The composition to be administered will, in carrier, diluent or excipient. The compound of structure (I) or any event, contain a therapeutically effective amount of a (II) is present in the composition in an amount which is compound of this disclosure, or a pharmaceutically or nutra effective to treat aparticular disease or condition of interest— ceutically acceptable salt thereof, for treatment of a disease or that is, in an amount Sufficient promote weight loss or any of condition of interest in accordance with the teachings of this the other associated indications described herein, and gener invention. ally with acceptable toxicity to a patient. Weight loss and 0109) A pharmaceutical or nutraceutical composition of other activity of compounds of structure (I) or (II) can be this disclosure may be in the form of a solid or liquid. In one determined by one skilled in the art, for example, as described aspect, the carrier(s) are particulate, so that the compositions US 2014/0004215 A1 Jan. 2, 2014 48 are, for example, in tablet or powder form. The carrier(s) may emulsion, cream, lotion, ointment, or gel base. The base, for be liquid, with the compositions being, for example, oral example, may comprise one or more of the following: petro syrup, injectable liquid or an aerosol, which is useful in, for latum, lanolin, polyethylene glycols, bee wax, mineral oil, example, inhalatory administration. diluents such as water and alcohol, and emulsifiers and sta 0110. When intended for oral administration, the pharma bilizers. Thickening agents may be present in a pharmaceu ceutical or nutraceutical composition is in either Solid or tical or nutraceutical composition for topical administration. liquid form, where semi-solid, semi-liquid, Suspension and If intended for transdermal administration, the composition gel forms are included within the forms considered herein as may include a transdermal patch or iontophoresis device. either solid or liquid. 0117 The pharmaceutical or nutraceutical composition of 0111. As a solid composition for oral administration, the this disclosure may be intended for rectal administration, in pharmaceutical or nutraceutical composition may be formu the form, for example, of a suppository, which will melt in the lated into a powder, granule, compressed tablet, pill, capsule, rectum and release the drug. The composition for rectal chewing gum, wafer, bar, or like form. Such a solid compo administration may contain an oleaginous base as a Suitable sition will typically contain one or more inert diluents or nonirritating excipient. Such bases include lanolin, cocoa edible carriers. In addition, one or more of the following may butter and polyethylene glycol. be present: binders such as carboxymethylcellulose, ethyl 0118. The pharmaceutical or nutraceutical composition of cellulose, cyclodextrin, microcrystalline cellulose, gum this disclosure may include various materials, which modify tragacanth or gelatin; excipients such as starch, lactose or the physical form of a solid or liquid dosage unit. For dextrins, disintegrating agents such as alginic acid, sodium example, the composition may include materials that form a alginate, Primogel, corn starch and the like; lubricants such as coating shell around the active ingredients. The materials that magnesium Stearate or Sterotex; glidants such as colloidal form the coating shell are typically inert, and may be selected silicon dioxide; Sweetening agents such as Sucrose or saccha from, for example, Sugar, shellac, and other enteric coating rin; a flavoring agent Such as peppermint, methyl salicylate or agents. Alternatively, the active ingredients may be encased in orange flavoring; and a coloring agent. a gelatin capsule. 0112. When the pharmaceutical or nutraceutical compo 0119 The pharmaceutical or nutraceutical composition of sition is in the form of a capsule, for example, a gelatin this disclosure in Solid or liquid form may include an agent capsule, it may contain, in addition to materials of the above that binds to the compound of this disclosure and thereby type, a liquid carrier Such as polyethylene glycol or oil. assists in the delivery of the compound. Suitable agents that 0113. The pharmaceutical or nutraceutical composition may act in this capacity include a monoclonal or polyclonal may be in the form of a liquid, for example, an elixir, syrup, antibody, a protein or a liposome. Solution, emulsion or Suspension. The liquid may be for oral administration or for delivery by injection, as two examples. 0.120. The pharmaceutical or nutraceutical composition of When intended for oral administration, a useful composition this disclosure in Solid or liquid form may include reducing contains, in addition to the present compounds, one or more the size of a particle to, for example, improve bioavailability. of a Sweetening agent, preservatives, dye? colorant and flavor The size of a powder, granule, particle, microsphere, or the enhancer. In a composition intended to be administered by like in a composition, with or without an excipient, can be injection, one or more of a surfactant, preservative, wetting macro (e.g., visible to the eye or at least 100 um in size), micro agent, dispersing agent, Suspending agent, buffer, stabilizer (e.g., may range from about 100 um to about 100 nm in size), and isotonic agent may be included. nano (e.g., may no more than 100 nm in size), and any size in 0114. The liquid pharmaceutical or nutraceutical compo between or any combination thereof to improve size and bulk sitions of this disclosure, whether they be solutions, Suspen density. sions or other like form, may include one or more of the I0121 The pharmaceutical or nutraceutical composition of following adjuvants: Sterile diluents such as water for injec this disclosure may consist of dosage units that can be admin tion, saline Solution, such as physiological saline, Ringer's istered as an aerosol. The term aerosol is used to denote a Solution, isotonic sodium chloride, fixed oils such as Syn variety of systems ranging from those of colloidal nature to thetic mono or diglycerides which may serve as the solvent or systems consisting of pressurized packages. Delivery may be Suspending medium, polyethylene glycols, glycerin, propy by a liquefied or compressed gas or by a Suitable pump system lene glycol or other solvents; antibacterial agents such as that dispenses the active ingredients. Aerosols of compounds benzyl alcohol or methyl paraben; antioxidants such as ascor of this disclosure may be delivered in single phase, bi-phasic, bic acid or sodium bisulfite; chelating agents such as ethyl or tri-phasic systems in order to deliver the active ingredient enediaminetetraacetic acid; buffers such as acetates, citrates (s). Delivery of the aerosol includes the necessary container, or phosphates and agents for the adjustment of tonicity Such activators, valves, Subcontainers, and the like, which together as sodium chloride or dextrose. The parenteral preparation may form a kit. One skilled in the art, without undue experi can be enclosed in ampoules, disposable Syringes or multiple mentation, may determine the most appropriate aerosol(s). dose vials made of glass or plastic. Physiological saline is a 0.122 The pharmaceutical or nutraceutical compositions generally useful adjuvant. An injectable pharmaceutical or of this disclosure may be prepared by methodology well nutraceutical composition is sterile. known in the pharmaceutical or nutraceutical art. For 0115. A liquid pharmaceutical or nutraceutical composi example, a pharmaceutical or nutraceutical composition tion of this disclosure intended for either parenteral or oral intended to be administered by injection can be prepared by administration should contain an amount of a compound of combining a compound of this disclosure with sterile, dis this disclosure such that a suitable dosage will be obtained. tilled water so as to form a solution. A Surfactant may be 0116. The pharmaceutical or nutraceutical composition of added to facilitate the formation of a homogeneous solution this disclosure may be intended for topical administration, in or Suspension. Surfactants are compounds that non-co which case the carrier may suitably comprise a solution, valently interact with the compound of this disclosure so as to US 2014/0004215 A1 Jan. 2, 2014 49 facilitate dissolution or homogeneous Suspension of the com therefore be described as “prodrugs'. All prodrugs of com pound in the aqueous delivery system. pounds of this invention are included within the scope of this 0123. The compounds of this disclosure, or their pharma disclosure. ceutically or nutraceutically acceptable salts, are adminis I0128. Furthermore, all compounds of this disclosure tered in a therapeutically effective amount, which will vary which exist in free base or acid form can be converted to their depending upon a variety of factors including the activity of pharmaceutically or nutraceutically acceptable salts by treat the specific compound employed; the metabolic stability and ment with the appropriate inorganic or organic base oracid by length of action of the compound; the age, body weight, methods known to one skilled in the art. Salts of the com general health, sex, and diet of the patient; the mode and time pounds of this disclosure can be converted to their free base or of administration; the rate of excretion; the drug combination; acid form by standard techniques. the severity of the particular disorder or condition; and the Subject undergoing therapy. I0129. In some embodiments, compounds of the present disclosure can be isolated from plant sources, for example, 0.124 Compounds of this disclosure, or pharmaceutically from those plants included in Table A and elsewhere through or nutraceutically acceptable derivatives thereof, may also be out the present application. Suitable plant parts for isolation administered simultaneously with, prior to, or after adminis of the compounds include leaves, bark, trunk, trunk bark, tration of one or more other therapeutic agents. Such combi stem, stem bark, twigs, tubers, root, root bark, bark Surface, nation therapy includes administration of a single pharma young shoots, rhizomes, seed, fruit, androecium, gynoecium, ceutical or nutraceutical dosage formulation which contains a calyx, stamen, petal, sepal, carpel (pistil), flower, or any com compound of this disclosure and one or more additional bination thereof. In some related embodiments, the com active agents, as well as administration of the compound of pounds are isolated from plant sources and synthetically this disclosure and each active agent in its own separate modified to contain any of the recited substituents. In this pharmaceutical or nutraceutical dosage formulation. For regard, synthetic modification of the compound isolated from example, a compound of this disclosure and another active plants can be accomplished using any number of techniques agent can be administered to the patient together in a single which are known in the art and are well within the knowledge oral dosage composition, such as a tablet or capsule, or each of one of ordinay skill in the art. agent can be administered in separate oral dosage formula tions. Where separate dosage formulations are used, the com 0.130. As noted above, the compounds of the present pounds of this disclosure and one or more additional active invention are Diels-Alder adducts of a chalcone and pre agents can be administered at essentially the same time, i.e., nylphenyl moiety. While not wishing to be bound by theory, concurrently, or at separately staggered times, i.e., sequen it is believed that the Diels-Alder reaction occurs as part of the tially; combination therapy is understood to include all these biosynthetic pathway in various plants, and thus certain regimens. embodiments of this disclosure include isolating the com pounds from plants and using as is or performing various 0.125. It is understood that in the present description, com synthetic modifications. However, in other embodiments the binations of substituents or variables of the depicted formulae compounds are prepared synthetically. For example, the fol are permissible only if such contributions result in stable lowing Reaction Scheme illustrates a method of making com compounds. pounds of this invention, i.e., compound of structure (I) of (II) 0126. It will also be appreciated by those skilled in the art using synthetic techniques: that in the process described herein the functional groups of intermediate compounds may need to be protected by Suitable protecting groups. Such functional groups include hydroxy, Reaction Scheme 1 amino, mercapto and carboxylic acid. Suitable protecting groups for hydroxy include trialkylsilyl or diarylalkylsilyl RIla (for example, t-butyldimethylsilyl, t-butyldiphenylsilyl or tri methylsilyl), tetrahydropyranyl, benzyl, and the like. Suitable R10a er protecting groups for amino, amidino and guanidino include
t-butoxycarbonyl, benzyloxycarbonyl, and the like. Suitable Y-X protecting groups for mercapto include —C(O)—R" (where R" is alkyl, aryl or arylalkyl), p-methoxybenzyl, trity1 and the like. Suitable protecting groups for carboxylic acid include alkyl, aryl orarylalkyl esters. Protecting groups may be added or removed in accordance with standard techniques, which are known to one skilled in the art and as described herein. The use of protecting groups is described in detail in Green, T. W. and P. G. M. Wutz, Protective Groups in Organic Synthesis 2\ay (1999), 3rd Ed., Wiley. As one of skill in the art would appre ciate, the protecting group may also be a polymer resin Such s , as a Wang resin, Rink resin or a 2-chlorotrityl-chloride resin. 0127. It will also be appreciated by those skilled in the art, although Such protected derivatives of compounds of this invention may not possess pharmacological activity as such, they may be administered to a mammal and thereafter metabolized in the body to form compounds of this disclosure which are pharmacologically active. Such derivatives may US 2014/0004215 A1 Jan. 2, 2014 50
-continued disclosure may be combined with one or more weight man 3. agement agents. A “weight management agent, as used R R 14a herein, refers to a biologically active compound, molecule, or composition that allows a Subject to manage their weight, which may involve maintaining a particular weight level. reducing weight gain or reducing weight. The biological activity of the weight management agents may include reduc ing or Suppressing appetite, altering metabolic levels, altering lipid metabolism, decreasing caloric intake, or the like. Exemplary weight management agents include anorectic agents, lipase inhibitors, cannabinoid receptor modulators, psychotropic agents, insulin sensitizers, stimulants, Satiety agents, or combinations thereof. 0.134. In certain embodiments, Diels-Alder adducts of a chalcone and prenylphenyl moiety of the present disclosure are used with at least one other weight management agent, Such as an anorectic agent, a lipase inhibitor, a cannabinoid receptor modulator, a psychotropic agent, an insulin sensi tizer, a stimulant, or a satiety agent. The weight management agents and the Diels-Alder adducts of a chalcone and pre nylphenyl moiety of the present disclosure may beformulated together or separately. In addition, the Diels-Alder adducts of a chalcone and prenylphenyl moiety of the present disclosure may be administered or taken by a subject simultaneously with, prior to, or after administration of the at least one other Weight management agent. I0135) In certain embodiments, the anorectic agent is Sibutramine, diethylpropion, benzphetamine, phendimetra zine, or catecholamine. In further embodiments, the lipase (IIb) inhibitor is a Mutamba extract, a Rosemary extract, carnosic acid, carnosol, lipostatin, tetrahydrolipostatin, Punica grana 0131 Representative compounds of structure (I) or (II) tum pericarp extract, Marchantia polymorphs whole plant (e.g., (Ib) or (IIb)) can be prepared synthetically according to extract, Panax japonicas extract or Platycodi radix extract. the general procedure illustrated in Reaction Scheme 1. Chal 0.136. In still further embodiments, the cannabinoid recep cone A and isoprenylphenyl B may be purchased from com tor modulator is a cannabinoid receptor agonist, antagonist, mercial sources, isolated from plant sources or prepared or inverse agonist. In yet further embodiments, the cannab according to procedures known in the art. Reaction of chal inoid receptor modulator is specific for cannabinoid receptor cone A with isoprenylphenyl B under appropriate Diels-Al 1 (CB1), cannabinoid receptor 2 (CB2), or both CB1 and der conditions (which are known in the art) results in com CB2. Such as a CB1 antagonist or inverse agonist. Exemplary pounds of structure (I) or (II). Compounds of structure (I) and cannabinoid receptor modulators include rimonabant, N-(pi (II) are regioisomers as a result of the asymmetry of chalcone peridin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4- A. The various substituents (i.e., the “R” groups) can be methyl-1H-pyrazole-3-carboxamide (AM 251), 1-(2,4- installed prior to Diels-Alder cyclization as depicted in Reac Dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-4- tion Scheme 1 or installed after cyclization. Methods for morpholinyl-1H-pyrazole-3-carboxamide (AM281), 4-6- modification of the above method and for adding various R methoxy-2-(4-methoxypheyl)1-benzofuran-3-carbonyl groups are well known in the art and within the grasp of one benzonitrile (LY 320135), Magnolia extract, magnolol, of ordinary skill in the art. honokiol, magnolol and honokiol, purinol, or Piper Longum 0.132. It is understood that one skilled in the art may be seed extract. In certain embodiments, Diels-Alder adducts of able to make these compounds by similar methods or by a chalcone and prenylphenyl moiety of the present disclosure combining other methods known to one skilled in the art. It is may be used with a second, third, fourth, or fifth Diels-Alder also understood that one skilled in the art would be able to adduct of a chalcone and a prenylphenyl moiety. In certain make, in a similar manner as described below, other com embodiments, the weight management agents includes two, pounds of structure (I) or (II) not specifically illustrated below three, four, five, six, seven, eight, nine, or ten Diels-Alder by using the appropriate starting components and modifying adducts of a chalcone and a prenylphenyl moiety. the parameters of the synthesis as needed. In general, starting 0.137 For example, certain embodiments Diels-Alder components may be obtained from sources such as Sigma adducts of a chalcone and prenylphenyl moiety of the present Aldrich, Lancaster Synthesis, Inc., Maybridge, Matrix Sci disclosure may be used with an anorectic agent and a lipase entific, TCI, and Fluorochem USA, etc. or synthesized inhibitor, or an anorectic agent and a cannabinoid receptor according to sources known to those skilled in the art (see, for modulator, or an anorectic agent and a psychotropic agent, an example, Advanced Organic Chemistry: Reactions, Mecha anorectic agent and an insulin sensitizer, oran anorectic agent nisms, and Structure, 5th edition (Wiley, December 2000)) or and a stimulant, or an anorectic agent and a satiety agent. In prepared as described in this invention. still further embodiments, Diels-Alder adducts of a chalcone 0133. In further embodiments, at least one Diels-Alder and prenylphenyl moiety of the present disclosure may be adduct of a chalcone and prenylphenyl moiety of the present used with a lipase inhibitor and a stimulant, or a cannabinoid US 2014/0004215 A1 Jan. 2, 2014
receptor modulator and a stimulant, or a cannabinoid receptor indica, Morus lhou, Morus japonica, Morus kagayamae, modulator and a lipase inhibitor. Any of the aforementioned Morus laevigata, Morus latifolia, Morus liboensis, Morus compositions may further comprise a satiety agent or a psy macroura, Morus mongolica, Morus multicaulis, Morus chotropic agent or an insulin sensitizer. notabilis, Morus rotundiloba, Morus Serrate, Morus hetero 0138. In certain embodiments, Diels-Alder adducts of a phyllus, Morus tillaefolia, Morus trilobata, Morus yunnan chalcone and prenylphenyl moieties used alone, in combina ensis, and Morus wittiorum. In certain embodiments, a Morus tion, or with another weight management agent have a pre extract is from Morus alba, or a Morus extract is a mixture of nylphenyl moiety that is a prenylated phenol, an isopreny extracts from one, two, three, four, or five different Morus lated flavonoid, a prenylated flavonoid, a prenylated species. A mixture of extracts may include extracts from two flavoinoid dimer, or a combination thereof. In certain embodi or more Morus species listed in Table A. ments, the Diels-Alder adducts of a chalcone and a pre 0141 Magnolia includes about 210 flowering plant spe nylphenyl moiety have a prenylphenyl moiety that is an iso cies in the subfamily Magnolioideae of the family Magnoli prenylated flavonoid such as an isoprenylated flavone, aceae. Magnolia, known as Hou Pu, can grow wild or under flavonol, flavanone, chalcone, isoflavone, isoflavanone, cultivation in many countries. Exemplary Magnolia species aurone, or stilbene. In certain embodiments, Diels-Alder includes Magnolia Obovata Thunb., Magnolia officinalis adducts of a chalcone and a prenylphenyl moiety have a Rehd. & Wilson, Magnolia rostrata W. W. Smith, Magnolia prenylated flavonoid that is a prenylated flavone, flavonol, tripetala, Magnolia globosa Hook f & Thoms, Magnolia flavanone, chalcone, isoflavone, isoflavanone, aurone, or stil Sieboldii K. Koch, Magnolia wilsonii (Finet. & Gagnep.) bene. In further embodiments, Diels-Alder adducts of a chal Rehd., Magnolia fraseri Walt. Magnolia macrophylla Michx. cone and a prenylphenyl moiety used alone, in combination, Magnolia decidua (Q. Y. Zheng) V. S. Kumar, Magnolia or with one or more otherweight management agents purified dolichogyna (Dandy ex Noot) Figlar & Noot. Magnolia from, isolated from, enriched for, or contained in a Morus duclouxii Finet & Gagnep. Magnolia figlarii V. S. Kumar, extract or a Milicia excelsa extract, Such as Albanin G, Magnolia fordiana, Magnolia emarginata Urb. & Ekman, Kuwanon G, Kuwanon M. Cathayanon A. Morusin, Morusi Magnolia hamori Howard, Magnolia pallescens Urb. & nol, Sanggenon C. Sanggenon D. Sanggenon O, or any com Ekman, Magnolia mahechae (Lozano) Govaerts. Magnolia bination thereof. ptaritepuiana Steyermark, Magnolia striatifolia Little, Mag 0.139. In further embodiments, the weight management nolia mexicana DC., Magnolia minor (Urb.) Govaerts, Mag agent used with Diels-Alder adducts of a chalcone and a nolia morii (Lozano) Govaerts, Magnolia marinensis (Lo prenylphenyl moiety is a psychotropic agent Such as a mood Zano) Govaerts, Magnolia neillii (Lozano) Govaerts, stabilizer, anti-depressant, or anti-convulsant. In still further Magnolia ovata (A.St.-Hil.) Spreng, Magnolia polyhypso embodiments, the weight managementagent used with Diels philla (Lozano) Govaerts, Magnolia quetzal, Magnolia Alder adducts of a chalcone and a prenylphenyl moiety is a rimachii (Lozano) Govaerts, Magnolia Sambuensis, Magno stimulant Such as caffeine, dicaffeoylquinic acid or dextro lia delavayi Franchet, Magnolia fistulosa (Finet & Gagnep) amphetamine, or one or more stimulant purified from, iso Dandy, Magnolia henryi Dunn, Magnolia nana Dandy, Mag lated from, enriched for, or contained in a Yerba mate extract, nolia Odoratissima, Magnolia grandiflora L., and Magnolia green tea extract, green coffee bean extract, Cola nut extract, guatemalensis. In certain embodiments, a Magnolia extractis Citrus aurantium fruit extract, Giacinia extract, Areca catechu from Magnolia officinalis, or a Magnolia extract is a mixture fruit/seed extract or dextroamphetamine. In even further of extracts from one, two, three, four, or five different Mag embodiments, the weight managementagent used with Diels nolia species. Alder adducts of a chalcone and a prenylphenyl moiety is an 0.142 Rosmarinus officinalis, commonly known as Rose insulin sensitizer Such as thiazolidinediones (e.g., rosiglita mary, is a woody, perennial herb with fragrant, evergreen, Zone, pioglitaZone), oxazolidinediones, isoxazolidinediones, needle-like leaves and white, pink, purple, or blue flowers, biguanides (e.g., metformin), selective mTOT (mitochondrial native to the Mediterranean region. Rosemary is a member of Target of Thiazolidinediones) modulators, cinnamon extract, the mint family Lamiaceae, which includes many other herbs. banaba extract, chromium, fish oil, acetic acid, D-chiro-inosi Other exemplary Rosemary species include Rosmarinus tol, or C-lipoic acid. In yet further embodiments, the weight tomentosus, Rosmarinus eriocalyx, and Rosmarinus palaui. management agent used with Diels-Alder adducts of a chal In certain embodiments, a Rosemary extract is from Rosmari cone and a prenylphenyl moiety is a satiety agent Such as nus officinalis, or a Rosemary extract is a mixture of extracts dodecanoic acid, glyceryl dodecanoate, glyceryl 1,3-didode from one, two, or three Rosemary species. canoate, glyceryl tridodecanoate, and derivatives or mixtures 0143. The plant origin for most commonly utilized Yerba thereof, or one or more satiety agent purified from, isolated mate is Ilex paraguariensis, which is a species of holly (fam from, enriched for, or contained in a hoodia extract, a pine nut ily Aquifoliaceae) well known as a source of the beverage extract, or a fiber Supplement. called mate. Yerba mate is native to subtropical South 0140. As noted herein, compounds of a Diels-Alder America in northeastern Argentina, Bolivia, Southern Brazil, adduct of a chalcone and a prenylphenyl moiety may be Uruguay and Paraguay. The genus is distributed throughout obtained by chemical synthesis or from a plant extract, Such the world's different climates. Exemplary Ilex species as a Morus or Milicia extract. For example, Morus is a genus includes American Holly (Ilex opaca), Carolina Holly (Ilex of flowering trees in the family Moraceae, which comprises ambigua), Chinese Holly (Ilex cornuta), Common Winter more than 30 species (known as mulberries) that grow wild or berry (Ilex verticillate), Dahoon (Ilex cassine), Deciduous under cultivation in many countries. Exemplary Morus spe Holly (Ilex decidua), English Holly (Ilex aquifolium), Aus cies include Morus alba L., Morus australis Poir; Morus tralia Holly (Ilex arnhemensis). Inkberry (Ilex glabra), Japa celtidifolia Kunth, Morus insignis, Morus mesozygia Stapf nese Holly (Ilex crenata), Large Gallberry (Ilex coriacea), Morus microphylla, Morus nigra L., Morus rubra L., Morus Smooth Winterberry (Ilex laevigata), Yaupon (Ilex vomito atropurpurea, Morus bombycis, Morus cathayana, Morus ria), Africa species (Ilex mitis), and Ilex canariensis Maca US 2014/0004215 A1 Jan. 2, 2014 52 ronesia, Ilex aquifolium). Ilex mucronate, formerly the type magnolol and honokiol mixture isolated from Magnolia plant species of Nemopanthus, is native to eastern North America. extract), and Yerba mate extract, wherein the Diels-Alder Nemopanthus was treated as a monotypic genus with eight adduct of a chalcone and a prenylphenyl moiety is Albanin G, species of the family Aquifoliaceae, now transferred to Ilex Kuwanon G, Kuwanon M. Cathayanon A. Morusin, Morusi based on molecular data (closely related to Ilex amelanchier). nol, Sanggenon C, Sanggenon D. Sanggenon O or any com In certain embodiments, a Yerba mate extract is from Ilex bination thereof. In further embodiments, a composition paraguariensis, or a Yerba mate extract is a mixture of comprising a Diels-Alder adduct of a chalcone and a pre extracts from one, two, three, four, or five Ilex species. nylphenyl moiety and a weight management agent comprises 0144 Guazuma, a genus of flowering trees in the family a Diels-Alder adduct of a chalcone and a prenylphenyl moi Malvaceae, is widely found in the Caribbean, South Ameri ety, Purinol (a high purity, such as at least 90% purity, or at can, Central America and Mexico. Mutamba is a common least 91%, 92%, 93%. 94%, or 95% purity) magnolol and name of Guazuma plant with various medicinal values in honokiol mixture isolated from Magnolia plant extract), and traditional herbal medicine. Exemplary condensed tannins of Yerba mate extract, wherein the Diels-Alder adduct of a chal this disclosure maybe extracted from different species of cone and a prenylphenyl moiety is Albanin G, Kuwanon G, Guazuma plant, including Guazuma commerSoniopsis, Gua Kuwanon M. Cathayanon A. Morusin, Morusinol, Zuma euguazuma, Guazuma gynophoricola, Guazuma Sanggenon C. Sanggenon D. Sanggenon O, or any combina blunei, Guazuma bubroma, Guazuma burbroma, Guazuma tion thereof, and the Yerba mate extract is an Ilex paraguay coriacea, Guazuma crimita, Guazuma grand flora, Guazuma ensis extract enriched for caffeine, dicaffeoylquinic acid, or guazuma, Guazuma invira, Guazuma iuvira, Guazuma lon both. gipedicellata, Guazuma parvifolia, Guazuma polybotra, 0147 In certain embodiments, a composition comprising Guazuma polybotrya, Guazuma rosea, Guazuma tomentosa, a Diels-Alder adduct of a chalcone and a prenylphenyl moiety Guazuma ulmifolia, and Guazuma utilis. In certain embodi comprises a Morus extract containing or enriched for at least ments, a Mutamba extract is from Guazuma ulmifolia, or a one Diels-Alder adduct of a chalcone and a prenylphenyl Mutamba extract is a mixture of extracts from one, two, three, moiety, a Magnolia extract, and a Mutamba extract, which four, or five Guazuma species. can be mixed or used together in a 2:1:5 weight ratio to a 0145. In certain embodiments, a composition comprising 2:1:10 weight ratio, respectively. In further embodiments, a a Diels-Alder adduct of a chalcone and a prenylphenyl moiety composition comprising a Diels-Alder adduct of a chalcone comprises a Morus extract containing or enriched for at least and a prenylphenyl moiety and a weight management agent one Diels-Alder adduct of a chalcone and a prenylphenyl comprises a Morus extract containing or enriched for at least moiety, a Magnolia extract, and a Yerba mate extract, which one Diels-Alder adduct of a chalcone and a prenylphenyl extracts can be mixed or used together in a 2:1:5 weight ratio moiety, a Magnolia extract, and a Mutamba extract, which to a 2:1:10 weight ratio, respectively. In further embodiments, extracts can be mixed or used together in a 2:1:5 weight ratio a composition comprising a Diels-Alder adduct of a chalcone to a 2:1:10 weight ratio, respectively. In any of the aforemen and a prenylphenyl moiety and a weight management agent tioned embodiments, a Morus extract is a Morus alba extract comprises a Morus extract containing or enriched for at least or extract enriched for Kuwanon G, Albanin G, Morusin, or one Diels-Alder adduct of a chalcone and a prenylphenyl any combination thereof, and a Magnolia extract is a Mag moiety, a Magnolia extract, and a Yerba mate extract, which nolia officinalis extract or extract enriched for magnolol. extracts can be mixed or used together in a 2:1:5 weight ratio honokiol, both, or a high purity mixture of both. In related to a 2:1:10 weight ratio, respectively. In any of the aforemen embodiments, a weight management agent is an anorectic tioned embodiments, a Morus extract is a Morus alba extract agent, lipase inhibitor, cannabinoid receptor modulator, psy or extract enriched for Kuwanon G, Albanin G, Morusin, or chotropic agent, insulin sensitizer, stimulant, satiety agent, or any combination thereof: a Magnolia extract is a Magnolia any combination thereof. In any of these embodiments, a officinalis extract or extract enriched for magnolol, honokiol, weight management agent is an anorectic agent. In any of both, or a high purity mixture of both; and a Yerba mate these embodiments, a weight management agent is a lipase extract is an Ilex paraguayensis extract is enriched for caf inhibitor. In any of these embodiments, a weight management feine, dicaffeoylquinic acid, or both. In related embodiments, agent is a cannabinoid receptor modulator. In any of these a weight management agent is an anorectic agent, lipase embodiments, a weight management agent is a psychotropic inhibitor, cannabinoid receptor modulator, psychotropic agent. In any of these embodiments, a weight management agent, insulin sensitizer, stimulant, Satiety agent, or any com agent is an insulin sensitizer. In any of these embodiments, a bination thereof. In any of these embodiments, a weight man weight management agent is a stimulant. In any of these agement agent is an anorectic agent. In any of these embodi embodiments, a weight management agent is a satiety agent. ments, a weight managementagentis alipase inhibitor. In any 0.148. In certain embodiments, a composition comprising of these embodiments, a weight management agent is a can a Diels-Alder adduct of a chalcone and a prenylphenyl moiety nabinoid receptor modulator. In any of these embodiments, a comprises a Diels-Alder adduct of a chalcone and a pre weight management agent is a psychotropic agent. In any of nylphenyl moiety, purinol, and Mutamba extract, wherein the these embodiments, a weight management agent is an insulin Diels-Alder adduct of a chalcone and a prenylphenyl moiety sensitizer. In any of these embodiments, a weight manage is Albanin G, Kuwanon G, Kuwanon M. Cathayanon A, ment agent is a stimulant. In any of these embodiments, a Morusin, Morusinol, Sanggenon C. Sanggenon D. Weight management agent is a Satiety agent. Sanggenon O, or any combination thereof. In further embodi 0146 In certain embodiments, a composition comprising ments, a composition comprising a Diels-Alder adduct of a a Diels-Alder adduct of a chalcone and a prenylphenyl moiety chalcone and a prenylphenyl moiety and a weight manage comprises a Diels-Alder adduct of a chalcone and a pre ment agent comprises a Diels-Alder adduct of a chalcone and nylphenyl moiety, Purinol (a high purity, such as at least 90% a prenylphenyl moiety, purinol, and Mutamba extract, purity, or at least 91%, 92%, 93%. 94%, or 95% purity, wherein the Diels-Alder adduct of a chalcone and a pre US 2014/0004215 A1 Jan. 2, 2014 nylphenyl moiety is Albanin G, Kuwanon G, Kuwanon M, composition comprising a Diels-Alder adduct of a chalcone Cathayanon A, Morusin, Morusinol, Sanggenon C, and a prenylphenyl moiety and a weight management agent Sanggenon D. Sanggenon O, or any combination thereof. comprises a Morus extract containing or enriched for at least 0149. In certain embodiments, a composition comprising one Diels-Alder adduct of a chalcone and a prenylphenyl a Diels-Alder adduct of a chalcone and a prenylphenyl moiety moiety, a Rosemary extract, and a mutamba extract, which comprises a Morus extract containing or enriched for at least extracts can be mixed or used together in a 2:5:5 weight ratio one Diels-Alder adduct of a chalcone and a prenylphenyl to a 2:5:10 weight ratio, respectively. In any of the aforemen moiety, a Rosemary extract, and a Yerba mate extract, which tioned embodiments, a Morus extract is a Morus alba extract can be mixed or used together in a 2:5:5 weight ratio to a or extract enriched for Kuwanon G, Albanin G, Morusin, or 2:5:10 weight ratio, respectively. In further embodiments, a any combination thereof. In related embodiments, a weight composition comprising a Diels-Alder adduct of a chalcone management agent is an anorectic agent, lipase inhibitor, and a prenylphenyl moiety and a weight management agent cannabinoid receptor modulator, psychotropic agent, insulin comprises a Morus extract containing or enriched for at least sensitizer, stimulant, Satiety agent, or any combination one Diels-Alder adduct of a chalcone and a prenylphenyl thereof. In any of these embodiments, a weight management moiety, a Rosemary extract, and a Yerba mate extract, which agent is an anorectic agent. In any of these embodiments, a extracts can be mixed or used together in a 2:5:5 weight ratio weight management agent is a lipase inhibitor. In any of these to a 2:5:10 weight ratio, respectively. In any of the aforemen embodiments, a weight management agent is a cannabinoid tioned embodiments, a Morus extract is a Morus alba extract receptor modulator. In any of these embodiments, a weight or extract enriched for Kuwanon G, Albanin G, Morusin, or management agent is a psychotropic agent. In any of these any combination thereof. In related embodiments, a weight embodiments, a weight management agent is an insulin sen management agent is an anorectic agent, lipase inhibitor, sitizer. In any of these embodiments, a weight management cannabinoid receptor modulator, psychotropic agent, insulin agent is a stimulant. In any of these embodiments, a weight sensitizer, stimulant, Satiety agent, or any combination management agent is a satiety agent. thereof. In any of these embodiments, a weight management 0152. In certain embodiments, a composition comprising agent is an anorectic agent. In any of these embodiments, a a Diels-Alder adduct of a chalcone and a prenylphenyl moiety weight management agent is a lipase inhibitor. In any of these comprises a (1) a Diels-Alder adduct of a chalcone and a embodiments, a weight management agent is a cannabinoid prenylphenyl moiety, carnosol, and mutamba extract or (2) a receptor modulator. In any of these embodiments, a weight Diels-Alder adduct of a chalcone and a prenylphenyl moiety, management agent is a psychotropic agent. In any of these carnosic acid, and mutamba extract; wherein the Diels-Alder embodiments, a weight management agent is an insulin sen adduct of a chalcone and a prenylphenyl moiety is Albanin G, sitizer. In any of these embodiments, a weight management Kuwanon G, Kuwanon M. Cathayanon A. Morusin, Morusi agent is a stimulant. In any of these embodiments, a weight nol, Sanggenon C, Sanggenon D. Sanggenon O or any com management agent is a satiety agent. bination thereof. In further embodiments, a composition 0150. In certain embodiments, a composition comprising comprising a Diels-Alder adduct of a chalcone and a pre a Diels-Alder adduct of a chalcone and a prenylphenyl moiety nylphenyl moiety and a weight management agent comprises comprises (1) a Diels-Alder adduct of a chalcone and a pre a (1) a Diels-Alder adduct of a chalcone and a prenylphenyl nylphenyl moiety, carnosol, and Yerba mate extract or (2) a moiety, carnosol, and mutamba extract or (2) a Diels-Alder Diels-Alder adduct of a chalcone and a prenylphenyl moiety, adduct of a chalcone and a prenylphenyl moiety, carnosic carnosic acid, and Yerba mate extract; wherein the Diels acid, and mutamba extract; wherein the Diels-Alder adduct of Alder adduct of a chalcone and a prenylphenyl moiety is a chalcone and a prenylphenyl moiety is Albanin G, Kuwanon Albanin G, Kuwanon G, Kuwanon M. Cathayanon A, G, Kuwanon M, Cathayanon A. Morusin, Morusinol, Morusin, Morusinol, Sanggenon C. Sanggenon D. Sanggenon C, Sanggenon D. Sanggenon O or any combina Sanggenon O or any combination thereof, and the Yerba mate tion thereof. extract is an Ilex paraguayensis extract is enriched for caf 0153. In certain embodiments, a composition comprising feine, dicaffeoylquinic acid, or both. In further embodiments, a Diels-Alder adduct of a chalcone and a prenylphenyl moiety a composition comprising a Diels-Alder adduct of a chalcone comprises a Morus extract containing or enriched for at least and a prenylphenyl moiety and a weight management agent one Diels-Alder adduct of a chalcone and a prenylphenyl comprises (1) a Diels-Alder adduct of a chalcone and a pre moiety, a Rosemary extract, and an Areca extract, which can nylphenyl moiety, carnosol, and Yerba mate extract or (2) a be mixed or used together in a 2:5:5 weight ratio to a 2:5:10 Diels-Alder adduct of a chalcone and a prenylphenyl moiety, weight ratio, respectively. In further embodiments, a compo carnosic acid, and Yerba mate extract; wherein the Diels sition comprising a Diels-Alder adduct of a chalcone and a Alder adduct of a chalcone and a prenylphenyl moiety is prenylphenyl moiety and a weight management agent com Albanin G, Kuwanon G, Kuwanon M. Cathayanon A, prises a Morus extract containing or enriched for at least one Morusin, Morusinol, Sanggenon C. Sanggenon D. Diels-Alder adduct of a chalcone and a prenylphenyl moiety, Sanggenon O or any combination thereof, and the Yerba mate a Rosemary extract, and an Areca extract, which extracts can extract is an Ilex paraguayensis extract is enriched for caf be mixed or used together in a 2:5:5 weight ratio to a 2:5:10 feine, dicaffeoylquinic acid, or both. weight ratio, respectively. In any of the aforementioned 0151. In certain embodiments, a composition comprising embodiments, a Morus extract is a Morus alba extract or a Diels-Alder adduct of a chalcone and a prenylphenyl moiety extract enriched for Kuwanon G, Albanin G. Morusin, or any comprises a Morus extract containing or enriched for at least combination thereof. In related embodiments, a weight man one Diels-Alder adduct of a chalcone and a prenylphenyl agement agent is an anorectic agent, lipase inhibitor, cannab moiety, a Rosemary extract, and a mutamba extract, which inoid receptor modulator, psychotropic agent, insulin sensi can be mixed or used together in a 2:5:5 weight ratio to a tizer, stimulant, Satiety agent, or any combination thereof. In 2:5:10 weight ratio, respectively. In further embodiments, a any of these embodiments, a weight management agent is an US 2014/0004215 A1 Jan. 2, 2014 54 anorectic agent. In any of these embodiments, a weight man Morus alba extract enriched for Kuwanon G, Albanin G, agement agent is a lipase inhibitor. In any of these embodi Morusin, or any combination thereof. In further embodi ments, a weight management agent is a cannabinoid receptor ments, a pharmaceutical or nutraceutical formulation com modulator. In any of these embodiments, a weight manage prising an extract mixture as described herein comprises from ment agent is a psychotropic agent. In any of these embodi about 0.5 weight percent (wt %) to about 5.0 wt % of the ments, a weight management agent is an insulin sensitizer. In major active ingredients, such as Kuwanon G, Albanin G, any of these embodiments, a weight management agent is a Morusin, or any combination thereof, in a Morus extract, Such stimulant. In any of these embodiments, a weight manage as a Morus alba extract or extract enriched for Kuwanon G, ment agent is a satiety agent. Albanin G. Morusin, or any combination thereof. 0154 In certain embodiments, a composition comprising 0157. In any of the aforementioned embodiments, a com a Diels-Alder adduct of a chalcone and a prenylphenyl moiety position comprising an extract mixture (optionally including comprises a (1) a Diels-Alder adduct of a chalcone and a a weight management agent) wherein one of the extracts is a prenylphenyl moiety, carnosol, and Areca extract or (2) a Guazuma ulmifolia (Mutamba) extract enriched for one or Diels-Alder adduct of a chalcone and a prenylphenyl moiety, more major active ingredients. In certain embodiments, a carnosic acid, and Areca extract; wherein the Diels-Alder Guazuma ulmifolia (Mutamba) extract is enriched for a pro adduct of a chalcone and a prenylphenyl moiety is Albanin G, cyanidin, procyanidin B2, procyanidin B5, procyanidin C1, Kuwanon G, Kuwanon M. Cathayanon A. Morusin, Morusi procyanidin dimers, procyanidin trimers, procyanidintetram nol, Sanggenon C, Sanggenon D. Sanggenon O or any com ers, procyanidin pentamers, procyanidin hexamers, con bination thereof. In further embodiments, a composition densed tannins, an oligomer of catechin or epicatechin, epi comprising a Diels-Alder adduct of a chalcone and a pre catechin, or any combination thereof. In further nylphenyl moiety and a weight management agent comprises embodiments, a pharmaceutical or nutraceutical formulation a (1) a Diels-Alder adduct of a chalcone and a prenylphenyl comprising an extract mixture as described herein comprises moiety, carnosol, and Areca extract or (2) a Diels-Alder from about 0.5 weight percent (wt %) to about 50 wt % of the adduct of a chalcone and a prenylphenyl moiety, carnosic major active ingredients in a Guazuma ulmifolia (Mutamba) acid, and Areca extract; wherein the Diels-Alder adduct of a extract, such as a procyanidin, procyanidin B2, procyanidin chalcone and a prenylphenyl moiety is Albanin G, Kuwanon B5, procyanidin C1, procyanidin dimers, procyanidin trim G, Kuwanon M. Cathayanon A. Morusin, Morusinol, ers, procyanidin tetramers, procyanidin pentamers, procyani Sanggenon C, Sanggenon D. Sanggenon O or any combina din hexamers, condensed tannins, an oligomer of catechin or tion thereof. epicatechin, epicatechin, or any combination thereof. 0155. In any of the aforementioned embodiments, a com 0158. In any of the aforementioned embodiments, a com position comprising an extract mixture (optionally including position comprising an extract mixture (optionally including a weight management agent) can be formulated with a phar a weight management agent) wherein one of the extracts is a maceutically or nutraceutically acceptable carrier, diluent or Magnolia extract enriched for one or more major active ingre excipient. In further embodiments, the pharmaceutical or dients, such as magnolol, honokiol, or both, or a high purity nutraceutical formulation comprises from about 0.5 weight magnolol, honokiol, or mixture thereof. In certain embodi percent (wt %) to about 90 wt %, about 0.5 wt % to about 80 ments, the Magnolia extract is a Magnolia officinalis extract wt %, about 0.5 wt % to about 75 wt %, about 0.5 wt % to enriched for magnolol, honokiol, or both, or a high purity about 70 wt %, about 0.5 wt % to about 50 wt %, about 1.0 wt magnolol, honokiol, or mixture thereof isolated from Mag % to about 40 wt %, about 1.0 wt % to about 20 wt %, about nolia plant extract that has at least about 90%, 91%, 92%, 1.0 wt % to about 10 wt %, about 3.0 wt % to about 9.0 wt %, 93%. 94%, or 95% purity. In certain embodiments, a phar about 5.0 wt % to about 10 wt %, about 3.0 wt % to about 6.5 maceutical or nutraceutical formulation comprising an wt % of the major active ingredients in an extract mixture, or extract mixture as described herein comprises from about 5.0 the like. In any of these embodiments, the pharmaceutical or weight percent (wt %) to about 10 wt % of the major active nutraceutical composition is formulated as a tablet, a capsule, ingredients in a Magnolia officinalis (Magnolia) extract, Such a powder, or granule. In related embodiments, a weight man as magnolol, honokiol, both, or a high purity mixture of both. agement agent is an anorectic agent, lipase inhibitor, cannab 0159. In any of the aforementioned embodiments, a com inoid receptor modulator, psychotropic agent, insulin sensi position comprising an extract mixture (optionally including tizer, stimulant, Satiety agent, or any combination thereof. In a weight management agent) wherein one of the extracts is a any of these embodiments, a weight management agent is an Yerba mate extract enriched for one or more major active anorectic agent. In any of these embodiments, a weight man ingredients. In certain embodiments, a Yerba mate extract is agement agent is a lipase inhibitor. In any of these embodi an Ilex paraguayensis extract is enriched for caffeine, dicaf ments, a weight management agent is a cannabinoid receptor feoylquinic acid, or both. In further embodiments, a pharma modulator. In any of these embodiments, a weight manage ceutical or nutraceutical formulation comprising an extract ment agent is a psychotropic agent. In any of these embodi mixture as described herein comprises from about 0.5 weight ments, a weight management agent is an insulin sensitizer. In percent (wt %) to about 5.0 wt % of the major active ingre any of these embodiments, a weight management agent is a dients in an Ilex paraguayensis (Yerba mate) extract. Such as stimulant. In any of these embodiments, a weight manage caffeine, dicaffeoylquinic acid, or both. ment agent is a satiety agent. 0160. In any of the aforementioned embodiments, a com 0156. In any of the aforementioned embodiments, a com position comprising an extract mixture (optionally including position comprising an extract mixture (optionally including a weight management agent) wherein one of the extracts is a a weight management agent) wherein one of the extracts is a Rosmarinus officinalis (Rosemary) extract enriched for one or Morus extract enriched for one or more major active ingredi more major active ingredients. In certain embodiments, a ents, such as Kuwanon G, Albanin G. Morusin, or any com Rosmarinus officinalis (Rosemary) extract is enriched for a bination thereof. In certain embodiments, a Morus extract is a carnosol, carnosoic acid, ursolic acid, or any combination US 2014/0004215 A1 Jan. 2, 2014
thereof. In further embodiments, a pharmaceutical or nutra agent, or any combination thereof. In any of these embodi ceutical formulation comprising an extract mixture as ments, a weight management agent is an anorectic agent. In described herein comprises from about 1.0 weight percent (wt any of these embodiments, a weight management agent is a %) to about 10 wt % of the major active ingredients in a lipase inhibitor. In any of these embodiments, a weight man Rosmarinus officinalis (Rosemary) extract, such as carnosol, agement agent is a cannabinoid receptor modulator. In any of carnosoic acid, ursolic acid, or any combination thereof. these embodiments, a weight management agent is a psycho 0161 In any of the aforementioned embodiments, a phar tropic agent. In any of these embodiments, a weight manage maceutical or nutraceutical formulation comprising an ment agent is an insulin sensitizer. In any of these embodi extract mixture (optionally including a weight management ments, a weight management agent is a stimulant. In any of agent) of a Morus extract, a Magnolia extract, and an Ilex these embodiments, a weight management agent is a satiety paraguayensis (Yerba mate) extract, will include from about agent. 0.5 weight percent (wt %) to about 5.0 wt % of the major 0163. In any of the aforementioned embodiments, a phar active ingredients in a Morus extract, from about 5.0 wt % to maceutical or nutraceutical formulation comprising an about 10 wt % of the major active ingredients in a Magnolia extract mixture (optionally including a weight management extract, and from about 0.5 wt % to about 5.0 wt % of the agent) of a Morus extract, a Rosmarinus officinalis (Rose major active ingredients in Yerba mate extract. In certain mary) extract, and an Ilex paraguayensis (Yerba mate) embodiments, a pharmaceutical or nutraceutical formulation extract, will include from about 0.5 weight percent (wt %) to comprises about 1% of the major active ingredients in a about 5.0 wt % of the major active ingredients in a Morus Morus extract (such as Kuwanon G, Albanin G. Morusin, or extract, from about 1.0 wt % to about 10 wt % of the major any combination thereof), about 7% of the major active ingre active ingredients in a Rosemary extract, and from about 0.5 dients in a Magnolia extract (Such as magnolol, honokiol, wt % to about 5.0 wt % of the major active ingredients in a both, or a high purity mixture of both), and about 1% of the Yerba mate extract. In certain embodiments, a pharmaceutical major active ingredients in a Yerba mate extract (such as or nutraceutical formulation comprises about 1% of the major caffeine, dicaffeoylquinic acid, or both). In any of the afore active ingredients in a Morus extract (such as Kuwanon G, mentioned embodiments, a Morus extract is a Morus alba Albanin G. Morusin, or any combination thereof), about extract or extract enriched for Kuwanon G, Albanin G, 4.5% of the major active ingredients in a Rosemary extract Morusin, or any combination thereof, and a Magnolia extract (such as carnosol, carnosoic acid, ursolic acid, or any combi is a Magnolia officinalis extract or extract enriched for mag nation thereof), and about 1% of the major active ingredients nolol, honokiol, both, or a high purity mixture of both. In any in a Yerba mate extract (such as caffeine, dicaffeoylquinic of these embodiments, the pharmaceutical or nutraceutical acid, or both). In any of the aforementioned embodiments, a composition is formulated as a tablet, a capsule, a powder, or Morus extract is a Morus alba extract or enriched extract. In granule. any of these embodiments, the pharmaceutical or nutraceuti 0162. In any of the aforementioned embodiments, a phar cal composition is formulated as a tablet, a capsule, a powder, maceutical or nutraceutical formulation comprising an or granule. In related embodiments, a weight management extract mixture (optionally including a weight management agent is an anorectic agent, lipase inhibitor, cannabinoid agent) of a Morus extract, a Magnolia extract, and a Guazuma receptor modulator, psychotropic agent, insulin sensitizer, ulmifolia (Mutamba) extract, will include from about 0.5 stimulant, Satiety agent, or any combination thereof. In any of weight percent (wt %) to about 5.0 wt % of the major active these embodiments, a weight managementagent is an anorec ingredients in a Morus extract, from about 5.0 wt % to about tic agent. In any of these embodiments, a weight management 10 wt % of the major active ingredients in a Magnolia extract, agent is a lipase inhibitor. In any of these embodiments, a and from about 0.5 wt % to about 50 wt % of the major active weight management agent is a cannabinoid receptor modula ingredients in a Mutamba extract. In certain embodiments, a tor. In any of these embodiments, a weight managementagent pharmaceutical or nutraceutical formulation comprises about is a psychotropic agent. In any of these embodiments, a 1% of the major active ingredients in a Morus extract (Such as weight management agent is an insulin sensitizer. In any of Kuwanon G, Albanin G. Morusin, or any combination these embodiments, a weight management agent is a stimu thereof), about 7% of the major active ingredients in a Mag lant. In any of these embodiments, a weight management nolia extract (such as magnolol, honokiol, both, or a high agent is a Satiety agent. purity mixture of both), and about 1% of the major active 0164. In further embodiments, a composition comprising ingredients in a Mutamba extract (such as a procyanidin, a Diels-Alder adduct of a chalcone and a prenylphenyl moiety procyanidin B2, procyanidin B5, procyanidin C1, procyani as provided herein (optionally including a weight manage din dimers, procyanidin trimers, procyanidin tetramers, pro ment agent) may be used in a method for treating or prevent cyanidin pentamers, procyanidin hexamers, condensed tan ing weight gain or obesity, promoting weight loss, appetite nins, an oligomer of catechin or epicatechin, epicatechin, or Suppression, modifying Satiety, modifying fat uptake, any combination thereof). In any of the aforementioned increasing metabolism to promote weight loss or prevent embodiments, a Morus extract is a Morus alba extract or weight gain, maintaining body weight, reducing body fat or extract enriched for Kuwanon G, Albanin G. Morusin, or any fatty tissues, increasing muscle or lean body mass, reducing combination thereof, and a Magnolia extract is a Magnolia hepatosteatosis, improving fatty liver, improving one or more officinalis extract or extract enriched for magnolol, honokiol, liver NASH scores, enhancing fatty acid metabolism in liver, both, or a high purity mixture of both. In any of these embodi promoting a healthy lipid profile (by, e.g., lowering LDL ments, the pharmaceutical or nutraceutical composition is cholesterol, lowering total cholesterol, lowering triglyceride, formulated as a tablet, a capsule, a powder, or granule. In or increasing HDL), promoting glucose metabolism, reduc related embodiments, a weight management agent is an ing fasting glucose levels, maintaining healthy glucose levels, anorectic agent, lipase inhibitor, cannabinoid receptor modu reducing caloric intake, improving caloric efficiency, reduc lator, psychotropic agent, insulin sensitizer, stimulant, Satiety ing food intake, reducing visceral fat, reducing waist circum US 2014/0004215 A1 Jan. 2, 2014 56 ference, reducing body-to-mass index (BMI), increasing or a pharmaceutically acceptable salt, stereoisomer or tau energy, increasing stamina, maintaining energy level while tomer thereof, wherein R'' and R'' are, at each occurrence, dieting, promoting thermogenesis reducing excess fluids, independently H, hydroxyl, halo, a gallic acid ester, a glyco side, Sulfhydryl, amino, aldehyde, C-2 alkyl, Calkoxy, reducing water retention while maintaining normal hydra Calkthio, Calkyamino, aryl, heteroaryl, aralkyl, alkyl tion, increasing muscle mass, improving fat-to-muscle mass carbonyl, aralkylcarbonyl, or a direct bond to an adjacent ratio, optimizing or improving body composition, optimizing subunit; R is, at each occurrence, independently Horanether or improving hormonal balance forappitite control, maintain bond to an adjacent subunit; R is, at each occurrence.inde ing normal insulin, leptin, ghrelin, PYY. GIP or enterostatin pendently H or a direct bond to an adjacent subunit; R is, at levels or functions, optimizing, managing or improving hor each occurrence, OH or an ether bond to an adjacent subunit; monal balance to control satiety, maintaining or managing and R,R,R, R and Rare, at each occurrence, inde healthy CCK peptide, GLP-1, bombesin, or somatostatin lev pendently H, hydroxyl, halo, a gallic acid ester, a glycoside, els or functions, maintaining healthy flora of intestinal tract, Sulfhydryl, amino, aldehyde, C-12 alkyl, C-12 alkoxy, C-12 alkthio, Calkyamino, aryl, heteroaryl, aralkyl, alkyl car optimizing, improving or managing digestion, inducing bonyl oraralkylcarbonyl, wherein at leastone of R',R,R, lipolysis, reducing intracellular triglyceride accumulation, R or R is a direct bond or ether bond to an adjacent subunit. reducing fat accumulation in adipose tissue or an adipocyte, In certain embodiments, oligomers of flavan-3-ol, with sub maintaining healthy adiponectin levels, managing or reduc units as set forth in structure (III) and wherein at least one of ing lipogenesis or weight gain associated with metabolism of R'", R', R, R or R is a direct bond or ether bond to an fructose, glucose or both, reducing or controlling oxidative adjacent subunit, are dimers, trimers, tetramers, pentamers, stress associated with an overweight or obese mammal (e.g., hexamers, heptamers, octamers, nonamers, decamers, or up by reducing reactive oxygen species or oxidative free radi to 30 flavan-3-ol subunits (such a 30-mer would have a total cals; improving ORAC (Oxygen Radical Absorption Capac molecular weight of about 10 Da). ity) values; maintaining a healthy level of glutathione, Super In further embodiments, two adjacent subunits may have one oxide dismutase, catalase, peroxidase or endogenous of the following structures (IIIa), (IIIb) or (IIIc): antioxidants; maintaining healthy oxidative homeostasis), controlling or managing systemic inflammation associated (IIIa) with an overweight or obese mammal (e.g., by promoting normal metabolism of arachidonic acid, maintaining a nor mal level of pro-inflammatory cytokines), managing mood stress or other mental disorders associated with an overweight or obese mammal, or any combination thereof. 0.165. In related embodiments, a weight management agent for use with the compositions to treat or improve the various weight-related conditions is an anorectic agent, lipase inhibitor, cannabinoid receptor modulator, psychotropic agent, insulin sensitizer, stimulant, Satiety agent, or any com bination thereof. In any of these embodiments, a weight man agement agent is an anorectic agent. In any of these embodi ments, a weight managementagentis alipase inhibitor. In any of these embodiments, a weight management agent is a can nabinoid receptor modulator. In any of these embodiments, a weight management agent is a psychotropic agent. In any of (IIIb) these embodiments, a weight management agent is an insulin sensitizer. In any of these embodiments, a weight manage ment agent is a stimulant. In any of these embodiments, a Weight management agent is a Satiety agent. 0166 In certain embodiments, provided herein is an iso lated oligomer or a composition (e.g., for weight management or weight loss) comprising a pharmaceutically acceptable excipient and an oligomer, wherein the oligomer comprises from two to thirty subunits, wherein the subunits have, at each occurrence, independently the following structure (III):
(III)
O US 2014/0004215 A1 Jan. 2, 2014 57
-continued rence, independently H, hydroxyl, halo, a gallic acid ester, a glycoside, Sulfhydryl, amino, aldehyde, C. alkyl, C alkoxy, C-2 alkthio, C-2 alkyamino, aryl, heteroaryl,
(IIIc) aralkyl, alkyl carbonyl or aralkylcarbonyl, wherein at least one of R". R', position 2, or position 4 is a direct bond or ether bond to an adjacent Subunit. In certain embodiments, oligomers of flavan-3-ol, with subunits as set forth in struc ture (IIId) and wherein at least one of R', R', position 2, or position 4 is a direct bond or ether bond to an adjacent subunit, are dimers, trimers, tetramers, pentamers, hexamers, heptam ers, octamers, nonamers, decamers, or up to 30 flavan-3-ol subunits (such a 30-mer would have a total molecular weight of about 10 Da). 0169. In further embodiments, two adjacent subunits may have one of the following structures (IIIe), (IIIf) or (IIIg):
(IIIe)
(0167 In any of the aforementioned embodiments, each of R" and R' is, at each occurrence, H or a direct bond to an adjacent subunit. In a further embodiment, R is, at each occurrence, Hand Ris, at each occurrence, hydroxyl; or R' is, at each occurrence, hydroxyl and R is, at each occur rence, H. In still further embodiments, each of R. R* and R is, at each occurrence, H. In certain embodiments, oligo mers of flavan-3-ol, with subunits as set forth in structure (IIIa), (IIIb), or (IIIc) are dimers, trimers, tetramers, pentam ers, hexamers, heptamers, octamers, nonamers, decamers, or up to 30 flavan-3-ol subunits (such a 30-mer would have a total molecular weight of about 10 Da). 0.168. In certain embodiments, provided herein is an iso lated oligomer or a composition (e.g., for weight management or weight loss) comprising a pharmaceutically acceptable excipient and an oligomer, wherein the oligomer comprises from two to thirty subunits, wherein the subunits have, at each occurrence, independently the following structure (IIId):
(IIIf
(IIId)
or a pharmaceutically acceptable salt, stereoisomer or tau tomer thereof, wherein R'' and R'' are, at each occurrence, independently H, hydroxyl, halo, a gallic acid ester, a glyco side, Sulfhydryl, amino, aldehyde, C. alkyl, C-2 alkoxy, Calkthio, Calkyamino, aryl, heteroaryl, aralkyl, alkyl carbonyl, aralkylcarbonyl, or a direct bond to an adjacent O subunit; and R. R. R. R. and Rare, at each occur US 2014/0004215 A1 Jan. 2, 2014 58
-continued formin), or a selective mTOT modulator. In yet further
(IIIg) embodiments, the stimulant is a Yerba mate extract, caffeine, green tea extract, green coffee bean extract, Cola nut extract, Citrus aurantium fruit extract, Giacinia extract, Areca catechu fruit/seed extract or dextroamphetamine. In further embodi ments, the Satiety agent is dodecanoic acid, glyceryl dode canoate, glyceryl 1.3-didodecanoate, glyceryl tridodecanoate and derivatives and mixtures thereof, or hoodia extract, pine nut extract, or a fiber Supplement. 0171 In certain embodiments, a composition comprises a Mutamba extract containing or enriched for one or more oligomers of flavan-3-ol as described herein, a Morus extract containing or enriched for at least one Diels-Alder adduct of a chalcone and a prenylphenyl moiety, and a Magnolia extract, which can be mixed or used together in a 5:2:1 weight ratio to a 10:2:1 weight ratio, respectively. In any of the aforementioned embodiments, a Morus extract is a Morus alba extract or extract enriched for Kuwanon G, Albanin G, Morusin, or any combination thereof, and a Magnolia extract is a Magnolia officinalis extract or extract enriched for mag nolol, honokiol, both, or a high purity mixture of both. Structures (IIIe) and (IIIf) are exemplary B-type linkages and 0172 In certain embodiments, a composition comprises a Structure (IIIg) is an example of an A-type linkage. More Mutamba extract containing or enriched for one or more specifically, Structure (IIIe) is a 4->8 linkage, wherein R", oligomers of flavan-3-ol as described herein, a Rosemary R or both are available as a direct bond or ether bond to one extract, and a Yerba mate extract, which can be mixed or used or more adjacent subunits up to total of 30 subunits, and togetherina 2:1:2 weight ratio to a 1:1:1 weight ratio, respec Structure (IIIe) is a 4->6 linkage, wherein R, R or both are tively. available as a direct bond or ether bond to one or more 0173 Instill further embodiments, one or more oligomers adjacent subunits up to total of 30 subunits. of flavan-3-ol or compositions of such oligomers of flavan 0170 In certain embodiments, the present disclosure pro 3-ol compounds as described herein, (optionally including a vides a composition comprising any of the aforementioned weight management agent) may be used in a method for oligomers of flavan-3-ol and at least one other weight man treating or preventing weight gain or obesity, promoting agement agent, Such as an anorectic agent, lipase inhibitor, weight loss, appetite Suppression, modifying Satiety, modify cannabinoid receptor modulator, psychotropic agent, insulin ing fat uptake, increasing metabolism to promote weight loss sensitizer, stimulant, or satiety agent. In further embodi or prevent weight gain, maintaining body weight, reducing ments, the anorectic agent is sibutramine, diethylpropion, body fat or fatty tissues, increasing muscle or lean body mass, benZphetamine, phendimetrazine, or catecholamine. In still reducing hepatosteatosis, improving fatty liver, improving further embodiments, the lipase inhibitor is a Rosemary one or more liver NASH scores, enhancing fatty acid metabo extract, carnosic acid, carnosol, lipostatin, tetrahydroliposta lism in liver, promoting a healthy lipid profile (by, e.g., low tin, Punica granatum pericarp extract, Marchantia polymor ering LDL cholesterol, lowering total cholesterol, lowering pha whole plant extract, Panax japonicas extractor Platycodi triglyceride, or increasing HDL), promoting glucose metabo radix extract. In yet further embodiments, the cannabinoid lism, reducing fasting glucose levels, maintaining healthy receptor modulator is a cannabinoid receptoragonist, antago glucose levels, reducing caloric intake, improving caloric nist, or inverse agonist, which may be specific for cannab efficiency, reducing food intake, reducing visceral fat, reduc inoid receptor 1 (CB1), cannabinoid receptor 2 (CB2), or both ing waist circumference, reducing body-to-mass index CB1 and CB2 (e.g., a a cannabinoid receptor 1 (CB1) antago (BMI), increasing energy, increasing stamina, maintaining nist or inverse agonist). In further embodiments, the cannab energy level while dieting, promoting thermogenesis reduc inoid receptor modulator is rimonabant, N-(piperidin-1-yl)- ing excess fluids, reducing water retention while maintaining 5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H normal hydration, increasing muscle mass, improving fat-to pyrazole-3-carboxamide (AM251), 1-(2,4-Dichlorophenyl)- muscle mass ratio, optimizing or improving body composi 5-(4-iodophenyl)-4-methyl-N'-morpholinyl-1H-pyrazole-3- tion, optimizing or improving hormonal balance for appitite carboxamide (AM281), 4-6-methoxy-2-(4-methoxypheyl) control, maintaining normal insulin, leptin, ghrelin, PYY. 1-benzofuran-3-carbonylbenzonitrile (LY 320135), a Morus GIP or enterostatin levels or functions, optimizing, managing extract, Diels-Alder adduct of a chalcone and a prenylphenyl or improving hormonal balance to control satiety, maintain moiety from plants (e.g., isolated from or contained in a ing or managing healthy CCK peptide, GLP-1, bombesin, or Morus alba extract, a Milicia excelsa extract, or is Albanin G, Somatostatin levels or functions, maintaining healthy flora of Kuwanon G, Kuwanon M. Cathayanon A. Morusin, Morusi intestinal tract, optimizing, improving or managing diges nol, Sanggenon C. Sanggenon D. Sanggenon O or a combi tion, inducing lipolysis, reducing intracellular triglyceride nation thereof), Magnolia extract, magnolol, honokiol, mag accumulation, reducing fat accumulation in adipose tissue or nolol and honokiol, purinol, or Piper Longum seed extract. In an adipocyte, maintaining healthy adiponectin levels, man still further embodiments, the psychotropic agent is a mood aging or reducing lipogenesis or weight gain associated with stabilizer, anti-depressant, or anti-convulsant. In even further metabolism of fructose, glucose or both, reducing or control embodiments, the insulin sensitizer is a thiazolidinedione ling oxidative stress associated with an overweight or obese (e.g., rosiglitaZone, pioglitaZone), a biguanide (e.g., met mammal (e.g., by reducing reactive oxygen species or oxida US 2014/0004215 A1 Jan. 2, 2014 59 tive free radicals; improving ORAC (Oxygen Radical epicatechin, epicatechin, or any combination thereof. In fur Absorption Capacity) values; maintaining a healthy level of ther embodiments, a pharmaceutical or nutraceutical formu glutathione, Superoxide dismutase, catalase, peroxidase or lation comprising an extract mixture as described hereincom endogenous antioxidants; maintaining healthy oxidative prises from about 0.5 weight percent (wt %) to about 50 wt % homeostasis), controlling or managing systemic inflamma of the major active ingredients in a Guazuma ulmifolia (Mu tion associated with an overweight or obese mammal (e.g., by tamba) extract. Such as a procyanidin procyanidin B2, pro promoting normal metabolism of arachidonic acid, maintain cyanidin B5, procyanidin C1, procyanidin dimers, procyani ing a normal level of pro-inflammatory cytokines), managing din trimers, procyanidin tetramers, procyanidin pentamers, mood stress or other mental disorders associated with an procyanidin hexamers, condensed tannins, an oligomer of overweight or obese mammal, or any combination thereof. catechin or epicatechin, epicatechin, or any combination 0.174. In related embodiments, a weight management thereof. agent for use with the compositions to treat or improve the 0178. In any of the aforementioned method ofuse embodi various weight-related conditions is an anorectic agent, lipase ments, a composition comprising an extract mixture (option inhibitor, cannabinoid receptor modulator, psychotropic ally including a weight management agent) wherein one of agent, insulin sensitizer, stimulant, Satiety agent, or any com the extracts is a Magnolia extract enriched for one or more bination thereof. In any of these embodiments, a weight man major active ingredients. In further embodiments, a pharma agement agent is an anorectic agent. In any of these embodi ceutical or nutraceutical formulation comprising an extract ments, a weight managementagentis alipase inhibitor. In any mixture as described herein comprises from about 5.0 weight of these embodiments, a weight management agent is a can percent (wt %) to about 10 wt % of the major active ingredi nabinoid receptor modulator. In any of these embodiments, a ents in a Magnolia extract, Such as magnolol, honokiol, both, weight management agent is a psychotropic agent. In any of or a high purity mixture of both. In certain of these embodi these embodiments, a weight management agent is an insulin ments, a Magnolia extract is a Magnolia officinalis extract sensitizer. In any of these embodiments, a weight manage enriched for magnolol, honokiol, or both, or a high purity ment agent is a stimulant. In any of these embodiments, a magnolol, honokiol, or mixture thereofisolated from a Mag Weight management agent is a Satiety agent. nolia officinalis plant extract having at least 90%, 91%, 92%, 0.175. In any of the aforementioned method ofuse embodi 93%.94%, or 95% purity. ments, a composition comprising an extract mixture (option 0179. In any of the aforementioned method ofuse embodi ally including a weight managementagent) can beformulated ments, a composition comprising an extract mixture (option with a pharmaceutically or nutraceutically acceptable carrier, ally including a Weight management agent) wherein one of diluent or excipient. In further embodiments, the pharmaceu the extracts is a Ilex paraguayensis (Yerba mate) extract tical or nutraceutical formulation comprises from about 0.5 enriched for one or more major active ingredients. In certain weight percent (wt %) to about 90 wt %, about 0.5 wt % to embodiments, an Ilex paraguayensis (Yerba mate) extract is about 80 wt %, about 0.5 wt % to about 75 wt %, about 0.5 wt enriched for caffeine, dicaffeoylquinic acid, or both. In fur % to about 70 wt %, about 0.5 wt % to about 50 wt %, about ther embodiments, a pharmaceutical or nutraceutical formu 1.0 wt % to about 40 wt %, about 1.0 wt % to about 20 wt %, lation comprising an extract mixture as described hereincom about 1.0 wt % to about 10 wt %, about 3.0 wt % to about 9.0 prises from about 0.5 weight percent (wt %) to about 5.0 wt % wt %, about 5.0 wt % to about 10 wt %, about 3.0 wt % to of the major active ingredients in an Ilex paraguayensis about 6.5 wt % of the major active ingredients in an extract (Yerba mate) extract, Such as caffeine, dicaffeoylquinic acid, mixture, or the like. In any of these embodiments, the phar or both. maceutical or nutraceutical composition is formulated as a 0180. In any of the aforementioned method ofuse embodi tablet, a capsule, a powder, or granule. ments, a composition comprising an extract mixture (option 0176). In any of the aforementioned method ofuse embodi ally including a weight management agent) wherein one of ments, a composition comprising an extract mixture (option the extracts is a Rosmarinus officinalis (Rosemary) extract ally including a weight management agent) wherein one of enriched for one or more major active ingredients. In certain the extracts is a Morus extract enriched for one or more major embodiments, a Rosmarinus officinalis (Rosemary) extract is active ingredients. In further embodiments, a pharmaceutical enriched for a carnosol, carnosoic acid, ursolic acid, or any or nutraceutical formulation comprising an extract mixture as combination thereof. In further embodiments, a pharmaceu described herein comprises from about 0.5 weight percent (wt tical or nutraceutical formulation comprising an extract mix %) to about 5.0 wt % of the major active ingredients in a ture as described herein comprises from about 1.0 weight Morus extract enriched for, for example, Kuwanon G, Alba percent (wt %) to about 10 wt % of the major active ingredi nin G. Morusin, or any combination thereof. In certain of ents in a Rosmarinus officinalis (Rosemary) extract, Such as these embodiments, a Morus extract is a Morus alba extract carnosol, carnosoic acid, ursolic acid, or any combination enriched for Kuwanon G, Albanin G. Morusin, or any com thereof. bination thereof. 0181. In any of the aforementioned method ofuse embodi 0177. In any of the aforementioned method ofuse embodi ments, a pharmaceutical or nutraceutical formulation com ments, a composition comprising an extract mixture (option prising an extract mixture (optionally including a weight ally including a weight management agent) wherein one of management agent) of a Morus extract, a Magnolia extract, the extracts is a Guazuma ulmifolia (Mutamba) extract and a Ilex paraguayensis (Yerba mate) extract, will include enriched for one or more major active ingredients. In certain from about 0.5 weight percent (wt %) to about 5.0 wt % of the embodiments, a Guazuma ulmifolia (Mutamba) extract is major active ingredients in a Morus extract, from about 5.0 wt enriched for a procyanidin procyanidin B2, procyanidin B5. % to about 10 wt % of the major active ingredients in a procyanidin C1, procyanidin dimers, procyanidin trimers, Magnolia extract, and from about 0.5 wt % to about 5.0 wt % procyanidin tetramers, procyanidin pentamers, procyanidin of the major active ingredients in Yerba mate extract. In hexamers, condensed tannins, an oligomer of catechin or certain embodiments, a pharmaceutical or nutraceutical for US 2014/0004215 A1 Jan. 2, 2014 60 mulation comprises about 1% of the major active ingredients embodiments, a Morus extract is a Morus alba extract or in a Morus extract (such as Kuwanon G, Albanin G. Morusin, extract enriched for Kuwanon G, Albanin G. Morusin, or any or any combination thereof), about 7% of the major active combination thereof, and a Magnolia extract is a Magnolia ingredients in a Magnolia extract (such as magnolol. officinalis extract or extract enriched for magnolol, honokiol, honokiol, both, or a high purity mixture of both), and about both, or a high purity mixture of both. In any of these embodi 1% of the major active ingredients in a Yerba mate extract ments, the pharmaceutical or nutraceutical composition is (such as caffeine, dicaffeoylquinic acid, or both). In any of the formulated as a tablet, a capsule, a powder, or granule. aforementioned embodiments, a Morus extract is a Morus alba extract or extract enriched for Kuwanon G, Albanin G, EXAMPLES Morusin, or any combination thereof, and a Magnolia extract is a Magnolia officinalis extract or extract enriched for mag nolol, honokiol, both, or a high purity mixture of both. In any Example 1 of these embodiments, the pharmaceutical or nutraceutical composition is formulated as a tablet, a capsule, a powder, or Preparation of Organic and Aqueous Extracts from granule. Morus alba 0182. In any of the aforementioned method ofuse embodi ments, a pharmaceutical or nutraceutical formulation com 0.184 Plant material from Morus alba L. root barks was prising an extract mixture (optionally including a weight ground to a particle size of no larger than two millimeters management agent) of a Morus extract, a Magnolia extract, (mm). Dried ground plant material (60 grams (g)) was then and a Guazuma ulmifolia (Mutamba) extract, will include transferred to an Erlenmeyer flask and Methanol: Dichlo from about 0.5 weight percent (wt %) to about 5.0 wt % of the romethane (1:1 volume ratio) (600 milliliters (mL)) was major active ingredients in a Morus extract, from about 5.0 wt added. The mixture was shaken for one hour, filtered and the % to about 10 wt % of the major active ingredients in a biomass was extracted again with Methanol: Dichlo Magnolia extract, and from about 0.5 wt % to about 50 wt % romethane (1:1 volume ratio) (600 mL). These organic of the major active ingredients in a Mutamba extract. In extracts were combined and evaporated under vacuum to certain embodiments, a pharmaceutical or nutraceutical for provide 3.55g of organic extract (OE). After organic extrac mulation comprises about 1% of the major active ingredients tion, the biomass was air dried and extracted once with ultra in a Morus extract (such as Kuwanon G, Albanin G. Morusin, pure water (600 mL). The aqueous solution was filtered and or any combination thereof), about 7% of the major active freeze-dried to provide 4.44 g of aqueous extract (AE). ingredients in a Magnolia extract (such as magnolol. 0185. Similar results were obtained using the same proce honokiol, both, or a high purity mixture of both), and about dure, but with the organic solvent being replaced with metha 1% of the major active ingredients in a Mutamba extract (Such nol or ethanol to provide a methanol extract (ME) or ethanol as a procyanidin procyanidin B2, procyanidin B5, procyani extract (EE), respectively. Other species and parts of plants din C1, procyanidin dimers, procyanidin trimers, procyanidin and marine sample were extracted using this same procedure. tetramers, procyanidin pentamers, procyanidin hexamers, condensed tannins, an oligomer of catechin or epicatechin, epicatechin, or any combination thereof). In any of the afore Example 2 mentioned embodiments, a Morus extract is a Morus alba extract or extract enriched for Kuwanon G, Albanin G, CB1 Binding Activity by Plant Extracts Morusin, or any combination thereof, and a Magnolia extract is a Magnolia officinalis extract or extract enriched for mag 0186 Cannabinoid receptor binding assays were used as a nolol, honokiol, both, or a high purity mixture of both. In any primary Screening method to identify CB1 antagonist com of these embodiments, the pharmaceutical or nutraceutical pounds. The assays were performed using methods adapted composition is formulated as a tablet, a capsule, a powder, or from by Reggio et al., J. Med Chem. 41:5177, 1998 (CB1 granule. receptor assay) and Munro et al., Nature 365:61, 1993 (CB2 0183 In any of the aforementioned method ofuse embodi receptor assay). Briefly, human cannabinoid receptor protein ments, a pharmaceutical or nutraceutical formulation com bound to Chem-1 membrane was used in modified HEPES prising an extract mixture (optionally including a weight (pH 7.4) buffer. A 10 microgram (ug) aliquot of Chem-1 management agent) of a Morus extract, a Rosmarinus offici membrane, radioactively labeled H CB1-ligand nalis (Rosemary) extract, and a Ilex paraguayensis (Yerba SR141716A (2 nanomolar (nM)) (CB1 receptor antagonist) mate) extract, will include from about 0.5 weight percent (wt and test extracts or positive control R(+)-WIN-55, 212-2 (10 %) to about 5.0 wt % of the major active ingredients in a uM) (CB1 receptor agonist) were incubated in Incubation Morus extract, from about 1.0 wt % to about 10 wt % of the Buffer (50 mM HEPES (pH 7.4), 5 mM MgCl, 1 mM CaCl2, major active ingredients in a Rosemary extract, and from 0.2% BSA) for 90 minutes at 37° C. After incubation, the about 0.5 wt % to about 5.0 wt % of the major active ingre membranes were filtered and washed four times and then the dients in a Yerba mate extract. In certain embodiments, a filters were counted to determine amount of H WIN-55, pharmaceutical or nutraceutical formulation comprises about 212-2 specifically bound to the CB1-membrane. 1% of the major active ingredients in a Morus extract (Such as 0187 Table 1 presents the CB1 binding assay results from Kuwanon G, Albanin G. Morusin, or any combination the OE and AES obtained from different plant parts of Morus thereof), about 4.5% of the major active ingredients in a alba: root barks, fruits, leaves, stem barks and stem. The Rosemary extract (Such as carnosol, carnosoic acid, ursolic greatest inhibition of CB1-ligand binding activity was found acid, or any combination thereof), and about 1% of the major in OE obtained from root barks with 91% inhibition attesting active ingredients in a Yerba mate extract (Such as caffeine, concentration of 100 ug/mL and 82% attesting concentration dicaffeoylquinic acid, or both). In any of the aforementioned of 10 ug/mL. US 2014/0004215 A1 Jan. 2, 2014
TABLE 1. was taken and combined (based on UV trace) for the CB1 inhibition assay. Column fractions having significant CB1 Inhibition of CB1 ligand binding activity by Extracts from binding activity were retained for further testing. Various Morus aiba Plant Parts
Plant Source Tested Extract Active Extract CB1 Inhibition* Example 4 Morus aiba OE, AEt. OE 91% (100 g/mL) Inhibition of CB1 Activity by Combined HTP (root barks) 82% (10 g/mL) Fractions from Morus alba Morus alba (fruits) EE Morus alba (leaves) EE", OE, AE, ME 0191 Organic extract of Morus alba root bark from Morus aiba AE Example 3 was further investigated by examining the com (stem barks) bined HTP fractions for inhibition of CB1 binding activity by Morus alba (stems) ME labeled CB1-ligand. The activity profile of combined HTP *Results are expressed as a percent inhibition relative to control radiolabeled SR141716A fractions indicates that more than one component in the frac ligand. tionated Morus alba organic extract might contribute to the *OE means ground plant material extracted with Methanol:Dichloromethane (1:1 volume ratio) as described in Example 1. inhibition of CB1-ligand binding activity. AE means OE extracted material that was air dried and extracted once with ultrapure water, filtered and freeze-dried to provide aqueous extract material as described in Example 1. 0.192 Table 3 presents the CB1 binding assay results from E means that the ground plant material was extracted with only ethanol, the combined HTP fractions of Morus alba root bark extract. 'ME means that the ground plant material was extracted with only methanol. TABLE 3 0188 These data show that organic extracts of Morus alba root bark contain moderately high levels of a component that CBl-ligand binding inhibition of combined Morus alba HTP fractions block the binding of the labeled CB1-ligand to a human CB1 receptor protein. In contrast, the other Morus alba plant tis HTPFraction Pools % CB1 Binding Activity Inhibition Sues tested in this experiment (fruits, leaves, stem bark, and Fraction 1 to 4 46% stems) all had undetectable levels of inhibition of CB1-ligand Fraction 5 to 36 88% Fraction 37 to 46 -55% binding to CB1 receptor. Fraction 47 to 84 85% 0189 The CB1 Binding assay also detected inhibition Fraction 85 to 95 78% activity from other species of plants listed in Table 2. The Fraction 96 67% common chemical components in those Moraceae and Fabaceae plants are Diels-Alder adducts of a chalcone and prenylphenyl moiety. The existence of Diels-Alder adducts of Example 5 a chalcone and prenylphenyl moiety is detectable in different plant tissues, such as roots, stems, rhizomes or whole plants. Isolation, Purification, and Identification of CB1-Binding Inhibitors from Morus alba Extracts TABLE 2 0193 An organic extract (11 g) from the root barks of CB1 binding activity from plant extracts containing prenylated flavonoids Morus alba, obtained as described in Example 1, was divided Family Latin Name CB1 Binding activity Plant parts and loaded separately onto two pre-packed flash columns (120 g silica, particle size 32-60 um, 4 cmx19 cm), and then Moraceae Morus aiba 82% at 10 g/ml Root barks eluted with Hexane, EtOAc and Methanol (as the mobile 91% at 100 g/ml Moraceae Miicia excelsa 29% at 10 g/ml Stem barks phase) at a flow rate of 20 mL/minutes. The gradients started 113% at 100 g/ml with 95% Hexane/EtOAC for 5 minutes, then increased Fabaceae Glycyrrhize glabra 48% at 10 g/ml Rhizome EtOAC from 5% to 100% over the duration of 25 minutes, and 67% at 100 g/ml then held at 100% EtOAc for additional five minutes, before increasing MeOH from 0% to 50% MeOH/EtOAC over a next period of 15 minutes, finally changed the elution solution to Example 3 100% MeOH and eluted the column for another 16 minutes. The total run time was 66 minutes and 88 fractions were generated for each column. The fractions were analyzed by High Throughput Purification (HTP) of Active Plant silica gel thin layer chromatography (TLC) and pooled Extracts together to generate eight column eluent pools. Each of the 0190. Organic extract material (400 mg) from the Morus eight eluent pools was then tested using the CB1 binding alba root bark extract obtained in Example 1 was loaded onto assay described in Example 2. The resulting CB1 binding a prepacked (2 cm IDx8.2 cm, 10 g silica gel) column. The assay data are shown in Table 4. column was then eluted using a Hitachi(R) High Throughput Purification (HTP) system with a gradient mobile phase of TABLE 4 (A) 50:50 volume ratio of EtOAc: Hexane and (B) Methanol from 100%. A to 100% B in 30 minutes at a flow rate of 5 CB1 activity by Silica Fraction Pools mL/min. The separation was monitored using a broadband Eluent Pool Number % CB1 Binding Activity Inhibition wavelength UV detector and the fractions were collected in a -43% 96-deep-well plate at 1.9 mL/well using a Gilson fraction -36% collector. The sample plate was dried under low vacuum and -5% centrifugation and then the samples were dissolved with 1.5 64% mL dimethylsulfoxide (DMSO) per well. A portion (100 uL) US 2014/0004215 A1 Jan. 2, 2014 62
TABLE 4-continued Compound 2 (10.5 mg) was identified as Albanin G, also known as Kuwanon H or Moracenin A, another Diels-Alder CB1 activity by Silica Fraction Pools adduct of a chalcone and prenylphenyl moiety by HRESIMS Eluent Pool Number % CB1 Binding Activity Inhibition (m/z) M-H =759; UV (MeOH): 265, 320 nm: 'C 71.9% NMR (126 MHz, METHANOL-d) ppm 16.35 (1C) 16.47 29% (1C) 20.96 (1C) 21.79 (1C) 23.32 (1C) 24.51 (1C) 24.53 (1C) 29% 33.74 (1C) 35.61 (1C) 36.81 (1C) 37.77 (1C) 97.19 (1C) 35% 102.27 (1C) 102.33 (1C) 104.24 (1C) 106.07 (1C) 106.53 (2C) 107.34 (1C) 112.37 (1C) 113.94 (1C) 114.35 (1C) 120. 0194 These data show that the highest level of inhibition 17 (1C) 121.60 (2C) 122.31 (2C) 123.25 (1C) 130.21 (2C) of CB1-ligand binding was in pool 4 (containing 1.4 g of 131.33 (2C) 132.96 (1C) 156.37 (3C) 157.07 (1C) 159.59 material) and pool 5 (1.7g of material) when tested in the CB1 (1C) 160.37 (1C) 161.23 (1C) 161.77 (1C) 161.96 (1C) 162. binding assay at a concentration of 20 ug/mL. 21 (1C) 182.45 (1C) 208.82 (1C). 0.195 The resulting best active best pool (containing 300 mg of material) was fractionated on a preparative C18 column (30 cmx250 cm) with a gradient mobile phase of water (A)
and methanol (B) over 60 minutes at a flow rate of 20 mL/minute to generate 22 fraction pools. Mass Spectrometry (MS) analysis showed that these pooled fractions of material contain three related compounds, described in more detail below, all of which had inhibitory activity in the CB1-binding assay described in Example 2. Compound 1 (28.2 mg) was identified as a Diels-Alder adduct of a chalcone and prenylphenyl moiety called Kuwanon G, also known as Moracenin B or Albanin F, by High Resolution Electron Spray Ionization Mass Spectroscopy (HRESIMS) (m/z) M+H=693.2329; UV (MeOH): 265, 320 nm: "H NMR (600 MHz, DMSO-d. 100° C.) & ppm 1.44 (s.3H) 1.52 (br. s. 3H) 1.58 (s, 3H) 1.92 (m, 2H)3.08 (d. 3H)3.56 (m. 2H)4.29 (d. J=10.02 Hz, 1H)4.48 (m, 1H) 5.07 (m, 1H) 5.14 (br. s. 1H) 5.93 (s. 2H) 5.96 (dd, J=8.35, 2.23 Hz, 1H) 6.02 (brs, 1H) 6.11 (d. J=2.23 Hz, 1H) 6.41 (dd, J-8.35, 2.23 HZ, 1H) 6.51 (s, 1H) 6.60 (m, 1H) 7.13 (d. J=8.35 Hz, 1H) 7.28 (brs, 1H); 'C NMR (126 MHz, METHANOL-d) & ppm 16.35 (1C) 21.78 (1C) 23.35 (1C) 24.53 (1C) 37.72 (1C) 97.14 (1C) 101.57 (1C) 102.22 (1C) 102.33 (1 C) 104.28 (1C) 106.55 (2C) 107.00 (1C) 107.21 (1C) 112.37 (1C) 114.47 Example 6 (1C) 120.27(1C) 121.62 (2C) 123.27 (1C) 131.05 (1C) 131. 35 (2C) 132.62 (1C) 132.99 (1C) 155.16 (1C) 155.56 (1C) Inhibition of CB1, CB2 and O-Glucosidase by 156.38 (1C) 159.66 (1C) 160.39 (2C) 161.13 (1C) 161.88 Kuwanon G and Albanin G Purified from Morus (1C) 164.51 (1C) 164.63 (1C) 182.46 (1C) 208.68 (1C). alba 0196. The CB1 binding assay, described in Example 2, was used to test the Kuwanon G and Albanin G compounds isolated and identified in Example 5. The Kuwanon G Alba nin G compounds were tested at concentrations ranging between 0.04 g/mL and 20 g/mL, to obtain a dose-response curve for each compound. The sample concentration was plotted against the percent inhibition and the ICso (defined as the concentration at which 50% inhibition of binding activity is achieved in relation to the control) was determined. CB1 assay data for each compound are shown in Table 5. 0.197 Inhibition of CB2 receptor-ligand binding activity of the purified Kuwanon G and Albanin G compounds was also examined using methods similar to those described in Example 2 for the CB1 receptor, with some modifications. Briefly, human cannabinoid CB2 receptor protein expressed in CHO-K1 cells were used in modified HEPES buffer (pH Kuwanon G 7.0). A 30 ug aliquot of CB2-membrane was mixed with tritium labeled nonspecific CB1 agonist H WIN-55,212-2 (2.4 nM) and test samples of Kuwanon G and Albanin G US 2014/0004215 A1 Jan. 2, 2014 compounds, or just the non-specific ligand R (+)-WIN-55, 212-2 (10 uM) (positive control) were incubated in incuba tion buffer (20 mM HEPES (pH 7.0), 0.5 mg/ml BSA) for 90 minutes at 37° C. After incubation, the membranes were filtered and washed; the filters were then counted to determine the amount of radiolabeled H. WIN-55, 212-2 that was specifically bound to the CB2-membrane. The CB2 assay data for each compound are shown in Table 5. 0198 Inhibition of rice C-glucosidase activity by each of the purified Kuwanon G and Albanin G compounds was measured as follows. Rice O-Glucosidase inhibition assay: the test compound or vehicle was pre-incubated with 77 mu/ml enzyme Rice C-glucosidase in MES buffer pH 6.3 for Sanggenon 15 minutes at 25°C. The reaction was initiated with addition of 2 mM p-nitrophenyl C-D-glucopyanoside, incubated for another 90 minute at 70° C., and then terminated by adding of 0201 Sanggenon was tested in the CB1 binding inhibition 1 M sodium carbonate. The end-product p-nitrophenol was activity assay as described in Example 2. The activity data are measured by spectrophotometer. The rice C-glucosidase set forth in Table 6. assay data for each Morus alba compound is shown in Table 5. TABLE 6 Inhibition of CB1 and CB2 by Sanggenon isolated from Milicia excelsa TABLE 5 CB1 CB1 ICso CB2 ICso Inhibition of CB1, CB2 and C-glucosidase by Kuwanon G and Albanin G Compound (20 g/ml) (IM) (IM) Sanggenon 96% 3.5 24 %. Inhibition Rice % Inhibition C-glucosidase CB1-Binding CB1 ICso CB2 ICso Compound (100 g/ml) (20 g/ml) (IM) (IM) 0202 These data show that Sanggenon is a potent inhibitor of CB1 ligand binding with CB1 selectivity over the CB2 Kuwanon G -22% 92% 10.1 28.9 receptor protein. Albanin G 66% 96% O.1 6.6 Example 8 0199 These data demonstrate that the two major active Preparation of Various Milicia excelsa Extracts compounds—Kuwanon G and Albanin G—are CB1 selective inhibitors and that the extract is free from any significant 0203 Milicia EtOAc extract 8 was produced as follows: 5 C-glucosidase inhibition. kg of dried Milicia excelsa stem barks were cut, crushed, and extracted with approximately 4-fold volume (20 L) of ethyl alcohol (Food grade, Korea Ethanol Supplies Company, Example 7 Korea) in water (v/v). The extraction solvent was treated at 80° C., for 4 hrs and the resulting extraction was filtered to CB1 and CB2 Binding Inhibition by Compounds obtain a Supernatant that was concentrated with evaporator at Purified from Milicia excelsa (African Teak) 40° C. The above-described extraction procedure was repeated two times. The resulting extraction solutions were 0200. The organic extract (8 g) from the stem barks of combined together and concentrated until the Volume become Milicia excelsa, obtained using the methods described in 1/25 of the original volumes. The concentrated solution was Example 1, was divided and loaded separately onto two pre then dried by vacuum freeze-drying to obtain 200 g of crude Milicia excelsa EtOH extract powder. 196 g of crude Milicia packed flash columns (120 g silica, particle size 32-60 um, 4 excelsa EtOH extract powderprepared in the above procedure cmx19 cm), then the column was eluted with the gradient as was suspended in 2 L of distilled water and the Suspension described in Example 5. A Diels-Alder adduct of a chalcone was vigorously mixed with 2 L of n-hexane to obtain an and prenylphenyl moiety was isolated from one of the active n-hexane soluble fraction and water-soluble fraction. The fractions and identified as Sanggenon C/D/O. The structure n-hexane soluble fraction was collected and the residual solu and spectroscopy data were as follows: ESIMS (m/z) tion was Subjected to a second n-hexane extraction. The M-2H 706: UV (MeOH): 265,320 nm: 'HNMR (500 above-described procedure was repeated four times and the MHz, METHANOL-d) ppm 1.55 (s, CH3 H) 1.58 (s, CH, resulting n-hexane Soluble fractions were combined and 3 H) 1.82 (m, CH3 H)2.28 (dd, J=18.65, 5.09 Hz, 1H)2.39 evaporated under vacuum to obtain 74.8g of n-hexane soluble (dd, J=17.80, 5.09 Hz, 1H)2.69 (m, 1H)2.94 (m, 1H)3.87 (d. extract 8-1 of Milicia excelsa stem bark. J=6.78 HZ, CH, 1H)4.16 (br. S., CH, 1H)4.49 (br. S., CH, 1H) 0204. The water-soluble fraction of Milicia excelsa stem 5.19 (br. S., 1H) 5.45 (br. S., 1H) 5.64 (s, 1H) 6.11 (d. J=2.26 bark prepared in above procedure was vigorously mixed with HZ, 1H) 6.17 (dd, J=8.48, 2.26 Hz, 1H) 6.23-6.34 (m, 3H) an equivalent Volume of ethyl acetate to obtain an ethyl 6.42 (dd, J=8.20, 1.70 Hz, 1H) 6.86 (d. J=8.19 Hz, 1H) 7.21 acetate soluble fraction and a water-soluble fraction. The (d. J=8.48 Hz, 1H) 8.08 (d. J=8.76 Hz, 1H). ethyl acetate soluble fraction was collected and the residual US 2014/0004215 A1 Jan. 2, 2014 64
Solution was subjected to the ethyl acetate extraction again. by that measured at 620 nm (ratio). The results are expressed This procedure was repeated four times. The ethyl acetate as a percent of the control response to 100 nMCP 55940 (full soluble fractions and water-soluble fractions were respec CB1 agonist). The standard reference agonist is CP 55940, tively evaporated under vacuum to obtain 63.9 g of ethyl which is tested in each experiment at several concentrations acetate soluble extract 8 and 35.34g of water-soluble extract to generate a concentration-response curve from which its 8-2 of Milicia excelsa stem bark. ECso value is calculated. Example 9 CB1 Antagonist Effect GPCR Functional Assay Test of O-Glucosidase and CB1 Activity in Morus (0209. The CHO-K1 cells are suspended in HBSS buffer alba Extracts complemented with HEPES 20 mM (pH 7.4), then distributed in microplates at a density of 10 cells/well and preincubated 0205 The Morus abla 70% EtOH extract and its enriched for five min at room temperature in the presence of one of the EtOAc fraction generated from water/EtOAc partition were following: the reference antagonist, AM 281, at 3 uM (basal evaluated for the activity against C-glucosidase in the assay control), HBSS (stimulated control), the reference antago described in Example 6. These data are displayed in Table 7. nist, for ICso determination, or the test plant extract com pounds. The reference agonist CP 55940 is also present at a TABLE 7 final concentration of 10 nM or is omitted from the reaction mixture for the basal control. Thereafter, the adenylyl cyclase CB1 and O-Glucosidase Inhibition Activity of Morus alba Extracts activator NKH 477 is added at final concentration of 3 uM. Ci-Glucosidase Inhibition Following 10 min incubation at 37°C., the cells are lysed and Sample (100 g/ml) CB1 Inhibition the fluorescence acceptor (D2-labeled cAMP) and fluores 70% EtOH 99% 64% (20 g/ml) cence donor (anti-cAMP antibody labeled with europium EtOAc Fraction -1.8% 58% (5 g/ml) cryptate) are added. After 60 min at room temperature, the fluorescence transfer is measured at 337 nm and 620 nm and 665 nm using a microplate reader (Rubystar, BMG). 0206. These data show that after EtOAc enrichment of The cAMP concentration is determined by dividing the signal Morus alba ethanol extract, the C-glucosidase inhibition measured at 665 nm by that measured at 620 nm (ratio). The activity of the ethanol extract was eliminated, while the bind results are expressed as a percent inhibition of the control ing inhibition activity of the extract (i.e., prenylated fla response to 10 nMCP 55940. The standard reference antago vonoids) in the CB1 receptor binding assay was retained. nist is AM 281, which is tested in each experiment at several concentrations to generate a concentration-response curve Example 10 from which its ICso value is calculated. The compounds were tested at six different concentrations (0.3, 1, 3, 10,30, and 150 In Vitro Study of CB1 Functional Activity of uM) to generate a dose curve. The results are set forth in Table Compounds Isolated from Morus alba and Other 8 below. Plants 0207. In vitro efficacy and CB1 binding specificity of TABLE 8 compounds isolated from Morus alba and Milicia excelsa were tested in the human CB1 receptor binding assay, and the CBI Agonist and Antagonist Activity agonist or antagonist function of the compounds was deter CB1 Agonist ECso CB1 Antagonist Inhibition mined by measuring the effect on cAMP or agonist-induced Compound (IM) (at 100M) cAMP modulation. Assays were performed by Ricerca Bio Kuwanon G 85 -1% Sciences LLC, (Concord, Ohio) on plant extract samples Albanin G ND 1796 essentially as described by Breivogel et al., J. Biol. Chem. Sanggenon 31 -14% 273:16865, 1998 and Gonsiorek et al., Mol. Pharmacol. 57:1045, 2000. 0210. The data presented in Table 7, suggest that the Kuwanon G and Sanggenon compounds are affecting the CB1 Agonist Effect GPCR Functional Assay CB1 receptor activity with agonist-like behavior instead of 0208 Briefly, CHO-K1 cells were suspended in HBSS antagonist. In contrast, Albanin G appears to have a modest buffer complemented with HEPES 20 mM (pH 7.4), then antagonist-like activity with the CB1 receptor. distributed in microplates at a density of 10 cells/well in the presence of one of the following: HBSS (basal control), a Example 11 reference agonist at 100 nM (stimulated control), a reference agonist (ECso determination) or a plant extract test com HPLC Quantification of Active Extracts from Morus pound. Thereafter, the adenylyl cyclase activator NKH 477 is alba added at a final concentration of 3 uM. Following a 10 minute incubation at 37°C., the cells are lysed and the fluorescence 0211 Extracts for raw materials were produced as fol acceptor (D2-labeled cAMP) and fluorescence donor (anti lows: 20 grams of plant powder mixed with Diatomaceous cAMP antibody labeled with europium cryptate) were added. earth was put into a 100 mL extraction cell. It was extracted After 60 minat room temperature, the fluorescence transfer is with solvent (100% EtOH or MeOH/CHCl at 1:1) using measured at ex=337 nm and em=620 nm and 665 nm using ASE 350 (Extraction condition: Heat=5 minutes, Static=5 a microplate reader (Rubystar, BMG). The cAMP concentra minutes, Flush=80 volume, Purge=900 seconds, Cycles=3, tion is determined by dividing the signal measured at 665 nm Pressure=1500 psi, Temperature—80° C., Solvent C=100% US 2014/0004215 A1 Jan. 2, 2014
Ethanol). After extraction, the solution was filtered and col TABLE 11-continued lected, then concentrated with evaporator at 50° C. to produce a solid extract. Kuwanon G and Albanin G Contents in Different Parts of Morus Plants 0212 Agilent HPLC/PDA system was used for the detec The content (%) tion and quantification of the Diels-Alder adducts of a chal of extract Extraction cone and prenylphenyl moiety Kuwanon G and Albanin in the Morus extracts. The C18 reversed-phase column (Phenom Morus aiba Part Kuwanon G AlbaninC Yield (%) enex, USA) was utilized as Luna 5um, 50 mm long and 4.6 Fine root 3.52 2.98 15.4 mm in diameter. A binary Purified water (mobile phase A) and Stem bark 0.97 O.61 10.2 Stem wood N.D N.D 3.2 acetonitrile (mobile phase B) gradient was used for elution. 5 Bark 3.48 1.26 24.6 The flow rate was set to 1 ml/min passing through the Luna Periderm O.09 ND 21.5 C18 column with a column temperature of 40°C. The UV detector was set to read absorbance at 270 nm.
TABLE 9 TABLE 12 Kuwanon G and Albanin G Content in Morus aiba Root Barks from Gradient Table of HPLC Analytical Method Different Geological Locations in Korea Time (min) Mobile phase A Mobile phase B Active content (% Extraction O.O 50 50 S.O 50 50 Vendor Name Kuwanon G Albanin G Yield (%) 3O.O 2O 8O 3O.S 50 50 Kyoungdong S.13 3.98 15.8 3S.O 50 50 Kyoundong 1.23 O.82 17.4 Asan 3.11 1.81 19.2 Seon-ilmulsan 1.99 O.S9 102 0213 Pure Kuwanon G, pure Albanin G, and Reference Kyoungdong O.80 N.D 11.05 Standard Material (RSM, Morus EtOAc fraction 17) were utilized as quantification standards. All extract samples were prepared in a concentration range from 3 mg/ml to 1 mg/ml TABLE 13 after Sonicating for approximately 15 minutes. The sample Solution was cooled in a flask to room temperature and filtered Kuwanon G and Albanin G Content in Morus aiba Root Barks through a 0.45 um nylon syringe filter and 10 ul of the sample purchased from Different Geological Locations in China. was injected into HPLC. The content (%). Extract Extraction 0214) Morus plants were collected from China and South Korea from different geological locations in both countries. Vendor Name Kuwanon G Albanin G Yield (%) The HPLC quantification of Kuwanon G and Albanin G con Sichuan 3.64 2.82 14.1 Hubei O.64 O.13 6 tent in different species, different plant parts, collected from Hunan O.14 O.18 5.65 different locations, and at differentage of plants, are listed in Guizhou 4.6O 3.09 14.1 Table 10 through Table 14. Yunnan O.67 O.33 8 Sichuan Xichang O.85 N.D 12.1 Sichuan Mian Yang 4.66 2.74 11 TABLE 10 Sichuan 3.41 2.73 14.5 Sichuan 3.44 2.67 15.1 Kuwanon G and Albanin G Content in Two Species of Morus Plants Hunan 4.6O 2.30 11.05 % of Extract Extraction Sample Kuwanon G AlbaninC Yield (%) TABLE 1.4 Morus aiba S.13 3.98 15.8 4.6 3.09 14.1 Kuwanon G and Albanin G Content in Different Age Moris Catayana 0.4 ND 9.7 of Morus aiba Root Barks.
Content% Extract Extraction
TABLE 11 Location Years old Kuwanon G Albanin G Yield (%) A. 2 8.85 6.12 12 Kuwanon G and Albanin G Contents in Different Parts of Morus Plants 3 3.49 2.40 8.9 B 2 2.49 1.00 8.6 The content (%) of extract Extraction Morus aiba Part Kuwanon G AlbaninC Yield (%) Example 12 1 Leaf O.O6 N.D 7.9 2 Fruit N.D N.D 23.1 3 Branch O.2O O.25 7.4 Preparation of Morus Alba 70% EtOH Extract 12 4 Leaf, Branch N.D N.D 7.3 Root bark 3.90 2.51 17.8 0215 Dried Morus alba roots and root barks (93.3 kg) Root wood O.15 N.D 5.4 were cut, crushed, and then extracted with approximately seven-fold volume (700 L) of 70% ethyl alcohol in water US 2014/0004215 A1 Jan. 2, 2014 66
(v/v); the extraction was carried out at 100° C. for 4 hrs. The n-hexane soluble and water soluble extracts were prepared ethanol solution was filtered to obtain the supernatant, which from 100 g of the crude Morus alba EtOH powder according was then concentrated with an evaporator under vacuum at to the method of Example 14. This resulted in production of 40° C. This extraction and concentration procedure was 13.7 g of the n-hexane soluble extract and 38 g of the water repeated two times. The extraction solutions were then com soluble extract of Morus alba. After solvent partition with bined together and concentrated until the volume become EtOAc, the final amount of Morus alba EtOAc fraction 15 1/25 of the original volume. The concentrated solution was obtained from this process was 47.6 g. The major active dried by vacuum freeze-drying to obtain 18.3 kg of Morus component content is listed in Table 16 of Example 20. alba 70% EtOH extract powder 12. The extraction yield was about 19.6% (w/w). The major active component content is Example 16 listed in Table 16 of Example 20. Preparation of Morus alba EtOAc Fraction 16 Example 13 0219 Morus EtOAc fraction 16 was produced using the extraction methods described in Examples 12 and 14.3 kg of Preparation of Morus alba EtOAc Fraction 13 dried Morus alba roots and root bark yielded 428 g of crude 0216. Morus alba EtOH extract 12 from Example 12 (15 Morus alba EtOH extract powder. Corresponding n-hexane kg) was extracted with approximately two-fold volume (300 soluble and water soluble extracts were prepared from 300 g L) of ethyl acetate (EP grade, Ducksan Chemical, Korea). of the crude Morus alba EtOH powder according to the meth Extraction was performed by homogenization of the extrac ods of Example 14. This resulted in production of 40.8g of the tion solution at 15,000 rpm for five minutes with homog n-hexane soluble extract and 92.7 g of the water-soluble enizer (IKAT25D, Germany). The well homogenized extrac extract of Morus alba. After solvent partition with EtOAc, the tion solution was then separated by centrifuge (Beckman final amount of Morus alba EtOAc fraction 16 obtained from J-20XP, Germany) at 3,000 rpm (rotorii JLA 8.1000) for five this process was 150.1 g. The major active component content minutes. The upper layer (EtOAc soluble layer) was filtered is listed in Table 16 of Example 20. by filterpaper (Hyundai Micro, No. 20, Korea) and the EtOAc solution was collected. The residue (precipitate material) col Example 17 lected from the centrifugation was re-extracted with two-fold volume (300 L) of ethyl acetate (EP grade, Ducksan Chemi Preparation of Morus alba EtOAc Fraction 17 cal, Korea). The re-extracted solution was agitated at 150 rpm 0220 Morus EtOAc fraction 17 was produced using the for 2 hours. The resulting mixture was then filtered (Hyundai extraction methods described in Examples 12 and 14.4kg of Micro, No. 20, Korea) to obtain an additional EtOAc extract dried Morus alba roots and root bark yielded 570 g of crude solution. The above-described procedure was repeated two Morus alba EtOH extract powder. Corresponding n-hexane times. The resulting three EtOAc extract solutions were then soluble and water soluble extracts were prepared from 570 g combined and concentrated by evaporator at 40°C. to obtain of the crude Morus alba EtOH powder according to the meth the final EtOAc extract 13. The yield was 3.04 kg from 15 kg ods of Example 14. This resulted in production of 80.5g of the of 70% EtOH extract. The major active component content is n-hexane soluble extract and 156 g of the water-soluble listed in Table 16 of Example 20. extract of Morus alba. After solvent partition with EtOAc, the Example 14 final amount of Morus alba EtOAc fraction 17, obtained from this process was 327 g. The major active component content Preparation of Morus alba EtOAc Fraction 14 is listed in Table 16 of Example 20. 0217 Morus alba EtOAc fraction 14 was produced from Example 18 EtOH extract using the extraction methods described in Example 12.4.5 kg of dried Morus alba roots and roots barks Preparation of Morus alba 70% EtOH Precipitate yielded 715 g of crude Morus alba EtOH extract powder. Extract 18 Corresponding n-hexane Soluble and water Soluble extracts were prepared from 691.4 g of the crude Morus alba EtOH 0221 Morus alba EtOH precipitate extract 18 was pro powder after solvent partition. This resulted in production of duced by follows: 634 kilograms (KG) of dried Morus alba 95.9 g of the n-hexane soluble extract and 263.8 g of the roots and root barks were cut, crushed and extracted with water-soluble extract of Morus alba. The water soluble approximately 7 fold volume (3600 liters (L) of 70% ethyl extract was further partitioned with an equivalent amount of alcohol in water (v/v); the extraction solvent was treated at ethyl acetate (EtOAc) to produce an EtOAc extract. The final 80°C., for 4 hrs; the residue was filtered to obtain the super amount of Morus alba EtOAc fraction 14 obtained from this natant which was then concentrated with an evaporator at 40° C. The above-described procedure was repeated three times. process was 331.8 g. The major active component content is The extraction solutions were then concentrated until the listed in Table 16 of Example 20. volume become about 1/30 the original starting volumes. Example 15 Then the concentrated solutions were combined to evaporate again in order to reduce Volume of concentrated Solution until 1790 volume of the original extraction solution. The concen Preparation of Morus alba EtOAc Fraction 15 trated solution was rested at room temperature for 24 hours 0218 Morus EtOAc fraction 15 was produced using the (hr) to allow separation into two layers (Supernatant and pre extraction methods described in Examples 12 and 14. Dried cipitate-layer). The precipitate was filtered and dried by Morus alba roots and root barks (2.0 kg) yielded 283.5g of vacuum freeze-drying to obtain Malba 70% EtOH precipitate crude Morus alba EtOH extract powder. Corresponding powder. A total of 24kg of the resulting product was obtained US 2014/0004215 A1 Jan. 2, 2014 67 from 634 kg of raw plant material. The extraction yield was components of Kuwanon G and Albanin G in the pilot and about 3.79% (w/w). The major active component content is production grade Mortis alba root bark extracts and fractions listed in Table 16 of Example 20. described in Examples 12-19.
Example 19 TABLE 16 Preparation of Morus alba 70% EtOH Precipitate Kuwanon G and Albanin G in Different Morus aiba (19-1), EtOH Combination (19-2), and EtOH Root Bark Extracts and Fractions Supernatant (19-3) Extracts % Total 0222 Morus alba EtOH precipitate extract was produced Sample Extract %. Kuwanon G % Albanin G Active as follows: 465 kg of dried Morus alba roots and root bark 12 2.88. 1.64 4.51 were cut, crushed, and extracted with approximately 10-fold 13 8.8O S.80 14.60 14 13.53 8.32 21.85 volume (4500 L) of 70% ethyl alcohol in water (v/v); the 15 1O.S1 6.95 1746 extraction solvent was treated at 80° C. for 4 hrs; the residue 16 10.93 7.38 18.31 was filtered to obtain the supernatant which was concentrated 17 (RSM) 9.95 6.65 16.60 with an evaporator at 40°C. Above-described procedure was 18 5.30 4.16 946 repeated three times. The extraction solutions were concen 19-1 6.21 4.33 10.54 trated until the volume become 1/30 the original volume. The 19-2 3.30 1.81 S.11 concentrated solutions were then combined and evaporated again to reduce the Volume of the concentrated Solution until 1790 volume of the original extraction solution was achieved. Example 21 The concentrated solution was left at room temperature for 24 hr to allow separation into a Supernatant and precipitate layer. Preparation of Rosemary Steam Distillated EtOH The precipitate layer was then dried by vacuum to obtain 12 Extract kg of Morus alba 70% EtOH precipitate extract powder. The 0226 Dried Rosemary leaf (2.6 kg) was distillated with extraction yield was about 2.6% (w/w). The supernatant layer approximately 6 fold volume (15 L) of water to remove essen was dried by vacuum drying to obtain 24 kg Morus alba 70% tial oils and volatile components at 100° C. for 5 hrs. After EtOH supernatant extract powder. The extraction yield for the steam distillation the retained residue was filtered to remove Supernatant extract was about 5.2%. the water distillated solution from the residue and then, the 0223. Morus alba 70% EtOH combination extract 19-2 residue was re-extracted with 6-fold volume (15 L) of ethyl was obtained by blending 2 kg of precipitate extract 19-1 and alcohol (95%) at 80°C. for 5 hrs. The resulting extraction was 4 kg of supernatant extract 19-3. The major active component filtered to obtain a Supernatant that was concentrated using an content in both Morus alba EtOH precipitate 19-1 and com evaporator at 40°C. The above-described extraction proce bination extract 19-2 is listed in Table 16 of Example 20. dure was repeated twice. The resulting Supernatants were Example 20 combined together and then concentrated with an evaporator at 40°C. The resulting concentrated solution was then dried HPLC Quantification of Active Content in Different by vacuum freeze-drying to obtain 357.1 g of Rosemary Morus alba Extracts steam distillated EtOH extract powder 21. The extraction yield was about 13.7% (w/w). Chemical analysis showed that 0224. The detailed HPLC quantification method for Rosemary extract 21 contained the following compounds Kuwanon G and Albanin G was described in Example 11. (percent weight): Carnosol. 2.97%; Carnosic Acid: 2.00% With the complexity of crude extracts, different elution sol and Ursolic acid: 18.49%; total actives of these three com vents, such as the following, could also be utilized: a binary pounds 23.80%. 0.1% phosphoric acid in purified water (mobile phase A) and acetonitrile (mobile phase B) gradient was used for elution Example 22 (Table 15). Preparation of Rosemary EtOH Extracts 22 and 22-1 TABLE 1.5 0227 Rosemary EtOH extract 22 was produced using Gradient table of HPLC Analytical Method essentially the methods described in Example 21. 29 kg of Time (min) Mobile phase A Mobile phase B dried Rosemary leaf was distillated with approximately 10-fold volume (300 L) of water to remove essential oils and O 33 67 5 33 67 volatile components at 100° C. for 2 hrs. The residue was next 55 24 76 filtered to remove water distillated solution from the residue 59 O 100 and then the residue was re-extracted with 10 fold volume 65 O 100 (300 L) of 95% ethyl alcohol at room temperature (20°C.) for 66 33 67 2 hrs. The resulting residue was filtered to obtain supernatant 75 33 67 that was concentrated with an evaporator at 40°C. The result ing residue was extracted again with a 10-fold volume (300 L) 0225. The flow rate 0.8 ml/min passing through the Luna of 70% ethyl alcohol (v/v) at room temperature (20°C.) for 2 C18 column with column temperature 35°C. The UV detec hrs. The resulting extract residue was filtered and the super tor was set up at 275 nm. The reference standard material natants retained and combined. The resulting Supernatant was (RSM, Morus EtOAc fraction 17) was extracted with DMSO then concentrated with an evaporator at 40°C. The resulting and utilized for calculation. Table 16 lists the major active concentrated solution was finally dried by vacuum freeze US 2014/0004215 A1 Jan. 2, 2014 68 drying to obtain 2.5 kg of Rosemary water distillated EtOH TABLE 17 extract powder 22. The extraction yield was about 8.6% (w/w). HPLC Actives profile for Carnosol, Carnosic acid and Rosemary Gradient Elution Scheme Ursolic acid of this extract are provided in Example 25, Table Time (min) Mobile phase A Mobile phase B 18. O 40 60 0228 Rosemary EtOH extract 22-1 was produced as fol 5 2O 8O lows: 58.68 kg of dried Rosemary leaf was distillated with 30 O 100 approximately 10 fold volume (600L) water to remove essen 34 O 100 tial oils and volatile components at 100° C. for 2 hrs; the 35 40 60 residue was removed from the water distillated solution by 40 40 60 filtration and then the residue was re-extracted with 10 fold volume (600 L) 95% ethyl alcohol at room temperature (20° 0232 Carnosol, Carnosic acid and Ursolic acid standards C.) for 2 hrs. The residue was filtered to obtain supernatant. were purchase from Sigma and dissolved in DMSO. Highest The residue was then extracted again with 10 fold volume level control concentration of carnosol and carnosic acid was (600 L) 70% ethyl alcohol (v/v) at room temperature (20° C.) made to 0.1 mg/ml. The highest level control concentration of for 2 hrs. The doubly extracted residue was filtered to obtain ursolic acid was made to 0.3 mg/ml and diluted to (0.065 Supernatant. Both Supernatants were combined and concen mg/ml or 0.02 mg/ml) using methanol. The test sample con trated with an evaporator at 40°C. Concentrated supernatants centration were adjusted to about 1 mg/ml in methanol in a were dried by vacuum freeze-drying to obtain Rosemary volumetric flask and sonicated until the sample dissolved water distillated EtOH extract powder 22-1. The yield was 5.0 (approximately 20 minutes). The sample flask was then kg Rosemary extract powder 22-1 obtained from 58.68 kg of cooled to room temperature, mixed well and filtered through raw material. The extraction yield was about 8.5% (w/w). a 0.45 um nylon syringe filter and then 10 ul of sample was injected into HPLC. HPLC results showed the Table 10. Example 23 TABLE 1.8 Preparation of Rosemary Water Distillated EtOH Extract 23 HPLC Results of Rosemary Leaf Extracts 0229 Rosemary EtOH extract was produced essentially as Total described in Example 22. 62 kg of dried Rosemary leaf Sample Extract Carnosol % Carnosic acid% Ursolic acid% % yielded 5.9 kg water distillated extract powder. The extraction 22 8.84 4.37 7.83 21.04 yield was about 9.5% (w/w). HPLC Actives profile for Car 23 6.52 5.32 10.47 22.31 nosol, Carnosic acid and Ursolic acid in this extract 23 are 24 4.07 2.69 14.53 21.30 provided in Example 25, Table 10. Example 24 Example 26 Preparation of Rosemary Steam Distillated EtOH Preparation of Yerba mate Ethyl Alcohol Extract 26 Extract 24 0233 Yerba mate (Ilex paraguariensis) EtOH extract was produced as follows: 1 kg of dried Ilex paraguariensis leaf. 0230 Rosemary steam distillated EtOH extract was pro was cut, crushed, and extracted with a 20-fold volume (20 L) duced essentially as described in Example 21.2 kg of dried of 95% ethyl alcohol at 85°C. for 4 hrs. The resulting residue Rosemary leaf yielded 317.7 g of water distillated extract was filtered to obtaina Supernatant that was concentrated with powder designated lotii RN348-3201. The extraction yield an evaporator at 40°C. The resulting residue was extracted a was about 15.8% (w/w). HPLC Actives profile for Carnosol, second time with 20-fold volume (20 L) of 95% ethyl alcohol Carnosic acid and Ursolic acid in this extract are provided in (v/v) at 85° C. for 4 hrs and filtered to obtain a second Example 22, Table 10. Supernatant which was concentrated with an evaporator at 40° C. The resulting concentrated cake was dried under vacuum Example 25 to obtain 260 g of Yerba mate EtOH extract powder. The extraction yield was about 26% (w/w). Analytical Results for Rosemary Leaf Extract 0231. The following analytical method was used to deter Example 27 mine the amount of Carnosol, Carnosic acid and Ursolic acid in Rosemary steam distillated ethanol extract 24 and water Preparation of Yerba mate Ethyl Alcohol Extract 27 distillated ethanol extracts 22 and 23. An Agilent HPLC/PDA 0234 Yerba mate EtOH extract was produced as follows: system was used with a C18 reversed-phase column (Phe 150 kg of dried Yerba mate (Ilex paraguayensis) leaf were cut, nomenex, USA, Luna 5um, 250 mmx4.6 mm), for detection crushed, and extracted with approximately 7-fold volume and quantitation of Carnosol, Carnosic acid and Ursolic acid. (1050 L) of 70% ethyl alcohol in water (v/v) and the extrac A binary 0.1% phosphoric acid in purified water (mobile tion solvent held at 100° C. for 4 hrs. The residue was filtered phase A) and acetonitrile (mobile phase B) gradient was used to obtain a Supernatant that was concentrated with an evapo for elution as set forthin Table 9. The column flow rate was set rator at 40°C. The above-described procedure was repeated at 1 ml/min passing through the Luna C18 column with a twice. The extraction solutions were then combined together column temperature of 40°C. The UV detector was set up to and concentrated until the volume become 1/25 of the original read absorbance at 210 nm. volume. The concentrated solution was dried by vacuum US 2014/0004215 A1 Jan. 2, 2014 69 freeze-drying to obtain 26.7 kg of Mate 70% EtOH extract Example 29 powder. The extraction yield is about 17.8% (w/w). HPLC analysis, as described in Example 29, determined that the Preparation of Magnolia Extracts 29 and 29A amount of Caffeine in Yerba mate extract 27 was 2.44% by weight. 0237 Magnolia (Magnolia officinalis) extract 29 was pro duced as follows. 70 kg of dried stem barks of Magnolia Example 28 officinalis was cut, crushed, and extracted with 70% ethyl alcohol and the extraction solvent treated at 80°C. for 4 hrs. Quantification of Caffeine in Yerba mate Leaf The resulting the residue was filtered to obtain a supernatant Extract that was concentrated with an evaporator at 40°C. The above described procedure was repeated two times. The extraction 0235. The following analytical method was used to deter Solutions were then combined together and concentrated until mine the amount of Caffeine in the Yerba mate leaf extracts. the volume become 1/25 of the original volume. Sodium The same Agilent HPLC/PDA system, including the C18 hydroxide (NaOH) was added into the concentrated solution reversed-phase column (Phenomenex, USA) described in to obtain a final concentration of 1% NaOH. After saponifi Example 25 was used for the detection of Caffeine and minor cation at 80° C. for 30 min, the solution was extracted with components. A binary 0.1% phosphoric acid in purified water 2-fold volume of Hexane with agitator for 1 hour. The result (mobile phase A) and methanol (mobile phase B) gradient ing Hexane soluble fraction was then collected and above was used for elution of Mate sample components as described described procedure repeated three times. The n-hexane in Table 11. The flow rate was set to 1 ml/min passing through soluble fractions were combined and evaporated under the Luna C18 column with a column temperature of 35° C. vacuum until the volume become 1/6 of the original volume. The UV detector was set to read absorbance at 275 nm. High purity Magnolia extract was obtained after re-crystalli Zation vacuum drying. 70 kg of dried stem barks of Magnolia TABLE 19 officinalis yielded 652 g of high purity Magnolia extract. The extraction yield is 0.93%. A second batch of Magnolia extract Yerba Mate Gradient Elution Scheme (29A) was also produced according to the same procedure. Time (min) Mobile phase A Mobile phase B Quantification of the active content in both extracts is pro vided in the following example. O 95 5 5 95 5 65 O 100 Example 30 75 O 100 8O 95 5 Analytical Method for Analysis of Magnolia stem 90 95 5 bark Extracts 0236. The quantification standard Caffeine was pur 0238. The following analytical method was used to deter chased from Sigma. Dicaffeoylquinic acid (DCYA) standards mine the amount of Magnolol and Honokiol in Magnolia were purchased from Chengdu Biopurify Phytohemicals stem bark extracts. The same Agilent HPLC/PDA system, Ltd., and dissolved in DMSO. The highest concentration level including the C18 reversed-phase column (Phenomenex, of caffeine and 4.5-DCYA was 0.05 mg/ml and diluted to L5 USA) described in Example 25 was used for the detection of from L1 (0.0031 mg/ml) using methanol. The highest con Magnolol and Honokiol. A binary purified water (mobile centration level of 3,4-DCYA was 0.02 mg/ml and diluted to phase A) and acetonitrile (mobile phase B) gradient was used L5 from L1 (0.00125 mg/ml) using methanol. The highest for detection of Magnolol and Honokiolas described in Table concentration level of 3.5-DCYA was 0.025 mg/ml and 13. The flow rate was set to 1 ml/min passing through the diluted to L5 from L1 (0.001.6 mg/ml) using methanol. Con Luna C18 column with a column temperature of 35° C. and centration of Yerba mate leaf extract samples were adjusted to absorbance was read at 290 nm. about 1 mg/ml in methanolina Volumetric flaskand Sonicated until dissolved (approximately 20 minutes), and then cooled TABLE 21 to room temperature, mixed well and filtered through a 0.45 Magnolia Gradient Elution Scheme um nylon Syringe filter. 10 Jul of sample was examined by HPLC. HPLC actives quantification results for Yerba mate Time (min) Mobile phase A Mobile phase B extract 27 and weight loss Compositions 1 and 3 (exemplified O.O 23 77 in Examples 38 and 39, respectively) are provided in Table 18.0 23 77 2O. 18.1 O 100 2S.O O 100 25.1 23 77 TABLE 20 3O.O 23 77 HPLC Quantification of Yerba Mate Extract and Weight Loss Compositions 0239. Magnolol and Honokiol standards were purchased 3-4 Total from Guangzhou Honsea Sunshine BioScience and Technol Sample Caffeine DCYA 3-5 DCYA 4-S DCYA % ogy co., Ltd. and extracted with methanol. The highest stan 27 2.44 O.77 1.24 2.09 6.55 dard concentration of Honokiol and Magnolol was 0.2 mg/ml Composition 1 18O O.S8 O.94 1.56 4.88 and diluted to L3 from L1 (0.05 mg/ml) using methanol. The Composition 3 1.29 O41 O.69 1.43 3.82 Magnolia stem bark extract sample concentration was adjusted to about 0.2 mg/mland combination sample concen tration was 2 mg/ml in methanol. A Volumetric flask was used US 2014/0004215 A1 Jan. 2, 2014 70 in sample preparation and Sonicated until dissolved (approxi 0243 The two residues from the above-extraction were mately 10 minutes), flask was cooled to room temperature then extracted again with about a 15-fold volume (30 L) of and QS with extraction method, mixed well and filtered 95% ethyl alcohol (v/v) at 85°C. for 4 hrs. The residue was through a 0.45 um nylon Syringe filter and 20 Jul sample was filtered to obtain a Supernatant that was concentrated with an analyzed by HPLC. The HPLC quantification results are pro evaporator at 40° C. The resulting concentrated cake was vided in Table 22. dried by vacuum drying to obtain 290 g of Mutamba EtOH extract powder 33-1 and the extraction yield was about 6.3% TABLE 22 (w/w). 0244. The Mutamba EtOH extract 33-2 was produced as HPLC Results of Magnolol and Honokiol in Magnolia Stem Bark follows: Mutamba EtOH powder extracts 33 and 33-1 were Extracts combined together and pulverized resulting in 660 g of Total% Mutamba EtOH extract 33-2. The final extraction yield was Sample Honokiol% Magnolol % (by weight) about 14.34% (w/w). HPLC analysis showed that this extract 29A 46.23 S1.54 97.77 contained 0.96% Procyanidin B2 and 0.62% Epicatechin. 29 S120 47.20 98.40 Example 34 Example 31 Preparation of Mutamba EtOH Extract Fractions 34, 34-1, and 34-2 Preparation of Areca catechu 70% EtOH Extract31 0245 Mutamba EtOH extract derived fractions 34, 34-1, 0240 Ground Areca catechu seed (7 kg) was divided into and 34-2 were produced as follows: 500 g of Mutamba stem two portions of 3 kg and 4 kg, crushed and place into two bark EtOH extract 33-2 from Example 33, was suspended in extraction units and extracted with about an 8-fold volume 5 L of distilled water and the suspension was mixed with 5 L (about 240 L and 320 L) of 70% ethyl alcohol in water (v/v) of ethylacetate vigorously to divide into an ethylacetate and the extraction solvent held at 90° C. for 4 hrs. After soluble fraction and a water-soluble fraction. The ethylac filtering the extract solution, the filtrate was concentrated etate soluble fraction was collected and the residual solution with an evaporator at 50° C. until only a water solution was subjected to a second ethylacetate extraction. The above remained, which was then collected and frozen at -70°C. The described procedure was repeated three times. The collected remaining crushed seed material was then extracted again as ethylacetate soluble fractions were pooled and then evapo before. The 1 and 2" frozen extract solutions were then rated in vacuo to give 117 g of ethylacetate soluble extract of dried in a freeze dryer device. The dried extracts were com Mutamba stem bark 34. HPLC analysis showed that this bined and ground into a fine powder to obtain 930 g. The final extract contained 2.57% Procyanidin B2 and 2.24% Epicat extraction yield was about 13.2% (w/w). echin. 0246 A water-soluble fraction of Mutamba stem bark Example 32 EtOH extract 33-2 prepared in Example 33 was vigorously mixed with an equivalent volume of butyl alcohol and water, Preparation of Mutamba (Guazuma ulmifoila) stem and allowed to separate into a butyl alcohol soluble fraction bark EtOH Extract 32 and water-soluble fraction. The butyl alcohol soluble fraction 0241 Mutamba EtOH extract32 was produced as follows: was collected and the residual solution was subjected to the 500 g of dried Mutamba (Guazuma ulmifolia) stem bark was butyl alcohol extraction again. This procedure was repeated cut, crushed and extracted with 20-fold volume (10 L) of 95% three times. The respective fractions were pooled and evapo ethyl alcohol at 85°C. for 4 hrs. The residue was filtered to rated in vacuo to obtain 260 g of butyl alcohol soluble extract obtain a Supernatant. The residue was then extracted a second 34-1 and 130 g of water-soluble extract 34-2 of Mutamba time as before and the two EtOH Supernatants combined stem bark. HPLC analysis showed that Mutamba extract frac together and concentrated with evaporator at 40° C. The tion 34-1 contained: 1.31% Procyanidin B2 and 0.86% Epi concentrated cake was dried by vacuum drying to obtain 70 g catechin. HPLC analysis was not performed on Mutamba of Mutamba stem bark EtOH extract powder. The extraction extract fraction 34-2. yield was about 14% (w/w). HPLC analysis showed that this extract contained 1.31% Procyanidin B2 and 0.86% Epicat Example 35 echin. Preparation of Mutamba EtOH Extract 35 Example 33 0247 Mutamba EtOH extract 35 was produced by fol lows: 920g of dried Mutamba stem bark was cut, crushed, and Preparation of Mutamba Extracts 33, 33-1, and 33-2 extracted with 20-fold volume (20 L) of 95% ethyl alcohol at 0242 Mutamba EtOH extract33 was produced as follows: 85°C. for 4 hrs. The residue was filtered to obtain a superna two different batches of 2.3 kg of dried Mutamba stem bark tant and the retained residue was then again extracted with 20 were cut, crushed and then each batch was extracted with L of 95% ethyl alcohol (v/v) and filtered as before. The 15-fold volume (30 L) of 95% ethylalcohol at 85°C. for 4 hrs. resulting Supernatants were combined together and concen The residue was filtered to obtain two batch supernatants. The trated with an evaporator at 40°C. The resulting concentrated two Supernatants were combined and concentrated with an cake was dried under vacuum to obtain 132 g of Mutamba evaporator at 40°C. The resulting concentrated cake was then EtOH extract powder 35. The extraction yield was about dried by vacuum to obtain 370 g of Mutamba EtOH extract 14.34% (w/w). HPLC analysis showed that extract 35 con powder 33. The extraction yield is about 8.04% (w/w). tained 1.03% Procyanidin B2 and 0.60% Epicatechin. US 2014/0004215 A1 Jan. 2, 2014
Example 36 filtered through a 0.45 um nylon syringe filter. HPLC analysis was performed by injecting a 10 ul sample into the HPLC. Preparation of Mutamba EtOH Extract 36 0248 Mutamba EtOH extract36 was produced as follows: 1. Belize 85.7 kg of dried Mutamba stem bark was cut, crushed, and extracted with approximately 8-fold volume (720 L) of 70% 0251 All plant materials were cut, crushed, and extracted ethyl alcohol in water (v/v) after incubation in the extraction with 70% EtOH and the procyanidin B2 and epicatechin solvent at 100 C for 4 hrs. The residue was filtered to obtain a contents analyzed by HPLC as described above. The HPLC retained Supernatant and the residue was then re-extracted results are presented in Table 24. twice more using the same extraction procedure. The retained Supernatants were then were combined together and concen TABLE 24 trated until the volume was 1/25 of the original starting vol ume. This concentrated Solution was then dried by vacuum Procyanidin B2 and Epicatechin Content freeze-drying to obtain 13.9 Kg of Mutamba stem bark 70% in Mutamba Trees from Belize EtOH extract powder 36. The extraction yield was about Extrac 16.2% (w/w). The extraction yield was about 14.34% (w/w). Mutamba Content (%) of Extract tion HPLC analysis showed that extract 36 contained 1.03% Pro cyanidin B2 and 0.60% Epicatechin. Tree Ext Tree Procyanidin Yield Sample ID Part B2 Epicatechin Total (%) Example 37 One year old B1 Stem O.67 O.94 1.61 8.9 female tree B2 Bark 1.18 1.06 2.24 17.1 Mature B3 Stem O.99 1.04 2.03 6.5 HPLC Quantification of Various Mutamba Extracts female tree B4 Bark 1.92 1.54 3.45 12.4 Female tree B5 Bark 4.49 2.21 6.70 17.1 0249. An Agilent HPLC/PDA system was used for the (3 m height) B6 Stem 1.02 1.03 2.05 7.5 detection and quantitation of procyanidin B2 and epicatechin Female tree B7 Bark 2.98 1.55 4.52 20.4 compounds in Mutamba plant extracts obtained from trees (3-4 m B8 Stem 1.01 1.03 2.03 9.3 grown in different locations, using different tree parts, differ height) ent tree sexes and different tree ages. A C18 reversed-phase Mature male B9 Bark 3.97 2.OO 5.97 15.1 column (Agilent, USA) was used (Zorbax eclipse XDB-C18, tree B10 Stem O.31 0.70 1.01 7.2 3.5um, 150 mmx4.6 mm. A binary column gradient was used for elution of material from the column. Mobile Phase A: 0.01% trifluoroacetic acid in purified water, and Mobile Phase B: acetonitrile gradient was used for elution (Table 23). 2. India The flow rate was set to 0.8 ml/min passing through the Luna C18 column with a column temperature of 35° C. The UV (0252 Five different Mutamba samples from different tree detector was set to record absorbance at 275 nm. parts were purchased from a vendor in India. The plant mate rials were cut, crushed, and extracted with MeOH/CH.Cl TABLE 23 (1:1 Volume ratio) and the procyanidin B2 and epicatechin contents analyzed by HPLC as described above. The HPLC Gradient Table of HPLC Analytical Method results are presented in Table 25. Time (min) Mobile Phase A Mobile Phase B TABLE 25 O 92 8 10 83 17 Procyanidin B2 and Epicatechin Content in Mutamba Trees from India 2O 80.6 19.4 25 77.4 22.6 Extract Content (%) of Extract Extraction 28 70.O 29.8 30 35 65 No. PART Procyanidin B2 Epicatechin Total Yield (%) 38 O 1OO 40 O 1OO I1-1 Leaf O O O 13.4 42 92 8 I1-2 Fruit O O O 12.7 45 92 8 I1-3 Fine stem O O O 8.2 I1-4 Stem bark O O O S.6 I1-5 Stem wood O O O 2.2 0250 Pure epicatechin reference sample was purchased from Sigma. Pure procyanidin B2 was purchased from Chengdu Biopurify Phytohemicals, Ltd. Both reference samples were dissolved in DMSO. Highest level concentra 3. Six Different Countries. tion range of epicatechin was 0.05 mg/ml and diluted to L5 from L1 (0.003 mg/ml) using 50% methanol in water. Highest 0253) Mutamba stem bark samples from six different level concentration of procyanidin B2 was 0.05 mg/ml and countries were purchased from a vendor. The plant materials diluted to L5 from L1 (0.003 mg/ml) using 50% methanol in were cut, crushed, and extracted with 100% EtOH (E) or 70% water. Concentration of the Mutamba extract samples were EtOH (70E) or MeOH/CHCl (1:1 volume ratio)(OE) and adjusted to 2 mg/ml in 50% methanol in water in a volumetric the procyanidin B2 and epicatechin contents analyzed by flask and Sonicated until dissolved (approximately 20 min HPLC as described herein. The HPLC results are presented in utes), and then cooled to room temperature, mixed well and Table 26. US 2014/0004215 A1 Jan. 2, 2014 72
TABLE 26 TABLE 28 Procyanidin B2 and Epicatechin Content in Mutamba Stem Bark Summary of Active Content of Composition 3 from Different Countries Rosemary Morus % Content/Extract Extraction % % % % Mate Extract Procyanidin Yield % Carnosic Ursolic Kuwanon Albanin % No. Country B2 Epicatechin Total (%) Solvent Sample Carnosol acid acid G G Caffeine U1 Peru 1.14 O.62 1.76 28 7OE Compo- 1.59 0.44 3.45 O.77 O.S3 1.29 M2 Mexico O.98 O.36 1.33 3 7OE sition 3 B11 Belize 1.31 0.75 2.06 13 7OE P99 Panama 1.31 O.86 2.17 E E10 England O.31 O.OS O.36 8.1 7OE I1-4 India O O O S.6 OE Example 40
Preparation of Morus. Acceleris. Loesyn. Bakutrol Example 38 Composition 6 Preparation of Magnolia. Morus. Yerba mate 0256 Four component (Morus.Acceleris. Loesyn. Composition 1 Bakutrol) composition 6 was produced as follows: 968.6 g of Morus alba root bark extract 20, 484.3 g of Panax ginseng 0254 Three component (Magnolia. Morus. Yerba mate) extract (Acceleris), and 387.4 g of standardized Aloe Composition 1 was produced by from the following three chromones in Aloe Vera inner leaf gel powder (Loesyn) were plant extract components: 1.02 kg of dried Magnolia extract combined in a capped one-gallonjar, then shaken and turned powder 29, 2.01 kg of Morus alba root bark extract powder in the jar to produce a uniform powder (Powder 1). 96.9 g of 18, and 10.08 kg of Yerba mate extract powder 27 were Psoralea seed extract (Bakutrol) was placed in a 2-liter bea blended with V-type blender (Seo-kang Engineering, Korea) ker, and gradually one-half of Powder 1 was added to the at 30 rpm for 1 hour. The final blending weight ratio was Baku.trol liquid until a granule-like mixture was achieved. Magnolia. Morus. Yerba mate of 1:2:10 and resulted in pro This mixture was then added to the remaining half of Powder duction of 12.65 kg of weight loss combination Composition 1 and mixed thoroughly. Finally, the complete mixture was 1. The major active compound profile in Composition 1 was put into a blender for pulverizing. The final combination determined by HPLC analysis as described in Examples 11, composition 6 had a blending weight ratio of Morus. Acce 20, 25, 28 and 30. The quantification results are shown in the leris. Loesyn. Bakutrol at 10:5:4:1. The active contents of Table 27. Composition 6 are shown in Table 29.
TABLE 27 TABLE 29 Summary of Active Components in Composition 1 Summary of Active Contents in Composition 6 Yerba Acceleris Magnolia Morus Mate Total Morus aiba root Ginsenoside Loesyn bark extract Bakutrol Honokiol Magnolol Kuwanon Albanin Caffeine Sample % % G% G% % (Rd, Rg3, % % % % Sample Rk1, RgS) Aloesin Kuwanon G Albanin G Bakuchiol Composition 1 3.88 3.91 1.09 O.77 18O Com- 10.02 O.80 1.57 O.88 3.34 position 6 Example 39 Example 41 Preparation of Morus. Rosemary. Yerba mate Composition 3 Preparation of Morus. Rosemary:Areca Composition 0255 Three component (Morus. Rosemary: Yerba mate) 9 Composition 3 was produced as follows: 2.4 kg of dried Morus root bark extract powder 18, 6.0 kg of dried Rosemary 0257 Three component (Morus. Rosemary:Areca) com extract powder 23, and 12.04 kg of dried Yerba mate extract position 9 was produced as follows: 153.2 g of dry Morus powder 27 were blended with v-type blender (Seo-kang Engi alba root bark extract 18,382.2 g of Rosemary extract powder neering, Korea) at 30 rpm for 1 hour. The final blending 22-1, and 765.8g of Areca catechu fruit extract powder 31, weight ratio was Morus. Rosemary: Mate at 2:5:10 and were blended together with a ribbon blender (Hankook P. M. resulted in 20.4 kg of Composition 3. The active contents in EMG, Korea) at 30 rpm for 1 hour to obtain 1.301 kg of the composition were quantified with the HPLC illustrated in UP609. The blending ratio of Morus. Rosemary:Areca was Examples 11, 20, 25, 28 and 30. The quantification results are 2:5:10 (weight ratio). The active contents of Composition 9 shown in the Table 28. are shown in Table 30. US 2014/0004215 A1 Jan. 2, 2014
TABLE 30 Example 44 Summary of Active Content in Composition 9 Preparation of Mutamba. Rosemary: Mate Composition 8 Rosemary Morus 0260 Three component (Mutamba. Rosemary: Mate) Carnosol Carnosic Ursolic Kuwanon Albanin Areca Composition 8 was produced as follows: 640.2 g of Mutamba Sample % acid 96 acid% G% G% stem bark extract powder 36, 320.1 g of Rosemary extract Compo- 2.14 2.01 S.12 O.63 O45 powder 22, and 640.2 g of Mate extract powder 27, were sition 9 blended with a ribbon-style blender (Seo-kang Engineering, Korea) at 30 rpm for 1 hour to obtain 1.52 Kg of combination Composition 8. The blending ratio of Mutamba. Rosemary: Example 42 Mate was 2:1:2 (weight ratio). Preparation of Magnolia. Morus. Mutamba TABLE 33 Composition 2 Summary of Active Ingredients for Composition 8 0258. Three component (Magnolia. Morus. Mutamba) Composition 2 was produced as follows: 1.02 Kg of dry Rosemary Mutamba Mate Magnolia extract powder 29, 2.04 Kg of dry Morus alba root Carnosol Carnosic Ursolic Procyan- Epicate- Caffeine bark extract powder 18, and 10.05 Kg of dry Mutamba stem Sample % acid 96 acid% idlin B2 chin % bark extract powder 36, were blended together with a V-type Compo- 1.64 O.94 3.56 O.S3 O45 2.44 blender (Seo-kang Engineering, Korea) at 30 rpm for 1 hour sition 8 to obtain 13.05 Kg of Composition 2. The blending ratio of Magnolia. Morus. Mutamba was 1:2:10 (weight ratio). TABLE 31 Summary of Active Ingredients for Composition 2 Magnolia Morus Mutamba Sample Honokiol% Magnolol % Kuwanon G% Albanin G% Procyanidin B2 Epicatechin Composition 4.06 4.19 1.01 O.71 1.10 O.81 2
Example 43 Example 45
Preparation of Mutamba. Rosemary. Mate Acute Food Intake in Sprague-Dawley Rats as a Composition 4 Measure of Appetite Suppression 0259 Three component (Mutamba. Rosemary. Mate) 0261 This example describes the acute food intake rat Composition 4 was produced as follows: 352.3 g of Mutamba animal model for evaluation of the effects of diet and test stem bark extract powder 36, 352.1 g of Rosemary extract compounds on amount and rate of food intake of rats after a powder 22, and 352.1 g of Mate extract powder 27, were fasting period. blended with a ribbon-style blender (Seo-kang Engineering, Korea) at 30 rpm for 1 hour to obtain 1.04 Kg of combination 0262 Method: Composition 4. The blending ratio of Mutamba. Rosemary: 0263 Male Sprague-Dawley (SD) rats (Koatech, Korea), Mate was 1:1:1 (weight ratio). eight weeks of age at the beginning of the experiment, were used in this study. During acclimation periods the animals TABLE 32 were maintained on a regular rat chow diet (2018S, Harlan, USA). The rats were housed in a climate-controlled room maintained on a 12 hr/12 hr reverse light/dark cycle. Rats Summary of Active ingredients for Composition 4 were administered 0.5% CMC (carboxymethyl cellulose) aqueous solution as vehicle or in combination with a test Rosemary Mutamba Mate composition 30 minutes prior to the start of the dark-phase feeding cycle. Experimental testing commenced at the onset Carnosol Carnosic Ursolic Procyan- Epicate- Caffeine of the 12hr dark cycle. Prior to initiation of a test cycle the rats Sample % acid 90 acid 90 idin B2 chin % were fasted overnight (less than about 16 hours) to enhance their hunger. Otherwise the animals had unlimited access to 45% high fat diet (Harlan, USA) and tap water. Food intake Compo- 2.11 1.79 2.30 O.47 O.32 O.71 was measured at 0, 1, 2, 4, 6, 8, 10 and 24 hours from the start sition 4 of the experiment to determine the acute food intake of each animal in a study, and total body weight was also measured at 2, 8, and 24 hours. US 2014/0004215 A1 Jan. 2, 2014 74
Example 46 study group between each Successive sample time point of the study. Milicia excelsa extract treatment groups also exhibited Acute Food Intake Study of Morus Alba Extract in reduced food intake at the 1 hr, 4 hr, 6 hr and 8 hr time points Sprague-Dawley (SD) Rats (Table 37). Treatment SD rats fed the Milicia excelsa extracts 0264. This Example presents a 24-hour food intake test were also reduced significantly in cumulative food intake for carried according to the Example 45 to determine the effect of 24 hour (Table 38). administration of Morus alba plant extract 15 on rat food intake. SD rats were administered Morus alba extract 15 TABLE 35 produced according to Example 15, in a solution of 0.5% CMC (carboxymethyl cellulose) 30 minutes prior to the start Body Weight in Non-Obese Fasting Rats Fed a High Fat Diet of dark-phase feeding cycle. The Morus alba extract was Dose Body weight (g administered at a dose of 250, 500 and 1000 mg/kg of animal weight, 7 animals per group. Group (mg/kg) Oh 2h 8h 24h 0265 Table 34 shows the food intake test results for rats Control Mean 2O3.34 209.74 219.06 217.22 treated with a single dose of Morus alba extract 15 at three SD 5.27 4.26 6.79 S.26 different amounts compared to control animals. TABLE 34 Cumulative Food Intake in Non-Obese Fasting Rats Fed a High Fat Diet Dose Cumulative Food Intake (hour
Group (mg/kg) 1 2 4 6 8 10 24
Control Mean 3.45 5.33 8.73 15.35 20.57 22.99 26.85 SD 1.20 1.OO 1.12 1.92 1.47 1.49 2.2O 250 Mean 1.43 2.94 5.86 9.77 13.56 16.68 24.16 SD O.98 1...SO 3.13 5.47 6.15 6.55 4.63 p value O.OO67 O.OO98 O.0763 O.O544 O.O376 O.0648 0.2338 Morus 500 Mean 1.72 2.97 5.23 8.37 12.28 14.O1 1993 aiba SD O.96 O.80 1.21 2.71 2.98 3.35 2.72 p value O.O315 O.OO29 O.OO33 O.OO70 O.OO71 O.OO87 O.OO6S 1,000 Mean O.74 1.26 3.12 6.06 9.95 15.11 1948 SD O.31 1.OO 2.12 2.97 1.57 2.41 3.06 p value O.OOO7 O.OOOS O.OO8O O.OO3S OOOOO O.OO31 O.OO88
0266 The data presented in Table 34 shows that all of the TABLE 35-continued Morus alba treatment groups exhibited a statistically signifi cant reduction in cumulative food intake. Further, a dose dependent reduction in food intake was observed in the first Body Weight in Non-Obese Fasting Rats Fed a High Fat Diet hour of food intake measurement through to completion of the study. These results demonstrate that Morus alba extract Dose Body weight (g) has a statistically significant effect on food intake in rats, which indicates that Morus alba extract can be used as a body weight control composition via inhibition of food intake. Group (mg/kg) Oh 2h 8h 24h Also, the reduced food intake from a single oral dose of Morus alba extract lasted more than 10 hours. Thus, it is Milicia 1,000 Mean 202.69 2O7.01 2OS.04 205.96 feasible to achieve a reduced appetite, enhanced Satiety, or excelsa SD 8.15 7.34 7.03 1249 reduced food or caloric intake by once or twice per day oral p value O868O O4610 O.OOSO O.O.309 administration of Morus alba extract. Example 47 TABLE 36 Acute Food Intake Study of Milicia excelsa Extract 8 Change in Body Weight Gain in Non-Obese Fasting Rats Fed High in SD Rats Fat Diet
0267. This 24-hour food intake study was conducted Dose Bodw weight gain according to the Example 45. SD rats were administered Milicia excelsa extract 8 produced according to Example 8, at Group (mg/kg) 2h 8h 24h a dosage of 1000 mg/kg of animal weight, in 0.5% CMC Control Mean 6.41 15.73 13.88 (carboxymethyl cellulose solution) solution 30 min prior to SD 1.69 4.36 1.92 the start of the dark-phase feeding cycle. Milicia 1,000 Mean 4.32 2.35 3.27 excelsa SD 2.46 6.44 11.00 0268. As shown the Table 35 and Table 36, body weight p O. 1028 O.OOO1 O.O144 and body weight gain were reduced significantly at the 8 hr value and 24 hour study time points. Body weight gain was deter mined by measurement of the weight difference for each US 2014/0004215 A1 Jan. 2, 2014 75
TABLE 37 Food Intake per Time Point in Non-Obese Fasting Rats Fed a High Fat Diet Dose Food Intake (g) per Time Point Group (mg/kg) 1 h 2h 4h 6h 8 10 h. 24h
Control Mean 3.09 2.07 2.76 4.19 3.51 1.33 6.34 SD 1.24 1.54 1.64 1.02 2.64 1.60 2.83 Miicia 1000 Mean O.80 O.94 O.35 O.70 O.98 1.OS 6.44 excelsa SD O41 0.75 OSO 1.07 1.81 1.78 S.09 p value 0.0002 0.0570 O.OO11 O.OOOO O.O236 O.7210 O.9562
TABLE 38 Cumulative Food Intake in Non-Obese Fasting Rats Fed High Fat Diet Dose Cumulative food intake (g Group (mg/kg) 1 h 2h 4h 6h 8 10 h. 24h
Control Mean 3.09 5.15 7.91 12.10 15.61 16.93 23.27 SD 1.24 1.96 1.57 2.01 3.75 3.23 1.57 Miicia 1OOO Mean O.80 1.74 2.09 2.79 3.77 4.82 11.26 excelsa SD O41 O.80 1.OS 2.04 3.SO 4.46 8.06 p value O.OOO2 O.OOO3 OOOOO O.OOOO OOOOO O.OOOO O.OO10
0269. These data show that Milicia excelsa extracts were Example 49 effective in significantly reducing body weight and body weight gain. In addition, SD rats fed a normal chow diet after High Fat Diet Induced Obesity (DIO) Rat Model fasting had reduced intake of food. Therefore, the present 0272 Male Spraue-Dawley rats, age 4-6 weeks (Orient result suggests that Milicia excelsa extract can be used as a Bio, Inc.; Seongnam, Korea) were housed individually in body weight controller via inhibition of food intake. Polycarbonate cages in a room with a 12 hr. 12 hr light-dark cycle and an ambient temperature of 24°C. All the rats were Example 48 fed a commercial chow diet for 1 week after arrival in the animal facility. Rats were then divided into normal and obe High Fat Diet Induced Obesity (DIO) Mouse Model sity groups and fed with normal diet (ND) and high fat diets (HFD) respectively. The HFD group was divided into mul (0270 C57CL/6J mice aged 4-6 weeks (Korea Research tiple treatment groups: a high fat diet vehicle group (HFD), an Institute of Bioscience & Biotechnology, Ohchang, Korea) orlistat (purchased as OTC drug Alli(R) positive control treat were housed in Polycarbonate cages (five mice per cage) in a ment group (ORI, 80 mg/kg of animal weight, 2 times/day). room with a 12 hr. 12 hr light-dark cycle and an ambient In some examples, sibutramine was used as a positive control temperature of 24°C. All the mice were fed a commercial (SIB, 3 mg/kg for rat studies and 10 mg/ml for mice studies). chow diet for 1 week after arrival in the animal facility. Mice 0273 Body weight was measured once or twice per week were then divided into normal and obesity groups and fed and feed intake was measured twice per week. Body weight with normal diet (ND) and high fat diets (HFD) respectively. gain was determined for each study group by measurement of The HFD group was divided into multiple treatment groups: the body weight difference for each study group between each a high fat diet vehicle group (HFD), an orlistat (purchased as successive week of the study. At the end of the experimental OTC drug AlliR) positive control treatment group (ORI, 40 period, following a 12-h fasting period, the animals were mg/kg of animal weight, 2 times/day) and optionally a anesthetized with ether, and blood was drawn from the Sibutramine positive control treatment group (10 mg/kg, 1 abdominal vein. Liver and kidney and adipose tissues (epid time/day). The HFD contained 340 g offat/kg of HFD (310 g idymal, retroperitoneal, perirenal adipose tissues) were lard plus 30 g soybean oil; Harlan Laboratories, USA). The HFD was formulated to provide 60% of the total energy removed from each animal, rinsed with physiological Saline, generated by the diet from fat by replacing carbohydrate and weighted. Serum concentrations of glucose, total choles energy with lard and Soybean oil, whereas the normal diet terol and LDL-cholesterol were determined using automatic (ND) group was fed a diet providing only 18% of the total diet analyzer (INTEGRA 400, Roche, Germany). Statistical sig energy from fat(Harlan Laboratories, USA). nificance of test results was performed by Student's t-test. 0271 Body weight was measured once each week and Example 50 feed intake was measured twice per week. At the end of the experimental period, following a 12 hr. fasting period, the Effect of Morus alba Ethanol Extract 19-2 on DIO animals were anesthetized with ether, and blood was drawn Mice from the abdominal vein. Liver and kidney and adipose tis (0274) Morus alba 70% ethanol extract 19-2 produced Sues (epididymal, retroperitoneal, perirenal adipose tissues) according to the Example 19 was orally administrated to DIO were removed from each animal, rinsed with physiological mice as described in the Example 48. The Morus alba extract saline, and weighed. Serum concentrations of glucose, total was administered at two dose levels: the G1 group at 500 cholesterol, triglycerides, and LDL-cholesterol were deter mg/kg of animal weight and the G2 group at 1000 mg/kg of mined using automatic analyzer (INTEGRA 400, Roche, animal weight. Animals were given an oral dose by gavage Germany). Statistical significance of test results was mea two times per day. Study results for measurement of animal sured using the Student's t-test. body weight are shown in Table 39. US 2014/0004215 A1 Jan. 2, 2014 76
TABLE 39
Effect of Morus alba extract 19-2 on Total Body Weight in DIO mice
Weeks
Group O 1 2 3 5 6 7
ND Mean 28.21 28.34 28.45 28.50 28.29 28.31 28.43 28.66 (Normal SD 2.032 2.OO6 2.277 2.417 2.414 2.449 2.425 2.413 Diet) p value OOOOO O.OOOO OOOOO O.OOOO OOOOO O.OOOO OOOOO O.OOOO HFD (High Mean 43.36 41.24 40.29 41.26 42.64 43.32 4S.O.S 46.23 Fat Diet) SD 2.414 2.118 2.712 3496 3.796 4.081 4.515 4.SSO ORI Mean 42.76 38.73 35.09 35.66 37.28 38.57 40.03 40.91 (40 mg/kg) SD 3.226 3.286 3.330 3.542 3.896 4.508 4.617 4.127 p value O.6889 O.O987 O.OOS4 O.OO88 O.0185 O.OS 16 O.OS30 O.O346 G1 Mean 42.86 40.58 38.76 39.55 40.82 42.44 44.10 45.22 (500 mg/kg) SD 2.921 2.492 3.219 3.662 4.658 5.306 S.949 6.166 p value O.72OO 0.5917 O3S48 O.3909 O4375 O.7321 0.7385 O.7281 G2 Mean 43.07 40.OS 38.02 36.98 37.56 38.29 39.18 39.66 (1 g/kg) SD O.835 O.287 O.O94 O.O14 O.O12 O.O24 O.O21 O.O17 p value O.8353 O.2868 O.0942 O.O136 O.O124 O.O242 O.O214 O.O171 p value: compare to HFD by t-test
0275. The data in Table 39 shows that the G1 group (low group. The positive control group (ORI, treated with orlistat dosage of Morus alba extract 19-2) did not exhibit a signifi at 40 mg/kg of body weight) showed Statistically significant cant difference body weight compared to the HFD group. In reductions in body weight at weeks 2, 3, 4, and 7 when contrast, the G2 group (high dosage of Morus alba extract compared with the HFD group. 19-2) exhibited statistically significant reductions in body 0276 Table 40 shows the effects on body weight gain of weight at weeks 3, 4, 5, 6 and 7 when compared with the HFD each group. TABLE 40
Effect of Morus alba on Body Weight Gain in Mice Fed High Fat Diet
Weeks
Group 1 2 3 4 5 6 7
ND Mean O.13 O.24 O.29 O.08 O.10 O.22 O.45 (Normal SD O.387 0.567 O.736 O.810 O.959 O.839 O.929 Diet) p value O.OOOO O.OOOO O.OOO2 O.1790 O.8322 O.1256 O.O3O2 HFD (High Mean -2.05 -3.37 -3.41 -2.14 -1.82 -0.22 1.24 Fat Diet) SD O.990 O.266 O.S48 1.214 1145 2.417 3.030 ORI Mean -4.00 -7.38 -6.70 -5O1 -3.81 -2.36 -1.42 40 mg/kg SD 0.571 O.839 1.227 1.409 1.609 1.175 1.259 p value O.OOO4 O.OOOO O.OOO1 O.OOOS O.OO32 O.OO59 O.OO41 G1 Mean -2.27 -4.28 -3.49 -2.22 -0.60 1.06 2.18 500 mg/kg SD O.921 1316 1.429 2.075 2.660 3.240 3.383 p value O.7247 O.O621 O.O887 O.1522 O.6745 0.6957 O.6660 G2 Mean -3.02 -S.O.S -6.09 -5.51 -4.78 -3.89 -3.41 1000 mg/kg SD O.936 1.076 1.244 O.728 1.084 1.511 2.136 p value O.OS21 O.OO18 O.OOO1 O.OOOO O.OOO1 O.OOO1 O.OOO2
p value: compare to HFD by t-test US 2014/0004215 A1 Jan. 2, 2014 77
0277. The data in Table 40 show that the change in weight trol treatment group (ORI) both showed a statistically signifi gain for the high dosage group (G2) and the positive control cant decrease in body weight gain per day of the study com group (ORI) both showed the greatest change in weight gain pared to the HFD group. In addition, both the G2 and ORI between week 2 and week 3 with the rate of weight loss groups also showed a statistically significant decrease in FER relative to the HFD group dropping from week 4 to week 7. as compared to the HFD group. Interestingly, the ORI and G2 0278 Table 41 shows the effects of the Morus alba extract groups also showed statistically significant changes in food on body weight gain, food intake, and the food efficiency ratio intake per day of the study, with the ORI showing an increase (FER), which is the average body weight gain per day over the in food intake compared to the HFD group, while the G2 study period, divided by the average food intake per day over group showed a decrease in average food intake per day of the the study period. study compared to the HFD group, Suggesting that the mecha TABLE 41 nism of action is different between the two compositions. 0280 Taken together, the data presented in this example Effect of Morus alba on Mice Fed a High Fat Diet indicate that the Morus alba 70% ethanol extract 19-2 when Average Body Average Food Food taken at a dose of 1000 mg/kg of subject body weight, twice Weight Gain Intake Efficiency per day, is effective in helping to control food intake and body Group (g day) (g/day) Ratio (FER) weight for Subjects on a high fat diet. ND Mean O.O10 2.973 O.OO3 (Normal Diet) SD O.O2O O.334 O.007 p value O.O3O2 O.OOOO O.O255 Example 51 HFD Mean O.O27 2.390 O.O11 (High Fat Diet) SD O.066 O.S8O O.O28 ORI Mean -0.031 2.711 -0.011 Effect of Morus alba Extract Precipitate 19-1 on (40 mg/kg) SD O.040 O.721 O.O15 DIO Mice p value O.OO41 O.O293 O.0043 G1 Mean O.047 2.332 O.O17 (500 mg/kg) SD O.074 O.611 O.O3O 0281 Morus alba precipitate 19-1, produced by precipi p value O666O O.6909 O.S321 tation from a concentrated 70% ethanol extract of Example G2 Mean -0.074 1934 -0.038 189, was orally administrated to DIO mice using the methods (1000 mg/kg) SD O.046 O.S.08 O.O24 p value O.OOO2 O.OO13 O.OOO1 described in Example 48. The Morus alba extract precipitate was administered at two dose levels: the G1 group at 250 FER (Feed efficacy ratio) = Body weight gain(g day). Food intake(g day) mg/kg of animal weight and the G2 group at 500 mg/kg of p value: compare to HFD by t-test animal weight. Animals were given an oral dose by gavage 0279. The data presented in Table 41 show that the high two times per day. Study results for measurement of animal dose Morus alba treatment group (G2) and the positive con body weight are shown in Table 42. TABLE 42
Effect of Morus alba extract precipitate 19-1 on Total Body Weight
Weeks
Group O 2 3 4 5 6 7
ND Mean 28.21 28.34 28.45 28SO 28.29 28.31 28.43 28.66 SD 2.032 2006 2.277 2.417 2.414 2.449 2.425 2.413 p value O.OOOO OOOOO O.OOOO OOOOO O.OOOO OOOOO OOOOO OOOOO HFD Mean 43.36 41.24 40.29 41.26 42.64 43.32 45.OS 46.23 SD 2.414 2.118 2.712 3496 3.796 4.081 4.515 4.SSO ORI Mean 42.76 38.73 35.09 35.66 37.28 38.57 40.03 40.91 40 mg/kg SD 3.226 3.286 3.330 3.542 3.896 4.508 4.617 4.127 p value O.6889 O.O987 O.OOS4 O.OO88 O.0185 O.OS16 O.OS30 O.O346 G1 Mean 43.10 39.97 37.09 36.78 39.00 40.83 41.77 42.82 250 mg/kg SD 2.990 2.678 2.290 2.576 2.788 3.162 3.383 3.930 p value O.8566 O.32SO O.O294 O.O153 0.0572 0.2155 O.1394 O.1473 G2 Mean 42.92 38.48 34.67 34.35 35.21 35.65 35.22 35.85 500 mg/kg SD 3.028 2.365 1806 1523 1688 2011 2.1.89 2.101 p value 0.7591 O.O330 O.OOOS O.OOO3 O.OOO4 O.OOO6 O.OOO2 O.OOO1
p value: compared to HFD by t-test US 2014/0004215 A1 Jan. 2, 2014 78
0282. As shown the Table 42, body weight was signifi TABLE 44-continued cantly decreased in a dose dependent manner in the Morus alba treatment groups. In particular, the G1 group (low dos Effect of Morus alba extract precipitate 19-1 on Mice Fed a High age of Morus alba extract precipitate 19-1) exhibited a sig Fat Diet nificant difference in body weight compared to the HFD group for week 2 and week 3. Further the weight gain trend, Average Body Average Food Efficiency as compared to the HFD group suggests a lowered rate of Weight Gain Food Intake Ratio weight gain compared to the HFD group even if the numbers Group (g/day) (g day) (FER) for weeks 4, 5, 6 and 7 failed to achieve statistical signifi HFD Mean O.062 2.390 O.O26 cance. Whereas the G2 group (high dosage of Morus alba (High Fat SD O.OS4 O.S8O O.O23 extract precipitate 19-1) exhibited statistically significant Diet) reductions in body weight at weeks 2 through week 7, when ORI Mean -0.031 2.711 -0.011 compared with the HFD group. The positive control group, (40 mg/kg) SD O.040 O.721 O.O15 ORI, (treated with orlistat) showed statistically significant p value O.OO41 O.O293 O.0043 reductions in body weight at weeks 2, 3, 4, and 7 when G1 Mean -OOO6 2.468 -OOO2 compared with the HFD group. (250 mg/kg) SD O.O60 O.682 O.O24 0283 Table 43 shows the effects on body weight gain in p value O.O372 O.S874 O.O3S1 each study group. TABLE 43 Effect of Morus alba Extract Precipitate 19-1 on Mice Fed a High Fat Diet
Weeks
Group 1 2 3 4 5 6 7
ND Mean O.13 O.24 O.29 O.08 O.10 O.22 O.45 SD O.387 0.567 O.736 O810 O.959 O.839 O.929 p value O.OOOO OOOOO O.OOO2 O.1790 O.8322 O.1256 O.O3O2 HFD Mean -2.12 -3.08 -2.10 -O.73 -0.04 1.69 2.87 SD O.689 O.88O 1.365 1.583 1897 2.339 2.504 ORI Mean -4.00 -7.38 -6.7O -501 -3.81 -2.36 -1.42 40 mg/kg SD 0.571 O.839 1.227 1409 1.609 1.175 1.259 p value O.OOO4 OOOOO O.OOO1 O.OOOS O.OO32 O.OOS9 O.OO41 G1 Mean -3.13 -6.02 -6.33 -4.11 -2.27 -1.34 -0.28 250 mg/kg SD 1.272 1916 2.678 2.375 2.316 2.654 2.757 p value O.O732 O.OO18 O.OO17 O.OOS9 O.O614 O.O352 O.O372 G2 Mean -4.44 -8.25 -8.57 -7.71 -7.27 -7.70 -7.07 500 mg/kg SD 1.399 1600 1.66S 1.594 1.315 1.414 1.582 p value O.OO11 OOOOO O.OOOO OOOOO O.OOOO OOOOO O.OOOO p value: compare to HFD by t-test
0284. The data in Table 43 show that both of the Morus TABLE 44-continued alba treatment groups and the positive control ORI group exhibited Statistically significant decreases in body weight Effect of Morus alba extract precipitate 19-1 on Mice Fed a High gains each week after the first week of treatment of as com Fat Diet pared to the HFD group. In addition, the data also show the weight loss effect of the Morus alba treatment groups is dose Average Body Average Food Efficiency dependent. Weight Gain Food Intake Ratio 0285 Table 44 shows the effects of the Morus alba extract Group (g/day) (g day) (FER) precipitate 19-1 on body weight gain, food intake, and the G2 Mean -0.154 1937 -O.O79 food efficiency ratio (FER), which is the average body weight (500 mg/kg) SD O.O34 O.S42 O.018 gain per day over the study period, divided by the average p value O.OOOO O.OO16 O.OOOO food intake per day over the study period. Feed efficacy ratio (FER) = Body weight gain (g day), Food intake(g day) p value: compare to HFD by t-test TABLE 44 Effect of Morus alba extract precipitate 19-1 on Mice Fed a High 0286 The data presented in Table 44 show that both of the Fat Diet Morus alba treatment groups (G1 and G2) and the positive Average Body Average Food Efficiency control treatment group (ORI) all showed statistically signifi Weight Gain Food Intake Ratio cant decreases in body weight gain per day of the study. The Group (g day) (g day) (FER) ORI and G2 treatment groups also showed a statistically significant decrease in the average amount of food intake per ND Mean O.O10 2.973 O.OO3 day as compared to the HFD group. In addition, the G1, G2. (Normal SD O.O2O O.334 O.007 and ORI treatment groups also showed a statistically signifi Diet) p value O.O3O2 OOOOO O.O255 cant decrease in FER as compared to the HFD group. US 2014/0004215 A1 Jan. 2, 2014 79
0287 Table 45 shows the effects of the Morus alba extract (G2) had statistically significant decreases in total glucose, precipitate 19-1 on several specific tissues that are known to cholesterol, and LDL-cholesterol as compared to the HFD have increased fat content in Subjects on a high fat diet. group. TABLE 45 Effects of Morus alba extract precipitate 19-1 on Organ Weight Epididymal Retroperi- Perirenal Total Group Liver Fat toneal Fat Fat Fat) ND Mean O.87 O.S8 O.14 O.09 O.81 (Normal Diet) SD O.102 O.189 O.O70 O.O32 O.28O p value O.OOS1 OOOOO OOOOO O.OOO1 OOOOO HFD Mean 1.44 1.98 O.S4 O.43 2.95 (High Fat Diet) SD O4O6 O464 O.O85 O.134 O.478 ORI Mean 1.12 1.92 O.47 O.33 2.73 (40 mg/kg) SD O.204 O.359 0.055 O.O94 O433 p value O.O842 O.8036 O.O791 0.1376 O.3649 G1 Mean 1.26 2.24 0.57 O.36 3.17 (250 mg/kg) SD O.247 0.237 0.075 O.121 O.252 p value O.3216 O.2006 O.S2O6 O.3072 O.2958 G2 Mean 1.06 1.42 O.35 O.19 1.97 (500 mg/kg) SD O.115 O.062 O.043 O.O36 O.117 p value O.O347 O.0118 O.OOO1 O.OO14 O.OOOS p value: compare to HFD by t-test
0288 The data in Table 45 show that there is a statistically 0291 Taken together, the data presented in this example significant difference between liver weight, epididymal fat, indicate that the Morus alba precipitate extract 19-1 when retroperitoneal fat, perirenal fat and total fat between the ND taken at a dose of 500 mg/kg of subject body weight, twice per control group (fed a normal nutritional diet having a moderate day, is effective in helping to control food intake and body caloric intact of fat) and the HFD group. Interestingly, the weight for Subjects eating a high fat diet. In addition, Subjects high dosage Morus alba extract precipitate group (G2) taking the 500 mg/kg dose of Morus alba precipitate extract showed statistically significant decreases in all categories of 19-1 also exhibited statistically significant improvements in fat measurement as compared with the HFD group, whereas blood chemistry end points and tissue fat levels as compared to control subjects in the HFD group. These results demon the ORI treatment group showed no statistically significant strate that Morus alba extracts enriched in the Diels-Alder changes in any of the measured values. adducts of a chalcone and prenylphenyl moiety Kuwanon G, 0289 Table 46 shows the effects of the Morus alba extract and Albanin G can be used to control body weight, lower food precipitate 19-1 on fasting glucose (F-Glu), total cholesterol intake, lower tissue fat content, lower blood glucose levels, (T-chol) and LDL-cholesterol (LDL-C) as measure in blood decrease total cholesterol and decrease LDL-cholesterol. samples obtained at the end of the study. Example 52 TABLE 46 Effect of Morus alba extract precipitate 19-1 on Biochemistry Effect of Morus Alba Ethyl Acetate Extract 16 on Parameters DIO Rats F-Glu T-Chol LDL-C 0292 Morus alba ethyl acetate extract 16 produced as Group (mg/dL) (mg/dL) (mg/dL) described in Example 16 was orally administrated to DIO rats ND Mean 125.40 107.00 S.OO using the methods described in Example 40. Study time SD 29.628 20.347 1793 period was 42 days. The Morus alba extract was administered p value O.OO16 O.OO85 O.OO60 HFD Mean 2S3.20 19360 9.08 to group G1 using a dose of 500 mg/kg of animal weight. SD 53.867 S2003 1689 Study animals were given an oral dose by gavage two times ORI Mean 222.43 134.57 4.27 per day. Study results for measurement of total animal body SD S1.OO6 31627 O.896 weight for all study groups are shown in Table 47. Table 48 p value 0.3375 O.O.338 O.OOO1 G1 Mean 28O14 173.57 5.59 shows the effects on body weight gain when calculated as the SD 39.01S 29.58O 1608 difference between a subject animal weighed on day 0 of the p value O.3361 O4134 O.OO46 study compared to the body weight measured at the end of G2 Mean 188.29 136.14 4.24 each time point day of the study. SD 36.523 37.936 1664 p value O.O313 O.OSO3 O.OOO6 0293. The data presented in Table 47 show that Morus alba ethyl acetate extract 16 when dosed at 500 mg/kg of animal p value: compare to HFD by t-test body weight, produced a statistically significant decrease in body weight from day 2 to day 14, day 21, and day 28 as 0290 The data in Table 46 show that low dosage Morus compared to the HFD group. The positive control SIB alba extract precipitate group (G1) had statistically signifi (Sibutramine dosed at 3 mg/kg) group, produced a statisti cant decrease in LDL-cholesterol compared to the HFD cally significant reduction in body weight at day 2 through the group. The high dosage Morus alba extract precipitate group remainder of the study (day 42). US 2014/0004215 A1 Jan. 2, 2014 80
TABLE 47 Effect of Morus alba Extract 16 (500 mg/kg of total subject weight) on Total Body Weight of DIO Rats Days
Group 3 4 7 10 14
ND Mean 325.08 332.10 336.67 339.58 340.88 353.66 360.07 371.43 (Normal SD 12.82 1298 14.15 14.35 14.96 18.07 18.48 1963 Diet) O.OOO O.OOO O.OOO O.OOO O.OOO O.OOO O.OOO O.OOO value HFD Mean 364.82 368.90 374SO 378.61 381.19 391.61 400.99 411.04 (High Fat SD 7.46 8.74 7.87 8.52 9.22 9.25 10.64 11.51 Diet) SIB Mean 364.15 368.06 356.78 357.21 358.78 368.11 377.69 388.70 (Sibtramine SD 10.40 10.28 11.60 13.06 13.82 15.94 17.24 1919 3 mg/kg) p O.870 O848 O.OO1 O.OO1 O.OO1 O.OO1 O.OO2 O.OO7 value GI Mean 364.35 366.90 366.10 364.73 362.01 373.26 385.46 393.89 (500 g/Kg) SD 9.34 8.58 8.71 10.97 1812 1856 14.42 15.84 O.901 O612 O.O36 O.OO6 O.O10 O.O15 O.O14 O.O13 value Days
Group 18 21 24 28 31 35 38 42
ND Mean 379.57 388.12 396.23 405.93 412.33 424.10 431.21 439.27 (Normal SD 20.17 20.99 22.32 22.64 22:42 21.69 22.59 21.27 Diet) p O.OOO O.OOO O.OOO O.OO OOOOO O.OOO O.OOO O.OOO value HFD Mean 42O.30 428.30 436.77 448.90 455.75 465.73 471.56 483.09 (High Fat SD 12.00 14.51 15.62 16.93 1748 16.38 16.59 16.34 Diet) SIB Mean 395.38 4O7.06 414.91 425.99 432.32 442.55 447.66 454.96 (Sibtramine SD 19. OS 21.34 20.48 20.94 22.49 23.56 25.53 25.35 3 mg/kg) p O.OO3 O.019 O.O16 O.O15 O.019 O.O21 O.O2S O.O10 value GI Mean 4O7.12 413.32 423.76 430.11 442.06 450.60 460.19 466.34 (500 g/Kg) SD 16.21 16.56 22.10 21.32 21.65 22.69 25.42 23.37 p 0.055 O.045 O.148 O.043 O.138 O. 106 O.254 O.O82 value p value: Compared to HFD group
TABLE 48 Effect of Morus alba Extract 16 (500 mg/kg of total subject weight) on Weight Gain in DIO Rats Days
Group 1 2 3 4 7 10 14 18
ND Mean 7.02 11.58 14.50 15.8O 28.57 34.99 46.34 54.49 (Normal Diet) SD 3.08 3.32 3.42 3.78 7.31 8.42 9.79 11.09 p value O.O21 O.163 O.6SO O.720 O.SO2 O.712 O.974 O.817 HFD Mean 4.07 9.67 13.78 16.37 26.79 36.16 46.21 SS.48 (High Fat Diet) SD 1.93 2.48 3.55 3.16 3.65 S.10 7.36 7.20 SIB Mean 3.91 -7.37 -6.94 -S.37 3.96 13.54 24.SS 31.23 (Sibtramine SD 1.89 3.49 6.76 6.91 9.81 10.77 12.83 12.49 3 mg/kg) p value 0.855 O.OOO O.OOO O.OOO O.OOO O.OOO O.OOO O.OOO G1 Mean 2.55 1.76 O.38 -2.34 8.92 21.11 29.54 42.77 (500 g/Kg)) SD 3.17 5.89 9.54 17.57 18.29 13.92 16.59 16.33 p value O.214 O.OO2 O.OO1 O.OO8 O.O13 O.OO8 O.O13 O.O43 Days
Group 21 24 28 31 35 38 42
ND Mean 63.04 71.15 80.84 87.25 99.02 106.13 114.19 (Normal SD 11.64 13.71 1431 14.17 13:46 14.72 1488 Diet) p value O.930 O.889 O604 0.559 0.757 O.925 O.S31 HFD Mean 63.48 71.95 84.07 90.93 100.90 106.73 118.26 (High Fat SD 10.13 1140 13.03 13.50 13.32 13.55 13.58 Diet) US 2014/0004215 A1 Jan. 2, 2014 81
TABLE 48-continued Effect of Morus alba Extract 16 (500 mg/kg of total subject weight) on Weight Gain in DIO Rats SIB Mean 42.91 50.75 61.84 68.17 78.40 83.51 90.81 (Sibtramine SD 14.03 13.21 13.27 14.71 15.22 17.02 16.74 3 mg/kg) p value O.OO2 O.OO1 O.OO 1 O.OO2 O.OO3 O.004 O.OO1 G1 Mean 48.97 59.41 65.76 77.71 86.25 95.84 101.99 (500 g/Kg)) SD 18.04 22.91 2214 22.76 23.59 25.92 23.90 p value O.044 O.145 O.04O O.136 O.109 O.259 O.O82 p value: Compared to HFD group
0294 The data in Table 48 show that the G1 Morus alba TABLE 50-continued ethyl acetate extract 16 treatment group exhibited statistically significant decreases in body weight gains from day 2 to day Effects of Morus alba extract 16 on Fat Deposit Weight in Rats Fed a 21, and then day 28 of the study as compared to the HFD High Fat Diet group. The positive control SIB (Sibutramine dosed at 3 Retro mg/kg) showed statistically significant decreases in body Epididymal peritoneal Perirenal Total weight gains from day 2 to completion of the study on day 42 Group Fat Fat Fat Fat as compared to the HFD group. HFD Mean 8.636 4.637 2.370 15.643 0295 Table 49 shows the effects of Morus alba extract (High Fat SD 1986 1463 O.799 4.179 Ethyl Acetate Extract 16 on DIO Rats for the following end Diet) points: average body weight gain per day of the study, average SIB Mean 6.698 3.190 1.562 11:450 (Sibtramine SD 1.064 O.701 O.399 1999 food intake per day of the study, and the food efficiency ratio 3 mg/kg) p value O.O17 O.O15 O.O13 O.O13 (FER) which is calculated as the average body weight gain G1 Mean S.911 2.648 1405 9.964 per day over the study period, divided by the average food (500 g/Kg)) SD O.404 O.624 O.242 1.183 intake per day over the study period. p value O.OO2 O.OO2 O.OO)4 O.OO2 p value: Compared to HFD group TABLE 49 0297 The data in Table 50 show that there is a statistically Effect of Morus alba ethyl acetate extract 16 on Rats Fed a High significant difference between Epididymal Fat, Retroperito Fat Diet neal Fat, Perirenal Fat and Total Fat between the ND control group, fed a normal diet having a moderate caloric intact of Weight gain Food intake Food Efficiency fat, and the HFD group. Morus alba extract treated group Group (g/day) (g day) Ratio (FER) (G1) and the positive control treatment group (SIB) showed ND Mean 2.720 16.460 O.16S statistically significant decreases in all categories of fat mea (Normal Diet) SD O.3SO O.6SO O.019 Surement as compared with the HFD group, demonstrating p value O.S31 O.O41 O.O59 that the Morus alba extract 16 is effective in reducing the HFD Mean 2.82O 15.42O O.183 amount of fat present in DIO rats. (High Fat Diet) SD O.32O O.7OO O.O21 SIB Mean 2.160 13.88O 0.155 0298 Table 51 shows the effects of the Morus alba extract (Sibtramine SD O.400 O.660 O.O24 16 on fasting glucose (F-Glu), total cholesterol (T-chol) and 3 mg/kg) p value O.OO1 O.007 O.O13 LDL-cholesterol (LDL-C) as measure in blood samples G1 Mean 2.430 16.300 O.149 obtained at the end of the study. (500 g/Kg)) SD 0.570 O.660 O.O33 p value O.O82 0.077 O.O15 TABLE 51 Food Efficiency Ratio (FER) = (body weight gain (g day), food intake (gday) p value: Compared to HFD group Effects of Morus alba extract 16 on Biochemistry Parameters in DIO Rats T-Chol Triglyceride Fasting Glucose 0296. The data in Table 49 show that the ethyl acetate Group (mg/dL) (mg/dL) (mg/dL) extract of Morus alba as administered to group G1 showed a statistically significantly lower Food Efficiency Ratio as com ND Mean 98.67 40.89 112.67 (Normal Diet) SD 10.89 17.61 10.57 pared to the HFD group. The SIB positive control treatment p value O.O27 0.753 O.OO1 group showed statically lower values than the HFD group for HFD Mean 113.40 44.50 135.70 Weight Gain per day, Food Intake per day, and Food Effi (High Fat Diet) SD 15.36 30.37 13.85 ciency Ratio. SIB Mean 106.60 41.10 136.00 (Sibtramine SD 10.83 23.50 12.01 3 mg/kg) p value O-269 O.783 O.959 TABLE SO G1 Mean 90.40 32.2O 113.80 (500 g/Kg)) SD 9.29 1428 11.33 Effects of Morus alba extract 16 on Fat Deposit Weight in Rats Fed a p value O.OO1 O.268 O.OO1 High Fat Diet p value: Compared to HFD group Retro Epididymal peritoneal Perirenal Total 0299. The data in Table 51 show that Morus alba extract Group Fat Fat Fat Fat 16 treatment group had statistically significant decrease in ND Mean 5.556 2.397 1468 9.42O Total-Cholesterol and Fasting Glucose as compared to the (Normal Diet) SD 1.300 O.S93 O.389 2.2O7 HFD group. The SIB treatment group showed no statistically p value O.OO1 O.OO1 O.OO7 O.OO1 significant changes compared to the HFD group for any of the measured endpoints. US 2014/0004215 A1 Jan. 2, 2014 82
0300. These data show that the Morus alba extract 16 Example 53 when taken at a dose of 500 mg/kg of body weight, twice per day, is effective in lowering the rate of body weight gain in Effect of Mutamba Ethanol Extract 32 on DIO Mice rats fed a high fat diet. In addition, subjects taking the 500 mg/kg dose of extract 16 also exhibited Statistically signifi 0301 Mutamba ethanol extract32, produced according to cant improvements in blood chemistry end points (Table 51) the Example 32, was orally administrated to DIO mouse and tissue fat levels (Table 50) as compared to control sub model as illustrated in the Example 48. The Mutamba extract jects in the HFD group. These results demonstrate that Morus was administered to treatment group G1 at 1000 mg/kg of alba extracts enriched in Kuwanon G and Albanin G can be animal weight per day. G1 treatment group animals were used to control body weight gain, lower tissue fat content, given an oral dose of 500 mg/kg by gavage two times per day. lower fasting blood glucose levels, and decrease total choles Study results for measurement of animal body weight are terol levels. shown in Table 52. TABLE 52 Effect of Mutamba Extract 32 on Total Body Weight in DIO Mice Weeks
Group O 1 2 3 4 5 6 7 8
ND Mean 27.98 28.04 28.12 28.56 27.99 28.05 28.37 28.67 29.39 (Normal SD 1.657 1.561 16O2 1982 1467 1517 1493 1.513 1977 Diet) p value OOOOO O.OO28 O.OO14 O.OOO O.OOO O.OOOS O.OOO3 O.OOO O.OOO HFD Mean 39.72 38.8O 39.56 40.78 418O 43.21 44.69 46.10 46.74 (High SD 3.576 4.OOS 3.672 3.301 3.178 3.776 3.612 3.085 3.144 Fat Diet) SIB Mean 39.70 34.24 33.96 36.OS 36.64 37.42 38.54 38.74 39.95 10 mg/ SD 3.575 1812 1948 1579 3.568 4.384 5.499 5.556 5.766 kg p value O.990 O.049 O.O29 O.O348 O.OSS2 O.O70 O.O82 O.O39 O.OS8 ORI Mean 39.96 37.37 37.05 37.41 37.60 38.12 40.04 41.77 42O6 40 mg/ SD 3.489 4190 4.401 4.659 4.860 5.563 6.028 5.998 6.246 kg p value O.920 O.S96 0.357 O.223 O.145 O.129 O.1769 O.1889 O.1730 G1 Mean 39.79 34.93 36.51 35.78 36.06 37.75 37.41 39.70 41.53 1000 mg/ SD 3.771 3.548 4.453 2.564 2.706 3.213 3.465 4.O39 4.074 kg p value O.978 O.1448 O.271 O.O281 O.O152 O.O393 O.O116 0.0226 O.OS35 p value: compare to HFD by t-test
0302. The data in Table 52 show that the Mutamba ethanol extract 32, 1000 mg/kg/day treatment group showed statisti cally significant decreases in body weight from week 3 through to week 7 of the study as compared to the HFD group. The positive control SIB (sibutramine dosed at 10 mg/kg) showed statistically significant decreases in total body weight from week 1, 2, 3 and 7 of the study as compared to the HFD group. 0303 Study results for measurement of animal body weight gain are shown in Table 53. TABLE 53 Effect of Mutamba extract 32 on Body Weight Gain in DIO Mice Weeks
Group 1 2 3 4 5 6 7 8
ND Mean O.O7 O.14 O.S8 O.O1 O.O8 O.39 O.69 1.41 (Normal SD O.393 O.S45 1.306 O.757 O.654 O606 0.779 1.018 Diet) p value O. 1193 O4309 O.4576 O.OOO9 O.OOOO O.OO15 O.OO11 OOOOO HFD Mean -O.93 -0.16 1.06 2.07 3.49 4.97 6.38 7.01 (High Fat SD 1.121 O941 O.606 1.097 1.142 1483 1.739 1863 Diet) SIB Mean -5.46 -4.84 -2.75 -2.17 -1.38 -O.27 -0.06 1.15 10 mg/kg SD 3.088 2.26O 1968 1997 2.462 3.360 3.216 3.563 p value O.O150 O.OO38 O.O262 O.OO47 O.OOS4 O.O159 O.OO62 O.O149 ORI Mean -2.59 -290 -2.SS -235 -1.84 O.08 1.81 2.10 40 mg/kg SD O.792 1346 1.714 1792 2.536 3.102 3.188 3.124 p value O.O268 O.OOS8 O.OO22 O.OO15 O.OO27 O.O130 O.0228 O.O166 G1 Mean -4.86 -3.28 -4.01 -3.73 -2.04 -2.38 -0.09 1.74 1000 mg/ SD 3.302 2.576 2.049 2.271 2.083 2.423 1974 1540 kg p value 0.0357 O.O345 O.OO38 O.OOO9 O.OOO8 OOOO4 O.OOO6 O.OO12 p value: compare to HFD by t-test US 2014/0004215 A1 Jan. 2, 2014
0304. The data in Table 53 show that the Mutamba ethanol 0308. The data in Table 55 show that the Mutamba, ORI extract 32, 1000 mg/kg/day treatment group and the positive and SIB treatment groups, all exhibited statistically signifi control treatment group SIB and ORI, all showed statistically cant decrease in LDL-cholesterol compared to the HFD significant decreases in body weight gain across all weeks of group. the study. 0309 Table 56 shows the effects of the Mutamba ethanol 0305 Study results for effects of treatments on body extract32 treatment on measurements in DIO mice on several weight gain, food intake, and the food efficiency ratio (FER) histopathological measures of fatty liver and the resulting are shown in Table 54. calculated Non-Alcoholic Staetohepatitis (NASH) score of the liver. TABLE 54 TABLE 56 Effect of Mutamba Extract 32 on DIO Mice Average Effect of Mutamba Extract 32 on Liver Pathology in DIO Mice Body Average Food Pathology Indications Weight Gain Food Intake Efficiency Ratio Group (g/day) (g day) (FER) Lobular ND Mean O.O2S 2.836 O.O09 Inflam- Hepatocellular Steatosis mation Ballooning NASH (Normal SD 1.018 O.253 O.018 Diet) p value O.OO13 O.OOO1 O.OO13 Group (0-3) (0-3) (0-2) (Sum) HFD Mean O.12S 2.536 O.049 ND Mean O.OO O.13 O.OO O.13 (High Fat SD 1863 O.213 O.O33 (Normal SD O.OOO O.354 O.OOO O.354 Diet) Diet) p value O.OOOO O.OOO4 O.OOOO O.OOS4 SIB Mean O.O21 2.724 O.OO8 HFD Mean 2.50 1...SO 1.00 S.OO (10 mg/kg) SD 3.563 O.676 O.064 (High Fat SD 1.OOO 0.577 O.OOO 1.414 p value O.O149 O.2868 O.O111 Diet) ORI Mean O.O38 2.826 O.O13 SIB Mean O.SO O.SO O.25 1.25 (40 mg/kg) SD 3.124 O410 O.OS6 (10 mg/kg) D 1.OOO 0.577 O.SOO 1.893 p value O.O166 O.O162 O.O093 p value O.O3OO O.0498 O.O240 O.O192 G1 Mean O.O31 2.631 O.O12 ORI Mean 120 160 O.60 3.40 (1000 mg/kg) SD 1540 O.667 O.O27 (40 mg/kg) SD O.837 O.894 O.S48 2.074 p value O.OO12 O.S819 O.OO10 p value O.O708 O.8529 O.1930 O.2315 G1 Mean O.80 1.20 O.OO 2.00 Feed efficacy ratio (FER) = Body weight gain (g/day), Food intake (g day) (1000 mg/kg) SD O.447 O.447 O.OOO 0.707 p value: compared to HFD by t-test p value O.O108 O.4O71 O.OOOO O.OO41 0306 The data presented in Table 54 show that average p value: compare to HFD by t-test body weight gain per day and food efficiency ratio (FER) endpoints were significantly lowered in the Mutamba and SIB 0310. The data in Table 56 show that there are statistically treatment groups, when compared to the high fat diet group. significant differences in the amount of Steatosis, Lobular The ORI treatment resulted in statistically significant inflammation, Hepatocellular ballooning and the NASH decreases in average body weight gain per day, average food score between the ND control group, fed a normal diet having intake per day and FER. a moderate caloric intact of fat, and the HFD group. Treat 0307 Study results for effects of treatments on blood bio ment group G1 showed statistically significant decreases in chemistry parameters are shown in Table 55. Steatosis, Hepatocellular ballooning and NASH score as TABLE 55
Effect of Mutamba Extract 32 on Biochemistry Parameters in DIO Mice
ALT AST T-chol LDL-C HDL-C TG Group (UL) (UL) (mg/dL) (mg/dL) (mg/dL) (mg/dL)
ND Mean 13.78 45.56 120.40 8.10 60.08 33.00 (Normal Diet) SD 1.171 1.137 10.431 1140 4.609 12.062 p value O.OS71 OO687 O.OOS9 O.O421 O.OS60 O.O968 HFD Mean 78.62 87.84 192.OO 11.42 70.78 48.40 (High Fat Diet) SD 54.749 38.225 32.458 2.853 9.671 13.777 SIB Mean 37.00 65.95 155.75 6.18 68.73 49.75 (10 mg/kg) SD 34.273 19436 53.786 2.636 15.400 16.091 p value O.2290 O.3357 O.2483 O.O254 O.8127 O.8958 ORI Mean 40.42 67.96 184.40 6.78 78.52 14360 (40 mg/kg) SD 23.183 14479 22.030 O.976 S.498 102.878 p value O.1887 O.3085 O.6763 O.OO88 O.1584 O. 1072 G1 Mean 34.34 55.60 18260 S.42 108.30 34.40 (1000 mg/kg) SD 11.243 6.784 8.905 O.672 51059 14.O29 p value O.1456 O1326 O.S62O O.OO79 0.1770 O.1500 p value: compare to HFD by t-test US 2014/0004215 A1 Jan. 2, 2014 84 compared with the HFD group. The SIB treatment group TABLE 58 showed statistically significant decreases in Steatosis, Lobu lar inflammation, Hepatocellular ballooning and NASH score Effect of Mutamba extract 33-2 on DIO Mice as compared with the HFD group. The ORI treatment group did not show any statistically significant changes compared to Body Weight Food the HFD group. These data demonstrate that the Mutamba Gain Food Intake Efficiency Ratio extract 32 was effective in reducing the amount of liver dam- Group (g/day) (g day) (FER) age present in mice fed a high fat diet. ND Mean O.OO3 2.395 O.OO1 0311. Overall, the data presented in this example show that (Normal SD O.018 O.2O6 O.007 Mutamba ethanol extract 32 was effective in lowering body Diet) p value OOOOO O4282 O.OOOO weight LDL blood cholesterol and fatty liver in mice fed a HFD Mean O.149 2.341 O.064 high fat diet. (High Fat SD O.O27 O.179 O.O12 Diet) Example 54 SIB Mean O.114 2624 O.043 (10 mg/kg) SD O.O34 0.372 O.O13 Effect of Mutamba Ethanol Extract 33-2 on DIO p value O.1399 O.0118 O.O458 Mice ORI Mean O.093 2.769 O.O34 (40 mg/kg) SD O.O27 O436 O.O10 0312 Mutamba ethanol extract 33-2, produced according p value O.O172 O.OO16 O.OO37 to the Example 33, was orally administrated to DIO mouse G1 Mean O.O87 2.337 O.O37 model as illustrated in the Example 48. The Mutamba extract (1000 mg/kg) SD O.O32 O.249 O.O14 was administered to treatment group G1 at 1000 mg/kg of G2 Ray o animal weight per day. G1 treatment group animals were (500 mg/kg) SD 00so 0.642 0.032 given an oral dose of Mutamba 33-2 at 1000 mg/kg by gavage p value O.8747 O.3421 O.6966 two times per day. G2 treatment group animals were given an oral dose of Mutamba ethanol extract 33-2 at 500 mg/kg by Feed efficacy ratio (FER) = Body weight gain (g day), Food intake (g day) gavage two times per day. Study results for measurement of p value: compare to HFD by t-test animal body weight gain are shown in Table 57. TABLE 57
Effect of Mutamba Extract 33-2 on Body Weight Gain in DIO Mice
Weeks
Group 1 2 3 4 5 6 7 8
ND Mean O.15 O.30 O.08 -0.13 -O.O7 O45 -0.10 O.14 (Normal SD O.S29 O.405 0.747 O.952 O.952 O.906 O.747 0.968 Diet) p value O.OS90 O.1707 O.OO3S O.OOO1 O.OOOO O.OOO1 O.OOOO O.OOOO HFD Mean O.85 O.81 2.12 4.11 547 6.79 7.55 8.21 (High Fat SD O.374 O.S96 O642 O.457 O.349 0.177 1.046 1493 Diet) SIB Mean -2.72 -2.39 -O.39 1.47 2.54 3.99 5.15 6.27 (10 mg/kg) SD 1.090 1286 1719 1.615 1.705 1931 2.051 1890 p value O.OOO4 O.OO26 O.O289 O.O166 0.0167 O.O312 O.O729 O.1399 ORI Mean -3.05 -2.81 -2.63 -1.28 O43 3.S2 3.72 S.13 (40 mg/kg) SD 1312 1.370 1.786 1.859 1706 1.384 1.445 1461 p value O.OOO7 O.OO18 O.OO16 O.OO21 O.OO21 O.OOST O.OO31 O.O172 G1 Mean O.39 1.02 2.19 3.13 3.99 4.29 5.19 4.78 (1000 mg/kg) SD 0.397 O.465 O.833 1.239 1.187 1616 2.104 1768 p value O.1143 O.S666 0.8935 0.1815 O.0486 O.O2S3 O.O819 O.O177 G2 Mean -1.65 -1.63 O.08 2.02 3.63 S.65 6.30 7.82 (500 mg/kg) SD O.843 1.730 2.212 2.686 3.249 3.895 4.567 4.408 p value O.OOO9 O.O321 O.1221 O.1568 0.2756 OSS10 O.S829 O.8747 p value: compare to HFD by t-test
0313. The data in Table 57 show that the Mutamba ethanol 0315. The data presented in Table 58 show that average extract 33-2, 1000 mg/kg/day treatment group showed statis- body weight gain per day and food efficiency ratio (FER) tically significant decreases in body weight gain at weeks 5, 6 endpoints were statistically significantly lowered in the and 8 of the study. The G2, 500 mg/kg/day treatment group Mutamba ethanol extract 33-2, 1000 mg/kg/day treatment showed statistically significant decreases in body weight gain at weeks 1, and 2 of the study. group comparedd to the highn1gn fatIal dietd1el group 0314 Study results for effects of treatments on body weight gain, food intake, and the food efficiency ratio (FER) 0316 Study results for effects of treatments on blood bio are shown in Table 58. chemistry parameters are shown in Table 59. US 2014/0004215 A1 Jan. 2, 2014 85
TABLE 59 Subject body weight showed significantly decreased body weight gain, Food Efficiency Ratio, and LDL-cholesterol. Effect of Mutamba extract 33-2 on Biochemistry Parameters in DIO Mice Therefore, the present results indicate that Mutamba extract T-chol LDL-C TG 33-2 can be used as a body weight and blood cholesterol Group (mg/dL) (mg/dL) (mg/dL) controller.
ND Mean 89.60 7.04 22.OO (Normal Diet) SD 9.659 2.304 7.071 Example 55 p value O.OOOO O. 1018 O.OO97 HFD Mean 213.25 10.13 37.OO Efficacy Study of Mutamba Fractions 34, 34-1, and (High Fat Diet) SD 21639 2.617 9.487 34-2 on a DIO Mouse Model SIB Mean 158.60 S.26 46.60 (10 mg/kg) SD S.O3O 1.389 15.126 p value O.O130 O.OO86 O.2631 0319 Mutamba fractions 34, 34-1, and 34-2 produced ORI Mean 182.40 7.42 412O according to the example 34 was orally administrated to DIO (40 mg/kg) SD 12.482 1.593 16.514 mice as described in Example 48. Three Mutamba fraction p value O.0306 0.0955 O4616 treatment groups were: G1 at 250 mg/kg of EtOA fraction 34: G Mean 168.00 6.14 22.60 G2 at 250 mg/kg of BuOH fraction 34-1; and G3 at 250 mg/kg (1000 mg/kg) SD 35.763 1.191 5.079 of Water fraction 34-2. The treatment article was given orally p value O.O628 O.O181 O.S.066 by gavaging 2 times per day. p value: compare to HFD by t-test 0320. The EtOA fraction of Mutamba treatment group 0317. The data in Table 59 show that Mutamba extract (G1) didn't show any effect on body weight. However, the 33-2, 1000 mg/kg/day treatment group decreased LDL-cho BuOH fraction treatment group (G2) showed significantly lesterolina statistically significant fashion as compared to the decreased body weight at week 4, 6, 7, and 8. The water HFD group. fraction treatment group (G3) showed significantly decreased 0318. The data presented in this example show that body weight at week 2 when compared to the HFD group Mutamba extract 33-2 when administered at 1000 mg/kg of (Table 60). TABLE 60
Effect of Mutamba Fractions 34, 34-1, and 34-2 on Body Weight in Mice Fed a High Fat Diet
Weeks
Group O 1 2 3 4 5 6 7 8
ND Mean 27.34 27.49 27.64. 27.42 27.21 27.27 27.79 27.24 27.48 SD 1.283 1.271 1242 1444 1831 1.700 1.6O1 1.465 1381 p O.OOOO OOOOO O.OOOO O.OOOO O.OOOO O.OOOO O.OOOO O.OOOO O.OOOO value HFD Mean 41.07 41.92 41.88 4318 45.18 46.54 47.85 48.62 49.27 SD 1955 2.295 2.486 2.132 2.295 2.110 1834 1.353 0.933 SIB Mean 38.41 3569 36.03 38.02 39.89 40.9S 42.40 43.57 44.69 SD 2.522 3.066 3.2OO 3.812. 3.817 4.185 4.412 45.34 4.322 p O.1290 O.O121 O.O2O2 O.O472 O.O458 0.0466 O.OSS6 O.O711 O.O788 value ORI Mean 39.76 36.71 36.95 37.14 38.49 40.19 43.28 43.49 44.90 SD 3.037 1792 1674 2.110 2.927 2.871 2.544 2.674 2.63S p O4846 O.OO64 O.OO92 O.OO38 O.OO74 O.OO79 0.0198 O.O1 OS O.O167 value G1 Mean 37.39 36.44 37.29 39.04 40.69 42.40 44.37 45.27 46.55 SD 4.593 S.S44. S.215 S.225 4.69S 4.013 3.926 3.721 3.519 p O.1823 O.1092 0.1531 O.1840 O.1263 0.1067 0.1487 0.1343 O.1807 value G2 Mean 38.00 38.93 38.73 38.17 38.32 40.16 40.92 41.91 42.36 SD 2.719 4.01O 4.241 S.OO7 S.136 S.224 3.599 3.378 3.001 p O. 1013 O.2294 0.2337 O.1067 O.O438 0.0572 O.O1O3 O.OO76 O.OO32 value G3 Mean 37.97 37.32 36.97 38.90 40.53 42.1S 43.60 44.40 45.38 SD 2.741 4.074 3.009 3.738 4.141 4.756 4.610 4.646 4.815 p O.1OOO O.O854 O.O346 (O.O827 O.O862 O.1334. O.1293 O.1264. O.1461 value
p value: compare to HFD by t-test US 2014/0004215 A1 Jan. 2, 2014 86
0321. The BuOH fraction 34-1 treatment group (G2) body weight gain was significantly decreased at weeks 6, 7, and 8. Otherwise, the EtOA fraction 34 treatment group (G1) showed significantly decreased body weight at week 1 and the water fraction 34-2 treatment group (G3) at week 2 when compared to HFD group (Table 61). TABLE 61 Effect of Mutamba fractions 34, 34-1, and 34-2On Body Weight Gain in Mice Fed a High Fat Diet Weeks
Group 1 2 3 4 5 6 7 8
ND Mean O.15 O.30 O.08 -0.13 -O.O7 O45 -0.10 O.14 SD O.S29 O4OS 0.747 O.952 O.952 O.906 O.747 0.968 p value O.OS90 O.1707 O.OO3S O.OOO1 O.OOOO O.OOO1 OOOOO O.OOOO HFD Mean O.85 O.81 2.12 4.11 5.47 6.79 7.55 8.21 SD O.374 O.S96 O.642 O457 O.349 0.177 1.046 1493 SIB Mean -2.72 -239 -0.39 1.47 2.54 3.99 5.15 6.27 SD O90 1.286 1719 1.615 1.705 1931 2.051 1890 p value O.OOO4 O.OO26 O.O289 O.O166 0.0167 0.0312 O.O729 O.1399 ORI Mean -3.05 -2.81 -2.63 -1.28 O.43 3.52 3.72 S.13 SD 312 1.370 1.786 1.859 1706 1.384 1445 1461 p value O.OOO7 O.OO18 O.OO16 O.OO21 O.OO21 O.OOSA O.OO31 O.O172 G1 Mean -0.95 -0.10 1.65 3.30 S.O1 6.98 7.88 9.16 SD 149 0.723 O.744 O.895 1.356 1.657 1.765 1.639 p value O.O2O4 O.O831 O.3539 O.1464 O.4988 O.8108 O.7554 0.3978 G2 Mean O.93 0.73 O.17 O.32 2.16 2.92 3.90 4.36 SD 467 1.768 2.894 3.226 2.843 1143 1.323 1868 p value O.917O O.9341 0.2109 OOS/S 0.0589 O.OO14 O.OO29 O.O124 G3 Mean -0.65 -1.00 O.93 2.56 4.18 S.63 6.43 7.41 SD S84 1.407 1.2O7 1661 2.288 2.369 2.S60 2.947 p value O.1004 O.O484 O. 1225 O. 1161 O.2782 O.3374 O.4428 O. 641O p value: compare to HFD by t-test
0322 There were no significant changes on FER when TABLE 62-continued mice were treated with the EtOA fraction 34 and water frac tion 34-1, but the BuOH fraction 34-1 treatment group Effect of Mutamba fractions 34, 34-1, and 34-2 on Mice Fed a High showed borderline changes (Table 62). Fat Diet FER TABLE 62 Body weight Food (Food Effect of Mutamba fractions 34, 34-1, and 34-2 on Mice Fed a High gain intake efficiency Fat Diet Group (g/day) (g day) ratio) FER G1 Mean O.167 2.532 O.066 SD O.O3O O.2O6 O.O12 Body weight R R p value O.3978 O.OO86 O.8046 gain 8Ke enciency G2 Mean O.O79 1978 O.040 Group (g/day) (g day) ratio) SD O.O34 O.158 O.O17 ND M O.OO3 2.395 O.OO1 p value O.O124 OOOOO O.OS13 in O.O18 O.2O6 O.OO7 G3 Mean O.13S 2.244 O.O60 p value O.OOOO O4282 OOOOO SD O.OS4 O.251 O.O24 HFD Mean O.149 2.341 O.064 p value O641O O.2182 0.7871 SIB Ran 8. 4. 8. FER (Feed efficacy ratio) = Body weight gain (g day), Food intake (g day) SD 0.034 0.372 0.013 p value: compare to HFD by t-test p value O.1399 O.0118 O.O458 ORI Man 9: E. 9. 0323. In the BuOH fraction 34-1 of Mutamba treatment p value 0.0172 0.0016 0.0037 group (G2), ALT, HDL-C and TG showed significant changes (Table 63). TABLE 63 Effect of Mutamba fractions 34, 34-1, and 34-2 on Biochemistry Parameters in DIO mice
ALT ALP T-chol LDL-C HDL-C TG Group (UIL) (UIL) (mg/dL) (mg/dL) (mg/dL) (mg/dL) ND Mean 15.30 264.62 89.60 7.04 42.58 22.00 SD 1681 62.45S 9.659 2.304 6.308 7.071 p value O.OO67 O.O390 OOOOO O.1018 O.OOO1 O.OO97 US 2014/0004215 A1 Jan. 2, 2014 87
TABLE 63-continued Effect of Mutamba fractions 34, 34-1, and 34-2 on Biochemistry Parameters in DIO mice
ALT ALP T-chol LDL-C HDL-C TG Group (UIL) (UIL) (mg/dL) (mg/dL) (mg/dL) (mg/dL) HFD Mean 122.8O 1812O 213.25 10.13 69.50 37.00 SD 32.072 11.240 21639 2.617 3.061 9.487 SIB Mean 67.24 156.68 158.60 S.26 64.78 46.60 SD 42.2O3 45.995 S.O.30 1.389 2.901 15.126 p value O.O668 O.3066 O.O130 O.OO86 0.0497 O.2631 ORI Mean 42.60 182.06 18240 7.42 78.34 41.2O SD 13.6O7 23.471 12482 1.593 5.176 16.514 p value O.OO14 O.94.87 O.O.306 O.O955 O.O2OO O4616 G2 Mean 57.66 162.96 1874O 8.18 75.00 33.40 SD 31.354 25.716 16.876 O.856 2.074 7.232 p value O.O181 O.2321 O.O830 O.1574 O.O146 O.OOS2 G3 Mean 72.22 175.16 1808O 9.58 68.36 34.00 SD 37.447 31.987 41.318 3.06S 11.010 16.O16 p value O.O697 O.7322 O.2013 O.7861 O.8483 O.1496 p value: compare to HFD by t-test
0324 Both the BuOH fraction 34-1 (G2) and the water TABLE 64-continued fraction 34-2 (G3) treatment groups showed a significantly decreased NASH score when compared with the high fat diet Effect of Mutmba fractions 34, 34-1, and 34-2 on Liver Pathology in (Table 64). Mice Fed a High Fat Diet Indications TABLE 64 Lobular Hepatocellular Steatosis Inf ti ballooni NSAH Effect of Mutmba fractions 34, 34-1, and 34-2 on Liver Pathology in Group (0-3)(8OSS 88Ol(0-3) allooning(0-2) (sum) Mice Fed a High Fat Diet G3 Mean 1.60 1.60 O.80 4.OO Indications SD O.S.48 O.S48 O.837 1871 p value 0.0141 O.O479 O.O871 O.O212 Lobular Hepatocellular lue: to HFD by t-test Steatosis Inflammation ballooning NSAH p value: compare to y t-tes Group (0-3) (0-3) (0-2) (Sum) 0325. As is evident from the results, the 250 mg/kg. BuOH ND Mean O.OO 1.OO O.OO 1.00 fraction 34-1 treatment group showed significantly decreased SD O.OOO O.OOO O.OOO O.OOO body weight gain, FER, TG and NASH score. Therefore, the p value 0.0000 O.OOO6 O.OOO1 OOOOO Mutamba BuOH extract 34-1 can be used as a body weight HFD Mean 2.75 2.50 1.75 7.00 and blood cholesterol and fatty liver controller. SD O.SOO O.577 OSOO O.816 Example 56 SIB Mean 1.20 1.60 O40 3.20 SD O.837 O.S48 O.S48 1304 Efficacy Study of Mutinaba EtOH Extract 32(in DIO p value O.O142 O.O479 O.OO66 O.OO15 Rats ORI Mean 1.OO 1.60 O.80 340 0326 Mutamba ethanol extract 32 produced according to SD 0.707 O.S48 O.837 1949 the example 32 was orally administrated by gavage to rats in p value 0.0042 O.O479 O.O871 O.O111 a DIO model (as described in Example 49) at a dosage of 500 G2 Mean 140 140 O.80 3.60 mg/kg twice a day. SD O.S.48 O.S48 O.837 1871 0327. In the Mutamba treatment group (G3), body weight p value O.OO66 O.O.222 O.O871 O.O108 was significantly decreased at days 87, 91, 94 and 98, when compared with the high fat diet group (Table 65). TABLE 65 Effect of Mutamba Ethanol Extract 32 on Body Weight in Rats Fed a High Fat Diet Days
Group O 3 7 10 14 17 21 24 28 31
ND Mean 320.66 335.39 351.46 362.OS 368.52 377.64 390.4O 394.90 4O1.13 408.06 SD 21.51 13.89 16.5S 17.24 16.7S 19.96 20.15 20.59 21.63 22.46 p value O.O26 O.OO9 O.O11 O.OO9 O.OO8 0.009 OOO9 O.O1 O O.OOS O.OO6 HFD Mean 343.82 361.82 381.42 393.40 403.22 413.10 426.37 431.33 442.03 451.15 SD 16.89 1561 18.18 17.87 20.45 1991. 20.62. 21.41 20.68 2252 G3 Mean 342.70 357.67 370.89 379.63. 386.71 395.49 4O7.OO 413.14. 417.6S 429.28 SD 15.73 17.54 18.61. 24.35 28.12 32.59 32.30 3430 41.27 38.57 p value O.890 O.661 (0.326 O.266 0.248 0.26O O.22O O.271 O.202 0.234
US 2014/0004215 A1 Jan. 2, 2014 89
0329 Weight gain (g/day) and Food efficiency ratio (FER) TABLE 68-continued were significantly lowered in the Mutinaba treatment group as compared to the high fat diet group (Table 67). Effects of Mutamba Ethanol Extract 32 on Absolute Organ Weights in Rats Fed a High Fat Diet TABLE 67 Epididymal Retroperitoneal Perirenal Group Fat Fat Fat Total Fat Effect of Mutamba Ethanol Extract 32 on DIO Rats HFD Mean O.O2O O.O13 O.007 O.O40 Weight Food FER SD O.OO3 O.OO2 O.OO1 O.OOS gain intake (Food efficiency G3 Mean O.O17 O.OO9 O.OOS O.O3O Group (g/day) (g day) ratio) SD O.OO3 O.OO2 O.OO1 O.OO6 p value O.OS4 O.OO6 O.O10 O.OO8 ND Mean 1.728 16.26S O.107 SD 0.255 O868 O.018 p value: Compared to HFD group p value O.007 O669 O.O10 HFD Mean 2.100 15.953 O.132 0331. These results show that both body weight and body SD O.O90 0.787 O.OOS G3 Mean 1704 15.693 O.109 weight gain were significantly decreased in the Mutamba SD O.133 O.709 O.OO6 treatment groups. At treatment group, FER (food efficiency p value O.OOO O.693 O.OOO ratio) and visceral fat weights were also significantly decreased. Therefore, the present results suggest that FER (Food efficiency ratio) = (body weight gain (g/day), food intake (giday) p value: Compared to HFD group Mutamba extract can be used as a body weight controller. Example 57 0330. In the Mutamba treatment group of absolute organ weight, Perirenal, Retroperitoneal and Total fat pad were Effect of Morus Alba Ethyl Acetate Extract 15 significantly decrease, when compared with the high fat diet Combined with Magnolia Extract 29 on DIO Mice group (Table 68). 0332 Morus alba ethyl acetate extract 15 produced as described in Example 15 and Magnolia extract 29 produced TABLE 68 according to Example 29 were combined and blended to a ratio of 2:1 by weight. The combination composition was Effects of Mutamba Ethanol Extract 32 on Absolute Organ Weights in orally administrated to DIO mice as described in Example 48 Rats Fed a High Fat Diet at a dosage of 300 mg/kg of animal weight (200 mg/kg. Morus Epididymal Retroperitoneal Perirenal and 100 mg/kg Magnolia). The study time period was seven Group Fat Fat Fat Total Fat weeks. Table 69 shows the shows the effects of the combina ND Mean O.O14 O.OO8 O.OOS O.O27 tion composition on body weight gain, that is, the change in SD O.OO)4 O.OO2 O.OO1 O.OO6 body weight in each study group as measured at the beginning p value O.OO7 O.OO1 O.OO)4 O.OO2 of the study compared to the weight measured on the day of each time point of the study. TABLE 69
Effect of Morus alba extract 15 Combined with Magnolia extract 29 on Body Weight Gain in Mice Fed a High Fat Diet
Weeks
Group 1 2 3 4 5 6 7
ND Mean -0.09 -0.25 O.09 O3S O.13 O.S6 0.79 SD O.2O2 O.S13 O418 O408 O.SOO O.S28 O.S43 p O.12SO 0.4003 0.2140 O.1935 0.0758, O.O3S1 O.O243 value HFD Mean -0.55 O16 1.45 1.95 3.29 4.40 5.77 SD O891 0.665 1.324 1.45S 1643 1.373 1476 ORI Mean -3.20 -4.88 -3.34 -2.61 -2.04 -1.65 -0.07 (40 mg/kg) SD O611 1463 1...SO2 1.859 1640 1.526 1.437 p O.OO11 O.OO28 O.OO72 O.O174 O.OO81 O.OO29 O.OO33 value G1 Mean -1.87 -2.78 -2.15 -1.62 -1.56 -1.48 -0.56 (300 mg/kg) SD 1.126 1.369 1.533 1546 1976 2.316 2.240 p O.1001 O.O144 OO152 O.O18O, O.O121 O.OO77 O.OOS1 value
p value: compare to HFD by t-test US 2014/0004215 A1 Jan. 2, 2014 90
0333. The data in Table 69 show that the animals treated TABLE 71-continued with a composition comprising Morus alba 15 combined with Magnolia extract 29 (treatment group G1) exhibited statisti Effect of Morus alba extract 15 Combined with Magnolia cally significant decreases in body weight gains from week 2 Extract 29 on Absolute Liver Weight and Weight of Fatty through to week 7 of the study as compared to the HFD group. Deposits in DIO Mice The positive control ORI (Orilistat) showed statistically sig Epid- Retro nificant decreases in body weight gains from week 1 through idymal peritoneal Perirenal Total to week 7 of the study as compared to the HFD group. Group Liver Fat Fat Fat Fat: 0334 Table 70 shows the effects of Morus alba extract 15 and Magnolia extract 29 combination composition on DOI ORI Mean 1.OS 1.92 0.55 O.21 2.68 mice for the following end points: average body weight gain 40 mg/kg SD O.137 0.230 O.O39 0.055 O.310 p value 0.2282 0.0236 0.0034 0.1729 O.O177 per day of the study, average food intake per day of the study, G1 Mean 1.23 1.73 O.S2 O.20 2.45 and the food efficiency ratio (FER) which is calculated as the 300 mg/kg SD O.274 O466 O.O76 O. 110 O.643 average body weight gain per day over the study period, p value 0.9286 0.0148 0.0036 O. 1649 O.O145 divided by the average food intake per day over the study period. *Total fat is sum of the three fat deposits (epididymal, retroperitoneal and perirenal fat) p value: compare to HFD by t-test TABLE 70 0337 The data in Table 71 show that there is a statistically Effect of Morus alba extract 15 Combined with Magnolia Extract 29 on DIO Mice significant difference in the weight of liver, epididymal fat, retroperitoneal fat, perirenal fat and total fat between the ND Body Weight Food Efficiency control group, fed a normal diet having a moderate caloric Gain Food Intake Ratio Group (g/day) (g day) (FER) intact offat, and the HFD group. In addition, treatment group G1 getting the composition of Morus alba extract 15 com ND Mean O.018 3.558 O.OOS SD O.O12 O.S60 O.OO3 bined with Magnolia extract 29, along with the positive con p value O.O243 O.3067 O.O242 trol treatment group (ORI), showed statistically significant HFD Mean O.128 3.342 O.O38 decreases in two fat deposits (Epididymal Fat and Retroperi SD O.O33 O.807 O.O10 toneal Fat) and Total Fat as compared with the HFD group. ORI Mean -0.002 3.961 O.OOO (40 mg/kg) SD O.O32 2.330 O.007 These data, demonstrate the composition of Morus alba p value O.OO33 O.2661 O.OOO6 extract combined with Magnolia extract is effective at reduc G1 Mean -0.012 2.261 -OOO6 ing the amount offat present in DIO mice. (300 mg/kg) SD O.OSO O.858 O.O22 p value O.OOS1 O.OO10 O.O189 0338 Table 72 shows the effects of the composition com Feed Efficacy Ratio (FER) = Body weight gain (g/day). Food intake (gday) prising Morus alba 15 combined with Magnolia extract 29 on p value: compare to HFD by t-test measurements in DIO mice of fasting glucose (F-Glu), total cholesterol (T-chol) and LDL-cholesterol (LDL-C) as mea 0335. The data in Table 70 show that the composition sure in blood samples obtained at the end of the study. having Morus alba extract 15 combined with Magnolia extract 29 (treatment group G1) showed a statistically signifi TABLE 72 cant effect on lowering body weight gain, food intake and lowering the Food Efficiency Ratio as compared to the HFD Effect of Morus alba Extract 15 Combined with Magnolia Extract 29 on group. The ORI positive control treatment group showed Biochemistry Parameters statistically lower values than the HFD group for Body F-Glu T-Chol Triglyceride LDL-C Weight Gain per day and Food Efficiency Ratio. Group (mg/dL) (mg/dL) (mg/dL) (mg/dL) 0336 Table 71 shows the effects of Morus alba extract 15 ND Mean 178.67 105.67 30.56 4.54 (Normal Diet) SD 56.934 17.349 11.886 1705 and Magnolia extract 29 combination composition on DIO p value OOOO4 O.OOOO 0.7005 O.O807 mice for total liver organ weight, the weights of three fatty HFD Mean 334.50 176.75 28.00 6.25 deposits: epididymal fat, retroperitoneal fat, perirenal fat, and (High Fat Diet) SD 30.957 10.996 6.976 O.480 total fat (the sum of the previous three fatty tissues). ORI Mean 225.50 134.00 62.50 3.30 (40 mg/kg) SD 52.208 30.299 64717 1134 p value O.O115 O.O379 O.3653 O.OO3O TABLE 71 G1 Mean 251.2O 145.60 24.00 2.28 (300 mg/kg) SD 51.237 16.652 7.071 O.96S Effect of Morus alba extract 15 Combined with Magnolia p value O.O2SO O.O150 0.4244 O.OOO1 Extract 29 on Absolute Liver Weight and Weight of Fatty Deposits in DIO Mice Epid- Retro 0339. The data in Table 46 show the combination compo idymal peritoneal Perirenal Total sition treatment group G1 and the positive control treatment Group Liver Fat Fat Fat Fat: group (ORI) showed statistically significant decreases in total ND Mean 1.OO O.S3 O.15 O.08 O.76 cholesterol, LDL-cholesterol, and fasting glucose. These SD O.145 0.150 O.O64 O.O17 O.222 p value 0.0410 0.0002 0.0000 O.O117 O.OOO1 data, demonstrate the Morus alba and Magnolia combina HFD Mean 122 2.60 O.69 O.31 3.60 tions composition when administered at 300 mg/kg of body SD O.215 0.424 O.OS1 O.118 O.S21 weight is effective in reducing cholesterol and fasting glucose levels in DIO mice. US 2014/0004215 A1 Jan. 2, 2014 91
Example 58 through to week 7 of the study as compared to the HFD group. The positive control ORI (orilistat dosed at 40 mg/kg) showed Effect of Morus alba Ethyl Acetate Extract 15 statistically significant decreases in total body weight from Combined with Yerba mate Extract 26 on DIO Mice week 2 through to week 7 of the study as compared to the HFD group. 0340 Morus alba ethyl acetate extract 15 produced as 0342. The data in Table 74 show that the animals in the described in Example 15 and Yerba mate extract 26 produced combination composition of Morus alba extract 15 and Yerba according to Example 26 were combined and blended to a mate extract 26 treatment group (G1) and the positive control ratio of 1:5 by weight. The combination composition was ORI group of animals both exhibited statistically significant orally administrated to DIO mice as described in Example 48 decreases in body weight gains from week 1 through to week at a dosage of 1200 mg/kg of animal weight (200 mg/Kg 7 of the study as compared to the HFD group. Morus and 1000 mg/Kg Yerba mate). The study time period 0343 Table 75 shows the effects of Morus alba extract 15 was seven weeks. Table 73 shows the effects of the combina and Yerba mate extract 26 combination composition on DIO tion composition on total body weight and Table 74 shows the mice for the following end points: average body weight gain effects of the combination composition on body weight gain. per day of the study, average food intake per day of the study, TABLE 73
Effect of Morus aiba extract 15 Combined with Yerba Mate extract 26 on Total Weight in DIO Mice
Weeks
Group O 1 2 3 4 5 6 7
ND Mean 28.60 28.51 28.34 28.69 28.94 28.73 29.16 29.39 (Normal SD 2.249 2.244 2.341 2.491 2.094 2.107 2.087 2.09S Diet) O.OOOO O.OOOO O.OOOO O.OOOO O.OOOO O.OOOO O.OOOO O.OOOO value HFD Mean 37.98 38.18 38.72 40.01 40.51 41.85 42.96 44.32 (High Fat SD 2.38.16 1983O 2.2782 15612 19172 1.7894 1.9183 2.0291 Diet) ORI Mean 38.40 35.20 33.58 35.12 35.84 36.41 36.8O 38.38 (40 mg/kg) SD 2.895 3.211 2.730 2.568 2.32O 2.416 2.534 2.257 O.8094. O.1510 O.O46S O.O343 O.O371 O.O226 O.O174 OO157 value G1 Mean 39.14 37.04 35.59 34.76 35.15 35.85 36.40 38.06 (1200 mg/kg) SD 2.98S 2.430 2.268 2564 2.496 2.590 2.669 2.696 p O.S169 O.4739 O.1085 O.O197 O.O195 O.O130 O.O104 O.O138 value p value: compare to HFD by t-test
TABLE 74
Effect of Morus aiba extract 15 Combined with Yerba Mate extract 26 on Weight Gain in DIO Mice Weeks
Group 1 2 3 4 5 6 7
ND Mean -O.09 -0.25 O.09 O.35 O.13 O.S6 0.79 (Normal SD O.2O2 O.S13 O418 O.408 OSOO O.S28 O.S43 Diet) p value O.12SO 0.4003 0.2140 O.1935 0.0758 O.O3S1 O.O243 HFD Mean -O.S.S O16 1.45 1.95 3.29 4.40 5.77 (High Fat SD O891 0.665 1.324 1.45S 1643 1.373 1476 Diet) ORI Mean -3.20 -4.88 -3.34 -2.61 -2.04 -1.65 -O.O7 (40 mg/kg) SD O611 1463 1...SO2 1.859 1.640 1.526 1.437 p value O.OO11 O.OO28 O.OO72 O.O174 O.OO81 O.OO29 O.OO33 G1 Mean -2.10 -3.55 -4.38 -3.99 -3.29 -2.74 -1.08 (1200 mg/kg) SD 0.655 1.671 2.413 2.464 2.567 2.878 2.484 p value O.O195 O.O116 O.OO92 O.OO97 O.OO79 O.OO76 O.OOS3 p value: compare to HFD by t-test
0341 The data in Table 73 show that the animals in the and the food efficiency ratio (FER) which is calculated as the combination composition of Morus alba extract 15 and Yerba average body weight gain per day over the study period, mate extract 26 treatment group (G1) exhibited statistically divided by the average food intake per day over the study significant decreases in total body weight from week 3 period. US 2014/0004215 A1 Jan. 2, 2014 92
TABLE 75 the Morus alba extract 15 and Yerba mate extract 26 combi nation composition treatment group G1 and the positive con Effect of Morus aiba extract 15 Combined with Yerba Mate extract 26 trol treatment group (ORI) showed statistically significant On DIO Mice decreases in Retroperitoneal Fat as compared with the HFD Food Efficiency group. These data, demonstrate the Morus alba and Yerba Weight Gain Food Intake Ratio mate combinations composition is effective in reducing the Group (g/day) (g day) (FER) amount of retroperitoneal fat present in DIO mice. 0347 Table 77 shows the effects of the Morus alba 15 and ND Mean O.018 3.558 O.OOS Yerba mate extract 26 combination composition on measure (Normal SD O.O12 O.S60 O.OO3 Diet) p value O.O243 O.3067 O.O242 ments in DIO mice of fasting glucose (F-Glu), total choles HFD Mean O.128 3.342 O.O38 terol (T-chol), triglyceride and LDL-cholesterol (LDL-C) as g Fat SD O.O33 O.807 O.O10 measure in blood samples obtained at the end of the study. Diet ORI Mean -O.OO2 3.961 O.OOO (40 mg/kg) SD O.O32 2.330 O.007 TABLE 77 p value O.OO33 O.2661 O.OOO6 G1 Mean -0.024 2.435 -O.O10 Effect of Morus alba and Yerba Mate Combination Composition on (1200 mg/kg) SD 0.055 O.475 O.O23 Biochemistry Parameters in DIO Mice p value O.OOS3 O.OO2O O.O143 F-Glu T-Chol Triglyceride LDL-C FER (Feed efficacy ratio) = Body weight gain (g/day), Food intake (g day) Group (mg/dL) (mg/dL) (mg/dL) (mg/dL) p value: compare to HFD by t-test ND Mean 178.67 105.67 30.56 4.54 0344. The data in Table 75 show that the Morus alba (Normal Diet) SD 56.934 17.349 11.886 1705 tract 15 and Yerba mate extract 26 combination composi- p value OOOO4 OOOOO O.700S 0.0807 ex comp HFD Mean 334.5O 176.75 28.00 6.25 tion treatment group G1 showed a statistically significant (High Fat Diet) SD 30.957 10.996 6.976 O.480 effect on lowering body weight gain, food intake and lower ORI Mean 225.50 134.00 62.50 3.30 Food Efficiency Ratio as compared to the HFD group. The (40 mg/kg) SD 52.208 30.299 64717 1134 ORI positive control treatment group showed statistically G1 Rele S. 1...." 8. g lower values than the HFD group for Body Weight Gain per (1200 mg/kg) SD(8. 42.275 14.170 3.209 0.667 day and Food Efficiency Ratio. p value O.O681 0.0102 O.OO15 O.OO32 (0345 Table 76 shows the effects of Morus alba extract 15 and Yerba mate extract 26 combination composition on DOI p value: compare to HFD by t-test mice for the weight of Retroperitoneal Fat. 0348 The data in Table 77 show the combination compo sition treatment group G1 and the positive control treatment TABLE 76 group (ORI) showed statistically significant decreases in total Effect of Morus alba and Yerba Mate Combination Composition on cholesterol, LDL-cholesterol, and fasting glucose. Retroperitoneal Fat in DIO Mice 0349. Overall, the data presented in this example show that the combination composition composed of Morus alba ethyl Group Retroperitoneal Fat acetate extract 15 (200 mg/Kg) and Yerba mate extract 26 ND Mean O.15 (1000 mg/Kg) was effective in lowering total body weight (Normal Diet) SD O.064 and the rate of body weight gain in mice fed a high fat diet. p value OOOOO HFD Mean O.69 Example 59 (High Fat SD O.OS1 Diet) Effect of Morus alba Ethyl Acetate Extract 14 ORI Mean 0.55 (40 mg/kg) SD O.O39 Combined with Rosemary Extract 21 on DIO Mice p value O.0034 0350 Morus alba ethyl acetate extract 14 produced as G1 Mean 0.57 described in Example 14 and Rosemary extract 21 produced (1200 mg/kg) SD O.08O according to Example 21 were combined and blended to a p value O.O281 ratio of 2:5 by weight. The combination composition was p value: compare to HFD by t-test orally administrated to DIO mice as described in Example 48 at a dosage of 700 mg/kg of animal weight (200 mg/Kg Morus 0346. The data in Table 76 show that there is a statistically and 500 mg/Kg Rosemary). The study time period was seven significant difference in the weight of retroperitoneal fat, weeks. Table 78 shows the effects of the combination com between the ND control group, fed a normal diet having a position on total body weight gain and Table 79 shows the moderate caloric intact offat, and the HFD group. In addition, effects of the combination composition on body weight gain. TABLE 78 Effect of Morus alba extract 14 Combined with Rosemary extract 21 on Weight Weeks
Group O 1 2 3 4 5 6 7
ND Mean 29.01 29.30 29.42 29.27 29.25 29.73 29.82. 30.11 (Normal SD 2.824 2.881 2.523 2.315 2.368 2.739 2.942 3.113 Diet) p O.OOO2 O.OOO2 O.OOO1 OOOOO O.OOOO O.OOOO O.OOOO O.OOOO value US 2014/0004215 A1 Jan. 2, 2014 93
TABLE 78-continued Effect of Morus alba extract 14 Combined with Rosemary extract 21 on Weight Weeks
Group O 1 2 3 4 5 6 7
HFD Mean 41.86 41.60 42.53 43.65 44.78 46.09 46.64 47.86 (High Fat SD 3.812 3.776 3.617 3.425 3.293 2.957 2.694 2.219 Diet) ORI Mean 42.39 38.83 36.64 36.48 38.57 40.07 40.95 42.99 (40 mg/kg) SD 3.489 4.433 4.681 3.948 4.221 4.690 4.775 4.910 p O.8079 0.2718 O.O349 O.OO73 O.O174 O.O238 0.0294 O.OS1.4 value G1 Mean 43.03 37.59 36.41 37.43 39.04 40.40 40.60 41.89 (700 mg/kg) SD 2.702 1930 2.597 3.630 4.103 4.381 4.527 4...SOS p O.S340 O.0312 O.OO46 O.OO91 O.O189 O.O.209 OO157 0.0156 value p value: compare to HFD by t-test
TABLE 79 Effect of combination with Morus alba extract 14 Combined with Rosemary extract 21 on Body Weight Fain in DIO Mice Weeks
Group 1 2 3 4 5 6 7
ND Mean O.29 O41 O.26 O.23 O.71 O.81 1.10 SD O.S30 O.704 O.66S 0.875 O.724 0.723 O.834 p value O.O779 0.5236 O.OOS6 OOOO7 O.OOO2 O.OOO3 O.OOOS HFD Mean -0.27 0.67 1.79 2.92 4.23 4.77 5.99 SD O.248 0.525 0.732 O.866 1.08O 1.378 1.933 ORI Mean -3.56 -5.75 -5.91 -3.82 -2.33 -1.44 O.60 40 mg/kg SD 1.391 1935 1852 1463 1.675 1.512 1.550 p value O.OOO2 O.OOOO O.OOOO O.OOOO O.OOOO O.OOOO O.OOO3 G1 Mean -5.44 -6.62 -5.60 -3.99 -2.63 -2.43 -0.67 700 mg/kg SD 1377 1.585 1.900 2.081 2.057 2.200 2.382 p value OOOOO O.OOOO O.OOOO O.OOOO O.OOOO O.OOOO O.OOO3 p value: compare to HFD by t-test
0351. The data in Table 78 show that the animals treated TABLE 8O with Morus alba extract 14 combined with Rosemary extract Effect of Morus aiba Extract 14 Combined with 21 (treatment group G1) exhibited Statistically significant Rosemary Extract 21 On DIO Mice decreases in total body weight from week 1 through to week 7 of the study as compared to the HFD group. The positive BodyGain Weight Food Intake EfficiencyFood Ratio control ORI (Orilistat dosed at 40 mg/kg) showed statistically Group (g/day) (g day) (FER) significant decreases in total body weight from week 2 ND Mean O.O23 3.046 O.007 through to week 6, but not weeks 1 and 7 of the study as SD O.O17 O.188 O.OO6 compared to the HFD group. p value O.OOOS OOOOO O.OOO3 HFD Mean O.12S 2.376 O.OS3 0352. The data in Table 79 show that the animals in treat- SD O.040 O4(OO O.O17 ment group (G1) who received Morus alba extract 14 com- 40ORI mg/kg MeanSD O.O12O.O32 2.711O456 O.OOSO.O12 bined with Rosemary extract 21 and the positive control ORI p value O.OOO3 O.OO69 O.OOO2 group both exhibited Statistically significant decreases in G1 Mean -0.014 2-18O -OOO6 body weight gain from week 1 through to week 7 of the study 700 mg/kg SD O.OSO O.474 O.O23 as compared to the HFD group. p value O.OOO3 O.1126 O.OOOS 0353 Table 80 shows the effects of Morus alba extract 14 Feed Efficacy Ratio (FER) = Body Weight Gain (g day), Food Intake (g/day) combined with Rosemary extract 21 combination composi p value: compare to HFD by t-test tion on DIO mice for the following end points: average body 0354. The data in Table 80 show that the Morus alba weight gain per day of the study, average food intake per day extract 14 combined with Rosemary extract 21 combination of the study, and the food efficiency ratio (FER) which is composition treatment group G1 showed a statistically sig calculated as the average body weight gain per day over the nificant effect on lowering average weight gain per day and study period, divided by the average food intake per day over lower Food Efficiency Ratio as compared to the HFD group. the study period. The ORI positive control treatment group showed statically US 2014/0004215 A1 Jan. 2, 2014 94 lower values than the HFD group for average weight gain per Morus alba extract 14 combined with Rosemary extract 21 day, average Food Intake per day and Food Efficiency Ratio. composition treatment group G1 and the positive control 0355 Table 81 shows the effects of Morus alba extract 14 treatment group (ORI) showed statistically significant combined with Rosemary extract 21 combination composi decreases in Perirenal Fat as compared with the HFD group. tion on DIO mice for the weight of Perirenal Fat. These data demonstrate the Morus alba and Rosemary com bination compositions are effective in reducing the amount of TABLE 81 perirenal fat present in DIO mice. 0357. Overall, the data presented in this example show that Effect of Morus alba 14 combined with Rosemary extract 21 the combination composition composed of Morus alba ethyl on Perirenal Fat in DIO mice acetate extract 14 (200 mg/Kg) and Rosemary extract 21 (500 Group Perirenal Fat mg/Kg) was effective in lowering total body weight and the ND Mean O.O8 rate of body weight gain in mice fed a high fat diet. (Normal SD O.O26 Diet) p value O.OOO1 Example 60 HFD Mean O.S8 (High Fat SD O.127 Efficacy Study of Mutamba Ethanol Extract 35 Diet) Combined with Morus alba EtOAC Fraction of ORI Mean O.43 (40 mg/kg) SD O.186 Ethanol Extract 15 in DIO Mice p value O.1369 G1 Mean O40 0358 Mutamba ethanol extract 35 produced according to (700 mg/kg) SD O.152 the example 35 and Morus alba EtOAc fraction of ethanol p value O.O479 extract 15 produced according to example 15 was blended in a ratio of 5:1. The combined composition was orally admin p value: compare to HFD by t-test istrated to DIO mice as described in the Example 48 at a 0356 The data in Table 81 show that there is a statistically dosage of 1200 mg/kg (G1) twice a day by gavage. significant difference in the weight of perirenal fat, between 0359 The treatment group (G1) shoed significantly the ND control group, fed a normal diet having a moderate decreased weight gain was at weeks 4, 5, 6 and 7 as compared caloric intact of fat, and the HFD group. In addition, the to the high fat diet group (Table 82). TABLE 82
Effect of Mutamba Extract 35 Combined with Morus aiba Extract 15 on Total Body Weight in DIO Mice Weeks
Group O 1 2 3 4 5 6 7
ND Mean 28.60 28.51 28.34 28.69 28.94 28.73 29.16 29.39 SD 2.249 2.244 2.341 2.491 2.094 2.107 2.087 2.095 p value OOOOO O.OOOO O.OOOO O.OOOO O.OOOO O.OOOO O.OOOO O.OOOO HFD Mean 37.98 38.18 38.72 40.01 40.51 41.85 42.96 44.32 SD 2.381 6 1983O 2.2782 15612 19172 1.7894 19183 2.O291 ORI Mean 38.40 35.20 33.58 35.12 35.84 36.41 36.8O 38.38 SD 2.895 3.211 2.730 2.568 2.32O 2416 2.534 2.257 p value O.8094 0.1510 O.O46S O.O343 O.O371 O.O226 O.O174 OO157 G1 Mean 37.22 35.36 34.49 35.24 35.09 34:12 34.70 35.24 SD 2.177 1.465 O.928 1.161 1.309 1.269 1.695 1570 p value O.9374 O.S169 O.4739 O.1085 O.O197 O.O.195 O.O130 OO104 p value: compare to HFD by t-test
0360. The treatment group (G1), the body weight gain was significantly decreased after the second week treatment of the experiment (Table 83) and such effect was lasted to the end of the treatment. TABLE 83
Effect of Mutamba Extract 35 Combined with Morus aiba Extract 15 on Weight Gain in DIO Mice Weeks
Group 1 2 3 4 5 6 7
ND Mean -O.09 -0.25 O.09 O.35 O.13 O.S6 0.79 SD O.2O2 O.S13 O.418 O408 O.SOO O.S28 O.S43 p value O.1250 0.4003 O.2140 O.1935 O.O758 O.O3S1 O.O243 HFD Mean -O.S.S O16 1.45 1.9S 3.29 4.40 5.77 SD O891 O.665 1324 14.SS 1643 1.373 1476 US 2014/0004215 A1 Jan. 2, 2014
TABLE 83-continued
Effect of Mutamba Extract 35 Combined with Morus aiba Extract 15 on Weight Gain in DIO Mice Weeks
Group 1 2 3 4 5 6 7
ORI Mean -3.20 -4.88 -3.34 -2.61 -2.04 -1.65 SD O611 1463 1. SO2 1.859 1640 1.526 1.437 p value O.OO11 O.OO28 O.OO72 O.O174 O.OO81 O.OO29 O.OO33 G1 Mean -1.86 -2.74 -1.98 -2.13 -3.10 -2.53 -1.98 SD 0.973 1483 1.258 O.901 2.036 1246 p value O.108O O.O378 O.OO67 O.OO11 O.OOS1 O.OOOS p value: compare to HFD by t-test
0361. The treatment group (G1) of FER was significantly TABLE 86 decreased, when compared with the high fat diet group (Table 84) Effect of Mutamba Extract 35 Combined with Morus aiba Extract 15 on Absolute Organ Weights in DIO Mice
TABLE 84 Retro Effect of Mutamba Extract 35 Combined with Epididymal peritoneal PeriRenal Total Morus aiba Extract 15 on DIO Mice Group Liver Fat Fat Fat Fat:
FER ND Mean 1.00 O.S3 O.15 O.08 O.76 Weight Gain Food intake (Food SD O.145 O.1SO O.064 O.O17 O.222 Group (g day) (g/day) efficiency ratio) p value 0.0410 O.OOO2 OOOOO O.O117 O.OOO1 HFD Mean 1.22 2.60 O.69 O.31 3.60 ND Mean O.018 3.558 O.OOS SD O.215 O424 O.OS1 O.118 O.S21 SD O.O12 O.S60 O.OO3 p value O.O243 0.3067 O.O242 ORI Mean 1.05 1.92 0.55 O.21 2.68 HFD Mean O.128 3.342 O.O38 SD 0.137 O.230 O.O39 0.055 O.310 SD O.O33 O.807 O.O10 p value 0.2282 O.O236 O.OO34 0.1729 O.O177 ORI Mean -OOO2 3.961 O.OOO G1 Mean 1.09 1.25 O.38 O.14 1.77 SD O.O32 2.330 O.007 SD O.O70 O.287 O.109 O.O29 O.410 p value O.OO33 O.2661 O.OOO6 p value 0.2515 OOOO4 OOOO4 O.O283 O.OOO3 G1 Mean -0.044 2.652 -0.017 SD O.O24 0.273 O.O09 *Total fat is sum of the three fatpads (epididymal, retroperitoneal and perirenal fat) p value O.OOO2 O.OOO4 O.OOO4 p value: compare to HFD by t-test FER(Feed efficacy ratio) = Body weight Gain (g day). Food Intake (g/day) p value: compare to HFD by t-test 0364 Overall, the data show that body weight and body weight gain were significantly decreased in DIO mice treated 0362. The treatment group (G1) of glucose, total choles with Mutamba extract 35 combined with Morus alba extract terol and LDL-C were significantly decreased as compared to 15. FER (food efficiency ratio) and fat weights were also the high fat diet group (Table 85). significantly decreased. Furthermore, fasting glucose, total cholesterol and LDL-cholesterol levels were significantly TABLE 85 decreased by sample treatment. Therefore, the present Effect of Mutamba Extract 35 Combined with Morus aiba Extract 15 example indicates that the combination of Mutamba extract on Biochemistry Parameters with Morus alba extract can be used as a body weight, glucose level, and cholesterol level controller. Glu T-chol TG LDL-C Group (mg/dL) (mg/dL) (mg/dL) (mg/dL) Example 61 ND Mean 178.67 105.67 30.56 4.54 SD 56.934 17.349 11.886 1.705 p value O.OOO4 OOOOO 0.7005 O.O807 HFD Mean 334-SO 176.75 28.00 6.25 Efficacy Study of Mutamba Ethanol Extract 35 SD 30.957 10.996 6.976 O480 Combined with Yerba mate Extract 26 in DIO Mice ORI Mean 225.50 134.OO 62.50 3.30 SD 52.208 30.299 64.717 1134 0365 Mutamba ethanol extract 35 produced according to p value O.O115 O.O379 O.3653 O.OO3O G1 Mean 194.80 116.2O 14.60 2.76 the example 35 and Mate extract 26 produced according to SD 33.596 33.752 8.173 O948 example 26 were blended in a ratio of 1:1. The dual combi p value O.OOO4 O.0114 O.O3S4 O.OOO3 nation composition was orally administrated to DIO mice as described in the example 48 at a dosage of 2000 mg/kg (G1) twice a day. 0363 Absolute weights of epididymal fat pads, retroperi toneal fat, perirenal fat and total fat pads were significantly 0366 Weight gain was significantly decreased in the treat decreased in the treatment group (G1) as compared to the high ment group (G1) after the third week of treatment (Table 87) fat diet group (Table 86). and that effect lasted to the end of the treatment. US 2014/0004215 A1 Jan. 2, 2014 96
TABLE 87 Effect of Mutamba Combined with Yerba Mate on Weight Gain in DIO Mice Weeks
Group 1 2 3 4 5 6 7
ND Mean -0.09 -0.25 O.09 O.35 O.13 O.S6 0.79 SD O.2O2 O.S13 0.418 O408 OSOO O.S28 O.S43 p value 0.1250 0.4003 0.2140 0.1935 0.0758 0.0351 O.O243 HFD Mean -0.55 O16 1.45 1.9S 3.29 4.40 5.77 SD O891 O66S 1324 14.SS 1643 1.373 1476 SIB Mean -3.63 -537 -4.69 -4.82 -4.25 -3.90 -2.27 SD 1.504 2.317 2.2SO 2.227 1862 2.276 2.960 p value 0.00885 0.0078 0.00553 0.00358 0.00119 0.00135 0.00511 ORI Mean -3.20 -4.88 -3.34 -2.61 -2.04 -1.65 -O.O7 SD O.611 1463 1.5O2 1.859 1.640 1.526 1.437 p value 0.0011 0.0028 0.0072 0.0174 0.0081 0.0029 O.OO33 G1 Mean -0.11 -1.28 -1.43 -2.25 -1.84 -2.03 -0.45 SD OS46 1.290 1.361 1794 2.060 2.241 1849 p value 0.3970 0.0709 0.0373 0.0141 0.01.04 0.0020 O.OO31 p value: compare to HFD by t-test
0367 FER was significantly decreased in the treatment 0368. These data show that weight gain and FER were group (G1) as compared with the high fat diet group (Table significantly decreased in the treatment group (G1). There 87). fore, the present example indicates that the combination of Mutamba extract and Yerba mate extract can be used as a TABLE 87 body weight controller. Effect of Mutamba Combined with Yerba Mate on DIO Mice Weight Gain Food Intake FER (Food Example 62 Group (g day) (g/day) efficiency ratio) ND Mean O.018 3.558 O.OOS SD 8. SS, 8. Efficacy Study of Mutamba Ethanol Extract 35 HFD R 0.12s 3.342 0.038 Combined with Magnolia Extract 29 in DIO Mice SD O.O33 O.807 O.O10 SIB Mean -0.051 3.S64 -0.014 0369 Mutamba ethanol extract 35 produced according to pSD value O.066O.OOS1 O.S3321.671 O.OO42O.018 theh examplele 35 and M,Magnolia Ii extract 29 produced accordingdi ORI Mean -OOO2 3.961 O.OOO to example 29 was blended in a ratio of 10:1. The dual com SD O.O32 2.330 O.007 p value O.OO33 O.2661 O.OOO6 bination composition was orally administrated to DIO mice G1 Mean -O.O10 2.212 -OOOS as described in the example 48 at a dosage of 1100 mg/kg SD O.O41 O.334 O.018 (G1) twice a day p value O.OO31 O.OOOO O.OO38 FER(Feed efficacy ratio) = body weight Gain (g/day). Food Intake (gday) 0370 Weight ga1n Was significantly decreased 1. the treat p value: compare to HFD by t-test ment group (G1) after the third week of treatment (Table 88) and such effect lasted until the end of the treatment. TABLE 88 Effect of Mutamba Extract Combined with Magnolia Extract on Weight Gain in DIO Mice
Weeks
Group 1 2 3 4 5 6 7
ND Mean -0.09 -0.25 O.09 O.35 O.13 O.S6 0.79 SD O.2O2 O.S13 O.418 O408 O.SOO O.S28 O.S43 p value 0.1250 0.4003 0.2140 0.1935 0.0758 0.0351 O.O243 HFD Mean -0.55 O16 1.45 1.9S 3.29 4.40 5.77 SD O891 O.665 1324 14.SS 1643 1.373 1476 SIB Mean -3.63 -537 -4.69 -4.82 -4.25 -3.90 -2.27 SD 1.504 2.317 2.2SO 2.227 1862 2.276 2.960 p value 0.00885 0.0078 0.00553 0.00358 0.00119 0.00135 0.00511 ORI Mean -3.20 -4.88 -3.34 -2.61 -2.04 -1.65 -O.O7 SD O.611 1463 1.5O2 1.859 1.640 1.526 1.437 p value 0.0011 0.0028 0.0072 0.0174 0.0081 0.0029 O.OO33