Compositions for regulating metabolism

Abstract

To provide a consumable composition including an extract containing a compound of Formula I for use in modulating metabolism and addressing the underlying pathogenesis of metabolic disorders such as nonalcoholic fatty liver disease, nonalcoholic steatohepatitis and type II diabetes mellitus.SOLUTION: The compounds herein are of use in methods of promoting and / or recovering healthy HNF4α function, mitigating the adverse effects of free fatty acids, modulating metabolism, and addressing the underlying pathogenesis of metabolic disorders such as NAFLD, nonalcoholic steatohepatitis (NASH) and T2DM.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This application claims priority to U.S. Provisional Patent Application No. 62 / 615,615, filed on 10 January 2018, the contents of which are incorporated herein by reference in their entirety. [Background technology]

[0002] The "Western diet" is associated with a global increase in metabolic disorders such as obesity, type 2 diabetes mellitus (T2DM), metabolic syndrome, non-alcoholic fatty liver disease (NAFLD), heart disease, and stroke. The interaction between genetic factors and environmental factors such as diet and lifestyle, particularly overnutrition and sedentary behavior, contributes to the progression and pathogenesis of these multifactorial diet-related diseases. Their prevalence is currently increasing dramatically. Nutrition is arguably the most important environmental factor regulating metabolic pathways and the expression of genes involved in various phenotypes associated with obesity, metabolic syndrome, and type 2 diabetes. Furthermore, the health benefits of nutrition can be regulated by gene variants.

[0003] A 70% ethyl alcohol extract of Tribulus terrestris has been suggested to provide a protective effect by inhibiting oxidative stress in a model of type 1 diabetes (i.e., streptozotocin-inducible diabetic rats) (Amin et al. (2006) Ann. NY Acad. Sci. Vol. 1084: pp. 391-401).

[0004] U.S. Patents 8,481,593 and 9,089,499 disclose para-coumaric acid derivatives, such as N-trans-feruloyltyramine, for use in topical and cosmetic compositions for the inhibition of human tyrosinase and the treatment of hyperpigmentation.

[0005] Acetone extract from the roots of Smilax aristolochiifolia is rich in N-trans-feruloyltyramine and has been suggested to be useful in preventing certain symptoms (e.g., hypertriglyceridemia, insulin resistance, blood pressure, and inflammation) in injury models associated with metabolic syndrome (Amaro et al. (2014) Molecules Vol. 19: pp. 11366-11384).

[0006] U.S. Patent Application Publication 2008 / 0132544 suggests the use of N-trans-feruloyltyramine isolated from pepper (Piper nigrum) in compositions for the treatment of visceral obesity, T2DM, insulin resistance syndrome, and metabolic syndrome. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] U.S. Patent No. 8,481,593 [Patent Document 2] U.S. Patent No. 9,089,499 [Patent Document 3] U.S. Patent Application Publication No. 2008 / 0132544 [Patent Document 4] U.S. Patent No. 6,391,651 [Patent Document 5] U.S. Patent No. 6,316,209 [Patent Document 6] U.S. Patent Application Publication No. 2004 / 0198656 [Non-patent literature]

[0008] [Non-Patent Document 1] Amin et al. (2006) Ann. NY Acad. Sci. Vol. 1084: pp. 391-401 [Non-Patent Document 2] Amaro (2014) Molecules Volume 19: 11366~84 pages [Non-licensed Document 3] King and Calhoun (2005), Phytochemistry, Volume 66 (No. 20): Pages 2468~73 [Non-licensed Document 4] Koら(2015), Internatl. J. Mol. Med. Volume 36 (No. 4): Pages 1042~8 [Non-licensed Document 5] Kiselyukら (2012) Chem Biol. Volume 19 (No. 7): Pages 806~818 [Non-licensed Document 6] Kiselyuk (2010), J. Biomol. Screen, Vol. 15 (No. 6): 663-70 [Non-licensed Document 7] Millarら, 2005, J. Lipid Res. Volume 46: Pages 2023~2028 [Non-licensed Document 8] Sanders and Griffin. 2016. Biol. Rev. Camb. Philos. Soc. 91 (No. 2): 452-468. [Non-licensed Document 9] Drelra (2006) Diabetes, Volume 55 (No. 12): Pages 3335-43 [Non-licensed Document 10] Wangら (2014) Curr. Diabetes Rev. Volume 10 (No. 2): Pages 131~145 [Non-licensed Document 11] Shiota and Printz (2012) Methods Mol. Biol. Volume 933: 103-23 [Non-licensed Document 12] Figlewiczら (1986) Peptides Volume 7: Pages 61~65 [Non-licensed Document 13] Yaswenら(1999)Nat. Med. 5 volumes: pages 1066~1070 [Non-licensed Document 14] Huszarら (1997) Cell 88 Volume: Pages 131~141 [Non-licensed Document 15] Butlerら (2000) Endocrinology 141 Volume (No. 9): 3518~21 pages [Non-licensed Document 16] Mulら (2011) Obesity (Silver Spring) Volume 20 (No. 3): Pages 612~21; [Non-licensed Document 17] Chenら (2000) Nat. Genet. Volume 26 (No. 1): Pages 97~102 [Non-licensed Document 18] Shepard (1993) J. Biol. Chem. Volume 268: Pages 22243~22246 [Non-licensed Document 19] Bruningら(2000) Science 289 Volume: Pages 2122~2125 [Non-licensed Document 20] Chatzigeorgiou, 2009, In ​​Vivo, Volume 28: 345-358 [Non-licensed Document 21] King, AJK, 2012. Br. J. Pharmacol. 166: 877-894. [Non-licensed Document 22] Savage (2009) Dis. Model Mech. 2 (Nos. 11-12): 554-62 pages [Non-licensed Document 23] Levin (1997), Am. J. Physiol. 273 (2 Pt 2): R725~30 pages [Non-licensed Document 24] Rogers and Blundell (1984) Neurosci. Biobehav. Rev. Volume 8 (No. 4): Pages 441~53 [Non-licensed Document 25] Clegg (2011) Physiol. Behav. Volume 103 (No. 1): Pages 10-6 [Non-licensed Document 26] Hariri and Thibault (2010) Nutr. Res. Rev. 23 (No. 2): 270-99 [Non-Patent Document 27] Kennedy et al. (2010) Disease Models and Mechanisms, Vol. 3 (Nos. 3-4): pp. 156-166 [Non-Patent Document 28] Leiter and Reifsnyder (2004) Diabetes Vol. 53, Supplement 1: pp. S4-11 [Non-Patent Document 29] Tschop and Heiman (2001) Exp. Clin. Endocrinol. Diabetes Vol. 109 (No. 6): pp. 307-3019 [Non-Patent Document 30] Leiter (2009) Methods Mol. Biol. Vol. 560: pp. 1-17 [Non-Patent Document 31] Hummel et al. (1972) Biochem. Genet. Vol. 7 (No. 1): pp. 1-13 [Non-Patent Document 32] Matsuoka et al. (2015) J. Biol. Chem. Vol. 290: pp. 7647-7657 [Non-Patent Document 33] Joost (2010) Results Probl. Cell Differ. Vol. 52: pp. 1-11 [Non-Patent Document 34] Lee et al. (2013) ACS Chem. Biol. Vol. 8 (No. 8): pp. 1730-1766 [Non-Patent Document 35] Porter et al. (1999) Br. J. Pharmacol. Vol. 128 (No. 1): pp. 13-20 [Non-Patent Document 36] Yin et al. (2011) Arterioscler. Thromb. Vasc. Biol. Vol. 31 (No. 2): pp. 328-336 [Non-Patent Document 37] Hayhurst et al. (2001) Mol. Cell Biol. Vol. 21 (No. 4) pp. 1393-1403 [Non-Patent Document 38] Martinez-Jimenez (2010) Mol. Cell. Biol. Volume 30 (No. 3): pp. 565-577 [Non-Patent Document 39] Gupta et al. (2005) J. Clin. Invest. Vol. 115 (No. 4) pp. 1006-1015 [Non-Patent Document 40] Kleiner et al. (2005) Hepatology Vol. 41 (No. 6): pp. 1313-1321 [Non-Patent Document 41] Brunt et al. (1999) Am. J. Gastroenterol. Vol. 94: pp. 2467-2474 [Non-Patent Document 42] Ludwig et al. (1980) Mayo Clin. Proc. Vol. 55: pp. 434-438 [Non-Patent Document 43] Neuschwander-Tetri and Caldwell (2003) Hepatology Vol. 37: pp. 1202-1219 [Non-Patent Document 44] Yeh (2004) Can. J. Cardiol. Vol. 20 (Supplement B): pp. 93-96B [Non-Patent Document 45] Geisel et al. (2003) Clin. Chem. Lab. Med. Vol. 41 (No. 11): pp. 1513-1517 [Non-Patent Document 46] Peddibhotla et al. (2013) ACS Med. Chem. Lett. Vol. 4: pp. 846-851 [Non-Patent Document 47] Inoue et al. (2002) J. Biol. Chem. Vol. 277: pp. 25257-2565 [Non-Patent Document 48] Carmiel-Haggai et al. (2005) FASEB J. Vol. 19: pp. 136-138 [Overview of the project] [Means for solving the problem]

[0009] The present invention provides a consumable composition comprising at least one carrier and an effective amount of extract containing a compound of formula I or its isomers, salts, homodimers, heterodimers, or conjugates.

[0010] [ka]

[0011] (In the formula, R 1 is present or absent, and if present, is a substituent on one or more ring atoms, independently of each ring atom, being a hydroxyl group, a halo group, a substituted or unsubstituted lower alkyl group, or a substituted or unsubstituted lower alkoxy group; The dashed lines may or may not be connected.

[0012] In some embodiments, the compound has the structure of formula II:

[0013] [ka]

[0014] (In the formula, R 2 R is present or absent, and if present, is a hydroxyl or methoxy group; 3 R is present or absent, and if present, it is a hydroxyl group; 4 (It may or may not be present, and if present, it is a hydroxyl or methoxy group.) It holds.

[0015] Preferably, the extract is an ethanol extract of a member of the genera Allium, Amoracia, Chenopodium, Fagopyrum, Annona, Piper, Eragrostis, Zea, Cannabis, Ipomea, Capsicum, Lycium, Solanum, or Tribulus. In some embodiments, the consumable composition is formulated as a dietary supplement, food ingredient or additive, medical food, nutritional supplement or pharmaceutical composition. Ideally, an effective amount of the composition would result in improvements in HNF4α activity, insulin-like growth factor levels, blood glucose levels, insulin levels, C-peptide levels, triglyceride levels, free fatty acid levels, blood uric acid levels, microalbuminuria levels, glucose transporter expression, adiponectin levels, total serum cholesterol levels, high-density lipoprotein levels, low-density lipoprotein levels, or a combination thereof. Furthermore, an effective amount would result in improvements in metabolism, liver function, fasting blood glucose levels, postprandial blood glucose levels, glycated hemoglobin (HbA1c), body weight, insulin sensitivity, serum lipid profile, or a combination thereof. [Brief explanation of the drawing]

[0016] [Figure 1] This figure shows the dose-response analysis of N-trans-caffeoyltyramine, N-trans-feruloyltyramine, and p-coumaroyltyramine in an assay to measure insulin promoter activity. Dimethyl sulfoxide (DMSO) and alberine (20 μM) were used as negative and positive controls, respectively. [Figure 2] This figure shows the effects of N-trans-caffeoyltyramine, N-trans-feruloyltyramine, and p-coumaroyltyramine on insulin mRNA levels, as determined by quantitative PCR. DMSO and alberin (20 μM) were used as negative and positive controls, respectively. [Figure 3]This figure shows the effects of N-trans-caffeioyltyramine, N-trans-feruloyltyramine, and coumaroyltyramine on HNF4α mRNA levels, as determined by quantitative PCR. DMSO and alberin (20 μM) were used as negative and positive controls, respectively. [Figure 4] This figure shows that the N-trans-caffeoyltyramine-mediated increase in insulin expression is inhibited by BI-6015, a well-known HNF4α antagonist. [Figure 5] This figure shows the effects of N-trans-caffeoyltyramine and N-trans-feruloyltyramine on estrogen-like activity. The assay was performed in the presence (1 μM) or absence (0 μM) of tamoxifen (Tam), with alberine and 7005 (CAS number 380336-90-3) (a well-known HNF4α transcription activator) as positive controls, and cis-feruloyltyramine and DMSO as negative controls. [Figure 6] This figure demonstrates that N-trans-caffeoyltyramine and N-trans-feruloyltyramine can reverse fat accumulation. T6PNE cells were pretreated for 1 day with 0.06 mM, 0.12 mM, or 0.25 mM palmitic acid, with the addition of 15 μM N-trans-caffeoyltyramine or a control (DMSO). Cells were harvested on days 3, 6, and 8 and stained with Nile Red and Oil Red O. Results are expressed as magnification changes in Nile Red staining: +palmitic acid / 10% FBS medium (without palmitic acid). [Figure 7] This figure shows that N-trans-caffeoyltyramine increases the nuclear expression of HNF4α in the liver. [Figure 8] This figure shows that the diameter of lipid droplets in the liver decreases with treatment using N-trans-caffeoyltyramine. [Figure 9]This figure shows the levels of blood samples, including alkaline phosphatase (ALP), alanine transaminase (ALT), γ-glutamyltransferase (GGT), biliary atresia, total bilirubin, albumin, blood urea nitrogen (urea), and cholesterol, in mice treated with N-trans-caffeoyltyramine or a control (DMSO). [Figure 10] This figure shows the triglyceride levels in the livers of mice fed a high-fat diet and treated with either N-trans-caffeoyltyramine or a control (DMSO). [Figure 11] This figure shows the effect of N-trans-caffeoyltyramine on HNF4α expression in the pancreas of mice fed a high-fat diet, compared to a control (DMSO). [Figure 12] This figure shows the effect of N-trans-caffeoyltyramine on HNF4α expression in the intestines of mice fed a high-fat diet, compared to a control (DMSO). [Figure 13]Tribulus terrestris seeds (1), hemp seeds and hulls (2), Annona seeds (3), soursop (Annona muricata) seeds (4), cherimoya leaves (5), corn stalks (6), goathead seeds (7), cherimoya hardwood (bark and core) (8), tomato ground pomace powder (9), potato peel (Solanum tuberosum) (yellow potato) (10), black pepper fruit (11), potato peel (purple potato) (12), potato peel (red potato) (13), tomato ground pomace (14), tomato extruded ground pomace pomace) (15), soursop (guanabana) leaves (16), garlic (Allium sativum) bulbs (17), potato (purple potato) skins (18), Anthyllis montana (A. montana) leaves (19), corn leaves (20), potato (purple potato) sprouts (21), cherimoya (cherimoya) seeds (22), leek (Allium Fistulosum (shallot) whole plant (23), potato (white potato) peel (24), cherimoya (cherimoya) green twig (25), hemp (taima) leaves (26), potato (white potato) peel (27), tomato seeds (28), tomato (beefsteak) whole fruit (29), soursop (guanabana) unripe fruit peel (30), soursop (guanabana) ripe raw fruit (31), custard apple (A.Squamosa (sweetsop) whole fruit (32), chili pepper (Capsicum annuum) (serrano pepper) fruit (33), potato (russet potato) peel (34), goji berry (Lycium barbarum) fruit (35), potato (purple potato) center (36), quinoa (Chenopodium quinoa) seeds (37), sweet potato (Ipomoea batatas) whole potato (38), sweet potato peel (39), horseradish (Armoracia rusticana) root (40), potato (Colorado potato) peel (41), buckwheat (Fagopyrum esculentum) hull (42), tree pepper (Capsicum This figure shows the amounts of N-trans-caffeoyltyramine, N-trans-feruloyltyramine, and p-coumaroyltyramine present in ethanol extracts from various sources, including frutescens (pepper) fruit (43), potato (purple potato) core (44), chili pepper (Thai chili) stem and leaves (45), soursop (guanabana) immature fruit (46), potato (yellow potato) core (47), and teff (Eragrostis tef) (teff) seeds (48) (percentage of extract, w / w). [Modes for carrying out the invention]

[0017] The present invention provides tyramine-containing hydroxycinnamides that modulate metabolism, particularly HNF4α activity, thereby mitigating the adverse effects of free fatty acids in both hepatocytes and pancreatic β-cells. The tyramine-containing hydroxycinnamides of the present invention are analogs of lead compounds identified in conventional screening assays for agents that modulate well-known signaling pathways. The tyramine-containing hydroxycinnamides exhibit dose-responsive HNF4α activity and upregulate insulin gene expression, as initially determined in T6PNE-engineered pancreatic cells. Furthermore, these compounds exhibit potent lipid-clearing activity in a hepatocyte (hepG2) lipid-loading model of fatty liver disease. While we do not wish to be constrained by theory, the tyramine-containing hydroxycinnamides of the present invention are thought to have a higher affinity for the HNF4α binding site than palmitic acid, a natural ligand that downregulates HNF4α activity, and thus regulate HNF4α activity. Genetic, functional genomics, transcriptome, and clinical evidence indicate that HNF4α agonists can improve overall metabolic health by enabling the body to maintain glucose and lipid homeostasis. Therefore, the compounds herein are useful in promoting and / or restoring healthy HNF4α function, mitigating the harmful effects of free fatty acids, regulating metabolism, and addressing the underlying pathogenesis of metabolic disorders such as NAFLD, non-alcoholic steatohepatitis (NASH), and T2DM. By using the compositions of the present invention, health and well-being are improved and promoted.

[0018] active compound The present invention provides plant-derived aromatic metabolites in which one or more acidic hydroxyl groups are attached to aromatic arenes, and the use of these in the regulation of metabolism. In one embodiment, the plant-derived aromatic metabolite is a structural analog of compound 1.

[0019] [ka]

[0020] In particular, the present invention includes tyramine-containing hydroxycinnamic acid amides having the structure of formula I, or isomers, salts, homodimers, heterodimers, or conjugates thereof.

[0021] [ka]

[0022] (In the formula, R 1 is present or absent, and if present, is a substituent on one or more ring atoms (e.g., at positions 2, 3, and / or 4), independently of each ring atom, being a hydroxyl group, a halo group, a substituted or unsubstituted lower alkyl group, or a substituted or unsubstituted lower alkoxy group; The dashed lines may or may not be connected.

[0023] With respect to the bases of this specification, the following inserted subscripts further define the bases as follows: "(C n The term "C1-C6 alkyl" defines the exact number (n) of carbon atoms in the group. For example, "C1-C6 alkyl" indicates an alkyl group having 1 to 6 carbon atoms (e.g., 1, 2, 3, 4, 5, or 6, or any range that can be derived from these (e.g., 3 to 6 carbon atoms)).

[0024] The term "lower alkyl" is intended to mean branched or unbranched saturated monovalent hydrocarbon groups containing 1 to 6 carbon atoms (i.e., C1-C6 alkyl), such as methyl, ethyl, propyl, isopropyl, tert-butyl, butyl, and n-hexyl.

[0025] Similarly, lower alkoxy groups are C1-C6 alkoxy groups having the structure -OR, where R is "alkyl" as further defined above. Specific alkoxy groups include, for example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, iso-butoxy, sec-butoxy, n-pentoxy, and 1,2-dimethylbutoxy.

[0026] The term "halo" is used herein to refer to chloro(Cl), fluoro(F), bromo(Br), and iodo(I) groups. In certain embodiments, the halo group is a fluoro group.

[0027] In any of the groups described herein, a substituent (e.g., a substituted lower alkyl group or a substituted lower alkoxy group) means that the available hydrogen is substituted with an alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, alkylaryl, heteroaralkyl, heteroarylalkenyl, heteroarylalkynyl, alkylheteroaryl, hydroxy, hydroxyalkyl, alkoxy, aryloxy, aralkoxy, alkoxyalkoxy, acyl, halo, nitro, cyano, carboxy, alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, alkylsulfonyl, arylsulfonyl, heteroarylsulfonyl, alkylthio, arylthio, heteroarylthio, aralkylthio, heteroaralkylthio, cycloalkyl, heterocyclyl, or glycosyl group.

[0028] Any undefined valency of an atom in the structure shown in this application implicitly indicates the hydrogen atom bonded to that atom.

[0029] In some embodiments, R 1 In certain embodiments, R is present and preferably represents independent substituents at the para and meta positions. 1 These are present and represent a para-position hydroxyl group and a meta-position hydroxy or lower alkoxy group. In certain embodiments, the tyramine-containing hydroxycinnamic amide having the structure of formula I is in a trans configuration.

[0030] In certain embodiments, the tyramine-containing hydroxycinnamide has the structure of formula II:

[0031] [ka]

[0032] (wherein R 2 is either present or absent, and when present, is a hydroxy or methoxy group; R 3 is either present or absent, and when present, is a hydroxy group; R 4 is either present or absent, and when present, is a hydroxy or methoxy group) has.

[0033] "Isomers" refer, inter alia, to optical isomers (e.g., essentially pure enantiomers, essentially pure diastereomers, and mixtures thereof), conformational isomers (i.e., isomers that differ only in the angle of at least one of their chemical bonds), positional isomers (especially tautomers), and geometric isomers (e.g., cis-trans isomers).

[0034] In certain embodiments, the tyramine-containing hydroxycinnamic acid amide is one of the following compounds.

[0035]

Chemical formula

[0036] Salts of the compounds disclosed herein may have the desired pharmaceutically active properties of the parent compound and may be formed from (1) inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, or phosphoric acid; or from acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo[2.2.2]-octa-2-ene-1-carboxylic acid, glucoheptonic acid, 3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid (1) refers to an acid addition salt formed from an organic acid such as lauryl sulfate, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, or muconic acid; or (2) refers to a compound containing a salt formed when an acidic proton present in the parent compound is substituted.

[0037] As is well known in the art, a homodimer is a molecule composed of two identical tyramine-containing hydroxycinnamide subunits. In contrast, a heterodimer is a molecule composed of two different tyramine-containing hydroxycinnamide subunits. Examples of homodimers of the present invention, but not limited to, include cross-linked N-trans-feruloyltyramine dimers, cross-linked N-trans-caffeoyltyramine dimers, and cross-linked p-coumaroyltyramine dimers. For example, see King and Calhoun (2005), Phytochemistry, vol. 66 (no. 20): pp. 2468-73, teaching the isolation of cross-linked N-trans-feruloyltyramine dimers from potato achene disease lesions. Conjugates of monomers of tyramine-containing hydroxycinnamide, such as lignanamides, with other compounds. Examples of conjugates include, but are not limited to, cannabisin A, cannabisin B, cannabisin C, cannabisin D, cannabisin E, and cannabisin F.

[0038] Sources of active compounds The compounds of the present invention can be obtained from any suitable plant species and / or plant raw materials known to contain the compound of formula I. Preferably, the compounds are provided as extracts containing the compound or as substantially pure compounds.

[0039] "Extract" refers to a composition containing a compound of formula I, separated from other undesirable substances present in the material of the natural source from which the extract was obtained. In some embodiments, the material of the natural source is a plant. Plant extracts can be obtained from any plant tissue, including whole plants; plant parts such as shoot vegetative organs / structures (e.g., leaves, stems and tubers), roots, flowers and floral organs / structures (e.g., bracts, sepals, petals, stamens, carpels, anthers and stalks), seeds (including embryos, endosperm and seed coats) or fruits (mature ovaries); plant tissues (e.g., vascular tissue, basal tissue, etc.); cells (e.g., guard cells, egg cells, etc.), or exudates, as well as their offspring and cultures or cell lines. Preferably, the extract contains a compound that is found to be generally recognized as safe for human consumption (GRAS). Thus, in certain embodiments, the extract is from an edible source. In this view, the extract is an edible extract.

[0040] Extracts can be prepared by freezing, grinding, macerating, grinding, fermenting, leaching, decoction, solvent extraction (e.g., partitioning) or precipitation, treatment with activated carbon, evaporation, filtration, and / or chromatographic fractionation of the material of the target source. In this view, the “extracts” of the present invention can be, but are not limited to, crude, fractionated, finely fractionated, separated, isolated, concentrated, or purified. The term “crude” means a compound or molecule that has not been completely separated from the components of the original composition in which the compound or molecule was present. In embodiments relating to fractionation or fine fractionation, molecules in the crude extract may be subjected to partial separation to provide a less crude extract containing other substances. In some embodiments, the compound is isolated. The term “isolated” means that, as with a crude extract, the compound or molecule is substantially concentrated or purified to the naturally occurring complex cellular environment in which it is found. When isolated molecules are concentrated or purified, the absolute level of purity is not necessarily important, and those skilled in the art can easily determine an appropriate level of purity depending on the intended use of the material. In some circumstances, the isolated molecules may form part of a composition (e.g., a somewhat crude extract containing numerous other substances) and may contain other components, for example. In other circumstances, the isolated molecules may be purified and, as determined, for example, by spectrophotometric analysis, NMR, or chromatography (e.g., LC-MS), to be essentially homogeneous.

[0041] Suitable solvents for preparing extracts include, for example, n-pentane, hexane, butane, chloroform, dichloromethane, diethyl ether, acetonitrile, water, butanol, isopropanol, ethanol, methanol, glacial acetic acid, acetone, norflurane (HFA134a), ethyl acetate, dimethyl sulfoxide, heptafluoropropane (HFA227), and subcritical or supercritical fluids such as liquid carbon dioxide and water, or any combination thereof in any proportion. When solvents such as those listed above are used, the resulting extracts typically contain nonspecific lipid-soluble materials. These nonspecific lipid-soluble materials can be removed by various processes, including cooling to a specified temperature, typically -20°C, followed by filtration or centrifugation to remove waxy ballast, and "dewaxing" with extraction and distillation using subcritical or supercritical carbon dioxide or a nonpolar solvent (e.g., hexane).

[0042] The concentrated extracts of the compounds of the present invention are ideally obtained by chromatographic fractionation. Chromatographic fractionation typically involves column chromatography and may be based on molecular size classification, charge, solubility, and / or polarity. Depending on the type of chromatographic method, column chromatography can be carried out using substrate materials consisting of, for example, dextran, agarose, polyacrylamide, silica, C18, C8, polyvinylpyrrolidone, polystyrene, Celite, and phenyl-hexyl, and may include solvents such as dimethyl sulfoxide, pyridine, water, dimethylformamide, methanol, physiological saline, ethylene chloride, chloroform, propanol, ethanol, isobutanol, formamide, methylene chloride, butanol, acetonitrile, isopropanol, tetrahydrofuran, dioxane, chloroform / dichloromethane, methanol, hexane, and ethyl acetate.

[0043] Typically, the product of the chromatographic step is recovered in multiple fractions and then examined for the presence of the desired compound using any suitable analytical technique (e.g., thin-layer chromatography, mass spectrometry, and ultraviolet absorption). Fractions in which the desired compound is concentrated may then be selected for further purification.

[0044] Alternatively, or in conjunction with chromatography, crystallization may be carried out to obtain high-purity tyramine-containing hydroxycinnamides. The solubility of the tyramine-containing hydroxycinnamides can be adjusted by changing the temperature and / or the composition of the solution, for example by removing ethanol, and / or adjusting the pH to promote precipitation, followed by filtering or centrifuging the precipitated crystals or oil. Other preferred methods, but not limited to, include liquid-liquid extraction, centrifugal partition chromatography, absorption onto a resin, or removal of impurities using a resin.

[0045] A “substantially pure” formulation of a compound is defined as a formulation having a chromatographic purity (of the desired compound) of greater than 95%, more preferably greater than 96%, more preferably greater than 97%, more preferably greater than 98%, more preferably greater than 99%, and most preferably greater than 99.5%, as determined by the area percentage of the HPLC profile.

[0046] The term “extract containing a compound” encompasses formulations having a chromatographic purity of at least 2%, preferably at least 5%, and more preferably at least 10% of the desired compound. Such extracts generally contain a larger proportion of impurities, non-target materials, and other molecules than “substantially pure” formulations.

[0047] In certain embodiments, the “extract containing the compound” is a product or substance of a “plant.” In this context, “plant” refers to “products containing plant materials, algae, microfungi, and combinations thereof.” The plant is defined by the method steps used to prepare the extract (e.g., by grinding, decoction, pressing, aqueous and / or ethanol extraction) and provides one or more compounds of interest in a quantified amount.

[0048] Ideally, the compounds of the present invention are extracted and / or purified from plants. Exemplary plant sources include, but are not limited to, plants in the genera, families, orders, genus, and species listed in Table 1.

[0049] [Table 1]

[0050] As an example, an extract containing N-trans-caffeoyltyramine can be obtained by crushing or finely grinding the dried fruit of Tribulus terrestris, subjecting the finely ground material to 80% ethanol at room temperature, filtering and concentrating the 80% ethanol extract, resuspending the concentrated extract in water, partitioning the aqueous solution with hexane, adding chloroform to the aqueous layer, and subjecting the chloroform layer to liquid chromatography using silica gel. See, for example, Ko et al. (2015), Internatl. J. Mol. Med. 36(4):1042-1048.

[0051] Extracts containing tyramine-containing hydroxycinnamide can be standardized using conventional techniques such as high-performance liquid chromatography (HPLC) or high-performance thin-layer chromatography (HPTLC). The term "standardized extract" refers to an extract that has been standardized by identifying characteristic components or physiologically active markers present in the extract. Characterization can be performed, for example, by analysis of spectral data such as mass spectrometry (MS), infrared (IR), ultraviolet (UV), and nuclear magnetic resonance (NMR) spectroscopy.

[0052] biological activity The biological activity of the compounds and / or extracts can be determined using one or more known biological in vitro assays, in vivo assays, and animal models, which are described in more detail below. Each of these assays provides a measure of the activity of the compounds of the present invention, which provides beneficial effects on cellular assessment items associated with metabolic disorders, including, but are not limited to, obesity, T2DM, heart disease, stroke, fatty liver disease (NAFLD), and non-alcoholic steatohepatitis (NASH).

[0053] Triglyceride assay in cultured hepatocytes. To measure triglyceride synthesis in cultured primary hepatocytes, fresh isolated hepatocytes (e.g., from rats) were cultured for 24 hours in standard medium (Dulbecco's modified Eagle medium with 0.25% bovine serum albumin) in or without monounsaturated and / or saturated fatty acids (e.g., palmitic acid (C16:0) or oleic acid (C18:1), or a 2:1 mixture of the two), and in or without the extract or compound of the present invention. Quantitative estimation of hepatic triglyceride accumulation was performed by extraction of hepatic lipids from cell homogenates using chloroform / methanol (2:1) and by enzymatic assay of triglyceride clumps using the ENZYCHROM® Triglyceride Assay Kit (Bioassay Systems, Hayward, CA).

[0054] Glucose consumption assay of adipocytes. Equivalent volume (5 x 10) 53T3-L1 adipocytes are seeded and cultured at 37°C in penicillin-streptomycin in DMEM containing normal D-glucose and 10% fetal bovine serum (FBS) under a humidified atmosphere of 95% air and 5% CO2. When the cells reach 100% confluence, the 3T3-L1 adipocytes are differentiated by treating the culture with 450 mg / dL D-glucose, 0.32 μM insulin, 0.5 mM 3-isobutyl-1-methylxanthine, and 1 μM dexamethasone for 2 days. Subsequently, the culture medium of the differentiated adipocytes is changed to DMEM containing 450 mg / dL D-glucose, with or without administration of the compound or extract of the present invention. After 24 hours, glucose consumption activity is determined by measuring the glucose concentration in the medium using insulin as a positive control. Protocols and assays for glucose uptake into cells are commercially available (e.g., ABCAM, Cambridge, MA;Promega, Madison, WI).

[0055] Insulin secretion activity. Insulin-secreting cells, such as rat RIN-m5F cells, are seeded in a 96-well plate and used in subconfluence after 24 hours of incubation. The cells are exposed to 100 μl of a quasi-toxic concentration of the compound or extract of the present invention and incubated at 37°C with 5% CO2 for 3 hours. Following treatment, the plate is centrifuged at 1000 g for 10 minutes, and the insulin concentration of the supernatant is determined using a solid-phase two-site enzyme immunoassay, such as the DRG Ultra-Sensitive Rat Insulin ELISA Kit (DRG International, Inc.).

[0056] Insulin promoter activity. T6PNE cells (Kiselyuk et al. (2012) Chem Biol. Vol. 19 (No. 7): pp. 806-818; Kiselyuk et al. (2010) J. Biomol. Screen Vol. 15 (No. 6): pp. 663-670) were seeded at a rate of 2000 cells per well in a 384-well tissue culture plate in the presence of 1 μM tamoxifen and 0.03 mM palmitic acid. After 24 hours of incubation, the compound or extract of the present invention was added to the cells. 48 hours after the addition of the compound or extract, the cells were fixed with 4% paraformaldehyde and stained with DAPI. Blue (DAPI) and green (human insulin promoter-driven GFP) channels were imaged.

[0057] Triglyceride assay in the liver. Mice are given the compound or extract of the present invention. Liver extracts are prepared by homogenizing them with 1 mmol / L EDTA in 0.25% sucrose. Lipids are extracted using chloroform / methanol (2:1 v / v) and suspended in 5% fatty acid-free bovine serum albumin. Triglyceride levels are measured using a triglyceride assay reagent (Sigma Chemical Co.).

[0058] In vivo hepatic triglyceride secretion. This assay employs the use of TRITON WR1339, which inhibits all lipoprotein lipases and therefore triglyceride clearance from blood (Millar et al., 2005, J. Lipid Res. Vol. 46: pp. 2023-2028). Mice are given the compound or extract of the present invention. Subsequently, 10% TRITON WR1339 per animal is injected intravenously (IV) into the mice, blood is collected, and triglycerides are evaluated at 0 minutes, 1 hour, and 2 hours. Plasma is separated and assayed for triglycerides. Triglyceride secretion rates are expressed as milligrams per kilogram per hour after normalization by the liver weight of the mouse.

[0059] De novo lipid synthesis assay. De novo lipid synthesis is thought to be involved in the pathogenesis of NAFLD (Sanders and Griffin. 2016. Biol. Rev. Camb. Philos. Soc. Vol. 91 (No. 2): pp. 452-468). Primary hepatocytes from animals treated with the compound or extract of the present invention are cultured overnight with 10% DMEM containing insulin (100 nM) and dexamethasone (1 μM). The cells are then incubated for 1 hour in 74 KBq / ml (2-14°C) sodium acetate (2.07 GBq / mmol). The cells are dissolved in 1N NaOH, acidified, and lipids are extracted with petroleum ether. Radioactivity is measured using a liquid scintillation counter.

[0060] Animal models of T2DM. Models of T2DM include, but are not limited to, leptin-deficient mice (ob / ob; Drel et al. (2006) Diabetes Vol. 55 (No. 12): pp. 3335-343; Wang et al. (2014) Curr. Diabetes Rev. Vol. 10 (No. 2): pp. 131-145), leptin receptor-deficient mice (db / db; Wang et al. (2014) Curr. Diabetes Rev. Vol. 10 (No. 2): pp. 131-145), obese Zucker rats (fa / fa; Shiota and Printz (2012) Methods Mol. Biol. Vol. 933: pp. 103-123), Wistar Kyoto rats (fa / fa; Figlewicz et al. (1986) Peptides Vol. 7: pp. 61-65), and proopiomelanocortin-deficient mice (POMC). - / -Yaswen et al. (1999) Nat. Med. Vol. 5: pp. 1066-1070), melanocortin 3 and 4 receptor knockout animals (Huszar et al. (1997) Cell Vol. 88: pp. 131-141; Butler et al. (2000) Endocrinology Vol. 141 (No. 9): pp. 3518-3521; Mul et al. (2011) Obesity (Silver Spring) Vol. 20 (No. 3): pp. 612-621; Chen et al. (2000) Nat. Genet. Vol. 26 (No. 1): pp. 97-102), animals overexpressing glucose transporter subtype 4 (Shepard et al. (1993) J. Biol. Chem. Examples include the 268th volume: pp. 22243-22246 and neuron-specific insulin receptor knockout mice (NIRKO mice; Bruning et al. (2000) Science 289th volume: pp. 2122-2125). Overviews of the use of such animal models are available (e.g., Chatzigeorgiou et al., 2009 In Vivo 28th volume: pp. 345-358; King, AJK 2012. Br. J. Pharmacol. 166th volume: pp. 877-894). These models are characterized by insulin resistance, hyperglycemia and hyperinsulinemia, symptoms similar to human T2DM. Animals are administered the compounds or extracts of the present invention, and the maximum tolerated dose and metabolic improvement are evaluated.

[0061] An animal model of lipodystrophy. Complete loss of adipose tissue (lipodystrophy) leads to metabolic changes similar to those in severe obesity and is associated with insulin resistance. Genetically modified mice lacking adipose tissue are characterized by bulimia, fatty liver, hypertriglyceridemia, insulin resistance, and T2DM (Savage (2009) Dis. Model Mech. Vol. 2 (Nos. 11-12): pp. 554-562). Due to the loss of functional adipose tissue, these mice are leptin-deficient and are useful for evaluating the effects of the compounds or extracts of the present invention on dysregulated metabolism. Such models are useful for demonstrating in vivo responses to the compounds of the present invention and for exploring key concepts such as dose-response.

[0062] A rat model of diet-induced obesity. Uninbred Sprague-Dawley rats are used as a polygeneic model of obesity (Levin et al. (1997) Am. J. Physiol. Vol. 273: R725-30). Similarly, rats fed a variety of palatable diets mimicking so-called human Western diets (cafeteria food) develop obesity due to bulimia (Rogers and Blundell (1984) Neurosci. Biobehav. Rev. Vol. 8 (No. 4): pp. 441-53). Likewise, animals exposed to a high-fat (HF) diet develop obesity and show decreased insulin and leptin sensitivity (Clegg et al. (2011) Physiol. Behav. Vol. 103 (No. 1): pp. 10-16; Hariri and Thibault (2010) Nutr. Res. Rev. Vol. 23 (No. 2): pp. 270-279). Such models are useful for demonstrating the in vivo reaction of the compounds of the present invention and for exploring key concepts such as dose-response relationships.

[0063] A mouse model of diet-induced obesity. Diet-induced obesity (DIO) mice are the standard for testing lipotoxicity in vivo (Kennedy et al. (2010) Disease Models and Mechanisms Vol. 3 (Nos. 3-4): pp. 156-166). Mice fed a high-fat diet develop abnormalities in both the liver and pancreas. Depending on the genetic background, mice develop insulin resistance with or without β-cell atrophy and complete diabetes when on a high-fat diet (Leiter and Reifsnyder (2004) Diabetes Vol. 53 Supplement 1: pp. S4-11; Tschop and Heiman (2001) Exp. Clin. Endocrinol. Diabetes Vol. 109 (No. 6): pp. 307-19). Mouse strains with different tendencies to express β-cell atrophy include NONcNZOl0 / LtJ (The Jackson Laboratory, Bar Harbor, ME), which expresses β-cell atrophy, and C57BL / 6J (The Jackson Laboratory, Bar Harbor, ME), which does not show β-cell loss. These models can be used to analyze the effects of a normal diet versus a high-fat diet ± test compounds. Approximately half of NONcNZOl0 / LtJ males develop diabetes and frequently develop islet atrophy when given a high-fat diet (Leiter (2009) Methods Mol. Biol. Vol. 560: pp. 1-17). Another strain that can be tested is the C57B1 / 6 background DIO mouse, which does not have a high tendency to express β-cell loss but is a good model of pre-T2D and obesity with elevated blood glucose and impaired glucose tolerance (Leiter (2009) Methods Mol. Biol. Vol. 560: pp. 1-17). C57Bl / 6KsJ db / db mice develop diabetes associated with β-cell decline (Hummel et al. (1972) Biochem. Genet. Vol. 7 (No. 1): pp. 1-13), and this has been shown to be correctable by MafA overexpression (Matsuoka et al. (2015) J. Biol. Chem. Vol. 290: pp. 7647-7657), suggesting the use of these in efficacy studies.Such models are useful for demonstrating the in vivo reaction of the compounds of the present invention and for exploring key concepts such as dose-response relationships.

[0064] An animal model of metabolic syndrome. New Zealand obese (NZO) mice are obese and have severe T2DM. Several gene-sensitive sites favorable to the development of steatosis and hyperglycemia have been identified in NZO mice. In addition to leptin receptor genes, several transcription factor genes have been identified as potential candidate genes, and some orthologues of these genes have been associated with human metabolic syndrome (Joost (2010) Results Probl. Cell Differ. Vol. 52: pp. 1-11). Such models are useful for demonstrating in vivo responses to the compounds of the present invention and for exploring key concepts such as dose-response.

[0065] Counterscreening. Counterscreening is often used to select compounds from a library to avoid off-target effects. In this invention, the activity of a compound as a modulator of HNF4α activity is a desirable target, even if other off-target effects may occur. Drugs that are traded for human use based on target effects other than HNF4α have subsequently been shown to have activity as HNF4α activators (alberine and benfluorex; Lee et al. (2013) ACS Chem. Biol. Vol. 8 (No. 8): pp. 1730-1736). Alberine is traded as a smooth muscle relaxant for gastrointestinal disorders, while benfluorex is traded as an appetite suppressant. It is well known that benfluorex is metabolized by cleaving its ester moiety to fenfluramine, a potent agonist of the serotonin 5-hydroxytryptamine 2 (5-HT2) receptor, and its effects were thought to be related to the activity of benfluorex as an appetite suppressant (Porter et al. (1999) Br. J. Pharmacol. Vol. 128 (No. 1): pp. 13-20). However, the modulation of the 5-HT2 receptor by benfluorex was associated with undesirable cardiopulmonary side effects. Therefore, based on these experiments using synthetic compounds, the compound or extract of the present invention was used, for example, in human 5-HT2 receptors in CHO-K1 cells. 2A , 5-HT 2B or 5-HT 2c Examine for off-target effects on 5-hydroxytryptamine receptor activation using a fluorescence imaging plate reader (FLIPR) assay, which enables rapid detection of elevated intracellular calcium levels in receptor-expressing cells. See, for example, Porter et al. (1999) Br. J. Pharmacol. 128(1):13-20. Other counterscreenings may be selected based on the first in vivo trial in which toxic effects can be associated with other off-target effects.

[0066] formulation Substantially pure compounds or extracts containing the compounds of the present invention can be provided in combination with a carrier in any preferred form for consumption by or administration to a subject. In this view, the compounds or extracts are added to consumables as exogenous components or additives. Preferred consumable forms include, but are not limited to, dietary supplements, food components or additives, medical foods, nutritional supplements or pharmaceutical compositions.

[0067] Food ingredients or additives are edible substances intended to directly or indirectly become ingredients or otherwise affect the characteristics of any food (including any substances intended to manufacture, produce, package, process, prepare, treat, pack, transport or hold food). Food products, in particular functional foods, are foods that are enhanced or concentrated during processing and contain additional complementary nutrients and / or beneficial components. Food products according to the present invention may be, for example, in the form of butter, margarine, sweet or savory spreads, condiments, biscuits, health bars, bread, cakes, cereals, candies, confectionery, soups, milk, yogurt or fermented dairy products, cheeses, juice-based and vegetable-based beverages, fermented beverages, shakes, flavored waters, teas, oils, or any other suitable food form.

[0068] Dietary supplements are products taken orally that contain the compounds or extracts of the present invention and are intended to supplement a diet. Nutritional fortifications are products derived from food sources that provide additional health benefits in addition to the basic nutritional value found in food. Pharmaceutical compositions are defined as any component of a drug product that is intended to produce pharmaceutical activity or other direct effects in the diagnosis, cure, alleviation, treatment or prevention of disease, or to affect the structure or any function of the body of a human or other animal. Dietary supplements, nutritional fortifications and pharmaceutical compositions can be found in a number of forms, such as tablets, coated tablets, pills, capsules, pellets, granules, soft gels, gel caps, liquids, powders, emulsions, suspensions, elixirs, syrups and any other forms suitable for use.

[0069] As used herein, the term "carrier" means a material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, magnesium talc, calcium stearate or zinc stearate, or stearic acid), or a solvent-encapsulating material involved in the transport or delivery of the compound of interest from one organ or part of the body to another organ or part of the body. Each carrier should be compatible with the other components of the formulation and harmless to the subject. Some examples of materials that can act as carriers include: (1) sugars such as lactose, glucose, and sucrose; (2) starches such as corn starch and potato starch; (3) cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, cellulose acetate, and hydroxypropylmethylcellulose; (4) tragacanth powder; (5) malt; (6) gelatin; (7) talc; (8) excipients such as cocoa butter and wax; (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols such as propylene glycol; (11) polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water. Examples include water; (17) isotonic saline solution; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffer solution; (21) polyester, polycarbonate and / or polyanhydrous; and (22) other non-toxic suitable substances used in conventional formulations.

[0070] In certain embodiments of the present invention, the consumable composition includes a compound or extract, a carrier, and a preservative for reducing or inhibiting microbial growth. The preservative is added in an amount of up to about 5% by mass of the film, preferably about 0.01% to 1% by mass. Preferred preservatives include sodium benzoate, methylparaben, propylparaben, sodium nitrite, sulfur dioxide, sodium sorbate, and potassium sorbate. Other suitable preservatives include, but are not limited to, edetates (such as disodium EDTA, also known as salts of ethylenediaminetetraacetic acid, i.e., EDTA).

[0071] To prepare a solid composition such as a tablet or capsule, the compound or extract is mixed with a carrier (e.g., conventional tableting components such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate, or gum) and other diluents (e.g., water) to form a solid composition. This solid composition is then subdivided into unit dosage forms containing an effective amount of the compound of the present invention. The tablets or pills containing the compound or extract can be coated, or otherwise formulated to provide a dosage form that offers the advantage of sustained action due to sustained release of the active compound from the solid matrix and / or potentially enhanced absorption.

[0072] Liquid forms into which the compounds or extracts of the present invention are incorporated for oral or parenteral administration include aqueous solutions, preferably flavor syrups, aqueous or oily suspensions, and flavor emulsions with edible oils and elixirs and similar vehicles. Suitable dispersants or suspending agents for aqueous suspensions include synthetic natural gums such as tragacanth, acacia, alginates, dextran, sodium carboxymethylcellulose, methylcellulose, polyvinylpyrrolidone, or gelatin. Liquid formulations for oral administration may take the form of solutions, syrups, or suspensions, or they may exist as dried products for reconstitution in water or other suitable vehicles before use. Such liquid formulations may be prepared by conventional means with a suspending agent (e.g., sorbitol syrup, methylcellulose, or hydrogenated edible fat), an emulsifier (e.g., lecithin or acacia); a non-aqueous vehicle (e.g., almond oil, oily ester, or ethyl alcohol); a preservative (e.g., methyl or propyl p-hydroxybenzoate or sorbic acid); and acceptable additives such as artificial or natural colorants and / or sweeteners.

[0073] A method for preparing a formulation or composition of the present invention includes the step of associating a compound or extract of the present invention with a carrier, optionally one or more appendages and / or an active ingredient. Generally, a formulation is prepared by homogeneously and essentially associating a compound or extract of the present invention with a liquid carrier or a finely divided solid carrier, or both, and then, if necessary, forming the product. Thus, a disclosed formulation consists of, or essentially consists of, a compound or extract described herein in combination with a suitable carrier.

[0074] When the compounds or extracts of the present invention are administered to humans and animals as pharmaceuticals, nutritional supplements, or dietary supplements, they can be given on their own or as a composition containing 0.1 to 99% (more preferably 10 to 30%) of the active ingredient in combination with, for example, an acceptable carrier.

[0075] Consumable products are consumed by the target and can provide less than 100 mg of the compounds described herein per day. In certain embodiments, the consumable provides tyramine-containing hydroxycinnamide between 10 and 60 mg / day. The effective dose can be established by methods well known in the art and depends on bioavailability, toxicity, etc.

[0076] While it is considered that individual tyramine-containing hydroxycinnamides may be used in the consumables of the present invention, it is further considered that two or more compounds or extracts may be combined in any relative amounts to provide conventional combinations of components containing two or more tyramine-containing hydroxycinnamides in a desired ratio, thereby enhancing the efficacy of the product and improving measures of sensory properties or some other qualities that are important for the end use of the product.

[0077] molecular target HNF4α (hepatocyte nuclear factor 4α) is a comprehensive nuclear transcription factor that regulates the expression of numerous genes involved in maintaining stable metabolism (homeostasis). In particular, HNF4α is expressed in both the liver (hepatocytes) and the pancreas (β-cells). HNF4α expression and transcriptional activity are reduced in human hepatocytes and human pancreatic β-cells in NAFLD and T2DM. HNF4α is the autosomal dominant monogenic form of diabetes and mutates in MODY1, providing human genetic evidence for its direct role in the pathogenesis of diabetes. HNF4α gene expression is downregulated in T2D. In addition, free fatty acids are elevated in overweight and obese individuals and inhibit HNF4α activity. Given that HNF4α haploinsufficiency causes diabetes and HNF4α is downregulated in T2D, restoring or increasing HNF4α activity to its normal wild-type state would bring overall health and therapeutic benefits.

[0078] HNF4α knockout rodent models exhibit a fatty liver phenotype, as well as decreased lipid synthesis, reduced de-novo cholesterol synthesis, decreased very low-density lipoprotein (VLDL) secretion and high-density lipoprotein (HDL) biosynthesis, and increased insulin intolerance. In addition, knockout mice show enhanced FFA uptake and reduced degradation via β-oxidation. This results in hypocholesterolemia, low serum triglyceride levels, and fatty liver. All of this indicates a significant dysregulation of lipid metabolism due to HNF4α deficiency (Yin et al. (2011) Arterioscler. Thromb. Vasc. Biol. Vol. 31 (No. 2): pp. 328-336; Hayhurst et al. (2001) Mol. Cell Biol. Vol. 21 (No. 4): pp. 1393-1403; Martinez-Jimenez (2010) Mol. Cell. Biol. Vol. 30 (No. 3): pp. 565-577). In comparison, increased HNF4α expression in the liver can increase the transcription of genes that promote hepatic FFA oxidation, ketone body production, and very low-density lipoprotein (VLDL) secretion as a means of dealing with excessive FFA accumulation (Martinez-Jimenez (2010) Mol. Cell. Biol. Vol. 30 (No. 3): pp. 565-577). Therefore, HNF4α provides a target for mitigating the adverse effects of FFA, which are characteristically elevated in NAFLD.

[0079] In T2DM, HNF4α plays a direct role in regulating genes involved in glucose transport and glycolysis. Without HNF4α in β-cells, rodents exhibit incomplete glucose-stimulated insulin secretion in β-cells, which means reduced insulin secretion (Gupta et al. (2005) J. Clin. Invest. Vol. 115 (No. 4): pp. 1006-15). HNF4α gene expression has been observed to be downregulated in individuals with T2DM, likely due to chronically elevated exposure to FFA. In particular, free palmitic acid (C16-saturated FA) has been shown to impair the function and viability of pancreatic β-cells and suppress normal insulin production caused by the action of HNF4α (Lee et al. (2013) ACS Chem. Biol. Vol. 8 (No. 8): pp. 1730-1736). Therefore, HNF4α provides a target for restoring the symptoms of T2DM.

[0080] Metabolic disorders The term “metabolic disorder” refers to a disorder or condition that occurs when the body is unable to properly metabolize carbohydrates, lipids, proteins, and / or nucleic acids. Therefore, in the context of this invention, disorders relating to metabolic abnormalities are encompassed by the term “metabolic disorder.” The term “metabolic disorder” includes, but is not limited to, insulin resistance, hyperglycemia, diabetes mellitus (especially T2DM), obesity, glucose intolerance, hypercholesterolemia, hyperlipoproteinemia, dyslipidemia, hyperinsulinemia, atherosclerotic disease, coronary artery disease, metabolic syndrome, hypertension, or disorders relating to glucose levels such as abnormal plasma lipoproteins, triglycerides, or pancreatic beta cell regeneration.

[0081] T2DM refers to a chronic disease or condition that occurs when the pancreas does not produce enough insulin, or when the body cannot effectively utilize the insulin produced by the pancreas. T2DM results in increased blood glucose levels (hyperglycemia). Based on studies that have established the relationship between plasma glucose concentration, measures of blood glucose exposure, and the risk of diabetic retinopathy, the following criteria are adopted for the diagnosis of diabetes: fasting blood glucose greater than or equal to 126 mg / dL (7.0 mmol / L); blood glucose greater than or equal to 200 mg / dL (11.1 mmol / L) two hours after ingestion of 75 g anhydrous glucose in an oral glucose tolerance test; or random blood glucose greater than 200 mg / dL (11.1 mmol / L) in a person with symptoms of diabetes. Other important definitions include impaired glucose tolerance, where, in an oral glucose tolerance test, blood glucose is equal to or greater than 140 mg / dL (7.8 mmol / L) but less than 200 mg / dL (11.1 mmol / L) after 2 hours; and impaired fasting glucose, where fasting blood glucose (FPG) is equal to or greater than 100 mg / dL (5.6 mmol / L) but less than 126 mg / dL. The compounds or extracts of the present invention are said to modulate metabolism by reducing one or more of the following levels to lower than those referenced herein: fasting blood glucose, blood glucose after 75 g anhydrous glucose ingestion, or random blood glucose levels. Another endpoint that can be monitored as part of the assessment of metabolic activity is the blood level of HbA1c, which is a measure of the mean blood glucose level over the past two to three months. HbA1c levels are used as a clinical indicator of the risk of diabetes, and increasing levels indicate an increased risk of T2DM. Therefore, a decrease in HbA1c can also be used to support an indicator of blood glucose control.

[0082] Obesity is a chronic, recurrent health risk defined by excess body fat. Body fat mass can be accurately measured using hydrodensitometry and dual-energy X-ray absorptiometry (DEXA). Body fat mass is calculated by dividing the weight in kilograms (kg / m²) by the squared height in meters. 2Body Mass Index (BMI), expressed as 30 kg / m², is simple, inexpensive to calculate, and correlates strongly with body fat mass in non-elderly individuals, and is therefore commonly used as a substitute for body fat mass. Obesity is defined by the National Institutes of Health as 30 kg / m². 2 This is defined as having a BMI of 18.5 kg / m² or higher. The relationship between BMI and mortality risk and major complications varies with age, sex, race, and smoking history, but generally, a BMI of 18.5 kg / m² is considered to be a significant factor. 2 From 24.9 kg / m 2 The lowest among the individuals, with a BMI of 25 kg / m² 2 From approximately 40 kg / m 2 The increase is curvilinear or linear. The compounds or extracts of the present invention are said to modulate metabolism by reducing mean and / or absolute body weight. Mean body weight is defined as the difference in the mean percentage reduction in baseline body weight between the group treated with the active product and the group treated with placebo. Absolute body weight is defined as the proportion of subjects in the group treated with the active product and the group treated with placebo who experienced a reduction of at least 5 percent of their baseline body weight. Secondary efficacy endpoints include, but are not limited to, blood pressure and pulse rate, lipoprotein lipids, fasting glucose and insulin, HbA1c (in T2DM), waist circumference, and improvement in quality of life.

[0083] NAFLD, or "fatty liver," is a metabolic disorder characterized by excessive accumulation of fat in the liver. NAFLD is primarily characterized by macrodriptery, and the presence of visible stipulation in >5% of hepatocytes is generally accepted as a practical definition of fatty liver (Kleiner et al. (2005) Hepatology Vol. 41: pp. 1313-1321). Non-alcoholic steatohepatitis, or NASH, is the most extreme form of NAFLD and is considered a major cause of cirrhosis of unknown etiology. Minimum diagnostic criteria for NASH include the presence of macrodriptery, inflammation, and swelling in >5% of hepatocytes, typically distributed mainly in the centrilobular region (acinar region 3) in adults. Steatohepatitis is not simply a condition involving inflammation and fatty degeneration, but rather a specific histological condition (Kleiner et al. (2005) Hepatology Vol. 41 (No. 6): pp. 1313-1321; Brunt et al. (1999) Am. J. Gastroenterol. Vol. 94: pp. 2467-2474; Ludwig et al. (1980) Mayo Clin. Proc. Vol. 55: pp. 434-438; Neuschwander-Tetri and Caldwell (2003) Hepatology Vol. 37: pp. 1202-1219). The compounds or extracts of the present invention are said to regulate metabolism by reducing the accumulation of fat in the liver to a measurable extent, thereby improving liver function.

[0084] The term metabolic syndrome refers to a group of tests and clinical findings that serve as markers for an increased risk of coronary heart disease, stroke, peripheral vascular disease, and / or T2DM. Risk factors associated with metabolic syndrome include, but are not limited to, abdominal obesity (i.e., excess adipose tissue in or around the abdomen), high triglycerides, low HDL cholesterol and high LDL cholesterol, blood pressure, atherosclerosis including elevated insulin resistance or glucose intolerance, prothrombotic conditions (e.g., high fibrinogen or plasminogen activator inhibitor-1 in the blood), and / or inflammatory conditions (e.g., elevated C-reactive protein in the blood). The compounds or extracts of the present invention are said to modulate metabolism by improving components of metabolic syndrome, ultimately leading to the prevention of the development of T2DM and a reduction in cardiovascular morbidity and mortality.

[0085] metabolic regulation The present invention also provides methods for regulating metabolism to alleviate, prevent, or treat metabolic disorders. According to such methods, an effective amount of the compound or extract of the present invention is administered to a subject in need of treatment to regulate the subject's metabolism, thereby addressing the underlying pathogenesis of one or more metabolic disorders and promoting the subject's health, well-being, and quality of life. As used herein, the term “subject” refers to an animal, preferably a mammal. In some embodiments, the subject is a pack animal, companion animal, livestock, laboratory animal, or zoological animal. In other embodiments, the subject is a human.

[0086] Subjects requiring treatment include those with observable symptoms of metabolic disorders (e.g., those with abnormal glucose or lipid metabolism), as well as those who do not have observable symptoms of metabolic disorders but are determined to be susceptible to developing metabolic disorders (i.e., those at risk of developing metabolic disorders). For example, according to the American Heart Association, metabolic syndrome (which increases the risk of heart disease, diabetes, stroke, and other health problems) is diagnosed when any three of the following five risk factors are present: high blood glucose; low levels of HDL ("good") cholesterol; high levels of triglycerides; large waist circumference or "apple-shaped" body; or high blood pressure.

[0087] Further examples include autoantibodies against insulin (IAAs); glutamate decarboxylase (GAD); and islet cell members of the tyrosine phosphate family known as IA-2, which have been identified as markers preceding the clinical development of T2DM. See, for example, U.S. Patents 6,391,651 and 6,316,209. Similarly, C-reactive protein (CRP), apolipoprotein CIII, and plasma homocysteine ​​levels have been identified as markers for identifying individuals at risk for high cholesterol (or hypercholesterolemia or hyperlipidemia). See, for example, U.S. Patent Application Publication 2004 / 0198656; Yeh (2004) Can. J. Cardiol. Vol. 20 (Supplement B): pp. 93-96B; and Geisel et al. (2003) Clin. Chem. Lab. Med. Vol. 41 (No. 11): pp. 1513-1517. Further factors that can be used alone or in combination to determine whether a subject is at risk of or prone to developing hypercholesterolemia include, but are not limited to, genetics (i.e., familial hypercholesterolemia), hypertension, smoking history, alcohol consumption, diabetes, obesity, physical inactivity, age and sex (i.e., postmenopausal women over 50 years of age), and stress.

[0088] As used herein, the term “effective dose” means the amount of a compound, extract, or formulation containing the compound or extract that is sufficient to significantly improve a disorder. Another important consideration when determining the effective dose to be used in humans is weighing the desired effect (benefit) against the risks associated with the use of the compound. The types of adverse effects observed and their likelihood are relevant to such a risk / benefit assessment. The fact that the effective dose may vary with the specific disorder being treated, e.g., diabetes or obesity, the end consumer’s age and health condition, the severity of the condition, the duration of treatment, the specific carrier used, and similar factors should also be considered.

[0089] Generally, a preferred daily dose of the compound or extract of the present invention is the lowest dose of the compound or extract that is effective in producing the desired benefit, in which case the effect is to improve fat and sugar metabolism, thereby supporting overall health and well-being. Such an effective dose generally depends on the factors described herein. For oral administration, the dose may be in the range of about 0.0001 mg to about 10 g per kilogram of body weight per day, about 5 mg to about 5 g per kilogram of body weight per day, about 10 mg to about 2 g per kilogram of body weight per day, or any other preferred dose. If desired, the effective daily dose of the compound or extract may be administered as two, three, four, five, six or more sub-doses, optionally in unit dosage forms, administered separately at appropriate intervals throughout the day. The preferred dosing is once per day.

[0090] The compounds or extracts of the present invention can be used alone or in combination with a specific diet (e.g., a low-glycemic index food) or standard treatment.

[0091] Administration of the compounds or extracts of the present invention modulates the metabolism of the subject, thereby addressing the underlying pathogenesis of one or more metabolic disorders and / or promoting the subject's health, well-being, and quality of life. Ideally, an effective amount of the compound or extract results in a measurable improvement in the level or activity of one or more metabolites. Examples include HNF4α activity, insulin-like growth factor levels (such as insulin-like growth factor 1 or IGF-1), blood glucose levels, insulin levels, C-peptide levels, triglyceride levels, free fatty acid levels, blood uric acid levels, microalbuminuria levels, glucose transporter expression, adiponectin levels, total serum cholesterol levels, high-density lipoprotein (HDL) levels, and / or low-density lipoprotein (LDL) levels.

[0092] More specifically, administration of the compounds or extracts of the present invention improves the serum lipid profile by improving metabolism, liver function, fasting blood glucose levels, postprandial blood glucose levels, glycated hemoglobin HbA1c, body weight, insulin sensitivity, lipid clearance, or a combination thereof. In certain embodiments, the use of the compounds or extracts of the present invention is preferably to prevent, slow, delay, or treat metabolic disorders such as T2DM, impaired glucose tolerance, impaired fasting blood glucose, hyperglycemia, postprandial hyperglycemia, hyperinsulinemia, NASH, NAFLD, or metabolic syndrome; to slow, delay, or treat the progression of prediabetes; to improve glycemic control and / or reduce fasting blood glucose, postprandial blood glucose, and / or glycated hemoglobin HbA1c; to prevent, slow, delay, or reverse the progression of impaired glucose tolerance, impaired fasting blood glucose, insulin resistance, or metabolic syndrome to T2DM; and to treat cataracts, or renal impairment, retinal impairment, neuropathy, tissue ischemia, diabetic foot lesions, dyslipidemia, arteriosclerosis, myocardial infarction, acute coronary syndrome, unstable angina, stable angina, stroke, and peripheral dysregulation. To prevent, slow, delay, prevent or treat complications of diabetes such as arterial occlusive disease, cardiomyopathy, heart failure, cardiac rhythm disorders, or microvascular or macrovascular diseases such as restenosis; to reduce body weight and / or body fat, or prevent or promote the increase of body weight and / or body fat; to prevent, slow, delay or treat diseases or conditions resulting from ectopic fat, especially abnormal accumulation of hepatic fat; to maintain and / or improve insulin sensitivity and / or treat or prevent hyperinsulinemia and / or insulin resistance; to reduce fat deposition; to prevent, slow, delay or reverse the progression of fatty liver to NASH; and / or to prevent, slow, delay or reverse the progression of NASH to cirrhosis, end-stage liver disease and / or hepatocellular carcinoma.

[0093] The following non-limiting embodiments are provided to further illustrate the present invention. [Examples]

[0094] Evaluation Indicators for Metabolic Activity: Materials and Methods Insulin and HNF4α expression. RNA was purified using the RNEASY® chromatographic separation and isolation kit (Qiagen) and converted to cDNA using qScript® cDNA SuperMix (Quanta Biosciences). Q-PCR was performed using the Opticon Real-Time System (MJ Research) and QPCR SuperMix (BioPioneer) with cDNA corresponding to 2 μg of RNA. Please note that all mRNA values ​​are normalized to 18S rRNA values ​​and are shown as multiplier changes exceeding vehicle-treated controls.

[0095] Counterscreening for estrogen-like activity. Estrogen-like activity was monitored by co-transduction of wild-type E47 or E47MER with a reporter plasmid containing a multimerized minimal promoter E-box 5' condensed with the firefly luciferase gene (4RTK-luc) (Kiselyuk et al. (2010) J. Biomol. Screen vol. 15 (no. 6): pp. 663-670). HeLa cells were transfused in 50 μl serum-free Dulbecco's modified Eagle medium per well using polyethyleneimine, 0.2 μg 4RTK-Luc plasmid, and either 0.3 μg human E47, E47MER, or pMSCVhph vector. For control of the effectiveness of the transduction, Renilla luciferase (pRL-TK) plasmid was included. The gene transfer conditions were as described in the PPRE-Luc reporter assay by Kiselyuk et al. (2010, J. Biomol. Screen, Vol. 15 (No. 6): pp. 663-670). Sixteen hours after gene transfer, the culture medium was changed and maintained for 48 hours with tamoxifen and / or the compound or vehicle (DMSO). Cells were then lysed and luciferase activity was assayed using the Promega DUAL-LUCIFERASE® reporter assay kit (Promega Corp., Madison, WI), and fluorescence was measured using a Veritas® microplate luminometer (Turner Biosystems, Sunnyvale, CA). Data were normalized to sea urchin luciferase (pRL-TK) and shown as a magnification change exceeding that of the vehicle alone.

[0096] Inhibition of HNF4αGFP expression. Using the insulin promoter assay described herein, the activity of HNF4α was evaluated in the presence of BI-6015 (0, 2.5, 5 μM) (Kiselyuk et al. (2012) Chem. Biol. Vol. 19 (No. 7): pp. 806-818), a well-known antagonist of HNF4α, in combination with N-trans-caffeoyltyramine (0, 5, 10, 20 μM).

[0097] [ka]

[0098] Liver microsome assay. A liver microsome stability assay was performed according to a well-known method (Peddibhotla et al. (2013) ACS Med. Chem. Lett. Vol. 4: pp. 846-851). Briefly, 3 μL of a 25 μM compound solution in acetonitrile was incubated with 123 μL of mouse, human, or rat liver microsomes (Xenotech, Kansas City, KS). After pre-incubation at 37°C for 10 minutes, 120 μL of an NADPH generating system (2 mM NADP) was added. +The enzymatic reaction was initiated by adding 10 mM glucose-6-phosphate, 0.4 U / ml glucose-6-phosphate dehydrogenase, and 5 mM MgCl2 in the presence of 100 mM potassium phosphate buffer (pH 7.4). The final concentration of each compound used was 1 μM. The microsomal concentration used was 1.0 mg / mL. The compounds were incubated in microsomes for 0, 5, 15, 30, and 60 minutes. The reaction was stopped by adding ice-cold ACN, and the reaction mixture was centrifuged at 10,000 g for 10 minutes, after which the supernatant was removed for analysis. A 10 μL portion of the resulting extract was injected into a Thermo HPLC system equipped with a PAL CTC plate sampler (96-well plate), a Dionex Ultimate 3000 binary pump (flow rate 0.600 mL / min), a Dionex Ultimate 3000 thermostat column compartment (temperature 40°C), and a Thermo Endura mass spectrometer (ESI source), using a Thermo Scientific Accucore C18 (2.6 μM, 2.1 x 50 mm) column. The gradient was started with 95% H2O (0.1% formic acid) and 5% ACN (0.1% formic acid) for the first 0.5 minutes, then with gradient conditions of 5–100% ACN (0.1% formic acid) for 0.5 to 3.5 minutes, followed by 95% H2O (0.1% formic acid) and 5% ACN (0.1% formic acid) for 0.5 minutes, and then another 1 minute until re-equilibrium at 95:5.

[0099] Lipid clearance (fatty denaturation assay) in HepG2 and T6PNE cells. The fatty denaturation assay was performed as described, with the exception of drug concentrations of 20 μM N-trans-caffeoyltyramine with 0.25 mM palmitic acid in the HepG2 cell line, and 10 μM N-trans-caffeoyltyramine, N-trans-caffeoyltyramine, or p-coumaroyltyramine with 0.25 mM palmitic acid in the T6PNE cell line (Kiselyuk et al. (2012) Chem. Biol. Vol. 19 (No. 7): pp. 806-818). Fatty denaturation was evaluated using the Oil Red O Method for Fats kit (Poly Scientific; Warrington, PA) according to the manufacturer's guidelines. In short, frozen tissue slides or fixed cells were incubated in solvent-free propylene glycol for 2 minutes, then in Oil Red O solution for 15 hours for slides or 1 hour for fixed cells, differentiated in 85% propylene glycol solution for 1 minute, washed twice with distilled water, and stained in hematoxylin for 10 seconds. The slides were then mounted in glycerin jelly mounting medium.

[0100] Alkaline phosphatase (ALP) quantification. Elevated serum ALP levels are thought to indicate liver dysfunction. Therefore, ALP was assayed according to a well-known method (Kiselyuk et al. (2012), Chem. Biol. Vol. 19 (No. 7): pp. 806-818). Briefly, blood was collected before slaughter and analyzed using a VetScan blood analyzer to measure alkaline phosphatase (ALP, IU / L), alanine aminotransferase (ALT, IU / L), gamma-glutamyltransferase (GGT, IU / L), bile acid (BA, μmol / L), total bilirubin (TBIL, mg / dL), albumin (ALB, g / dL), serum urea nitrogen (BUN, mg / dL), and cholesterol (CHOL, mg / dL).

[0101] Triglyceride (TG) quantification. TG levels were assayed using a triglyceride colorimetric assay kit (Cayman Chemicals; Ann Arbor, MI) according to the manufacturer's instructions.

[0102] Lipid droplet size analysis. All slides were scanned at 20x magnification using the Aperio Scanscope FL system (Aperio Technologies Inc.; Vista, CA). Appropriate dyes were selected, illumination levels were calibrated using a pre-configured procedure, parameters were saved, and applied to all slides. The resulting digital images represent all tissue sections. Sections were evaluated for image quality. All obtained images were then placed in a dedicated project folder and stored on a designated local server. Selected areas of the slides were selected using the Aperio Imagescope (version 12, Aperio Technologies Inc.). For analysis, slides were inspected, all tissue areas were selected, and analyzed using the web-based Image Scope viewer. Slides were quantified using the "Color Deconvolution v9" algorithm (version 11, Aperio Technologies Inc.) for oil red staining. The algorithm was optimized using a pre-configured procedure to maximize the ratio of strong red-positive oil droplet signals to noise, subsequent macros were saved, and applied to all slides.

[0103] HNF4α immunostaining in organ samples. Samples were collected from mice, fixed in 4% paraformaldehyde, and embedded in paraffin or OCT frozen medium (Sakura Finetek; Torrance, CA). 5 μm thick slides were washed four times with PBS and treated with 0.3% Triton® in PBS for 10 minutes. Antigen retrieval was performed using CitriSolv® (Fisher Scientific; Waltham, MA) for 10 minutes at sub-boiling temperature. After washing with PBS for 10 minutes, the slides were incubated at room temperature for 60 minutes in a blocking solution with 5% normal donkey serum (Jackson Immuno Research; West Grove, PA). Cells were fixed in 4% paraformaldehyde for 15 minutes at 4°C, washed with PBS, treated with 0.3% Triton® in PBS for 10 minutes, and blocked as previously described for slide samples.

[0104] Primary antibodies were used. HNF4α antibodies were used (#sc-6556, Santa Cruz Biotechnology; Santa Cruz, CA and #3113, Cell Signaling Technology; Danvers, MA). For fluorescence imaging, samples were incubated with anti-mouse, rabbit, or goat labeled with ALEXA FLOUR® 488 green fluorescent dye or rhodamine, and the nuclei were counterstained with DAPI (4',6-diamidino-2-phenylindole). A control using a secondary antibody alone was used to ensure the specificity of immunohistochemistry. Fluorescently labeled sections were analyzed using a conventional inverted microscope (Olympus, PlanFl 40x / 0.60) or a confocal microscope equipped with a krypton / argon laser.

[0105] Determination of bioavailability. Male C57BL / 6 mice were administered N-trans-caffeoyltyramine or N-trans-feruloyltyramine via intravenous injection, intraperitoneal injection, or oral administration (3 mice for each route) (Table 2).

[0106] [Table 2]

[0107] Blood samples were collected from each mouse at 0.25, 0.5, 1, 2, 4, 6, and 24 hours after administration. Eight μL fractions of blood were used for analysis. 200 μL of internal standards containing 100 ng / mL labetalol, 100 ng / mL dexamethasone, 100 ng / mL tributamide, 100 ng / mL verapamil, 100 ng / mL glybride, and 100 ng / mL celecoxib were added to ACN. The mixture was then vortex-mixed and centrifuged at 12000 rpm for 15 minutes at 4°C to form microspheres of precipitated proteins. 4 μL of the supernatant was injected for LC-MS / MS analysis. Bioavailability (%) was expressed as AUC. 0-inf (% AUC Extra <20% or AUC 0-last (% AUC Extra The calculation was performed using nominal doses with a value of >20%.

[0108] pH stability evaluation. Individual stock solutions were prepared in DMSO at a concentration of 10 mg / mL. Four different buffer solutions were prepared to achieve solutions with pH values ​​of 2, 7.4, 8.5, and 10. For each pH assay, 5 μL of stock solution was added to 245 μL of buffer solution in a 2 mL tube, vortexed, and incubated in a 37°C water bath. At each time point, a 50 μL fraction was taken, neutralized, and analyzed by HPLC at 280 nm using a DAD detector. The magnification change of the peak region at 280 nm was analyzed at the first and last time points, and at 0.5 and 72 hours, respectively. [Examples]

[0109] Evaluation of the compound's activity as an HNF4α agonist Considering the role of HNF4α in maintaining healthy metabolism in humans, the test compounds were screened for their activity as HNF4α agonists (either direct or indirect). Using a well-known insulin promoter reporter assay, Kiselyuk et al. (2010. J. Biomol. Screen vol. 15(no. 6): pp. 663-670) screened a library of compounds for their activity in promoting insulin activation. They identified compound 1 as an insulin activator (Kiselyuk et al. (2012) Chem. Biol. vol. 19(no. 7): pp. 806-818), and the compound was subsequently shown to possess HNF4α agonist activity in an ornithine transcarbamoylase (OTC) promoter assay. The OTC promoter is well known to react with HNF4α in transient transfection assays (Inoue et al. (2002) J. Biol. Chem. vol. 277: pp. 25257-65).

[0110] To identify plant compounds with similar biological activity to this synthetic agent (compound 1), bioinformatics were employed to predict a target subset with desired HNF4α agonist activity from a complete set of known plant compounds. Several algorithms, combined with training data (i.e., positive data), were used to build models for key properties of the positive data that predict the desired biological activity. More specifically, a set of 18 synthetic compounds (e.g., compound 1) with known capabilities to influence HNF4α activity were included in the positive dataset. These structures were used to search a database of plant compounds for chemical structures with similar structural properties. Several metrics were used to measure concept-based similarity from either graph theory or information theory, either individually or in combination.

[0111] Plant compounds with structural and top-10 percentile similarity to 18 targets were selected, and compounds predicted to be potential agonists of HNF4α activity were screened in an HNF4α assay, taking into account their chemical structure characteristics. The screening identified a class of plant tyramine-containing hydroxycinnamides (i.e., N-trans-caffeoyltyramine, N-cis-caffeoyltyramine, N-trans-feruloyltyramine, and p-coumaroyltyramine) capable of acting as HNF4α modulators. In particular, N-trans-caffeoyltyramine was determined to be approximately an order of magnitude more potent than alverine in activating HNF4α (Figure 1). Due to the hydroxyl derivatization of both phenyl rings, N-trans-caffeoyltyramine is less lipophilic and therefore expected to be more bioavailable. Overall, the increased efficacy and anticipated enhanced bioavailability suggest that N-trans-caffeoyltyramine and other tyramine-containing hydroxycinnamic amides are expected to be more desirable compounds for use in the methods described herein.

[0112] Secondary experiments were conducted to demonstrate that these compounds directly regulate HNF4α activity. In particular, it was demonstrated that insulin (Figure 2) and HNF4α (Figure 3) gene expression are upregulated (e.g., as determined by quantitative PCR analysis) in the presence of N-trans-caffeoyltyramine and N-trans-feruloyltyramine. In addition, p-coumaroyltyramine also upregulates insulin and HNF4α gene expression, while cis-feruloyltyramine, N-coumaroyldopamine, N-trans-feruloyloctopamine, and p-coumaroyloctopamine are found to be inactive. Furthermore, using an insulin promoter assay, the N-trans-caffeoyltyramine-mediated increase in insulin expression is inhibited by BI-6015, a well-known HNF4α antagonist (Figure 4). In addition, it was shown that N-trans-caffeoyltyramine and N-trans-feruloyltyramine did not exhibit estrogen-like activity (Figure 5).

[0113] Using human, rat, and mouse liver microsomes, in vitro pharmacognosy showed that N-trans-caffeoyltyramine is stable, and its higher bioactivity in humans may be due to a longer half-life in human cells compared to mouse liver microsomes (Table 3). In human microsomes, the clear main pathway of in vivo transformation was oxidation of the left-sided aryl ring.

[0114] [Table 3]

[0115] Analysis of HepG2 hepatocytes treated with N-trans-caffeoyltyramine (20 μM) or N-trans-feruloyltyramine (20 μM) showed that these compounds can remove harmful fats from the liver and further inhibit fat accumulation in HepG2 hepatocytes treated with 0.25 mM palmitic acid, as evidenced by oil red O staining for fats. Similar inhibition of fat accumulation was observed in T6PNE cells treated with 0.25 mM palmitic acid and 10 μM N-trans-caffeoyltyramine, 10 μM N-trans-feruloyltyramine, or 10 μM p-coumaroyltyramine. N-trans-caffeoyltyramine reduced lipid accumulation when palmitic acid was added before compound administration (Figure 6).

[0116] In addition to performing assays to demonstrate the beneficial effects of the compound of the present invention, initial safety / toxicity assays were conducted. The results of these analyses are presented in Table 4.

[0117] [Table 4] [Examples]

[0118] Efficacy in diet-induced obese mice In addition to demonstrating the in vitro efficacy of the compounds of the present invention, experiments were conducted in vivo in an animal model of human disease, namely diet-induced obesity in mice. The experiments were carried out to establish feeding and treatment plans, drug administration and administration plans, and to present evidence of the beneficial effects of N-trans-caffeoyltyramine on glucose and lipid homeostasis, fatty liver, β-cell function, and hepatocyte function. Twelve mice (10 weeks old) were fed a high-fat diet for 4 weeks to induce obesity. After 4 weeks, and while on the high-fat diet, six mice were administered 5% DMSO or 120 mg / kg N-trans-caffeoyltyramine intraperitoneally twice daily for 14 days. One hour after the last intraperitoneal infusion of DMSO or N-trans-caffeoyltyramine, the animals were sacrificed and blood and organ (liver, kidney, intestine, and pancreas) samples were collected. Organ samples were subjected to histological, RNA, triglyceride, and protein analysis. In particular, the mice in this study showed no toxic effects at any of the doses tested. The treated mice exhibited consistent levels of activity, attention, grooming, and appetite with the control group. None of the treated mice showed weight loss, disease, or abnormal behavior compared to the control group.

[0119] The results showed that N-trans-caffeioyltyramine treatment reduced lipid accumulation and significantly increased HNF4α expression in the liver (P=0.0042), particularly nuclear expression of HNF4α (Figure 7). Immunostaining results showed that N-trans-caffeioyltyramine increased HNF4α activity. In addition, lipid droplet diameter in the liver decreased in animals treated with N-trans-caffeioyltyramine (Figure 8). Furthermore, alkaline phosphatase (Figure 9) and triglyceride (Figure 10) levels were significantly reduced in mice treated with N-trans-caffeioyltyramine. The reduction in hepatic lipid and droplet diameter, and the reduction in alkaline phosphatase, demonstrate the beneficial effects of increased HNF4α activity. Alkaline phosphatase and triglyceride levels are often part of blood tests in humans, and elevated levels are indicators of poor liver function, obesity, and metabolic syndrome. Therefore, alkaline phosphatase and triglyceride levels would provide useful markers for evaluating the effects of tyramine-containing hydroxycinnamides in humans administered the compounds of the present invention. In the pancreas, HNF4α expression was increased in animals treated with N-trans-caffeoyltyramine compared to DMSO-controlled mice (Figure 11). Similarly, administration of N-trans-caffeoyltyramine increased HNF4α expression in the intestines (Figure 12).

[0120] These in vivo data demonstrated a correlation between HNF4α expression and hepatic fat levels. In addition, the results showed that N-trans-caffeoyltyramine increased HNF4α activity in vivo, resulting in beneficial effects on lipid, triglyceride, alkaline phosphatase, and HNF4α levels. [Examples]

[0121] Evaluation of compound-related toxicity Considering the need to weigh the benefits and risks of the compounds of the present invention, in vivo toxicity studies in laboratory animals (e.g., mice, rats, dogs) are typically performed. Such studies are typically conducted in accordance with Good Laboratory Practice (GLP) rules to ensure reliability and reproducibility for regulatory purposes. When the compounds are administered to humans for periods ranging from several weeks to several months to several years, chronic toxicity studies (studies lasting from six months to one year) are typically performed. For compounds used in food, oral toxicity studies are recommended.

[0122] The purpose of chronic toxicity studies is to determine the toxicological profile of the test compound. In the first phase of the study, the study is conducted in rats. A total of 160 Sprague Dawley rats (80 males and 80 females), each approximately 5–7 weeks old and weighing between 80–100 g, were randomly selected and assigned to treatment groups by weight so as not to significantly differ in mean body weight between groups. The test compound or extract was orally administered to the rats at dose levels of 0.5, 1, and 2 g / kg body weight per day for a continuous period of 180 days. The animals were observed daily for any clinical signs of toxicity (e.g., behavioral changes, skin and fur appearance, feeding and drinking, etc.) and mortality. At the end of the experiment, the animals were subjected to hematological, biochemical, and histopathological evaluation using standard toxicological methods. [Examples]

[0123] Isolation of tyramine-containing hydroxycinnamides from plant sources Ethanol extracts were prepared from various plant species and their plant tissues. Individual compounds were identified in the extracts by extracting dried plant powder material with 95% aqueous ethanol. The ethanol extracts were concentrated, adsorbed onto Celite, and dry-loaded onto a C18 solid-phase extraction column. The extracts were desalted by washing them with 2 columns of water, collecting and discarding the water. The compounds were eluted with 2 columns of methanol, and the extracts were concentrated and dried. Before analysis, the extracts were resuspended in 1:1 acetonitrile:water. Before analysis, calibration curves were prepared using synthetic standards of known concentrations. A list of the sources used in the analysis is shown in Table 5 below. The plants are listed in descending order for each compound, with the plant producing the highest amount of the compound at the top of the list and the plant producing the lowest amount at the bottom.

[0124] [Table 5A]

[0125] [Table 5B]

[0126] The amounts of N-trans-caffeoyltyramine, N-trans-feruloyltyramine, and p-coumaroyltyramine present in a specific ethanol extract (percentage of extract, w / w) were determined. Quantification of the compounds was performed by normalizing the results by the weight of the ethanol extract. The results of these analyses are shown in Figure 13. [Examples]

[0127] Efficacy of the test compound in an animal model of NAFLD Other well-established animal models exist to investigate the benefits of compounds in NAFLD, such as diet-induced obesity mouse models.

[0128] Animals and Diet. Adult male Sprague-Dawley rats (250-300 grams) were obtained. Custom-prepared diets included a control, a high-fat only diet, and a high-fat diet containing the test compound or extract. The control diet was a low-fat diet in which 12% of the total calories from fat came from corn oil, with the majority of the fat being linoleic acid. The high-fat (HF) diet contained 60% of the total calories from lard and 2% from corn oil, and the diet was rich in oleic acid and saturated fatty acids palmitic acid and stearic acid. Such high-fat diets have been previously used to induce NAFLD in rats (Carmiel-Haggai et al. (2005) FASEB J. 19: pp. 136-138). Seven rats were randomized into each of the four groups and fed the diet for four weeks. Group I: control diet; Group II: HF diet; Group III: HF + 0.5% compound / extract diet; Group IV: HF + 1% compound / extract. Rats were placed on a 12-hour day / night cycle and provided with ad libitum feeding and drinking water. At the end of the 4-week period, rats were fasted for 16-18 hours, anesthetized, and blood and liver samples were collected for biochemical and histological analysis.

[0129] Serum and liver triglycerides and cholesterol. Serum triglycerides and total cholesterol are measured using a commercially available assay kit (e.g., Wako Diagnostics; Richmond, VA). Total lipids are extracted from liver samples with a chloroform-methanol mixture (2:1) (approximately 0.25 g) and washed with a 0.73% sodium chloride solution. The organic and aqueous phases are separated by centrifugation at 2000 rpm for 10 minutes. The organic phase containing total lipids is completely dried under nitrogen, and the lipid extract is reconstituted in isopropanol. Triglycerides and total cholesterol are measured using fractional amounts of the lipid extract with an assay kit (e.g., from Wako Diagnostics).

[0130] Measurement of serum and hepatic thiobarbiturate-reactive substances (TBARS). Serum and hepatic TBARS are measured as indicators of lipid peroxidation products.

[0131] Liver histology. Liver specimens are fixed in 10% formalin and embedded in paraffin. Sections (5 μm) are stained with hematoxylin and eosin and evaluated by pathologists blinded to the experimental group and condition. Sections are subjected to semi-quantification to assess fatty degeneration.

[0132] Statistical analysis. Data are expressed as mean + SE. Statistical analysis of the groups is performed using a two-tailed Student's t-test, and p < 0.05 is considered statistically significant. [Examples]

[0133] Evaluation of the safety and efficacy of test compounds in the treatment of NASH in subjects with T2DM. The objective of the study is to evaluate whether the test compound or extract of the present invention can improve liver health and hepatic fat mass compared to placebo in subjects with T2DM and NASH. The study will also include evaluation of serum alanine aminotransferase (ALT) levels and (when measured by MRI-induced proton density lipid percentage or MRI-PDFF) determination of whether the test compound or extract is more effective than placebo in reducing hepatic fat mass. Comparison of serum ALT levels and hepatic fat mass between the test compound or extract treatment and the placebo treatment will be performed at 24 weeks (or observation after the last baseline) in adult subjects with NASH and T2DM.

[0134] A secondary objective of the study is to evaluate the effects of the test compound or extract of the present invention on liver health compared to placebo treatment. The monitored endpoints include serum AST levels after 24 weeks of treatment; glycated hemoglobin (HbA1c) levels after 24 weeks; and the level of liver fibrosis as measured using transient elastography with FibroScan. Together, the results will allow for an evaluation of the safety and tolerability of the test compound and extract treatments compared to placebo treatment.

[0135] Furthermore, several exploratory objectives are included in the study design. For example, the effect of the test compound or extract on the target immune profile can be evaluated based on fluorescence-activated cell sorting (FACS) analysis, which measures changes from baseline in: 1) high-sensitivity C-reactive protein (hsCRP) and erythrocyte sedimentation rate (ESR); 2) serum levels of tumor necrosis factor alpha (TNF-α) from baseline; transforming growth factor (TGF) beta; 3) levels of interleukin (IL)-2, -4, -6, -10, and -12; 4) levels of interferon (IFN) gamma; and 5) changes from baseline in immunological markers such as differentiation antigen group 3 (CD3), CD4, CD8, CD25, CD40, CD56, CD69, CD127, forkhead box P3 (FOXP3+), IL17, and retinoic acid-associated orphan receptor-γt (RORγt). Further exploratory objectives include evaluating the effects of the test compound or extract on blood inflammatory markers (TNF-α, fibroblast growth factor 19 (FGF-19)), liver fibrosis or cell death markers (cytokeratin-18 (CK-18), soluble Fas (sFas)), and oxidative stress markers such as hydroxyeicosatetraenoic acid (HETE), hydroxyoctadecadienoic acid (HODE), oxoeicosatetraenoic acid (oxoETE), oxooctadecadienoic acid (oxoODE), and ox-non-alcoholic steatohepatitis (oxNASH). Furthermore, the study will evaluate the effects of the test compound or extract using the homeostasis model assessment of insulin resistance (HOMA IR) to measure insulin; evaluate the effects of the test compound or extract on serum lipid profiles (triglycerides, high-density lipoprotein (HDL), low-density lipoprotein (LDL), and total cholesterol); and evaluate the effects of the test compound or extract on GLP-1 and adiponectin.

[0136] Safety or tolerability endpoints will be evaluated after 24 weeks of treatment with the test compound or extract. Endpoints include the number and severity of any reported adverse events; physical examination findings, clinical laboratory values ​​(serological, hematological, and urinalysis), and 12-lead electrocardiogram (ECG) from baseline to study completion; and the number of subjects who discontinued the study before protocol completion. Clinical safety laboratory results will be collected and measured at the following time points during the study: days 1 and 3, and weeks 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24 (or at early discontinuation).

[0137] A total of 80 patients with T2DM and NASH were randomized into two groups: one group received placebo once daily (n=40), and the other group received 80 mg of the test compound or extract once daily (n=40).

[0138] The test compound or extract is administered at a dose of 80 mg per day, but the dose may be adjusted based on the patient's tolerance, or it may be set to a fixed amount for the duration of the study regardless of tolerance. [Examples]

[0139] Evaluation of the safety and efficacy of test compounds in the treatment of NASH in obese patients. The study is carried out according to the method of Example 7, with the only difference being that the inclusion criteria for subjects include the requirement that subjects are obese, defined as having a BMI of ≥30 instead of T2DM. [Examples]

[0140] Evaluation of the safety and efficacy of test compounds in the treatment of NAFLD in subjects with T2DM. The objective of this study is to determine whether the test compound or extract can improve liver fat volume and liver health compared to placebo in subjects with both T2DM and NAFLD by evaluating magnetic resonance imaging-derived proton density fat fraction (MRI-PDFF) after 24 weeks of treatment.

[0141] The secondary objectives of this study are: 1) to evaluate the effect of the test compound or extract treatment on liver health compared to placebo by assessing serum ALT levels after 24 weeks of treatment; 2) to evaluate the effect of the test compound or extract treatment on liver health compared to placebo by assessing serum AST levels after 24 weeks of treatment; 3) to evaluate the effect of the test compound or extract treatment on glycated hemoglobin (HbA1c); 4) to evaluate the effect of the test compound or extract treatment on liver fibrosis as measured using transient elastography with FibroScan; and 5) to evaluate the overall safety and tolerability of the test compound or extract treatment compared to placebo. The exploratory objectives of this study are listed in Example 7.

[0142] A total of 80 patients with T2DM and NAFLD were randomized into two groups, as described in Example 7: one group received placebo once daily (n=40), and the other group received a dose of 80 mg of the test compound or extract once daily (n=40). Patients were screened at visit 1 between day -28 and day -2. At screening, patients underwent a screening procedure intended to ensure they met the inclusion / exclusion criteria, including abdominal MRI to quantitatively measure liver fat mass. Patients who met the inclusion / exclusion criteria based on the screening evaluation returned to the study center on day -1 for baseline evaluation (visit 2). At the baseline visit, the inclusion / exclusion criteria were confirmed, and baseline laboratory values, physical examination findings, and ECG results were also evaluated.

[0143] Subjects are required to have a certified histological report that records and evaluates the degree of fatty degeneration, intralobular inflammation, hepatocyte ballooning, and fibrosis to confirm the diagnosis of NAFLD.

[0144] At the 24-week follow-up visit (or early termination), all subjects will undergo a treatment completion evaluation, including MRI-based liver fat imaging and clinical laboratory safety assessment.

Claims

1. At least one carrier, and N-trans-caffeoleyltyramine, and N-trans-ferloyltyramine Timer shell extract containing An orally consumable composition comprising, An oral composition in the form of a powder or granules.

2. The orally consumable composition according to claim 1, wherein N-trans-caffeoyltyramine or N-trans-feruloyltyramine is in the form of a pharmaceutically acceptable salt.

3. An orally consumable composition according to claim 1 or 2, which is in a suitable form selected from the group including nutritional supplements, food ingredients, food additives, medical foods, nutritional supplements, or pharmaceutical compositions.

4. An orally consumable composition according to claim 3, selected from the group including seasonings, biscuits, health bars, bread, cakes, cereals, candies, or confectionery.

5. The orally consumable composition according to claim 3, which is a single dosage form and is set for administration of less than 100 mg per day.

6. The orally consumable composition according to claim 1, wherein the N-trans-caffeoyltyramine and N-trans-feruloyltyramine are provided in doses ranging from 10 to 60 mg / day.

7. The orally consumable composition according to claim 1, further comprising at least one preservative and an active ingredient.

8. The orally consumable composition according to claim 1, wherein the timeberry shell extract containing N-trans-caffeoyltyramine and N-trans-feruloyltyramine is in the form of an ethanol extract.

9. The orally consumable composition according to claim 1, further comprising p-coumaroyltyramine.

10. The orally consumable composition according to claim 1 or 2, wherein the carrier is selected from the group consisting of sugars, starches, cellulose and its derivatives, waxes, oils and esters.

11. The orally consumable composition according to claim 10, wherein the cellulose and its derivatives are carboxymethylcellulose, ethylcellulose, cellulose acetate, and hydroxypropylmethylcellulose.

12. The consumable composition according to claim 1, provided in an effective amount that improves HNF4α activity, insulin-like growth factor levels, blood glucose levels, insulin levels, C-peptide levels, triglyceride levels, free fatty acid levels, blood uric acid levels, microalbuminuria levels, glucose transporter expression, adiponectin levels, total serum cholesterol levels, high-density lipoprotein levels, low-density lipoprotein levels, or a combination thereof.

13. A consumable composition according to claim 1, provided in an effective amount that improves metabolism, liver function, fasting blood glucose levels, postprandial blood glucose levels, glycated hemoglobin HbA1c, body weight, insulin sensitivity, serum lipid profile, or a combination thereof.

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