Metabolic health promoting components

Abstract

An example of a composition for promoting metabolic health can include a combination of cyanidin and delphinidin in an amount sufficient to treat metabolic disorders.Furthermore, an example of a method for treating a condition or disorder related to metabolic health in a subject can include reducing endotoxin levels compared to baseline endotoxin levels, and adjusting cardiometabolic biomarkers related to lipid or glucose metabolism from abnormal cardiometabolic baseline levels to normal levels.Exemplary methods can also include reducing liver inflammation, treating high-fat diet-induced colon dysfunction, reducing the expression of toll-like receptors TLR4 and TLR2, reducing plasma endotoxin levels, reducing the expression of NADPH oxidase NOX1, and increasing tight junction proteins in the colon of a subject.

Description

[Technical Field]

[0001] Priority Data This application claims priority to U.S. Provisional Patent Application No. 63 / 437,111, filed January 4, 2023, and U.S. Provisional Patent Application No. 63 / 503,692, filed May 22, 2023, each of which is incorporated by reference in its entirety into this application. [Background technology]

[0002] background Consumption of a high-fat and / or high-carbohydrate diet can lead to obesity and obesity-related chronic inflammation. If these dietary patterns are maintained for extended periods, such as weeks, months, or years, chronic inflammation can lead to impaired function and disease in organs such as the liver and colon. Excessive intake of fat and carbohydrates can also cause short-term effects immediately after ingestion. These effects include inflammation, oxidative stress, and hyperglycemia. These short-term effects after food intake are collectively referred to as "postprandial metabolic disorders." High-fat and high-carbohydrate diets, commonly known as "Western diets," have become common in many societies around the world. Therefore, if measures are not taken to correct the effects of these dietary patterns, inflammation and disease associated with the Western diet may become even more widespread. [Brief explanation of the drawings]

[0003] [Figure 1] FIG. 1 shows a schematic diagram of the chemical structure of cyanidin. [Figure 2] FIG. 2 shows a schematic diagram of the chemical structure of delphinidin. [Figure 3] FIG. 3 shows a schematic representation of the chemical structure of petunidin. [Figure 4] FIG. 4 shows a schematic representation of the chemical structure of peonidin. [Figure 5] FIG. 5 shows a schematic representation of the chemical structure of malvidin. [Figure 6]FIG. 6 graphically depicts groups of mice in a study according to one embodiment of the present disclosure. [Figure 7] FIG. 7 graphically illustrates food intake in mice according to one embodiment of the present disclosure. [Figure 8] FIG. 8 graphically illustrates weight gain in mice according to one embodiment of the present disclosure. [Figure 9] FIG. 9 graphically illustrates percent fat mass according to one embodiment of the present disclosure. [Figure 10] FIG. 10 graphically illustrates lean body mass according to one embodiment of the present disclosure. [Figure 11] FIG. 11 graphically illustrates liver lipid levels according to one embodiment of the present disclosure. [Figure 12] FIG. 12 graphically illustrates free fatty acid levels according to one embodiment of the present disclosure. [Figure 13] FIG. 13 graphically illustrates proteins according to one embodiment of the present disclosure. [Figure 14] FIG. 14 graphically illustrates protein expression according to one embodiment of the present disclosure. [Figure 15] FIG. 15 graphically illustrates staining of liver tissue according to one embodiment of the present disclosure. [Figure 16] FIG. 16 graphically illustrates hepatocyte injury patterns according to one embodiment of the present disclosure. [Figure 17] FIG. 17 graphically illustrates endotoxin concentration according to one embodiment of the present disclosure. [Figure 18] FIG. 18 graphically illustrates plasma endotoxin concentrations according to one embodiment of the present disclosure. [Figure 19] FIG. 19 graphically illustrates proteins according to one embodiment of the present disclosure. [Figure 20] FIG. 20 graphically illustrates the expression of TLR4 and TLR2 according to one embodiment of the present disclosure. [Figure 21] FIG. 21 graphically illustrates the percentage of phosphorylated proteins in total proteins according to one embodiment of the present disclosure. [Figure 22] FIG. 22 graphically illustrates NF-κB-DNA binding according to one embodiment of the present disclosure. [Figure 23] FIG. 23 graphically illustrates protein levels according to one embodiment of the present disclosure. [Figure 24] FIG. 24 graphically illustrates AP-1-DNA binding according to one embodiment of the present disclosure. [Figure 25] FIG. 25 graphically illustrates the percentage of phosphorylated proteins in total proteins according to one embodiment of the present disclosure. [Figure 26] FIG. 26 graphically illustrates HIF-1-DNA binding according to one embodiment of the present disclosure. [Figure 27] FIG. 27 graphically illustrates protein expression according to one embodiment of the present disclosure. [Figure 28] FIG. 28 graphically illustrates the concentration of 4-HNE protein adducts according to one embodiment of the present disclosure. [Figure 29] FIG. 29 graphically illustrates plasma LBP concentrations in the high fat and control groups according to one embodiment of the present disclosure. [Figure 30] FIG. 30 graphically illustrates plasma LBP concentrations in a control group, a control group supplemented with anthocyanins, a high fat group, and a high fat group supplemented with anthocyanins, according to one embodiment of the present disclosure. [Figure 31] FIG. 31 graphically illustrates colonic TLR-4 and MyD88 protein levels in control and high-fat groups according to one embodiment of the present disclosure. [Figure 32] FIG. 32 graphically illustrates TLR-4, TLR-2, and MyD88 protein levels in a control group, a control group supplemented with anthocyanins, a high-fat group, and a high-fat group supplemented with anthocyanins, according to one embodiment of the present disclosure. [Figure 33] FIG. 33 graphically illustrates levels of occludin, claudin, and ZO-1 in the control and high-fat groups, according to one embodiment of the present disclosure. [Figure 34] FIG. 34 graphically illustrates levels of occludin, claudin, and ZO-1 in a control group, a control group supplemented with anthocyanins, a high-fat group, and a high-fat group supplemented with anthocyanins, according to one embodiment of the present disclosure. [Figure 35] FIG. 35 graphically illustrates MLCK and MLC phosphorylation in control and high-fat groups, according to one embodiment of the present disclosure. [Figure 36] FIG. 36 graphically illustrates MLCK, MLC phosphorylation, and MYPT-1 phosphorylation in a control group, a control group supplemented with anthocyanins, a high-fat group, and a high-fat group supplemented with anthocyanins, according to one embodiment of the present disclosure. [Figure 37] FIG. 37 graphically illustrates occludin protein levels in control and lipopolysaccharide (LPS)-treated Caco-2 cells, according to one embodiment of the present disclosure. [Figure 38] FIG. 38 graphically illustrates the results of an MTT assay showing cell viability of control cells and cells treated with LPS alone and in combination with cyanidin, delphinidin, PCA, and GA, according to one embodiment of the present disclosure. [Figure 39] FIG. 39 graphically illustrates TEER and paracellular transport of FITC-dextran for control cells and cells treated with LPS alone or in combination with cyanidin, delphinidin, PCA, and GA, according to one embodiment of the present disclosure. [Figure 40] FIG. 40 graphically illustrates levels of occludin, ZO-1, and claudin-1 in control cells and cells treated with LPS alone or in combination with cyanidin, delphinidin, PCA, and GA, according to one embodiment of the present disclosure. [Figure 41] FIG. 41 graphically depicts the expression of MLCK, phosphorylation of MYTP-1, and phosphorylation of MLC in control cells and cells treated with LPS alone or in combination with cyanidin, delphinidin, PCA, and GA, according to one embodiment of the present disclosure. [Figure 42] FIG. 42 graphically illustrates the expression of NOX1 and 4-HNE in control cells and cells treated with LPS alone or in combination with cyanidin, delphinidin, PCA, and GA, according to one embodiment of the present disclosure. [Figure 43]FIG. 43 graphically illustrates DHE oxidation in control cells and cells treated with LPS alone or in combination with cyanidin, delphinidin, PCA, and GA, according to one embodiment of the present disclosure. [Figure 44] FIG. 44 graphically illustrates the phosphorylation of PI3K, Akt, p65, ERK, and JNK expression in the control and high-fat diet groups, according to one embodiment of the present disclosure. [Figure 45] FIG. 45 graphically illustrates phosphorylation of PI3K and Akt in a control group, a control group supplemented with anthocyanins, a high fat group, and a high fat group supplemented with anthocyanins, according to one embodiment of the present disclosure. [Figure 46] FIG. 46 graphically illustrates the phosphorylation of IKK and p65 in a control group, a control group supplemented with anthocyanins, a high fat group, and a high fat group supplemented with anthocyanins, according to one embodiment of the present disclosure. [Figure 47] FIG. 47 graphically illustrates the phosphorylation of p38, ERK, and JNK in a control group, a control group supplemented with anthocyanins, a high fat group, and a high fat group supplemented with anthocyanins, according to one embodiment of the present disclosure. [Figure 48] FIG. 48 graphically illustrates the levels of NOX1, NOX2, NOX4, iNOS, and 4-HNE in a control group, a control group supplemented with anthocyanins, a high-fat group, and a high-fat group supplemented with anthocyanins, according to one embodiment of the present disclosure. [Figure 49] FIG. 49 graphically illustrates NOX1 in the control and high-fat groups after 4 and 8 weeks, according to one embodiment of the present disclosure. [Figure 50] FIG. 50 graphically illustrates colon weight to colon length ratio and crypt length in the control group, the control group supplemented with anthocyanins, the high fat group, and the high fat group supplemented with anthocyanins, according to one embodiment of the present disclosure. [Figure 51] FIG. 51 graphically illustrates the number of goblet cells per crypt and PAS-stained colon samples after 4 and 8 weeks in the control group, the control group supplemented with anthocyanins, the high-fat group, and the high-fat group supplemented with anthocyanins, according to one embodiment of the present disclosure. [Figure 52] FIG. 52 graphically depicts mRNA levels of Klf4, Muc2, and Tff3 in the control and high-fat groups, according to one embodiment of the present disclosure. [Figure 53] FIG. 53 graphically illustrates mRNA levels of Klf4, Muc2, and Tff3 in a control group, a control group supplemented with anthocyanins, a high fat group, and a high fat group supplemented with anthocyanins, according to one embodiment of the present disclosure. [Figure 54] FIG. 54 is a flow chart illustrating a recruitment and screening strategy according to one embodiment of the present disclosure. [Figure 55] Figure 55A graphically illustrates plasma endotoxin concentrations in subjects administered a placebo or an anthocyanin-containing blend (ACRB), and Figure 55B graphically illustrates LPS-binding protein concentrations in subjects administered a placebo or an ACRB, according to one embodiment of the present disclosure. [Figure 56] Figure 56A is a graph showing the change in plasma triglycerides over time in subjects administered a placebo or an ACRB according to one embodiment of the present disclosure; Figure 56B is a graph showing the change in total cholesterol over time in subjects administered a placebo or an ACRB according to one embodiment of the present disclosure; and Figure 56C is a graph showing the plasma glucose concentration over time in subjects administered a placebo or an ACRB according to one embodiment of the present disclosure. [Figure 57]Figure 57A graphically illustrates IL-8 mRNA levels in subjects administered a placebo or ACRB, according to one embodiment of the present disclosure. Figure 57B graphically illustrates IL-18 mRNA levels in subjects administered a placebo or ACRB, according to one embodiment of the present disclosure. Figure 57C graphically illustrates IL-1β mRNA levels in subjects administered a placebo or ACRB, according to one embodiment of the present disclosure. Figure 57D graphically illustrates TNFα mRNA levels in subjects administered a placebo or ACRB, according to one embodiment of the present disclosure. Figure 57E graphically illustrates TLR4 mRNA levels in subjects administered a placebo or ACRB, according to one embodiment of the present disclosure. Figure 57F graphically illustrates NOX2 mRNA levels in subjects administered a placebo or ACRB, according to one embodiment of the present disclosure. Figure 57G graphically illustrates NOX4 mRNA levels in subjects administered a placebo or ACRB, according to one embodiment of the present disclosure. [Figure 58] Figure 58A graphically depicts IKK, JNK, and PTP1B in subjects administered placebo or ACRB according to one embodiment of the present disclosure. Figure 58B graphically depicts IKK phosphorylation in subjects administered placebo or ACRB according to one embodiment of the disclosure. Figure 58C graphically depicts JNK phosphorylation in subjects administered placebo or ACRB according to one embodiment of the disclosure. And Figure 58D graphically depicts PTP1B in subjects administered placebo or ACRB.

[0004] These figures are provided to illustrate various aspects of the invention and are not intended to limit the scope with respect to dimensions, materials, configurations, arrangements, or proportions unless specifically limited in the claims. DETAILED DESCRIPTION OF THE INVENTION

[0005] Although these exemplary embodiments are described in sufficient detail to enable those skilled in the art to practice the invention, it should be understood that other embodiments are possible and that various modifications can be made to the invention without departing from the spirit and scope of the invention. Accordingly, the following detailed description of embodiments of the invention is not intended to limit the scope of the invention, which is defined by the claims, but is presented solely for the purposes of describing the features and characteristics of the invention, setting forth the best mode for carrying out the invention, and providing a full description to enable those skilled in the art to practice the invention. Accordingly, the scope of the invention is defined solely by the appended claims.

[0006] definition In describing and claiming the present invention, the following terminology will be used.

[0007] As used herein and in the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms unless the context clearly dictates otherwise. For example, the reference to "an anthocyanidin" is intended to include one or more such substances, and the reference to "administering" is intended to include one or more such steps.

[0008] In this specification, the terms "about" and "approximately" are used to provide flexibility, for example, when indicating that a particular value at the endpoint of a numerical range is "slightly above the endpoint" or "slightly below the endpoint." The degree of flexibility for a particular variable can be easily determined by one of ordinary skill in the art based on the context. In addition, when a numerical value is used in conjunction with the term "about" in this specification, it is understood that the exact numerical value itself is provided as if "about" were not used.

[0009] As used herein, the term "substantially" refers to the complete or nearly complete extent or degree of an action, characteristic, property, state, structure, item, or result. The exact degree of permissible deviation from absolute perfection may vary depending on the specific context. However, the degree of near perfection is generally such that it produces the same overall result as if absolute and complete perfection were achieved. "Substantially" also applies when used in a negative sense, referring to the complete or nearly complete absence of an action, characteristic, property, state, structure, item, or result.

[0010] As used herein, the term "substantially" when used with respect to a specified characteristic or circumstance refers to a degree of deviation that is measurable from that characteristic or circumstance. The exact degree of deviation that is permitted may vary depending on the particular context.

[0011] As used herein, the terms "negligible" or "clinically negligible" refer to the degree of effect when a composition is administered to a subject. For example, if the degree of effect does not cause a clinical change in the subject, the result is considered to be clinically negligible.

[0012] As used herein, "soluble" refers to a substance that can be dissolved in a solvent to form a single solution. A substance is defined as "soluble" in a particular solvent if it can dissolve in an amount of at least 5 grams per liter of that solvent.

[0013] As used herein, a "concentrate" refers to an extract obtained from a source that contains at least the same amount of active fractions, compounds, or other components in a smaller volume than the source itself. As an example, a "concentrate" may be a dry powder obtained from the components without the use of any solvents in the concentration process.

[0014] Comparative terms such as "more effectively," "greater," "improved," and "enhanced" may be used to indicate a result or characteristic of a formulation or process that achieves or exists a measurably better or more positive result than the comparator. In some cases, the comparator may be set to the prior art or the state of the characteristic that existed prior to administration of the formulation or method.

[0015] As used herein, terms such as "comprises," "comprising," "containing," and "having" have the meanings ascribed to them in U.S. Patent Law, including "includes," "including," and are generally construed as open-ended terms. "Consisting of" or "consists of" are closed terms, including only the components, structures, steps, or the like specifically recited in conjunction with these terms, as well as those subject to U.S. Patent Law. "Consisting essentially of" or "consists essentially of" have the meaning generally ascribed to them in U.S. Patent Law. Notably, although these terms are generally closed terms, exceptions are permitted to include additional items, materials, components, steps, or elements that do not materially affect the basic and novel characteristics or function of the article with which they are used. For example, trace elements whose presence in a composition does not affect its properties or characteristics are permissible under the phrase "consisting essentially of," even if they are not explicitly recited in the list of items following that term. When using descriptions that specify open-ended terms such as "comprising" or "including," it is understood that the phrase "consisting essentially of" should be given as if expressly recited, just as when the phrase "consisting of" is specified, and vice versa.

[0016] A "dosage unit" refers to a single compositional unit that can be administered to a subject or patient and contains a sufficient amount of active ingredient to achieve or contribute to a therapeutic effect. In some embodiments, a "dosage unit" is a unit that can be easily processed and packaged and remains a physically and chemically stable unit dose, containing either the active ingredient itself or a mixture of the active ingredient and a solid or liquid pharmaceutical vehicle material. Furthermore, the terms "dosage" and "dose" can refer to such a dosage unit. Alternatively, a "dosage" or "dose" may include multiple dosage units that provide a desired amount of active ingredient to be administered to a subject at a single time. Multiple dosages or doses may be used according to a dosing schedule and utilized to establish a dosing regimen.

[0017] An "effective amount," "therapeutically effective amount," or "therapeutically effective rate" refers to the amount or delivery rate necessary to achieve a therapeutic effect for the disease or condition to which the active ingredient is delivered, while remaining non-toxic, so that the active ingredient exerts a therapeutic effect in treating the disease or condition. It is understood that the ability of an active ingredient to exert its intended action can be affected by various biological factors. Thus, an "effective amount," "therapeutically effective amount," or "therapeutically effective rate" may depend in some cases on such biological factors. Furthermore, while the achievement of a therapeutic effect may be measured by a physician or other qualified medical professional using known evaluation methods, the achievement of a therapeutic effect may be subjective due to individual differences and response to treatment. Determining a therapeutically effective amount or delivery rate is well within the ordinary skill of one of ordinary skill in the art of pharmacy and medicine.

[0018] As used herein, the term "extract" refers to a substance prepared using a solvent such as ethanol, water, steam, superheated water, methanol, hexane, liquid chloroform, liquid CO2, liquid N2, propane, supercritical CO2, or a combination thereof. As used herein, the term "extract" can refer to a liquid extract or a product obtained by further processing the liquid form, such as a dried powder or other solid form. Extracts can take a variety of forms, including solids, liquids, granules, shreds, and distillates. They can also be prepared by any number of procedures or protocols, including shredding, grinding, pulverizing, boiling, steaming, soaking, extraction, percolation, and gassing. Furthermore, appropriate reagents, such as water, alcohol, steam, and other organic substances, can be used. Extracts typically have a specific purity percentage, which can range from relatively low to high purity. In some embodiments, the extract can be a plant extract made from a specific part of the source, such as the peel, pulp, leaves, flowers, fruits, kernels, or seeds, or can be made from the entire source. Additionally, the extract may contain one or more active fractions or active ingredients, and the purity of the extract may be controlled by or be a function of the extraction process or protocol.

[0019] As used herein, the terms "formulation" and "composition" are used interchangeably and refer to a combination of at least two components, in some embodiments, at least one of which is an active agent or has the property of exerting a physiological activity when administered to a subject.

[0020] The components of the formulations or compositions described or described herein are assumed to be expressed as weight percent (wt%) unless otherwise specified. Additionally, component amounts given in percentage form are assumed to be expressed as weight percent (e.g., w / w%) ratios.

[0021] "Intestinal hyperpermeability" refers to a condition in which permeability of the gastrointestinal system is higher than normal (i.e., in an average subject), and can sometimes refer to increased permeability of the stomach, small intestine, and / or large intestine.

[0022] As used herein, a "linear inhibitory effect" or "dose response" refers to a linear decrease in secretion or biosynthesis along a dose-response curve at all concentrations of an inhibitor. For example, inhibition at low concentrations followed by loss of inhibition or increased secretion at higher concentrations indicates the absence of a linear inhibitory effect.

[0023] The term "or" is used in the inclusive sense of "and / or" and not in the exclusive sense of "either / or."

[0024] As used herein, " pharmaceutically acceptable " generally refers to a material that is suitable for administration to a subject in combination with an active ingredient or component. For example, a " pharmaceutically acceptable carrier " can be a substance or material that can be appropriately combined with an active ingredient to form a composition or formulation that is suitable for administration to a subject. The excipients, diluents, and other components used in preparing compositions or formulations for administration to a subject can be used in conjunction with this term.

[0025] "Prevent" and variations thereof refer to preventative measures against a particular undesirable physiological condition. The preventative measures can be partial or complete. Partial preventative measures may result in a delay in the onset of the physiological condition. Those skilled in the art will recognize the desirability of delaying the onset of a physiological condition and will recognize that a subject at risk for a particular physiological condition should be administered the compositions of the present invention to delay its onset. For example, those skilled in the art will recognize that obese subjects are at increased risk for coronary artery disease. Thus, those skilled in the art will administer the compositions of the present invention to improve the gut microbiota of obese individuals.

[0026] As used herein, "subject" refers to an individual receiving treatment. In one embodiment, the subject can be a mammal. In another embodiment, the subject can be a human. In another embodiment, the subject can be a domesticated animal or livestock.

[0027] The terms "treat," "treating," or "treatment," as used herein and as understood by those skilled in the art, refer to an approach for obtaining beneficial or desired results in a treated subject, including, but not limited to, clinical results. Beneficial or desired results include, but are not limited to, alleviation or amelioration of one or more signs or symptoms of a condition, reducing the extent of disease progression, stabilizing the disease state (i.e., not worsening), slowing or inhibiting disease progression, improvement or palliation of the disease state, reducing disease recurrence, and partial or complete remission (whether detectable or not). "Treat," "treating," and "treatment" can refer to extending survival as compared to expected survival in the absence of treatment and may be prophylactic. Such prophylactic treatment is also referred to as prevention or prophylaxis of a disease or condition. Prophylaxis may be partial or complete. Partial prophylaxis may result in a delay in the onset of a physiological condition.

[0028] As used herein, "solvent" refers to a gas, aqueous, or organic liquid having the necessary properties to extract solid materials from plant products. Examples of solvents include, but are not limited to, water, steam, superheated water, methanol, ethanol, ethyl acetate, hexane, chloroform, liquid CO2, liquid N2, propane, or combinations of these materials.

[0029] In this specification, a plurality of items, structural elements, compositional elements, and / or materials may be presented in common lists for convenience. However, these lists are to be construed as though each item in the list were individually identified and considered a separate and unique item. Thus, the individual items in the list are not to be construed as equivalents to the other items in the list, or as de facto equivalents of the other items solely based on the fact that they are presented in a common grouping in the list.

[0030] Concentrations, amounts, and other numerical data may be presented in range format herein. Such range format is used merely for convenience and brevity and should not be construed as including only the numerical values ​​expressly recited as the limits of the range. Rather, all individual numerical values ​​or subranges within the range should be construed as including each as if expressly recited. For example, a numerical range of about 1 to about 4.5 should be construed to include not only the explicitly recited range limits of 1 to about 4.5, but also individual numerical values ​​such as 2, 3, and 4, and subranges such as 1 to 3 and 2 to 4. The same principle applies to ranges reciting only a single number (e.g., "less than about 4.5"), which should be construed as including all values ​​and ranges expressly recited above. Furthermore, this interpretation applies regardless of the breadth of the range or the type of property being described.

[0031] Any steps recited in a method or process claim may be performed in any order and are not limited to the order recited in the claims. A means-plus-function or step-plus-function limitation applies only if all of the following conditions are met in the particular claim limitation: a) "means-for" or "step-for" is expressly recited; and b) the corresponding function is expressly recited. The structure, material, or acts supporting the means of the means-plus-function are expressly recited in the specification. Therefore, the scope of the present invention should be determined solely by the appended claims and their legal equivalents, rather than by the descriptions and examples set forth herein.

[0032] Technology Examples Reference will now be made to illustrated embodiments and specific language will be used in describing them, without intending to limit the scope of the technology. Additional features and advantages of the technology will become apparent from the detailed description considered in conjunction with the accompanying drawings.

[0033] Given the general examples provided in the Summary above, when describing compositions or methods of treatment using compositions herein, individual or separate descriptions are considered applicable to one another, whether or not they are explicitly discussed in the context of a particular example or embodiment. For example, when any component of a composition is described herein, the description also includes methods of treatment or other methods of using those compositions, and vice versa.

[0034] Furthermore, various modifications and combinations may be derived from the present specification and drawings, and therefore the accompanying drawings should not be construed as limiting.

[0035] A dietary pattern with a high fat content is associated with a state of chronic inflammation that affects organ function. In the case of the liver, increased inflammation and lipid deposition can lead to the development of nonalcoholic fatty liver disease (NAFLD).

[0036] Chronic inflammation resulting from a high-fat / high-carbohydrate diet and / or obesity is thought to be due, in part, to increased levels of bacterial lipopolysaccharide (LPS) in the circulating blood. This increase, defined as metabolic endotoxemia, may result from increased translocation of LPS from the intestinal lumen into the circulation. The underlying mechanisms may include (i) paracellular transport of LPS due to increased permeability of the intestinal barrier, (ii) cotransport of LPS incorporated into nascent chylomicrons through intestinal epithelial cells, and (iii) increased LPS production due to alterations in the gut microbiota.

[0037] Toll-like receptors (TLRs) are involved in the pathophysiology of multiple chronic liver diseases. At the molecular level, LPS binds to TLR4, activating multiple signaling cascades, including NF-κB and mitogen-activated kinase (MAPK), and promoting the transcription of chemokines and proinflammatory cytokines. The liver normally maintains low levels of TLR4 expression and processes LPS without causing significant inflammation. Upon exposure to high concentrations of LPS, the liver upregulates TLR4 expression, and subsequent activation can lead to liver inflammation and fibrosis. If this condition becomes chronic, it can ultimately lead to tissue insulin resistance and NAFLD.

[0038] For these reasons, a long-term high-fat and high-carbohydrate diet may lead to chronic metabolic health problems. In addition to the consequences of long-term dietary patterns, a high-fat and high-carbohydrate diet may also have short-term health effects, such as immediately after consuming a high-fat and high-carbohydrate meal. Following meal ingestion, physiological postprandial responses may occur for normal nutrient absorption and metabolism. However, excessive intake of fat and / or carbohydrates can trigger a series of short-term events known as postprandial metabolic disorders. These events include abnormalities in glucose and lipid metabolism, endotoxemia, inflammation, and oxidative stress. Postprandial metabolic disorders have been associated with other pathologies, including increased risk of cardiovascular disease and mortality, NAFLD, and accelerated progression of type 2 diabetes.

[0039] A single high-fat and / or high-carbohydrate meal can induce postprandial hyperglycemia, postprandial hypertriglyceridemia, and / or endotoxemia. Therefore, a single meal can be a useful model for studying the effects of various interventions to prevent or alleviate postprandial metabolic disorders. The mechanisms by which a high-fat meal acutely increases endotoxemia and subclinical inflammation may include the following: (1) Under high-fat loading, transient damage to the intestinal epithelium allows passive diffusion of LPS through the paracellular space. This damage can be repaired as little as 1 hour after ingestion of a high-fat meal. (2) LPS micellization and incorporation into chylomicrons, along with absorption of other lipids via the lymphatic system, delivers LPS directly to the liver. (3) Mast cell activation by dietary fat triggers an inflammatory response involving activation of myosin light chain kinase (MLCK), induction of TNFα, and ultimately damage to tight junctions, thereby opening a pathway for LPS to diffuse directly through the intestinal barrier.

[0040] In addition to these mechanisms, chronic consumption of a high-fat Western diet may cause a shift in the balance of the microbiota, resulting in the dominance of LPS-producing pathogenic bacteria instead of commensal bacteria. Pathogenic bacteria produce higher levels of LPS, which can impair the integrity of the intestinal mucosa, trigger inflammation, and ultimately damage tight junctions. This can impair intestinal barrier function and increase LPS release into the circulation. Therefore, consumption of a high-fat diet may lead to endotoxemia due to increased intestinal permeability and fat absorption, resulting in adverse health effects.

[0041] Postprandial dyslipidemia may also contribute to both inflammation and insulin resistance. From the perspective of glucose metabolism, inflammation-associated oxidative stress can activate the mitogen-activated kinases c-jun N-terminal kinase (JNK) and IκB kinase (IKK), resulting in downstream activation of the transcription factor NF-κB. Activation of both JNK and IKK, as well as increased expression of NF-κB-regulated protein tyrosine phosphatase 1B (PTP1B), can downregulate the insulin signaling pathway and cause insulin resistance.

[0042] Anthocyanidins (ACs) comprise a group of flavonoids with a basic flavonoid structure of C6-C3-C6, bearing a positive charge on the heterocyclic ring. Different hydroxylation patterns characterize different ACs, and in nature they exist primarily as glycosylated derivatives (anthocyanins). AC intake has been associated with improvements in conditions with significant pro-inflammatory components, such as type 2 diabetes, cardiovascular disease, and ulcerative colitis. However, in terms of mechanism of action, different ACs exhibit distinct biological activities. Thus, in their 3-O-glucoside form, cyanidin and delphinidin are more effective than other ACs, such as malvidin, petunidin, and peonidin, in suppressing tumor necrosis factor alpha (TNFα)-induced activation of the inflammatory NF-κB signaling pathway and preventing permeability of Caco-2 cell monolayers, which are used as a model of the intestinal epithelium. Similarly, the 3-O-glucosides of cyanidin and delphinidin are more effective than the 3-O-glucosides of malvidin and peonidin in stimulating the release of glucagon-like peptide 1 (GLP-1) from enteroendocrine cells.

[0043] Dietary strategies aimed at reducing metabolic endotoxemia may help alleviate liver inflammation and disease associated with a Western diet. In this regard, supplementation with a cyanidin- and delphinidin-rich extract (CDRE) has been observed to prevent high-fat diet (HFD)-induced metabolic endotoxemia and attenuate inflammation in mice fed an HFD for 14 weeks. Furthermore, a 4-week CDRE supplementation study demonstrated its efficacy in reversing HFD-induced endotoxemia and associated liver inflammation. This CDRE supplementation period prevented the increase in endotoxemia caused by a high-fat diet and attenuated the overexpression of TLR4 and TLR2 in the liver and the activation of downstream inflammatory cascades (NF-κB, HIF-1, AP-1) and upstream MAPK (p38) and extracellular signal-regulated kinase (ERK) 1 / 2. Therefore, increased intake of cyanidin and delphinidin may help alleviate the deleterious effects of regular consumption of a high-fat diet, namely metabolic endotoxemia and liver inflammation.

[0044] Gastrointestinal (GI) health is also important for maintaining sustained overall health. However, the GI can also be adversely affected by a high-fat diet. In particular, a high-fat diet (HFD) can cause intestinal inflammation, impaired barrier function, and dysbiosis, affecting other aspects of GI immunity. The cyanidin and delphinidin composition described in this study may prevent or reduce the harmful GI effects of an unhealthy diet, thus providing direct benefits to human systemic and metabolic health.

[0045] The GI barrier regulates interactions between resident immune cells, luminal contents, GI microbiota, and the host. Increased intestinal permeability plays a central role in the development of diseases associated with HFD and obesity. GI barrier function is maintained by tight junctions (TJs), protein complexes that connect adjacent epithelial cells. TJs regulate water and ion transport while preventing the paracellular transfer of intraluminal food and microbial toxins into the circulatory system. Barrier dysfunction allows the paracellular transfer of intraluminal bacterial lipopolysaccharide (LPS) into the blood, causing metabolic endotoxemia and inducing systemic inflammation. Mechanistically, HFD intake induces increased NADPH oxidases NOX1 and NOX4 in the intestine and activation of the oxidative stress-sensitive mitogen-activated kinases (MAPKs) ERK1 / 2 and the transcription factor NF-κB, thereby inducing TJ opening. These cascades are activated by LPS interaction with toll-like receptor (TLR)-4, promoting a cycle involving local inflammation, oxidative stress, and increased barrier permeability. In parallel, HFD exerts additional detrimental effects on goblet cells, negatively impacting GI immune protection.

[0046] Flavonoids are a large family of bioactive molecules that can regulate GI tract function and, through such actions, may maintain health. Given differences in chemical structure and metabolism, these compounds affect the GI tract differently. Among flavonoids, compounds belonging to the anthocyanin (AC) subfamily can have GI anti-inflammatory and / or barrier-protective effects in rodent models of HFD-induced obesity and inflammatory bowel disease, as well as in vitro. Supplementation with cyanidin and delphinidin for 14 weeks can prevent HFD-induced intestinal permeability, endotoxemia, altered mucus secretion, and altered microbiota profiles in mice. The ability of these ACs to protect the intestinal barrier from HFD-associated and inflammation-induced barrier hyperpermeability may be due to the regulation of epithelial cell redox homeostasis and signaling and the preservation of TJs. The ability of flavonoids, particularly ACs, to mitigate the detrimental effects of an HFD on the GI tract may contribute to mitigating the adverse consequences of a Western diet on systemic and metabolic health, including insulin resistance and hepatic steatosis.

[0047] Short-term (4 weeks) supplementation of HFD-fed mice with a cyanidin- and delphinidin-rich extract (CDRE) prevented endotoxemia and hepatic activation of pro-inflammatory signaling pathways downstream of TLR-4. This may be due to the preservation of intestinal barrier function by AC. CDRE supplementation may also alleviate the deleterious consequences of HFD on the colonic barrier and goblet cell physiology. TLR-4 and redox-regulated signaling pathways were shown to be involved in the preservation of TJ structure and dynamics by AC in mice and Caco-2 cells.

[0048] Therefore, the present disclosure describes compositions and methods for promoting the metabolic health of subjects.This includes reducing liver inflammation, reducing colon dysfunction, reducing endotoxemia, and other effects related to metabolic health.The compositions described in the present disclosure can comprise a combination of anthocyanidins.Specifically, the combination of cyanidin and delphinidin can be comprised in said composition.

[0049] Figure 1 shows the chemical structure of cyanidin. Cyanidin can exist in a composition as a separate chemical compound having this chemical structure, or it can exist as a glycoside, in which the cyanidin molecule is linked to a sugar molecule. The sugar molecule can be a variety of sugars, such as galactose, glucose, arabinose, or xylose.

[0050] The chemical structure of delphinidin is shown in Figure 2. Like cyanidin, delphinidin can be included in the composition as a separate chemical compound or as a glycoside in which delphinidin is bound to a sugar.

[0051] The compositions described herein can contain additional anthocyanidins.Non-limiting examples include petunidin, peonidin, and malvidin.The chemical structures of these compounds are shown in Figure 3, Figure 4, and Figure 5, respectively.These compounds can also exist as independent chemical compounds or in the form of glycosides bound to sugar molecules.

[0052] As used herein, "anthocyanin" refers to a class of compounds containing anthocyanidins and sugar molecules bound to the anthocyanidins, also known as anthocyanidin glycosides. The anthocyanidins referred to herein include cyanidin, delphinidin, petunidin, peonidin, and malvidin. These anthocyanidins can naturally occur in fruits and vegetables as various glycoside compounds depending on the type of sugar bound to the anthocyanidin. Examples of sugars that can be bound to anthocyanidins include glucose, rutinose, galactose, arabinose, sambubiose, and other sugars. Various glycosides of anthocyanidins and anthocyanidins themselves (without bound sugars) can exhibit similar physiological effects. Therefore, when referring to "cyanidin," it should be interpreted as including not only cyanidin alone without any sugar bound to it, but also cyanidin glycosides, in which any sugar is bound to cyanidin. Similarly, references to delphinidin, petunidin, peonidin, malvidin, and anthocyanidins in general should be construed to include these molecules alone or in their glycoside form.

[0053] Anthocyanidins, particularly a combination of cyanidin and delphinidin, can be included in metabolic health-promoting compositions. The combination of cyanidin and delphinidin can be present in an amount sufficient to treat metabolic disorders. In some examples, cyanidin can be included in the composition in an amount of 14% to 20% by weight, and delphinidin in an amount of 10% to 14% by weight. In other examples, cyanidin can be included in an amount of 16% to 17% by weight, and delphinidin in an amount of 12% to 13% by weight. The total amount of anthocyanins in the composition can be 30% to 35% by weight in some examples. These values ​​can be based on the total weight of the composition. As noted above, some examples may include petunidin, peonidin, and / or malvidin. In certain examples, peonidin can be included in an amount of 1.5% to 2% by weight, petunidin can be included in an amount of 0.3% to 0.8% by weight, and malvidin can be included in an amount of 0.5% to 1% by weight.

[0054] Cyanidin and delphinidin are derived from various sources. In a specific example, cyanidin and delphinidin are derived from black rice, blueberry, blackcurrant, crowberry, bilberry, black chokeberry, or a combination thereof. In a more specific example, the combined source of cyanidin and delphinidin can be derived from black rice, blackcurrant, and bilberry.

[0055] The black rice component may be a black rice extract in some examples. The black rice component may comprise about 50% to about 70% by weight of the active fraction of the composition. The active fraction may be the total weight of the black rice, blueberry, blackcurrant, crowberry, bilberry, and black chokeberry components in some examples. In yet another example, the black rice component may comprise about 55% to 65% by weight, about 57.5% to about 62.5% by weight, or about 60% by weight of the active fraction of the composition. In some cases, the black rice component has a standardized anthocyanin content, which may range from about 15% to about 25% by weight, or from about 17.5% to about 22.5% by weight, or about 20% by weight. The black rice component may be derived from rice (Oryza sativa L.).

[0056] The blackcurrant ingredient can be a blackcurrant extract. The blackcurrant ingredient can comprise about 15% to about 45% by weight, or about 25% to about 35% by weight, or about 27.5% to about 32.5% by weight, or about 30% by weight of the active fraction of the composition. Furthermore, the blackcurrant ingredient has a standardized anthocyanin content, which can range from about 25% to about 35% by weight. In a specific example, the blackcurrant ingredient can have a standardized anthocyanin content of about 30% by weight. In another specific example, the blackcurrant ingredient can be derived from blackcurrant (Ribes nigrum).

[0057] The bilberry component can be a bilberry extract. The bilberry component can comprise about 5% to about 15% by weight of the active fraction of the composition. Alternatively, the bilberry component can comprise about 7.5% to about 12.5% ​​by weight of the active fraction of the composition. Alternatively, the bilberry component can comprise about 10% by weight of the active fraction of the composition. In some examples, the bilberry component can have a standardized anthocyanin content of about 25% to about 45% by weight, or about 30% to about 40% by weight. In certain cases, the bilberry component can contain 36% by weight of anthocyanins as measured by HPLC or 25% by weight of anthocyanins as measured by UV. Furthermore, the bilberry component can be derived from Vaccinium myrtillus.

[0058] In various examples, the term "active fraction" may refer to the total amount of anthocyanin-containing extracts in a composition. For example, if a composition contains a combination of black rice extract, black currant extract, and bilberry extract, the active fraction may refer to the total amount of black rice extract, black currant extract, and bilberry extract in the composition. As in the above example, a certain fraction of the extract may be composed of anthocyanin compound molecules themselves. For example, the extract may have a standardized anthocyanin content of about 15% to about 45% by weight.

[0059] In some examples, the composition can include a black rice component, a blackcurrant component, and a bilberry component in a weight ratio of about 6:3.5:1 to about 6:1.5:1. In another example, the ratio can be in the range of about 6:2:0.5 to about 6:2:2.5. In yet another example, the ratio can be in the range of about 4:2:1 to about 8:2:1. In yet another example, the ratio is about 6:2:1. In yet another example, the weight ratio is A:B:C, where A is the weight percentage of the black rice component, B is the weight percentage of the blackcurrant component, and C is the weight percentage of the bilberry component. The value of A can range from 4 to 8, or 4 to 6, or 6 to 8, or 5 to 7. The value of B can range from 1.5 to 3.5, or 2 to 3, or 1.5 to 2.5, or 2.5 to 3.5. The value of C is in the range of 0.5 to 2.5, or 1 to 2.5, or 0.5 to 1.5, or 1.5 to 2.5.

[0060] The compositions described herein may further comprise a pharmaceutically acceptable carrier. The carrier may include various inactive ingredients, including sweeteners, preservatives, flavorings, thickeners, or combinations thereof. In a specific example, the carrier may include oil, lecithin, gelatin, glycerin, or wax. Non-limiting examples of usable oils include olive oil, canola oil, coconut oil, soybean oil, sunflower oil, linseed oil, and combinations thereof. Non-limiting examples of lecithins include sunflower lecithin, soybean lecithin, egg yolk lecithin, and combinations thereof. Non-limiting examples of waxes include beeswax, paraffin wax, carnauba wax, and combinations thereof. In yet another example, the composition may include a coating agent, an isotonic agent, an absorption retardant, a binder, an adhesive, a lubricant, a disintegrant, a colorant, a flavoring, a sweetener, an adsorbent, a surfactant, an antioxidant, a vitamin, a mineral, a protein, a lipid, a carbohydrate, or a combination thereof.

[0061] The dosage form of the composition includes softgels, tablets, powders, beverages, gummies, or other oral dosage forms. In another example, the oral dosage form can be in the form of a capsule, tablet, softgel, lozenge, sachet, powder, beverage, syrup, suspension, or food. As an example, the oral dosage form can be incorporated into a liquid beverage such as water, milk, juice, or soda. In another example, the oral dosage form can be prepared as a nutritional drink. The nutritional drink can be provided as a premix formulation or as a powder mix that can be added to a beverage. In another example, the powder mix can be in the form of granules. In another example, the composition can be a powder that can be sprinkled on food.

[0062] In a specific example, the oral dosage form can be a powder that can be dispersed and / or dissolved in water or other beverages. The powder can include a black rice component, a blackcurrant component, a bilberry component, and a powdered carbohydrate. The powdered carbohydrate can include starch, sugar, maltodextrin, or a combination thereof.

[0063] In one example, the oral dosage form can be designed to be administered to a subject once daily. In one example, the oral dosage form can be designed to be administered to a subject in the morning. In another example, the oral dosage form can be administered in the afternoon or evening. Furthermore, the oral dosage form can be administered during or after a meal. In yet another example, the dosage form can be designed to be administered intermittently. For example, the dosage form can be administered for 2 days on and 1 day off, 3 days on and 2 days off, 3 days on and 4 days off, 4 days on and 3 days off, 5 days on and 2 days off, or 6 days on and 1 day off, and each of these regimens can be designed to be repeated continuously for a certain period of time. In another example, the dosage regimen can be alternating daily on and off. The duration of administration can also be variable. For example, the dosage form can be designed to be administered for 2 weeks, 3 weeks, 1 month, 6 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 1.5 years, or indefinitely.

[0064] In some examples, the black rice ingredient can be included in the oral dosage form in an amount of about 400 mg to about 800 mg. The blackcurrant ingredient can be included in the oral dosage form in an amount of about 150 mg to about 350 mg. The bilberry ingredient can be included in the oral dosage form in an amount of about 50 mg to about 250 mg. The oral dosage form may contain a total amount of anthocyanins of about 215 mg to about 415 mg. In a particular example, the oral dosage form may contain about 245 mg of anthocyanins. In another particular example, the oral dosage form may contain about 215 mg of anthocyanins.

[0065] The present disclosure also describes various methods in which the above-described compositions can be used. In one example, a method for treating a disease or disorder related to metabolic health in a subject can include reducing endotoxin levels compared to baseline endotoxin levels and adjusting cardiometabolic biomarkers related to lipid or glucose metabolism from abnormal cardiometabolic baseline levels to normal levels. In a specific example, the subject can be on a high-fat diet. A high-fat diet can be defined as consuming at least 35% of total calories in the form of fat. However, the subject's high-fat diet can include more fat, such as at least 40%, at least 45%, at least 50%, at least 55%, or at least 60% of total calories in the form of fat.

[0066] In some cases, the subject's body mass index (BMI) may be less than 18.5, or between 18.5 and 24.9, or between 25.0 and 29.9, or between 30.0 and 34.9, or between 35 and 39.9, or a BMI of 40 or greater.

[0067] In yet another example, the method can include reducing the endotoxin level compared to a reference endotoxin level, which can be a reduction of more than 5%, or more than 10%, or more than 20%, or more than 30%, or more than 40%, or more than 50% relative to the reference endotoxin level.

[0068] The method can also include reducing the level of inflammation compared to a baseline level of inflammation, where the reduction in the level of inflammation is achieved by reducing cytokine levels compared to a baseline level or reducing NF-B levels compared to a baseline level.

[0069] In another example, the method includes modulating a cardiometabolic biomarker related to lipid metabolism. The modulation can be achieved by reducing triglyceride levels by more than 5%, more than 10%, more than 20%, more than 30%, more than 40%, or more than 50% compared to baseline levels. Alternatively, modulating the cardiometabolic biomarker can include reducing cholesterol levels by more than 5%, more than 10%, more than 20%, more than 30%, more than 40%, or more than 50% compared to baseline levels.

[0070] Additionally, the method can include decreasing GTT AUC by more than 5%, more than 10%, more than 20%, more than 30%, more than 40%, or more than 50% compared to baseline levels as a means of adjusting a cardiometabolic biomarker related to glucose metabolism. Alternatively, the biomarker related to glucose metabolism can be adjusted by decreasing HbA1c serum levels by more than 0.05, 0.1, 0.15, 0.2, or 0.25 mm / L compared to baseline levels. In another alternative example, the biomarker related to blood glucose metabolism is adjusted by decreasing glucose serum levels measured 2-5 hours after the subject ingests a high-fat meal compared to baseline levels.

[0071] In other examples, the method can include modulating the insulin biomarker by decreasing the ITT AUC by more than 5%, more than 10%, more than 20%, more than 30%, more than 40%, or more than 50% compared to a reference level. Alternatively, the insulin biomarker can be modulated by decreasing IKK phosphorylation levels, JNK1 / 2 phosphorylation levels, or a combination thereof by more than 5%, more than 10%, more than 20%, more than 30%, more than 40%, or more than 50% compared to a reference level.

[0072] The method can further include reducing intestinal barrier permeability compared to baseline levels. In some instances, the intestinal barrier permeability can be reduced by more than 10% as measured using transepithelial electrical resistance (TEER). TEER can be measured using a Millicell-ERS Resistance System (Millipore, Bedford, MA) that includes a dual-electrode volt-ohm meter. TEER is calculated using the following formula: TEER = (Rm - Ri) × A, where Rm is the transmembrane resistance of the intestinal barrier membrane, Ri is the specific resistance of the cell-free medium, and A is the surface area of ​​the membrane cm. 2 Intestinal barrier permeability can also be measured by paracellular transport of FITC-dextran. The intestinal barrier permeability measured by FITC-dextran paracellular transport can be reduced by more than 10%, more than 20%, more than 50%, more than 100%, more than 200%, or more than 300%. Intestinal barrier permeability can also be measured as endotoxin concentration. Intestinal barrier permeability measured by endotoxin concentration can be reduced by more than 10%, more than 20%, or more than 50% in some cases.

[0073] The method can also include altering the relative amount of bacteria in the intestine. In some examples, the method can include reducing the ratio of Firmicutes to Bacteroides by more than one or more of 10%, 20%, 50%, 100%, 200%, 300%, 400%, or 500% compared to a baseline ratio. In yet another example, the method can include increasing the level of Akkermansia by more than one or more of 10%, 20%, 50%, 100%, 200%, 300%, 400%, or 500%.

[0074] The methods described herein also include reducing the level of oxidative stress compared to a baseline level, or reducing the level of bacterial infection compared to a baseline level, or reducing the level of viral infection compared to a baseline level.

[0075] The method can further include increasing a liver biomarker from a baseline level. For example, one or more of CPT-1A, acyl-CoA oxidase, insulin sensitivity, insulin secretion, glucose uptake, or a combination thereof can be increased. The method can also include decreasing a liver biomarker from a baseline level. For example, RBP4, SREBP-1C, lipid accumulation, blood lipids, oxidative stress, or a combination thereof can be decreased. The method can also include increasing an adipose tissue biomarker from a baseline level. For example, AMPK, GLUT4, ACC1, LPL, insulin sensitivity, insulin secretion, glucose uptake, fatty acid oxidation, or a combination thereof can be increased. The method can also include decreasing an adipose tissue biomarker from a baseline level. For example, FAS, hyperglycemia, fatty acid synthesis, gluconeogenesis, serum lipids, or a combination thereof can be decreased.

[0076] The method can further include increasing a skeletal muscle biomarker from a baseline level, such as increasing G6PD, hexokinase, CHO metabolism, glucose uptake, insulin receptor sensitivity, or a combination thereof. The method can also include decreasing a skeletal muscle biomarker from a baseline level, such as decreasing PEPCK.

[0077] Additionally, the method can include increasing a pancreatic biomarker from a baseline level, such as increasing beta cell function, glucose uptake, or a combination thereof. In other examples, the method can include decreasing a pancreatic biomarker from a baseline level, such as decreasing JNK, IL-1-β, IL-6, TNF-α, blood lipid levels, hyperglycemia, oxidative stress, or a combination thereof.

[0078] In various examples, the metabolic condition or disorder can include a condition or disorder related to postprandial metabolic abnormalities, blood pressure, blood glucose, insulin sensitivity, abdominal fat, cholesterol, triglycerides, liver function, or a combination thereof. In a particular example, the condition or disorder is related to blood pressure, and the method includes reducing systolic or diastolic blood pressure by more than 10%, more than 20%, or more than 50% compared to baseline levels. In another example, the metabolic disease or disorder is related to abdominal fat, and the method includes reducing the amount of visceral adipose tissue (VAT) by more than 3%, more than 5%, or more than 10% compared to pre-treatment levels after a selected period of time.

[0079] Another example includes a method for reducing liver inflammation in a subject. This method can include administering an effective amount of a combination of cyanidin and delphinidin to the subject. In a specific example, the subject is obese, and liver inflammation is reduced without adjusting high-fat diet-induced biomarkers. High-fat diet-induced biomarkers include cholesterol, triglycerides, fatty acids, glucose, or insulin. Liver inflammation is reduced without reducing one or more of these biomarkers. In another example, the method can include treating high-fat diet-induced colonic dysfunction in an obese subject without adjusting high-fat diet-induced biomarkers. Colonic dysfunction can include endotoxemia, changes in colonic structure, loss of colonic barrier integrity, reduction of colonic goblet cells, or a combination thereof.

[0080] In another example, the method can include administering an effective amount of a combination of cyanidin and delphinidin to a subject, thereby reducing the expression of TLR4 in the subject. The expression of TLR4 is reduced in the liver or colon, or both, of the subject. In yet another example, the method can include administering an effective amount of a combination of cyanidin and delphinidin to a subject, thereby reducing the expression of toll-like receptor TLR2 in the liver of the subject.

[0081] In yet another example, the method can include reducing endotoxin levels in the plasma of a subject by administering to the subject an effective amount of a combination of cyanidin and delphinidin.

[0082] As another example, the method can include increasing the amount of tight junction proteins in the colon of a subject by administering to the subject an effective amount of a combination of cyanidin and delphinidin, which can include occludin, ZO-1, claudin 1, or a combination thereof.

[0083] In yet another example, the method can include decreasing expression of NADPH oxidase NOX1 in the colon of a subject by administering an effective amount of a combination of cyanidin and delphinidin.

[0084] Embodiment In some examples, the techniques can include the following numbered embodiments: 1. A composition for promoting metabolic health, comprising a combination of cyanidin and delphinidin in amounts sufficient to treat metabolic disorders.

[0085] 2. The composition of any one of embodiments 1-60, wherein the source of the combination of cyanidin and delphinidin is derived from a black rice component, a blueberry component, a blackcurrant component, a crowberry component, a bilberry component, a black chokeberry component, or a combination thereof.

[0086] 3. The composition of any one of embodiments 1-60, wherein the source of the combination of cyanidin and delphinidin is derived from the black rice component, the blackcurrant component, and the bilberry component.

[0087] 4. The composition of any one of embodiments 1-60, wherein the black rice component is a black rice extract.

[0088] 5. The composition of any one of embodiments 1-60, wherein said black rice component comprises about 50% to about 70% by weight of the active fraction of said composition.

[0089] 6. The composition of any one of embodiments 1-60, wherein said black rice ingredient comprises about 55% to about 65% by weight of the active fraction of said composition.

[0090] 7. The composition of any one of embodiments 1-60, wherein the black rice ingredient comprises about 57.5% to about 62.5% by weight of the active fraction of the composition.

[0091] 8. The composition of any of embodiments 1-60, wherein the black rice ingredient comprises about 60% by weight of the active fraction of the composition.

[0092] 9. The composition of any of embodiments 1-60, wherein the black rice ingredient comprises about 62% by weight of the active fraction of the composition.

[0093] 10. The composition of any of embodiments 1-60, wherein the black rice ingredient has a standardized anthocyanin content in the range of about 15% to about 25% by weight.

[0094] 11. The composition of any of embodiments 1-60, wherein the black rice ingredient has a standardized anthocyanin content in the range of about 17.5% to about 22.5% by weight.

[0095] 12. The composition of any of embodiments 1-60, wherein the black rice ingredient has a standardized anthocyanin content of about 20% by weight.

[0096] 13. The composition of any one of embodiments 1-60, wherein the black rice component is derived from rice species (Oryza sativa L.).

[0097] 14. The composition of any one of embodiments 1-60, wherein the blackcurrant component is a blackcurrant extract.

[0098] 15. The composition of any one of embodiments 1-60, wherein the blackcurrant component comprises from about 15% to about 45% by weight of the active fraction of the composition.

[0099] 16. The composition of any one of embodiments 1-60, wherein the blackcurrant component comprises about 25% to about 35% by weight of the active fraction of the composition.

[0100] 17. The composition of any one of embodiments 1-60, wherein the blackcurrant component comprises about 20% to about 25% by weight of the active fraction of the composition.

[0101] 18. The composition of any one of embodiments 1-60, wherein the blackcurrant component comprises from about 27.5% to about 32.5% by weight of the active fraction of the composition.

[0102] 19. The composition of any one of embodiments 1-60, wherein the blackcurrant component comprises about 30% by weight of the active fraction of the composition.

[0103] 20. The composition of any one of embodiments 1-60, wherein the blackcurrant component comprises about 23% by weight of the active fraction of the composition.

[0104] 21. The composition of any one of embodiments 1-60, wherein the blackcurrant component has a standardized anthocyanin content in the range of about 25% to about 35% by weight.

[0105] 22. The composition of any one of embodiments 1-60, wherein the blackcurrant component has a normalized anthocyanin content of about 30% by weight.

[0106] 23. The composition of any one of embodiments 1-60, wherein the blackcurrant component is derived from blackcurrant (Ribes nigrum).

[0107] 24. The composition of any one of embodiments 1-60, wherein the bilberry component comprises a bilberry extract.

[0108] 25. The composition of any one of embodiments 1-60, wherein the bilberry component is in the range of about 5% to about 15% by weight of the active fraction of the composition.

[0109] 26. The composition of any one of embodiments 1-60, wherein the bilberry component is in the range of about 7.5% to about 12.5% ​​by weight of the active fraction of the composition.

[0110] 27. The composition of any one of embodiments 1-60, wherein the bilberry component is about 10% by weight of the active fraction of the composition.

[0111] 28. The composition of any one of embodiments 1-60, wherein the bilberry component is about 15% by weight of the active fraction of the composition.

[0112] 29. The composition of any one of embodiments 1-60, wherein the bilberry component has a standardized anthocyanin content in the range of about 25% to about 45% by weight.

[0113] 30. The composition of any one of embodiments 1-60, wherein the bilberry component has a standardized anthocyanin content in the range of about 30% to about 40% by weight.

[0114] 31. The composition of any one of embodiments 1-60, wherein the bilberry component comprises 36% by weight anthocyanins as measured by HPLC or 25% by weight anthocyanins as measured by UV.

[0115] 32. The composition of any one of embodiments 1-60, wherein the bilberry component is derived from Vaccinium myrtillus.

[0116] 33. The composition of any one of embodiments 1-60, wherein the ratio of the black rice component, the blackcurrant component, and the bilberry component ranges from about 6:3.5:1 to about 6:1.5:1.

[0117] 34. The composition of any one of embodiments 1-60, wherein the ratio of the black rice component, the blackcurrant component, and the bilberry component ranges from about 6:2:0.5 to 6:2:2.5.

[0118] 35. The composition of any one of embodiments 1-60, wherein the ratio of the black rice component, the blackcurrant component, and the bilberry component ranges from about 4:2:1 to 8:2:1.

[0119] 36. The composition of any one of embodiments 1-60, wherein the ratio of the black rice component, the blackcurrant component, and the bilberry component is about 6:2:1.

[0120] 37. The composition of any of embodiments 1-60, further comprising a pharmaceutically acceptable carrier.

[0121] 38. The composition of any of embodiments 1-60, further comprising a sweetener, a preservative, a flavoring agent, a thickener, or a combination thereof.

[0122] 39. The composition of any one of embodiments 1-60, wherein the composition is in an oral dosage form.

[0123] 40. The composition of any one of embodiments 1-60, wherein the oral dosage form comprises a softgel, tablet, powder, beverage, or gummy.

[0124] 41. The composition of any one of embodiments 1-60, wherein the black rice ingredient ranges from about 400 mg to about 800 mg of the oral dosage form.

[0125] 42. The composition of any one of embodiments 1-60, wherein the blackcurrant component comprises from about 150 mg to about 350 mg of the oral dosage form.

[0126] 43. The composition of any one of embodiments 1-60, wherein the bilberry component comprises from about 50 mg to about 250 mg of the oral dosage form.

[0127] 44. The composition of any one of embodiments 1-60, wherein the oral dosage form comprises from about 215 mg to about 415 mg of anthocyanin.

[0128] 45. The composition of any one of embodiments 1-60, wherein the oral dosage form comprises about 245 mg of anthocyanin.

[0129] 46. ​​The composition of any one of embodiments 1-60, wherein the oral dosage form comprises about 215 mg of anthocyanin.

[0130] 47. A composition for reducing inflammation, comprising a combination of cyanidin and delphinidin in an amount sufficient to reduce inflammation, and a carrier comprising an oil, lecithin, gelatin, glycerin, or wax.

[0131] 48. The composition of any one of embodiments 1-60, wherein the carrier comprises the oil.

[0132] 49. The composition of any one of embodiments 1-60, wherein the oil comprises olive oil, canola oil, coconut oil, soybean oil, sunflower oil, flaxseed oil, or a combination thereof.

[0133] 50. The composition of any one of embodiments 1-60, wherein the carrier comprises the lecithin.

[0134] 51. The composition of any one of embodiments 1-60, wherein the lecithin comprises sunflower lecithin, soybean lecithin, egg yolk lecithin, or a combination thereof.

[0135] 52. The composition of any one of embodiments 1 to 60, wherein the carrier comprises the gelatin.

[0136] 53. The composition of any one of embodiments 1-60, wherein the carrier comprises the glycerin.

[0137] 54. The composition of any one of embodiments 1-60, wherein the carrier comprises the wax.

[0138] 55. The composition of any one of embodiments 1-60, wherein the wax comprises beeswax, paraffin wax, carnauba wax, or a combination thereof.

[0139] 56. The composition of any of embodiments 1-60, wherein the cyanidin is present in an amount of 14% to 20% by weight, and the delphinidin is present in an amount of 10% to 14% by weight.

[0140] 57. The composition of any of embodiments 1-60, wherein the cyanidin is present in an amount of 16% to 17% by weight, and the delphinidin is present in an amount of 12% to 13% by weight.

[0141] 58. The composition of any one of embodiments 1-60, wherein the total amount of anthocyanins in the composition is 30% to 35% by weight.

[0142] 59. The composition of any of embodiments 1-60, further comprising peonidin, petunidin, and malvidin.

[0143] 60. The composition of any of embodiments 1-60, wherein the peonidin is present in an amount of 1.5% to 2% by weight, the petunidin is present in an amount of 0.3% to 0.8% by weight, and the malvidin is present in an amount of 0.5% to 1% by weight.

[0144] 61. An embodiment of a method for treating a condition or disorder related to metabolic health in a subject, comprising: A method comprising the steps of reducing endotoxin levels compared to baseline endotoxin levels, and adjusting cardiometabolic biomarkers related to lipid or glucose metabolism from abnormal cardiometabolic baseline levels to normal levels.

[0145] 62. The method of any one of embodiments 61 to 95, wherein the subject is on a high-fat diet.

[0146] 63. The method of any of embodiments 61-95, wherein the subject's body mass index (BMI) is less than about 18.5, 18.5-24.9, 25.0-29.9, 30.0-34.9, 35-39.9, or greater than 40.

[0147] 64. The method of any one of embodiments 61-95, The method further comprising reducing the endotoxin level by more than any one or more of 5%, 10%, 20%, 30%, 40%, 50%, or a combination thereof, compared to a baseline endotoxin level.

[0148] 65. The method of any one of embodiments 61-95, reducing cytokine levels relative to baseline levels; or reducing the level of NF-B relative to a baseline level; The method further comprises the step of reducing the inflammation level relative to a baseline inflammation level by

[0149] 66. The method of any one of embodiments 61-95, reducing triglyceride levels by more than one or more of 5%, 10%, 20%, 30%, 40%, 50%, or a combination thereof, compared to baseline levels; or reducing cholesterol levels by more than one or more of 5%, 10%, 20%, 30%, 40%, 50%, or a combination thereof, relative to a baseline level; The method further comprises adjusting the cardiometabolic biomarkers related to lipid metabolism by

[0150] 67. The method of any one of embodiments 61-95, reducing the GTT AUC by more than one or more of 5%, 10%, 20%, 30%, 40%, 50%, or a combination thereof, relative to a baseline level; or reducing the HbA1c serum concentration by more than one or more of 0.05, 0.1, 0.15, 0.2, 0.25 mmol / L, or a combination thereof, relative to a baseline level; or reducing the glucose serum concentration relative to a baseline glucose serum concentration level measured within 2 to 5 hours after the subject ingests a high-fat meal; The method further comprises adjusting the cardiometabolic biomarker related to glucose metabolism by

[0151] 68. The method of any one of embodiments 61-95, modulating an insulin biomarker by reducing the ITT AUC by more than one or more of 5%, 10%, 20%, 30%, 40%, 50%, or a combination thereof; or modulating an insulin biomarker by decreasing IKK phosphorylation levels, JNK1 / 2 phosphorylation levels, or a combination thereof by more than one or more of 5%, 10%, 20%, 30%, 40%, 50%, or a combination thereof; The method further comprises:

[0152] 69. The method of any one of embodiments 61-95, Reducing intestinal barrier permeability relative to baseline levels The method further comprises:

[0153] 70. The method of any of embodiments 61-95, wherein the intestinal barrier permeability is reduced by more than 10% as measured using TEER.

[0154] 71. The method of any of embodiments 61 to 95, wherein the intestinal barrier permeability is reduced by more than one or more of 10%, 20%, 50%, 100%, 200%, 300%, or a combination thereof, as measured using FITC-dextran paracellular transport.

[0155] 72. The method of any of embodiments 61 to 95, wherein the intestinal barrier permeability is reduced by more than one or more of 10%, 20%, 50%, or a combination thereof, as measured using endotoxin concentration.

[0156] 73. The method of any of embodiments 61-95, further comprising reducing the Firmicutes to Bacteroidetes ratio by more than one or more of 10%, 20%, 50%, 100%, 200%, 300%, 400%, 500%, or a combination thereof, compared to a reference ratio.

[0157] 74. The method of any of embodiments 61-95, further comprising increasing the level of Akkermansia by more than one or more of: 10%, 20%, 50%, 100%, 200%, 300%, 400%, 500%, or a combination thereof.

[0158] 75. The method of any one of embodiments 61-95, reducing the level of oxidative stress compared to a baseline level; or reducing the level of bacterial infection compared to a baseline level; or Reducing the level of viral infection compared to a baseline level The method further comprises:

[0159] 76. The method of any one of embodiments 61-95, increasing liver biomarkers from baseline levels, including increases in one or more of CPT-1A, acyl-CoA oxidase, insulin sensitivity, insulin secretion, glucose uptake, or a combination thereof; or reducing said liver biomarkers from baseline levels, including a reduction in one or more of RBP4, SREBP-1C, lipid accumulation, blood lipids, oxidative stress, or a combination thereof. The method further comprises:

[0160] 77. The method of any one of embodiments 61-95, increasing adipose tissue biomarkers from baseline levels, including increases in one or more of AMPK, GLUT4, ACC1, LPL, insulin sensitivity, insulin secretion, glucose uptake, fatty acid oxidation, or a combination thereof; or reducing said adipose tissue biomarkers from baseline levels, including a reduction in one or more of FAS, hyperglycemia, fatty acid synthesis, gluconeogenesis, serum lipids, or a combination thereof. The method further comprises:

[0161] 78. The method of any one of embodiments 61-95, increasing skeletal muscle biomarkers from baseline levels, including increases in one or more of G6PD, hexokinase, carbohydrate metabolism, glucose uptake, insulin receptor sensitivity, or a combination thereof; or decreasing the skeletal muscle biomarker from baseline levels, including decreasing PEPCK. The method further comprises:

[0162] 79. The method of any one of embodiments 61-95, increasing a pancreatic biomarker from baseline levels, including an increase in one or more of beta cell function, glucose uptake, or a combination thereof; or reducing said pancreatic biomarkers from baseline levels, including a reduction in one or more of JNK, IL-1β, IL-6, TNF-α, blood lipid levels, hyperglycemia, oxidative risk, or a combination thereof. The method further comprises:

[0163] 80. The method of any one of embodiments 61-95, wherein the metabolic condition or disorder is postprandial metabolic disorder.

[0164] 81. The method of any of embodiments 61-95, wherein the metabolic condition or disorder comprises a condition or disorder related to one or more of blood pressure, blood glucose, insulin sensitivity, abdominal fat, cholesterol, triglycerides, liver health, or a combination thereof.

[0165] 82. In any of the methods of embodiments 61-95, the metabolic condition or disorder is related to blood pressure; The method further comprising reducing one or more of the systolic blood pressure or the diastolic blood pressure by more than one or more of 10%, 20%, 50%, or a combination thereof, compared to the baseline level.

[0166] 83. In any of the methods of embodiments 61-95, the metabolic condition or disorder is associated with abdominal fat; The method further comprising reducing the amount of visceral adipose tissue (VAT) by more than one or more of 3%, 5%, 10%, or a combination thereof, compared to pre-treatment levels after a predetermined period of time.

[0167] 84. A method for reducing liver inflammation in a subject, comprising administering to the subject an effective amount of a combination of cyanidin and delphinidin.

[0168] 85. The method of any of embodiments 61-95, wherein the subject is obese and the liver inflammation is reduced without modulating high-fat diet-induced biomarkers.

[0169] 86. The method of any of embodiments 61-95, wherein the high-fat diet-induced biomarker is cholesterol, triglycerides, fatty acids, glucose, or insulin.

[0170] 87. A method for treating high-fat diet-induced colonic dysfunction in obese subjects without modulating high-fat diet-induced biomarkers.

[0171] 88. The method of any of embodiments 61-95, wherein the high-fat diet-induced biomarker is cholesterol, triglycerides, fatty acids, glucose, or insulin.

[0172] 89. The method of any of embodiments 61-95, wherein the colon dysfunction comprises endotoxemia, altered colonic architecture, loss of colonic barrier integrity, and loss of colonic goblet cells.

[0173] 90. A method for reducing the expression of toll-like receptor TLR4 in the liver of a subject, comprising administering to the subject an effective amount of a combination of cyanidin and delphinidin.

[0174] 91. A method for reducing the expression of toll-like receptor TLR2 in the liver of a subject, comprising administering to the subject an effective amount of a combination of cyanidin and delphinidin.

[0175] 92. A method for reducing endotoxin concentration in the plasma of a subject, comprising administering to the subject an effective amount of a combination of cyanidin and delphinidin.

[0176] 93. A method for increasing tight junction proteins in the colon of a subject, comprising administering to the subject an effective amount of a combination of cyanidin and delphinidin.

[0177] 94. A method for reducing the expression of NADPH oxidase NOX1 in the colon of a subject, comprising administering an effective amount of a combination of cyanidin and delphinidin.

[0178] 95. The method of any one of embodiments 61-95, wherein the method comprises administering the composition of any one of embodiments 1-60. [Example]

[0179] Example 1 - Liver inflammation study A series of experiments using mice showed that short-term (i.e., a few weeks) supplementation with cyanidin and delphinidin could prevent and / or reverse high-fat diet-induced endotoxemia and associated liver inflammation in mice.

[0180] Cholesterol and triglyceride concentrations were measured using kits purchased from Wiener Lab Group (Rosario, Argentina). Glucose and alanine aminotransferase levels were measured using kits purchased from Sigma-Aldrich Co. (St. Louis, MO). Insulin concentrations were measured using kits purchased from Crystal Chem Inc. (Downs Grove, IL). Antibodies against monocyte chemoattractant protein-1 (MCP-1) (#2029), TNFα (#11948), phosphorylated (Ser176 / 180)-IKKα / β (#2697), IKKα (#2682), phosphorylated (Ser536)-p65 (#3033), p65 (#8242), TLR2 (#13744), phosphorylated (Thr180 / Tyr182)-p38 (#9211), phosphorylated (Thr202 / Tyr204)-ERK (#4370), ERK (#9102), and β-actin (#12620) were obtained from Cell Signaling Technology (Danvers, MA). Oligonucleotides for NF-κB (sc-2505) and AP-1 (sc-2501), as well as Col1A1 (collagen type I α1 chain) (sc-293182), fibronectin (sc-271098), TLR4 (sc-293072), nitric oxide synthase 2 (iNOS) (sc-649), p38α / β (sc-7149), and HSC-70 (sc-1059) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against 4-hydroxynonenal (4-HNE) (ab46545), NOX1 (ab55831), NOX4 (ab216654), and gp91phox (ab129068), as well as a free fatty acid concentration measurement kit, were purchased from Abcam, Inc. (Cambridge, MA). Hematoxylin and eosin solution was purchased from Thermo Fisher Scientific Inc. (Piscataway, NJ). Plasma endotoxin concentrations were measured using a kit from Abbexa LLC (Houston, TX).Reagents for the T4 polynucleotide kinase and EMS assays were purchased from Promega (Madison, WI). [γ-32P]ATP was purchased from PerkinElmer (Waltham, MA). PVDF membranes and Clarity Western ECL substrate were obtained from Bio-Rad (Hercules, CA). All other chemical reagents were purchased from Sigma-Aldrich Co. (St. Louis, MO). Cyanidin and delphinidin-rich extract (CDRE) was provided by NSE Products, Inc. (Provo, UT). Determination of AC blend composition: Polyphenols present in CDRE were measured by HPLC. The composition of CDRE is shown in Table 1. [Table 1]

[0181] All procedures followed the laboratory animal care standards outlined in the NIH Guide for the Care and Use of Laboratory Animals, and experimental protocols were approved by the University of California, Davis, Animal Use and Care Administrative Advisory Committee prior to implementation. Procedures were performed under the supervision of the University of California, Davis, Animal Resource Services. Mice were purchased from The Jackson Laboratories (Sacramento, CA). Sixty mice (5-week-old male C57BL / 6J mice, weighing 20-25 g) were acclimated to a control diet for 1 week, followed by 4 weeks of feeding either: (i) a diet in which approximately 10% of total calories came from fat (control, group C); or (ii) a diet in which approximately 60% of total calories came from fat (lard) (group HF). After 4 weeks of diet administration, 10 mice from each group were euthanized, and the remaining 20 mice were divided into two subgroups (10 mice each). One subgroup continued on the control diet and HFD, while the other subgroup was supplemented with CDRE, receiving a total of 50 mg of cyanidin (28.9 mg) + delphinidin (21.1 mg) per kg body weight (CDRE-supplemented control diet, CA group; CDRE-supplemented HFD, HFA group) (Figure 6). The amount of CDRE was calculated to represent a achievable intake / supplementation dose in humans and adjusted for mice using the scaling criteria of Reagan-Shaw et al. (S. Reagan-Shaw, M. Nihal and N. Ahmad, Dose translation from animal to human studies revisited, FASEB J., 2008, 22, 659-661). Based on this calculation, a human-equivalent dose of 50 mg of cyanidin and delphinidin per kg of body weight for a 70 kg adult would be equivalent to 50 g of fresh blackcurrants. This calculation is based on reported AC content in foods. Body weight and food intake were measured weekly throughout the study. After two weeks of dietary intake, blood samples (20 μl) were collected from the submandibular vein.Body composition was measured at 4 and 8 weeks after diet initiation using EchoMRI (EchoMRI LCC, Houston, TX) the day before euthanasia. After 4 or 8 weeks of dietary treatment, mice were euthanized by cervical dislocation, and blood was collected from the submandibular vein into EDTA tubes. Plasma was obtained after centrifugation at 3,000 g for 15 minutes at room temperature. Different adipose tissue pads and livers were harvested and weighed. Tissues were either subjected to histopathological processing or flash-frozen in liquid nitrogen and stored at -80°C for further analysis.

[0182] Lipid triglyceride, cholesterol, and free fatty acid contents were measured using various methods. Liver triglyceride content was analyzed after extraction and saponification using a method previously reported by Weber et al. (TE Weber, SL Trabue, CJ Ziemer, BJ Kerr, Evaluation of elevated dietary corn fiber from corn germ meal in growing female pigs, J. Anim. Sci., 2010, 88, 192-201), with slight modifications. Briefly, 100 μl aliquots of 10% (w / v) liver homogenate were mixed with 300 μl of a 1:2 KOH (30% w / v):ethanol (v:v) solution and allowed to evaporate overnight at 55°C. The next day, 1 ml of 50% (v / v) ethanol was added, and the mixture was centrifuged at 10,000 g for 5 min at room temperature. 200 μl of the resulting supernatant was mixed with 15 μl of 1M MgCl2 and placed on ice for 10 minutes. The mixture was then centrifuged at 10,000 g for 5 minutes at room temperature. Ten μl of the supernatant was used to measure triglyceride content using the enzymatic triglyceride kit TG Color GPO / PAP AA (Wiener Lab, Rosario, Argentina). Liver cholesterol content was determined using the Bligh and Dyer method (A. Sundermann, L.F. Eggers, and D. Schwudke, in Encyclopedia of Lipidomics, Springer, Netherlands, Dordrecht, ed. M.R. Wenk, 2016), with slight modifications. Briefly, a 300 μl aliquot of 10% (w / v) liver homogenate was mixed with 900 μl of chloroform:methanol (2:1, v:v) solution. After centrifugation at 3,000 g for 10 min, the supernatant was discarded and the lower layer was collected and evaporated for 1 h 30 min using an Eppendorf Vacuum Cleaner (Eppendorf, Hamburg, Germany). After evaporation, the pellet was resuspended in 50 μl of methanol + 0.05% (v / v) Tween-20 solution and sonicated.Ten microliters of the lipid suspension was analyzed for cholesterol content using the enzymatic total cholesterol kit Cholestat (Wiener Lab, Rosario, Argentina). Free fatty acid content was measured according to the manufacturer's instructions (Free Fatty Acid Quantitative Assay Kit, Abcam Inc., Cambridge, MA). Briefly, 10 mg of liver was homogenized in 200 μl of 1% (v / v) Triton X-100 in chloroform and incubated on ice for 20 minutes. The extract was centrifuged at 14,000 g for 5 minutes at room temperature. The collected organic phase was first air-dried at 50°C and then vacuum-dried for 30 minutes. The dried lipids were resuspended in 200 μl of the fatty acid assay buffer provided with the kit and vortexed vigorously for 5 minutes. Free fatty acid concentration was determined by a fluorometric assay according to the manufacturer's instructions.

[0183] Plasma total cholesterol, triglyceride, free fatty acid, alanine aminotransferase (ALT), glucose, and insulin concentrations were measured according to the manufacturer's guidelines. Plasma endotoxin concentrations were measured using kits from Abbexa LCC (Houston, TX) according to the manufacturer's protocol.

[0184] Liver tissue samples were fixed overnight in 4% (w / v) neutral paraformaldehyde solution. The samples were then washed twice in phosphate-buffered saline, dehydrated, and embedded in paraffin for histological analysis. Five-micrometer-thick sections were taken from the paraffin blocks and mounted on glass slides. Hematoxylin and eosin (H&E) staining was performed according to standard procedures. Sections were examined using an Olympus BX46 microscope (10x eyepiece, 22 fields of view) (Olympus America Inc., Center Valley, PA). The pathologist was blinded to the identity of all samples. After observation, specimens were classified into discrete histological categories, classified as "patterns of liver injury." These patterns were as follows: (i) normal, zone 1 pallor, and zone 3 pallor: no significant changes in hepatocytes; minimal to macrovesicular steatosis; (ii) microvesicular: enlarged hepatocytes with tiny lipid droplets within the cytoplasm; and (iii) hypertrophy / ballooning: defined as slightly to markedly enlarged hepatocytes with distinct cytoplasm. Images were numerically scored according to Kleiner et al.

[0185] Livers were homogenized using a BeadMill (Thermo Fisher Scientific Inc., Piscataway, NJ). Aliquots of the total homogenate containing 30 μg of protein were denatured in Laemmli buffer, separated by 8-15% polyacrylamide gel electrophoresis under reducing conditions, and electrotransferred to PVDF membranes. The membranes were blocked with 5% (w / v) bovine serum albumin or nonfat dry milk for 1 h and then incubated overnight at 4°C in the presence of the corresponding primary antibody (1:1000 dilution). After 90 min of incubation at room temperature in the presence of the corresponding secondary antibody (HRP conjugated) (1:10,000 dilution), the conjugates were visualized by enhanced chemiluminescence. Images were acquired using a Bio-Rad ChemiDoc Imager, and bands were quantified using Image Lab Software (Bio-Rad, Hercules, CA).

[0186] DNA binding of NF-κB and AP-1 was assessed in liver nuclear fractions. EMSA was performed by end-labeling oligonucleotides containing the NF-κB consensus sequence with [γ-32P]ATP. The oligonucleotides were end-labeled with T4 polynucleotide kinase and purified on a Chroma Spin-10 column. Samples were incubated with the labeled oligonucleotides (20,000–30,000 cpm) in 1x binding buffer [5x binding buffer: 50 mM Tris-HCl buffer (pH 7.5), 20% (v / v) glycerol, 5 mM MgCl2, 2.5 mM EDTA, 2.5 mM DTT, 250 mM NaCl, and 0.25 mg / ml poly(dI-dC)] at room temperature for 20 minutes. Products were separated by electrophoresis in 6% (w / v) non-denaturing polyacrylamide gels using 0.5 TBE (45 mM Tris / borate, 1 mM EDTA, pH 8.3) as the run buffer. The gels were dried, and radioactivity was quantified using a Phosphoimager 840 (Amersham Pharmacia Biotech. Inc., Piscataway, NJ).

[0187] Data were analyzed by one-way analysis of variance (ANOVA) using Statview 5.0 (SAS Institute Inc., Cary, NC). Fisher's least significant difference test was used to compare differences in means between groups. Repeated measures ANOVA with Tukey-Kramer multiple comparison test was used to analyze changes in body weight and food intake. A P value of <0.05 was considered significant. Data are presented as mean ± SE.

[0188] The mean daily food intake during the final 4 weeks of the study was significantly lower in mice fed the HFD compared to mice fed the control diet throughout the study (Figure 7). During the same period, the mean daily calorie intake was similar in both groups (Table 2). After 8 weeks of feeding the corresponding diet, HFD consumption increased body weight by 17% (Table 2). [Table 2]

[0189] Weight gain in the HF group began to be significantly higher compared to the control group after 3 weeks of HFD feeding (Figure 8). CDRE supplementation during the final 4 weeks did not affect food intake or weight gain in either control- or HFD-fed mice. Mice fed the HFD had 30% and 40% higher body fat mass at 4 and 8 weeks, respectively, and 7% and 13% lower lean mass at 4 and 8 weeks, respectively. CDRE supplementation did not affect body fat mass or lean mass (Figures 9 and 10). Accordingly, at 8 weeks, the weights of subcutaneous, epididymal, visceral, and retroperitoneal fat pads were 48–92% higher in the HF group compared to the C group (Table 3). CDRE supplementation did not affect any of the above parameters. [Table 3]

[0190] After 8 weeks of dietary intake, plasma total cholesterol and triglyceride concentrations in the HF group were 15% and 25% higher, respectively, than in the C group (Table 2). CDRE supplementation did not affect the increase in total cholesterol. Plasma triglyceride levels in the HFA group were not significantly different from those in the other three groups. Plasma fatty acid levels were not affected by dietary treatment. Plasma glucose and insulin levels were 24% and 67%, respectively, higher in the HF group than in the C group, and HOMA-IR was 78% higher in HF mice, indicating a state of insulin resistance. CDRE supplementation did not change these parameters.

[0191] This study also investigated the effects of HFD and CDRE supplementation on the development of fatty liver (steatosis) and liver damage. Liver weight was 10% lower in the HF and HFA groups compared with the control group. Plasma alanine aminotransferase concentrations, measured as an indicator of liver damage, were similar between groups (Table 2). After 8 weeks of HFD feeding, hepatic triglyceride content was 15% higher in the HF group compared with the C group. CDRE supplementation did not prevent hepatic TG accumulation. After 8 weeks of HFD feeding, hepatic cholesterol and free fatty acid content were similar between all groups (Figures 11 and 12). Protein levels of Col1A1 and fibronectin, markers of liver fibrosis, were similar between all groups (Figures 13 and 14).

[0192] Hepatocellular injury caused by factors such as fat and toxins can induce liver enzyme induction, leading to microvesicular steatosis and hypertrophy / ballooning. These clinical features contribute to the development and progression of NAFLD. After hematoxylin / eosin staining of the liver, changes in the pattern of hepatocellular injury were assessed and scored as follows: (i) microvesicular steatosis, a form of steatosis that is relatively rare in human NAFLD but more pronounced in rodent NAFLD; (ii) hepatocellular hypertrophy / ballooning, characterized by enlarged hepatocytes with distinct cytoplasm. The proportion of hepatocytes containing microvesicular steatosis and hepatocellular hypertrophy was significantly higher in the high-fat group compared with the control group (Figures 15 and 16), suggesting the early stages of fatty liver (steatosis) and liver injury. CDRE supplementation did not affect these parameters.

[0193] HFD consumption can cause endotoxemia, which can induce liver inflammation and promote diseases such as NAFLD. Therefore, plasma LPS levels were also evaluated. A trend toward significantly higher plasma endotoxin concentrations (41%, P = 0.078) was observed after 4 weeks of HFD consumption compared with the control group (Figure 17). After 8 weeks of HFD consumption, plasma endotoxin concentrations in the HF group were significantly higher than those in the control group (44%, P < 0.04) (Figures 17 and 18). CDRE supplementation abolished HFD-associated endotoxemia (Figure 18).

[0194] Upregulation of TLR receptors is frequently observed in association with endotoxemia. Four weeks after HFD feeding, liver protein levels of TLR4 and TLR2 did not change significantly. However, after 8 weeks of feeding this diet, they were 45% and 42% higher in HF mice compared with group C (Figure 19). Four weeks of CDRE supplementation prevented the upregulation of hepatic TLR2 and TLR4 (Figure 20).

[0195] Activation of TLR4 and TLR2 receptors activates proinflammatory signaling cascades, including NF-κB, MAPK, and AP-1. NF-κB activation was assessed by measuring the phosphorylation of IKK and p65 by Western blot and NF-κB-DNA binding in nuclear fractions by EMSA. Four weeks after HFD administration, no changes were observed in p65 phosphorylation at Ser536. After 8 weeks of dietary feeding, IKKα / β phosphorylation and p65 phosphorylation at Ser176 / 180 were 27% and 38%, respectively, higher in the HF group compared with the C group (Figure 21). CDRE supplementation of HFD-fed mice prevented both increases. Accordingly, NF-κB-DNA binding in nuclear fractions was significantly higher in the HF group compared with the C and HFA groups (Figure 22). NF-κB regulates the transcription of genes related to inflammatory responses. Among these, liver protein levels of the chemokine MCP-1, cytokine TNFα, and inducible nitric oxide synthase (iNOS) were evaluated (Figure 23). MCP-1 and TNFα levels were already elevated in the HF group compared with the C group 4 weeks after HFD administration (43% and 1.25-fold higher, respectively). After 8 weeks, MCP-1 and TNFα protein levels were 3-fold and 2.3-fold higher in the HF group compared with the C group. CDRE supplementation attenuated the HFD-induced increase in MCP-1 but did not affect the increase in TNFα. At 8 weeks, iNOS levels were higher in the HF group compared with the C and CA groups, but no significant differences were observed between the HFA group and the HF and CA groups.

[0196] AP-1 is activated by the MAPK family, including p38, ERK1 / 2, and JNK. After 8 weeks of HFD feeding, AP-1 activation, as measured by EMSA, was 50% higher in the HF group compared with all other groups (Figure 24). A positive correlation was observed between NF-κB and AP-1-DNA binding activity (P = 0.001, r = 0.63). From the upstream kinases of AP-1, only ERK1 / 2 phosphorylation at Thr202 / Tyr204 was significantly higher in the HF group than in mice fed a control diet after 4 weeks of HFD feeding. After 8 weeks, hepatic phosphorylation levels of p38 at Thr180 / Tyr182 and ERK1 / 2 at Thr202 / Tyr204 were 92% and 34%, respectively, higher in the HF group compared with the control group (Figure 25). Supplementation with CDRE for 4 weeks prevented the HFD-induced increase in p38 phosphorylation and restored ERK1 / 2 phosphorylation levels to control values. JNK phosphorylation at Thr183 / Tyr185 was not affected by HFD feeding (data not shown).

[0197] The transcription factor HIF-1 is also involved in LPS-mediated inflammatory responses. HIF-1-DNA binding was 40% higher in HFD-fed mice compared with the control and HFA groups (Figure 26). Positive correlations were observed between HIF-1 DNA binding activity and NF-κB (P = 0.001, r: 0.56) and AP-1 DNA binding activity (P = 0.0004, r: 0.63).

[0198] Oxidative stress is one of the major factors in the development of NAFLD. Expression of NADPH, NOX1, NOX2 (gp91phox), and NOX4 was investigated. Protein levels of NOX1 and NOX4 were elevated in the HF and HFA groups compared with the control group (69% and 53%, respectively), whereas no change was observed in NOX2 (Figure 27). Oxidative damage to cellular components was assessed by Western blot analysis of adducts of the lipid oxidation product 4-hydroxynonenal (4-HNE) with liver proteins. The levels of 4-HNE-protein adducts were similar between the groups (Figure 28). This indicates an early stage of NAFLD development following 8 weeks of HFD feeding.

[0199] These results indicate that short-term supplementation with cyanidin and delphinidin can prevent and / or reverse high-fat diet-induced endotoxemia and associated liver inflammation in mice. Overall, CDRE supplementation did not reverse or prevent HFD-induced adiposity, fatty liver, or elevated circulating triglyceride, cholesterol, glucose, and insulin levels, but it did normalize metabolic endotoxemia and reduce liver inflammation.

[0200] Metabolic endotoxemia was already elevated in mice after 4 weeks of HFD feeding and remained elevated even after 8 weeks of diet continuation. CDRE supplementation restored LPS plasma concentrations to control levels. Normalization of plasma LPS by CDRE suggests that endotoxemia associated with a Western diet and / or obesity may be alleviated by dietary modifications that include the consumption of AC-rich fruits and vegetables and / or AC supplementation. At the molecular level, a cyanidin- and delphinidin-rich blend may promote a healthier microbiota profile and alleviate endotoxemia by reducing paracellular transport of LPS caused by loss of intestinal barrier function and the intercellular transport of LPS with absorbed lipids. However, current data do not allow us to identify the mechanisms involved. The beneficial effect of AC in reversing endotoxemia is important for the liver because LPS causes liver inflammation, which, in combination with hepatic steatosis, can progress to NAFLD and further develop into nonalcoholic steatohepatitis (NASH).

[0201] The inflammatory state associated with endotoxemia is reflected by high levels of TLR2 and TLR4 in the liver after 8 weeks of HFD feeding. The absence of high levels of TLR4 after 4 weeks is likely due to the liver maintaining low levels of TLR4 expression and processing LPS without inflammation. However, 8 weeks of high LPS exposure increased TLR4 expression. TLR2 expression also increased after 8 weeks of HFD feeding, but this increase was suppressed by CDRE supplementation. The relationship between these two receptors and LPS is complex. Although LPS is not a ligand for TLR2, TLR2 is required for LPS-induced TLR4 activation in the kidney. Therefore, preventing HFD-induced overexpression of both TLR2 and TLR4 is likely central to the ability of cyanidin and delphinidin to reduce hepatic inflammation.

[0202] Consistent with previous findings, HFD was associated with elevated levels of pro-inflammatory and pro-fibrotic cascades downstream of TLR2 and TLR4, namely, NF-κB, p38, ERK1 / 2, AP-1, and HIF-1. These signaling pathways interact in a manner that exacerbates inflammation. CDRE supplementation completely prevented or reversed the activation of these cascades. Regarding the molecular expression of the inflammatory process and associated oxidative stress, 8 weeks of HFD feeding induced elevated levels of MCP-1, TNFα, iNOS, and the NADPH oxidases NOX1 and NOX4. Meanwhile, CDRE supplementation attenuated the elevation of MCP-1 but did not improve other parameters. This discrepancy may be partially explained by differences in the timing of protein expression during the inflammatory process and / or the signaling regulation of these proteins. Furthermore, different cell types are involved in liver inflammation, which may explain the different responses to cyanidin and delphinidin supplementation. Kupffer cells are thought to be involved in the hepatic inflammatory response to LPS because they are responsible for removing LPS that reaches the liver via the portal vein and initiating TLR4-mediated inflammation. Indeed, Kupffer cell ablation suppresses bone marrow macrophage recruitment and attenuates steatohepatitis. Therefore, the reduction in MCP-1 expression in HFD-fed mice supplemented with CDRE for 4 weeks may be highly relevant in attenuating macrophage recruitment and the amplification of hepatitis.

[0203] Example 2 - Colonic Physiology Studies Another series of experiments was conducted to characterize the effect of CDRE supplementation in mitigating the deleterious effects of HFD in terms of colonic barrier function and goblet cell physiology. These experiments characterized the involvement of TLR-4 and redox-regulated signaling pathways in AC-mediated maintenance of TJ structure and dynamics.

[0204] IκB kinase (IKK) α (#2682), phosphorylated (Ser176 / 180)-IKKα / β (#2697), p65 (#8242), phosphorylated (Ser536)-p65 (#3033), TLR-2 (#13744), phosphorylated (Thr180 / Tyr182)-p38 (#9211), extracellular signal-regulated kinase (ERK1 / 2) (#9102), phosphorylated (Thr202 / Tyr204)-ERK1 / 2 (#4370), and c-jun Antibodies against N-terminal kinase (JNK) 2 (#9258), PI3K (#5405), phosphorylated (Ser473)-Akt (#4060), Akt (#9279), myosin light chain (MLC) (#8505), phosphorylated (Thr18 / Ser19)-MLC (#3674), MyD88 (#4283), myosin phosphatase target subunit 1 (MYPT1) (#8574), phosphorylated (Thr696)-MYPT1 (#5163), and β-actin (#12620) were obtained from Cell Signaling Technology (Danvers, MA). Antibodies against TLR-4 (sc-293072), nitric oxide synthase 2 (iNOS) (sc-649), phosphorylated (Thr183 / Tyr185)-JNK1 / 2 / 3 (sc-6254), and HSC-70 (sc-1059) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against 4-hydroxynonenal (4-HNE) (ab46545), NADPH oxidase (NOX)1 (ab55831), NOX4 (ab216654), and gp91phox (ab129068), as well as kits for measuring free fatty acids and LPS-binding protein (LBP), were purchased from Abcam, Inc. (Cambridge, MA). ZO-1 (33-9100), occludin (33-1500), claudin-1 (71-7800), MLC kinase (MLCK) (PA5-15176), phosphorylated (Thr18 / Ser19)-MLC (PIPA5117239), and Periodic Acid Schiff (PAS) solution were obtained from Thermo Fisher Scientific Inc. (Piscataway, NJ).PVDF membranes and Clarity Western ECL substrates were obtained from Bio-Rad (Hercules, CA). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and all other chemicals were purchased from Sigma-Aldrich Co. (St. Louis, MO). CDRE was provided by NSE Products, Inc. (Provo, UT), and its chemical composition is described in a previous publication

[21] .

[0205] All procedures were performed in accordance with the standards of laboratory animal care outlined in the NIH Guide for the Care and Use of Laboratory Animals. The experimental protocol was approved by the University of California, Davis, Animal Use and Care Management Advisory Committee prior to implementation. Procedures were performed with the assistance of the University of California, Davis, Animal Resource Services. Sixty mice (male C57BL / 6J, 20–25 g) were purchased from Jackson Laboratories (Sacramento, CA) and acclimated to a control diet for 1 week, after which they were fed either: i) a diet in which approximately 10% of total calories came from fat (control, group C); or ii) a diet in which approximately 60% of total calories came from fat (lard) (group HF) for 4 weeks. After 4 weeks of dietary treatment, 10 mice from each group were euthanized, and the remaining 20 mice were divided into two subgroups (10 mice per subgroup): one continued to receive the control and high-fat diets, while the other was supplemented with 50 mg of AC per kg body weight for 4 weeks (control diet supplemented with AC, group CA; HFD supplemented with AC, group HFA). Two weeks after the start of dietary treatment, blood samples (20 μl) were collected from the submandibular vein. After 4 or 8 weeks of dietary treatment, mice were euthanized by cervical dislocation, and blood was collected from the submandibular vein into EDTA tubes. Plasma was obtained after centrifugation at 3,000 x g for 15 minutes at room temperature. Colons were harvested, measured, and weighed, and sections were either processed for histological analysis or flash-frozen in liquid nitrogen and stored at -80°C for further analysis.

[0206] Plasma LPS-binding protein (LBP) levels were measured using a kit from Abcam (Cambridge, MA, USA) according to the manufacturer's protocol.

[0207] Proximal colon samples were fixed overnight in 4% (w / v) neutralized paraformaldehyde solution. Samples were washed twice in phosphate-buffered saline, dehydrated, and embedded in paraffin for histological analysis. 5-μm-thick sections were taken from the paraffin blocks and mounted on glass slides. Periodic Acid Schiff staining (PAS) was performed according to standard procedures. Three to five photographs were taken per animal, and 10 to 12 crypts per photograph were observed using a Leica DMI3000 B microscope (Leica Microsystems Inc., Buffalo Grove, IL) equipped with a 20x eyepiece. Goblet cell number and crypt length were assessed and analyzed using NIH Image J software. The investigators were blinded to the identity of all samples.

[0208] Colon samples were homogenized using a BeadMill 24 (Thermo Fisher Scientific Inc., Piscataway, NJ). Aliquots of total homogenate containing 30–40 μg of protein were denatured in Laemmli buffer, reductively separated by 8–15% polyacrylamide gel electrophoresis, and electrotransferred to PVDF membranes. The membranes were blocked for 5 min with EveryBlot blocking buffer (Bio-Rad, Hercules, CA) and then incubated overnight at 4°C with the corresponding primary antibody (1:750 or 1:1,000 dilution). After 90 min at room temperature with a secondary antibody (HRP-conjugated) (1:10,000 dilution), the label was visualized by enhanced chemiluminescence. Images were captured using a Bio-Rad ChemiDoc Imager, and bands were quantified using Image Lab Software (Bio-Rad, Hercules, CA).

[0209] For quantitative PCR studies, RNA was isolated from colons using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. cDNA was generated using high-capacity cDNA reverse transcriptase (Applied Biosystem, Grand Island, NY). Klf4, Muc2, and Tff3 mRNA levels were assessed by quantitative PCR (iCycler, Bio-Rad, Hercules, CA) using the following primers: Klf4:F:5'-AGCCACCCACACTTGTGACTATG-3' R:5'-CAGTGGTAAGGTTTCTCGCCTGTG-3' Muc2:F:5'-CCATTGAGTTTGGGAACATGC-3' R: 5'-TCGGCTCGGTGTTCAGAG-3' Tff3:F:5'-TAATGCTGTTGGTGGTCCTG-3' R: 5'-CAGCCACGGTTGTTACACTG-3' 18S:F:5'-AGTCCCTGCCCTTTGTACACA-3' R: 5'-GATCCGAGGGCCTCACTAAAC-3'

[0210] Ct values ​​were normalized to the housekeeping gene 18S. Gene expression was determined using the 2-ΔΔCt method (X. Rao, X. Huang, Z. Zhou, X. Lin, An improvement of the 2^(-delta CT) method for quantitative real-time polymerase chain reaction data analysis, Biostat. Bioinforma. Biomath. 3 (2013) 71-85).

[0211] Caco-2 cells were cultured in minimum essential medium (MEM) supplemented with 10% (v / v) fetal bovine serum (FBS), antibiotics (50 U / ml penicillin and 50 μg / ml streptomycin), 1% (v / v) 100X non-essential amino acids, and 1 mM sodium pyruvate at 37°C in a 5% (v / v) CO2 atmosphere. The medium was changed every 3 days during cell growth and differentiation. Cells were used between passages 5 and 20. For experiments, cells were differentiated for 19–21 days in inserts or for 10–12 days in dishes. For experiments in dishes, cells were incubated with LPS (100 ng / ml) for 72 hours in the absence or presence of 1 μM cyanidin 3-O-glucoside, delphinidin 3-O-glucoside, protocatechuic acid (PCA), or gallic acid (GA). For experiments with inserts, Caco-2 cells were treated for 72 hours with LPS (100 ng / ml) in both the upper and lower chambers, in the absence or presence of 1 μM cyanidin 3-O-glucoside, delphinidin 3-O-glucoside, PCA, or GA in the upper chamber. All experiments were performed in 2% (v / v) FBS- and phenol red-free MEM, and the medium was changed every 24 hours.

[0212] The permeability of Caco-2 monolayers was assessed by measuring the transepithelial electrical resistance (TEER) and paracellular transport of FITC-dextran. For both methods, experiments yielded TEER values ​​between 350 and 450 Ω.cm. 2The incubation period was initiated between 19 and 21 days. For assessment of TEER and FITC-dextran permeability, Caco-2 cell monolayers were incubated for 72 hours in the absence or presence of LPS (100 ng / ml) and the 3-O-glucosides of cyanidin and delphinidin, PCA, and GA, as previously described. LPS, the different ACs, and metabolites were replaced daily. After 72 hours of incubation, TEER and FITC-dextran permeability were assessed. TEER was measured using a Millicell-ERS Resistance System (Millipore, Bedford, MA) containing a dual-electrode volt-ohm meter. TEER was calculated as follows: TEER = (Rm - Ri) × A (where Rm is the transmembrane resistance of the intestinal barrier membrane, Ri is the specific resistance of the cell-free medium, and A is the surface area of ​​the membrane cm). 2 The clearance (CL) of FITC-dextran (4 kDa) from the apical to the basolateral side was calculated using the following formula: fFITC / (FFITC / A), where fFITC is the flux of FITC-dextran (fluorescence units / h), FFITC is the fluorescence of FITC-dextran in the upper chamber at time zero (fluorescence units / nl), and A is the surface area of ​​the membrane (1 cm). 2 ) Arbitrary units (AU) were calculated based on the TEER or CL values ​​of untreated (control) cells.

[0213] Cell viability was assessed by the MTT assay, based on the conversion of MTT to formazan crystals by viable cells. Cells were cultured in 96-well plates for 10 days. Caco-2 cells were treated with LPS (100 ng / ml) for 72 hours in the presence or absence of cyanidin and delphinidin 3-O-glucosides, PCA, and GA. After 72 hours, 5 μl of a 0.5 mg / ml MTT solution in PBS was added to 100 μl of medium and incubated at 37°C for 2 hours. The reaction was stopped by adding 100 μl of 0.01 N HCl containing 10% (w / v) SDS, and the plate was incubated overnight. Absorbance (λ570–λ690 nm) was measured using a Biotek Synergy H1 plate reader (BioTek Instruments, Winooski, VT) and normalized to the value for control (untreated) cells.

[0214] Data were analyzed by one-way analysis of variance (ANOVA) or Student's t-test using Statview 5.0 (SAS Institute Inc., Cary, NC). Fisher's least significant difference test was used to compare differences in means between groups. For dynamic analysis of LBP, two-way ANOVA was used to evaluate the experimental effects of time (0, 2, 4, and 8 weeks), treatment (C or HF), and their interaction. If significant differences were observed, Tukey's Honest Significant Difference (HSD) post-hoc test for multiple comparisons was used to identify differences between means. A P value of <0.05 was considered significant. Data are presented as mean ± SE.

[0215] As previously reported, mice fed an HFD for 8 weeks developed obesity, insulin resistance, and hyperlipidemia in this group, and CDRE supplementation during the final 4 weeks did not prevent these symptoms. LBP, a known marker of metabolic endotoxemia, is a protein that binds LPS and transports it to a soluble or membrane-bound form at C14, which then binds to TLR-4. Plasma LBP levels showed a significant interaction between the C and HF groups over time (0–8 weeks) (p<0.01). Thus, similar to plasma LPS, plasma LBP concentrations in the HF group fed an HFD for 8 weeks were significantly higher (95%) than those in the control group at the corresponding time points. Figure 29 shows plasma LBP concentrations in the control group (C) and the high-fat group (HF). CDRE supplementation during the final 4 weeks of HFD feeding attenuated the increase in plasma LBP concentrations caused by HFD feeding. FIG. 30 shows plasma LBP concentrations in the control group (C), the anthocyanin-supplemented control group (CA), the high-fat group (HF), and the anthocyanin-supplemented high-fat group (HFA).

[0216] After 4 weeks of HFD feeding, colonic TLR-4 and downstream MyD88 protein levels were not significantly affected (Figure 31). However, consistent with increased exposure to LPS, after 8 weeks of HFD feeding, colonic TLR-4 and MyD88 levels in the HF group were 24% and 48%, respectively, higher than those in the C group. These increases were prevented by 4 weeks of CDRE supplementation (Figure 32). Colonic TLR-2 levels were not affected by HFD feeding or CDRE supplementation.

[0217] Exposure to high levels of LPS induces increased permeability, which may be secondary to upregulation of TLR-4-induced and redox-sensitive signaling. Therefore, we investigated parameters of TJ structure and function in the colon affected by HFD. Expression of the TJ proteins occludin, ZO-1, and claudin-1 in the colon was assessed by Western blot. After 4 weeks of dietary intake, HF mice showed a 39%, 33%, and 52% decrease in occludin, ZO-1, and claudin-1 levels, respectively, compared with control mice (Figure 33). This decrease in TJ protein expression continued after 8 weeks of HFD intake. Accordingly, HF mice showed a 29%, 23%, and 23% decrease in occludin, ZO-1, and claudin-1 protein levels, respectively, compared with control mice. CDRE supplementation during the final 4 weeks of treatment restored occludin, ZO-1, and claudin-1 protein levels to control values ​​(Figure 34).

[0218] TJ dynamics are regulated by MLCK, a kinase that phosphorylates MLC, leading to TJ opening and increased paracellular permeability. MLCK expression and MLC phosphorylation were unchanged after 4 weeks of HFD feeding (Figure 35). After 8 weeks, HFD increased MLCK expression by 23% and MLC phosphorylation by 41%, which were prevented by AC supplementation (Figure 36). Next, we assessed the phosphorylation level of MYPT-1, a subunit of MLC phosphatase (MLCP), an enzyme that dephosphorylates MLC. MYPT-1 phosphorylation at Thr696 was not affected by 8 weeks of HFD feeding or CDRE supplementation.

[0219] To assess whether LPS / TLR-4 regulation is involved in preventing AC-induced barrier hyperpermeability, the ability of the 3-O-glucosides of cyanidin and delphinidin, as well as their gut microbiota metabolites PCA and GA, to prevent LPS-induced hyperpermeability of Caco-2 cell monolayers was investigated. To first determine the optimal incubation time for inducing TJ changes by LPS, cells were treated with LPS (100 ng / ml) for 24 to 96 hours. Cells treated with LPS for 72 hours showed a 29% decrease in occludin protein levels compared to untreated controls (Figure 37). This treatment did not affect Caco-2 cell viability (Figure 38). TEER and FITC-dextran paracellular transport were measured to assess monolayer permeability. Incubation for 72 hours in the presence of LPS added to the upper and lower chambers resulted in a 22% decrease in TEER and a 45% increase in FITC-dextran paracellular transport compared to untreated cells (Figure 39). Addition of 1 μM cyanidin, delphinidin 3-O-glucoside, PCA, and GA to the upper chamber prevented the LPS-induced decrease in TEER and increase in FITC-dextran transport.

[0220] Consistent with the loss of barrier integrity, after 72 hours of incubation, LPS reduced the protein levels of occludin, ZO-1, and claudin-1 by 27%, 24%, and 24%, respectively (Figure 40). Cyanidin and delphinidin 3-O-glucoside at 1 μM concentrations were effective in preventing the LPS-induced reduction in all TJ proteins tested. PCA (1 μM) blocked the observed reduction in ZO-1 and claudin-1, but not occludin. GA (1 μM) treatment restored claudin-1 levels but did not prevent the reduction in occludin and ZO-1 protein.

[0221] TJ dynamics can be altered by LPS. In this regard, LPS, AC, and its metabolites did not affect MLCK expression in Caco-2 cells (Figure 41). After 72 hours of incubation with LPS, a significant increase (33%) in MLC phosphorylation levels was observed. This was associated with a 37% increase in MYPT-1 phosphorylation at Thr696, which inhibits the phosphatase that dephosphorylates MLC. At 1 μM concentrations, cyanidin 3-O-glucoside, delphinidin 3-O-glucoside, PCA, and GA prevented the LPS-induced increase in MYPT-1 and MLC phosphorylation. Therefore, the ability of AC and its metabolites to attenuate LPS-induced hyperpermeability may be due in part to the preservation of tight junction structure and the inhibition of MLC phosphorylation.

[0222] Given the detrimental effects of LPS on the intestinal monolayer and the involvement of redox imbalance in the development of intestinal permeability, investigations were then directed toward the ability of the 3-O-glucosides of cyanidin and delphinidin, PCA, and GA, to regulate NOX1 expression and protein / lipid oxidative modifications in LPS-treated Caco-2 cells. It was found that incubation with LPS for 72 hours did not increase NOX1 protein expression but did increase the level of 4-HNE-protein adducts by 33% (Figure 42). At 1 μM concentrations, the 3-O-glucosides of cyanidin and delphinidin, PCA, and GA, were effective in preventing the LPS-induced increase in 4-HNE-protein adducts. Oxidative damage to cellular components may be due to increased production of oxidants. Therefore, the oxidation of the DHE probe in LPS-treated Caco-2 cells was next assessed. After 72 hours of incubation, a 30% increase in DHE oxidation was observed compared to untreated (control) cells. Cyanidin 3-O-glucoside, PCA, and GA, but not delphinidin 3-O-glucoside, attenuated the LPS-induced increase in DHE oxidation (Figure 43).

[0223] Upregulation of TLR-4 may be associated with downstream activation of PI3K / Akt, NF-κB, and MAPKs. These signaling cascades are involved in regulating intestinal tight junction (TJ) function. After 4 weeks of HFD feeding, no changes were observed in the activation of PI3K / Akt, p65, ERK1 / 2, and JNK1 / 2 (Figure 44). However, all of these pathways were activated after 8 weeks of HFD feeding. Specifically, PI3K protein levels and downstream Akt phosphorylation at Ser473 were 26% and 33%, respectively, higher in the HF group compared with the C group (Figure 45). Both events were prevented by CDRE supplementation. Next, IKK and p65 phosphorylation were evaluated as parameters of NF-κB pathway activation. After 8 weeks of HFD feeding, phosphorylation of IKK at Ser176 / 180 and phosphorylation of p65 at Ser536 were 37% and 41%, respectively, higher in HFD-fed mice compared with controls (Figure 46). These changes were prevented by CDRE supplementation. Among MAPKs, ERK1 / 2 and JNK1 / 2, but not p38, were found to be activated in the colon of mice fed an HFD for 8 weeks. Specifically, phosphorylation of ERK1 / 2 at Thr202 / Tyr204 and phosphorylation of JNK1 / 2 at Thr183 / Tyr185 were 38% and 31%, respectively, higher in the HF group compared with the C group (Figure 47). CDRE supplementation completely prevented these increases.

[0224] Cellular redox homeostasis was also investigated, including 、 O2 -The evaluation of the expression of enzymes involved in HO, HO, and NO production, namely NADPH oxidase and iNOS, was included. Of these enzymes, only NOX1 protein levels were 55% higher in the HF group compared to the control group after 8 weeks of HFD consumption, which was prevented by CDRE supplementation (Figure 48). This increase in NOX1 expression was not observed after 4 weeks of HFD consumption (Figure 49). NOX2, an isoform abundant in macrophages but negligible in epithelial cells, was not affected by HFD or CDRE supplementation. This is consistent with the lack of effect of HFD on the levels of the macrophage marker F4 / 80. In mice fed the control diet, CDRE supplementation significantly reduced NOX2 protein levels by 34%.

[0225] Regarding oxidative damage to cellular components, 4-HNE-protein adducts, which reflect both lipid and protein oxidation, were not affected in the colon by 8 weeks of HFD feeding and / or CDRE supplementation (Figure 48).

[0226] HFD feeding can induce changes in colonic morphology. This was confirmed by assessing the colon weight / length ratio and colonic crypt length, indicators of tissue inflammation. HFD feeding caused a significant increase in the colon weight / length ratio (16% at week 4 and 12% at week 8) and a decrease in crypt length (19% at both time points) (Figure 50). Four weeks of CDRE supplementation restored these values ​​to the levels of control mice.

[0227] Goblet cells play an important role in maintaining barrier function. HFD consumption altered goblet cell numbers and functional parameters. Accordingly, histological analysis by PAS staining in mice fed an HFD for 4 and 8 weeks revealed a 19% and 23% decrease in the number of goblet cells per crypt, respectively, compared with control animals (Figures 51 and 52). CDRE supplementation restored goblet cell numbers in HFD-fed mice. The mRNA levels of goblet cell differentiation markers (Kruppel-like factor 4, Klf4), mucin production (Muc2), and intestinal mucosal repair (trefoil factor 3, Tff3) were assessed by qPCR. No significant changes in mRNA levels were observed after 4 weeks of HFD consumption. After 8 weeks, Klf4 mRNA levels were significantly lower (33%) in HFD-fed mice compared with control animals, which was prevented by CDRE supplementation (Figure 53). While a HFD had no significant effect on Muc2 RNA levels at 4 and 8 weeks, CDRE supplementation resulted in 64% higher Muc2 mRNA levels compared to the HF group. After 8 weeks of HFD feeding, Tff3 mRNA levels were significantly lower (57%) in the HF group compared to the C group. CDRE supplementation not only alleviated the HFD-induced downregulation of Tff3 mRNA levels, but also significantly higher (48% compared to the C group) compared to all other groups.

[0228] Short-term supplementation with cyanidin and delphinidin reversed or attenuated HFD-induced changes in colonic permeability, redox balance, and immune defense parameters. The beneficial effects of AC in protecting the intestinal barrier against fat-induced damage are primarily mediated through TLR-4 and redox regulatory mechanisms. In the colon, HFD feeding caused impaired TJ structure and function. This was associated with increased expression of NOX1 and TLR4, resulting in oxidative stress and activation of TLR-4-regulated signaling pathways (NF-κB, PI3K / Akt, ERK1 / 2, and JNK1 / 2), which regulated TJ opening. All of these events were prevented or reversed by 4 weeks of AC supplementation. Subsequent mechanistic analysis of Caco2 cells treated with LPS supports the role of cyanidin, delphinidin, and their major microbial metabolites in preventing monolayer permeability, regulating redox homeostasis, and modulating redox and TLR-4-regulated signaling. Furthermore, HFD also affected colonic goblet cell number and function, but these changes were reversed by CDRE supplementation.

[0229] These results indicate the development of metabolic endotoxemia between weeks 4 and 8 after HFD feeding. Supporting the involvement of endotoxemia in HFD-associated colonic dysfunction, TLR-4 levels and downstream signaling cascades, namely NF-κB (IKK, p65), MAPKs ERK1 / 2 and JNK, and the PI3K / Akt pathway, were elevated at week 8 after HFD feeding. Meanwhile, the decrease in TJ proteins and abnormal goblet cell function parameters at week 4 of HFD feeding suggest that these events precede endotoxemia and involve TLR4- and redox-mediated changes. The results demonstrate the efficacy of CDRE in alleviating both altered parameters at weeks 4 and 8 of HFD feeding, and these changes appear to be driven via distinct mechanisms. Four weeks of CDRE supplementation prevented endotoxemia and suppressed a series of events involving TLR-4 and downstream signaling, supporting the pharmacological potential of cyanidin and delphinidin in preventing the progression of high-fat diet-induced impairment of colonic barrier function and immune homeostasis. The early stage of HFD-induced colonic injury may be partly due to increased intestinal bile acids. In this regard, deoxycholic acid promotes permeability of Caco-2 cell monolayers through increased oxidative stress production by NADPH oxidase and mitochondria and activation of redox signaling, which is inhibited by the flavan-3-ol (-) epicatechin.

[0230] Intestinal barrier function is maintained by TJs, which restrict the passage of molecules other than water and ions across epithelial cells. Four weeks after HFD feeding, abnormalities in colonic barrier structure were confirmed by decreased content of key TJ protein structural components, namely, occludin, ZO-1, and claudin-1. After 8 weeks, other events related to TJ opening were affected: i) activation of TJ regulatory signaling cascades, namely, NF-κB, ERK1 / 2, and Akt; and ii) increased expression of MLCK and increased phosphorylation of MLC (which leads to actin-myosin ring contraction). CDRE supplementation restored all HFD-altered barrier parameters and the downstream events mentioned above. Cyanidin and delphinidin, assayed as O-glucosides, and their microbial metabolites, GA and PCA, prevented LPS-induced permeability in Caco-2 cell monolayers. These results support the notion that cyanidin and delphinidin, but not other minor components in CDRE, are the primary components protecting the colonic epithelial barrier from HFD-induced damage.

[0231] The ability of AC to suppress redox and TLR-4-regulated signaling (NF-κB, MAPK, PI3K / Akt) may be involved in its ability to restore or prevent lipid- and LPS-induced impairment of barrier structure and function. During TJ dynamics, phosphorylation of MLC by MLCK induces contraction of the peripheral junction actin-myosin ring, subsequently leading to TJ opening. This can also be triggered by inhibitory phosphorylation of MLC phosphatase (MYPT-1) followed by increased MLC phosphorylation. NF-κB, MAPK, and PI3K / Akt regulate different events related to TJ dynamics. In this regard, NF-κB and ERK1 / 2 promote MLCK gene transcription, NF-κB regulates ZO-1 expression and junctional localization, and ERK1 / 2 phosphorylates and inactivates MYPT-1. JNK1 / 2 activation indirectly causes barrier dysfunction through its pro-inflammatory effects. Activation of the PI3K / Akt pathway increases barrier permeability, in part by downregulating claudin-1 expression. It also increases the expression of the pore-forming protein claudin-2. Mechanistically, observations in mice and Caco-2 cells differed, with increased MLC phosphorylation in both models. However, in HFD mice, this was primarily due to MLCK overexpression, whereas in cells, it was due to MYPT1 inactivation. This further supports the involvement of additional mechanisms in HFD-induced barrier dysfunction, in addition to LPS / TLR-4-mediated mechanisms.

[0232] Exposure to high fat levels affects gastrointestinal redox homeostasis, in part through upregulation of NOX1. Although distributed throughout the gastrointestinal tract, NOX1 is abundantly expressed in colonic epithelial cells. While NOX1 is a key factor maintaining gastrointestinal physiology, its overactivation is also associated with pathology. In this regard, increased NOX1 expression has been shown in animal models of colitis-associated tumors. Both TLR-4 and NOX1 transcripts are upregulated in patients with Crohn's disease and colorectal cancer, suggesting a link between TLR-4 upregulation and NOX1 overexpression in human GI diseases. Indeed, in response to the luminal microbiota, TLR-4 activation leads to NOX1 transcription, which is driven in part by NFκB. Supporting this link, we observed that HFD consumption upregulates both TLR-4 and NOX1. In this context, the ability of CDRE to suppress TLR-4 / NF-κB / NOX1 upregulation may be considered as a strategy to restore colonic redox homeostasis and barrier function.

[0233] HFD consumption also affects goblet cells, a major component of the colonic immune system. Indeed, in mouse colons, an HFD reduces goblet cell number and proliferation. A 4- and / or 8-week HFD diet reduced goblet cell population and the mRNA levels of proteins produced by these cells, namely, Klf4, which is involved in goblet cell differentiation, and Tff3, which regulates mucus viscosity and mucosal repair. CDRE supplementation not only restored the levels of these goblet cell-produced proteins, but also increased Tff3 mRNA levels above control levels. This latter effect is likely due to AC's ability to help repair HFD-induced colonic mucosal damage. Tff3 regulates the rapid migration of surface epithelium across the basement membrane and is essential for mucosal repair. In the presence of inflammation or epithelial damage, Tff3 promotes GI mucosal repair. Overall, the beneficial effects of AC on goblet cell number and function are crucial for mitigating the detrimental effects of a high-fat diet on the GI immune system.

[0234] In summary, CDRE prevented or reversed HFD-induced changes in colonic structure, barrier integrity, and goblet cell number and differentiation. These results suggest the involvement of mechanisms related to TLR-4 activation, elevated NOX1, and loss of redox homeostasis, and these changes were attenuated by AC in vitro and / or in vivo. Furthermore, CDRE supplementation reversed the changes in TJ structure that preceded endotoxemia. Thus, the consumption of an AC-containing diet, particularly one rich in cyanidin and delphinidin, may protect against the deleterious effects of a high-fat diet on the colon.

[0235] Figure details for Example 2 are explained below: Figures 29, 30, and 32: Effect of CDRE supplementation on LPS-binding protein and colonic TLR and MyD88 expression in mice fed a control diet and a high-fat diet. Mice were fed different diets as described in the methods. The following parameters were measured at 0, 2, 4, and / or 8 weeks of the corresponding diet: A, B - Plasma LPS-binding protein (LBP) concentration. A - Dynamics of HFD-induced plasma LBP changes, and B - Plasma LBP concentration at 8 weeks. C - TLR-2, TLR-4, and MyD88 protein levels in the colon were measured by Western blot. Bands were quantified, and values ​​were referenced to HSC-70 levels (loading control), while CA, HF, and HFA results were referenced to control group values ​​(C). Results are shown as the mean ± SE of 9–10 mice / group. A- * There was a significant difference from C at the corresponding time point (p<0.05), B- * Significantly different compared to all other groups. C - Values ​​with different symbols are significantly different (p<0.05, one-way ANOVA and two-way ANOVA).

[0236] Figures 33, 34, and 36: Effect of CDRE supplementation on HFD-induced changes in tight junction proteins and events related to tight junction opening. Mice were fed different diets as described in the methods. A, B - Levels of tight junction proteins, namely occludin, ZO-1, and claudin-1, were measured by Western blot in the colon 4 and 8 weeks after the corresponding treatment. C - After 8 weeks of feeding the corresponding diet, MLCK expression, MYPT-1 (Thr696), and MLC (Thr18 / Ser19) phosphorylation in the colon were assessed by Western blot. Bands were quantified, and values ​​for occludin, ZO-1, claudin-1, and MLCK were referenced to HSC-70 levels (loading control), while values ​​for MYPT-1 and MLC were referenced to total protein levels. Results for CA, HF, and HFA were referenced to the control group value (C). Results are shown as the mean ± SE of 9–10 mice per group. * Significantly different compared to other groups (p<0.05, one-way ANOVA). Values ​​with different symbols are significantly different.

[0237] Figures 39, 40, and 41: Effects of cyanidin, delphinidin, and their metabolites on LPS-induced intestinal permeability, TJ protein levels, and TJ regulatory events in Caco-2 cell monolayers. Caco-2 cells were treated with LPS for 72 hours and cultured in the presence or absence of cyanidin-3-O-glucoside, delphinidin-3-O-glucoside, protocatechuic acid (PCA), and gallic acid (GA) as described in the methods. A - Permeability of Caco-2 cell monolayers was assessed by measuring TEER and paracellular transport of FITC-dextran. The following parameters were measured by Western blot: B - TJ protein levels (occludin, ZO-1, and claudin-1); and C - MLCK protein levels, MYPT-1 (Thr696), and MLC phosphorylation (Thr18 / Ser19). Bands were quantified, and values ​​for occludin, ZO-1, claudin-1, and MLCK were referenced to HSC-70 levels (loading control), while values ​​for MYPT-1 and MLC were referenced to total protein levels. *Significantly different compared to other groups. Results were referenced to control, untreated cell values ​​(C). Results are shown as the mean ± SE of 9–10 independent experiments. Values ​​with different symbols are significantly different (p<0.05, one-way ANOVA).

[0238] Figures 42 and 43: Effect of cyanidin, delphinidin, and their metabolites on oxidative stress parameters in LPS-treated Caco-2 cell monolayers. Caco-2 cells were treated for 72 hours with or without LPS and 1 μM cyanidin- and delphinidin-3-O-glucosides, procatechuic acid (PCA), and gallic acid (GA), as described in the Methods. A - Expression of NOX1 and 4-HNE was assessed by Western blot. B - Oxidation of the fluorescent probe DHE was assessed as described in the Methods. NOX1 and 4-HNE bands were quantified, and values ​​were referenced to HSC-70 levels (loading control). DHE (oxidized DHE) fluorescence was normalized to the absorbance of sulforhodomin B (SRB). *Significantly different compared to other groups. Results were referenced to control, untreated cell values ​​(C). Results are shown as the mean ± SE of 9–10 independent experiments. * Significantly different compared to other groups (p<0.05, one-way ANOVA). Results were referenced to control, unspiked cell values ​​(C).

[0239] Figures 45, 46, 47: Effect of CDRE supplementation on PI3K / Akt, NF-κB, and MAPK pathways in the colon of HFD-fed mice. Mice were fed different diets as described in the methods. At 8 weeks on the corresponding diets, the following parameters were measured in the colon: A - PI3K protein level and Akt phosphorylation (Ser473). B - NF-κB activation was assessed by measuring the phosphorylation of IKK (Ser176 / 180) and p65 (Ser536). C - MAPK activation was assessed by measuring the phosphorylation of p38 (Thr180 / Tyr182), ERK1 / 2 (Thr202 / Tyr204), and JNK1 / 2 (Thr183 / Tyr185). Bands were quantified, and PI3K values ​​were expressed based on HSC-70 levels (loading control), and Akt, IKK, p65, p38, ERK1 / 2, and JNK1 / 2 values ​​were expressed based on total protein levels. All results for CA, HF, and HFA were expressed based on control values ​​(C). Results are shown as the mean ± SE of 9–10 animals per group. * Significantly different compared to all other groups (p<0.05, one-way ANOVA).

[0240] Figure 48: Effect of CDRE supplementation on colonic oxidative stress and inflammation parameters in HFD-fed mice. Mice were fed different diets as described in the methods. At week 8 on the corresponding diet, the following parameters were measured by Western blot in the colon: NOX1, gp91phox (NOX2), NOX4, iNOS, and 4-HNE-protein adducts. Bands were quantified, and values ​​were presented based on HSC-70 levels (loading control). Results for CA, HF, and HFA were presented based on the control group value (C). Results are presented as the mean ± SE of 9–10 animals per group. Values ​​with different symbols are significantly different (p<0.05, one-way ANOVA).

[0241] Figures 50, 51, and 53: Effect of CDRE supplementation on HFD-induced colonic structural abnormalities and goblet cell number and function. Mice were fed different diets as described in the methods. After 4 and / or 8 weeks of corresponding treatment, the following parameters were measured: A - colon weight / colon length, B - crypt length, C - goblet cell number, and D - Images of colonic Periodic Acid-Schiff (PAS) histological staining of the colon after 8 weeks. E - Genes involved in goblet cell differentiation, namely Klf4, Muc2, and Tff3, were evaluated by qPCR at 8 weeks of dietary treatment, and values ​​were normalized to 18S (housekeeping gene). Results for E-CA, HF, and HFA were referenced to control group values ​​(C). All results are shown as the mean ± SE of 9 mice / group. Values ​​with different superscripts are significantly different (p<0.05, one-way ANOVA).

[0242] Figures 31, 44, and 49: Effects of 4 and 8 weeks of HFD feeding on TLR-4 and downstream pathways in the colon. Mice were fed a control diet or an HFD for 4 or 8 weeks. A - TLR4 and MyD88 protein levels in the colon; B - PI3K protein levels and Akt phosphorylation; C - p65 phosphorylation; D - ERK1 / 2 and JNK1 / 2 phosphorylation; and E - NOX1 protein levels were measured by Western blot. Bands were quantified, and values ​​were referenced to total protein levels (Akt, p65, ERK1 / 2, and JNK1 / 2) or HSC-70 (TLR4, MyD88, PI3K, and NOX1). Results for the HF group were referenced to the control group value (C) at the corresponding time point. Results are shown as the mean ± SE of 6–9 mice per group. * Significantly different from control (p<0.05, one-way ANOVA).

[0243] Figure 35: Effect of 4 and 8 weeks of HFD feeding on MLCK expression and MLC phosphorylation. Mice were fed a control or high-fat diet for 4 or 8 weeks. MLCK expression and MLC (Thr18 / Ser19) phosphorylation were assessed by Western blotting in tissues collected from the colon. Bands were quantified, and MLCK values ​​were referenced to HSC-70 levels (loading control) or total MLC protein levels. Results were referenced to control values ​​(C). Results are shown as the mean ± SE of 6–8 mice / group. * Significantly different compared to group C. Values ​​with different symbols are significantly different (p<0.05, one-way ANOVA).

[0244] Figures 37 and 38: Effect of LPS treatment on occludin protein levels and cell viability in Caco-2 cells. A - Caco-2 cells were treated with LPS for 24, 48, 72, and 96 hours. Changes in occludin protein over time were assessed by Western blot. B - Caco-2 cells were treated with LPS alone or in the presence of 1 μM cyanidin- and delphinidin-3-O-glucosides, protocatechuic acid (PCA), and gallic acid (GA) for 72 hours, as described in the methods. Cell viability was assessed by MTT assay, as described in the methods. Occludin bands were quantified, and values ​​were referenced to HSC-70 levels (loading control). MTT results were referenced to the values ​​of control, untreated cells (C). Results are shown as the mean ± SE of 7–8 independent experiments. * Significantly different from the corresponding time point. ** Significantly different compared to 24 hours (p<0.05, one-way ANOVA).

[0245] Figure 52: Effect of 4 and 8 weeks of HFD feeding on colonic crypt length, goblet cell number, and function. Mice were fed a control diet or an HFD for 4 or 8 weeks. A - Images of Periodic Acid-Schiff (PAS) tissue staining of the colon after 4 weeks of control diet and HFD feeding. B - Genes involved in goblet cell differentiation, namely Klf4, Muc2, and Tff3, were evaluated by qPCR, and values ​​were normalized to 18S (housekeeping gene). Results for the HF group were shown relative to the control group value (C) at the corresponding time point. Results are shown as the mean ± SE of 6–9 mice per group. * Significantly different from the control group (p<0.05, one-way ANOVA).

[0246] Example 3 - Randomized, Placebo-Controlled Crossover Study of the Effect of an Anthocyanin-Rich Blend on Inflammatory and Metabolic Responses to a High-Fat Meal in Healthy Human Subjects This study investigated the beneficial effects of supplementation with an anthocyanin (AC)-rich powder blend (ACRB) on inflammation-related parameters and on lipid and carbohydrate metabolism-related parameters. A 1000 kcal high-fat diet (HFM) (reference) was administered concomitantly with the ACRB. In a study cohort of healthy subjects, the ACRB attenuated acute inflammation and metabolic disorders associated with HFM intake.

[0247] Materials and Methods research design This study was a randomized, double-blind, placebo-controlled, crossover intervention comparing the effects of ACRB or placebo supplementation. Each intervention (visit) took place 5 hours after ingestion of HFM and supplements, with a washout period of 7 to 30 days between visits. This study (registered at http: / / www.clinicaltrials.gov as NCT03309982) was conducted at the Regal Facility at the University of California, Davis (UCD) in accordance with the guidelines of the Declaration of Helsinki. All procedures were approved by the UCD IRB Administration and the UCD Social and Behavioral Sciences Committee. Written informed consent was obtained from study volunteers. The clinical intervention was conducted from December 2017 to March 2018.

[0248] Research Subjects BMI > 21 or 29.9 kg / m between the ages of 19 and 35 2Fewer than 25 healthy volunteers were recruited from the Davis / Sacramento area to participate in this study. Exclusion criteria included a systolic blood pressure of 160 mmHg or higher or a diastolic blood pressure of 95 mmHg or higher, fasting blood glucose of less than 50 mg / dL or more than 100 mg / dL, fasting serum triglycerides of more than 150 mg / dL, a strict or noncompliant diet (e.g., vegetarian), current use of herbal supplements, use of anti-inflammatory drugs or drugs that interfere with insulin metabolism, regular participation in endurance exercise activities, current smoker or tobacco product user within the past year, heavy alcohol intake or substance abuse / dependence, history of stroke, liver, kidney, or thyroid disease or cancer, malabsorption or gastrointestinal disorders or surgery, severe eating disorders, depression, anxiety, or other psychiatric disorders, diarrhea or oral antibiotic use within the past 4 weeks, weight change (more than 5%) in the past 8 weeks, and allergy or hypersensitivity to any component of ACRB or HFM. Furthermore, to be included in the final data analysis, participants had to be confirmed by the principal investigator (PI) to have a healthy metabolic status in all determined parameters.

[0249] Sample size estimation The number of participants was determined by a power calculation using data to determine the effect of a high-fat diet on plasma endotoxin levels. Based on this data, the sample size to detect a mean change in mean endotoxin level of 0.12 U / ml was calculated to be 24 participants with a power of 0.80 and a type I error of 0.05.

[0250] Recruitment and screening Recruitment and screening were conducted according to the Integrated Criteria for Clinical Trial Reporting strategy shown in Figure 54. Briefly, female and male volunteers who expressed interest in the study were provided with information about the study design and procedures. If volunteers agreed to participate, a screening telephone interview was conducted to assess potential eligibility. Volunteers who expressed interest and met the basic requirements of the inclusion and exclusion criteria were invited to an in-person visit (Visit 0). Participants were asked to participate in a fasting state (≥12 h). After written informed consent was explained and obtained, anthropometric parameters were recorded, finger-prick blood samples were collected, and glucose and triglycerides (TG) were measured using a CardioCheck® analyzer (PTS Diagnostic, Indianapolis, IN, USA). Participants who met the inclusion and exclusion criteria were invited to participate in the clinical trial and scheduled for two in-person visits separated by a washout period (7–28 days).

[0251] clinical intervention At Visit 0, participants received the following dietary restrictions and guidelines: i) avoiding polyphenol-rich foods within 24 hours before each study visit; ii) consuming a similar low-fat dinner the night before each meal (before the start of a 12-hour fast); and iii) assessing adherence to the dietary instructions by completing a 3-day food diary (on the provided form) before Visits 1 and 2. On study visit days (Visits 1 and 2), blood samples were collected by fingerstick upon arrival to measure glucose and triglycerides (TG) using a CardioCheck® analyzer to ensure participants were fasting and to confirm compliance with the inclusion / exclusion criteria. Weight and blood pressure were also measured. Participants were instructed to receive either placebo or ACRB according to the randomization plan. The powder was packaged in sealed, unbranded (numbered) black sachets and coded to allow research staff to blind to treatment. Research staff dissolved the powder in 200 ml of water and provided it to each participant along with the prepared HFM. Participants were instructed to consume HFM within 15 minutes after consuming the ACRB supplement drink. The HFM (320 g) consisted of an English muffin bun, sausage, egg, and cheese. Carotenoid-free palm oil was added to achieve the desired total dietary fat content. The total energy content of the HFM was 1,026 kcal, containing 70.5 g fat (29.8 g saturated fat), 270 mg cholesterol, 65 g carbohydrates, 5.2 g sugars, and 33 g protein. 62% of the total energy came from fat, 25% from carbohydrates, and 13% from protein. Venous blood samples were collected at time 0 (baseline) and 0.5, 1, 2, 3, and 5 hours after HFM ingestion. Serum and plasma were separated and stored frozen for future analysis. Additional blood samples were collected at time 0 and 3 hours and processed for isolation of chylomicrons and peripheral blood mononuclear cells (PBMCs).

[0252] Composition of test product and placebo The anthocyanin-rich supplement tested was a blend of anthocyanin-rich plant extract powders (hereafter referred to as Anthocyanin-Rich Blend, ACRB). Each 4g ACRB powder consisted of 1g of anthocyanin-rich extracts (150mg bilberry extract, 230mg blackcurrant extract, and 620mg black rice extract) and 3g of a maltodextrin mixture. The placebo powder (4g) consisted of 3.85g of the same maltodextrin mixture contained in ACRB, plus 125mg Red No. 40 and 25mg Blue No. 1. The ACRB and placebo were manufactured by Deseret Laboratories, Inc. (St. George, UT) exclusively for NSE Products, Inc. (Pharmanex) (Provo, UT). Polyphenols in ACRB were measured by HPLC in Dr. Mary Ann Lila's laboratory at North Carolina State University.

[0253] biochemical analysis Blood samples were collected in EDTA / sodium citrate tubes and non-anticoagulant tubes. After collection, they were centrifuged at 3,000 × g for 15 minutes at room temperature. The following parameters were measured using ELISA kits according to the manufacturer's instructions: LPS (Abbexa, Texas, USA), LPS-binding protein (LBP), free fatty acids (FFA) (Abcam, California, USA), gastric inhibitory polypeptide (GIP), glucagon-like peptide-1 (GLP-1), glucagon-like peptide-2 (GLP-2), insulin, leptin, adiponectin (Crystal Chem, Illinois, USA), and ghrelin (BioVendor, North Carolina, USA). Concentrations of triglycerides (TG), total cholesterol (Chol), HDL (Wiener Lab, Santa Fe, Argentina), and glucose (Sigma-Aldrich, Missouri, USA) were measured by colorimetric assay according to the manufacturer's instructions. LDL levels were calculated using the Friedewald formula.

[0254] Isolation of peripheral blood mononuclear cells (PBMCs) Blood was collected into sodium citrate-containing cell preparation tubes (BD Vacutainer CPT) (BD, New Jersey, USA). Immediately after collection, blood samples were centrifuged at 1800 × g for 30 minutes at room temperature. After centrifugation, plasma was separated, aliquoted, and stored at −80°C until biochemical analysis. PBMCs remaining in the tubes were centrifuged at 300 × g for 15 minutes at room temperature and washed twice with phosphate-buffered saline (PBS). The pellet was suspended in lysis buffer (Thermo Fisher Scientific, Massachusetts, USA) or TRIzol™ Reagent (Invitrogen, California, USA) for subsequent protein or RNA extraction.

[0255] Western blot analysis Activation of the NF-κB (p-IKK / IKK, Cell Signaling Technology, MA, USA) and insulin resistance (p-JNK / JNK and PTP1b, Cell Signaling Technology, MA, USA) pathways was assessed by Western blot. Proteins were extracted from PBMCs using lysis buffer (Thermo Fisher Scientific, MA, USA) containing protease and phosphatase inhibitors. Thirty micrograms of extracted total protein was denatured in Laemmli buffer, separated by 10% (w / v) polyacrylamide gel electrophoresis under reducing conditions, and electroblotted onto a PVDF membrane. The membrane was blocked with 5% (w / v) nonfat milk for 1 hour and then incubated overnight at 4°C with the corresponding primary antibody (1:1,000 dilution) in 5% (w / v) BSA solution. Subsequently, the cells were incubated in the presence of the corresponding secondary antibody (HRP-labeled) (1:10,000 dilution) at room temperature for 90 minutes, and then visualized with the ECL system using a Phosphor Imager 840 (Amersham Pharmacia Biotech. Inc., New Jersey, USA).

[0256] RNA extraction and quantitative PCR (qPCR) analysis Inflammatory status and NADPH oxidase were assessed by qPCR. RNA was extracted from PBMCs using TRIzol™ reagent (according to the manufacturer's instructions). RNA was converted to cDNA using high-capacity cDNA reverse transcriptase (Applied Biosystems, NY, USA). RNA expression of IL-1β, IL-8, IL-18, TNFα, TLR-4, NOX2, and NOX4 was assessed by qPCR (iCycler, Bio-Rad, CA, USA). β-actin was used as a housekeeping gene. Relative fold changes in gene expression were calculated by 2. -ΔΔΔCt Calculated by the method.

[0257] Measurement of AC and AC metabolites in serum Assay Development. A broad-spectrum quantitative MS / MS assay was used and validated to detect 150 analytes. These analytes were quantified against 107 authenticated commercial and synthetic standards. Metabolites were purified from 100 μL of human serum by 96-well plate solid-phase extraction (SPE; Strata™-X polymeric reversed-phase, microelution 2 mg / well). SPE-processed samples were chromatographically separated and quantified by Exion ultra-high performance liquid chromatography (UHPLC)-tandem mass spectrometry (SCIEX QTRAP 6500+ESI-MS / MS). Samples were injected onto a Kinetex® PFP UPLC column (1.7 μm, 100 Å, 100 mm × 2.1 mm; Phenomenex®) with an oven temperature maintained at 37°C. Mobile phases A and B consisted of 0.1% vv formic acid in water and 0.1% vv formic acid in acetonitrile (respectively), with a binary gradient from 2% B to 90% B over 30 min, with a flow rate gradient varying from 0.55 mL / min to 0.75 mL / min. MS / MS scans were performed in Analyst® (v.1.6.3, SCIEX) using advanced scheduled multiple reaction monitoring (ADsMRM) with polarity switching, and quantification was performed using the MultiQuant™ (v.3.0.2, SCIEX) software platform.

[0258] safety Blackcurrant Extract: Blackcurrant extract is a dark purple powder derived from blackcurrant berries. It is standardized to 30% anthocyanins. Blackcurrants are available in the human diet, and blackcurrant berries can be used as a flavor component in liqueurs, as a food flavoring, and as a food. As part of the modern diet, blackcurrants can be consumed in a variety of forms, including fresh, dried, and frozen fruit, fruit powder, jams, jellies, and extracts in supplements.

[0259] Each packet of the plant polyphenol blend may contain 300 mg of blackcurrant extract, which may contain 90 mg of anthocyanins. Blackcurrants may provide approximately 175 mg of anthocyanins per cup (112 grams).

[0260] Blackcurrant extract has been administered in human clinical trials with no reported side effects. 120 healthy men and women were supplemented with 300 mg of anthocyanins from bilberry and blackcurrant extracts standardized for anthocyanins for three weeks, with no reported side effects. 58 diabetic patients were also given 320 mg of anthocyanins from bilberry and blackcurrant for 24 weeks, with no reported adverse events from either the placebo or anthocyanin product.

[0261] Black rice extract Black (purple) rice extract is black brown rice standardized to 20% anthocyanins. Rice (Oryza sativa L., Poaceae) is consumed as a staple food in many Asian countries. Rice varieties include long-grain white rice, long-grain brown rice, wild rice, basmati, brown basmati, and jasmine rice. Some rice varieties have white pericarp, but dinner varieties with black (purple) or red pericarp also exist.

[0262] Each packet of the plant polyphenol blend contains 600 mg of black (purple) rice extract, which may provide 120 mg of anthocyanins. The high-anthocyanin black rice may be Suwon variety #415, which may contain 470 mg of anthocyanins in the form of cyanidin-3-glucoside per 100 g of rice grains.

[0263] Black rice has been consumed for centuries with no reported side effects. Patients with dyslipidemia received either black rice extract (200 mg, containing 86.4 mg of anthocyanins) or placebo for 12 weeks. Male subjects (n=8) received 500 mg of isotope-labeled cyanidin-3-glucoside, a form of anthocyanin found in black rice. No adverse effects were associated with a single dose of isotope-labeled cyanidin-3-glucoside, although metabolites remained in the circulation for ≤48 hours.

[0264] bilberry extract Bilberry extract, deep purple in color, is obtained from fresh wild bilberry fruit (Vaccinium myrtillus) native to Europe and can be standardized to 36% anthocyanins (HPLC). Bilberry is available as the Vaccinium myrtillus species.

[0265] Each packet of plant polyphenol blend contains 100 mg of bilberry extract, resulting in 36 mg of anthocyanins. Bilberries can be consumed fresh or dried, and fresh or dried berries can be used to brew bilberry tea. The anthocyanin content ranges from 300 to 700 mg per 100 g of fresh fruit, depending on growing conditions and berry ripeness.

[0266] No adverse effects have been reported when bilberry extract is taken in clinical studies. 120 healthy men and women were given 300 mg of anthocyanins from bilberry and blackcurrant extracts standardized for anthocyanins for three weeks, and no adverse events were reported. 58 diabetic patients were given 320 mg of anthocyanins derived from bilberry and blackcurrant for 24 weeks, and no adverse events associated with the intake were reported during the intervention period, regardless of whether they took a placebo or anthocyanin product. Colon cancer patients were given 500 mg, 1000 mg, or 2000 mg of anthocyanins from bilberry extract for seven days before surgery. No adverse events related to the intake of bilberry extract were reported, and the study was safe and well tolerated.

[0267] anthocyanin Each packet of plant polyphenol blend contains 245 mg of anthocyanins from a blend of bilberry, blackcurrant, and black rice extracts. Anthocyanins are found in legumes, berries, fruits, vegetables, and red wine. Daily intakes of anthocyanins range widely, from 180 to 215 mg (33) and from 0 to 364.5 mg (0). A polyphenol blend with an anthocyanin content of 245 mg may be a safe level. One cup of fresh blueberries contains approximately 240 mg of anthocyanins (USDA Nutrient Database, Fresh Blueberries), and one cup of fresh blackcurrants contains approximately 176 mg of anthocyanins (USDA Nutrient Database, Fresh European Blackcurrants).

[0268] Anthocyanins can be supplemented without adverse observations or side effects. Twenty-five colon cancer patients scheduled for surgery received 500 mg, 1,000 mg, or 2,000 mg of anthocyanins derived from bilberry extract for seven days before surgery. Daily intake of all seven doses was safe and well tolerated by patients. The patient receiving the highest dose reported dark stools during supplementation, an expected result due to the natural dark color of anthocyanins. No adverse events were associated with the consumption of anthocyanin products.

[0269] Subjects (n=10) with moderately impaired blood glucose tolerance received a single dose of a product containing 50 mg of delphinidin derived from maqui berry. No adverse observations or side effects were observed in the patients. Male subjects (n=8) received 500 mg of isotope-labeled cyanidin-3-glucoside. Although metabolites remained in the circulation for ≤48 hours, no adverse effects were associated with the single dose of isotope-labeled cyanidin-3-glucoside.

[0270] result Anthocyanidin-rich blend composition The total anthocyanidin content in ACRB was 320.4 mg / g. Glucosides of cyanidin and delphinidin accounted for a large proportion of the anthocyanidins, accounting for approximately 52% and 38% of the total anthocyanidins, respectively.

[0271] Participant flowchart and baseline characteristics As shown in Figure 54, of the 82 subjects interviewed, 27 were enrolled, randomized to the intervention group, and scheduled for Visit 1 and Visit 2. Two of these participants did not complete the study (one was unable to complete the HFM intake within 15 minutes at Visit 1). The third participant was excluded from the analysis because his postprandial plasma triglycerides and insulin were outside the normal range, indicating a metabolic abnormality suggestive of insulin resistance or metabolic syndrome. The participants' baseline characteristics were within the normal range and did not change significantly between Visit 1 and Visit 2 (data not shown). No adverse events due to the intervention were reported.

[0272] The experimental layout was a "restricted" two-treatment, two-period crossover, allowing for separate evaluation of treatment, period, and sequence effects. Overall, no period effects were observed for the parameters evaluated. Sequence effects were observed for five response variables (plasma endotoxin, GIP, GLP-2, ghrelin, and adiponectin). These sequence and treatment effects will be disclosed in detail in follow-up reports of the individual measurements.

[0273] Efficacy of ACRB intake on postprandial endotoxemia One of the outcomes of this study was to evaluate the changes in blood LPS levels induced by HFM after treatment with ACRB or placebo. The ACRB treatment and time effects were significant (p = 0.046 and p < 0.001, respectively), but their interaction was not significant (p = 0.51). After removing outliers, the significance of the treatment effect (p = 0.050) and the interaction (p = 0.14) changed. ACRB treatment resulted in lower mean endotoxin levels compared to placebo treatment.

[0274] The iAUC index was calculated using the individual time-point data values ​​for each blood parameter. After excluding two outliers (Studentized residuals), the effects of treatment (p = 0.0380) and sequence (p = 0.0002) were significant. The treatment effect indicated that ACRB attenuated HF meal-induced endotoxemia, as evidenced by a lower increase in plasma LPS in participants receiving ACRB compared with placebo. As shown in Figure 55A, when participants received ACRB, the iAUC values ​​were reduced by 46% compared with placebo (0.68 ± 0.18 to 0.37 ± 0.12 EU / mL x 5 h). In the analysis of the placebo-ACRB treatment sequence, the iAUC was 1.03 ± 0.31 in the placebo group and 0.59 ± 0.31 EU / mL x 5 h in the ACRB group. Cmax values ​​for endotoxin were significantly different compared to baseline levels, p<0.01.

[0275] Further evidence of HFM-induced endotoxemia was the increase in iAUC of plasma LPS-binding protein content after HFM ingestion. The treatment effect was significant (p = 0.0314). As shown in Figure 55B, ACRB treatment reduced the increase in LPS-binding protein from 15.9 ± 3.0 to 8.2 ± 2.0 (ng / mL × 5 hours) (paired t-test, p = 0.02).

[0276] Effect of ACRB intake on postprandial cardiometabolic parameters Secondary outcomes included changes in cardiometabolic biomarkers related to lipid and glucose metabolism. The increase in plasma TG after HFM ingestion followed the dynamics shown in Figure 56A. Baseline TG levels were 58.2 ± 4.5 mg / dL and 61.9 ± 6.3 mg / dL in the placebo and ACRB groups, respectively, reaching maximum values ​​of 122.6 ± 5.5 mg / dL and 112 ± 9.5 mg / dL 2 hours after HFM ingestion. As shown in Figure 56A, the AUC value, which represents the increase in plasma TG levels over a 5-hour period, was 21% lower when participants received the ACRB compared to those receiving the placebo (paired t-test, p = 0.029). Four participants had no postprandial plasma TG response and were considered outliers and excluded from the analysis. Subsequently, postprandial plasma TG increases after ACRB treatment were reduced by 37% compared with placebo (146.2 ± 15.4 vs. 203.9 ± 21.1, p = 0.006; n = 19).

[0277] The increase in total cholesterol associated with HFM was mild, regardless of whether participants received an ACRB or placebo, as shown in Figure 56B. Baseline cholesterol was 158.4 ± 2.9 mg / dL in the placebo group and 162.3 ± 2.7 mg / dL in the ACRB group, with peak values ​​of 172.3 ± 2.9 mg / dL and 167.7 ± 2.7 mg / dL, respectively, 5 hours after HFM ingestion. The increase in cholesterol at 5 hours was reduced to 55% of the placebo value by ACRB administration (paired / unpaired t-test p = 0.055). Plasma HDL cholesterol, LDL cholesterol, and free fatty acids were unaffected by ACRB ingestion.

[0278] As shown in Figure 56C, peak postprandial plasma glucose was observed 30 minutes after HFM ingestion and returned to baseline 2–3 hours after HFM ingestion. The 5-hour postprandial increase in plasma glucose was attenuated by 40% with ACRB administration compared to placebo (p=0.016; paired t-test). Concurrently, ACRB treatment reduced the postprandial increase in plasma GIP. HFM-associated increases in plasma insulin, leptin, adiponectin, ghrelin, GLP-1, and GLP-2 were similar whether subjects consumed placebo or ACRB.

[0279] Efficacy of ACRB intervention on outcomes related to inflammation, lipid, and glucose metabolism Other endpoints measured in PBMCs 3 h postprandially included TNFα, IL-8, IL-18, IL-1β, TLR4, NOX2, NOX4, IKK, JNK1 / 2, and PTP1B. As shown in Figure 57D, HFM intake caused a significant increase in TNFα mRNA levels (45%, p = 0.005), and also showed a trend toward an increase in IL-8 mRNA (51%, p = 0.09), as shown in Figure 57A. The HFM-associated increase in TNFα mRNA was attenuated by ACRB intake (p = 0.11 compared to baseline). Meanwhile, ACRB administration did not affect the increase in IL-8 mRNA. Intake of HFM, placebo, or ACRB did not affect the mRNA levels of IL-18 (Figure 57B), IL-1β (Figure 57C), or TLR4 (Figure 57E). As shown in Figure 57F, NOX2 mRNA levels were similar before and after HFM in placebo- and ACRB-treated groups. On the other hand, as shown in Figure 57G, after HFM ingestion, NOX4 mRNA levels increased by 74% (p=0.038), but were attenuated by ACRB treatment (p=0.25).

[0280] As shown in Figures 58A-58D, HFM intake increased IKK (Ser178 / 180) and JNK1 / 2 (Thr183 / Tyr185) phosphorylation by 86% and 140%, respectively (p=0.038 and p=0.009) in PBMCs from placebo-treated participants. On the other hand, ACRB treatment attenuated the postprandial increase in IKK phosphorylation, as shown in Figure 58B, and abolished JNK1 / 2 phosphorylation, as shown in Figure 58C. The difference between groups was significant for JNK1 / 2 phosphorylation (p=0.009), but not for IKK phosphorylation (p=0.44). As shown in Figure 58D, no changes in PTP1B expression were observed in PBMCs by HFM or ACRB.

[0281] The described features, structures, or characteristics may be combined in any suitable manner in one or more examples. In the preceding description, numerous specific details, including examples of various configurations, have been provided to provide a thorough understanding of examples of the described technology. However, those skilled in the art will recognize that certain details may be omitted or that other methods, components, devices, etc. may be used to practice the technology. In other instances, well-known structures or operations have not been shown or described in detail so as not to obscure aspects of the technology.

[0282] The above detailed description explains the invention with reference to specific embodiments. However, it should be understood that various changes and modifications can be made without departing from the scope of the invention as defined in the appended claims. The detailed description and accompanying drawings should be considered merely illustrative and not restrictive, and all such changes and modifications are intended to be included within the scope of the invention as described and defined herein.

Claims

1. A composition for promoting metabolic health comprising a combination of cyanidin and delphinidin in an amount sufficient to treat a metabolic disorder.

2. 10. The composition of claim 1, wherein the source of the combination of cyanidin and delphinidin is derived from a black rice component, a blueberry component, a blackcurrant component, a crowberry component, a bilberry component, a black chokeberry component, or a combination thereof.

3. 3. The composition of claim 2, wherein the source of the combination of cyanidin and delphinidin is derived from the black rice component, the blackcurrant component, and the bilberry component.

4. 4. The composition of claim 3, wherein the black rice component is a black rice extract.

5. 4. The composition of claim 3, wherein the black rice ingredient comprises about 50% to about 70% by weight of the active fraction of the composition.

6. 4. The composition of claim 3, wherein the black rice ingredient comprises about 55% to about 65% by weight of the active fraction of the composition.

7. 4. The composition of claim 3, wherein the black rice ingredient comprises from about 57.5% to about 62.5% by weight of the active fraction of the composition.

8. 4. The composition of claim 3, wherein the black rice ingredient comprises about 60% by weight of the active fraction of the composition.

9. 4. The composition of claim 3, wherein the black rice ingredient comprises about 62% by weight of the active fraction of the composition.

10. 4. The composition of claim 3, wherein the black rice ingredient has a standardized anthocyanin content ranging from about 15% to about 25% by weight.

11. 4. The composition of claim 3, wherein the black rice ingredient has a standardized anthocyanin content ranging from about 17.5% to about 22.5% by weight.

12. 4. The composition of claim 3, wherein the black rice ingredient has a standardized anthocyanin content of about 20% by weight.

13. 4. The composition of claim 3, wherein the black rice component is derived from rice (Oryza sativa L.).

14. 4. The composition of claim 3, wherein the blackcurrant ingredient is a blackcurrant extract.

15. 4. The composition of claim 3, wherein the blackcurrant ingredient comprises from about 15% to about 45% by weight of the active fraction of the composition.

16. 4. The composition of claim 3, wherein the blackcurrant ingredient comprises from about 25% to about 35% by weight of the active fraction of the composition.

17. 4. The composition of claim 3, wherein the blackcurrant ingredient comprises from about 20% to about 25% by weight of the active fraction of the composition.

18. 4. The composition of claim 3, wherein the blackcurrant ingredient comprises from about 27.5% to about 32.5% by weight of the active fraction of the composition.

19. 4. The composition of claim 3, wherein the blackcurrant ingredient comprises about 30% by weight of the active fraction of the composition.

20. 4. The composition of claim 3, wherein the blackcurrant ingredient comprises about 23% by weight of the active fraction of the composition.

21. 4. The composition of claim 3, wherein the blackcurrant component has a standardized anthocyanin content ranging from about 25% to about 35% by weight.

22. 4. The composition of claim 3, wherein the blackcurrant component has a standard anthocyanin content of about 30% by weight.

23. 4. The composition of claim 3, wherein the blackcurrant component is derived from blackcurrant (Ribes nigrum).

24. 4. The composition of claim 3, wherein the bilberry component comprises a bilberry extract.

25. 4. The composition of claim 3, wherein the bilberry component is in the range of about 5% to about 15% by weight of the active fraction of the composition.

26. 4. The composition of claim 3, wherein the bilberry component is in the range of about 7.5% to about 12.5% ​​by weight of the active fraction of the composition.

27. 4. The composition of claim 3, wherein the bilberry component is about 10% by weight of the active fraction of the composition.

28. 4. The composition of claim 3, wherein the bilberry component is about 15% by weight of the active fraction of the composition.

29. 4. The composition of claim 3, wherein the bilberry component has a standardized anthocyanin content ranging from about 25% to about 45% by weight.

30. 4. The composition of claim 3, wherein the bilberry component has a standardized anthocyanin content in the range of about 30% to about 40% by weight.

31. 4. The composition of claim 3, wherein the bilberry component comprises 36% by weight of anthocyanins as measured by HPLC or 25% by weight of anthocyanins as measured by UV.

32. 4. The composition of claim 3, wherein the bilberry component is derived from Vaccinium myrtillus.

33. 4. The composition of claim 3, wherein the ratio of the black rice component, the blackcurrant component, and the bilberry component ranges from about 6:3.5:1 to about 6:1.5:

1.

34. 4. The composition of claim 3, wherein the ratio of the black rice component, the blackcurrant component, and the bilberry component ranges from about 6:2:0.5 to 6:2:2.

5.

35. 4. The composition of claim 3, wherein the ratio of said black rice component to said blackcurrant component to said bilberry component ranges from about 4:2:1 to 8:2:

1.

36. 4. The composition of claim 3, wherein the ratio of said black rice component, said blackcurrant component, and said bilberry component is about 6:2:

1.

37. The composition of claim 3, further comprising a pharmaceutically acceptable carrier.

38. 4. The composition of claim 3, further comprising a sweetener, a preservative, a flavoring agent, a thickener, or a combination thereof.

39. The composition of claim 3, wherein the composition is in an oral dosage form.

40. 40. The composition of claim 39, wherein the oral dosage form comprises a softgel, tablet, powder, beverage, or gummy.

41. 40. The composition of claim 39, wherein the black rice ingredient ranges from about 400 mg to about 800 mg of the oral dosage form.

42. 40. The composition of claim 39, wherein the blackcurrant component comprises from about 150 mg to about 350 mg of the oral dosage form.

43. 40. The composition of claim 39, wherein the bilberry component comprises from about 50 mg to about 250 mg of the oral dosage form.

44. 40. The composition of claim 39, wherein the oral dosage form comprises from about 215 mg to about 415 mg of anthocyanin.

45. 40. The composition of claim 39, wherein the oral dosage form comprises about 245 mg of anthocyanin.

46. 40. The composition of claim 39, wherein the oral dosage form comprises about 215 mg of anthocyanin.

47. A composition for reducing inflammation, comprising a combination of cyanidin and delphinidin in an amount sufficient to reduce inflammation, and a carrier comprising an oil, lecithin, gelatin, glycerin, or wax.

48. 48. The composition of claim 47, wherein the carrier comprises the oil.

49. 49. The composition of claim 48, wherein the oil comprises olive oil, canola oil, coconut oil, soybean oil, sunflower oil, flaxseed oil, or a combination thereof.

50. 48. The composition of claim 47, wherein the carrier comprises the lecithin.

51. 51. The composition of claim 50, wherein the lecithin comprises sunflower lecithin, soybean lecithin, egg yolk lecithin, or a combination thereof.

52. 48. The composition of claim 47, wherein the carrier comprises gelatin.

53. 48. The composition of claim 47, wherein the carrier comprises glycerin.

54. 48. The composition of claim 47, wherein the carrier comprises the wax.

55. 55. The composition of claim 54, wherein the wax comprises beeswax, paraffin wax, carnauba wax, or a combination thereof.

56. 48. The composition of claim 47, wherein the cyanidin is present in an amount of 14% to 20% by weight and the delphinidin is present in an amount of 10% to 14% by weight.

57. 57. The composition of claim 56, wherein the cyanidin is present in an amount of 16% to 17% by weight and the delphinidin is present in an amount of 12% to 13% by weight.

58. 57. The composition of claim 56, wherein the total amount of anthocyanins in the composition is 30% to 35% by weight.

59. 48. The composition of claim 47, further comprising peonidin, petunidin, and malvidin.

60. 60. The composition of claim 59, wherein the peonidin is present in an amount of 1.5% to 2% by weight, the petunidin is present in an amount of 0.3% to 0.8% by weight, and the malvidin is present in an amount of 0.5% to 1% by weight.

61. 1. A method of treating a condition or disorder related to metabolic health in a subject, comprising: A method comprising the steps of reducing endotoxin levels compared to baseline endotoxin levels, and adjusting cardiometabolic biomarkers related to lipid or glucose metabolism from abnormal cardiometabolic baseline levels to normal levels.

62. 62. The method of claim 61, wherein the subject is on a high fat diet.

63. 62. The method of claim 61, wherein the subject has a body mass index (BMI) of less than about 18.5, between 18.5 and 24.9, between 25.0 and 29.9, between 30.0 and 34.9, between 35 and 39.9, or greater than 40.

64. 62. The method of claim 61, The method further comprises reducing the endotoxin level by more than any one or more of 5%, 10%, 20%, 30%, 40%, 50%, or a combination thereof, compared to a baseline endotoxin level.

65. 62. The method of claim 61, reducing cytokine levels relative to baseline levels; or reducing NF-B levels relative to baseline levels; The method further comprises the step of reducing the inflammation level relative to a baseline inflammation level by

66. 62. The method of claim 61, reducing triglyceride levels by more than one or more of 5%, 10%, 20%, 30%, 40%, 50%, or a combination thereof, compared to baseline levels; or reducing cholesterol levels by more than one or more of 5%, 10%, 20%, 30%, 40%, 50%, or a combination thereof, relative to a baseline level; The method further comprises adjusting the cardiometabolic biomarkers related to lipid metabolism by

67. 62. The method of claim 61, reducing GTT AUC by more than one or more of 5%, 10%, 20%, 30%, 40%, 50%, or a combination thereof, relative to a baseline level; or reducing the HbA1c serum concentration by more than one or more of 0.05, 0.1, 0.15, 0.2, 0.25 mmol / L, or a combination thereof, relative to a baseline level; or reducing the glucose serum concentration relative to a baseline glucose serum concentration level measured within 2 to 5 hours after the subject ingests a high-fat meal; The method further comprises adjusting the cardiometabolic biomarker related to glucose metabolism by

68. 62. The method of claim 61, modulating the insulin biomarker by reducing the ITT AUC by more than one or more of 5%, 10%, 20%, 30%, 40%, 50%, or a combination thereof; or modulating an insulin biomarker by decreasing IKK phosphorylation levels, JNK1 / 2 phosphorylation levels, or a combination thereof by more than one or more of 5%, 10%, 20%, 30%, 40%, 50%, or a combination thereof; The method further comprises:

69. 62. The method of claim 61, Reducing intestinal barrier permeability relative to baseline levels The method further comprises:

70. 70. The method of claim 69, wherein the intestinal barrier permeability is reduced by more than 10% as measured using TEER.

71. 70. The method of claim 69, wherein the intestinal barrier permeability is reduced by more than one or more of 10%, 20%, 50%, 100%, 200%, 300%, or a combination thereof as measured using FITC-dextran paracellular transport.

72. 70. The method of claim 69, wherein the intestinal barrier permeability is reduced by more than one or more of 10%, 20%, 50%, or a combination thereof, as measured using endotoxin concentration.

73. 62. The method of claim 61, further comprising reducing the Firmicutes to Bacteroidetes ratio by more than one or more of 10%, 20%, 50%, 100%, 200%, 300%, 400%, 500%, or a combination thereof compared to a reference ratio.

74. 62. The method of claim 61, further comprising increasing the level of Akkermansia by more than one or more of 10%, 20%, 50%, 100%, 200%, 300%, 400%, 500%, or a combination thereof.

75. 62. The method of claim 61, reducing the level of oxidative stress compared to a baseline level; or reducing the level of bacterial infection compared to a baseline level; or Reducing the level of viral infection compared to a baseline level The method further comprises:

76. 62. The method of claim 61, increasing liver biomarkers from baseline levels, including increases in one or more of CPT-1A, acyl-CoA oxidase, insulin sensitivity, insulin secretion, glucose uptake, or a combination thereof; or reducing said liver biomarkers from baseline levels, including a reduction in one or more of RBP4, SREBP-1C, lipid accumulation, blood lipids, oxidative stress, or a combination thereof. The method further comprises:

77. 62. The method of claim 61, increasing adipose tissue biomarkers from baseline levels, including increases in one or more of AMPK, GLUT4, ACC1, LPL, insulin sensitivity, insulin secretion, glucose uptake, fatty acid oxidation, or a combination thereof; or Reducing said adipose tissue biomarkers from baseline levels, including reducing one or more of FAS, hyperglycemia, fatty acid synthesis, gluconeogenesis, serum lipids, or a combination thereof. The method further comprises:

78. 62. The method of claim 61, increasing skeletal muscle biomarkers from baseline levels, including increases in one or more of G6PD, hexokinase, carbohydrate metabolism, glucose uptake, insulin receptor sensitivity, or a combination thereof; or decreasing the skeletal muscle biomarker from baseline levels, including decreasing PEPCK. The method further comprises:

79. 62. The method of claim 61, increasing a pancreatic biomarker from baseline levels, including an increase in one or more of beta cell function, glucose uptake, or a combination thereof; or reducing said pancreatic biomarkers from baseline levels, including reducing one or more of JNK, IL-1β, IL-6, TNF-α, blood lipid levels, hyperglycemia, oxidative risk, or a combination thereof. The method further comprises:

80. 62. The method of claim 61, wherein the metabolic condition or disorder is postprandial metabolic disorder.

81. 62. The method of claim 61, wherein the metabolic condition or disorder comprises a condition or disorder related to one or more of blood pressure, blood glucose, insulin sensitivity, abdominal fat, cholesterol, triglycerides, liver health, or a combination thereof.

82. 62. The method of claim 61 , wherein the metabolic condition or disorder is related to blood pressure; The method further comprising reducing one or more of the systolic blood pressure or the diastolic blood pressure by more than one or more of 10%, 20%, 50%, or a combination thereof, compared to the baseline level.

83. 62. The method of claim 61 , wherein the metabolic condition or disorder is associated with abdominal fat; The method further comprising reducing the amount of visceral adipose tissue (VAT) by more than one or more of 3%, 5%, 10%, or a combination thereof, compared to pre-treatment levels after a predetermined period of time.

84. 1. A method of reducing liver inflammation in a subject, comprising administering to said subject an effective amount of a combination of cyanidin and delphinidin.

85. 85. The method of claim 84, wherein the subject is obese and the liver inflammation is reduced without modulating high-fat diet-induced biomarkers.

86. 86. The method of claim 85, wherein the high-fat diet-induced biomarker is cholesterol, triglycerides, fatty acids, glucose, or insulin.

87. A method for treating high-fat diet-induced colonic dysfunction in obese subjects without modulating high-fat diet-induced biomarkers.

88. 88. The method of claim 87, wherein the high-fat diet-induced biomarker is cholesterol, triglycerides, fatty acids, glucose, or insulin.

89. 88. The method of claim 87, wherein the colon dysfunction comprises endotoxemia, altered colonic architecture, loss of colonic barrier integrity, and loss of colonic goblet cells.

90. 1. A method for reducing expression of toll-like receptor TLR4 in the liver of a subject, comprising administering to said subject an effective amount of a combination of cyanidin and delphinidin.

91. 1. A method for reducing expression of toll-like receptor TLR2 in the liver of a subject, comprising administering to said subject an effective amount of a combination of cyanidin and delphinidin.

92. 1. A method for reducing endotoxin concentration in the plasma of a subject, comprising administering to the subject an effective amount of a combination of cyanidin and delphinidin.

93. 1. A method for increasing tight junction proteins in the colon of a subject, comprising administering to the subject an effective amount of a combination of cyanidin and delphinidin.

94. 1. A method for reducing expression of NADPH oxidase NOX1 in the colon of a subject, the method comprising administering an effective amount of a combination of cyanidin and delphinidin.

95. 61. A composition according to any preceding claim for use in treating a disease or disorder related to the metabolic health of a subject.

96. 96. The composition of claim 95, wherein the condition or disorder is one or more of liver inflammation, high-fat diet-induced colonic dysfunction, overexpression of toll-like receptor TLR4, overexpression of toll-like receptor TLR2, elevated endotoxin levels, overexpression of NADPH oxidase NOX1, or a combination thereof.

97. 1. Use of a combination of cyanidin and delphinidin in the manufacture of a medicament useful for treating a condition or disorder related to the metabolic health of a subject.

98. 98. The use of claim 97, wherein the condition or disorder is one or more of liver inflammation, high-fat diet-induced colonic dysfunction, overexpression of toll-like receptor TLR4, overexpression of toll-like receptor TLR2, elevated endotoxin levels, overexpression of NADPH oxidase NOX1, or a combination thereof.

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