KR101275344B1 - Method of using punicic acid to enhance immune response and prevent metabolic disorders - Google Patents

Abstract

In accordance with the present invention, oral or parenteral administration of a therapeutically effective amount of funic acid to an animal prevents or ameliorates immunoinflammatory diseases such as inflammatory bowel disease, enhances the development of the immune system, and enhances CD4 ⁺ and CD8 ⁺ T lymphocytes. Methods to enhance immune responses in animals, including mammals and humans, to maintain or increase levels, increase immune function, increase immune response to viruses, and prevent or improve metabolic syndrome, type 2 diabetes and obesity This is described.

Funic acid, pomegranate, intestinal disease, immune response, type 2 diabetes

Description

METHOD OF USING PUNICIC ACID TO ENHANCE IMMUNE RESPONSE AND PREVENT METABOLIC DISORDERS}

FIELD OF THE INVENTION The present invention relates generally to methods of using funic acid to enhance immune responses while minimizing the deleterious side effects of immunoactivation in animals, including mammals and humans, and preventing immunoinflammatory or metabolic diseases. More specifically, the present invention utilizes funic acid to enhance immune response, enhance the development of the immune system, prevent or improve immunoinflammatory and metabolic diseases (ie metabolic syndrome, obesity and type 2 diabetes), CD4 To maintain or increase ⁺ and CD8 ⁺ T cell levels and to improve resistance to viral diseases.

Cross-Reference to Related Applications

This application is filed with U.S. Patent Application No. 11 / XXX, XXX filed on January 7, 2005 (patent document No. 34548.126) and PCT / US2005 / XXXXX filed on January 7, 2005. 35 USC of Patent Attorney Document No. 34548.128 As part of a continuous application under §120, these two applications also provide 35 U.S.C. U.S. Provisional Patent Application No. 60 / 537,617, filed Jan. 20, 2004 under §119 (e), is a priority, the entirety of which is hereby incorporated by reference.

A complete list of references cited herein is provided in the preceding section of the claims.

Justice

The following definitions are used throughout this application.

Antigen: Any substance to which an antibody molecule or T cell receptor can specifically bind.

ANOVA: analysis of variance. An operation that divides the total deviation in a data set into specific elements based on the source of the deviation. This process has been used to determine whether the numerical difference between treatment groups is statistically significant.

Adipogenesis: The process by which new adipocytes or adipocytes are produced.

CD19: Differentiation group 19. Protein expressed on the surface of B cells.

CD4: differentiation population 4. Co-receptors found on the surface of helper T cells.

CD3: differentiation population 3. Co-receptors found on the surface of all T cells.

CD8: differentiation population 8. Co-receptors found on the surface of cytotoxic T cells.

CD4 ⁺ T cells: helper T cells. Cells that secrete proteins that regulate the activity of other immune cells on antigens.

CD8 ⁺ T cells: cytotoxic T cells. Cells capable of incapacitating or destroying abnormal cells, including tumor cells, virus infected cells and cells infected with bacteria in the cells.

Con A: Concanavalin A. Mitogen causing proliferation of lymphocytes. Stimulating lymphocytes with Con A has been widely used to investigate the effects of dietary intervention on immune function.

cRPMI: The complete Roswell Park Memorial Institute badge. Cell medium formulated to contain all the nutrients needed for long-term cell survival. Detailed compositions will be described in Media and Reagent Preparation.

DSS: dextran sodium sulfate. A chemical administered by drinking water (2.5% weight / volume) that kills the epithelial cells of the colon and causes colon inflammation. The DSS colitis model is an established model of experimental IBD.

Epitope: The portion of an antigen present in major histocompatibility complex 1 or 2 that the antibody or T cell receptor specifically recognizes.

FACS: Fluorescence-activated cell sorting. Special buffers used for flow cytometry to prevent internalization of cell surface markers. Detailed compositions will be described in Media and Reagent Preparation.

Glycemia: Concentration of glucose in the blood.

Hyperglycemia: increased concentration of glucose in the blood above the normal range.

Hyperinsulinemia: Increased concentration of insulin in the blood above the normal range.

IBD: Inflammatory Bowel Disease. Intestinal immunoinflammatory disease characterized by two clinical signs: Crohn's disease (CD) and ulcerative colitis (UC).

Insulinosis: concentration of insulin in the blood.

mAb: monoclonal antibody. Antibodies formed by B cells in response to specific antigenic stimulation that binds to specific sites of a protein.

Nutraceuticals: Compounds with certain medical and nutritional benefits.

PBS: phosphate buffered saline. Buffer solution used for cell culture. Cells can be resuspended in PBS for a short time before being transferred to a more nutritious buffer. Detailed compositions will be described in Media and Reagent Preparation.

TCR: T cell receptor. A protein expressed on the surface of T lymphocytes, which is always co-expressed with a CD3 molecule that recognizes a particular antigen. After recognizing the antigen, the T cell receptor activates lymphocytes and initiates an immune response to the antigen.

Type 2 diabetes or non-insulin dependent diabetes mellitus: A more common type of diabetes caused by the cell's nonresponsiveness to the action of insulin. If the cells do not respond to insulin, the cells cannot take glucose from the blood, which in turn results in glucose toxicity. In addition, cells are deprived of energy resulting from the oxidation of glucose.

Description of Prior Art

An immune response is the body's defense mechanism against foreign substances that invade to cause infection and / or disease. The function of the immune system is a complex process that requires the combined effort of several types of cells, including white blood cells. The immune response begins when the antigen-presenting cell encounters a foreign antigen. Antigen cells degenerate foreign antigens and arrange portions of the antigens (ie, viruses, bacteria or other foreign objects) called epitopes on their surfaces.

One particular T cell clone, the only one of many different clones of the body's T cells (subgroup of white blood cells), recognizes the displayed antigen and binds to the donor cell. This binding regulates the production of proteins called cytokines by antigen presenting cells such as interleukin-1 (IL-1) and tumor necrosis factor (TNF). This protein communicates between antigen presenting cells and T cells. T cells also produce other proteins such as interleukin-2 (IL-2) and gamma interferon (IFN-γ). As part of the ongoing process, IL-2 commands other T cells and killer T cells to proliferate.

Proliferating helper T cells release a substance that allows B cells to proliferate and produce antibodies. Antibodies released by B cells bind to antigens on the surface of freely moving viruses and other pathogens. As the infection is suppressed, the activated T and B cells are stopped by the suppressor T cells. However, several "memory cells" remain behind and react quickly when the same pathogen attacks again.

Because of the complexity of the body's immune response, further research is needed to find new ways to boost the immune system to enhance the animal's immune response. Ideally, new methods of improving the immune system will work to prevent or reduce the harmful side effects associated with immune and / or inflammatory responses.

In addition, current research suggests that other immune diseases, such as inflammatory bowel disease (IBD), may be helpful for further research into the immune system. Inflammatory bowel disease (IBD) is a chronic, recurrent disease of unknown origin, affecting more than one million Americans and millions of people worldwide. IBD is a common cause of chronic disease in large parts of the patient population. IBD appears in two different forms: ulcerative colitis (UC) and Crohn's disease (CD). Although the two conditions look very similar clinically, UC is often accompanied by inflammation of the colon and rectum as opposed to the upper gastrointestinal tract. On the other hand, since CD affects larger areas of the digestive tract of the upper intestine, it is more likely to cause malabsorption and chronic vitamin and nutritional deficiencies.

People with IBD suffer from symptoms such as chronic visceral inflammation, diarrhea, bleeding, abdominal pain, fever, joint pain, and weight loss. These symptoms can range from mild to severe. IBD may develop initially from mild discomfort gradually and unknowingly, or suddenly appear acute.

Current IBD therapeutics include corticosteroids such as 6-methylprednisolone and budesonide, and azathioprine, 6-mercaptopurine, cyclosporine, cyclosporine, And immunosuppressive agents such as methotrexat (Lichtenstein et al., 2003). New therapies still under development include the FDA-approved anti-tumor necrosis factor (TNF-α) (Camilleri, 2003; Liechtenstein et al., 2003; Bassaganya-Riera et al., 2004 ). Current treatments for IBD have improved, but have not been successful enough. Moreover, conventional treatments often have significant side effects for the user. Therefore, there is still a need for new prophylactic and / or therapeutic methods for treating immunoinflammatory diseases such as IBD.

There is also a need for new ways to improve the immune response of mammals using nutritional products. Further research into the immune system suggests the involvement of nutrients to prevent immunosuppression.

There is also a need for new methods to enhance immune responses in immune suppressed people such as infants, children and the elderly. Such people have a limited ability to respond to viral and bacterial infections, thereby reducing their resistance to viral and bacterial diseases. Therefore, there is a need to find new methods, including nutritional methods, to enhance the immune response of these vulnerable people.

There is also a need for new methods of treating or preventing metabolic disorders such as metabolic syndrome or syndrome X and type 2 diabetes. In Western society, the prevalence of obesity leads to several metabolic diseases such as type 2 diabetes, cardiovascular disease, hypertension, and hyperlipidemia, all of which are considered syndrome X. About 15 million Americans have type 2 diabetes, and a million are newly diagnosed and cost $ 13 billion each year. Several elements of metabolic syndrome (eg insulin resistance and obesity) appear to be associated with immunoinflammatory disorders. This connection between immunity, inflammation and metabolism is particularly strong in abdominal adipose tissue. Vohl et al. Found that the expression of proinflammatory cytokines and their receptors (e.g. IL-6 and IL-1R) was increased in human mastoid adipose tissue as compared to subcutaneous fat tissue (Bol et al., 2004). ). Gemfibrozil, an agonist of the peroxysome's proliferative-activated receptor alpha, has been approved for the treatment of cardiovascular disease and leads to a cardioprotective effect that cannot be fully explained by its effect on the blood lipid profile ( Rubins et al., 2000; Moller et al., 2003). Therefore, other mechanisms are also involved in improving cardiovascular disease. The regulation of immune function and inflammation by gemfibrozil provides an explanation for the strong health benefits it provides. In addition, gemfibrozil is an example of successful intervention using an approach that works not only for metabolism but also for immune function and inflammation for metabolic disease.

New treatments for type 2 diabetes include agonists of prolifer-activated receptor gamma of peroxysomes, such as thiazolidinedione (TZD). However, questions have been raised about the safety of this treatment due to adverse cardiovascular (blood stagnation and congestive heart failure) and liver (fatty liver) side effects. Thus, there is a need to find new and safe ways to prevent or treat metabolic syndrome, type 2 diabetes and obesity, including nutritional methods that act on molecular networks located at the interface between immune, inflammatory and metabolic actions.

Treatment with funic acid may be one of such new and safe methods. Funic acid is a non-toxic, natural edible ingredient that has been known to humans for centuries and has been orally effective for humans. Punic acid is found naturally in the seeds of pomegranate, Punica granatum, which accounts for over 60% of the oil. Momordica balsamina is another medicinal plant belonging to the family Cucumber and having a concentration similar to that of funic acid. The presence of funic acid in pomegranate seeds and other medicinal plants is well known in the art. The benefits of pomegranate against cancer are also well known in the art. For example, methods for treating medical disorders using pomegranate fruit are common. However, methods of enhancing mammalian immune responses using funic acid are not. For example, Murad US Pat. No. 6,630,163 teaches how to treat skin diseases using fruit extracts comprising pomegranate. The method involves administering at least one fruit extract in a therapeutically effective amount to neutralize free radicals in the skin. This method aims to treat skin diseases. However, this method does not teach the use of pomegranate oil or funic acid to enhance the immune response as a method of treating disorders of the immune system and gastrointestinal tract. In addition, no teaching was made regarding the use of funic acid to promote the development and function of the immune system or the prevention of metabolic diseases.

Shehadeh, US Pat. No. 6,030,622 teaches a method of making a plant extract composition comprising an arum extract, a pomegranate extract, a tea extract and a hibiscus extract. The pomegranate ingredient used to produce this extract is the fruit peel, but the fruit peel does not contain punic acid and the punic acid is only in the seed. In addition, the extraction methods used (aqueous and ethanolic) are designed to extract the water-soluble components, not oils or funic acids.

Lansky's U.S. Pat.Nos. 6,060,063 and 5,891,440 also produce phytoestrogen oral supplements extracted with an aqueous solvent from pomegranate seeds and prepared with shizandra berry, Chinese asparagus. Roots, and optionally plant extracts comprising Chinese licorice and Chinese angelica root, are taught to mix with pomegranate seed extract. However, this method does not teach the use of funic acid to enhance an immune response that prevents metabolic diseases in mammals.

Japanese Patent No. 2000238566 to Murase and Imamura teaches a method for cloning genes involved in the synthesis of catalic and funic acids. While such techniques may be useful for the production of enzymes of funic acid, they do not teach the use of funic acid to enhance mammalian immune responses or to prevent metabolic diseases.

Other patents, such as British Patents 1466418 and 758724, also teach methods of polymerizing conjugated trienic fatty acids and esters and improving synthetic resins using funic acid. However, no patent teaches the medical effects of funic acid on the immune system.

In addition, the use of pomegranate seed oil appears to be useful for the proliferation and survival of human breast adenocarcinoma (MCF-7) cells, but only in in vitro studies using topical administration (Kim et al. 2002). Another study shows the use of pomegranate oil in the treatment of tumors in a mouse model of chemically induced skin cancer (Hora et al., 2003).

Summary of the Invention

     In response to the above-mentioned needs, the present invention provides a method for enhancing the immune response of a desired animal, including mammals and humans. The method comprises administering a therapeutically effective amount of a compound selected from the group consisting of carboxylic acid, esters thereof, pharmaceutically suitable salts thereof, metabolites thereof, and combinations thereof. This compound may be administered to the animal in a single dose or multiple doses. This method takes advantage of the natural properties of funic acid to enhance the immune response of animals including mammals and humans.

The methods provided herein provide fungic acid as a prophylactic or therapeutic compound for increasing immune responses and treating immunoinflammatory diseases (ie, IBD, allergic and autoimmune diseases) and metabolic diseases (ie, obesity and type 2 diabetes). Show that you can use Funic acid is also a potential as adjuvant therapy aimed at further increasing the efficiency of other pharmacological treatments currently used to prevent or improve IBD.

Therefore, a method of treating an immune disorder in an animal is provided, wherein the animal is administered an amount of funic acid effective to treat the immune disorder. Although any form of carboxylic acid can be used, in a preferred embodiment the free acid form of the carboxylic acid is used.

In a preferred embodiment of the invention, the funic acid compound is administered orally to the animal. The funic acid compound can be administered alone or in combination with a pharmaceutically suitable carrier.

The funic acid compound can also be administered parenterally, by injection or rectal. The funic acid compound may be administered alone or in combination with a pharmaceutically suitable excipient.

In another embodiment of the invention, a therapeutically effective amount of the funic acid compound may be administered to the animal in combination with a nutritional food supplement. Such supplements include, but are not limited to, infant formula, children's products, senior formula, milk, cheese, kefir, cereal bars, weight controls, energy bars, other human foods, functional foods, and animal feed. .

Punic acid can also be administered in combination with other active ingredients such as vitamins or other fatty acids.

The effective amount of the funic acid compound depends on the needs of the animal. For example, in one embodiment, an amount effective to enhance an immune response in an animal is provided.

In another embodiment, an animal is administered to the animal in an amount effective to enhance an immune response against a viral antigen, particularly an influenza antigen.

In another embodiment, an amount of a funic acid compound is administered that is effective to enhance the development of the animal's immune system. The amount of funic acid is preferably an amount effective to enhance the development of primary and secondary lymphoid organs and immune cells, including but not limited to thymus, spleen, lymph nodes, intestinal related lymphoid tissue in the intestine and other mucosal surfaces.

In another embodiment, an animal is administered to the animal in an amount effective for treating an inflammatory bowel disease (IBD). In particular, it is administered in an amount effective to prevent or ameliorate clinical signs and intestinal lesions associated with IBD.

In another embodiment, the amount of the funic acid compound effective to treat type 2 diabetes and obesity is administered to the animal. In particular, it is administered in an amount effective to normalize impaired glucose tolerance, prevent hyperglycemia, prevent hyperinsulinemia, and minimize abdominal fat accumulation.

In another embodiment, the animal is administered an amount of the funic acid compound effective to increase CD4 ⁺ and CD8 ⁺ lymphocyte levels. The amount of the funic acid compound is an amount effective to increase the level of T cells while preventing an exacerbated immune response, allergy, hypersensitivity, or autoimmune response.

In another embodiment, the animal is administered to the animal in an amount effective to prevent immunosuppression in the animal, including mammals and humans. In particular, infants, children, the elderly and other at-risk populations are administered in an amount effective to prevent immunosuppression.

The present invention also provides a method of preventing or treating a viral and / or bacterial infection in a mammalian subject comprising the step of orally or parenterally administering a stable and effective amount of funic acid. Preferably, the method comprises treating viral and / or bacterial infection by increasing immune function of the subject by increasing immune function.

Also provided by the present invention is the use of funic acid in the preparation of a composition for preventing or treating a viral and / or bacterial infection, preferably comprising increasing immune function to a subject.

The invention also relates to the provision of the use of funic acid in the preparation of a composition for preventing or ameliorating intestinal immune or inflammatory diseases.

A further aspect of the invention relates to the use of funic acid in the preparation of a composition for increasing the development of the immune system, the use of the funic acid in the manufacture of a medicament for increasing immune function, viral and / or bacterial infections. It relates to the use of funic acid for use in preventing or treating, preventing or ameliorating intestinal immune or inflammatory diseases, enhancing the development of the immune system or enhancing immune function.

The present invention also relates to methods for improving the nutritional quality of infant formulas, elderly formulas, milk, cheese, kefir, cereal bars, weight control, other human foods, and animal feeds. Adding a safe and effective dosage of the nic acid.

The formulations of the funic acid disclosed herein are typically presented in unit dosage form and may be prepared by any method well known in the pharmaceutical or nutrition arts. Possible formulations include, but are not limited to, capsules, cassettes, tablets, boluses, lozenges, each containing a predetermined amount of funic acid.

Advantageously, there is no upper limit to the amount of funic acid that can be administered to the animal in need. In addition, the methods of the present invention can be administered to animals of all ages and all health conditions, such as mammals and humans. For example, sick people such as elderly people, obese people, diabetics, sick people or very young people can benefit from the present invention, and even healthy people without a history of immunosuppression. In addition, the methods of the present invention can be administered in a variety of ways, thus providing a variety of effective means for enhancing immune responses and preventing metabolic diseases in mammals.

The scope of the invention will become apparent from the following detailed description of the preferred embodiments of the invention in conjunction with the accompanying drawings.

FIG. 1A is a graph illustrating the effect of a control diet of dietary funic acid and the same calorie on weight loss during Experiment 1. FIG.

FIG. 1B is a graph illustrating the effect of a control diet of dietary funic acid and the same calories on rectal bleeding during Experiment 1. FIG.

1C is a graph illustrating the effect of a control diet of dietary funic acid and the same calorie on disease activity index during Experiment 1. FIG.

FIG. 2A is a photograph of a 20-fold magnification of hematoxylin and eosin (H & E) -stained paraffin-inserted colon tissue recovered from mice fed funic acid during Experiment 1. FIG.

FIG. 2B is a photograph of 20-fold magnification of H & E-stained paraffin-inserted colon tissue recovered from mice ingesting a funic acid diet during Experiment 1. FIG.

FIG. 2C is a photograph of H & E-stained paraffin-inserted colon tissue recovered from mice fed the control diet during Experiment 1. FIG.

 FIG. 2D is a picture of H & E-stained paraffin-inserted colon tissue recovered from mice fed the control diet during Experiment 1. FIG.

FIG. 2E is a photograph of 40-fold magnification of H & E-stained paraffin-embedded colon tissue recovered from mice fed a funic acid diet during Experiment 1. FIG.

FIG. 2F is a photograph of 40-fold magnification of H & E-stained paraffin-inserted colon tissue recovered from mice fed a funic acid diet during Experiment 1. FIG.

FIG. 3A is a graph illustrating the effect of funic acid on blood glucose concentrations in mice fed a diet from Experiment 4. FIG.

FIG. 3B is a graph illustrating the effect of funic acid on blood glucose concentration in mice fed high fat diet from Experiment 4. FIG.

Funic acid

As used herein, the term punic acid refers to conjugated linoleic acid isomers, non-toxic salts, active esters, active isomers, active metabolites, containing cis-9, trans-11, cis-13 double bonds in the C ₁₈ carbon chain, Constituent lipids containing funic acid and mixtures thereof. Punic acid is also known as trichosanic acid and is the seed oil of Punica granatum (Punicaceae, pomegranate) and Trichosantes angina (Cucurbitaceae, snake cucumber) Found in Funic acid makes up about 86% of the oil in pomegranate seeds. For example, non-toxic salts include alkyl esters having 1 to 6 carbon atoms in the alkyl group, as well as mono-, di- and tri-glycerides thereof, and mixtures thereof. The active isomers of the funic acid are geometric isomers, such as eleostearic acid (cis-9, trans-11, trans-13 octadecatriic acid) and nontoxic salts thereof (e.g. sodium, potassium, calcium and magnesium Salts) and active esters thereof (eg, alkyl esters having 1 to 6 carbon atoms in the alkyl group, as well as mono-, di- and tri-glycerides and mixtures thereof).

The funic acid may be a substantially pure single chemical compound or a mixture of one or more punic acid compounds described above. The term "substantially pure" means that the purity is at least 90% by weight, preferably at least 95% by weight, such as 98% by weight, more preferably about 99% by weight, and 100% by weight. The funic acid may be in the form of an extract available or obtained directly from pomegranate seed oil or from one or more purification steps below.

Funic acid has the ability to extremely strongly resist the oxidation, inflammation and destruction functions of the free radicals of oxygen. Funic acid is shown not only to prevent sclerosis but also to delay the progression of cancer in the body. Punic acid acts as an antioxidant that physiologically lowers plasma cholesterol. In addition, funic acid has an ex vivo inhibitory effect on human platelet coagulation and arachidonic acid metabolism. In short, funic acid has the potential to be widely used in the pharmaceutical and health care, food and cosmetic industries, for example.

The funic acid used in the process described above may be in free acid form or may be chemically linked via an ester bond. In natural form, the funic acid is stable to heat. Funic acid can be used in its natural oil state or in dried and powdered form. In addition, the free acid form of the funic acid can be converted to its nontoxic salts such as sodium, potassium or calcium salts by reacting a chemical equivalent of the free acid form with an alkali hydroxide at basic pH.

administration

 In the process of the invention, therapeutically effective amounts of the funic acid compound are administered to animals, including mammals and humans. In a preferred embodiment, even though the funic acid compound is administered orally or parenterally, other forms of administration such as pharmaceutical compounds or aerosols are contemplated.

For oral administration, an effective amount of funic acid can be administered, for example, in solid, semisolid, liquid or gaseous form. Specific examples include tablets, capsules, powders, granules, solutions, suspensions, syrups, and elixirs materials. However, the funic acid compound is not limited to this form.

In order to formulate the funic acid of the present invention into tablets, capsules, powders, granules, solutions or suspensions, it is preferred that the funic acid compound is mixed with a binder, disintegrant and / or lubricant. If desired, the composition formed can be mixed with diluents, buffers, infiltration agents, preservatives and / or flavoring agents using known methods. Examples of binders include crystalline cellulose, cellulose derivatives, corn starch, and gelatin. Examples of disintegrants include corn starch, potato starch, and sodium carboxymethylcellulose. Examples of lubricants include talc and magnesium stearate. Also additives such as lactose and mannitol may be used.

For parenteral administration, the funic acid compounds of the invention can be administered rectally or by injection. For parenteral administration, suppositories can be used. Suppositories can be prepared by mixing a pharmaceutically suitable excipient with the carboxylic acid of the invention, which melts at body temperature but remains solid at room temperature. Examples of these include, but are not limited to, cacao butter, carbon wax or polyethylene glycol. The resulting composition can be molded into the desired shape using methods known in the art.

In injection administration, the funic acid compounds of the invention can be administered transdermally, subcutaneously, intravenously or intramuscularly. Such injectable medical drugs are prepared by dissolving, suspending or emulsifying the funic acid of the present invention by methods known in aqueous or non-aqueous solvents, such as vegetable oils, glycerides of synthetic resin acids, esters of high fatty acids, or propylene glycol. Can be prepared. If desired, additives such as solubilizers, osmotic agents, emulsifiers, stabilizers or preservatives may also be added.

Pharmaceutically suitable solvents can be used to formulate the funic acid of the invention in suspension, syrup or elixir.

The funic acid of the present invention may also be used with additive compounds having other pharmaceutically suitable activities for the manufacture of medical drugs.

The funic acid of the present invention can also be administered in the form of aerosols or inhalants by filling the liquid or fine powder in the form of a non-pressurized container, such as an aerosol container or nebulizer, together with a gas or liquid spray and, if necessary, a known adjuvant (e.g., an expanding agent). Can be. For example, pressurized gas of dichlorofluoromethane, propane or nitrogen can be used as the nebulizer.

Punic acid can be administered to animals in need, including mammals and humans, as pharmaceutical or veterinary compositions such as tablets, capsules, solutions or emulsions. In a preferred embodiment of the invention, the free acid form of the funic acid is administered. However, it is also contemplated in the present invention to administer other forms of funic acid, including but not limited to, esters thereof, pharmaceutically suitable salts thereof, metabolites thereof, and combinations thereof in a single dosage form or multiple dosage forms.

Punic acid can also be administered to animals as needed as a nutritional additive, ie as a food or nutritional supplement.

The terms “prophylaxis or treatment” or “treatment or amelioration” and similar terms used herein include prophylactic and complete or partial treatment. These terms include any other changes to the patient's condition that reduce the symptoms, ameliorate the symptoms, reduce the severity of the symptoms, reduce the incidence of the disease, or improve the therapeutic outcome.

The funic acid is preferably used and / or administered in the form of a composition. Suitable compositions are preferably pharmaceutical compositions, foods or food supplements. Such compositions provide a convenient form for delivering the funic acid. With regard to oxidation, the compositions of the present invention may comprise an amount of antioxidant effective to increase the stability of the funic acid.

The amount of funic acid is preferably from about 0.001 g to about 20 g (more preferably from 0.1 g to 10 g, such as from 0.5 g to 5 g) of the peronic acid or derivative thereof per day. Suitable compositions can be formulated according to the above.

Preferred compositions according to the invention are foods. The food product (including animal feed) preferably contains a fatty phase containing funic acid. Food is optionally used as a blend with supplemental fats. For example, the blend may comprise 0.3 to 95% by weight (preferably between 2 and 80% by weight, most preferably 5 to 40% by weight) of funic acid; And 99.7 to 5% by weight (preferably 98 to 20% by weight, most preferably 95 to 60% by weight) of auxiliary fats such as cocoa butter, cocoa butter equivalents, palm oil or a fraction thereof, oils of palm kernels or Fractions thereof, internal-esterified mixtures of said fats and fractions thereof; Or liquid oils such as sunflower oil, sunflower oil of high heritage, soybean oil, rapeseed oil, cottonseed oil, fish oil, safflower oil, safflower oil of high heritage and corn oil. Examples of suitable foods include margarine, fat continuous or water continuous or dual continuous spreads, low fat spreads, confectionary products such as chocolate, chocolate coating or chocolate peeling, or confectionery peeling, ice cream, ice cream coatings, ice cream inclusions, dressings, mayonnaise, Cheese, cream substitutes, dry soups, beverages, cereal bars, sauces, snack bars, dairy products, clinical nutritional products and infant formulas.

Another example of a composition is a pharmaceutical composition such as multiparticulates, powders, elixirs, syrups, suspensions and solutions, including tablets, pills, capsules, caplets, granules, beads, pellets and microencapsulated particles. The pharmaceutical composition comprises a pharmaceutically acceptable diluent or carrier and is preferably suitable for parenteral administration (eg oral). Oral dosage compositions may be in solid or liquid form and may be in tablet, powder, suspension, and syrup forms. Optionally, the composition may comprise one or more flavoring and / or coloring agents.

Pharmaceutically acceptable carriers suitable for use in such compositions are well known in the art of pharmacy. The composition of the present invention may contain 0.1 to 99% by weight of the funic acid. Compositions of the present invention are generally prepared in unit dosage form. Preferably, the unit dose of funic acid is 1 mg to 1000 mg (more preferably 100 mg to 750 mg). Excipients used in the preparation of such compositions are excipients known in the art.

Further examples of the product form of the composition are food supplements such as gelatin, starch, modified starch, starch derivatives such as soft gels or hard capsule forms comprising encapsulating material selected from the group consisting of glucose, sugar, lactose and fructose. The encapsulating material may optionally contain crosslinking agents, polymerizers, stabilizers, antioxidants, light absorbers for protecting the photosensitive filler, preservatives and the like. Preferably, the unit dose of funic acid in the food supplement is 1 mg to 1000 mg (more preferably 100 mg to 750 mg).

The method of the present invention administers a therapeutically effective amount of the funic acid compound to the animal in need. The effective amount of the funic acid depends on the form of the compound to be administered, the duration of administration, the mode of administration (eg oral or parenteral), the age of the animal and the condition of the animal.

For example, the amount of funic acid effective to enhance an immune response in an animal ranges from 10 to 10,000 mg / kg / day. Preferred effective amounts of funic acid are from 100 to 5,000 mg / kg / day, more preferably from 10 to 100 mg / kg / day. An effective amount of funic acid of about 35 to 40 mg / kg / day is planned by the method of the present invention, with 38 mg / kg / day being the preferred dosage. As long as the food of the recipient contains the necessary fatty acids, the upper limit of the effective amount to be administered is not of great importance because the funic acid is relatively non-toxic.

The amount of funic acid effective to treat and prevent IBD, including UC and CD, is from 50 to 500 mg / kg / day and the preferred dosage is from 100 to 150 mg / kg / day.

An effective amount of funic acid is most effective at enhancing the immune response of the animal when administered for a period ranging from 7 to 100 days (preferably 15 to 50 days, most preferably 30 to 42 days).

The most effective amount of funic acid to treat type 2 diabetes and obesity is 40 to 500 mg / kg / day, with a preferred dose of 100 to 150 mg / kg / day.

An effective amount of the funic acid compound of the present invention is administered in a nutritional, medical or veterinary composition, with a preferred dosage ranging from about 0.01% to 2.0% by weight of the food or nutritional product.

The invention may include the treatment of a disease or condition caused by a bacterial or viral infection or a bacterial or viral infection. Examples of bacterial infections include but are not limited to Staphylococcus aureus, Aeromonas hydrophila, Legionella pneumophila or Bacillus laterosporus, for example. Infections caused by microorganisms from the genus Staphylococcus, Aeromonas, Legionella, Bacillus or Micrococcus. Viral infections include, for example, Picornavirus, togavirus, paramyxovirus, Ortomyxovirus, Rhabdovirus, Reovirus, retrovirus (Retrovirus, Bunyavirus, Coronavirus, Arenavirus, Parvovirus, Papovavirus, Adenovirus, Herpevirus and Fox Includes those caused by one of the Poxviruses.

Examples of diseases or disorders that lack an immune system include diseases or disorders resulting from treatment with drugs (eg, chemotherapy), malnutrition and infection (eg, viruses such as HIV and AIDS).

Preparation of Phenic Acid

 Pomegranate rich pomegranate oil has already been isolated from pomegranate seeds and has been described as an anticarcinogen (Kim et al. 2002; Hora et al. 2003). Pomegranate seeds were separated from their juice jars, washed with water and dried in a convection dryer. Oil extraction was performed by a "cold press" at 80 ° C using a type 40A electric screw press (Skeppata Maskin, Orebro, Sweden). The resulting oil contained 480 mg of funic acid per gram of oil. Phenic acid can also be obtained by enzyme biosynthesis using methods known in the art (Iwabuchi et al., 2003), (Horsch et al., 2002).

However, the solvent extraction method is recommended because the enzyme biosynthesis and low temperature press method of the carboxylic acid extraction are insufficient (eg, 10% yield). Prior to solvent extraction, the seeds were steam heated to reduce enzymatic hydrolysis to improve processing. After heating, the seeds were finely ground and used for solvent extraction. A prescribed liquid solvent, such as hexane, is needed to soak the seeds multiple times for 12-20 hours with stirring, filtration and solvent evaporation. Alternatively, the pomegranate rich pomegranate oil is a CO ₂ known in the art. It can be regenerated by a supercritical extraction method.

Since the oil is more sensitive to the oxidation process upon release from the seed, it is preferable to carry out the extraction under a nitrogen blanket to avoid contact with air. In addition, the oil is nitrogen cleaned and stored with one or various antioxidants in the dark at 4 ° C. or at −20 ° C. for long term storage. Oil from the pomegranate seeds is extracted in an amount of 18% by weight of the weight of the seeds during this process.

The practice of the present invention is further illustrated by the following experiment. All publications, patents, and patent applications mentioned in this experimental section are incorporated herein by reference. While the invention has been described above in connection with specific preferred embodiments, and many details have been set forth for the purpose of illustration, those skilled in the art will recognize that the invention allows further embodiments and the specific embodiments described herein. It will be appreciated that the following detailed description may vary significantly without departing from the basic principles of the invention.

The invention is illustrated in accordance with the following non-limiting examples and is not intended to be limited to this range in any way. All percentages and parts and ratios in the examples and herein are by weight unless otherwise indicated.

Experiment 1

purpose

To determine the effect of funic acid on IBD and colitis related diseases.

Way

In the first experiment, eleven C57BL6 mice received 38 g of 0 g or 0.6 g of funic acid per 100 g of food. In the control diet, foods were made in the same calorie by replacing punic acid with linoleic acid (by weight). On day 32 of the experiment (7 days before killing the mouse), mice were challenged with 2.5% DSS, 36,000-44,000 moles / weight (ICN Biomedicals, Aurora, Ohio) in drinking water to challenge the mouse. I was. Animal models of IBD, including the DSS colitis model, are a means of detecting the immunological onset of IBD (Strober et al. 2002) and a stable method of testing the effectiveness of prophylactic and / or therapeutic treatment of novel compounds such as funic acid. One important feature of the DSS challenge is the ability to break the epithelial cell barrier and promote increased cell exposure to normal luminal and mucosal microflora (Strobe et al. 2002). After the DSS challenge, the mice were weighed daily and blinded to examine clinical signs of disease associated with colitis (perianal contamination, rectal bleeding, diarrhea and hair). The disease activity index and rectal bleeding scores were calculated using already published composite clinical scores (Saubermann et al. 2000; Bassagana-Liera et al. 2004).

After killing the mice, colon, brain, kidney, liver and spleen were collected from the mice. The colon was weighed and scored by blind observation of the weight of the visual lesion after a grade of 0-3 (eg 0 = no lesion, 3 = severe lesion). The colon contents were washed by gently rinsing the colon with sterile 1 × PBS using a mouse cabbage needle connected to a 5 mL syringe. Other tissues were added directly to formalin beakers. All samples were assigned the following information: 1) mouse number; 2) date of collection; 3) experiment number; 4) type of solvent; And 5) type of tissue. Colon, brain, kidney and liver fragments were fixed in 10% buffered neutral formalin (Fisher, Atlanta, GA), inserted into paraffin and cut (6 μm) and hematoxylin and eosin for histology Staining (H & E) (AML Labs, Baltimore, MD). Tissue slides were examined with an Olympus microscope (Olympus America Inc., Va., Virginia) and Adobe Photoshop Elements 2.0 (Adobe Systems Inc) ., San Jose, CA).

The colon was graded using a composite tissue score including 1) vesicle damage and 2) regeneration, 3) degeneration / hyperplasia, 4) intrinsic mucosal vascular changes, 5) submucosal changes, and 6) the presence of inflammatory infiltrates. The fragments were graded in the range of 0 to 4 for each of the categories described above and the data presented as defined composite scores. For vesicle injury, 0 = none; 1 = 1/3 of reference; 2 = 2/3 of the reference; 3 = only superficial epithelium intact; 4 = total vesicle and epithelial loss. For epithelial erosion, 0 = no erosion; 1 = weak focal erosion; 2 = weak multipath erosion; 3 = significant ulcer over the entire colon; 4 = colon tissue loss. For mucosal thickness, 0 = normal thickness; 1 = increase in weak thickness; 2 = weak to severe thickness increase; 3 = increase in severe thickness; 4 = mucosa obstructing the lumen of the intestine. For regeneration, 4 = no tissue healing; 3 = traces on the surface epithelium; 2 = regeneration with vesicle deficiency; 1 = almost complete regeneration; 0 = complete regeneration or normal tissue. For alteration / hyperformity, 0 = none; 1 = weak goblet cells; 2 = severe goblet cell degeneration; 3 = immature cells present in goblet cell degeneration and base, extending to 2/3 of the gland; 4 = immature cells present at base, extending beyond two-thirds of gland. For vascular change, 0 = none; 1 = weak capillary dilatation in the intrinsic mucosa; 2 = weak capillary dilatation under the intrinsic mucosa and submucosa; 3 = severe capillary swelling; 4 = unique mucosal bleeding. For intrinsic mucosal leukocyte infiltration, 0 = normal infiltration (same matrix of plasma cells and lymphocytes with some neutrophils); 1 = weak increase in neutrophils in the intrinsic mucosa; 2 = weak increase in prolonged neutrophils into the submucosal layer; 3 = severe neutrophil residence under the intrinsic mucosa and / or submucosa; 4 = severe neutrophil residence and migration of plasma cells of the intrinsic mucosa and / or submucosa. Brain, kidney and liver were collected to assess the safety of fenic acid administration.

Statistical analysis was performed by analysis of variance (ANOVA). ANOVA was carried out using the general linear model procedure of SAS (SAS 1988), as described above (Bassagana-Riera et al. 2003). The difference in probability value ( P <0.05) is considered statistically significant.

result

The mortality rate associated with colonic inflammation in the control group was 50% on day 6 of the 2.5% DSS challenge, whereas the mortality rate was 0% in mice fed with funic acid. The average onset of clinical disease in control mice was one day after initiation of the DSS challenge, whereas in control-fed mice, clinical disease did not appear until six days after the DSS challenge. On average, rectal bleeding began on day 4 of the DSS challenge in control-fed mice, whereas rectal bleeding did not appear on day 7 of the DSS challenge in mice fed the funic acid supplement. The DSS challenge resulted in weight loss, colitis-related clinical disease, and rectal bleeding, which appeared faster and more severely in the control mice than in the mice fed the funic acid supplement. FIG. 1A illustrates the effect of dietary funic acid (0.6 g / 100 g) and the same calorie control on weight loss during a representative 7 day DSS challenge (2.5%, weight / volume) of experimentally induced IBD. 1B illustrates the effect of dietary funic acid (0.6 g / 100 g) and the same calorie control on rectal bleeding during the 7 day DSS challenge (2.5%, weight / volume). FIG. 1C illustrates the effect of dietary funic acid (0.6 g / 100 g) and the same calorie control on disease activity index during the 7-day DSS challenge (2.5%, weight / volume). Mice received a funic acid supplement for 32 days prior to the DSS challenge. Colitis-related diseases were improved. Prevention or amelioration of clinical signs of colon inflammation correlates with lower colon weight and less severe scores during visual analysis of lesions in mice fed funic acid.

Specifically, after the DSS challenge, the average visual score (rank of 0 to 3) in the colon of the control diet mice was 3, while the score in the mice fed the funic acid was 1.4. The average colon weight was 0.958 g in mice fed the control diet, whereas the average colon weight was 0.153 g in mice fed the funic acid. Lower colon weight and weaker lesions can be caused by reduced inflammatory cell migration into the colon. Histopathological examination of H & E-stained slides of the colon under the microscope revealed no major lesions in mice fed FUNIC, whereas mice on the control diet had thickened colonic mucosa, epithelial erosion, and infiltration of inflammatory cells. appear. At 20 magnification, loss of epithelial surface was observed in FIG. 2C when compared to FIG. 2A. At 40 magnification, inflammatory cell infiltration, epithelial flattening and epithelial erosion were observed in FIGS. 2E and 2F. 2B shows thickening of the colon mucosa without erosion and infiltration. 2D showed that epithelial cells maintain their normal columnar shape.

As a score of the pathological results, it is shown that the severity of colon lesions in the control diet mice is greater than in the mice fed the funic acid (Table 1).

Tissue score after 7 days challenge with 2.5% DSS Item Counterpart Phenic Acid Diet P value Mucosa thickness 4.00 ± 0.42 ^* 1.83 ± 0.24 0.0146 Epithelial erosion 4.00 ± 0.12 ^* 0.00 ± 0.00 0.0001 Inflammatory Cell Infiltration 4.00 ± 0.30 ^* 0.50 ± 0.17 0.0001 Vascular changes 0.50 ± 0.20 0.50 ± 0.35 0.1927 Deformation / Hyperplasia 3.50 ± 0.50 ^* 0.50 ± 0.28 0.0061 Vesicle damage 4.00 ± 0.45 0.33 ± 0.26 0.0009 The values are the least square mean ± standard error of the mean.
* Means the values are statistically different.

The goal of a prophylactic or therapeutic approach of IBD is generally accepted to include inducing substantial clinical improvement (Liechtenstein et al. 2003). Oral administration of funic acid has resulted in significant clinical and pathological improvement. In addition, visual and histological (no data) analysis of major organs is associated with metabolism (ie, liver and kidneys), and the brain shows no abnormalities, suggesting that the dose being used is effective and safe. . No adverse events were observed during the study.

Experiment 2

purpose

To investigate the effects of funic acid on immune system improvement, lymphocyte count and lymphocyte function.

Way

Tour, dose and punic acid preparation were the same as in Experiment 1.

Medium and Reagent Preparation

After preparation of each medium or buffer, the following information was attached to the final product: 1) name of the product, 2) date of manufacture, 3) expiration, 4) recommended storage, 5) initials of the technician, and 6) sterile.

The first medium prepared was cRPMI. The formulation is based on an intercellular medium using a bicarbonate buffer system and altered amounts of amino acids and vitamins. cRPMI medium was used for the culture of normal human and tumor leukocytes. When properly supplemented, cRPMI has a wide range of uses to support the growth of many types of cultured cells, including new human lymphocytes. cRPMI was prepared in the following manner

cRPMI:

content ingredient 400 mL RPMI 1640 50 mL Fetal Bovine Serum 5 mL Sodium Pyruvate 5 mL Non-Essential Amino Acids 10 mL Essential amino acid 5 mL L-glutamine 5 mL Penicillin-streptomycin 12.5 mL 1M Hepes Buffer

The composition of cRPMI is known in the art (Bassagana-Riera et al. 2001). All components were combined under a sterile Lamina flow hood (Nuaire, Flymouth, Minnesota) and sterilized by filtration using a 0.22 micron membrane filter (Fisher, Atlanta, GA) with a 60 micron prefilter. Prepared medium was prepared in ethoxaclin-washed (Sigma, Montana St. Louis), autoclaved, sterile screwcap treated glass bottle (50 mL) (30 min at 121 ° C., 15 PSI) Labeled and stored at 4 ° C.

FACS and PBS buffers were prepared according to the following concentrations:

FACS buffer:

content ingredient 500 mL 1X PBS 5 mL Fetal Bovine Serum 1 mL 10% Sodium Azide

After combining these, the FACS buffer was labeled with k and stored at 4 ° C.

PBS (1X):

content ingredient 1,000.00 mL Deionized Water ^* 8.00 g Sodium chloride 1.15 g Sodium phosphate 0.20 g Potassium chloride 0.20 g Potassium phosphate Deionized water was obtained from an E-pure water purification system (Barnstead Int., Dubuque, Louisiana).

PBS (10X):

content ingredient 1,000.00 mL water 80.00 g Sodium chloride 11.50 g Sodium phosphate 2.00g Potassium chloride 2.00g Potassium phosphate

After combining the components and stirring for 30 minutes, the solution was autoclaved at 121 ° C. for 30 minutes at 15 PSI (Brinkman, Tutnauer, Jerusalem, Israel). Concentrated hydrochloric acid or sodium bicarbonate was added to adjust the pH to 7.2 (AR15 PH / MV / TEMP system, Accumet, Arvada, Colorado).

A total of 22 C57BL6 mice were used in Experiment 2. All mice were born on November 3, 2003, weaned on November 16, 2003, and were given an experimental diet from December 19, 2003 to January 15, 2004. A total of 10 mice received a control diet and 12 mice received a food supplemented with funic acid (0.6 g punic acid / 100 g food). Mice were weighed weekly. Mice were euthanized and blood from each mouse was collected and stored at −20 ° C. for regenerated serum. Thymus, spleen and colon lymph nodes were harvested and weighed and collected in cRPMI under sterile conditions. Colon and liver samples were then collected in RNA (Ambion Inc, Austin, TX) and frozen at −80 ° C. for gene expression analysis. Brain, kidney, liver, colon and ileum were also collected in 10% buffered neutral formalin solution and treated as described in Experiment 1 for tissue evaluation. The remainder of the dead mice were stored in 50 mL conical tubes and frozen at −20 ° C. for body composition and fatty acid analysis. All samples generally attached the following information: 1) mouse number; 2) date of collection; 3) experiment number; 4) type of solvent; And 5) type of tissue. Tissue slides were examined under an Olympus microscope (Olympus Americas, Inc. Varus, Virginia). Images were collected using FlashBus FBG software (Integral Technologies, Indianapolis, Indiana) and processed with Adobe Photoshop Elements 2.0 (Adobe Systems Inc., San Jose, Calif.).

Spleen, thymus and colon lymph nodes were received at complete RPMI on ice. Tissue was gently broken between the two frozen ends of two glass slides in a Petri dish containing 3 mL cRPMI to obtain a single cell suspension from each tissue. Single cell suspensions were transferred from Petri dishes to 15 mL polypropylene tubes (Fisher, Atlanta, GA) and washed by centrifugation in an Eppendorf 5810 R centrifuge (Westbury, New York) at 200 x g for 5 minutes at 4 ° C. Discard the supernatant by tilting the tube. Cell pellets were crushed by tapping the tube against the solid surface. The total number of splenocytes, thymic cells, and colon lymph node leukocytes in each mouse was counted on the Z1 Coulter Single Particle Counter (Miami, Florida). Briefly, 10 mL of isoton II diluent solution (Beckman Coulter, Miami, FL) was added to the counting vial followed by 40 μL of cell suspension, 4 drops of Zapaglobin (Beckman Coulter). , Miami, FL), and 10 mL of additional isotone solution were added. The volume in which cells were resuspended was measured using 5 mL sterile pipettes. The total amount of cells in each tissue and mouse was determined using the concentration (cell count / mL) and total volume in each tube.

Flow Cytometry

Flow cytometry provides an efficient and sensitive quantitative method for analyzing the phenotype of cells containing lymphocytes. Cells express proteins in the surface membrane that can be used to infer specific functional properties such as the ability to proliferate and produce antibodies, including cells in a particular population or subpopulation. Monoclonal antibodies (mAb) bind to these proteins (eg, CD4, CD3, CD8, alphabeta-TCR, gammadelta-TCR, and CD 19) in a species-specific manner. For flow cytometry applications, the mAb is labeled with different fluorescent dyes (eg, phycoerythrin (PE); fluorescein isothiocyanate (FITC), chromium (Cy)). Flow cytometry is currently used both in the study of human immunodeficiency virus-induced lymphatic deficiency, leukemia and lymphoma (eg, immunophenotyping and measurement of lymphocyte activity, etc.) and in monitoring of transplant rejection.

To evaluate the effects of funic acid on CD4 ⁺ , CD8 ⁺ T-cell receptor (TCR) alphabeta ⁺ , TCRgammadelta ⁺ , and CD19 ⁺ lymphocytes in the spleen, thymus, and colon lymph nodes, ingested funic acid Splenocytes, thymic cells and colon lymph node lymphocytes recovered from mice and mice fed control diets were examined by flow cytometry. A description of the use of flow cytometry is described in Experiment 1. The splenocyte suspension was free of red blood cells by osmotic lysis (1 × PBS solution was prepared by adding 4.5 mL of sterile deionized water for 1-2 seconds and then adding 0.5 mL of 10 × PBS solution). Cells were washed again by centrifugation at 200 × g for 5 minutes at 4 ° C. in Eppendorf 5810R centrifugation (Westbury, NY). The tube was tilted and the supernatant drained. Cell pellets were broken by tapping the tube against the solid surface. FACS buffer (1 mL) was added to each tube. Splenocytes, thymic cells, and colon lymph node-derived mononuclear cells were counted on a Z1 coulter single particle counter (Miami, Florida) and adjusted to 2 × 10 ⁶ cell counts / mL. Splenocytes, thymic cells and colon lymph nodes were analyzed by flow cytometry for CD4, CD8, CD3, and TCR alphabeta according to the foregoing (Bassagana-Riera et al. 2001; Bassagana-Riera et al. 2002; Bassagana-Riera et al. 2003). The expression of TCRgammadelta and CD19 molecules was analyzed. Briefly, 100 μl of splenocytes, thymic cells and colon lymph node lymphocyte suspensions were added to a round bottom micro titration plate (Becton Dickinson, Lincoln Park, NY) and 50 μl of primary antibody solution in FACS buffer. Stained with. Anti-mouse CD8-PE (1: 200 dilution); Anti-mouse CD4-FITC (1: 500 dilution); Anti-mouse CD3-cychrome (1: 500 dilution); Anti-mouse alphabeta-TCR-PE (1: 200 dilution), anti-mouse gammadelta-TCR-FITC (1: 500 dilution); And anti-mouse CD19-PE (1: 500 dilution). CD4 and CD8 expression was examined in two-color assays and CD19 was examined in monochrome assays. Data were collected using a Coulter EPIC XL-MCL flow cytometer (Miami, Florida). Data analysis was performed using CellQuest software (BD Biosciences, San Diego, Calif.).

Lymphocyte Blastogenesis Test

cRPMI medium was prepared as above. Wells of 96 well flat round bottom microtiter plates (Becton Dickinson, Lincoln Park, NY) were seeded with 2 × 10 ⁵ mononuclear cells at a total volume of 200 μl per well. Wells contained Concanavalin A (5 μl / ml, Con-A; Sigma), or medium alone (non-irritant). Titration plates were incubated for 5 days at 37 ° C. under 5% CO ₂ wetting atmosphere. After 5 days, 0.5 μCi of methyl- [ ³ H] C thymidine (specific radioactive 6.7 Ci mmol- ¹ ; Amersham Life Science, Arlington Height, Ill.) Was added to each well in 10 μl of medium. The plates were added and incubated again for 20 hours. Well contents are collected from a fiber filter with a PHD cell collector (Skatron Instruments Inc., Sterling, VA), and a liquid scintillation counter (Beckman Instruments, Illinois). Radioactivity measured by Saumburg). The stimulation index (SI) was calculated by dividing the number of stimulated wells / minute by the number of samples in three by the number / minute of unstimulated wells. Lymphocyte embryonic development analysis was performed according to the inventors already described (Bassagana-Riera et al. 2001; Bassagana-Riera et al. 2002; Bassagana-Riera et al. 2003).

Statistical analysis was performed by analysis of variance (ANOVA). ANOVA was conducted using the general linear model procedure of SAS (SAS 1988), as described above (Bassagana-Riera et al., 2003). The difference in probability values ( P <0.05) was considered statistically significant.

result

Lymph nodes are secondary lymphoid organs that are immune to viruses and bacteria or that other foreign antigens are initiated. Except for gamma delta T cells, immature lymphocytes recirculate from blood to lymph nodes and from lymph nodes to tissues. This may be because lymph nodes with denser populations initiate stronger and more effective immune responses against foreign antigens. These results demonstrate that the total number of lymphocytes in the lymph nodes of mice fed funic acid is greater ( P <0.0043) than the lymph nodes from mice fed the control diet. Although the weight of the lymph nodes was also greater (223.33 vs. 261.66 mg) than in mice fed funic acid, it was not statistically significant. In addition, phenotypic analysis of lymphocyte subpopulations of lymph nodes shows that largely expanded lymphocyte subpopulations include CD4 ⁺ T cells, CD8 ⁺ T cells and TCR alphabeta (αβ) ⁺ T cells. The number of B cells in the mice fed the funic acid was not significantly larger than the mice fed the control diet. These data suggest that animals that ingested punic acid induce T cell responses that are more effective than in animals that did not ingest funic acid (including humans). These findings seem to mean regulating immune system improvement, general immune health and resistance to infectious diseases. Table 2 illustrates the difference between lymph nodes recovered from mice fed either funic acid or a control diet.

Phenotype and Improvement Analysis of Colon Lymph Nodes Item Counterpart Phenic Acid Diet P value Weight (mg) 223.33 ± 3.79 261.66 ± 3.28 0.4544 Lymphocytes (x10 ⁶ / mL) 11.03 ± 1.77 18.64 ± 1.53 ^* 0.0043 CD8 ⁺ T cells 2.69 ± 0.46 4.53 ± 0.41 ^* 0.0083 CD4 ⁺ T Cells 4.10 ± 0.64 6.65 ± 0.58 ^* 0.0087 CD3 ⁺ T Cells 6.07 ± 0.94 10.24 ± 0.85 ^* 0.0043 CD19 ⁺ T Cells 3.80 ± 0.80 5.98 ± 0.72 ^* 0.0602 TCR αβ ⁺ cells 6.59 ± 1.14 11.78 ± 1.08 ^* 0.0042 The values are the least square mean of the mean ± standard error.
^* Is to illustrate that the values are statistically different

Spleens are also another crucial organ of the immune system. Events in the spleen indicate changes that occur in the recirculation of lymphocytes in the blood. The weight of the spleen in mice fed funic acid was much greater than in mice fed the control diet ( P <0.151). The spleen is larger due to the greater number of αβ T cells ( P <0.0352). This lymphocyte population includes CD4 ⁺ and CD8 ⁺ T cells (Table 3).

Analysis of the phenotype and improvement of the spleen Item Counterpart Phenic Acid Diet P value Weight (mg) 752.22 ± 3.65 884.54 ± 3.30 ^* 0.0151 Total number of lymphocytes (x10 ⁶ / mL) 148.87 ± 7.89 144.21 ± 7.13 0.6662 CD8 ⁺ T cells 13.19 ± 0.80 12.72 ± 0.73 0.6737 CD4 ⁺ T Cells 16.08 ± 1.22 17.04 ± 1.10 0.5697 CD3 ⁺ T Cells 29.25 ± 1.97 30.05 ± 1.78 0.7675 CD19 ⁺ T Cells 106.59 ± 4.99 102.22 ± 5.42 0.5960 TCR αβ ⁺ cells 29.37 ± 2.13 33.12 ± 1.93 ^* 0.0352 The values are the least square mean of the mean ± standard error.
^* Is to illustrate that the values are statistically different

The thymus is the main organ of the immune system where T cell precursors originate in mature bone marrow and differentiate into mature T cells, which can be released into the bloodstream and are present in secondary lymphoid organs such as the spleen and lymph nodes. Significantly greater weight of the thymus in mice ingested with toxic acid means that the phoenix acid advantageously improves thymic cells. The main population attributed to this larger thymus size and cytoplasm was CD4 ⁺ CD8 ⁺ double positive thymic cells. These thymic cells represent a stage of maturation prior to the development of mature CD4 ⁺ or CD8 ⁺ T cells. Some of the double positive lymphocytes will differentiate into CD4 ⁺ T cells and other CD8 ⁺ T cells. These data demonstrate that funic acid contributes to thymic improvement and thus to the immune system (Table 4).

Phenotype and improvement analysis of the thymus Item Counterpart Phenic Acid Diet P value Weight (mg) 611.11 ± 5.41 845.00 ± 4.68 ^* 0.0041 Lymphocytes (x10 ⁶ / mL) 63.09 ± 11.04 69.18 ± 9.56 0.6814 CD8 ⁺ Thymic Cells 1.36 ± 0.21 1.34 ± 0.18 0.9447 CD4 ⁺ CD8 ⁺ Thymic Cells 56.48 ± 10.09 63.25 ± 8.73 0.6178 CD4 ⁺ Thymic Cells 2.39 ± 0.33 2.51 ± 0.28 0.7885 TCR αβ ⁺ Thymic Cells 7.39 ± 1.40 9.12 ± 1.21 0.3617 The values are the least square mean of the mean ± standard error.
^{* Indicates} that the figures are statistically different

Tables 1-4 show that funic acid improves the immune system (ie immune organ weight and / or cytoplasm) in mammals and increases the number of T cells (ie, CD4 ⁺ and CD8 ⁺ ). Table 5 examines whether funic acid modulates the function of these lymphocytes. In particular, Table 5 illustrates the proliferation capacity of lymphocytes in response to stimulation of T cell mitogen Con A. These results show that the proliferative capacity of splenocytes recovered from mice fed FUNIC acid is significantly greater than that recovered from mice fed the control diet. This finding is used to show that T cells from PUA-ingested mice will grow faster and respond to pathogens during infection. The same numerical trend can be observed compared to the proliferative capacity of lymphocytes derived from lymph nodes. However, this numerical difference in lymph nodes is not statistically significant.

 Proliferation of Lymphocytes in Response to Stimulation with Con A Item Counterpart Phenic Acid Diet P value Count / min, spleen (cRPMI) 58.66 ± 12.66 73.17 ± 10.96 0.3974 Count / min, spleen (cRPMI & Con A) 694.00 ± 293.78 1905.27 ± 254.42 ^* 0.0057 Irritation index, spleen 14.25 ± 3.65 25.20 ± 3.16 ^* 0.0354 Count / min, lymph node (cRPMI) 35.05 ± 4.35 43.12 ± 3.76 0.1772 Count / min, lymph nodes (cRPMI & Con A) 516.51 ± 186.03 929.06 ± 161.10 0.1100 Irritation index, lymph node 14.83 ± 4.16 22.40 ± 3.61 0.1856 The values are the least square mean of the mean ± standard error.
^* Is to illustrate that the values are statistically different

Experiment 3

purpose

To determine the effect of funic acid on cellular immune responses to influenza virus antigens after vaccination.

Way

A total of 25 C57BL6 mice were used in Experiment 3. Ten were fed the control diet and 15 were fed a diet supplemented with punic acid (0.6 g punic acid / 100 g food). All mice received a control or funic acid supplement for 9 weeks before being immunized with influenza virus (VR-1469) consisting of 100 μg of the UV-inactivated influenza virus antigen in a Freunds Incomplete adjuvant. Mice were killed on day 15 of food supplementation. Spleens were received in sterile cRPMI on ice. Single cell suspensions were obtained from each tissue as described in Experiment 1. Single cell suspensions were transferred from Petri dishes to 15 mL polypropylene tubes (Fisher, Atlanta, GA) and washed by centrifugation at 200 × g for 5 minutes at 4 ° C. in an Eppendorf 5810 R centrifuge (Westbury, NY). The tube was tilted to drain the supernatant. Cell pellets were broken by tapping the tube against the solid surface. Spleen cells were counted on a Z1 coulter single particle counter (Miami, Florida). Briefly, 10 mL of isotone II dilution (Beckman Coulter, Miami, FL) was counted vials, 40 μL of cell suspension, 4 drops of Zap-oglobin (Beckman Coulter, Miami, FL). ), And then added to an additional 10 mL of isotone solution and used for functional analysis.

Carboxy fluorine succinimidyl ester (CFSE) proliferation assay

2 x 10 ⁷ splenocytes were isolated and subjected to CFSE proliferation assay (molecular probe). Cells were centrifuged for 5 minutes (200 × g) and the supernatant was aspirated. One cell of CFSE vial was diluted to 10 mM (Component A) in 90 μl of dimethyl sulfoxide (Component B) while the cells were centrifuged. 10 mM CFSE solution was diluted to 1 μM. A total of 2 × 10 ⁷ splenocyte pellets were resuspended in 1 mL of 5 μM CFSE solution and incubated in the dark for 10 minutes at 37 ° C. in 5% CO ₂ wet atmosphere. Cells were repellet in 3 mL of RPMI-1640 containing 10% fetal calf serum, resuspended and pre-warmed at 37 ° C. The cells were then incubated in the dark for another 15 minutes at 37 ° C. in 5% CO ₂ wet atmosphere. 3 mL of RPMI-164 containing 10% fetal calf serum was added, the cell suspension was centrifuged for 5 min (200 xg) and the supernatant was aspirated using a sterile Pasteur pipette. The last two steps were repeated once again and the cells were resuspended in cRPMI. CFSE-stained splenocytes were counted and the cell concentration was adjusted to 2 x 10 ⁶ splenocytes / mL cRPMI. Cell suspensions (100 μl) were added to a 96 well flat round bottom microtiter plate containing 100 μl of medium (not stimulated) or 10 μg / ml of the VR-1469 influenza virus antigen. Samples were treated ex vivo with six bees for each animal. Cells were incubated for 5 days at 37 ° C. under 5% CO ₂ wet atmosphere. As the cells split, CFSE membrane staining decreased, resulting in reduced average fluorescence intensity. This property is used to distinguish between proliferative and non-proliferative lymphocytes based on average fluorescence intensity. After 6 days, cells cultured from the above six wells were treated in vitro and mice were collected to prepare immunophenotypes. 100 μl of CFSE-stained splenocytes were added to a round bottom microtiter plate (Becton Dickinson, Lincoln Park, NY) and stained with 50 μl of primary antigen solution in FACS buffer. Anti-mouse CD8-PE (1: 200 dilution); Anti-mouse CD4-biotin (1: 500 dilution); Anti-mouse CD3-cychrome (1: 500 dilution); Anti-mouse beta-TCR-PE (1: 200 dilution), anti-mouse gammadelta-TCR-FITC (1: 500 dilution); And anti-mouse CD19-PE (1: 500 dilution). Staining of CD4 versus CD8 requires an extra step of adding streptavidin-cychrome that binds to biotin. Cell surface phenotype was examined in three color assays. Data collection was performed using a Coulter EPIC XL-MCL flow cytometer (Miami, Florida).

result

The results of Experiment 2 show that dietary funic acid supplementation enhances the ability of lymphocytes to proliferate (reproduce) in response to general mitogen stimulation (stimulation that causes proliferation), but the results of Experiment 3 show that the fungic acid is affected by influenza virus (flu The ability of lymphocytes to respond to viruses after being vaccinated) is also shown. Experiment 3 examined the ability of punic acid to modulate the immune response to influenza virus antigens and was characterized by one of the cellular targets of punic acid action (ie, CD8 ⁺ T cells). By examining influenza virus-specific proliferation of lymphocyte acetone, it was found that funic acid enhances the proliferative capacity of CD8 ⁺ T cells in response to influenza virus (Table 6). This finding is consistent with the results already reported in Experiment 2, showing the effects on viral disease resistance and prevention of common colds and influenza in vaccinated animals.

Acet-specific proliferation of lymphocytes in response to stimulation with influenza virus antigens
Item Not vaccinated Vaccinated
P value contrast Funic acid contrast Funic acid Relative Proliferation Index CD8 ⁺ T Cells 0.78 ± 0.18 0.57 ± 0.36 0.60 ± 0.40 1.15 ± 0.37 ^* 0.06 Relative Proliferation Index CD4 ⁺ T Cells 0.88 ± 0.31 0.68 ± 0.48 0.76 ± 0.58 1.10 ± 0.63 0.33 Relative Proliferation Index B Cells 1.00 ± 0.04 1.15 ± 0.63 0.96 ± 0.57 1.16 ± 0.27 0.90 The values are the least square mean of the mean ± standard error.
^{* Indicates} that the figures are statistically different

Table 3 also shows long term dietary supplementation studies (105 days). During the study, mice were monitored for adverse effects or clinical signs. No negative adverse effects associated with dietary funic acid supplementation were observed during the study. At the end of the study, the brain, kidneys, liver, heart and lungs were collected and fixed to formalin for tissue analysis. H & E stained slides show no microscopic lesions in these organs. Thus, funic acid effectively regulates immune function, but was also stable during the 105 day replenishment period.

Experiment 4

purpose

To determine the effect of funic acid on the improvement of obesity and type 2 diabetes caused by high fat foods. In particular, mice with high-fat foods were examined whether fengic acid could normalize impaired glucose tolerance, prevent hyperglycemia and hyperinsulinemia, and reduce abdominal fat accumulation.

Way

In Western countries, metabolic syndrome or syndrome X (ie diabetes, obesity, cardiovascular disease, hypertension and hyperlipidemia) is steadily increasing. Adequate and urgent needs are needed for the improvement of nutritional based therapeutic or prophylactic adjustments using orally active natural compounds. A total of 50 C57BL6 mice were used in Experiment 4. Twenty-five mice ate a control diet and 25 mice ate a food supplemented with punic acid (0.6 g punic acid / 100 g food). In the first 32 days of the experiment, all foods contained 7% fat, or 0.02 total cholesterol, and they obtained 14.5% calories from fat by replacing linoleic acid (by weight) instead of funic acid in the control formula (Table 7). This food is defined as a diet and formulated to have the same calories between treatment groups. On the 32nd day of the experiment, 20 mice in each group contained 19.6% fat, ie 0.2% total cholesterol, which obtained 40.1% calories from fat by replacing lard (by weight) in place of punic acid in the control high fat diet. One high fat diet was consumed (Table 8). In addition, the high fat diet was formulated to be the same calories among the treatment groups. The remaining mice in each group (n = 5) were fed a diet. On day 78 of the experiment, mice were killed and blood collected and fasted using the Accu-Check Instant Plus System (Roche Diagnostics Corporation, Indianapolis, Indiana). Glucose concentrations were analyzed immediately or stored for subsequent analysis of insulin concentrations in plasma. Abdominal white adipose tissue and scapula brown adipose tissue were collected and weighed and stored at -80 ° C for RNA analysis. Liver, lung, kidney, pancreas and heart were visually examined for abnormalities (total lesions), fixed to phosphate-completed formalin (10%) and treated as described in Experiment 1 for tissue evaluation. All samples were affixed with the following information: 1) mouse number; 2) date of collection; 3) experiment number; 4) type of solvent; And 5) type of tissue.

Composition of Diet ¹ ingredient Counterpart PUA expression casein 200 200 L-cysteine 3 3 Corn starch 397.486 397.486 Maltodextrin 132 132 Sugar 100 100 Cellulose 50 50 Mineral Mix (AIN-93) ² 35 35 Vitamin Mix (AIN-93) ³ 10 10 Choline Bitartrate 2.5 2.5 Tert-Butylhydroquinone ⁴ 0.014 0.014 soy milk 60 60 Linoleic acid 10 - Pomegranate oil - 10 1. Provide about 7% fat and 0.02 total cholesterol, and 14.5% calories were obtained from fat.
2. 3 g nicotinic acid, 1.6 g calcium pantothenate, 0.7 g pyridoxine HCl, 0.6 g thiamine HCl, 0.6 g riboflavin, 0.2 g folic acid, 0.02 g D-biotin, 2.5 g vitamin B ₁₂ (0.1% in mannitol) per kg of food , 15 g DL-alpha tocopheryl acetate (500 IU / g), 0.8 g Vitamin A palmitate (500,000 IU / g), 0.2 g Vitamin D ₃ (cholecalciferol, 500,000 IU / g), 0.075 g Vitamin K ( Filoquinone), and 974.705 g sugar.
3. 357g calcium carbonate, 196g potassium phosphate monobasic, 70.78g potassium citrate, 74g sodium chloride, 46.6g potassium sulfate, 24.3g magnesium oxide, 6.06g iron citrate, 1.65g zinc carbonate, 0.63g manganese carbonate, 0.31g per kg of food Cupric carbonate, 0.01 g potassium iodide, 0.01025 g sodium serene, 0.00795 g ammonium pararollidate, 1.45 g sodium meta-silicate, 0.275 g potassium sulphate, 0.0174 g lithium chloride, 0.0815 g boric acid, 0.0635 g Sodium fluoride, 0.0318 g nickel carbonate, hydroxide, tetrahydrate, 0.0066 g ammonium vanadate, and 220.716 g sugar.
4. Antioxidants

Composition of the Highland Diet ¹ ingredient Counterpart PUA expression casein 232 232 L-cysteine 3.0 3.0 DL-methionine 3.5 3.5 Corn starch 137 137 Maltodextrin 150 150 Sugar 162.595 162.595 Cellulose 50 50 cholesterol 1.9 1.9 Mineral Mix (AIN-93) ² 40.60 40.60 Dibasic calcium phosphate 4.64 4.64 Vitamin Mix (AIN-93) ³ 16.24 16.24 Choline Bitartrate 5 5 Tert-Butylhydroquinone ⁴ 0.02 0.02 Vitamin K, Phyloquinone 0.005 0.005 soy milk 30 30 lard 163.5 153.5 Pomegranate oil - 10 1. Provide about 19.6% fat and 0.2% total cholesterol, and 40.1% calories were obtained from fat.
2. 3 g nicotinic acid, 1.6 g calcium pantothenate, 0.7 g pyridoxine HCl, 0.6 g thiamine HCl, 0.6 g riboflavin, 0.2 g folic acid, 0.02 g D-biotin, 2.5 g vitamin B ₁₂ (0.1% in mannitol) per kg of food , 15 g DL-alpha tocopheryl acetate (500 IU / g), 0.8 g Vitamin A palmitate (500,000 IU / g), 0.2 g Vitamin D ₃ (cholecalciferol, 500,000 IU / g), 0.075 g Vitamin K ( Filoquinone), and 974.705 g sugar.
3. 357g calcium carbonate, 196g potassium phosphate monobasic, 70.78g potassium citrate, 74g sodium chloride, 46.6g potassium sulfate, 24.3g magnesium oxide, 6.06g iron citrate, 1.65g zinc carbonate, 0.63g manganese carbonate, 0.31g per kg of food Cupric carbonate, 0.01 g potassium iodide, 0.01025 g sodium serene, 0.00795 g ammonium paramolybdate, 1.45 g sodium meta-silicate, 0.275 g potassium sulphate, 0.0174 g lithium chloride, 0.0815 g boric acid, 0.0635 g Sodium fluoride, 0.0318 g nickel carbonate, hydroxide, tetrahydrate, 0.0066 g ammonium vanadate, and 220.716 g sugar.
4. Antioxidants

Sugar load test

The glucose load test was performed in a 78 day experiment. Animals were fasted overnight (14 hours). Mice were injected intraperitoneally with D-glucose (1 mg / kg body weight) and blood samples were collected from the tail vein before injection (0 min) and at 15, 30 and 60 minutes post injection.

Measurement of Serum Insulin Concentration

Serum insulin concentrations were measured using a commercially available enzyme-linked immunosorbent assay kit (Linco Research, St. Charles, Montana).

statistics

Data were analyzed by analysis of variance (ANOVA). ANOVA was conducted using the general linear model procedure of SAS (SAS Institute, Inc., Cary, North Carolina), as described above (Bassagana-Riera et al. 2004). The difference in probability values ( P <0.05) was considered significant.

result

Excessive abdominal fat accumulation and insulin resistance are key features that represent metabolic syndrome. Glucose load testing is a standard method of assessing glucose homeostasis in vivo. By using the glucose load test, glucose tolerance did not differ significantly between the two groups who consumed the diet and found that the diabetic phenotype was not developed (FIG. 3A). However, the ability to normalize the impaired glucose tolerance of the mice fed the control high fat diet was significantly reduced compared to the mice fed the high fat diet supplemented with funic acid (FIG. 3B).

Weight and Plasma Fasting Glucose and Insulin Concentrations of Abdominal White Adipose Tissue and Scapula Brown Adipose Tissue in Mice Fed Dietary and High Fat Diet Supplemented with Control or Punic Acid (PUA) ¹
Item Diet Notice SEM ²
ANOVA
P value contrast PUA contrast PUA White adipose tissue, g 0.865 ^b 0.801 ^b 1.478 ^a 0.830 ^b 0.06 0.0001 Brown adipose tissue, g 0.130 0.138 0.129 0.124 0.008 0.08 Glucose (mg / dL) 153.8 ^b 121.40 ^b 301.30 ^a 219.5 ^b 11.3 0.0001 Insulin (ng / mL) 0.932 ^b 0.669 ^b 2.254 ^a 1.229 ^b 0.23 0.01 1. The least square mean values in the row for a particular tissue with different superscripts are significantly different ( P <0.05)
2. Standard Error of Least Squared Mean

In addition, mice fed the control high fat diet had higher hyperglycemia and hyperinsulinemia than mice fed the high fat diet supplemented with funic acid or mice fed the diet (Table 9). Accordingly, FUNIC acid supplementation prevents or improves the progression of hyperglycemia, reduces hyperinsulinemia, and normalizes impaired glucose tolerance in mice fed high fat diet (Table 9). This finding is of clinical importance for the prevention and treatment of type 2 diabetes, metabolic syndrome and their complications (cardiovascular disease, seizures, retinopathy, nephropathy and amputation).

Hyperglycemia and hyperinsulinemia observed in mice fed the control high-fat diet correlated with an increase in abdominal white adipose tissue deposition (Table 9). However, no differences in brown adipose tissue weight were observed between groups. Compared with mice fed the control diet, the reduced abdominal obesity observed in mice fed high fat diet supplemented with funic acid can be caused by suppressed fat production and increased fat consumption. Fatty liver or visceral enlargement has not been observed in mice fed food supplemented with punic acid, and reduced abnormal obesity does not seem to be caused by reduced lipogenesis, which may be due to increased fatty acid consumption. All these suggest that funic acid can be used for the treatment and prevention of insulin resistance, abdominal obesity and metabolic syndrome.

It is to be understood that the present invention is not limited to the specific structures and arrangements illustrated and described herein, but includes all modifications within the scope of the following claims.

Citations

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Bassagana-riera and Hontesilas et al. (2002). "Long-term influence of lipid nutrition on the induction of CD8 (+) responses to viral or bacterial antigens." Vaccine 20 (9-10): 1435-44]

Bassagana-riera and RM Pogranichniy et al. (2003). "Conjugated Linoleic Acid Ameliorates Viral Infectivity in a Pig Model of Virally Induced Immunosuppression." J Nutr 133: 3204-3214.

Bassagana-riera and JK Reynolds et al. (2004). "Activation of PPAR gamma and delta by conjugated linoleic acid mediates protection from experimental inflammatory bowel disease." Gastroenterology 127 (3): 777-91]

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Claims (73)

A compound selected from the group consisting of funic acid, non-toxic salts of funic acid, C ₁ -C ₆ alkyl esters of funic acid, monocarboxylic acids, funic acid di-glycerides, funic acid tri-glycerides, and mixtures thereof A composition for enhancing an immune response, enhancing the development of the immune system, or treating or preventing intestinal immunoinflammatory diseases in an animal comprising a. The composition of claim 1 comprising the compound in an amount of 10 mg to 10,000 mg. The composition of claim 1 for increasing CD4 ⁺ and CD8 ⁺ T cell levels in an animal. The composition of claim 1 for treating or preventing viral infections, bacterial infections, or both viral and bacterial infections. The composition of claim 1 for increasing the immune response to the viral antigen. The composition of claim 5, wherein the viral antigen is an influenza viral antigen. The composition of claim 1, wherein the immune system comprises primary and secondary lymphoid organs and immune cells. 8. The composition of claim 7, wherein the primary and secondary lymphoid organs are thymus, spleen, lymph node, bronchial related lymphoid tissue and intestinal related lymphoid tissue. delete delete The composition of claim 1, wherein the intestinal immunoinflammatory disease is inflammatory bowel disease. delete delete delete delete The composition of claim 1, wherein the funic acid is in its free acid form. The composition of claim 1 further comprising a pharmaceutically acceptable carrier. The composition of claim 17, wherein the pharmaceutically acceptable carrier is for parenteral administration. The composition of claim 17, wherein the pharmaceutically acceptable carrier is for oral administration. The composition of claim 17, wherein the pharmaceutically acceptable carrier is in the form of a capsule, cachet, tablet, bolus or lozenge. The composition of claim 17, wherein the pharmaceutically acceptable carrier is for injection. The composition of claim 17, wherein the pharmaceutically acceptable carrier is for rectal administration. The composition of claim 22, wherein the pharmaceutically acceptable carrier is a suppository. The composition of claim 1 wherein the animal is a mammal. The composition of claim 24, wherein the mammal is a human. The composition of claim 1 further comprising at least one vitamin or fatty acid. A compound selected from the group consisting of funic acid, non-toxic salts of funic acid, C ₁ -C ₆ alkyl esters of funic acid, monocarboxylic acids, funic acid di-glycerides, funic acid tri-glycerides, and mixtures thereof A supplemental nutritional product for enhancing an immune response, enhancing the development of the immune system, or improving intestinal immune disease in an animal, comprising. The nutritional product of claim 27, wherein the nutritional product is a fiber bar, energy bar, cereal, bread, milk, cheese, low carb food, infant formula, children's product, yogurt, kefir, aged formula, weight control, sports nutrition A supplemental nutritional product selected from the group consisting of formulas, fruit drinks and foods. A compound selected from the group consisting of funic acid, non-toxic salts of funic acid, C ₁ -C ₆ alkyl esters of funic acid, monocarboxylic acids, funic acid di-glycerides, funic acid tri-glycerides, and mixtures thereof Animal feed for improving the immune response, to enhance the development of the immune system, or to improve the intestinal immune disease, including in the animal. 30. The animal feed of claim 29, wherein the animal feed is a dog feed, a cat feed or a horse feed. delete The composition of claim 1, wherein the composition is a pharmaceutical composition in the form of a tablet, capsule, solution or emulsion. A compound selected from the group consisting of funic acid, non-toxic salts of funic acid, C ₁ -C ₆ alkyl esters of funic acid, monocarboxylic acids, funic acid di-glycerides, funic acid tri-glycerides, and mixtures thereof To improve the immune response in the animal, to enhance the development of the immune system, or to improve intestinal immune disease, Margarine, continuous fat spread, continuous water spread, double continuous spread, low fat spread, confectionery products, chocolate, chocolate coating, chocolate peeling, confectionery peeling, ice cream, ice cream coating, ice cream inclusions, dressing, mayonnaise, cheese, cream substitute , Foods selected from the group consisting of dry soups, beverages, cereal bars, sauces, snack bars, dairy products, clinical nutritional products and infant formulas. The composition of claim 1, wherein the composition is included in a soft gel or hard capsule comprising an encapsulating material selected from the group consisting of gelatin, starch, modified starch, starch derivative, glucose, sugar, lactose and fructose. The composition of claim 11 wherein the inflammatory bowel disease is selected from the group consisting of Crohn's disease and ulcerative colitis. delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete

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