WO2004022725A2 - Chia seeds - Google Patents

Abstract

The disclosed invention is directed to improving the potency, efficacy, and use of PUFA-rich seeds and PUFA-rich seed-based compositions and to separating out active components from chia.

Description

980002000140

CHIA SEEDS

Related Applications

[0001] The application claims priority to U.S. Provisional Patent Application No, 60/409,084, filed September 9, 2003, which is hereby incoφorated by reference in its entirety.

Field of the Invention

[0002] The field of the invention is pharmaceuticals and nutraceuticals.

Background

[0003] Chia is the common name for a variety Salvia species. One of the most popular varieties of chia is Salvia hispa ica or Spanish sage. Chia grows naturally on sandy slopes and open desert areas below 4000 feet. Chia seeds were in use by ancient Aztecs, Mexicans and habitants of Southern California and Arizona as food supplement for energy, endurance and strength needed under extreme conditions such as heat, dryness, short-term food and water deficiency. Historian Harrison Doyle, who in the early 20^th century lived with various tribes, wrote "it was nothing for tribesmen to run 24 hours on tablespoon of chia seeds and gourd of water." Chia seeds contain soluble fibers that form edible gels, gums and mucilage.

[0004] "The magic of Chia", a book with almost 100 recipes for chia seeds, explains that the nutrition-packed seeds typically swell by seven to nine times in water or in stomach acids, so a feeling of fullness discourages excess eating. The seeds contain all essential amino acids, vitamins, and trace mineral such as boron, magnesium calcium zinc, and the most important and beneficial omega-3 essential fatty acid as well as omega-6 fatty acid.

[0005] During the last 20 years chia seeds have become a subject of scientific investigations. For example, chia seeds have been studied for use as food supplements for various animals. For example, Ayerza and colleagues published several reports showing the beneficial effects of chia seeds on growth, body mass, fat content in animals fed. According to these studies, 6.2% reduction in weight was observed in animals with a diet supplemented with 20% of chia seeds. In addition, all animals fed with chia seeds showed lower levels of saturated fatty acids as well as the saturated:polyunsaturated fatty acids and omega-6:omega-3 ratios.

[0006] The composition of chia seeds has been well described. Briefly, it was found that chia seed oil amount varying between 32-39%, with the oil offering one of the highest natural 980002000140 source of alpha-linolenic acid known (60-63%). Linolenic acid (unsaturated omega-3 fatty acid) belongs to group of poly-unsaturated fatty acids (PUFA). PUFA play an important role in human physiology but they cannot be endogenously synthesized, oils containing omega-3 fatty acids have been shown to have several health beneficial effects, for example to reduce the risk of cardiovascular diseases. Chia seeds possess the highest percentage of PUFA, alpha-linolenic and linoic (i.e., 83%), of all crops.

[0007] Linolenic acid (LNA) called also 18:3 omega-3 or omega-3 fatty acid has three double bonds at the 3, 6, and 9 carbon position, and omega-3 means that first double bond is at carbon 3. For comparison, linoleic acid (LA) is also an 18 carbon fatty acid with two double bonds, at 6 and 9 carbon position. Therefore is called also omega-6 fatty acid. Both LNA and LA as well as other poly-unsaturated fatty acids (PUFAs) are very unstable if not frozen or protected from the light.

[0008] Health and beneficial balance in the diet between LNA and LA preferably should be about 1 :2. Unfortunately, typical diets usually rely heavily on foods that contain these two fatty acids in a ratio of about 1 :10. Some commonly used cooking oils also contain improper LNA to LA ratios. For example, sunflower oil has a LNA to LA ratio of 0: 65, safflower oil has a ratio of 0:75, corn oil has a ratio of 0:59, olive oil has a ratio of 0:8, canola oil has a ratio of 1 :4, and soybean oil has a LNA to LA ratio of 1:7.

[0009] Individuals deficient in LNA or LA will typically suffer from a wide spectrum of maladies. Table 1 lists a number of very serious symptoms associated with LNA or LA deficiencies. It has also been reported that these symptoms are reversible if LNA and/or or LA was provided in food.

Table 1 Symptoms of LNA and LA deficiency

LA Deficiency LNA Deficiency

Eczema-like skin eruption Growth retardation

Loss of hair Weakness

Liver degeneration Impairment of vision and learning

Kidney degeneration Motor incoordination

Excessive water loss through skin Behavioral changes

Behavioral disturbances tingling sensation in arms and legs

Susceptibility to infections Hypertension 980002000140

LA Deficiency LNA Deficiency

Failure of wound healing Tissue inflammation

Arthritis-like conditions Immune dysfunction

Heart and circulatory problems Edema

[0010] Chia seeds are higher in protein content and amino acid content than most traditionally utilized grains. For example, chia seeds contain approximately 19-23% protein, which is higher that the protein concentration in wheat (14%), corn (14%), rice (8.5%), oats (15.3%) and barley (9.2%). Chia seeds also contain all the 20 amino acids, as opposed to traditional grains which typically lack two or more amino acids. Chia seeds are also a good source of vitamins B as well as calcium, magnesium, phosphorus, boron, potassium, zinc and copper.

[0011] Water and methanol extracts of chia seeds have been pressed and the oils extracted have demonstrated a strong anti-oxidizing activity due to the presence of chlorogenic acid, caffeic acid, myricetin, quercetin, and kaemferol flavonols.

[0012] Once the oil has been extracted from chia seeds, the material that remains contains 50-60% of soluble fibers. Chia seeds possesses 5% soluble fiber, which appears as mucilage when is placed in water, and is useful as a dietary fiber. The importance of chia seeds is not only from its nutritional value, but also because of its thickening nature, which is important within the cosmetology industry, and other applications.

[0013] Chia seeds have great potency to improve health. However, 20% of chia seeds in food provided per day is needed to be used for longer time to improve significantly fat composition, to reduce body weight at least in animals. In case of human beneficial effects are observed with 3-20g of chia seeds per dose.

Summary of the Invention

[0014] The disclosed invention is directed to improving the potency, efficacy, and use of seeds of PUFA-rich seeds and compositions made from PUFA-rich seeds and to separating out active components from PUFA-rich seeds.

[0015] Various objects, features, aspects and advantages of the present invention will become more apparent from the following detailed description. 980002000140

Brief Description of the Drawings

[0016] Figure 1 is a graphical representation of the effects of chia seed extracts on GSK-3 alpha/beta kinase activity as measured in vitro in C2C12 muscle cells.

[0017] Figure 2is a graphical representation of the effects of a chia seed extracts on mTOR activity as measured in vitro in C2C12 muscle cells.

[0018] Figure 3 a graphical representation of the effects of a chia seed extracts on total glucose uptake as measured in vitro in C2C12 muscle cells.

[0019] Figure 4 shows the effect of of chia preparations (water and ethanol extraction) on activity of acetyolo-CoA-Carboxylase (AC) in C2C12 muscle cells in vitro (two independent experiments). More intensive bands indicate stronger activation of the enzyme. Legend: CH/E- ethanol extract, CH/H - water fraction, O-untreated control. These results show that CH/H at concentration 10 and 30uL/ml is more potent in stimulation of AC than CH E.

[0020] Figure 5 shows that chia preparation modulate activity of endothelial Nitric Oxide Synthetase (eNOS) in C2C12 cells in muscle cells in vitro following the treatment for 30 minutes. Legend: CH/E - ethanol extract, CH/H - water fraction, tATB- total alpha-tubulin, O- untreated control. The results show that both preparations (CHJE and CH/H) significantly stimulate phosphorylation of eNOS.

[0021] Figure 6 is a graphical representation of the effect of ethanol fraction of chia seeds stimulating production of Nitric Oxide in C2C12 muscle cells following 30 and 60 minutes of the treatment as measured by colorimetric assay

Detailed Description of the Invention

[0022] The disclosed invention relates to polyunsaturated fatty acid (PUFA) rich seed compositions, methods for preparing seeds, and methods for using the seed compositions.

[0023] Preparation of freeze-dried extract of PUFA-rich seeds and reduction of volume will increase concentration of active principles thus generating more potent and efficacious preparations. Moreover, important and active principles in PUFA-rich seeds are very unstable in liquid form (linoleic and linoic acid, short-and long fatty acids, PUFAs) and photosensitive. Therefore there is still a need to provide new preparation of PUFA-rich seeds having improved stability and efficacy, for example in the form of solid preparations. On the other hand, PUFA- rich seeds contains many unidentified chemical entities with health beneficial potency thus, identification of the chemical entities in seeds such as small and/or large molecules, polar 980002000140 and/or un-polar compounds potent in modulating metabolism of fat, glucose, cholesterol, muscle and hormones offers a possibility to discover new potent substances for use in the field of nutraceuticals and pharmaceuticals.

PUFA-rich seeds

[0024] One example of PUFA-rich seeds are the seeds from a variety of Salvia species. Exemplary Salvia species include, Salvia aethiopis L.; Salvia amissa Epling; Salvia apiana Jepson; Salvia argentea L.; Salvia arizonica Gray; Salvia azurea Michx. ex Lam.; Salvia pitche i Torr. ex Benth.; Salvia ballotiflora Benth; Salvia brandegeei Munz; Salvia carduacea Benth.; Salvia chapmanii Gray; Salvia clevelandii (Gray) Greene; Salvia coccinea; Salvia columbariae Benth; Salvia davidsonii Greenm.; Salvia disjuncta Fernald; Salvia divinorum Epling & Jativa; Salvia dolichantha (Cory) Whitehouse; Salvia dorrii (Kellogg) Abrams; Salvia carnosa Dougl. ; Salvia elegans Vahl; Salvia engelmannii Gray; Salvia eremostachya Jepson; Salvia farinacea Benth. ; Salvia earlei Woot. & Standl.; Salvia farinacea Benth. var. latifolia Shinners; Salvia funerea M.E. Jones; Salvia glutinosa L.; Salvia greatae Brandeg.; Salvia greggii Gray; Salvia henry i Gray; Salvia hispanica L.; Salvia lemmonii Gray; Salvia microphylla Benth. var. wislizeni Gray; Salvia leptophylla Benth; Salvia leucantha Cav.; Salvia leucophylla Greene; Salvia longistyla Benth; Salvia lycioides Gray; Salvia ramosissima Fern.; Salvia lyrata L.; Salvia mellifera Greene; Salvia micrantha Vahl; Salvia micrantha Vahl var. blodgettii (Chapman) Epling; Salvia blodgettii Chapman; Salvia micrantha Vahl var. micrantha; Salvia microphylla Benth; Salvia grahamii Benth; Salvia miniata Fernald; Salvia misella Kunth; Salvia mohavensis Greene; Salvia munzii Epling; Salvia nemorosa L.; Salvia nutans L.; Salvia occidentalis Sw.; Salvia ofβcinalis L.; Salvia pachyphylla Epling ex Munz; Salvia parryi Gray; Salvia penstemono ides Kunth & Bouche; Salvia pinguifolia (Fern.) Woot. & Standl.; Salvia potus Epling; Salvia chia Fern.; Salvia pratensis L.; Salvia reflexa Hornem.; Salvia lancifolia Poir.; Salvia regla Cav.; Salvia riparia Kunth; Salvia privoides Benth; Salvia roemeriana Scheele; Salvia sclarea L.; Salvia serotina L.; Salvia sonomensis Greene; Salvia spathacea Greene; Salvia splendens Sellow ex Roemer & J.A. Schultes; Salvia subincisa Benth.; Salvia summaA. Nels.; Salvia virgata auct. non Jacq.; Salvia texana (Scheele) Torr.; Salvia thomasiana Urban; Salvia tiliifolia Vahl; Salvia urticifolia L.; Salvia vaseyi (Porter) Parish; Salvia verbenacea L.; Salvia verticillata L. ; and Salvia vinacea. Preferred species from which PUFA-rich seeds may be obtained in Salvia hispanica and Salvia columbariae. Other types of 980002000140

PUFA rich seeds are flaxseeds, sunflower seeds, soy beans (seeds), hemp seeds, safflower seeds, cottonseeds, rapeseeds, and the like.

[0025] A PUFA-rich seed typically contains 10%, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 99% PUFA.

[0026] PUFA-rich seeds should be of sufficient nutritional quality as to warrant description as an article of food. For example, a chia seed of sufficient nutritional value will possess at least 50% of the fatty acid and amino acid concentrations found in a wild type Salvia spp. plant. Other types of seeds, such as flax seeds can also be used with the teachings provided below.

Preparation of Seeds for Processing

[0027] PUFA-rich seeds, such as chia seeds are isolated from the host plant and prepared for subsequent use. The potency of the seeds can be enhanced by increasing concentration of active principles components per mass of seed (per gram of seeds or per dose). Properly prepared, seeds can be extracted from whole, pressed, or crushed seeds using polar and/or non-polar solvent. PUFA-rich seeds can be presoaked in polar or non-polar solvent prior to extraction. Dried PUFA-rich seeds can be ground in the dark and at low temperature for direct use or for formulation such as capsules, tablets, or as an additive for drinks, foods, gels, smoothes, and bars. Identification and separation of an active chemical entities present in the PUFA-rich seeds by separating out at least one active component, preferably by fractionation using methods like liquid chromatography. PUFA-rich seeds preparations can also be combined with one or more other active components in a synergistic or additive combination with other extracts, food supplements, nutrients, minerals, vitamins or pharmaceuticals.

[0028] Preparation of PUFA-rich seeds typically involves desiccating the seeds prior to use. Desiccation serves to preserve labile components of the seeds prior to use. A preferred method of desiccation involves freeze drying the seeds prior to use. Freeze-drying (lyophilization) is a commonly used method in the United States for preparation of lyophilized fruits, vegetables and other goods. Lyophilized seeds have a number of advantages over non-lyophilized seeds, for example, lyophilized seeds have a reduced volume, demonstrate increased stability or shelf-life, have a high re-hydration capacity, a preserved nutritional value, and demonstrate an increased concentration per mass of active components.

[0029] Water content of seeds when stored is an important factor when determining storage life. Two commonly used methods of seed drying are vacuum freeze drying and drying over desiccants. The process of freeze drying begins with the freezing of the PUFA-rich seeds. For 980002000140 example, seeds can be placed in a freezer at the appropriate temperature, pressure and length of time to freeze the seeds. Typically, the seeds are frozen at a temperature of -1 to -80°C. When frozen, seeds are transferred to the lyophilization chamber and spread out preferably in an even layer on a drying surface. The seeds are then subjected to vacuum for a period sufficient to remove the water from the seeds. Typically this period of time is preferably about 1 to 240 hours, 2 to 120 hours, 4 to 80 hours, or 6 to 72 hours, to remove the water from the seeds. Preferably, these steps are done in dark or under reduced light conditions to prevent decomposition of photosensitive components in the seeds. Moreover, all ingredients unstable at room or higher temperature are safe as the starting materials are frozen before lyophilization is initiated.

[0030] An alternative a method of desiccation utilizes a desiccant such as silica gel. For example, 200 grams of seeds can be folded into nylon netting and placed on top of a wire mesh in a desiccator positioned above approximately 2 kilograms of silica gel. Optionally, fans may be used to circulate the air within the desiccator to facilitate the drying of the seeds. Typically, the drying procedure is performed at or below room temperature (25°C) for 1 to 10 days or longer, if necessary.

[0031] Water content of seeds can be assayed using a variety of methodologies. For example, the water content of seeds can be measured gravimetrically according to International Seed Testing Association rules (ISTA, 1985). Briefly, the dry weights of the seeds are determined by heating ground PUFA-rich seeds at 130-133°C for 2 hours, or whole seeds at 103-105°C for 17 hours.

[0032] Desiccated PUFA-rich seeds can also be subjected to a variety of extraction procedures. For example, PUFA-rich seeds can be treated in a solvent to facilitate the extraction process. A variety of solvents can be used to pre-soak the PUFA-rich seeds. Solvents include, water, ethanol, methanol, and glycerol. Use of these solvents facilitates fatty acid extraction from the PUFA-rich seeds prior to freezing and subsequent freeze-drying.

Formulations

[0033] Preparation of PUFA-rich seeds, one or more fractions or active components of chia seeds can be synergistically combined with other active compositions. Contemplated such compositions include nutrients, drugs, metabolites or substances needed for formulation. Examples for nutrients are plant or animal extracts, vitamin, lipids, amino acids, sugars, flavonoids, isoflavonoids, statins, beta-glucans, and minerals. Preferred drugs are prescription 980002000140 or non-prescription drugs including painkillers, anti-inflammatory, psychopharmacologicals, anti-lipidemics and so forth. Preferred metabolites are nucleic acids, enzymes, ribose, inosine and other nucleoside and nucleotide analogs, or substances found in plant like plant hormones, or in animal cells and involved in the process of energy metabolism, mobility function or reproductive function or anti-aging function.

[0034] Of course, inactive compositions may also be added to any PUFA-rich seed preparation to improve formulation, odor or consistency, or in other way to improve potency, safety and quality.

[0035] It is contemplated that chia seeds preparations described herein may be provided in fluid or non-fluid forms. Contemplated fluid forms include aqueous solutions having co-solvent other than methanol. Preferred co-solvents are glycerol and penthanol. Fluid formulations may further include emulsions or liposome preparations. Emulsions preferably comprise one or more emulsifiers (e.g., bile acids) or microemulsions. Liposomes preferably comprise transferosomes, nanosomes, or inside -out liposomes. It is still further contemplated that fluid formulations may comprise gels, creams, lotions, pastes, or other non-liquids.

[0036] It is contemplated that solid formulations of PUFA-rich seeds, or components or fractions prepared from chia seeds may comprise any dehydrated or precipitated form of chia seeds preparation. For example, the chia seeds can be processed into granulated forms of lyophilized powdered seeds. Preferred dehydration methods include freeze-drying, vacuum drying, and boiling. Preferred precipitation methods include ammonium sulfate, acetone, and ethanol precipitation.

[0037] Fluid or solid forms of PUFA-rich seed preparations may be administered in many ways. Fluid forms may be injected, inhaled, ingested, topically applied or transdermally delivered. Preferred injections are intramuscular, intravenous or subcutaneous injections. Preferred inhalation embodiments are aerosols or sprays. Preferred ingestion forms include tablets, capsules, syrups or powders. Preferred topical application method includes eye drops, mouthwashes, tampons, creams, lotions, soaps, liquids for bath etc. Preferred transdermal delivery method includes occlusive dressing and electrophoresis.

[0038] The term "pharmaceutically acceptable carrier" encompasses any of the standard pharmaceutical carriers, diluents, buffers, excipients, solid fillers, and the like, including (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar— 980002000140. agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, cetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; (10) coloring agents: (11) phosphate-buffered saline solution (12) emulsions, such as an oil/water or water/oil emulsion; (12) adjuvants; and (13) sterile aqueous solutions, for example. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some additional examples of materials which can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations. Additional suitable pharmaceutical carriers and their formulations are described in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Publishing Co., Easton, 19th ed. 1995, the entirety of which is herein incorporate by reference) as well as in US Patent 6,416,965, the entirety of which is herein incorporated by reference). As one of skill in the art would recognize, a "carrier" that comprises substantial amounts of unknown compounds and/or impurities is not preferred for pharmaceutical use. Thus, preferred pharmaceutically acceptable carriers are, therefore, those in which such unknown compounds and/or impurities comprise, in total, less than about 25% of the carrier by weight. More preferred pharmaceutically acceptable carriers are those in which such unknown compounds and/or impurities comprise, in total, less than about 20% of the carrier by weight. Still more preferred pharmaceutically acceptable carriers are those in which such unknown compounds and/or impurities comprise, in total, less than about 20% of the carrier by weight. Still more preferred pharmaceutically acceptable carriers are those in which such unknown compounds and/or impurities comprise, in total, less than about 15% of the 980002000140 carrier by weight. Still more preferred pharmaceutically acceptable carriers are those in which such unknown compounds and/or impurities comprise, in total, less than about 10% of the carrier by weight. Still more preferred pharmaceutically acceptable carriers are those in which such unknown compounds and/or impurities comprise, in total, less than about 5% of the carrier by weight. Especially preferred pharmaceutically acceptable carriers are those in which such unknown compounds and/or impurities comprise, in total, less than about 1% of the carrier by weight.

[0039] The term "therapeutically effective amount" as used herein refers to an amount that provides therapeutic effects for a given condition and administration regime. The amount will vary with the condition being treated, the stage of advancement of the condition, and the type and concentration of formulation applied. Appropriate amounts in any given instance will be readily apparent to those skilled in the art or capable of determination by routine experimentation.

[0040] The term "patient" as used herein refers to a mammal which is being treated prophylactically and/or for a condition having visible and/or otherwise measurable symptoms. Preferably the patient is a human. In a preferred embodiment, the patient is a patient receiving treatment under the direction of a healthcare professional. Preferred embodiments also include patients receiving self-directed treatment.

[0041] The term "coadministered" as used herein means that at least two of the compounds of the invention are administered during a time frame wherein the respective periods of pharmacological activity overlap. In a preferred embodiment, a compound is administered at a time when the pharmacological activity of a previously administered compound is at least half- maximal. In another preferred embodiment, when at least two compounds of the invention are administered, the duration of time between administering the first and second compounds does not exceed the half-life

of the first administered compound.

[0042] The term "unit dosage" as used herein refers to a physically discrete unit, suitable for oral or parenteral administration, containing an individual quantity of the active component in association with a pharmaceutically acceptable carrier or diluent, the quantity of the active component being such that at least one unit or severable fraction of a unit is required for a single therapeutic administration. In the case of severable units, such as scored tablets, at least one severable fraction such as one-half or one-quarter of the unit may be all that is required for a single therapeutic administration. It will be appreciated that the term "unit dosage" does not include mere powders or solutions except when the powders or solutions have been prepared so 980002000140 as to be suitable for oral administration, e.g., in capsules, cachets, pills, tablets, lozenges or other measured forms suitable for oral ingestion, or have been prepared so as to be suitable for parenteral administration, e.g., in vials of a solution suitable for parenteral injection. The compounds of the invention may be administered alone or in combination with pharmaceutically acceptable carriers or diluents, in either single or multiple doses. Pharmaceutical compositions are preferably administered in unit dosage form.

[0043] The terms "parenteral administration" and "administered parenterally" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.

[0044] The terms "systemic administration," "administered systemically," "peripheral administration" and "administered peripherally" as used herein mean the administration of a compound, drug or other material other than directly into the central nervous system, such that it enters the patient's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration.

[0045] In another aspect, a preferred embodiment of the present invention provides a pharmaceutical and/or nutraceutical that comprises a therapeutically effective amount of PUFA- rich seeds formulated together with one or more pharmaceutically acceptable carriers, including additives and/or excipients and/or diluents.

[0046] As described in detail herein, the pharmaceutical compositions of the present invention may be specially formulated for administration in solid or liquid form, including preferred embodiments adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, boluses, powders, granules, pastes for application to the tongue; (2) parenteral administration, for example, by subcutaneous, intramuscular or intravenous injection as, for example, a sterile solution or suspension; or (3) topical application, for example, as a cream, ointment or spray applied to the skin.

[0047] Formulations of the present invention include those suitable for oral, nasal, topical (including buccal and sublingual) and/or parenteral administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the 980002000140 particular mode of administration. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred per cent of the weight of a single dosage form, active ingredient will range from about 0.01 per cent to about ninety-nine percent of the total weight, more preferably from about 0.05 per cent to about 90 per cent, more preferably from about 0.1 per cent to about 90 per cent, more preferably from about 0.5 per cent to about 85 per cent, more preferably from about 1 per cent to about 80 per cent, more preferably from about 5 per cent to about 70 per cent, most preferably from about 10 per cent to about 30 per cent. In preferred embodiments, the weight of active ingredient will constitute at least a certain percentage of the total weight of the single dosage form, the certain percentage being selected from one or more or the following: about 0.01%, about 0.05%, about 0.1%, about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, about 7.5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or about 99%. In certain preferred embodiments, the weight of active ingredient will constitute no more than a certain percentage of the total weight of the single dosage form, the certain percentage being selected from one or more or the following: about 0.01%, about 0.05%, about 0.1%, about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, about 7.5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or about 99%.

[0048] Methods of preparing these formulations or compositions include the step of bringing into association a contemplated compound (or compounds) of the present invention with the carrier and/or one or more accessory ingredients. In preferred embodiments, the contemplated compound is substantially pure prior to being brought into association with the carrier and/or one or more accessory ingredients.

[0049] In general, the formulations are prepared by uniformly and intimately bringing into association a contemplated compound of the present invention with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

[0050] An oral route of administration is preferred for the pharmaceuticals, including nutraceuticals, of the invention. Formulations of the invention suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and 980002000140 acacia) and/or as bronchoaveolar lavages for intended delivery systems to the lung and the like, each containing a predetermined amount of a compound of the present invention as an active ingredient. A compound of the present invention may also be administered as a bolus, electuary or paste.

[0051] In a preferred embodiments to prepare an orally administerable solid dosage form (e.g. capsules, tablets, pills, dragees, powders, granules and the like), an active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any other pharmaceutically acceptable carriers such as: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, cetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; (10) coloring agents: (11) phosphate-buffered saline solution (12) emulsions, such as an oil/water or water/oil emulsion; (12) adjuvants; and (13) sterile aqueous solutions, for example. In the case of capsules, tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.

[0052] A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.

[0053] The tablets and other solid dosage forms of the pharmaceutical compositions of the present invention, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in 980002000140 varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. They may be sterilized by, for example, filtration through a bacteria- retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes. The active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients.

[0054] Liquid dosage forms for oral administration of the compounds of the invention include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.

[0055] Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.

[0056] Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar— agar and tragacanth, and mixtures thereof.

[0057] The ointments, pastes, creams and gels may contain, in addition to an active compound of this invention, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

[0058] Pharmaceutical compositions of this invention suitable for parenteral administration comprise one or more compounds of the invention in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable 980002000140 solutions or dispersions just prior to use, which may contain antioxidants, buffers, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.

[0059] Examples of suitable aqueous and non-aqueous carriers which may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0060] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

[0061] In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally-administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.

[0062] Injectable depot forms are made by forming microencapsule matrices of the subject compounds in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissue.

[0063] When the compounds of the present invention are administered to humans or other animals, they can be given per se or as a pharmaceutical composition comprising the active ingredient(s) and a pharmaceutically acceptable carrier. In preferred embodiments, the active ingredient or ingredients represent, in total, about 0.01 to about 99.5% by weight (more 980002000140 preferably, about 0.5 to about 90% by weight) of the total formulation, for example. In particularly preferred embodiments, the active ingredient represents at least about 0.01%, about 0.05%, about 0.1%, about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, about 7.5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or about 99% by weight of the pharmaceutical composition.

[0064] The preparations of the present invention may be given by routes including oral, parenteral, and topical routes. They are of course given by forms suitable for each administration route. Oral administration is preferred. And the preferred form for oral administration is tablet or capsule.

[0065] Regardless of the route of administration selected, the compounds of the present invention, which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present invention, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art.

[0066] Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

[0067] The selected dosage level will depend upon a variety of factors including the activity of the particular compound of the present invention employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

[0068] A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds of the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

[0069] In general, a suitable daily dose of a compound of the invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above. Generally, doses of the compounds of this invention for a patient, when used for the indicated effects, will range from 980002000140 about 0.0001 to about 100 mg per kilogram of body weight per day, more preferably from about 0.01 to about 50 mg per kg per day, and still more preferably from about 0.1 to about 40 mg per kg per day.

[0070] In preferred embodiments, a unit dose of the compound of this invention for a patient, when used for the indicated effects, will range from about 0.001 mg to about 1000 mg, more preferably from about 0.01 mg to about 500 mg, still more preferably from about 0.01 mg to about 100 mg, still more preferably from about 0.01 mg to about 10 mg, still more preferably from about 0.01 mg to about 1 mg. In preferred embodiments, a unit dose of the compound of this invention for a patient, when used for the indicated effects, will range from about from about 0.1 mg to about 10 mg. In preferred embodiments, a unit dose of the compound of this invention for a patient, when used for the indicated effects, will range from about from about 0.1 mg to about 1 mg. In preferred embodiments, a unit dose of the compound of this invention for a patient, when used for the indicated effects, will range from about from about 1 mg to about 10 mg. In preferred embodiments, a unit dose of the compound of this invention for a patient, when used for the indicated effects, will range from about from about 10 mg to about 25 mg. In preferred embodiments, a unit dose of the compound of this invention for a patient, when used for the indicated effects, will range from about from about 25 mg to about 50 mg. In preferred embodiments, a unit dose of the compound of this invention for a patient, when used for the indicated effects, will range from about from about 50 mg to about 100 mg. In preferred embodiments, a unit dose of the compound of this invention for a patient, when used for the indicated effects, will be selected from one or more of about 0.001 mg, about 0.01 mg, about 0.1 mg, about 0.25 mg, about 0.5 mg, about 0.75 mg, about 1 mg, about 2 mg, about 2.5 mg, about 5 mg, about 7.5 mg, about 10 mg, about 15 mg, about 20 mg, about 50 mg, about 100 mg, about 200 mg, about 500 mg and about 1,000 mg.

Uses for Seed Preparation

[0071] Preparation of PUFA-rich seeds have a variety of uses. For example, chia seed preparations can be used to mediate or improve symptoms of fatty acid deficiencies listed in Table 1. PUFA-rich seed preparations can be typically combine capability to increase volume if taken orally, contain PUFA and other ingredients intact and preserve active principles from decomposition due to lack of water (dry form). Pre-soaked chia seeds in water or buffer for 1 minute to 24 hrs to stimulate gel and gum formation from the seeds. These pre-soaked seeds can be subsequently frozen, freeze dried, and used topically as well as systemically. 980002000140

[0072] PUFA-rich seeds can be fermented to provide additional organic substances in the preparation that were not present in the same relative quantities as those found in an unfermented seed. Grounded chia seeds and/or crashed in dark and at low temperature with or without subsequent soaking, before further freeze-drying or use for making capsules, tablets and/or other formulations for oral and/or topical administration. Optionally, freeze-dried material produced as described above could be also used to form capsules, tablets and other formulations for oral and/or topical administrations.

[0073] Roasted PUFA-rich seeds for preparation of coffee-like drinks and based on whole roasted chia seeds, roasted and grounded chia seeds, or grounded before roasting. Roasting could be done at normal air pressure, and/or in vacuum or in increased air pressure in order to modulate roasting temperature and roasting time. Also freeze-dried chia seeds could be used for roasting.

[0074] Sprouted PUFA-rich seeds for further processing such as freeze-drying, extraction, grounding and/or extraction

[0075] All preparation methods and resulting preparations mentioned above can be used without freeze-drying or as freeze-dried material for food supplements to improve health, taste, smell or nutritional values of foods and drinks in solid, liquid or semi-solid forms, or as a source to identify active ingredients for development of new nutraceuticals and/or pharmaceuticals.

[0076] Consumption of PUFA-rich seeds can provide a number of general benefits. For example, chia seed consumption enhances physical and mental strength, improves chronic fatigue conditions, has a calming effect on a subject and can improve digestion and upset stomach conditions.

[0077] Consumption of PUFA-rich seeds may slow down aging process. Without being limited to a particular mechanism, PUFA-rich seed consumption may serve as caloric-restriction mimetic. PUFA-rich seeds may also reduce or prevent free radicals mediated damage by limiting free radical generation produced through mitochondrial activity. PUFA-rich seed consumption may also decrease insulin use or modulate the activity of insulin transduction pathway in insulin-sensitive and insensitive tissues, thus modulating glucose utilization. Accordingly, PUFA-rich seed consumption may prevent or treat hyperglycemia and diabetes.

[0078] Improving muscle functioning (short- and long-term), building muscle mass and/or improving utilization and storage of energy in muscle and in the body. For example, chia seed consumption may prevent muscle and bone mass loss by modulating activity and expression of myostatin and by other mechanisms. Thus, PUFA-rich seeds may improve osteoarthritis, 980002000140 osteoporosis conditions, hypertension, blood cholesterol levels, and serve to improve heart function and heart muscle metabolism.

[0079] PUFA-rich seeds have properties that stimulate the regeneration process in liver, skin, kidney and other tissues and organs. Thus, PUFA-rich seeds can prevent liver and kidney damage caused by alcohol, radiation and drugs.

[0080] The seeds can also influence the recruitment, growth, and differentiation of stem cells (bone marrow as well as stem cells isolated from other tissues and organs). Improving skin conditions related to damage, infection, aging, UN exposure and inflammation. Similarly, PUFA-rich seeds can modulate hair metabolism and growth.

[0081] Chia seeds can modulate intracellular processing of fatty acids such as elongation, desaturation, beta-oxidation, omega-oxidation in microsomes, exchanged with membrane phospholipids, participation in eicosanoid synthesis, oxidation in peroxisomes for energy production. They can also modulate and improving activity of hormones such as glucocorticosteroids, melanocortins, thyroids, insulin, IGF-1, glucagons, growth hormone, all of which affect the metabolism of the host.

[0082] PUFA-rich seeds can modulate metabolism and activity of prostaglandins, enkefalins and endorphins as well maintain hormonal and/or metabolic homeostasis. By modulating Th-1, Th-2 cytokine profile, PUFA-rich seeds can control immunogenic activity towards cancer and/or bacterial, fungal and/or virus infections; modulating of secretion of cytokines such as IL-2 and/or 11-12 and others from various types of cells.

[0083] The PUFA-rich seed containing compositions disclosed herein have utility in controlling and treating fat accumulation and obesity. The compositions are particularly useful in controlling fat deposition in different organs and tissues. Without being limited to a particular mechanism, initial investigation into the biological effects of PUFA-rich seeds and PUFA-rich seed extracts on fat metabolism suggest that stearoyl-CoA desaturase-1 (SCD-1) and possibly other types of SCD such as delta5 and deltaό gene expression and activity may be affected by PUFA-rich seeds. Omega-3 fatty acids are known to modulate SCD activity. Because PUFA- rich seeds contain significant amounts of omega-3 fatty acids, it is expected that consumption of the PUFA-rich seed products will modulate SCD activity. Other mechanisms that may be affected by PUFA-rich seeds include the PUFA-rich seed modification of expression and activity of leptin gene product, expression and activity of transcription factors involved in regulation of lipid metabolism and/or energy metabolism such as but not limited to SREBP (sterol regulatory element-binding proteins) and HΝF-4 (hepatic-nuclear factor 4). 980002000140

[0084] Modulating of expression and activity of genes involved in the mechanism of lipid, sugar and energy metabolism such as but not limited to the LXR-alpha (liver X receptor alpha), S14 genes, FAS (fatty acid synthetase), Malic enzyme (ME), Glucose-6-phosphate dehydrogenase, Acetyl-CoA corboxylase (ACC), L-PK, ATP citrate-lyase, cytosolic phosphoenolpyruvate carboxykinase (PEPCK), Lipoprotein lipase, The uncoupling proteins 1-3 (UCP1-3), Glucose transporters, Carnitine palmoyltransferase 1 (CPT-1) and other CPT types, Acyl-CoA oxidase (AOX), Cytochrome P450-4A1, ACS, HMG-CoA reductase and synthase, PPARs (peroxisome proliferator-activated receptor, and Malonyl-Coa decarboxylase, may be affected by PUFA-rich seed consumption. Modulating expression and or activity of fatty acids tranporters including but not limited to FAT-CD-36, mitochondrial aspartate aminotransferase, caveolin, adipose differentiation-related protein, and FABP (fatty acid binding protein). Also, modulation of levels of metabolites such Malonyl-CoA and aminoglucose may also be affect by PUFA-rich seed consumption. In addition, PUFA-rich seeds may affect activity of phosphatase PTP-lbeta known as crucial enzyme for maintaining high activity of insulin-stimulated pathway. Currently, PTP-lBeta inhibitors are under development for treatment of diabetes.

[0085] PUFA-rich seeds and extracts therefrom also have utility in suppressing appetite. Without being limited to a particular mechanism, it is thought that PUFA-rich seeds may directly or indirectly influence cerulenin, ghrelin, PYY-3-36, cholecystokinin activity or by other mechanisms including limitation of nutrient absorption following oral administration and/or mediated by central nerve system and/or by NPY and NPY-Iike peptide.

[0086] PUFA-rich seed consumption is also useful for its affects on the metabolism. For example, chia seeds and chia seed extracts have utility as a caloric-restriction mimetic. Chia seeds are also thought to increase energy expenditure, increase fatty acid oxidation, and modulate mitochondrial metabolic activity.

[0087] PUFA-rich seed preparations may modulate activity of nitric oxide synthetase, glucose uptake, AMPK (AMP-dependent kinase), and mTOR activity. For example, Chia seed extracts were shown in the Examples discussed below to modulate nitric oxide synthetase, glucose uptake, and mTOR activity.

[0088] The following examples are offered to illustrate but not to limit the invention. 980002000140

Example 1 Chia seeds analysis

[0089] Chia seeds preparations (products) mentioned above can be separated into fractions or components. The separation into fractions or components may be done by chromatographic or chemical methods. Chromatographic methods include ion exchange, size inclusion, end reverse and thin chromatography. Preferred ion exchange chromatography matrixes are DEAE- DMAE, DE52 chromatography. Preferred size exclusion matrixes are Sephadex 50, 100, or 200, or molecular weight cut-off filters. Preferred reverse reversed phase matrixes are nucleosil C12, C16, and C18.

[0090] Chemical separation generally includes extraction between two or more solvents, and may advantageously also include precipitation with additional compounds. A particularly preferred separation involves fractional distillation with hexane and water, discarding one of the phases. Another preferred separation involves precipitation by mixing with ammonium sulfate or acetone. Other preferred buffer for fractionation and making extracts is ammonium buffer compose of formic acid and ammonium formate at different ratio to provide a broad pH range during the process of extraction.

[0091] Chemical identification of new chemical structures and subsequent preparative separation includes Mass Spectrum analysis, Liquid- and/or gas-chromatography combined with Mass spectrophotometer (LC-MS or GS-MS), it also includes electron spray method for detection combined with mass spectroscopy to identify number and molecular weight of compounds within chia seeds preparation listed under 1-7 above.

Example 2 Amount of omega-3 and other polyunsaturated fatty acids in various formulations of Chia seeds

[0092] In Experiments 1 and 2 discussed below, various chia seed formulations are prepared and analyzed to determine the quantity of omega-3 and other polyunsaturated fatty acids present in each.

[0093] The method for saponification and methylation of fatty acids for gaschromatographic analysis follow that reported by Mecalfe, et al, Anal. Chem. (1966) 38:514-515). The weighed sample is placed in a 20 mL vial. Four mL of 0.5N methanolic sodiumhydroxide are added to the vial , which is then heated in a water bath at ~80°C for 5 mins. After a brief cooling period, 3 mL of BF3 in methanol are added to methylate the sample. After another five minutes of 980002000140 heating in the water bath, the sample vial is allowed to cool. Two mL of a saturated solution of sodium chloride and 10 mL of hexane is added to the vial. The samples are then mixed with a vortex mixer for one minute. The hexane fraction is transferred by pipet into a 20 mL vial containing ~10 mg of sodium sulfate to dry the sample. The hexane solution is removed for gas chromatographic analysis and may be concentrated by evaporation. An injection volume of 1 μL of the hexanesolution is typically analyzed.

[0094] Samples are analyzed with a Shimadzu GC-17A gas chromatograph (GC) equipped with an autosampler and an autoinjector. Analysis of fatty acid methyl esters is based on areas calculated with Shimadzu Class VP 4.3 software. The column is from J&W Scientific, DB-23 (123-2332): 30 m (length), I.D. (mm) 0.32 wide bore, film thickness of 0.25 μM. Fatty acid identification is made by the use of comparison of retention times with those of authentic standards.

[0095] The conditions for the GC follow: Column temperature ramping by holding at 120°C for one minute followed by an increase of 5°C/min from 120-240°C. The temperature of the injector and flame ionization detector is 250°C. A split(8:l) injection mode is used. The carrier gas is helium with a column flow rate of 2.5 mL/min.

Experiment 1

[0096] Samples tested include ground and freeze dried chia seeds (Fr 1); freshly ground chia seeds after ethanol extraction (Fr 2); freshly ground chia seeds after water extraction (Fr 3); freshly ground seeds (Fr 4); the ethanol extract from Fr 2 (Fr5); and the water extract from Fr 3 (Fr6). Regarding the ethanol and water extractions, fifty (50) grams of seeds are ground and extracted with either 200 ml of ethanol or 500 ml of water. All samples of the chia seeds are based on grounded seeds of chia, freeze-dried or freshly grounded.

Table 2 Amount of Omega 3 and Omega 6 in samples mentioned above.

Sample Total fat% in

%Omega - -3 %omega -6 samples

Frl 16.73 4.71 28.4

Fr2 13.04 3.80 20.7

Fr3 13.18 3.95 21.1

Fr4 13.88 4.10 22.1

Fr5 0.12x5 dilution 0.03x5 0.19x5

Fr6 0.18x10 0.08x10 0.34x10 980002000140

[0097] These results show that freeze-dried grounded seeds (Frl) contain more omega-3 than freshly grounded seeds (Fr4). It also shows that remaining seeds after water or ethanol extraction are lower in omega-3. Ethanol and water after extraction contains very low amount of omega-3, although water extraction appeared to be more efficient in extracting omega-3 fatty acid.

Experiment 2

[0098] Chia seed extracts were also tested to determine the impact of temperature on the seeds or seed extract. In this experiment the samples tested included: Freshly ground chia seeds were incubated at 45°C for 2 hours (Frl); freshly ground chia seeds were incubated at 45°C for 3 hours (Fr2); freshly ground chia seeds were incubated at 45°C for 3 hours (Fr3); ground and freeze dried chia seeds (Fr4); freshly ground chia seeds (Fr5); ground and freeze dried chia seeds after 1 hour at 45°C (Fr6); ground and freeze dried chia seeds after 2 hours at 45°C (Fr7); and ground and freeze dried chia seeds after 3 hours at 45°C (Fr8).

Table 3 Amount of Omega 3 and Omega 6

Sample %omega-3 %Omega-6 %Tota il Fat

Frl 2.7 0.9 4.4

Fr2 0.5 0.2 0.8

Fr3 0.1 0.0 0.1

Fr4 16.0 5.4 26.6

Fr5 14.0 4.5 22.7

Fr6 10.5 3.3 16.9

Fr7 2.9 0.9 4.7

Fr8 0.4 0.2 0.8

[0099] These results show that omega-3 and total fat% in grounded and freeze-dried chia is more stable at 45°C than in freshly grounded seeds.

Example 3 In vitro biological activity of chia seeds

[00100] Chia seeds possess several useful biological activities after grounding and freeze- drying. For example, chia speeds are known to contain very high level of polyunsaturated fatty acids (PUFA), especially omega-3 fatty acid as well as many other constituents. Both, grounded and freeze-dried seeds and extracts were tested for its biological activities in vitro to provide 980002000140 evidences that procedures used in such preparations do not diminish the biological potency of chia seed extracts.

[00101] Chia seeds from Salvia hispanica were used in the experiments described in this example. Fresh seed were ground and freeze dried, resulting in a powdered form of the chia seeds. Approximately 100 grams of seeds were extracted with 200 mL of ethanol to produce the ethanol extract used in the experiments discussed below. Approximately 20 grams of seeds were extracted with 100 mL water to produce the water extract used in the experiments discussed below.

[00102] Chia is described by ancient natives of California, Arizona and Mexico to improve performance of muscles under extreme conditions such as heat and lack of water. In addition, chia seeds seem to suppress the need for food under extreme conditions. Thus, it was of interest to test chia seed preparation for muscle metabolism in general. To do so, the activity of GSK-3- alpha/beta kinase (Figure 1) and mTOR kinase (prevent muscles from protein degradation and atrophy) (Figure 2) were tested in response to chia seed preparations.

[00103] Figure 1 shows the effect of the chia seeds extracted with water (CH-H) and ethanol (CH-E) on GSK-3-alpha/beta kinase. Interestingly, GSK-3-alpha/beta kinase enzyme activity was observed to increase at low dose (0.3 uL/mL) up to 4 times over untreated control, and higher concentration shows rather inhibitory effect. This results show that extract used in this experiment are potent in modulation of phosphorylation of GSK3-alpha/beta over untreated control following 30 minutes of the treatment.

[00104] Figure 2 shows the effect of chia seed extracts on mTOR kinase. mTOR kinase activity has been correlated to muscle maintenance, specifically to muscle protein degradation and atrophy. These results collected from two independent experiments (21-2 and 22-3) show up to 5 times increased phosphorylation of mTOR at concentration 3ul/mL. Third bar presents average values obtain from two experiments.

Example 4 Glucose uptake and chia seed extracts

[00105] The effect of chia seed extracts on glucose uptake was measure by using fluorescent analog of glucose, 2-NBDG from Molecular Probes Inc. All cells were treated for 30 minutes with tested compounds in culture medium SkBM from Clonetics with 25mM Glucose. After washing, cells were transferred to HBSA (Hepes-buffered Saline), pH 7.0 with 50uM of 2- 980002000140

NBDG without glucose. One minute later, cells were washed in ice-cold PBS and fixed in -20C 70% ethanol. Fluorescence was measured at 480/530 (excitation/emission). Presented results present the level of fluoresce over untreated control.

[00106] Figure 3 shows the effect of chia extracts on total glucose uptake in C2C12 myotubes in vitro. Ethanol extract was used in this experiment in comparison to Rosiglitasone which is well known anti-diabetic drug. In this experiment, C2C12 cells were treated with Chia extract or with Rosiglitazone for 30 minutes only at concentration indicated. The result show that glucose uptake is significantly induced (up to 2-times) at low concentration of chia preparation used in this experiment.

Example 5 Western blot analysis

[00107] C2C12 muscle cells were cultured in SKBM-2 mediums provided by Clonetics. 48 hrs after cell plating, medium was changed to SKBM medium to stimulate differentiation of the cells to myotubes. When differentiated, the myotubes were transferred to PBS supplemented with 5mM glucose for three hrs before the treatment.

[00108] Analysis of C2C12 cells for the level of phosphorylated eNOS, ACC, GSK- 3alpha/beta, and for secretion of nitric oxide secretion was performed in the same experimental system. The cells were treated for 30 minutes at 37 °C. After the treatment, the cells were washed with ice-cold PBS and lysed with 80ul of lysis buffer/well (M-PER buffer from Pierce supplemented with protease and phosphatase inhibitor mix (Calbiochem) for 10 minutes on ice. Next, the plates were transferred to -20 °C to improve^'the lysis of the cells. Next cells were sonicated for 5 minutes and lysate was transferred to Eppendorf tubes and centrifuged at 14,000rpm for 10 minutes. Supernatants were collected in fresh Eppendorf tubes and kept on ice to measure the amount of total proteins. 3μl of each lysate was used to measure the protein concentration using standard Bradford method (Biorad). Subsequently, 20μg per sample of sample protein was used for Western analysis using NuPage 10% Bis/Tris gels (Invitrogen). After exposure of membranes to primary and secondary antibodies the level of phosphorylation of ACC, eNOS, GSK-3alpha/beta and mTOR was detected using ECL-Plus (Amersham) following producer's instruction. Chemilumiscent signals were detected by using ChemiDoc system from Biorad. Intensity of detected signals were analyzed and measured using Quantity One software (Biorad). 980002000140

[00109] The effect of chia seed extracts (water and ethanol extraction) on activity of acetyolo- CoA-carboxylase (AC) in C2C12 muscle cells in vitro was studied using Western blot analysis. Two independent experiments are shown in Figure 4. More intensive bands indicate stronger activation of the enzyme. As shown, CH/E indicates chia seed-ethanol extract, CH/H indicates chia seeds-water extract, and O-indicates the untreated control. The results of these experiments indicates that CH/H at concentration 10 and 30uL/ml was more potent in stimulation of AC than CH/E.

[00110] The effect of chia seed extracts on endothelial nitric oxide synthetase (eNOS) activity in C2C12 cells in vitro following the treatment for 30 minutes is shown in Figure 5. As shown, CH/E indicates chia seed-ethanol extract, CH/H indicates chia seeds-water extract, O-indicates the untreated control, and tATB- total alpha-tubulin. The results shown indicate that both preparations CH/E and CH/H significantly stimulate phosphorylation of eNOS.

[00111] Figure 6 shows the effect of ethanol fraction of Chia on nitric oxide production in C2C12 muscle cells following 30 (Exp I) and 60 (Exp II) minutes of the treatment as measured by colorimetric assay. Collected results show a dose-dependent stimulation of nitric oxide release from the treated C2C12 cells. This result may suggest that chia preparations may stimulate muscle performance due to increased nitric oxide level. In addition, increased glucose uptake level described in Fig 3 could be explain, in part, by the presence of increased level of Nitric oxide in muscle. Such effect is described in the literature and shows that nitric oxide may stimulate glucose uptake independently of insulin. If so, chia seed preparations could be used for improvement of diabetic conditions particularly those related to insulin resistance.

[00112] Collected results indicate that Chia preparations modulate activates of these kinases in the dose-dependent manner following 30 minutes of the treatment. More accurately, these results show that chia preparations used in this research contain active principles with potency to modulate activity of ACC, GSK-3alpha/beta, mTOR, eNOS and to stimulate nitric oxide release from muscle cells and to increase glucose uptake to muscle cells under the same experimental conditions in vitro as described under Materials and Methods.

Example 6 Skin improvement

[00113] A subject presenting eczema-like skin eruption consumes a preparation of freeze dried chia seeds containing approximately 1 kg of chia seeds per day as a dietary supplement for 980002000140 thirty days. The symptoms of the eczema-like skin eruptions decrease as the subject metabolizes the freeze dried chia seed containing dietary supplement.

Example 7 Appetite suppression

[00114] A subject consumes approximately thirty freeze dried chia seeds and a glass of water shortly after awakening from a night's sleep, on an empty stomach and in the evening before sleep. After consumption of the seeds, the subject consumes approximately 50-250 ml of water. No other food stuffs are consumed. The subject experiences few or no urges to eat. Thus, consumption of the chia seeds results in appetite suppression in the subject.

Example 8 Reduced cholesterol in humans

[00115] Consumption of chia seeds has been reported as being capable of reducing cholesterol levels in certain animals. A base line cholesterol level is established for a human subject, who then consumes approximately 100 mg of whole chia seeds three times daily with water for thirty days. At the end of the thirty day period, the subject again tested to establish the serum cholesterol level. After the diet supplementation, the subject experiences a reduction of cholesterol levels.

Example 9 Stimulatory qualities of chia seeds

[00116] Chia seeds have an energy boosting effect on humans. A human subject consumes a shake containing 500 mg of powdered chia seeds. After consuming the shake, the subject experiences a caffeine-like energy boosting effect.

Example 10 Chia seed consumption before exercise to increase muscle performance

[00117] Chia seeds have a boosting effect on human muscle performance. A human subject consumes a shake containing 750 mg of powdered chia seeds. After consuming the shake, the subject runs 5 kilometers and feels less fatigued than she would had she not consumed the shake containing the powdered chia seeds. 980002000140

Example 11 Treating depression with chia seed preparation

[00118] . Consumption of chia seeds can reduce symptoms of depression. A human subject suffering from symptoms of depression consumes approximately 100 mg of freeze dried chia seeds three times daily with water for thirty days. At the end of the thirty day period, the subject feels the symptoms of the depression have been alleviated.

Example 12 Treating diabetes with chia seed preparation

[00119] . . Consumption of chia seeds restores glucose homeostasis. A human subject suffering from diabetes consumes approximately 300 mg of freeze dried chia seeds three times daily with water for thirty days. At the end of the thirty day period, the subject is tested for the ability to metabolize glucose and is found to have had her glucose homeostasis restored.

Example 13 Chronic inflammation and chia seed preparation

[00120] A subject suffering from chronic bowel inflammation consumes 300 mg of chia seeds three times per day. After two weeks of consuming chia seeds, the subject experiences a reduction in bowel inflammation.

[00121] Modifications of the above-described modes for carrying out the invention that are obvious to persons of skill in the art are intended to be within the scope of the following claims. All publications, patents, and patent applications cited in this specification are incorporated herein by reference as if each such publication, patent or patent application were specifically and individually indicated to be incoφorated herein by reference.

980002000140

References

1. Ayerza R, Coates W, Lauria M. Chia seed (Salvia hispanica L.) as an omega-3- fatty acid source for broilers: influence on fatty acid composition, cholesterol and fat content of white and dark meats, growth performance, and sensory characteristics. Poult Sci, 81(6): 826-37, 2002.

2. Ayerza R, Coates W. Chia seeds: new source of omega-3 -fatty acids, natural antioxidants and dietetic fiber. Southwest Center for Natural Products Research $ Commercialization. Office of Arid Lands Studies, Tucson, AZ, 2001.

3. Ayerza R, Coates W. Dietary levels of chia: influence on hen weight, egg production and sensory quality, for two strains of hens. Br Poult Sci, 43(2):283-90, 2002.

4. Ayerza R, Coates W. Dietary levels of chia: influence on yolk cholesterol, lipid content and fatty acid composition for two strains of hens. Poult Sci, 79(5):724-39, 2002.

5. Ayerza R, Coates W. New industrial crops: Northwestern Argentina Regional Project. 45-51, 1996.

6. Ayerza R, Coates W. The omega-3 enriched eggs: the influence of dietary alpha- linoleic fatty acid source combination on egg production and composition. Canadian Journal of Animal Science, 81(3):355-362, 2001.

7. Ayerza R. Fatty acid composition, protein and oil content of chia (Salvia hispanica L.) grown in Peru, Columbia and Argentina. Third European Symposium on Industrial Crops and Products, Elsevier, Reims, France 9 p, 1996.

8. Ayerza R. Oil content and fatty acid composition of Chia (Salvia Hispanica L.) from five northwestern locations in Argentina. Journal of The American Oil Chemists ' Society, 72:1079-1081, 1995.

9. Bushway A. A. Belya P.R, Bushway R. J. Chia seed as a source of oil, polysaccharide and protein. Journal of Food Science, 46:1349-1356, 1981.

10. Taga M.S, Miller E. E, Pratt D.E. Chia seeds as a source of natural lipid antioxidants. Journal of American Oil Chemists ' Society, 61:928-931, 1984. 980002000140

11. Yinrong Lu, L. Yeap Foo. Polyphenolics of Salvia - review. Phytochemistry, 59: 1 17-140, 2002.

12. Cordain L, Eaton SB, Brand Miller J, Mann N, Hill K. The paradoxical nature of hunter-gatherer diets: meat-based, yet non-atherogenic. European Journal of Clinical Nutrition, 56, suppl l:S42-S52, 2002.

13. Hollmann W, Struder HK. Brain, psyche and physical activity. World Congress on Medicine and Health 21 July - 31 August, 2000.

14. Langfort J, Pilis W, Zarzeczny R et al. Effects of low-carbohydrate-ketogenic diet on metabolic and hormonal responses to graded exercise in men. Journal of Physiology and Pharmacology, 47(2): 361-371, 1996.

15. Cabrera Ch. Herbal Medicine: Delivery and Dosage: A look at some traditional herbal preparations and applications. Nutrition Science News, February 1997.

16. Kirkpatrick E, Noogt J, Blanch HW. Separation of polyunsaturated fatty acids using silver ion extraction. Marine Bioproducts Engineering Center, University of Hawaii at Manoa and University of Berkeley, 2000.

17. SCF. Opinion of the Scientific Committee on Food on the safety in use of isopropyl alcohol and isobutyl acetate for the extraction of beta-carotene from Blakeslea trispora.Opinion expressed on 5 March, 2003.

18. Cases S, Stone SJ, Zhou P, Yen E, Tow B, Lardizabal KD, Noelker T, Farese RV Jr. Cloning of DGAT2, a second mammalian diacylglycerol acyltransferase, and related family members. Journal of Biological Chemistry, 276(42): 38870-38876, 2001.

19. Zou J, Wei Y, Jako C, Kumar A, Selvaraj G, Taylor DC. The Arabidopsis thaliana TAG1 mutant has a mutation in a diacylglycerol acyltransferase gene. The Plant Journal, 19(6): 645-653, 1999.

20. Brzustowicz MR, Cherezov N, Zerouga M, Caffrey M, Stillwell W, Wassail SR. Controlling membrane cholesterol content. A role for polyunsaturated (docosahexaenoate) phospholipids. Biochemistry, 41 :12509-12519, 2002. 980002000140

21. Kim HJJ Miyazaki M, Ntambi JM. Dietary cholesterol opposes PUFA-mediated repression of the stearoyl-CoA desaturase-1 gene by SREBP-1 independent mechanism. Journal of Lipid Research, 43: 1750-1757, 2002.

22. Sutherland WH, De Jong SA, Walker RJ, Williams SM. Release of cholesterol from cell membranes to postprandial plasma from mildly hypercholesterolemic subjects: the effect of melas rich on olive and safflower oils. Metabolism 51(10):1306-12, 2002.

23. Eggesba JB, Hagve TA, Barsum K, Hamastmark AT, Hjermann I, Kierulf. Lipid composition of mononuclear levels of serum HDL cholesterol. Scand J Clin Lab Invest 56(3):199-210, 1996.

24. Cowett RM (Editor). Principles of perinatal-noenatal metabolism. Springer- VerlagNew Yorklnc, ISBN 0-387-97499-7, 1991.

25. Drewnowski A. Taste preferences and food intake. Annu Rev Nutr 17:237-53, 1997.

26. Haag M. Polyunsaturated fatty acids: their cellular role and clinical applications (Part 1 and 2). Medicine Journal - Medpharm Publications, May 2002.

27. Hanson RW. Nutrient control of gene transcription minireview series. Journal of Biological Chemistry, 275(40):30747, 2000.

28. Simopoulos AP. Essentail fatty acids in health and chronic disease. Amercican Journal of Clinical Nutrition, 70(suppl): 560S-9S, 1999.

29. Simopoulos AP. Omega-3 fatty acids in health and disease and in growth and development. American Journal of Clinical Nutrition, 54:438-63, 1991.

30. Eaton SB, Strassman Bl, Nesse RM et al. Evolutionary Health Promotion. Preventive Medicine 34:109-118, 2002.

31. Anselmino C. Omega-3 et benefice sante. English translation prepared by Hornstra G. Maastricht University, 2002.

32. Anderson GJ, Connor WE. On the demonstration of omega-3 essentail fatty acid deficiency. Am J Clin Nutr 49(4):585-587, 1989. 980002000140

33. Campbell KL, Roudebush P. Effects of four diets on serum and cutaneous fatty acids, transepidermal water losses, skin surface lipids, hydration and condition of the skin and haircoat of dogs. 11th Proceedings Annual Members ' Meeting AA VD&ACVD Meeting: pp80-84, 1995.

34. Campbell KN. Fatty acid supplementation and skin disease. Veterinary Clinics of North Amercia, Small Animal Practise, 20(6): 1475-86, 1995.

35. Cederholm TF, Berg AB, Johansson EK, Hellstroem KH. Low levels of essential fatty acids are related to impaired delayed skin hypersensitivity in malnourished chronically ill elderly people. European Journal of Clinical Investigation 24(9): 615, 1999.

36. Galland L. Increased requirements for essential fatty acids in atopic individuals: a review with clinical description. J Am Coll Nutr 5(2):213-228, 1986.

37. Ito K, Kikuchi S, Yamada M, Torii S, Katagiri M. Effect of the alpha-linolenic acid enriched diet on atopic dermatisis. A pilot study on 6 outpatients. Japanese Journal of Pediatric Allergy and Clinical Immunology 6(3): 87-91 , 1992.

38. Mahadik SP, Shendarkar NS, Scheffer RE, Mukherjee S, Correnti EE. Utilization of precursor essential fatty acids in culture by skin fibroblasts from schizophrenic patients and normal controls. Prostaglandins Leukot Essent Fatty Acids, 55(l-2):65-70, 1996.

39. Moore SA, Hurt E, Yoder E, Sprecher H, Spector AA. Docosahexaenoic acid synthesis in human skin fibroblasts involves peroxisomal retroconversion of tetracosahexaenoic acid. J Lipid Res 36(l l):2433-2443, 1995.

40. Ramalingaswami V, Sinclair HM. The relation of deficiencies of vitamin A and essential fatty acids to follicular hyperkeratosis in the rat. British J of Derm, 1953.

41. Strieker WE, Anorve-Lopez E, Reed CE. Food skin testing in patients with idiopathic anaphylaxis. Journal of Allergy and Clinical Immunology 77(3):516-519, 1986.

42. Ziboh VA, Miller CC, Cho Y. Metabolism of polyunsaturated fatty acids by skin epidermal enzymes: generation of antiinflammatory and antiproliferative metabolites. Am J Clin Nwtr 71(lsuppl):361 S-366S, 2000. 980002000140

43. Caughey GE, Mantzioris E, Gibson RA, Cleland LG, James MJ. The effect on human tumor necrosis factor α and interleukin lβ production of diets enriched in n-3 fatty acids from vegetable oil or fish oil. American Journal of Clinical Nutrition 63:116-22, 1996.

44. Classen N, Coetzer H, Steinmann ML, Kruger MC. The effect of different N- 6/N-3 essential fatty acid ratios on calcium balance and bone in rats. Prosta Leukot Essent Fatty Acids 53(1): 13, 1995.

45. Delion S, Chalon S, Guilloteau D, Besnard JC, Durand G. Linoleic acid deitary deficiency alters age-related changes of dopaminergic and serotoninergic neurotransmission in the rat frontal cortex. J Neurochem 66:1582, 1996.

46. Goodwin JS, Webb DR. Regulation of the immune response by prostaglandins. Clin Immunol Immunopathol 15(1): 106-122, 1980.

47. Jeffery NM, Sanderson P, Sherrington EJ, Newsholme EA, Calder PC. The ratio of n6 to n3 polyunsatirated fatty acids in the rat diet alters serum lipid levels and lymphocyte function. Lipids 3\(l):131, 1996.

48. Li Y-P, Schwartz RJ, Waddell ID, Holloway BR, Reid MB. Skeletal muscle myocytes undergo protein loss and reactive oxygen-mediated NF-κB activation in response to tumor necrosis factor α. FASEB J 12:871-880, 1998.

49. Matsuzaki J, Kuwamura M, Yamaji R, Inui H, Nakano Y. Inflammatory responses to lipopolysaccharide are supressed in 40% energy-restricted mice. Journal of Nutrition 131 :2139-2144, 2001.

50. Vaughn DM, Reinhardt GA, Swaim SF, Lauten SD, Garner CA, Boudreaux MK, Spano JS, Hoffman CE, Conner B. Evaluation of effects of dietary n-6 to n-3 fatty acid ratios on leukotriene B synthesis in dog skin and neutrophils. Vet Dermatol 5(4):163-173, 1994.

51. Went NJ, Bolt AG, Whitehouse M. Anti-flammatory liniment with linolenic acid- containing oils. PTC International; American Chemical Society, July 12 1990.

52. Bagga D, Wang L, Farias-Eisner R, Glaspy JA, Reddy ST. Differential effects of prostaglandin derived from omega-6 and omega-3 polyunsaturated fatty acids on COX-2 expression and IL-6 secretion. Proc Natl Aced Sci USA 100(4): 1751-6, 2003. 980002000140

53. Bolster DR, Crozier SJ, Kimball SR, Jefferson LS. AMP-activated protein kinase suppresses protein synthesis in rat skeletal muscle trough down-regulated mammalian target of rapamycin (mTOR) signaling. Journal of Biological Chemistry, 277(27):23977-23980, 2002.

54. Barber MC, Ward RJ, Richards SE, Salter AM et al. Ovine adipose tissue monounsaturated fat content is correlated to depot-specific expression of the stearoyl-CoA desaturase gene. Journal of animal science 78(l):62-68, 2000.

55. Ben AH, Lasky D, Ntambi JM. Cloning and characterization of the human stearoyl-CoA desaturase gene promoter: transcriptional activation by sterol regulatory element binding protein and repression bby polyunsaturated fatty acids and cholesterol. Biochemical and biophysical research communications 284(5): 1194-1198, 2001.

56. Cohen P, Miyazaki M, Socci ND et al. Role of stearoyl-CoA desaturase in leptin -mediated weight loss. Science 297(5579): 240 suppl. 2002.

57. Duplus E, Glorian M, Forest C. Fatty acid regulation of gene transcription. Journal of Biological Chemistry 275(40):30749-30752, 2000.

58. Lefevre P, Tripon E, Plumelet C, Douaire M, Diot Ch. Effects of polyunsaturated fatty acids and clofibrate on chicken stearoyl-CoA desaturase 1 gene expression. Biochemical and Biophysical Research Communications, 280:25-31, 2001.

59. Miyazaki M, Kim YC, Ntambi JM. A lipogenic diet in mice with a disruption of the stearoyl-CoA desaturase 1 gene revelas a stringent requirement of endogenous monounsaturated fatty acids for triglyceride synthesis. Jorunal of Lipid Research 42(7): 1018- 1024, 2001.

60. Miyazaki M, Kim Y-Ch, Gray-Keller MP, Attie AD, Ntambi JM. The biosynthesis of hepatic cholesterol esters and triglycerides is impaired in mice with a disruption of the gene for stearoyl-CoA desaturase 1. Journal of Biological Chemistry 275(39): 30132- 30138, 2000.

61. Miyazaki M, Man WC, Ntambi JM. Targeted disruption of stearoyl-CoA desaturase 1 gene in mimce causes atrophy of sebaceous and meibomian glands and depletion of wax esters in the eyelid. Journal of Nutrition 131(9):2260-2268, 2001. 980002000140

62. Mziaut H, Korza G, Ozols J. The N terminus of microsomal Δ9 stearoyl-CoA desaturase contains the sequence determinant for its rapid degradation. PNAS 97(16):8883-8, 2000.

63. Ntambi JM et al. Loss of stearoyl-CoA desaturase functions protects mice against adiposity. PNAS Early Edition (online), Aug.12, 2002.

64. Ntambi JM, Miyazaki M, Stoehr JP, Lan H, Kendziorski ChM, Yandell BS, Song Y, Cohen P, Friedman JM, Attie AD. Loss of stearoyl-CoA desaturase-1 function protects mice against adiposity. Proc. Natl. Acad. Sci. USA 99(17):11482-11486, 2002.

65. Ntambi JM. Dietary regulation of stearoyl-CoA desaturase 1 gene expression in mouse liver. JBiol Chem., 267(15):10925-10930, 1992.

66. Ntambi JM. Regulation of stearoyl-CoA desaturase by polyunsaturated fatty acids and cholesterol. Journal of Lipid Research, 40:1549-1558, 1999.

67. Park Y, Storkson JM, Ntambi JM et al. Inhibition of hepatic stearoyl-CoA desaturase activity by trans-10, cis-12 conjugated linoleic acid and its derivatives. Biochimica et Biophysica Acta 1486:285-292, 2000.

68. Zhang L, Ge L, Tran T, Stenn K, Prouty SM. Isolation and characterization of th ehuman stearoyl-CoA desaturase gene promoter: requirement of a conserved CCAAT cis- element. Biochemical Journal 357(1): 183-193, 2001.

69. Chajes V, Sattler W, Stranzl A, Kostner GM. Influence n-3 fatty acids on the growth of human breats cancer cells in vitro: relationship to peroxides and vitamin-E. Breast Cancer Res Treat 34(3): 199-212, 1995.

70. Hardman. Omega-3 fatty acids to augment cancer therapy. Journal of Nutrition 132(1 1):S3508-3512, 2002.

71. Norrish AE, Skeaff CM, Arribas GLB, Sharpe SJ, Kackson RT. Prostate cancer risk and consumption of fish oils: a dietary biomaker-based case-control study. British Journal of Cancer 81(7): 1238- 1242, 1999. 980002000140

72. Orengo IF, Black HS, Kettler AH, Wolf JE Jr. Influence of dietary menhaden oil upon carcinogenesis and variuos cutaneous responses to UV radiation. Photochemistry and Photobiology 49(7): 71-78, 1989.

73. Saker KE, Cowing BE, Herbein JH. Influence of PUFA on a prospective cancer biomaker in feline adipose. Paper presented at International Symposium on Predictive Oncology and Intervention Strategies: Paris, France February 9-12, 2002.

74. Leaver HA, Bell HS, Rizzo MT, Ironside JW, Gregor A, Wharton SB, Whittle IR. Antitumour and pro-apoptotic actions of highly unsaturated fatty acids in glioma. Prostaglandins Leukot Essent Fatty Acids 66( 1 ): 19-29, 2002.

75. Huang YS, Mills DE, Ward RP, Horrobin DF, Simmons VA. Effects of essential fatty acids depletion on tissue phospholipid fatty acids in spontaneously hypertensive and normotensive rats. Lipids 24(7):565-571, 1989.

76. Eaton SB, Strassman Bl, Nesse RM, et al,. Evolutionary Health Promotion. Preventive Medicine 34:109-118, 2002.

77. Avula CP, Fernandes G. Modulation of antioxidant enzymes and apoptosis in mice by dietary lipids and treadmill exercise. J Clin Immunol 19:35-44, 1999.

78. Campbell KL, Dorn GP. Effects of oral sunflower oil and olive oil on serum and cutaneous fatty acid concentrations in dogs. Research in Veterinary Science 53(2)"172-178, 1992.

79. Cunnane SC, Stitt PA, Gangulli S, Amstrong JK. Raised omega-3fatty acid levels in pigs fed flax. Canadian Journal of Animal Science 70(1 ):251-254, 1990.

80. Harris WS, Gouping L, Rambjor GS, Walen Al, Ontko JA, Cheng Q, Windsor SL. Influence of n-3 fatty acid supplementation on the endogenous activities of plasma lipases. American Journal of Clinical Nutrition 66:254-60, 1997.

81. Harris WS, Rambjor GS, Windsor SL, Diederich D. N-3 fatty acids and urinary excretion of nitric oxide metabolites in humans. American Journal of Clinical Nutrition 65:459- 64, 1997. 980002000140

82. Harris WS. N-3 fatty acids and serum lipoproteins: animal studies. American Journal of Clinical Nutrition 65(suppl):S1611-S16, 1997.

83. Harris WS. N-3 fatty acids and serum lipoproteins: human studies. American Journal of Clinical Nutrition 65(suppl):S1645-S54, 1997.

84. Jolly CA and Fernandes G. Dietary N-3 fatty acids and calorie restriction in autoimmune disease: inlflience in different immune compartments. Current Organic Chemistry 4:945-957, 2000.

85. Lim BO, Jolly CA, Zaman K, Fernandes G. Dietary n-6 and n-3 fatty acids and energy restriction modulate mesenteric lymph node lymphocyte function in autoimmune-prone (NZBxNZW)Fl mice. JNwtr 130: 1557-64, 2000.

86. Marsen TA, Pollok M, Oette K, Baldamus CA. Pharmacokinetics of omega-3- fatty acids during ingestion of fish oil preparations. Prostaglandins Leukotrienes and Essential Fatty Acids 46:191-196, 1992.

87. Muthukumar AR, Jolly CA, Zaman K, Fernandes G. Calorie restriction and n-3 fatty acid supplementation modulate proinflammatory cytokines and polymeric Ig receptor expression in the submandibular glands of autoimmune prone (ΝZBxΝZW)Fl mice. J Clin Immunol 20:354-361, 2000.

88. Ayerza R, Coates W. Dietary levels on yolk cholesterol, lipid content and fatty acid composition for two starins of hens. Poult Sci 79(5):724-39, 2000.

89. Louet JF, Le May C, Pegorier J-P, Decaux J-F, Girard J. Regulation of liver carnitine palmitoyltransferase I gene expression by hormones and fatty acids. Biochem Soc Trans 29:310-316, 2001.

90. Leverve X, Piquet MA, Fontaine E, Keriel C, Rigoulet M. Polyunsaturated fatty acid (pufa) deficiency affects mitochondrial oxidative phosphorylation and hepatocyte metabolism. Laboratory of Fundamental and Applied Bioenergetics, University of Grenoble.

91. Berger A, Mutch DM, German JB, Roberts MA. Dietary effects of arachidonate- rich fungal oil and fish oil on murine hepatic and hippocampal gene expression. Lipids Helath Dis \(\):2, 20 2. 980002000140

92. Berger A, Mutch DM, German JB, Roberts M-A. Unraveling lipid metabolism with microarrays:effects of arachidonate and docosahexaenoate acid on murine hepatic and hippocampal gene expression. Genome Biology 3(7):preprint0004.1-0004.53, 2002.

93. Gil A, Pita M, Martinez A, Molina JA, Sanchez Medina F. Effect of dietary nucleotides on the plasma fatty acids in at-term neonates. Human Nutrition: Clinical Nutrition 40C:185-195, 1986.

94. Boza J, Jimenez J, Faus MJ, Gil A. Influences of postnatal age and dietary nucleotides on plasma fatty acids in the weanling rat. Journal of Parenteral andEnteral Nutrition 16(4):322-6, 1992.

95. De Lorgeril M, Salen P. Diet as preventive medicine in cardiology. Current Opinion in Cardiology 15(5):364-370, 2000.

96. Dyeberg, Jorn et al. Marine n-3 fatty acids, wine intake, and heart rate variability in patients referred for coronary angiography. Circulation 103(5):651-657, 2001.

97. Goristein S, Zemser M, Berliner M, Goldstein R et al. Moderate beer consumption and positive biochemical changes in patients with coronary atherosclerosis. Journal of Internal Medicine 242:219-224, 1997.

98. Taga MS, Miller EE, Pratt DE. Chia seed as a source of natural lipid antioxidants. Journal of the American Oil Chemists ' Society 61:928-931, 1984.

99. Christensen, Schmidt, Madesn, Trring, Skou. Heart rate variability and n-3 polyunsaturated fatty acids in patients with diabetes mellitus. Journal of Internal Medicine 249(6):545-552, 2001.

100. Attele AS. Ginseng berry extract treats obesity. Diabetes 51 : 1851-1858, 2002.

101. Hays JH. Substituting saturated fat for starch may help with glycemic control and weight loss. EndocrPract 8: 177-183, 2002.

102. Westman EC. Low-carb diet reduced weight by 10%. American Journal of Medicine 113:30-36, 2002. 980002000140

103. MCGarry JD. Banting lecture:dysregulation of fatty acid metabolism in the etiology of type 2 diabetes. Diabetes 51(1):7-18, 2002.

104. Adams PB, Lawson S, Sanigorski A, Sinclair AJ. Arachidonnic acid to eicosapentaenoic acid ratio in blood correlates positively with clinical symptoms of depression. Zι wώ 31:S157-S161, 1996.

105. Conquer JA et al. Fatty acids analysis of blood plasma of patients with Alzheimer's diseases types of dementia, and cognitive impairment. Lipids 35:1305-1312, 2000.

106. Emsley R et al. Randomized, placebo-controlled study of ethyl-EPA as supplement in schizophrenia. American Journal of Psychiatry 159:1596-1598, 2002.

107. Stoll AL et al. Omega-3 fatty acids in bipolar disorder: a review. Prostaglandins Leukot Essent Fatty Acids 60(5-6):329-37, 2000.

108. Stoll AL et al. Omega-3 fatty acids in bipolra disorder: a preliminary double- bllind controlled trial. Arch Gen Psychiatry 56(5):407-12, 1999.

109. Friedman J. Fat in all the wrong places. Nature 415:265-69, 2002.

110. Minokoshi Y et al. Leptin stimulates fatty acid oxidation by activating AMP- activated protein kinase. Nature 415:339-343, 2002.

111. Shimomura I, Hammer R, Ikemoto S, Brown M, Goldstein J. Leptin reverses insulin resistance and diabetes mellitus in mice with congenital Hpodystrophy. Nature 401 :73- 76, 1999.

112. Barclay WR. Fermentation process for producting long chain omega acids with euryhaline microorganisms. US Patent 6451567.

113. Cappelli P, Liberato L, Stuard S, Ballone E, Albertazzi A. N-3 polyunsaturated fatty acid supplementation in chronic progressive renal disease. Journal ofNephrology 10(3):157-162, 1997.

114. Minokoshi Y, Kim YB, Peroni OD, Fryer LGD, Muller C, Carling D, Kahn BB. Leptin stimulates fatty-acid oxidation by activating AMP-activated protein kinase. Nature 415:339-343, 2002. 980002000140

1 15. Kersten S, Desvergne B, Wahli W. Roles of PPARs in health and disease. Nature 405:421-424, 2000.

116. Shimomura I, Hammer RE, Ikemoto S, Brown MS, Goldstein JL. Leptin reverses insulin resistance and diabetes mellitus in mice with congenital Hpodystrophy. Nature 401:73- 76, 1999.

117. Bolster DR, Crozier SJ, Kimball SR, Jefferson LS. AMP-activated protein kinase suppresses protein synthesis in rat skeletal muscle trough dwon-regulated mammalian target of rapamycin (mTOR) singaling. The Journal of Biological Chemistry 277(27):23977-23980, 2002.

118. Gao X, Zhang Y, Arrazola P, Hino O, Kobayashi T, Yeung RS, Ru B, Pan D. Tsc tumour suppressor proteins antagonize amino-acid-TOR signaling. Nature Cell Biology 4(9):699-704, 2002.

119. Inoki K, Li Y, Zhu T, Wu J, Guan K-L. TSC2 is phosphorylated and inhibited by Akt and suppresses mTOR signalling. Nature Cell Biology 4(9):648-657, 2002.

Claims

980002000140Claims

1. A PUFA-rich seed of suitable nutritional value that has been freeze dried.

2. The seed of claim 1, wherein the seed has also been ground into a powder.

3. The seed of claim 1, wherein the seed lacks a glutinous coat.

4. The seed of claim 1, wherein the seed has been prepared for incoφoration into a food stuff.

5. The seed of claim 5, wherein the seed has been prepared for incorporation into an aqueous solution.

6. The seed of claim 1, an alcohol extract of the seed.

7. The seed of claim 6, wherein the alcohol is ethanol.

8. The seed of claim 1, wherein the seed is selected from the group consisting of a chia seed, a flaxseeds, sunflower seeds, soy beans (seeds), hemp seeds, safflower seeds, cottonseeds, and rapeseeds.

9. A method of increasing polyunsaturated fatty acids in a diet comprising:

providing a PUFA-rich seed to a subject in need of an increased amount of polyunsaturated fatty acids.

10. The method of claim 9, wherein the seed are selected from the group consisting of a chia seed, a flaxseeds, sunflower seeds, soy beans (seeds), hemp seeds, safflower seeds, cottonseeds, and rapeseeds.

11. The method of claim 9, wherein the PUFA-rich seed is freeze dried.

12. A method of increasing muscle-related enzymatic activity comprising: 980002000140 providing a PUFA-rich seed to a subject, wherein at least one muscle-related enzyme activity is increased.

13. The method of claim 12, wherein the enzyme is GSK-3.

14. A method of modulating enzymatic activity comprising providing a PUFA-rich seed containing composition to an enzyme, wherein the activity of the enzyme is modulated.

15. The method of claim 14, wherein the enzyme is selected from the group consisting of nitric oxide synthetase, ACC, glucose uptake, mTOR.

16. The method of claim 14, wherein the PUFA-rich containing composition comprises fresh PUFA-rich seeds.

17. The method of claim 14, wherein the PUFA-rich containing composition comprises freeze dried PUFA-rich seeds.