Cell hydration composition comprising cyclodextrin

Abstract

To provide a composition that interacts with a biological cell line, comprising a biologically active molecule as well as a biomolecule surface, a cell component, and a water molecule having a specific density.SOLUTION: According to the present invention, a composition comprises a biologically active component constructed to increase the activity of a biological cell line by increasing the hydration of one or more components of the cell line. The biologically active component may comprise a first carbohydrate clathrate subcomponent, which increases the hydrogen bond structure of water, and a second solute subcomponent. The biologically active component may comprise an inclusion complex composed of a clathrate component and a complexing compound. The clathrate subcomponent may comprise amylose or cyclodextrin. According to the present invention, there are also provided a beverage and method for improving the hydration of cells in an animal, such as a human.SELECTED DRAWING: Figure 11

Description

[Technical field]

[0001] The present invention generally relates to the regulation of biological cell activity, particularly cell activity that is dependent on the state of hydration. More specifically, the present invention relates to a biologically active component that is constructed to increase the activity of a biological cell system by increasing the hydration of one or more components of the cell system. The biologically active component may include a first carbohydrate clathrate subcomponent that increases the hydrogen bond structure of water. The present invention further relates to the delivery of biological compounds in vivo to modify the physiological activity of a mammal. [Background technology]

[0002] Water molecules interact primarily through hydrogen (H) bonds and through the alignment of dipole moments. For example, bonds between adjacent water molecules are reinforced or stabilized by the alignment of the bond axes with the next nearest neighbors. In liquid water, such alignment propagates into the surrounding aqueous medium, establishing a molecular structure on the submicrometer scale.

[0003] Examples of products and methods of use of cyclodextrins as clathrates to form inclusion complexes with bioactive guest molecules to improve the solubility and / or bioavailability of pharmaceutical compounds are described in U.S. Pat. Nos. 7,115,586 and 7,202,233, and U.S. Patent Application Publication Nos. 2004 / 0137625 and 2009 / 0227690, the complete disclosures of which are incorporated herein by reference for all purposes.

[0004] Examples of products and methods of use containing clathrates that bind hydrophobic biomolecules are described in U.S. Pat. Nos. 6,890,549, 7,105,195, 7,166,575, 7,423,027, and 7,547,459; U.S. Patent Application Publication Nos. 2004 / 0161526, 2007 / 0116837, 2008 / 0299166, and 2009 / 0023682; Japanese Patent Application JP 60-094912; Suzuki and Sato, "Nutritional significance of cyclodextrins: indigestibility and hypolipemic effect of α-cyclodextrin," J. Nutr. Sci. Vitaminol. (Tokyo, Japan), the complete disclosures of which are incorporated herein by reference for all purposes. 1985;31:209-223); and Szejtl et al., Staerke / Starch, 27(11), 1975, pp. 368-376.

[0005] U.S. Patent Application Publication No. 2009 / 0110746 describes chemical agents that have the property of increasing the water diffusivity of dissolved molecular oxygen (O2) in the human body, and that may include a cyclodextrin as a second "carrier" component to improve the solubility of a first pro-oxygenating agent, where the cyclodextrin is not intended as an agent that directly alters water diffusivity, tissue oxygenation, water structure, or cellular hydration. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] U.S. Patent No. 7,115,586 [Patent Document 2] U.S. Patent No. 7,202,233 [Patent Document 3] US Patent Application Publication No. 2004 / 0137625 [Patent Document 4] US Patent Application Publication No. 2009 / 0227690 [Patent Document 5] U.S. Patent No. 6,890,549 [Patent Document 6] U.S. Patent No. 7,105,195 [Patent Document 7] U.S. Patent No. 7,166,575 [Patent Document 8] U.S. Patent No. 7,423,027 [Patent Document 9] U.S. Patent No. 7,547,459 [Patent Document 10] US Patent Application Publication No. 2004 / 0161526 [Patent Document 11] US Patent Application Publication No. 2007 / 0116837 [Patent Document 12] US Patent Application Publication No. 2008 / 0299166 [Patent Document 13] US Patent Application Publication No. 2009 / 0023682 [Patent Document 14] Japanese Patent Application No. JP 60-094912 [Patent Document 15] US Patent Application Publication No. 2009 / 0110746 [Non-patent literature]

[0007] [Non-Patent Document 1] Suzuki and Sato, "Nutritional significance of cyclodextrins: indigestibility and hypolipemic effect of α-cyclodextrin" J. Nutr. Sci. Vitaminol. (Tokyo 1985; 31:209-223) [Non-Patent Document 2] Szejtl et al., Staerke / Starch, 27(11), 1975, pp. 368-376 [Non-Patent Document 3] Hinrichs, W. et al., "An Amylose Antiparallel Double Helix at Atomic Resolution," Science, (1987), 238(4824): 205-208 [Non-Patent Document 4] Lindner and Saenger (Carbohydr. Res., 99:103, 1982) [Non-Patent Document 5] Segtan et al. (Anal. Chem. 2001;73, 3153-3161) [Non-Patent Document 6] R. Giangiacomo(Food Chemistry, 2006, 96.3. 371-379) Summary of the Invention [Means for solving the problem]

[0008] A composition that interacts with a biological cell system comprising a biologically active molecule along with a biomolecular surface, cellular components and water molecules having a specific density, the composition comprising a biologically active component configured to increase the activity of the biological cell system by increasing the hydration of one or more components of the cell system. [Brief description of the drawings]

[0009] [Figure 1] FIG. 1 shows a chemical bond model of β-cyclodextrin, a cyclic oligosaccharide with seven α[1-4] linked glucose units. [Diagram 2] FIG. 1 shows a structural model of a cyclodextrin with an overall toroidal topology. [Diagram 3] FIG. 1 shows a structural model of cyclodextrin, including the arrangement of glucosyl hydroxyl groups along the edge of a toroid. [Figure 4] FIG. 1 shows the calculated molecular dynamic distribution of water molecules around a β-cyclodextrin molecule 1 picosecond after initial contact. [Diagram 5]FIG. 5 shows the calculated molecular dynamics distribution of the water molecules of FIG. 4, including the more organized open water structure, 1000 picoseconds after initial contact. [Figure 6] FIG. 6 shows a thresholded image of the distribution of water molecules shown in FIGS. 4 and 5. [Figure 7] FIG. 1 shows a comparison of NIR derivative spectra, including specific wavelength ranges, for a water sample containing dissolved cyclodextrin and a water sample containing no dissolved cyclodextrin. [Figure 8] FIG. 1 shows a comparison of NIR derivative spectra, including specific wavelength ranges, for a water sample containing dissolved cyclodextrin and a water sample containing no dissolved cyclodextrin. [Figure 9] FIG. 1 shows a comparison of NIR derivative spectra, including specific wavelength ranges, for a water sample containing dissolved cyclodextrin and a water sample containing no dissolved cyclodextrin. [Figure 10] FIG. 1 shows a comparison of NIR derivative spectra, including specific wavelength ranges, for a water sample containing dissolved cyclodextrin and a water sample containing no dissolved cyclodextrin. [Figure 11] FIG. 1 shows a comparison of seed germination kinetics in water containing varying amounts of cyclodextrin, amino acids, and cyclodextrin / amino acid inclusion complexes. [Figure 12] FIG. 1 shows a comparison of seed germination kinetics in water containing varying amounts of cyclodextrin, vitamins, and cyclodextrin / vitamin inclusion complexes. [Figure 13] FIG. 1 shows a comparison of seed germination rates in water containing varying amounts of hydrating active ingredients according to the present disclosure. [Figure 14] FIG. 1 shows a comparison of the longevity of nematodes in media containing varying amounts of cyclodextrin as an active ingredient for hydration according to the present disclosure. [Figure 15] FIG. 1 shows a comparison of nematode longevity in media containing varying amounts of derivatized cyclodextrin as an active ingredient for hydration according to the present disclosure. [Figure 16]FIG. 1 shows a comparison of the longevity of nematodes in media containing varying amounts of cyclodextrin inclusion complexes as active ingredients for hydration according to the present disclosure. [Figure 17] FIG. 1 shows the mortality frequency of nematodes in media with and without a cyclodextrin inclusion complex as the active ingredient of hydration according to the present disclosure. [Figure 18] FIG. 1 shows population survival curves for nematodes living in media with and without a cyclodextrin inclusion complex as the active ingredient of hydration according to the present disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] Water structure is intentionally increased or organized by adding one or more solutes or suitable molecular aggregates whose surface can strongly compete with water molecules for hydrogen bonding and / or dipole orientation. In particular, factors and agents that strengthen water molecular interactions and increase water structure thereby change the hydration or solvation of additional molecular surfaces. Thus, a first solution additive that increases water structure can increase hydration interactions (e.g., bond strength and kinetics) with the molecular surface of a second solution component, or can decrease such interactions, depending on the hydrogen bonding surface properties of the second component.

[0011] Furthermore, factors that modify water structure generally change the average distance between water molecules, thereby increasing or decreasing the density of water. For example, when the temperature of water is reduced below its freezing point, the hydrogen bonds between water molecules overcome the kinetic energy of the water molecules, resulting in an increase in water structure, which reduces the density of frozen water by approximately 9%. Similarly, in liquid water, an increase in the strength of water's hydrogen bonds increases the average distance between water molecules, which is observed as an increase in specific volume (i.e., a decrease in density). A decrease in the density of liquid water can increase the diffusion rate of dissolved solutes. Thus, aqueous additive components that reduce the density of water can increase the diffusion rate of co-dissolved solutes.

[0012] Chaotropes, as used herein, are water-containing solute additives that function in aqueous solutions to disrupt hydrogen bond networks, thereby reducing water structure. Chaotropes are generally less polar than water molecules and have weaker hydrogen bond potentials. Chaotropes can preferentially bind to non-polar solutes and particles, thereby increasing the solubility of non-polar solutes.

[0013] Kosmotropes, as used herein, are solutes that promote strong and extended hydrogen bond networks in aqueous solutions, thereby increasing and / or stabilizing the submicrometer scale structure of water molecular interactions. Kosmotropes that have hydrogen bond chemical potentials greater than that of water and / or have dipole moments greater than that of water can increase the hydrogen bond network between water molecules. In addition, kosmotropes can increase hydration interactions at molecular surfaces, which may contain binding sites between molecules, by strengthening the hydration structure. Thus, kosmotropes can be used as aqueous solution additives to stabilize molecular interactions.

[0014] Additionally, kosmotropes can increase the effective chemical activity of dissolved cosolutes. Increasing the strength of hydrogen-bonding interactions between water molecules causes water to adopt a more open configuration with a lower specific density and a larger specific volume. Thus, the addition of a kosmotrope to an aqueous solution can increase the diffusivity of one or more of the dissolved cosolute species or compounds by causing a decrease in density. Increasing the diffusivity of a solute species or compound can increase its reactivity, chemical potential, effective concentration, and availability.

[0015] As discussed herein, clathrate components are amphiphilic carbohydrate compounds that have an exterior surface that is hydrophilic and that hydrogen bonds strongly with water, and an interior surface that is less hydrophilic The interior surface of the clathrate is capable of selectively binding to molecular structures that are relatively non-polar, or less hydrophilic than water.

[0016] An inclusion complex, as used herein, is a chemical complex formed between two or more compounds, where a first compound (also referred to as a host) has a structure that defines a partially enclosed space into which molecules of a second compound (also referred to as a guest) fit and bind to the first compound. The host molecule, which may be referred to as a clathrate, may bind reversibly or irreversibly with the guest molecules.

[0017] Biological cells, as used herein, are self-replicating, functional metabolic units of living organisms that can live as single-celled organisms or as subunits of multicellular organisms and include lipid membrane structures that contain a functional network of interacting biomolecules such as proteins, nucleic acids, and sugars. Biological cells include prokaryotic cells, eukaryotic cells, and dissociated cells from multicellular organisms, which may include cultured cells previously obtained from multicellular organisms.

[0018] A biological system, as used herein, is a functionally interconnected network of biological cells and / or subcellular components, which may include living cells, non-living cells, organelles, and / or biomolecules.

[0019] A biologically active molecule, as used herein, is a molecular compound that has a functional activity in a biological cellular system.

[0020] A biomolecule, as used herein, is a molecular compound synthesized by a biological cell. Biomolecules include compounds normally synthesized by cells, as well as compounds synthesized by genetically engineered cells, and chemically synthesized copies of compounds derived from cells.

[0021] A biomolecular surface, as used herein, is the outer atomic boundary of a biomolecule and may include biochemical interaction surfaces such as binding sites.

[0022] A cellular component, as used herein, is a functional element of a biological cell and includes biomolecules, biomolecular complexes, organelles, polymeric structures, membranes and membrane-bound structures, and may further include functional pathways and / or networks, such as sequences of molecular events.

[0023] The density of a substance is the mass per unit volume of that substance under specific conditions of temperature and pressure.

[0024] The specific volume of a substance is the volume per unit mass of the substance, e.g., m 3 The specific volume of a substance is equal to the reciprocal of its density.

[0025] A biologically active component, as used herein, is a molecular entity that modifies (increases or decreases) the activity of a biological cell system.

[0026] A bioactive agent, as used herein, is a substance that, when added to a biological cell system, or cellular component, causes a change in the biological activity of that system, or component.

[0027] Water structure, as used herein, refers to the network of hydrogen bonds that hold and organize the orientation of water molecules in the liquid and solid states. Water structure, as used herein, increases when the hydrogen bonds between water molecules at a given temperature are strengthened and decreases when the hydrogen bonds between water molecules at a given temperature are weakened.

[0028] Interactions between cellular components, as used herein, refer to chemical bonds between biomolecular surfaces. Such interactions may include binding between two biomolecules, such as a ligand and its specific receptor. Alternatively, such interactions may include binding between a biomolecule and a cellular organelle, such as a cell membrane.

[0029] Extracellular signals, as used herein, are biomolecules that can modify (increase or decrease) the activity of a cell when applied to the outside of the cell. Extracellular signals can bind to components of the cell membrane (outer membrane) of the cell, or can pass through the cell membrane to regulate activity within the cell. Extracellular signals can include, but are not limited to, components of the extracellular matrix; cell membrane components, such as glycoproteins and glycolipids; antigens; and diffusible biomolecules, such as nitric oxide.

[0030] An intracellular messenger, as used herein, is a component inside a biological cell that has an active state in which it functions as an intermediate signal transduction of an extracellular signal to an intracellular target.

[0031] A pharmacological agent, as used herein, is a synthetic chemical that binds to a biomolecule or biomolecular complex, thereby altering its activity.

[0032] The present invention includes active compositions that increase the activity of a biological cell system by increasing the hydration of one or more components of the cell system.

[0033] The active composition for modifying cellular hydration preferably comprises a first carbohydrate clathrate component that increases the hydrogen bond structure of water.In some examples, the active composition preferably comprises a first carbohydrate clathrate component that increases the hydrogen bond structure of water and a second solute compound that may be a bioactive agent.In some examples, the active composition preferably comprises an inclusion complex formed between the clathrate component and a complexing compound that may be a bioactive agent.

[0034] Biological cells are multicompartment structures that contain chemically active water-based chambers and lipid-based membranes. The structure and activity of cells derive from highly selective chemical binding associations between their biomolecular components, such as lipids, structural proteins, enzymatic proteins, carbohydrates, salts, nucleotides, and other metabolic and signaling biomolecules. The strength and specificity of biomolecular binding reflects the complementary chemical topology at the binding interface. Hydrophilic and / or hydrophobic surfaces generally dominate the chemical topology of biomolecular binding interfaces. In aqueous systems, hydrophobic and hydrophilic interactions are substantially driven by competitive hydration interactions with water molecules at concentrations greater than 50M.

[0035] Cellular hydration, as used herein, refers to the interactions between water molecules and biomolecular components of a cellular system. Cellular hydration can be altered by changing the strength and / or kinetics of hydrogen bonds between water molecules and biomolecular surfaces.

[0036] Aqueous solution additives that modify water structure can alter the strength, kinetics, and / or specificity of binding between cellular components by altering the hydration of binding surfaces of biomolecules. For example, kosmotropic water-containing additives that increase water structure can alter the strength, kinetics, and / or specificity of binding between a secreted intercellular signaling factor and its cognate receptor located in the plasma membrane of a potential target cell, thus biasing the outcome of a cellular signaling network.

[0037] Clathrates suitable as active ingredients for cell hydration according to the invention include amylose and cyclodextrins. Amylose is a linear polysaccharide of D-glucose units. As shown in FIG. 1, cyclodextrins are macrocyclic oligosaccharides of D-glucose units linked by α(1-4) interglucose linkages. Amylose and cyclodextrins are easily prepared in large quantities from hydrolyzed starch. Preparation of cyclodextrins involves enzymatic conversion, most commonly using the enzyme cyclodextrin-glycosyltransferase produced by Bacillus strains.

[0038] Cyclodextrins can vary by the number of glucose units contained in the ring, as shown in Figure 2. Cyclodextrin species include α-cyclodextrin (6 units), β-cyclodextrin (7 units), γ-cyclodextrin (8 units), and δ-cyclodextrin (9 units). Parent cyclodextrins, as used herein, are natural, chemically underivatized α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin, which have 18 (α-), 21 (β-), and 24 (γ-) free, unmodified hydroxyl groups, respectively.

[0039] Cyclodextrins generally have a toroidal topology, resembling a truncated cone or the shape of one half of an open-ended cylinder, as shown generally in Figure 3. Cyclodextrins can therefore be described as including an outer chemical surface that includes the exterior surface and the edge of the cylinder, and an inner chemical surface that surrounds an internal cavity (inside the cylinder).

[0040] The exterior surface of cyclodextrin contains a high density of hydrophilic chemical groups that hydrogen bond with water. In particular, the hydroxyl groups of the parent α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin structures are all concentrated at the ends of the cyclodextrin cylinder. More specifically, the hydroxyl (-OH) chemical groups of cyclodextrin are located along the edges of the cylinder and their orientation is sterically restricted. The hydroxyl group at the C(6) position of glucose can be referred to as the first OH group and points in a counterclockwise direction relative to the narrow open end of the cyclodextrin cylinder. The hydroxyl group at the C(2) position of glucose can be referred to as the second hydroxyl group and is at a clockwise angle relative to the wide open end of the cyclodextrin cylinder.

[0041] The high density and restricted orientation of cyclodextrin hydroxyl groups creates strong hydrogen-bonding surfaces, particularly at both ends of the cyclodextrin cylinder. Physicochemical analysis and solvation modeling of cyclodextrins indicate that water molecules in close proximity to the cyclodextrin are fixed in position and have low angular (rotational) mobility. Usefully, each type of cyclodextrin has different cylinder diameters and numbers of hydroxyl groups, and different numbers and mobilities of strongly bound water molecules.

[0042] The hydrogen-bonding activity of cyclodextrin compounds can be propagated to the surrounding aqueous medium. Dynamic modeling of cyclodextrin molecules introduced into a defined population of water molecules at standard temperature and pressure induces nanosecond water reorganization throughout the volume, as shown in Figures 4 and 5. Figure 4 shows the population distribution 1 picosecond (ps) after the start of the mixing simulation, and Figure 5 shows the redistribution of the same population at 1000 ps (1 nanosecond), where the water molecules adopt a more open conformation.

[0043] In some instances, cyclodextrins may function as active components in cellular hydration through their kosmotropic activity, which increases the binding structure of water, thereby modifying the hydration of biomolecular surfaces by increasing hydrogen bonds between water molecules, thereby altering the strength, kinetics, and / or specificity of bonds between cellular components.

[0044] In some instances, cyclodextrins may function as active ingredients in cellular hydration through their kosmotropic activity, which increases the water bond structure, leading to open water structures with lower specific density (i.e., higher specific volume) due to stronger hydrogen bonds between water molecules, and also increasing the diffusion rate of bioactive molecules. Such examples may include soluble bioactive molecules such as enzymes, enzyme substrates, nutrients, metabolites, cytokines, neurotransmitters, hormones, extracellular signals, intracellular messengers, or pharmacological agents.

[0045] The active ingredient of cell hydration, which increases the diffusion rate in water, can regulate one of many biological processes that are limited by the rate of change of the concentration of bioactive ingredients.For example, the clearance of neurotransmitters from synaptic clefts is generally diffusion-limited, including the passive diffusion of glutamate from excitatory synapses in mammalian brains, and the active catabolism of acetylcholine by the diffusion-limited enzyme acetylcholinesterase in vertebrate neuromuscular synapses.Similarly, the activity of electrically excitable cells, such as muscle cells, is generally coordinated by the diffusion-limited change in the concentration of intracellular second messenger signal calcium.

[0046] The cell hydration activity of cyclodextrins can be modified, either increased or decreased, by forming inclusion complexes with complexing agents. The interior surface of cyclodextrins lacks hydroxyl groups and is less hydrophilic than the surrounding aqueous environment, thereby preferentially binding cosolute molecules with low hydrophilicity and hydrogen bonding potential.

[0047] When ingested by an animal, carbohydrate clathrate compositions that increase the hydrogen bond structure of interstitial and intracellular fluids can improve cellular hydration, including the hydration structure of cell membrane surfaces and the solvation of biomolecules that aid in healthy cell function. Improving cellular hydration can support healthy cell function, for example, by increasing the in-, out-, and / or diffusivity of solutes, nutrients, waste products, cytokines, metabolites, and other molecular agents that support cell function, differentiation, repair, growth, and survival, as well as by stabilizing cell membranes in sensitive tissues such as muscle and nerve.

[0048] In some instances, the carbohydrate inclusion complex ingested by the animal can increase the hydrogen bond structure of water, thereby improving cell hydration and / or diffusivity of cellular components. In some instances, the carbohydrate inclusion complex ingested by the animal can be dissociated to release free (i.e., uncomplexed) cyclodextrin clathrate components, which increase the hydrogen bond structure of water, thereby improving cell hydration and / or diffusivity of cellular components. In some instances, the carbohydrate inclusion complex can increase water structure without dissociation, improving cell hydration. In some instances, the carbohydrate inclusion complex can be dissociated into clathrate components to increase water structure and cell hydration, and the complexing agent can further increase water structure and / or provide other beneficial properties, such as nutrition or flavor.

[0049] The carbohydrate clathrate composition of the present invention can be provided in various forms, including being formed into solid powder, tablet, capsule, caplet, granule, pellet, wafer, powder, instant beverage powder, effervescent powder, or effervescent tablet.Some carbohydrate clathrate compositions can also be formed as or incorporated into water-containing beverages or other foods.Such carbohydrate clathrate compositions can be inclusion complexes that remain reasonably stable during storage, and thus do not dissociate from the complexing agent and form stronger complexes with other compounds, which reduces the kosmotropic activity of the complex and thereby reduces its ability to improve cellular hydration.

[0050] The present disclosure also provides methods for improving cellular hydration in animals, such as humans. For example, some methods may include (a) preparing a beverage by dissolving an inclusion complex formed by a carbohydrate clathrate component and a complexing agent that can dissociate from the carbohydrate clathrate component under physiological conditions, and (b) orally ingesting the beverage to an animal, where the carbohydrate clathrate component dissociates from the complexing agent and the strength, extent, and kinetics of hydrogen-bonded water structures on the biomolecular surface of the cell are modified.

[0051] I. Carbohydrate Clathrate Compositions The carbohydrate clathrate component may comprise any suitable carbohydrate, including, but not limited to, α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, methylated β-cyclodextrin, 2-hydroxypropylated β-cyclodextrin, water soluble β-cyclodextrin polymers, partially acetylated α-, β- and γ-cyclodextrin, ethylated α-, β- and γ-cyclodextrin, carboxyalkylated β-cyclodextrins, quaternary ammonium salts of α-, β- and γ-cyclodextrin, amylose (e.g., acetylated amylose), and mixtures thereof.

[0052] In preferred embodiments, carbohydrate clathrates can be selected based on their kosmotropic activity to increase water structure, alone or in combination with other solutes. Preferred cyclodextrin kosmotropes may include α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, 2-hydroxypropyl-cyclodextrin, carboxymethylated cyclodextrin, and quaternary ammonium cyclodextrin.

[0053] Cyclodextrin derivatives may include alkylated derivatives, hydroxyalkylated derivatives, alkoxyalkylated derivatives, acetylated derivatives, quaternary ammonium salt derivatives, carboxyalkylated derivatives, maltosylated derivatives, and glucosylated derivatives. The alkyl groups of the cyclodextrin derivatives may be straight or branched, and the main chain may be 1 to 3 carbons in length, with a total of 1 to 6, preferably 1 to 3, carbon atoms. Some non-limiting examples of cyclodextrin derivatives may include methylated beta-cyclodextrin, 2-hydroxypropylated beta-cyclodextrin, water-soluble beta-cyclodextrin polymers, partially acetylated alpha-cyclodextrin, beta-cyclodextrin, and / or gamma-cyclodextrin, ethylated alpha-cyclodextrin, beta-cyclodextrin, and / or gamma-cyclodextrin, carboxyalkylated beta-cyclodextrin, quaternary ammonium salts of alpha-cyclodextrin, beta-cyclodextrin, and / or gamma-cyclodextrin, and mixtures of any combination of these derivatives together or in combination with one or more cyclodextrins. Exemplary mixtures of cyclodextrins may include combinations of alpha-cyclodextrin, beta-cyclodextrin, and / or gamma-cyclodextrin in a weight ratio range of about 1:1:1 to 2:2:1, respectively. The cyclodextrin may be in hydrated crystalline and / or amorphous form, including, but not limited to, hydrated and / or amorphous forms of α-cyclodextrin, β-cyclodextrin, and / or γ-cyclodextrin, and mixtures thereof.

[0054] When the carbohydrate clathrate composition is in a solid form, the cyclodextrin component may be present in a concentration range of about 10-90% w / w, or about 15-70% w / w, or about 15-60% w / w. Preferably, the cyclodextrin component may be present in a concentration range of about 10-50% w / w, or about 15-40% w / w. More preferably, the cyclodextrin component may be present in a concentration range of about 20-25% w / w.

[0055] When the carbohydrate clathrate composition is in the form of a water-containing beverage, the cyclodextrin component may be present in a concentration range of about 0.01-75% w / v, or about 0.05-50% w / v, or about 0.1-25% w / v. Preferably, the cyclodextrin component may be present in a concentration range of about 0.1-10% w / v. More preferably, the cyclodextrin component may be present in a concentration range of 0.1-5% w / v.

[0056] The carbohydrate clathrate composition preferably comprises a clathrate capable of forming inclusion complexes with various complexing agents, such as amino acids, vitamins, flavorants, odorants, colorants, etc. Non-exclusive examples of carbohydrate clathrate components that can be combined with a complexing agent to form an inclusion body include α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, 2-hydroxypropyl-cyclodextrin, carboxymethylated cyclodextrin, quaternary ammonium cyclodextrin, amylose, amylose derivatives, or any desired mixtures thereof.

[0057] The cyclodextrin clathrate component can further be selected based on the desired binding characteristics with the selected complexing agent. Non-limiting examples of acceptable cyclodextrins include α-cyclodextrin, β-cyclodextrin and γ-cyclodextrin in commercially available and government regulatory approved forms. The number of glucose units determines the internal dimensions of the cavity and its volume, and can also determine the selectivity in forming inclusion complexes with guest molecules. The selected complexing agent, when bound to a host cyclodextrin or other host carbohydrate clathrate, can modify the physicochemical properties of the complexed host to increase its kosmotropic activity.

[0058] When the clathrate component is in the form of an amylose component, the amylose component may contain glucose units expressed as a degree of polymerization (DP) in the range of DP=10-900, more preferably DP=20-200, and most preferably DP=30-80. Amylose derivatives may include, but are not limited to, acetylated amylose. Preferably, the amylose component may have a structure that includes α1,4-linked D-glucopyranose in a helical arrangement that defines a central cavity for binding hydrophobic molecules. For example, the A-starch helix and the B-starch helix of V-amylose may include parallel, left-handed double helices that define a central cavity. The helices of the amylose inclusion complex may be stabilized by hydrophobic forces created by host-guest interactions, intermolecular hydrogen bonds between glucoses of adjacent amylose, and intramolecular hydrogen bonds formed by adjacent turns in the helix. See Hinrichs, W. et al., "An Amylose Antiparallel Double Helix at Atomic Resolution," Science, (1987), 238(4824): 205-208, the complete disclosure of which is incorporated herein by reference for all purposes. The amylose clathrate component can be used to form inclusion complexes with complexing agents having low molecular weight, such as, but not limited to, flavors, colors, vitamins, amino acids, and / or amines.

[0059] When the composition containing the amylose clathrate component is in a solid form, the amylose component may be present in a concentration range of preferably about 10-90% w / w, or about 15-70% w / w, or about 15-60% w / w. More preferably, the amylose component may be present in a concentration range of about 10-50% w / w, or about 15-40% w / w. Most preferably, the amylose component may be present in a concentration range of about 20-25% w / w. When the composition containing the amylose clathrate component is in the form of a water-containing beverage, the amylose component may be present in a concentration range of preferably about 0.1-75% w / v, or about 1-50% w / v, or about 1-25% w / v.

[0060] II. Complexing Agents In some examples, the clathrate compositions disclosed herein may optionally contain complexing agents, which may include one or more amino acids, vitamins, flavors, odorants, and / or other nutritional ingredients, as well as combinations or mixtures of these agents. The carbohydrate clathrate compositions may further include one or more carbonation forming components for use in forming beverage products.

[0061] Complexing agents can be strongly complexed with the clathrate components to increase kosmotropic activity and thereby affect cellular hydration, or they can be weakly complexed with the clathrate components such that they can dissociate from the clathrate components to increase water structure with the free clathrate components.

[0062] Non-limiting examples of amino acids suitable for forming inclusion complexes with the carbohydrate clathrate compositions of the present disclosure include aspartic acid, arginine, glycine, glutamic acid, proline, threonine, theanine, cysteine, cystine, alanine, valine, tyrosine, leucine, isoleucine, asparagine, serine, lysine, histidine, ornithine, methionine, carnitine, aminobutyric acid (alpha-, beta-, and gamma-isomers), glutamine, hydroxyproline, taurine, norvaline, sarcosine, salts thereof, and mixtures thereof. Also included are N-alkyl C1-C3 derivatives and N-acylated C1-C3 derivatives of these amino acids, as well as mixtures of any amino acid or derivatives thereof. Preferred complexing amino acids that can be included in cyclodextrins to increase water structure and cellular hydration include L-arginine, L-lysine, N-methyllysine, and L-carnitine.

[0063] Non-limiting examples of vitamins include nicotinamide (vitamin B3), niacinamide, niacin, pyridoxal hydrochloride (vitamin B6), ascorbic acid, edible ascorbic acid esters, riboflavin, pyridoxine, thiamine, vitamin B9, folic acid, folate, pteroyl-L-glutamic acid, pteroyl-L-glutamate, salts thereof, and mixtures thereof. Preferred vitamins that can be included in the cyclodextrin to increase water structure and cellular hydration include nicotinamide and niacinamide.

[0064] Non-limiting examples of flavorings include apple, apricot, banana, grape, black currant, raspberry, peach, pear, pineapple, plum, orange, and vanilla flavorings. Examples of flavor-related compounds include butyl acetate, butyl isovalerate, allyl butyrate, amyl valerate, ethyl acetate, ethyl valerate, amyl acetate, maltol, isoamyl acetate, ethyl maltol, isomaltol, diacetyl, ethyl propionate, methyl anthranilate, methyl butyrate, pentyl butyrate, and pentyl pentanoate. Flavorings are added to the selected cyclodextrin component at a concentration of about 10 to 800 M. -1 , preferably 30 to 150M -1 , more preferably 40 to 100 M -1 The binding constant can be chosen to be weakly bound, with a binding constant in the range of

[0065] Non-limiting examples of other flavor improving ingredients can include polyol additives such as erythritol, maltitol, mannitol, sorbitol, lactitol, xylitol, inositol, isomalt, propylene glycol, glycerol (glycerin), threitol, galactitol, palatinose, reduced isomaltooligosaccharides, reduced xylooligosaccharides, reduced gentiooligosaccharides, reduced maltose syrup, and reduced glucose syrup.

[0066] Non-limiting examples of colorants include colorants that are known to be highly water-soluble and have low lipophilicity.Examples of colorants with these properties are betalains, such as betacyanins and betaxanthins, including vulgaxanthin, miraxanthin, portulaxanthin and indicaxanthin; anthocyanidins, such as aurantidin, cyanidin, delphinidin, europinidin, luteolinidin, pelargonidin, malvidin, peonidin, petunidin and rosinidin, and all the corresponding anthocyanins (or glucosides) of these anthocyanidins; and phenolic curcuminoids, such as turmeric colorants, including curcumin, demethoxycurcumin and bisdemethoxycurcumin.

[0067] All of the above examples of amino acids, vitamins, flavorings and related compounds may be in appropriate salt or hydrated form.

[0068] The complexing agent can be selected to form an inclusion complex with the selected clathrate component. The complexing agent can bind to the clathrate component as a guest molecule in the cavity of the clathrate molecule and / or can form so-called outer sphere complexes in which the selected weak complexing agent is bound to the clathrate molecule at a position at or near the edge of the clathrate. For example, the selected weak complexing agent can be bound to the cyclodextrin molecule at or near the primary and / or secondary hydroxyl groups at the edge of the cyclodextrin ring. Some complexing agents that form outer sphere complexes with the selected cyclodextrin can reduce or prevent the self-aggregation of dissolved, hydrated cyclodextrin molecules by shielding the intermolecular hydrogen bonds that form between two adjacent cyclodextrin molecules in water.

[0069] When the carbohydrate clathrate composition is a solid, the complexing agent may be present in a concentration range of about 1-50% w / w. Preferably, the complexing agent may be present in a concentration range of about 1-40% w / w or about 1-25% w / w. More preferably, the complexing agent may be present in a concentration range of about 5-15% w / w.

[0070] When the carbohydrate clathrate composition is in the form of a water-containing beverage, the complexing agent may be present in a concentration range of about 0.1-25% w / v or about 1-20% w / v. Preferably, the complexing agent may be present in a concentration range of about 1-15% w / v or about 1-10% w / v or about 3-8% w / v. More preferably, the complexing agent may be present in a concentration range of about 5-8% w / v.

[0071] III. Inclusion Complexes As mentioned above, the inclusion complex may comprise a clathrate host molecule complexed with one or more complexing agents. In the form of a solid product, such as a solid powder or tablet, the inclusion complex exhibits some unique properties compared to a solid composition that essentially contains the same components but without the formation of a preliminary inclusion complex. An inclusion complex is essentially a chemical entity with non-covalent hydrogen bonds formed between the clathrate molecule and a weak complexing agent molecule. In its solid state, the inclusion complex has the potential to dissociate into the clathrate component for increasing water structure and the complexing agent that can further increase water structure or provide other beneficial properties, such as nutrition or flavor, when the inclusion complex is introduced into an aqueous environment, such as when dissolved in a water-containing beverage or when ingested.

[0072] In the form of a solid product, the clathrate components and the one or more complexing agents may be substantially in the form of an inclusion complex as described above. It is preferred that more than about 25% of the clathrate components are complexed with the one or more complexing agents in the form of an inclusion complex. It is increasingly preferred that more than 35%, more than 45%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, and more than 95% of the clathrate components are complexed, in that order.

[0073] IV. Carbonate-forming components Some clathrate compositions may include carbonation-forming components that, when dissolved in an aqueous environment, produce carbonation, or effervescence, which can advantageously inhibit self-aggregation of the clathrate molecules, thereby structuring the water and increasing the surface area of ​​the clathrate for increased cellular hydration.

[0074] Non-limiting examples of carbonation forming components include sodium carbonate, sodium bicarbonate, potassium carbonate and potassium bicarbonate. Preferred carbonation forming components include sodium carbonate and sodium bicarbonate.

[0075] When the carbohydrate clathrate composition is in a solid form, the carbonation forming component may be present in a concentration range of about 1-60% w / w or about 5-60% w / w. Preferably, the carbonation forming component may be present in a concentration range of about 5-45% w / w or about 10-45% w / w. More preferably, the carbonation forming component may be present in a concentration range of about 10-15% w / w.

[0076] When the carbohydrate clathrate composition is in the form of an aqueous beverage, the carbonation component may be present in a concentration range of about 1-30% w / v or about 1-25% w / v. Preferably, the carbonation component may be present in a concentration range of about 2-15% w / v or about 2-10% w / v. More preferably, the carbonation component may be present in a concentration range of about 2-5% w / v.

[0077] V. Other Ingredients Some compositions may further include other ingredients that affect the flavor and / or nutritional value of the composition. These additional ingredients may include, but are not limited to, one or more of the following: flavor additives, nutritional ingredients, and / or various hydroxyl acids that act as clathrate aggregation inhibitors in the formulation. Non-limiting examples of such other ingredients may include citric acid, ascorbic acid, sodium chloride, potassium chloride, sodium sulfate, potassium citrate, europium chloride (EuCl3), gadolinium chloride (GdCl3), terbium chloride (TbCl3), magnesium sulfate, alum, magnesium chloride, maltodextrin, mono-, di-, tri-basic sodium or potassium salts of phosphoric acid (e.g., inorganic phosphates), hydrochloride salts (e.g., inorganic chlorides), sodium hydrogen sulfate. Non-limiting examples of hydroxyl acids that prevent aggregation of cyclodextrin may include isocitrate, citric acid, tartaric acid, malic acid, threonic acid, their salts, and mixtures thereof. These hydroxyl acids may also exhibit some nutritional benefits.Other non-limiting examples of additional optional ingredients, such as flavor additives, that can be used include suitable organic salts, such as choline chloride, sodium alginate, sodium glucoheptonate, sodium gluconate, potassium gluconate, guanidine hydrochloride, glucosamine hydrochloride, amiloride hydrochloride, monosodium glutamate (MSG), adenosine monophosphate, magnesium gluconate, potassium tartrate (monohydrate), and sodium tartrate (dihydrate).

[0078] Preferred other ingredients can include, for example, citric acid, ascorbic acid, and maltodextrin.

[0079] When the carbohydrate clathrate composition is in a solid form, the one or more other components may each be present in a concentration range of about 1-30% w / w or about 1-25% w / w. Preferably, the one or more other components may each be present in a concentration range of about 1-20% w / w or about 1-15% w / w. More preferably, the one or more other components may each be present in a concentration range of about 2-5% w / w.

[0080] When the carbohydrate clathrate composition is in the form of a water-containing beverage, the one or more other ingredients may be present in a concentration range of about 1-20% w / v or about 1-15% w / v. Preferably, the one or more other ingredients may be present in a concentration range of about 1-10% w / v or about 1-5% w / v. More preferably, the one or more other ingredients may be present in a concentration range of about 1-3% w / v.

[0081] VI. Ratio of ingredients In addition to the above discussion of the types and amounts of the various components that may be used in the carbohydrate clathrate compositions disclosed herein, it is also noted that the relative amounts of these components may be discussed as well. The weight ratio of clathrate component to complexing agent may be within the range of about 5:1 to 1:10, and more preferably within the range of about 2:1 to 1:5. The ratio may be within the range of about 2:1 to 1:2, more preferably, within the range of about 1:1 to 1:2.

[0082] With respect to other possible ingredients, such as flavor ingredients, carbonation-forming ingredients, and other ingredients mentioned above, the weight ratio of the clathrate component to each of the other ingredients may independently be within the range of about 25:1 to 1:25, or about 10:1 to 1:10, or about 5:1 to 1:5, or in some cases about 2:1 to 1:2, as well as 1:1.

[0083] VII. Preferred Embodiments The preferred embodiments of the carbohydrate clathrate compositions disclosed herein are provided by way of example and are not intended to limit the scope of the disclosure in any way. EXAMPLES

[0084] Effect of cyclodextrins on the molecular dynamics of water structure. The simulated water-solvated cyclodextrin molecular system was modeled using HyperChem® 5.11 software (HyperCube Inc, Gainesville, FL) with input parameters taken from the single crystal analysis of cyclohepta-amylose dodecahydrate clathrate (or β-cyclodextrin) reported by Lindner and Saenger (see Carbohydr. Res., 99:103, 1982) in a periodic solvent box of water (3.1x3.1x3.1 nm 2 ) containing a total of 984 water molecules. 3 ) was used to create the model. Molecular transformations and atom types were adjusted and properly formatted using TinkerFFE 4.2 (TINKER Software Tools for Molecular Design, version 5.0, Jay William Ponder, Washington University, St. Louis, MO). Molecular mechanics and dynamics calculations were performed using Tinker 5.0 software after preliminary optimization using the truncated Newton-Raphson method with the Linux x86-64 operating system (Slamd 64 v12.2).

[0085] Molecular dynamics simulations were performed using MM3 Force Field molecular mechanics software at a constant temperature (298 K) for 120 picoseconds (psec), with steps of 0.1 femtoseconds (fsec). Records were generated by dumping intermediate structures every 100,000 steps (equivalent to an elapsed time of 10 psec).

[0086] Observations: At time zero of each simulation, the standard water solvent box contained one β-cyclodextrin clathrate molecule and a population of 984 uniformly distributed water molecules. Figures 4 and 5 show a representation of the central portion of the solvent box at a particular elapsed time during one representative simulation. It will be appreciated that the positions and orientations of the water molecules are shown as (bent) rods, and the β-cyclodextrin is shown as a van der Waals surface. Furthermore, it will be appreciated that Figures 4 and 5 show the volume of the solvent box, thus compressing the three-dimensional molecular distribution into two dimensions.

[0087] Figure 4 shows the central part of the solvent box at 1 psec of simulation time. In particular, at 1 psec of simulation time, the water molecules immediately next to β-cyclodextrin have acquired relatively static (stable) positions through hydrogen bonding with the cyclodextrin. Such water molecules can be referred to as the first hydration layer. However, the distribution of most of the water molecules in the solvent box remains generally similar to the initial unstructured distribution (1 psec earlier).

[0088] Figure 5 shows the simulation after an elapsed time of 1000 psec (i.e., 1 nsec). At 1000 psec, the water molecules immediately adjacent to β-cyclodextrin continue to occupy relatively static (stable) positions. However, compared to 1 psec (Figure 4), the water molecules beyond the first hydration layer have acquired a more open microstructure.

[0089] The differences in water structure can be more easily observed without the perspective-shaded illustrations included in Figures 4 and 5. Figure 6 shows an alternative view of the distribution of water molecules shown in Figures 4 (left side, labeled 1 psec) and 5 (right side, labeled 1000 psec), which was created by the following method: the image files of Figures 4 and 5 with 256 tone levels (8 bits) were opened in Photoshop 9.0 (Adobe, Inc), scaled to 300 dpi, and thresholded at tone level 207; the images were cropped using the circle selection tool to make the outer annulus diameters equal, the corners of the outer squares filled with black (tone level 0), and then cropped further to make the interior of the annulus black, which contains almost no cyclodextrin molecules. The outer and inner dimensions of the annulus were applied equally to the images being compared. The resulting thresholded representation qualitatively indicates that the water molecules surrounding the central (closed) cyclodextrin molecule at 1000 psec have a more open and congruent structure (eg, right panel of FIG. 6).

[0090] To quantitatively evaluate the changes in water microstructure shown in Figures 4-6, molecular density was estimated by measuring the open paths through the indicated volumes, a method similar to mean free path analysis, in which the mean free path in a defined volume of a molecular substance is inversely related to the density of the molecules. In particular, open paths between water molecules are indicated by white pixel elements, and the number of open paths in a volume is easily quantified using the histogram tool in Photoshop 9.0 to count the number of white pixel elements. Applied to the panel in Figure 6, a measured 2% increase was calculated for open paths at 1000 psec elapsed time compared to open paths at 1 psec elapsed time. For comparison, freezing pure water reduced the density by 9%. Since path length is inversely proportional to molecular density, the analysis shows that dissolved cyclodextrin reduces the density of the aqueous solution by increasing the organization of water molecules.

[0091] In summary, the results show that rapid (psec) hydrogen bonding adhesion between the hydroxyls on the outer surface of β-cyclodextrin and water molecules is followed by a slower (nanosecond) propagation of water molecular reorientation throughout the solvent box, resulting in a more open water structure. The measurements further show that cyclodextrin can sufficiently increase hydrogen bonding between water molecules around the water volume to reduce the density of water. EXAMPLES

[0092] Effect of Cyclodextrin Additives on Water Binding Detected by IR Spectroscopy. For physical microstructural tests related to the detection of water, water-sugar interactions, and the effect of sugars on increasing and decreasing water structure, infrared (IR) spectroscopy, and in particular near-infrared (NIR) spectroscopy, as reported, for example, by Segtan et al. (see Anal. Chem. 2001;73, 3153-3161) and R. Giangiacomo (see Food Chemistry, 2006, 96.3. 371-379), was used preferentially.

[0093] The hydration binding energies in pure water and in solutions of the same water containing cyclodextrin compounds were assayed using IR spectroscopy in the near- and mid-infrared regions. A cuvette with a short optical length was used to minimize attenuation due to water absorption in order to record a linear signal throughout the entire wavelength range.

[0094] Spectra in the NIR region were registered on a FOSS NIR System, Inc. 6500 spectrometer and Sample Transport Module (STM) using a 1 mm-es cuvette. Transmission spectra were collected from 1100 to 2498 nm using a lead sulfide (PbS) detector and Vision2.51 software (2001; FOSS NIRSystems, Inc.).

[0095] Spectra were obtained from 2500 to 15385 nm (4000–650 cm) using a Perkin-Elmer Spectrum 400 FT-NIR / FT-IR spectrometer and a UATR (Universal Attenuated Total Reflectance; ZnSe-diamond crystal, 1× flat-top plate) sample handling unit. -1 Measurements were performed at 24 °C using a triglycine sulfate (TGS) detector and Spectrum ES 6.3.2 software (PerkinElmer, 2008).

[0096] Three water samples were used in this study. The first water sample (USA I) was obtained from the United States and was purified by reverse osmosis, carbon filtration, exposure to ultraviolet light, membrane filtration at 0.2 micron, absolute filtration type, and ozonation. The second water sample (USA II) was also obtained from the United States. The third water sample (BP I) was obtained from Budapest, Hungary. Capillary electrophoresis revealed similar ionic components with different concentrations between the three waters.

[0097] The following cyclodextrins were added to the above water samples at concentrations ranging from 0.1% to 5% w / v: α-Cyclodextrin (αCD, also called ACD), Lot. No. CYL-2322. β-Cyclodextrin (βCD, also called BCD), Lot. No. CYL-2518 / 2. γ-Cyclodextrin (γCD, also called GCD), Lot. No. CYL-2323. 2-Hydroxypropyl-β-cyclodextrin (HPβCD, HPBCD), DS*=3.5, Lot. No. CYL-2232. 2-Hydroxypropyl-γ-cyclodextrin (HPCD, HPGCD), DS*=4.8, Lot. No. CYL-2258. Carboxymethyl-β-cyclodextrin (CMBCD), Lot. No. CYL-2576. Quaternary ammonium-β-cyclodextrin (QABCD).

[0098] In some examples, various inclusion complexes were formed between cyclodextrin and complexing bioactive agents, including the amino acids L-arginine and L-carnitine and the vitamin niacinamide (also known as nicotinamide). All reagents were of analytical purity. In some examples, L-arginine and nicotinamide were added in free form or in cyclodextrin-complexed (molecularly entrapped) form to evaluate the independent and co-dependent activity of cyclodextrin with the bioactive agent. The concentrations of the above additives in free form and in the form of cyclodextrin inclusion complexes ranged from 0.1% to 5.0% w / v.

[0099] Observations: Figure 7 shows the second derivative NIR spectra for the wavelength range 900-1200 nm. The results show that the water-bonding interactions are significantly modified by the addition of QABCD, and even more significantly by the addition of CMBCD and HPBCD.

[0100] Figure 8 shows the second derivative NIR spectra shown from 1200 to 1500 nm. The results show that the water-bonding interactions are significantly modified by the addition of QABCD and HPBCD, and even more significantly by the addition of CMBCD.

[0101] Figure 9 shows the second derivative NIR spectra shown from 1620 to 1710 nm. The results show that the water-bonding interactions are significantly modified by the addition of CMBCD, QABCD, and HPBCD.

[0102] Figure 10 shows the second derivative NIR spectra shown from 2170 to 2370 nm. The results show that the water-bonding interactions are significantly modified by the addition of CMBCD and HPBCD, and even more significantly by the addition of QABCD.

[0103] As shown in Figures 7-10, the addition of cyclodextrin alters the molecular bonding interactions in the aqueous medium. In particular, referring to Figure 9, refined NIR differential spectra in the wavelength range of 1620-1770 nm show that the modifications related to carbon-hydrogen bonds involve the CH3-, CH2- and CH- groups of the cyclodextrin additive. The significant spectral changes that occurred in each of the cyclodextrin-treated water samples indicate that the cluster system in the bulk water was dominated by the altered microstructure of hydrogen bonds. This effect was greatest in the water samples treated with charged quaternary ammonium-β-cyclodextrin (QABCD), for example, as shown in Figures 9 and 10. EXAMPLES

[0104] Accelerating the germination of plant embryos Wheat seeds (Triticum aestivum) were germinated with USA I water, USA II water, and BP I water as described in Example 2. The germination rate with unsupplemented (control) water was compared to the germination rate with the same water variously supplemented with cyclodextrin components and / or bioactive agents as active components for cell hydration. For each condition, 10 seeds were continuously placed in contact with the water in a Petri dish and kept at 25° C. with a 12-hour light / dark cycle. Photometric images were recorded 1-6 days after sowing. The percentage of germinated seeds was calculated and compared as a function of time and the concentration of additive applied.

[0105] Water samples for germinating seeds were used alone without additives, or containing cyclodextrin, or clathrate inclusion complexes of cyclodextrin with L-arginine or nicotinamide (both from Sigma Chemical Co.; St. Louis, MO), or L-carnitine (Lonza AG; Switzerland). Additives were included at 0.1% (w / v) and 5.0% (w / v). Additive solutions were freshly prepared on the day of initiation of germination.

[0106] The parent cyclodextrins α-cyclodextrin (ACD), β-cyclodextrin (BCD), and γ-cyclodextrin (GCD) were obtained from Wacker Chemie (Munich, Germany). The following derivatized cyclodextrins were obtained from Cyclolab Ltd. (Budapest, Hungary): hydroxypropylated beta-cyclodextrin (DS~3) (HPBCD), carboxymethylated beta-cyclodextrin (DS~3.5) (CMBCD), 2-hydroxy-3-N,N,N-trimethylamino)propyl-β-cyclodextrin chloride (DS~3.6) (QABCD).

[0107] Observations Germination kinetics in control water and additive-amended water under identical conditions was quantified as the percentage of seeds with sprouts. Each determination consisted of 100 seeds for each parameter. The results are reported in Table 1 below and in Figures 11-13.

[0108] A) Cyclodextrin / L-Arg inclusion complex increases seed germination.

[0109] [Table 1]

[0110] Table 1 shows the comparative effects of 0.5% w / v α-CD, 0.5% w / v L-arginine (L-Arg), and 0.5% w / v α-CD / L-arginine inclusion complex, each dissolved in USA I water, on wheat seed germination. The results tabulated above show that the wheat seed germination rate is much higher in water containing 0.5% (w / v) inclusion complex between α-cyclodextrin and L-arginine (αCD / L-Arg inclusion complex) compared to pure water lacking any additives (control). Furthermore, the results in Table 1 show that the wheat seed germination rate in water containing the inclusion complex between α-cyclodextrin and L-arginine (αCD / L-Arg inclusion complex) is much higher compared to water containing 0.5% (w / v) α-cyclodextrin (αCD) alone as an additive, and also compared to water containing 0.5% (w / v) L-arginine (L-Arg) alone as an additive. Thus, the results show that the complex of α-cyclodextrin and L-arginine has a synergistic effect on increasing the seed germination rate that is not exhibited by any of the individual components of the complex used as a sole additive. The results in Table 1 are also shown in Figures 11 and 13.

[0111] B) Cyclodextrin / nicotinamide inclusion complexes increase seed germination.

[0112] [Table 2]

[0113] Table 2 shows the comparative effect of 0.5% w / v α-cyclodextrin, 0.5% w / v nicotinamide, and 0.5% w / v α-cyclodextrin / nicotinamide inclusion complex (αCD / nicotinamide inclusion complex), each dissolved in USA I water, on wheat seed germination. The results tabulated above show that the wheat seed germination rate is much higher in water containing the inclusion complex between α-cyclodextrin and nicotinamide (αCD / nicotinamide inclusion complex) compared to pure water lacking any additives (control). Furthermore, the results in Table 2 show that the wheat seed germination rate is much higher in water containing the inclusion complex between α-cyclodextrin and nicotinamide (αCD / nicotinamide inclusion complex) compared to water containing α-cyclodextrin (αCD) alone as an additive, and also compared to water containing nicotinamide alone as an additive. Thus, the results indicate that α-cyclodextrin and nicotinamide, when used as an inclusion complex, have synergistic bioactivity that significantly increases seed germination rates. No such bioactivity was demonstrated by any of the individual components of the complex used as the sole additive. The results in Table 2 are also shown in Figures 12 and 13.

[0114] C) Using USA II water and BP I water for germination, qualitatively similar results were obtained to those reported in Tables 1 and 2 and Figures 11-13. Thus, in particular, L-arginine-containing or nicotinamide-containing cyclodextrin inclusion complexes, when dissolved in USA II water or BP I water, respectively, significantly increased wheat seed germination rates as shown above using USA I water.

[0115] D) Shoot length (the rate at which shoots grow during germination) did not differ between conditions with statistically significant confidence intervals (P<0.05). This result indicates that cyclodextrins, particularly cyclodextrin inclusion complexes, can be selectively used as active ingredients in cell hydration to promote the rate of seed germination without necessarily also affecting the rate of shoot growth. EXAMPLES

[0116] Extending C. elegans life span in water with altered hydration C. elegans nematodes were grown in Petri dishes containing regular liquid nutrient medium prepared with USA I water (control) (described in Example 2) lacking any other additive components, or instead with the same water supplemented with parental α-cyclodextrin, β-cyclodextrin, or γ-cyclodextrin, and / or bioactive agents as active components for cell hydration. 50±3 worms were transferred to each dish. Each condition was repeated in triplicate. The experiment was repeated with USA II water and BP I water described in Example 2.

[0117] Additives to water: A. Parent α-cyclodextrin, β-cyclodextrin and γ-cyclodextrin added. B. L-arginine and nicotinamide added. C. Addition of an inclusion complex of cyclodextrin with L-arginine and nicotinamide.

[0118] Observations: The results recorded are shown in Tables 3-5 below and further illustrated in Figures 14-18.

[0119] [Table 3]

[0120] Table 3 reports the percentage of animals surviving to middle age (10 days), senior (15 days) and geriatric age (18 days) in media containing variable amounts of parent α-cyclodextrin, β-cyclodextrin and γ-cyclodextrin as active components for cell hydration. In this example, the parent cyclodextrin was added to the nutrient medium dissolved in USA I water at a concentration of 0.1% w / v.

[0121] Consistent with all previous studies, in this example, normal nematode animals survived for 2 weeks in normal medium. Each of the parent cyclodextrins significantly increased survival (percentage of survival) at high life span ages (10-15 days) of nematodes. Furthermore, α-cyclodextrin and γ-cyclodextrin significantly increased the number of animals surviving to old age, i.e., after 15 days. The results are also shown graphically in FIG. 14, which compares the cumulative percentage of animals surviving to 15 and 18 days in media containing each of the parent cyclodextrins as additives. The results indicate that parent cyclodextrins, particularly α-cyclodextrin and β-cyclodextrin, can be used as active ingredients in cell hydration to improve biological functions of living animals.

[0122] Biological mechanisms that support aging may include improvements in a broad range of cellular activities during aging, or by selectively activating cellular activity pathways that slow aging. Clathrate-induced water structures, hydration of cellular components, and increased diffusivity of bioactive cellular components, including inter- and intra-cellular signals, may all contribute to the overall effect of cyclodextrins on organismal survival.

[0123] [Table 4]

[0124] Table 4 reports the percentage of animals surviving to middle age (10 days), senior (15 days), and geriatric age (18 days) in media containing variable amounts of derivatized α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin as active components for cell hydration. In this example, derivatized cyclodextrins were added to the nutrient medium dissolved in USA I water at 0.1% w / v.

[0125] As listed in Table 4, HP-, carboxymethyl-, and quaternary ammonium-derivatives of β-cyclodextrin had only a minor effect on the initial survival of nematodes up to 10 days. In contrast, a significant increase in survival was observed at aged age (15 days), but not at aged age (18 days). The results are also shown graphically in FIG. 15, comparing the cumulative percentage of animals surviving up to 15 and 18 days in media containing each of the derivatized cyclodextrins as additives. The results indicate that derivatized cyclodextrins can be used as active ingredients in cell hydration to improve biological functions in living animals.

[0126] [Table 5]

[0127] Table 5 reports the percentage of animals surviving to middle age (10 days), senior (14 days), and geriatric age (18 days) in nutrient media dissolved in USA I water variably supplemented with cyclodextrin inclusion complex at 0.1% w / v as the active ingredient for cell hydration. In this example, the inclusion complex contained α-cyclodextrin and a bioactive agent, specifically L-arginine, L-carnitine, or niacinamide.

[0128] As in the previous example, nematode animals survived for 2 weeks in unsupplemented medium. The α-cyclodextrin complex with L-arginine and niacinamide more than doubled the survival of nematodes at advanced ages (14 days) and further enabled a small but significant number of animals to survive to advanced ages where no animals survived in nutrient medium alone. In contrast, the α-cyclodextrin complex with L-carnitine had little or no significant effect on nematode survival. Similarly, L-arginine and nicotinamide added to the medium alone, without α-cyclodextrin, had little effect on nematode survival. The results are also shown graphically in Figure 16, comparing the cumulative percentage of animals surviving up to 14 and 18 days in medium containing each cyclodextrin inclusion complex as an additive. The results show that α-cyclodextrin inclusion complexes, especially those with L-arginine and niacinamide, can be used as active ingredients in cellular hydration to improve biological functions in living animals.

[0129] As further shown in FIG. 17, inclusion complexes of α-cyclodextrin and L-arginine (data series A; 1:1 complex, dissolved at 0.1% w / v in medium made with USA I water) and α-cyclodextrin and niacinamide (data series B; 1:1 complex, dissolved at 0.1% w / v in medium made with USA I water) can reduce nematode mortality. FIG. 17 shows the number of dead animals each day for each medium condition, with the control data series being medium made with USA I water, lacking further additives or supplements. The results indicate that complexed forms of α-cyclodextrin can be used as an active component of cell hydration to delay the death of live animals.

[0130] Figure 18 instead presents the data of Figure 17 as survival curves for animals growing in regular medium with USA I water (control), or medium supplemented with a 1:1 inclusion complex of α-cyclodextrin and L-arginine (sample 1); or medium supplemented with a 1:1 inclusion complex of cyclodextrin and niacinamide (sample 2). Thus, the delayed death shown in Figure 17 results in survival to older ages, with the mean age of survival (50% survival) increasing from approximately 13 days in regular medium to approximately 14 days in medium containing cyclodextrin inclusion complex as the active component of cell hydration, representing an 8% increase in lifespan.

[0131] While the present invention has been shown and described with reference to the foregoing operational principles and preferred embodiments, it will be apparent to those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and the present invention is intended to embrace all such alternatives, modifications and variations that fall within the scope of the appended claims.

Claims

1. A composition for interacting with a biological cell system, comprising a bioactive molecule along with a biomolecular surface, a cellular component, and water molecules having a specific density, comprising: A composition comprising a biologically active component adapted to increase the activity of a biological cell-based system by increasing the hydration of one or more components of the cell system.

2. 2. The composition of claim 1, wherein the biologically active component comprises a first carbohydrate clathrate subcomponent that increases the hydrogen bonding structure of water.

3. The composition of claim 2 , wherein the biologically active component comprises a second solute subcomponent.

4. The composition of claim 3, wherein the second solute subcomponent is a bioactive agent.

5. The composition of claim 1 , wherein the biologically active component comprises an inclusion complex composed of a clathrate component and a complex-forming compound.

6. The composition of claim 5 , wherein the complexing compound is a bioactive agent.

7. 3. The composition of claim 2, wherein the clathrate subcomponent comprises a compound selected from the group consisting of amylose and cyclodextrin.

8. The composition of claim 2 , wherein the clathrate subcomponent comprises a cyclodextrin.

9. 9. The composition of claim 8, wherein the cyclodextrin is constructed to exhibit kosmotropic activity that increases water binding structure.

10. The composition of claim 9, wherein the cyclodextrin is constructed to cause an increase in hydrogen bonds between water molecules, which increases the hydration of biomolecular surfaces of the biological cell system, thereby changing interactions between cellular components of the biological cell system.

11. 10. The composition of claim 9, wherein the cyclodextrin is constructed to strengthen hydrogen bonds between water molecules, resulting in an open water structure with a lower specific density, and increasing the diffusion rate of the bioactive molecule.

12. 12. The composition of claim 11, further comprising the bioactive molecule selected from the group consisting of an enzyme, an enzyme substrate, a nutrient, a metabolite, a cytokine, a neurotransmitter, a hormone, an extracellular signal, an intracellular messenger, and a pharmacological agent.

13. 8. The composition of claim 7, wherein the cyclodextrin is selected from the group consisting of α-, β- and γ-cyclodextrin, methylated α-, β- and γ-cyclodextrin, 2-hydroxypropylated β-cyclodextrin, water soluble β-cyclodextrin polymers, partially acetylated α-, β- and γ-cyclodextrin, ethylated α-, β- and β-cyclodextrin, carboxyalkylated β-cyclodextrins, quaternary ammonium salts of α-, β- and γ-cyclodextrin, amylose (e.g., acetylated amylose), and mixtures thereof.

14. The composition of claim 7, further comprising a complexing agent, said clathrate subcomponent being capable of forming an inclusion complex with said complexing agent.

15. 15. The composition of claim 14, wherein the complexing agent is selected from the group consisting of amino acids, vitamins, flavors, odorants, and colorants.

16. 16. The composition of claim 15, wherein the clathrate subcomponent is selected from the group consisting of α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, 2-hydroxypropyl-cyclodextrin, carboxymethylated cyclodextrin, quaternary ammonium cyclodextrin, amylose, amylose derivatives, and mixtures thereof.

17. A composition for interacting with a biological cell system, comprising a bioactive molecule along with a biomolecular surface, a cellular component, and water molecules having a specific density, comprising: a first carbohydrate clathrate component capable of increasing the hydrogen bond structure of water; a second solute subcomponent; and Including, A composition wherein said clathrate component and said solute component are capable of increasing the activity of a biological cell system by increasing the hydration of one or more components of the cell system.

18. 18. The composition of claim 17, wherein the clathrate subcomponent comprises a compound selected from the group consisting of amylose and cyclodextrin.

19. 20. The composition of claim 17, wherein the second solute subcomponent is a bioactive agent.

20. 18. The composition of claim 17, further comprising a complex-forming compound, wherein the clathrate compound and the complex-forming compound form an inclusion complex.

21. 21. The composition of claim 20, further comprising the bioactive molecule selected from the group consisting of an enzyme, an enzyme substrate, a nutrient, a metabolite, a cytokine, a neurotransmitter, a hormone, an extracellular signal, an intracellular messenger, and a pharmacological agent.

22. 20. The composition of claim 18, wherein the clathrate component is a cyclodextrin selected from the group consisting of α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, methylated β-cyclodextrin, 2-hydroxypropylated β-cyclodextrin, water soluble β-cyclodextrin polymers, partially acetylated α-, β- and γ-cyclodextrin, ethylated α-, β- and γ-cyclodextrin, carboxyalkylated β-cyclodextrins, quaternary ammonium salts of α-, β- and γ-cyclodextrin, amylose (e.g., acetylated amylose), and mixtures thereof.

23. 23. The composition of claim 22, wherein the complexing agent is selected from the group consisting of amino acids, vitamins, flavorings, odorants, and colorants.

24. 20. The composition of claim 18, wherein the clathrate subcomponent is selected from the group consisting of α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, 2-hydroxypropyl-cyclodextrin, carboxymethylated cyclodextrin, quaternary ammonium cyclodextrin, amylose, amylose derivatives, and mixtures thereof.

25. A beverage that interacts with a biological cell system that includes a bioactive molecule along with a biomolecular surface, cellular components, and water molecules having a specific density, a water-containing component selected from the group consisting of non-carbonated liquids and carbonated liquids; a first carbohydrate clathrate component capable of increasing the hydrogen bond structure of water; The second solute subcomponent and Including, A beverage in which the clathrate component and the solute component are capable of increasing the activity of the biological cell system by increasing the hydration of one or more components of the cell system.

26. 26. The beverage of claim 25, wherein the clathrate subcomponents comprise a compound selected from the group consisting of amylose and cyclodextrin.

27. 27. The beverage of claim 26, wherein the second solute subcomponent is a bioactive agent.

28. 28. The beverage of claim 27, further comprising a complex-forming compound, wherein the clathrate compound and the complex-forming compound form an inclusion complex.

29. 29. The beverage of claim 28, further comprising a flavoring selected from the group consisting of apple, apricot, banana, grape, black currant, raspberry, peach, pear, pineapple, plum, orange, and vanilla flavors, and examples of flavor-related compounds include butyl acetate, butyl isovalerate, allyl butyrate, amyl valerate, ethyl acetate, ethyl valerate, amyl acetate, maltol, isoamyl acetate, ethyl maltol, isomaltol, diacetyl, ethyl propionate, methyl anthranilate, methyl butyrate, pentyl butyrate, pentyl pentanoate, erythritol, maltitol, mannitol, sorbitol, lactitol, xylitol, inositol, isomalt, propylene glycol, glycerol (glycerin), threitol, galactitol, palatinose, reduced isomaltooligosaccharides, reduced xylooligosaccharides, reduced gentiooligosaccharides, reduced maltose syrup, and reduced glucose syrup.

30. 29. The beverage of claim 28, further comprising a colorant selected from the group consisting of betalains, betacyanins, betaxanthins, vulgaxanthins, miraxanthins, portulaxanthins, indicaxanthins, anthocyanidins, aurantidinins, cyanidins, delphinidins, europinidins, luteoridinins, pelargonidins, malvidins, peonidins, petunidins, rosinidins, glucosides of the corresponding anthocyanins or anthocyanidins, turmeric colorants, phenolic curcuminoids, curcumin, demethoxycurcumin, and bisdemethoxycurcumin.

31. Nicotinamide (vitamin B 3 ), niacinamide, niacin, pyridoxal hydrochloride (vitamin B 6 ), ascorbic acid, edible ascorbate, riboflavin, pyridoxine, thiamine, vitamin B 9 30. The beverage of claim 28, further comprising a vitamin selected from the group consisting of folic acid, folate, pteroyl-L-glutamic acid, pteroyl-L-glutamate, salts thereof, and mixtures thereof.

32. 30. The beverage of claim 28, further comprising an amino acid or amine.

33. The amino acids are aspartic acid, arginine, glycine, glutamic acid, proline, threonine, theanine, cysteine, cystine, alanine, valine, tyrosine, leucine, isoleucine, asparagine, serine, lysine, histidine, ornithine, methionine, carnitine, aminobutyric acid (alpha-, beta-, and gamma-isomers), glutamine, hydroxyproline, taurine, norvaline, sarcosine, salts thereof, mixtures thereof, and N-alkylated C-amino acids of these amino acids. 1 ~C 3 and N-acylated C 1 ~C 3 33. The beverage of claim 32, wherein the amino acid is selected from the group consisting of any amino acid, derivative thereof, and a mixture of any amino acid or derivative thereof.

34. 1. A method for improving cellular hydration in an animal, such as a human, having a body in which physiological conditions occur and in which there are biological cellular systems that contain biomolecular surfaces, cellular components and water molecules with a specific density as well as biologically active molecules, comprising: preparing a beverage by dissolving an inclusion complex formed by a carbohydrate clathrate component and a complexing agent capable of dissociating from said carbohydrate clathrate component under physiological conditions; orally ingesting the beverage to an animal, whereupon the carbohydrate clathrate components dissociate from the complexing agent and the strength, extent, and kinetics of hydrogen-bonded water structures on the biomolecular surface of cells are altered; The method includes:

35. 35. The method of claim 34, further comprising the step of selecting the clathrate component from the group consisting of amylose and cyclodextrin.

36. 36. The method of claim 35, wherein the selecting step comprises selecting the cyclodextrin from the group consisting of α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, methylated β-cyclodextrin, 2-hydroxypropylated β-cyclodextrin, water soluble β-cyclodextrin polymers, partially acetylated α-, β- and γ-cyclodextrin, ethylated α-, β- and β-cyclodextrin, carboxyalkylated β-cyclodextrins, quaternary ammonium salts of α-, β- and γ-cyclodextrin, amylose (e.g., acetylated amylose), and mixtures thereof.

37. 35. The method of claim 34, further comprising the step of selecting the complexing agent from the group consisting of amino acids, vitamins, flavorings, odorants, and colorants.

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