Method for producing and testing functional water with enhanced cell membrane permeability and its application

Abstract

A method for making functional water with enhanced cell membrane permeability is provided. The method includes preparing a ceramic by melting natural clay and iron-containing sand, immersing the ceramic in water at a predetermined mass ratio of ceramic to water at a predetermined temperature for a first predetermined time, and sonicating the ceramic in water at a predetermined sonication power at a predetermined temperature for a second predetermined time. The sonicated water is then passed through a column packed with ceramic at a predetermined temperature to obtain functional water. The water flows through the column at a predetermined mass ratio of water flow to ceramic or a third predetermined time for a unit volume of water to pass through a unit mass of ceramic. The ceramic induces a structural change in at least some of the water molecules in the water.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED PATENT APPLICATIONS This PCT application claims priority under 35 U.S.C. § 119(e) to and the benefit of U.S. Provisional Patent Application No. 63 / 322,378, filed March 22, 2022, by Yungfeng Lu, and entitled “MAKING METHODS AND APPLICATIONS OF FUNCTIONAL WATER WITH ENHANCED CELL-PENETRATING CAPABILITY,” which is hereby incorporated by reference in its entirety.

[0002] The present invention relates generally to water, and in particular to a method for producing and testing functional water having enhanced cell membrane permeability, and its applications. [Background technology]

[0003] The description of the background art provided herein is intended to generally present the context of the present invention. The subject matter discussed in the Background Art section should not be presumed to be prior art merely as a result of its mention in the Background Art section. Similarly, the problems mentioned in the Background Art section, or problems related to the subject matter of the Background Art section, should not be presumed to have been previously recognized in the prior art. The subject matter in the Background Art section merely represents different approaches, which may also be inventions in themselves.

[0004] Water is a vital component of all living organisms. Humans are mostly water, accounting for about 75 percent of body weight in infants, 50-60 percent in adult men and women, and as low as 45 percent in the elderly. All chemical reactions and physiological processes, from the uptake and processing of nutrients to the elimination of metabolic waste products, occur in aqueous solutions. The average adult takes in about 2500 mL of aqueous fluids per day, mostly through the digestive tract, and produces 230 mL through metabolism. Approximately the same volume of water leaves the body each day, mostly in urine, via evaporation from the skin, and from air exhaled from the lungs.

[0005] According to the Centers for Disease Control and Prevention (CDC), drinking water can prevent dehydration, a condition that can lead to unclear judgment, mood swings, overheating of the body, constipation, and kidney stones. Besides water, there are many beverage options that can be part of a healthy diet. In particular, functional water is water that has been enriched with supplements such as vitamins, minerals, fruits, and herbs that provide additional health and other functional benefits. The market for functional water has grown rapidly over the past few decades as a result of consumer preferences shifting from sugary and carbonated drinks to water, and as a result of the desire for more than just hydration. In particular, alkaline water, electrolytic reduced water, and high-concentration hydrogen water have attracted great interest due to their potential anti-inflammatory and antioxidant properties. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Tadao Kozumi and Yoshichika Kitagawa, “water structure changes induced by ceramics can be detected by increased permeability through aquaporin”, Biochemistry and Biophysics Reports (5, 2016, pp. 353-358) Summary of the Invention [Problem to be solved by the invention]

[0007] Thus, there exists a heretofore unmet need in the art to address the aforementioned deficiencies and inadequacies. [Means for solving the problem]

[0008] In one aspect, the present invention relates to a method for making aquaporin water with enhanced cell membrane permeability. In one embodiment, the method includes the steps of preparing ceramic by melting natural clay and iron-containing sand, immersing the ceramic in water at a predetermined mass ratio of ceramic to water at a predetermined temperature for a first predetermined time, sonicating the ceramic in water at a predetermined sonication power at a predetermined temperature for a second predetermined time, and flowing the sonicated water through a column packed with ceramic at a predetermined temperature to obtain aquaporin water, where the water flows through the column at a predetermined mass ratio of water flow to ceramic or for a third predetermined time for a unit volume of water to pass through a unit mass of ceramic. The ceramic induces a structural change in at least some of the water molecules in the water.

[0009] In one embodiment, the water molecules of the aquaporin water have an HOH bond angle α, and the HOH bond angle α1 of the altered water molecule is greater than the normal HOH bond angle α0 of the water molecule before treatment. In one embodiment, the HOH bond angle α1 is about 120°, and the normal bond angle α0 is 104.45°.

[0010] In one embodiment, the method further includes steps of preparing a culture medium by adding a growth-enhancing substance to the aquaporin water and filtering the aquaporin water to remove sediments and bacteria in the aquaporin water, preparing a control medium by adding a growth-enhancing substance to deionized water, adding live subject cells to the culture medium and control medium, respectively, after preparing the culture medium and the control medium, and verifying multiple capabilities of the aquaporin water by measuring the cells in the culture medium and the control medium using a corresponding kit to determine the capabilities of the aquaporin water.

[0011] In one embodiment, the capabilities of aquaporin water include increasing cell viability, protecting cells from oxidative damage, maintaining cell telomere length, and down-regulating survival genes associated with inflammation and aging.

[0012] In one embodiment, the ceramic is a Tadami ceramic, the predetermined mass ratio of ceramic to water is 0.01-0.1 grams / ml of ceramic to water, the predetermined temperature is room temperature, the first predetermined time is 18-36 hours, the predetermined sonication power is 100 watts, the second predetermined time is 3-7.5 minutes, the predetermined mass ratio of water flow to ceramic is 0.01 liters / minute / gram, and the third predetermined time is 0.01 seconds for 1 liter of water to pass through 1 gram of ceramic.

[0013] In one embodiment, the growth enhancers include Minimum Essential Medium (MEM) as the base medium, and fetal bovine serum (FBS) and penicillin / streptomycin (P / S) as supplements.

[0014] Another aspect of the present invention relates to aquaporin water prepared by the method disclosed above. In a particular embodiment, the water molecules of the aquaporin water have an HOH bond angle α, and the HOH bond angle α1 of the changed water molecule is larger than the normal HOH bond angle α0 of the water molecule before the treatment. In one embodiment, the HOH bond angle α1 is about 120°, and the normal bond angle α0 is 104.45°.

[0015] The aquaporin water prepared by the method disclosed above can be applied in various applications. For example, a further aspect relates to a method for enhancing removal of metabolic waste products in a living subject, comprising preparing aquaporin water as described above and providing the aquaporin water to the living subject. In certain embodiments, the aquaporin water is configured to enhance removal of metabolic waste products from cells of the living subject. In one embodiment, the metabolic waste products include hydrogen peroxide.

[0016] Further aspects relate to methods comprising preparing aquaporin water as described above and providing the aquaporin water to a living subject, hi certain embodiments, the aquaporin water may be configured to be anti-inflammatory, to increase viability of cells of a living subject, to maintain telomere length of cells of a living subject, and / or to downregulate genes in a living subject.

[0017] In another aspect, the present invention relates to a method for making functional water with enhanced cell membrane permeability. In one embodiment, the method includes the steps of preparing ceramic by melting natural clay and iron-containing sand, immersing the ceramic in water at a predetermined mass ratio of ceramic to water at a predetermined temperature for a first predetermined time to obtain functional water, sonicating the ceramic in water at a predetermined sonication power at a predetermined temperature for a second predetermined time, and flowing the sonicated water through a column packed with ceramic at a predetermined temperature to obtain functional water, wherein the water flows through the column at a predetermined mass ratio of water flow to ceramic or for a third predetermined time for a unit volume of water to pass through a unit mass of ceramic. The ceramic induces structural changes in at least some of the water molecules of the water.

[0018] In one embodiment, the method further comprises confirming the capacity of the functional water by adding live subject cells to the functional water as a culture medium, adding live subject cells to deionized water as a control medium, and measuring the cells in the culture medium and the control medium using a corresponding kit to determine the capacity of the functional water. In one embodiment, the capacity of the functional water includes increasing cell viability, protecting cells from oxidative damage, maintaining cell telomere length, and down-regulating genes of the live subject associated with inflammation and aging.

[0019] In one embodiment, the method further comprises adding a growth enhancing substance to the culture medium before adding the cells to the functional water, and filtering the culture medium to remove sediments and bacteria in the functional water. In one embodiment, the growth enhancing substance includes Minimum Essential Medium (MEM) as the basal medium, and Fetal Bovine Serum (FBS) and Penicillin / Streptomycin (P / S) as supplements.

[0020] In one embodiment, the ceramic is a Tadanoumi ceramic and the functional water is an aquaporin water. In one embodiment, the predetermined mass ratio of ceramic to water is 0.01-0.1 grams / ml of ceramic to water. In one embodiment, the predetermined temperature is room temperature, the first predetermined time is 18-36 hours, the predetermined sonication power is 100 watts, the second predetermined time is 3-7.5 minutes, the predetermined mass ratio of water flow to ceramic is 0.01 liters / minute / gram, and the third predetermined time is 0.01 seconds for 1 liter of water to pass through 1 gram of ceramic.

[0021] Another aspect of the present invention relates to aquaporin water prepared by the method disclosed above. In a particular embodiment, the water molecules of the aquaporin water have an HOH bond angle α, and the HOH bond angle α1 of the changed water molecule is larger than the normal HOH bond angle α0 of the water molecule before the treatment. In one embodiment, the HOH bond angle α1 is about 120°, and the normal bond angle α0 is 104.45°.

[0022] A further aspect of the present invention relates to an aqueous solution comprising the aquaporin water disclosed above, wherein the changed water molecules of the aqueous solution have a volume V1, the unchanged water molecules of the aqueous solution have a volume V0, and the aqueous solution has a volume V, where V=V1+V0 and V1 / V0>1.

[0023] The functional water prepared by the method disclosed above can be applied in various applications. For example, a further aspect relates to a method for enhancing removal of metabolic waste products in a living subject, comprising preparing functional water as described above and providing the functional water to the living subject. In certain embodiments, the functional water may be configured to enhance removal of metabolic waste products from cells of the living subject. In one embodiment, the metabolic waste products include hydrogen peroxide.

[0024] A further embodiment relates to an aqueous solution comprising a quantity of first water molecules in a first volume V1 having a first average HOH bond angle α1 between the two H-0 bonds of each of the first water molecules, and a quantity of second water molecules in a second volume V0 having a second average HOH bond angle α0 between the two H-0 bonds of each of the second water molecules.

[0025] In one embodiment, the first average HOH bond angle α1 is greater than the second average HOH bond angle α0. In one embodiment, the first average HOH bond angle α1 is 120° and the second average HOH bond angle α0 is about 104.45°.

[0026] In one embodiment, the aqueous solution has a volume V, where V=V1+V0 and V1 / V0>1.

[0027] These and other aspects of the present invention will become apparent from the following description of the preferred embodiments taken in conjunction with the following drawings, although variations and modifications of said aspects may be effected without departing from the spirit and scope of the novel concepts of the present invention.

[0028] The accompanying drawings illustrate one or more embodiments of the invention and, together with the written description, serve to explain the principles of the invention. Wherever possible, the same reference numbers are used throughout the drawings to refer to the same or like elements of an embodiment. [Brief description of the drawings]

[0029] [Figure 1] FIG. 2 is a flow chart of a method for producing functional water with enhanced cell membrane permeation ability according to a particular embodiment of the present invention. [Diagram 2] FIG. 2 shows a flow chart of a process for verifying the capabilities of functional water produced by the method shown in FIG. 1 in accordance with certain embodiments of the present invention. [Diagram 3] FIG. 1 is a schematic diagram illustrating the shape of a typical water molecule, in accordance with certain embodiments of the present invention. [Figure 4A]FIG. 2 shows a schematic and qualitative illustration of a potential relationship between the energy spectrum and the HOH bond angle of a water molecule according to a particular embodiment of the present invention, based on a proposed model. [Figure 4B] FIG. 1 shows a proposed three-dimensional model showing the volume and effective area of ​​a water molecule related to an HOH bond angle according to certain embodiments of the present invention. [Diagram 5] Figures showing the characteristics of AQP water with improved aquaporin permeability: (A) shows the relative AQP permeability (AQP pf) between AQP water made from distilled water (dw) and dw; (B) shows photographs of clay (top) and Tadanoumi ceramic (bottom); and (C) shows the proliferation of human skin cells in control medium and ceramic-treated medium. [Figure 6] 1A and 1B show (A) X-ray diffraction (XRD) pattern and (B) thermogravimetric analysis (TGA) of Tadanoumi ceramic according to certain embodiments of the present invention. [Figure 7] (A) Relative cell viability of AML-12 cultured in DI water and AQP water, and (B) relative cell death of HepG2 cultured in DI water and AQP water, with P value: *<0.05. [Figure 8] (A) Relative T / S ratios of cells cultured in media made from DI water or AQP water as determined by qPCR, P value: *<0.05, and (B) RTL ratios of cells cultured in media made from DI water or AQP water as determined by qPCR. [Figure 9] FIG. 1 shows the fold change in gene expression in cells cultured in AQP water versus cells cultured in control water as determined by RT-PCR. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] The present invention will now be described more fully hereinafter with reference to the accompanying drawings, in which exemplary embodiments of the invention are shown. The present invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. Like reference numerals refer to like elements throughout.

[0031] The terms used herein have their ordinary meaning in the art throughout, within the context of the present invention and in the specific context in which each term is used. Certain terms used to describe the present invention are discussed below or elsewhere in the specification to provide practitioners with further guidance regarding the description of the present invention. For convenience, certain terms may be highlighted, for example using italics and / or quotation marks. The use of highlighting does not affect the scope and meaning of the term. The scope and meaning of a term is the same in the same context whether it is highlighted or not. It is understood that the same thing may be described in more than one way. As a result, alternative words and synonyms may be used for any one or more of the terms discussed herein, and whether a term is detailed or discussed herein does not have any special significance. Synonyms of certain terms are described. The recitation of one or more synonyms does not exclude the use of other synonyms. The use of examples anywhere in this specification, including examples of any term discussed herein, is merely illustrative and in no way limits the scope and meaning of the present invention or of any exemplified term. Likewise, the present invention is not limited to the various embodiments shown herein.

[0032] As used herein and throughout the claims that follow, the meaning of "a," "an," and "the" will be understood to include plural references unless the context clearly indicates otherwise. Also, when an element is described as being "on" another element, it will be understood that the element may be directly on the other element, or intervening elements may be between the element and the other element. Conversely, when an element is described as being "directly on" another element, there are no intervening elements present. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0033] It will be understood that, although terms such as first, second, third, etc. may be used herein to describe various elements, components, regions, layers, and / or portions, these elements, components, regions, layers, and / or portions should not be limited by these terms. These terms are only used to distinguish one element, component, region, layer, or portion from another element, component, region, layer, or portion. Thus, a first element, component, region, layer, or portion discussed below may be described as a second element, component, region, layer, or portion without departing from the teachings of the present invention.

[0034] Furthermore, relative terms such as "lower" or "bottom" and "upper" or "top" may be used herein to describe the relationship of one element shown in a figure to another element. It will be understood that such relative terms encompass different orientations of the device in addition to the orientation shown in the figures. For example, if a device in one of the figures is flipped over, an element described as being on the "lower" side of the other element would be oriented on the "upper" side of the other element. The exemplary term "lower" may thus encompass both an orientation of "lower" and "upper", depending on the particular orientation of the figure. Similarly, if a device in one of the figures is flipped over, an element described as being "below" or "beneath" the other element would be oriented "above" the other element. The exemplary term "below" or "beneath" may thus encompass both an orientation of upper and lower.

[0035] It will be further understood that terms such as "comprises and / or comprising," or "includes and / or including," or "has and / or having," or "carry and / or carrying," or "contain and / or containing," or "involve and / or involving" are open ended, i.e., meant to include without limitation. As used herein, these terms specify the presence of a referenced feature, region, integer, step, operation, element, and / or component, but do not exclude the presence or addition of one or more other features, regions, integers, steps, operations, elements, components, and / or groups thereof.

[0036] Unless otherwise defined, all terms used herein (including technical and scientific terms) have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Terms such as those defined in commonly used dictionaries should be interpreted to have a meaning consistent with their meaning in the context of the relevant art and this specification, and will not be interpreted in an idealized or overly formal sense unless expressly defined in this specification.

[0037] As used herein, "around," "about," "approximately," or "nearly" generally means within 20 percent, preferably within 10 percent, and more preferably within 5 percent of a given value or range. Numerical quantities given herein are approximations, meaning that the terms "around," "about," "approximately," or "approximately" can be inferred unless expressly stated.

[0038] As used herein, the phrase "at least one of A, B, and C" should be construed to mean the logical (A or B or C) using a non-exclusive logical OR. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0039] The following description is merely exemplary in nature and is in no way intended to limit the invention, its application, or uses. The broad teachings of the invention can be implemented in various forms. Thus, while the invention includes certain embodiments, the true scope of the invention should not be limited thereto, since other modifications will become apparent upon reading the drawings, the specification, and the following claims. For clarity, the same reference numbers are used in the drawings to identify similar elements. It should be understood that one or more steps of a method can be performed in different orders (or simultaneously) without changing the principles of the invention.

[0040] As mentioned above, the market for functional water has grown rapidly over the past few decades. One type of functional water that has been produced can be aquaporin (AQP) water.

[0041] Aquaporins (AQPs): AQP water is a new class of functional water that speeds up the transport of water through cell membranes. Cells are the basic structural and functional units of life and contain a cytoplasm surrounded by a cell membrane. The cell membrane provides a protective barrier around the cell while regulating which substances or molecules can or cannot pass through it. Aquaporins regulate the transport of ions through the cell membrane, such as calcium ions (Ca 2+ ) channel, potassium ion (K + ) channel, sodium ion (Na + ) channel, and proton (H + Similar to ion channels such as ATP channels, the transport of water molecules, either individually or in clusters, through cell membranes is mediated through water channels, also called aquaporins, which are driven by osmotic pressure across the cell membrane. Aquaporins are integral membrane proteins present in all living organisms and form pores in the cell membrane to facilitate the transport of water between cells. The existence of water channels was speculated 60 years ago, and it was not until 1992 that the first aquaporin, aquaporin 1, was discovered by Peter Agre of Johns Hopkins University, who in 2003 was awarded the Nobel Prize in Chemistry for his pioneering work in the discovery of water channels.

[0042] Currently, 13 aquaporins (AQPO~12) have been identified in mammals and are conventionally classified into three subgroups: 1) orthodox AQPs (AQPO, 1, 2, 5, 6, and 8), which are primarily for water transport; 2) aquaglyceroporins (AQP3, 7, 9, and 10), which transport neutral solutes such as glycerol, urea, ammonia, and carbon dioxide in addition to water; and 3) superaquaporins (AQP11 and 12), whose details have yet to be elucidated. In addition, hydrogen peroxide (H2O2) and water (H2O) have similar dipole moments and hydrogen bonding strengths. Like water, hydrogen peroxide also transports through cell membranes via aquaporins, including AQP3, 8, 9, and 11, a new aquaporin subset named peroxyporins. It is important to note that hydrogen peroxide is toxic. Effective removal of hydrogen peroxide from the body is essential for reducing oxidative stress, anti-aging, and the prevention and treatment of many diseases.

[0043] AQP Water®. AQP water is a novel class of functional water made according to certain embodiments of the present invention. Compared with ordinary water or other functional waters, AQP water has the unique ability to allow water to pass through aquaporins more effectively and cross cell membranes, resulting in more rapid hydration and effective removal of compounds that are toxic to the human body, such as hydrogen peroxide.

[0044] A certain aspect of the present invention relates to a method for preparing functional water with enhanced cell membrane permeability. For example, Fig. 1 shows a flow chart of a method for preparing functional water with enhanced cell membrane permeability according to some embodiments of the present invention. It should be noted that unless otherwise stated in this disclosure, the steps of this method may be arranged in different orders, and therefore are not limited to the order shown in Fig. 1.

[0045] As shown in FIG. 1, in the method of making functional water, in process 110, ceramic is prepared by melting natural clay and iron-containing sand. In certain embodiments, the ceramic can also be Tadanoumi Ceramic®, which is discussed further below. Then, in process 120, the ceramic is immersed in water at a predetermined mass ratio of ceramic to water at a predetermined temperature for a predetermined time, and the ceramic in the water is sonicated at a predetermined sonication power at a predetermined temperature for a second predetermined time. During this process, the ceramic induces a structural change in at least one or some water molecules of the water, reducing the cluster size of the water. In certain embodiments, when the functional water is AQP water, the predetermined mass ratio of ceramic to water can be in the range of 0.01 to 0.1 grams / ml of ceramic to water. In one embodiment, the predetermined mass ratio of ceramic to water can be in the range of 0.05 to 0.1 grams / ml of ceramic to water. In one embodiment, the predetermined temperature is room temperature, the first predetermined time is 18-36 hours, the predetermined sonication power is 100 watts, and the second predetermined time is 3.0-7.5 minutes. For example, the predetermined time can be 24 hours and the second predetermined time can be 5 minutes.

[0046] Once the water has been sonicated, in process 130, the sonicated water is flowed through a column packed with ceramic (which is the same type of ceramic as that immersed in the water) at a predetermined temperature to obtain functional water. Specifically, the sonicated water flows through the column at a predetermined mass ratio of water flow to ceramic or for a third predetermined time for a unit volume of sonicated water to pass through a unit mass of ceramic. In one embodiment, the predetermined mass ratio of water flow to ceramic is 0.01 liters / minute / gram. In one embodiment, the third predetermined time is 0.01 seconds for 1 liter of water to pass through 1 gram of ceramic.

[0047] Once the functional water is obtained, the capabilities of the functional water can be confirmed in process 140. In one embodiment, the capabilities of the functional water that can be confirmed can include increasing cell viability, protecting cells from oxidative damage, maintaining telomere length of cells, and down-regulating genes of interest in survival associated with inflammation and aging.

[0048] Figure 2 shows a flow chart of a process for confirming the capability of the functional water produced by the method shown in Figure 1 according to a certain embodiment of the present invention. It should be noted that unless otherwise stated in this disclosure, the steps of the process may be arranged in different orders and therefore are not limited to the order shown in Figure 2.

[0049] As shown in Fig. 2, in process 210, a culture medium is prepared by adding growth enhancing substances to functional water and filtering the functional water to remove sediments and bacteria in the functional water. In one embodiment, the growth enhancing substances include Minimum Essential Medium (MEM) as a basal medium, and Fetal Bovine Serum (FBS) and Penicillin / Streptomycin (P / S) as supplements. In process 220, a control medium is prepared by adding growth enhancing substances to deionized water. After preparing the culture medium and the control medium, in process 230, live target cells are added to the culture medium and the control medium respectively to carry out a confirmation process. In process 240, the capacity of the functional water is determined by measuring the cells in the culture medium and the control medium using the corresponding kit.

[0050] AQP water was developed by Tadao Kozumi (Cosmic Co., Ltd., Hiroshima, Japan) and Yoshichika Kitagawa (Akita Prefectural University, Japan) in a paper entitled "Water structure changes induced by ceramics can be detected by increased permeability through aquaporin." (1)This was first reported in Biochemistry and Biophysics Reports (5, 2016, pp. 353-358), which was published in Biochemistry and Biophysics Reports (5, 2016, pp. 353-358), and is incorporated herein by reference in its entirety. Initially, AQP water was prepared by simply soaking Tadanoumi Ceramic® (see FIG. 4(B)) in distilled water or tap water at room temperature for 24 hours (4 grams of ceramic in 50 mL of water, equivalent to 0.08 grams / ml of ceramic in water). In contrast, in the method shown in FIG. 1, the AQP water of the present invention is obtained by further sonicating the ceramic in water after the soaking time in addition to the soaking process, and then passing the sonicated water through a column packed with ceramic to filter the water. In this case, the structural change of water molecules and the reduction in the water cluster size induced by Tadanoumi ceramic may be more stable, thus enhancing the stability of the resulting AQP water.

[0051] 3 shows a schematic shape of a typical water molecule according to a certain embodiment of the present invention. As described above, a structural change of the water molecule is induced by carrying out the method for producing functional water. Specifically, as shown in FIG. 3, first, a typical water molecule 300 in a normal state 300a has an HOH bond angle α0 (which is the angle defined between two H-0 bonds 310) and a bond length L0 (which is the length between the hydrogen and oxygen along each H-0 bond 310), and in a normal water molecule, the normal bond angle α0 is 104.45° on average, and the bond length L0 is 0.9584 Å. However, in the functional water obtained through the process as described above according to a certain embodiment of the present invention, at least one or some water molecules in the state 300a are induced or changed to the state 300b, in which the HOH bond angle of the functional water molecule in the state 300b changes from α0 to α, where α ≠ α0, and the bond length L of the functional water molecule in the state 300b may remain almost unchanged or may change accordingly. In other words, the structure of the water molecule 300 is changed or modified during the process of making the functional water as described above.

[0052] In a particular embodiment, when water molecules are treated via the method for producing functional water as described above, it has been observed that the HOH bond angle α of aquaporin water molecules is, on average, about α=α1=120.00°. From such observations, a possible and plausible model for the change in the HOH bond angle has been proposed. (2) Specifically, FIG. 4A shows, based on the proposed model, a potential relationship between the energy spectrum of a water molecule and the HOH bond angle according to a certain embodiment of the present invention, in a schematic and qualitative manner. As shown in FIG. 4A, in which the proposed energy spectrum E of a water molecule related to the HOH bond angle is shown in a schematic and qualitative manner, point A is the first minimum of the energy spectrum curve 400. In normal water, the water molecule remains in the energy ground state corresponding to point A on the energy spectrum curve 400, characterized by energy E0, and the corresponding normal bond angle α0 is 104.45°. In the preparation of functional water according to a certain embodiment of the present invention, the energy of the water molecule may change to pass through peak point B (which is a relative maximum point), and then reach the next relative minimum point C of the energy spectrum curve 400, where the corresponding HOH bond angle α1 is about 120°, so that the changed or altered water molecule may be retained and remain stable for a certain period of time (e.g., more than one week). It should be noted that the illustrated energy spectrum curve 400 may include multiple relative energy minima, such that there may be additional relative minima points D and F, where the HOH bond angle α at each point is greater than the normal bond angle α of 104.45°, respectively. In other words, in certain embodiments where water molecules have been altered or modified, the average HOH bond angle α of the altered or modified water molecules may be greater than the normal HOH bond angle α of 104.45° (e.g., points C, D, and F).

[0053] With the altered or modified water molecules, it has been observed that functional water may be readily consumable by the human body and may provide significant health benefits to humans, which may be further explained by the plausible model presented above. (2) Specifically, FIG. 4B shows a proposed three-dimensional model illustrating the volume and effective area of ​​a water molecule related to the HOH bond angle according to a certain embodiment of the present invention. As partially shown in FIG. 4B, a water molecule is formed by an oxygen atom and two hydrogen atoms, and both H-0 bonds are in the same plane (hereinafter, XZ plane in the three-dimensional coordinate system), and the water molecule is formed in three-dimensional space when the HOH bond area rotates simultaneously in the XZ plane and the YZ plane. Since the normal HOH bond angle α0 of a normal water molecule is 104.45° and the HOH bond angle α1 of a changed or modified water molecule is increased to about 120°, the effective area (shown as a patterned area in FIG. 4B) decreases due to the increase in the bond angle (specifically, the effective area A0 of a normal water molecule is about 0.4447 Å). 2 and the effective area A1 of the modified water molecule is about 0.3977 Å 2 (F = α·S), based on the zeroth order approximation, the effective volume and corresponding surface area S of the water molecule are also correspondingly reduced. This reduces the surface tension of the changed or modified water molecule, which is given by F = α·S. Thus, dangerous free radicals generated by the human body can easily attach to and / or be carried by the modified water molecules and removed from the body's cells. On the other hand, the changed or modified water molecules of the functional water may provide enhanced permeation ability due to their smaller "effective volume". The functional water made according to certain embodiments of the present invention thus contains a first amount of changed or modified water molecules in a first volume V1 with a first HOH bond angle α1 and a second amount of normal water molecules in a second volume V0 with a second HOH bond angle α0, where α1>α0. To be effective, the ratio of V1 / V0 must be greater than 1. In other words, V1 / V0>1.

[0054] The inherent ability of AQP water to pass through human aquaporins more effectively was demonstrated in oocytes expressing human aquaporins AQP1, 2, 3, or 5. Briefly, oocytes in culture medium were transferred to water. The water passed through the aquaporins and caused the cells to swell by osmotic forces. The cell swelling rate was then measured and used to calculate the osmotic water permeability (Pf). Compared to tap water, the Pf of tap water treated with Tadanoumi ceramic was found to be increased by 26%, 48%, and 21% in oocytes expressing AQP2, AQP3, and AQP5, respectively (statistical significance p<0.05). Consistently, as shown in Figure 5(A), compared to distilled water, the Pf of distilled water treated with Tadanoumi ceramic was increased by 2.2-fold, 1.2-fold, 1.3-fold, and 1.6-fold in oocytes expressing AQP1, AQP2, AQP3, and AQP5, respectively.

[0055] Meanwhile, AQP water was also found to promote the proliferation of normal human skin cells. For illustration, Tadanoumi Ceramic® (1 g of ceramic in 50 mL of medium, equivalent to 0.02 g / ml of ceramic in water) was first placed in a cell culture medium at room temperature for 24 hours. The fully grown cells were trypsinized and 1.0 × 10 5 The cells were used at a concentration of 10 ...

[0056] Due to the ability of AQP water to pass through human aquaporin more effectively and promote the proliferation of normal human skin cells, AQP water can be applied as an aqueous solution for consumption.For example, the aqueous solution containing at least 75% mass ratio of the AQP water disclosed above can be used as an aqueous solution for consumption for humans.

[0057] Tadanoumi Ceramic®. Tadanoumi Ceramic® is uniquely produced in Hiroshima, Japan, by melting natural clay and iron-bearing sand. Figure 6 shows (A) the X-ray diffraction (XRD) pattern and (B) the thermogravimetric analysis (TGA) of Tadanoumi ceramic according to a certain embodiment of the present invention. As shown in Figure 6(A), the crystal structure was investigated using x-ray diffraction (XRD, Miniflex II, Rigaku Corporation, Cu Ka radiation 1.5406 Å, 40 kV, and current 30 mA, scan rate 5° per minute). Tadanoumi ceramic exhibits a diffraction pattern that does not match any known pattern in the Joint Committee on Powder Diffraction Standards (JCPDS) database. Tadanoumi ceramic is dense (non-porous) and has a surface area of ​​0.8 cm2 as measured by nitrogen adsorption-desorption at 77 K. 2 / g, which is negligible. Thermal stability was measured by thermogravimetric analysis (TGA, Netzsch STA 449 F3 Jupiter, air flow rate 50 L / min, heating rate 10 °C / min), as shown in Figure 6(B). Tadanoumi ceramics show negligible mass loss up to 1000 °C, indicating high thermal stability. In addition, Tadanoumi ceramics do not contain any detectable heavy metals, as confirmed by x-ray fluorescence spectroscopy technique.

[0058] The functional water prepared by the method disclosed above can be applied in various applications. For example, a further aspect relates to a method for enhancing the removal of metabolic waste in a living subject, comprising preparing functional water as described above and providing the functional water to the living subject. In certain embodiments, the functional water may be configured to enhance the removal of metabolic waste from cells of the living subject, to increase the viability of cells of the living subject, to maintain telomere length of cells of the living subject, and / or to be anti-inflammatory, to downregulate genes of the living subject. In one embodiment, the metabolic waste includes hydrogen peroxide.

[0059] Ability to increase cell viability and protect cells from oxidative damage: The ability of AQP water to increase cell viability was examined in AML-12 cells, a mouse hepatic cell line derived from the liver of transgenic mice. Figure 7 shows (A) P values: * Figure 7 shows the relative cell viability of AML-12 cultured in DI water and AQP water, which is <0.05, and (B) the relative cell death of HepG2 cultured in DI water and AQP water. As shown in Figure 7(A), control culture medium was prepared by dissolving Eagle's MEM (EMEM) powder (Sigma Aldrich) in deionized water (indicated as DI-Water) and supplemented with 10% FBS (Gibco) and 1% P / S (Genesee). Experimental culture medium was prepared by dissolving EMEM powder (Sigma Aldrich) in AQP water (indicated as AQP-Water) and supplemented with 10% FBS (Gibco) and 1% P / S (Genesee). Culture medium was filtered through a 0.22 μm PES vacuum filtration system (Genesee) to remove all precipitates and bacteria. AML-12 cells were seeded in 96-well plates at 10,000 cells / well. The cells were cultured in DI-Water and AQP-Water for 24 hours, respectively, and the cell viability was measured using a Cell-titer blue kit (Sigma). The relative viability of cells cultured in AQP-Water was calculated using the following formula:

[0060]

number

[0061] As shown in FIG. 7(A), cells cultured in AQP-Water showed 115% relative cell viability compared to cells in DI-Water (statistical significance p<0.05), suggesting that AQP water increases cell viability.

[0062] The ability of AQP-Water to protect cells from oxidative damage was investigated using HepG2, an immortal cell line derived from human hepatocellular carcinoma. As shown in Figure 6(B), control culture medium was prepared by dissolving EMEM powder (Sigma Aldrich) in deionized water (indicated as DI-Water) and supplemented with 10% FBS (Gibco) and 1% P / S (Genesee). Experimental culture medium was prepared by dissolving EMEM powder (Sigma Aldrich) in AQP-activated water (indicated as AQP-Water) and supplemented with 10% FBS (Gibco) and 1% P / S (Genesee). The culture medium was filtered through a 0.22 μm PES vacuum filtration system (Genesee) to remove all precipitates and bacteria. HepG2 cells were seeded in 96-well plates at 10,000 cells / well. The cells were first cultured in medium DI-Water and AQP-Water for 24 h, respectively. The cells were then cultured for another 24 hours with the addition of H2O2 to the medium at a final concentration of 250 μM. Cell viability was measured using a Cell-titer blue kit (Sigma). The relative cell viability of cells cultured in AQP-Water with H2O2 treatment was calculated using the following formula:

[0063]

number

[0064] The viability of cells cultured in AQP-Water was slightly higher than that in DI-Water (74% vs. 71%).

[0065] Capacity in maintaining telomere length: Telomeres, the terminal segments of linear chromosomes, are nucleoprotein complexes consisting of highly conserved tandem arrays of G-rich repeats. Telomeres maintain genomic stability by preventing the ends of chromosomes from being recognized as broken DNA and triggering inappropriate responses associated with DNA damage. Upon cell division and / or oxidative stress, telomere length is compromised until they reach a significantly shorter length, at which point the permanent cell cycle is arrested and cells cease division. As an indicator of aging and oxidative stress, telomere length is recognized as a biomarker of aging and risk factors for age-related diseases.

[0066] The ability of AQP water to maintain telomere length was examined in HEK-293, a human epithelial cell line derived from fetal kidney. Control culture medium, designated as control water, was prepared by dissolving DMEM powder (Sigma Aldrich) in deionized water supplemented with 10% FBS (Gibco) and 1% P / S (Genesee). AQP culture medium, designated as AQP water, was prepared by dissolving DMEM powder (Sigma Aldrich) in AQP water supplemented with 10% FBS (Gibco) and 1% P / S (Genesee). Culture medium was filtered through a 0.22 μm PES vacuum filtration system (Genesee) to remove all precipitates and bacteria.

[0067] HEK-293 cells were cultured in control water and AQP water in T25 culture flasks, respectively, and subcultured every 3 days. 5 × 10 cells subcultured for 5, 10, 15, and 20 passages, denoted as P5, P10, P15, and P20, respectively, were cultured in control water and AQP water in T25 culture flasks, respectively, and subcultured every 3 days. 5Cellular genomic DNA from the cells was extracted using a DNA extraction kit (Qiagen). Telomere length was measured by quantitative polymerase chain reaction (qPCR) method using the telomere primer pair telg (5'-ACACTAAGGTTTGGGTTTGGGTTTGGGTTTGGGTTAGTGT-3') and telc (5'-TGTTAGGTATCCCTATCCCTATCCCTATCCCTAACA-3'). Genomic gene number was measured by qPCR using the beta globin primer pair Fwd 5'-GTGCGAGAGCGTCAGTATTAAG-3' and Rev 5'-TCCCTGCTTGCCCATACTA-3'.

[0068] Figure 8 shows (A) P value: * (A) Relative T / S ratios of cells cultured in media made from DI water or AQP water, determined by qPCR, of <0.05, and (B) RTL ratios of cells cultured in media made from DI water or AQP water, determined by qPCR. Specifically, qPCR tests were performed using a thermal cycling program consisting of stage 1 (95°C for 15 min), stage 2 (2 cycles of 94°C for 15 s and 49°C for 15 s), and stage 3 (32 cycles of 94°C for 15 s, 62°C for 10 s, and 74°C for 15 s, with signal acquisition). Standard curves of Cq values ​​against DNA concentration were constructed for telomere and genomic DNA, respectively, using MyiQ software (Bio-Rad iQ5 2.0 Standard Edition Optical System Software). The concentrations of telomere (T) and genomic DNA (S) in the samples were then calculated based on their Cq values, respectively. The T / S ratio was used to quantify the changes in telomeres during cell proliferation. RTL (relative telomere length) was also calculated by normalizing the concentration of telomeres of cells cultured in AQP water to the concentration in control water.

[0069] FIG. 8(A) shows the T / S ratio of cells cultured in control water or AQP water after 0, 5, 10, 15, and 20 passages. The cells showed a consistent decrease in T / S ratio with increasing passages, indicating the shortening of telomere length during culture. Compared to cells in control water, cells cultured in AQP water showed a significantly slower rate of decrease in T / S ratio. FIG. 8(B) shows the relative telomere length (RTL) of cells cultured in AQP water versus cells cultured in control water. Consistent with the findings in FIG. 6(B), the RTL value increased with increasing passages. P5 showed an RTL close to 1.0, while P10, P15, and P20 showed significantly higher RTL of 1.56, 1.76, and 2.05, respectively, indicating that AQP water can effectively slow down the telomere shortening process. Compared to control water, AQP water increased telomere length by approximately 56%, 76%, and 105% in P10, P15, and P20 cells, respectively. These results support the ability of AQP water to slow down the telomere shortening process.

[0070] Ability in downregulating genes associated with inflammation and aging: Nuclear factor-κB (NF-κB) has a key role in inflammatory diseases. NF-κB activation induces the expression of various proinflammatory genes, regulation of inflammatory cytokine production, recruitment of leukocytes, and formation of inflammasomes. Deregulated NF-κB activation is a hallmark of chronic inflammatory diseases such as diabetes, cancer, and neurodegenerative diseases. For many chronic diseases, oxidative stress is another important risk factor. The Keap1-Nrf2 [Kelch-like ECH-related protein 1-nuclear factor (erythroid-derived 2)-like 2] regulatory pathway is the main inducible defense against oxidative stress, Nrf2 mediates the antioxidant response, and its inhibitor, Keap1, acts as a sensor for oxidative stress.

[0071] The ability of AQP water in down-regulating inflammation and aging-related genes was investigated in HEK-293, a human epithelial cell line derived from fetal kidney. Control culture medium, designated as control water, was prepared by dissolving DMEM powder (Sigma Aldrich) in deionized water supplemented with 10% FBS (Gibco) and 1% P / S (Genesee). AQP culture medium, designated as AQP water, was prepared by dissolving DMEM powder (Sigma Aldrich) in AQP-activated water supplemented with 10% FBS (Gibco) and 1% P / S (Genesee). The culture medium was filtered through a 0.22 μm PES vacuum filtration system (Genesee) to remove all precipitates and bacteria.

[0072] HEK-293 cells were cultured in control water and AQP water in T25 culture flasks, respectively, and subcultured every 3 days for 27 passages. 5 Cellular RNA from the cells was extracted using an RNA extraction kit (Qiagen). The isolated cellular RNA was converted to cDNA using a cDNA reverse transcription kit (Qiagen) via reverse transcription polymerase chain reaction (RT-PCR) method using random primers. Representative genes related to inflammation and aging were monitored by the polymerase chain reaction (RT-PCR) method using the primer pairs NF-kB (Fwd 5'-GCACCCTGACCTTGCCTATTT-3' and Rev 5'-GTCCCAGGCGCCTTGTGAAGC-3'), Nrf2 (Fwd 5'-GAGACAGGTGAATTTCTCCCAAT-3' and Rev 5'-TTTGGGAATGTGGGCAAC-3'), and Keap1 (Fwd 5'-GGAGGACCACACCAAGCAAGC-3' and Rev 5'-GGATGAAGCCAGCACCACCTTG-3'). The GAPDH gene was measured using primers Fwd 5'-GCACCGTCAAGGCTGAGAAC-3' and Rev 5'-TGGTGAAGACGCCAGTGGA-3' as an internal control.

[0073] PCR tests were performed using a thermal cycle program consisting of stage 1 (95°C for 15 min), stage 2 (2 cycles of 94°C for 15 s and 49°C for 15 s), and stage 3 (32 cycles of 94°C for 15 s, 62°C for 10 s, and 74°C for 15 s, followed by signal acquisition). Gene expression levels of GAPDH (glyceraldehyde-3-phosphate dehydrogenase), NF-kB, Nrf2, and Keap1 were calculated using MyiQ software (Bio-Rad iQ5 2.0 Standard Edition Optical System Software). The DNA amounts of cells cultured in AQP water and control water were calculated based on their Cq values ​​and normalized with GAPDH expression.

[0074] FIG. 9 shows the fold change in gene expression in cells cultured in AQP water versus cells cultured in control water, as determined by RT-PCR. Specifically, FIG. 9 shows the fold change in gene expression in cells cultured in AQP water for 27 passages compared to those in control water. Compared to cells cultured in control water, AQP water suppressed gene expression of NF-kB by 5-fold, indicating an anti-inflammatory effect. Furthermore, cells cultured in AQP water showed a 0.5-fold decrease in Nrf2 expression. Consistently, AQP water enhanced expression of Keap1, an inhibitor of Nrf2, by 0.7-fold compared to control water. This result suggests that AQP water may help resolve oxidative stress during cellular aging.

[0075] Potential Functions and Benefits of AQP Water: The studies presented above suggest that AQP water passes through aquaporins faster than normal water and exhibits antioxidant and anti-inflammatory capabilities as well as the ability to maintain telomeres. The mechanism of action of AQP water remains difficult to understand and requires further research. Kozumi and Kitagawa proposed that the enhanced permeation of AQP water may be due to Tadanoumi ceramics, which induced structural changes in at least one or some water molecules, reducing the size of water clusters. Based on the role of aquaporins in enabling the transport of water and metabolites through cell membranes, the observed function of AQP water may be due to the faster permeation of AQP water, which results in more effective removal of metabolic waste products such as hydrogen peroxide.

[0076] The AQP water prepared by the method disclosed above can be applied in various applications.For example, a further aspect relates to a method for enhancing the removal of metabolic waste in a living subject, comprising preparing AQP water as described above and providing the AQP water to the living subject.In a particular embodiment, the AQP water is configured to enhance the removal of metabolic waste from cells of the living subject.In one embodiment, the metabolic waste includes hydrogen peroxide.

[0077] Hydrogen peroxide is a major member of reactive oxygen species (ROS) that is mainly generated as a by-product of cellular metabolism via the electron transport chain in mitochondria and cytochrome P450. Another major source is the enzymatic reaction mediated by oxidases (e.g., NAPDH oxidase) that are ubiquitous in various cells, especially phagocytes and endothelial cells. In humans and other organisms, hydrogen peroxide is mainly eliminated and decomposed into water and oxygen by catalase. Catalase is the most abundant antioxidant enzyme, ubiquitously present in the liver, red blood cells, and alveolar epithelial cells.

[0078] Hydrogen peroxide not only serves as a weapon against pathogens (e.g. bacteria) due to its cytotoxicity, but also as a signaling molecule for many physiological processes. Unbalanced production and scavenging of hydrogen peroxide leads to local or systemic accumulation of hydrogen peroxide, resulting in oxidative stress and cellular apoptosis through DNA damage, lipid peroxidation, and protein oxidation. Expanded investigations have linked ROS to diseases such as cancer, insulin resistance, diabetes, cardiovascular disease, atherosclerosis, aging, gout, and chronic inflammation. Enabling effective transport of hydrogen peroxide through cell membranes and its subsequent decomposition into water and oxygen is important for reducing oxidative stress and maintaining healthy homeostasis. In this context, we believe that AQP water is a truly unique class of functional water that may provide significant benefits to human health.

[0079] The functional water as described above can be applied in various applications. For example, in a further aspect, the functional water can be used to prepare an aqueous solution. In a particular embodiment, for example, the aqueous solution can include a quantity of first water molecules (i.e., functional water in which the water molecules are changed or modified) in a first volume V1, with a first average HOH bond angle α1 (i.e., about 120°) between the two H-0 bonds of each of the first water molecules, and a quantity of second water molecules (i.e., normal water molecules) in a second volume V0, with a second average HOH bond angle α0 (e.g., 104.45°) between the two H-0 bonds of each of the second water molecules. In one embodiment, the first average HOH bond angle α1 (e.g., 120°) is greater than the second average HOH bond angle α0 (i.e., 104.45°). In one embodiment, the aqueous solution has a volume V, where V=V1+V0, and V1 / V0>1.

[0080] The foregoing description of exemplary embodiments of the invention has been presented for purposes of illustration and description only, and is not intended to be exhaustive or to limit the invention to the precise form disclosed. Many modifications and variations are possible in light of the above teaching.

[0081] The embodiments have been chosen and described to enable others skilled in the art to utilize the invention and its various embodiments and to explain the principles of the invention and their practical application, and various modifications may be suitable for the particular uses contemplated. Alternative embodiments will become apparent to those skilled in the art to which the invention pertains without departing from the spirit and scope of the invention. Thus, the scope of the invention is defined by the appended claims rather than the foregoing description and the exemplary embodiments described therein.

[0082] Certain references, which may include patents, patent applications, and various publications, are cited and discussed in the description of the present invention. Citation and / or discussion of such references is provided solely to clarify the description of the present invention and is not an admission that any such reference is "prior art" to the invention described herein. All references cited and discussed herein are incorporated herein by reference in their entirety and to the same extent as if each reference was individually incorporated by reference. [Explanation of symbols]

[0083] 300 water molecules 300a Normal state 310 H-0 bond 400 Energy Spectrum Curve

[0084] (References) TIFF2025510134000004.tif20170

Claims

1. A method for producing aquaporin water having enhanced cell membrane permeability, comprising: preparing a ceramic by melting natural clay and iron-containing sand; immersing the ceramic in water at a predetermined mass ratio of ceramic to water at a predetermined temperature for a first predetermined time period and sonicating the ceramic in water at a predetermined sonication power at a predetermined temperature for a second predetermined time period; Flowing the ultrasonically treated water through a ceramic-packed column at a predetermined temperature to obtain aquaporin water, the water flowing through the column at a predetermined mass ratio of water flow to ceramic or for a third predetermined time for a unit volume of water to pass through a unit mass of ceramic. Including, The method, wherein the ceramic induces a structural change in at least a portion of the water molecules.

2. The water molecule of aquaporin water has an HOH bond angle α, and the HOH bond angle of the changed water molecule is α 1 However, the normal HOH bond angle α of the water molecule before treatment 0 The method of claim 1 , wherein

3. HOH bond angle α 1 is about 120°, and the normal bond angle α 0 The method of claim 2, wherein the angle is 104.45°.

4. 2. The method of claim 1, Preparing a culture medium by adding a growth enhancing substance to the aquaporin water and filtering the aquaporin water to remove sediments and bacteria in the AQP water; preparing a control medium by adding growth enhancing substances to deionized water; Preparing the culture medium and the control medium, and then adding live subject cells to the culture medium and the control medium, respectively; Determine the aquaporin water capacity by measuring cells in culture medium and control medium using the corresponding kit. A process for confirming the multiple capabilities of aquaporin water by Further comprising:

5. The capabilities of aquaporin water are: Increased cell viability, Protecting cells from oxidative damage, Maintenance of telomere length in cells, and Downregulation of survival target genes associated with inflammation and aging 5. The method of claim 4, comprising:

6. 2. The method of claim 1, wherein the ceramic is Tadanoumi ceramic, the predetermined mass ratio of ceramic to water is 0.01 to 0.1 grams / ml of ceramic to water, the predetermined temperature is room temperature, the first predetermined time is 18 to 36 hours, the predetermined sonication power is 100 watts, the second predetermined time is 3 to 7.5 minutes, the predetermined mass ratio of water flow to ceramic is 0.01 liters / minute / gram, and the third predetermined time is 0.01 seconds for 1 liter of water to pass through 1 gram of ceramic.

7. As a growth enhancer, Minimum essential medium (MEM) as the basal medium, and Fetal bovine serum (FBS) and penicillin / streptomycin (P / S) as supplementary substances The method of claim 1, comprising:

8. The water molecule of aquaporin water has an HOH bond angle α, and the HOH bond angle of the changed water molecule is α 1 However, the normal HOH bond angle α of the water molecule before treatment 0 The aquaporin water prepared by the method of claim 1,

9. HOH bond angle α 1 is about 120°, and the normal bond angle α 0 The aquaporin water according to claim 8, wherein the temperature is 104.45°.

10. An aqueous solution comprising the aquaporin water according to claim 8, The volume of water molecules changed in the aqueous solution is V 1 having The unchanged water molecules in an aqueous solution have a volume V 0 having The aqueous solution has a volume V, and V=V 1 +V 0 And V 1 / V 0 > 1, Aqueous solution.

11. 1. A method for enhancing metabolic waste removal in a living subject, comprising: A step of preparing the aquaporin water defined in claim 1; and Providing aquaporin water to a living subject, the aquaporin water configured to enhance removal of metabolic waste products from cells of the living subject. A method comprising:

12. 12. The method of claim 11, wherein the metabolic waste product comprises hydrogen peroxide.

13. 1. A method for increasing the viability of cells in a living subject, comprising: A step of preparing the aquaporin water defined in claim 1; and Providing aquaporin water to a living subject, the aquaporin water configured to increase the viability of cells of the living subject. A method comprising:

14. 1. A method for protecting cells of a living subject from oxidative damage, comprising: A step of preparing the aquaporin water defined in claim 1; and Providing aquaporin water to a living subject, the aquaporin water configured to be antioxidant to protect the living subject's cells from oxidative damage. A method comprising:

15. 1. A method for maintaining telomere length in a cell of a living subject, comprising: A step of preparing the aquaporin water defined in claim 1; and Providing aquaporin water to a living subject, the aquaporin water configured to maintain telomere length in cells of the living subject. A method comprising:

16. 1. A method for downregulating genes associated with inflammation and aging in a living subject, comprising: A step of preparing the aquaporin water defined in claim 1; and Providing aquaporin water to a living subject, the aquaporin water configured to be anti-inflammatory so as to downregulate genes in the living subject. A method comprising:

17. A method for producing functional water having enhanced cell membrane permeability, comprising the steps of: preparing a ceramic by melting natural clay and iron-containing sand; immersing the ceramic in water at a predetermined mass ratio of ceramic to water at a predetermined temperature for a first predetermined time to obtain functional water; and ultrasonicating the ceramic in water at a predetermined ultrasonic treatment power at a predetermined temperature for a second predetermined time; A step of passing the ultrasonically treated water through a ceramic-packed column at a predetermined temperature to obtain functional water, the water passing through the column at a predetermined mass ratio of water flow to ceramic or for a third predetermined time for a unit volume of water to pass through a unit mass of ceramic. Including, The method, wherein the ceramic induces a structural change in at least some of the water molecules of the water.

18. 18. The method of claim 17, Adding live target cells to the functional water as a culture medium; Adding live subject cells to deionized water as a control medium; and Measure the cells in the culture medium and the control medium using the corresponding kit to determine the capacity of the functional water. The method further comprises a step of confirming the capability of the functional water by

19. The capabilities of functional water are as follows: Increased cell viability, Protecting cells from oxidative damage, Maintenance of telomere length in cells, and Downregulation of survival target genes associated with inflammation and aging 20. The method of claim 18, comprising:

20. The method according to claim 18, further comprising the steps of: Adding a growth enhancing substance to the functional water as a culture medium; A step of filtering the culture medium to remove sediments and bacteria in the functional water. The method further comprises:

21. As a growth enhancer, Minimum essential medium (MEM) as the basal medium, and Fetal bovine serum (FBS) and penicillin / streptomycin (P / S) as supplementary substances 20. The method of claim 18, comprising:

22. 18. The method of claim 17, wherein the ceramic is Tadanoumi ceramic, the functional water is aquaporin water, the predetermined mass ratio of ceramic to water is 0.01 to 0.1 grams / ml of ceramic to water, the predetermined temperature is room temperature, the first predetermined time is 18 to 36 hours, the predetermined ultrasonic treatment power is 100 watts, the second predetermined time is 3 to 7.5 minutes, the predetermined mass ratio of water flow to ceramic is 0.01 liters / minute / gram, and the third predetermined time is 0.01 seconds for 1 liter of water to pass through 1 gram of ceramic.

23. The water molecule of aquaporin water has an HOH bond angle α, and the HOH bond angle of the changed water molecule is α 1 However, the normal HOH bond angle α of the water molecule before treatment 0 The aquaporin water prepared by the method of claim 17, wherein the aquaporin water has a molecular weight of 1.0 or more.

24. HOH bond angle α 1 is about 120°, and the normal bond angle α 0 The aquaporin water according to claim 23, wherein the temperature is 104.45°.

25. 24. An aqueous solution comprising the aquaporin water of claim 23, The volume of water molecules changed in the aqueous solution is V 1 having The unchanged water molecules in an aqueous solution have a volume V 0 having The aqueous solution has a volume V, and V=V 1 +V 0 And V 1 / V 0 > 1, Aqueous solution.

26. 1. A method for enhancing metabolic waste removal in a living subject, comprising: A step of preparing the functional water defined in claim 17; and Providing functional water to a living subject, the functional water configured to enhance removal of metabolic waste products from cells of the living subject. A method comprising:

27. 27. The method of claim 26, wherein the metabolic waste product comprises hydrogen peroxide.

28. The first average HOH bond angle α between each of the two H-0 bonds of the first water molecule 1 A first volume V 1 a quantity of first water molecules, and The second average HOH bond angle α between the two H-0 bonds of each of the second water molecules 0 A second volume V 0 of a certain amount of second water molecules 4. An aqueous solution comprising:

29. First average HOH bond angle α 1 is the second average HOH bond angle α 0 30. The aqueous solution of claim 28, wherein the aqueous solution is greater than

30. First average HOH bond angle α 1 is 120°, and the second average HOH bond angle α 0 30. The aqueous solution of claim 29, wherein the pH is about 104.45°.

31. It has a volume V, and V=V 1 +V 0 And V 1 / V 0 29. The aqueous solution of claim 28, wherein:

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