Water-soluble substituent-based novel compound having high solubility

Abstract

The present invention relates to: a water-soluble substituent-based novel compound having high solubility and a method for preparing same. The novel compound having high solubility of the present invention has repeating units represented by chemical formula 1, and has an excellent anti-inflammatory effect and an excellent effect on diseases caused by oxidative stress and neuroinflammatory diseases, and thus is useful as a health food or a pharmaceutical composition. [Chemical formula 1]

Description

Novel highly soluble compounds based on water-soluble substituents

[0001] The present invention relates to novel highly soluble compounds having water-soluble groups, such as short-chain fatty acids or phenolic functional groups, as substituents, and to the uses thereof.

[0002] Unless otherwise indicated in this specification, the contents described in this section are not prior art for the claims of this application, and are not to be recognized as prior art simply because they are included in this section.

[0003] Pharmaceuticals and foods are indispensable substances for human health and their application to the human body is essential; however, in the case of synthetic substances, it is difficult to manufacture active materials, and in particular, for natural products, there are problems in maintaining toxicity and quality within standard specifications at a consistent level.

[0004] As such, the creation of new substances in the fields of medicine and food can be considered one of the most urgent tasks to be resolved among the science and technology issues facing Korea; however, the impact of pesticide residues on the human body caused by the excessive addition of pesticides to food is becoming increasingly serious. Furthermore, as reported in the media, pesticide usage significantly exceeds the limits of usage standards, and the resulting health damage from food consumption poses a major threat to health, affecting not only adults but also infants and fetuses through secondary infections.

[0005] However, antioxidant agents and excessive pesticides are still being used, and various residual pesticides, such as those from the spraying of formalin, pose a significant threat to health. Amidst this, multifaceted research utilizing the antibacterial and antifungal effects of useful natural materials, such as chitosan, has recently been actively conducted.

[0006] Chitosan, known as a biocompatible material, is a linear polysaccharide obtained by chemically deacetylating the polymeric chemical structure of chitin—which is mainly found in the exoskeletons of crustaceans such as crabs—using an alkaline solution or by removing N-acetyl groups using enzymes (deacetylation), thereby converting the ends of the side chains of the chitosan polymer network to -NH2. The amine and hydroxyl groups of chitosan form hydrogen bonds in interactions between the internal or external structures of the chitosan polymer, resulting in excellent bonding strength.

[0007] Chitosan polymers have many primary amino groups, which serve as useful functional groups for inducing various chemical bonds and can provide bioactivity to the polymer. The advantages of chitosan include enzymatic biodegradability, blood compatibility, biocompatibility, and non-toxicity when applied to the body. Furthermore, because it is the second most abundant natural polymer in nature after cellulose, it offers the advantages of being easy to obtain as a medical material and being low-cost.

[0008] However, there are limitations to utilizing the activity of chitosan when used as is. Furthermore, since it is not easy to develop technologies to derivatize chitosan into other components to activate functions or improve efficacy, current applications remain limited to using chitosan polymers in their raw form and mixing them with other active ingredients.

[0009] Although some chitosan derivatives are known, their presence is negligible, and there is a problem in that their applicability is extremely limited.

[0010] {Prior Art Literature}

[0011] [Patent Literature]

[0012] (Patent Document 1) Korean Published Patent No. 10-2020-0054485

[0013] The present invention aims to solve the problem of the prior art described above by providing a novel compound with new activity using the active ingredients of natural substances that have previously been used as active materials.

[0014] Therefore, the objective of the present invention is to provide a new highly soluble compound based on a water-soluble substituent.

[0015] Another objective of the present invention is to provide a highly soluble compound of a novel chitosan derivative structure based on a water-soluble substituent by introducing a short-chain fatty acid or a phenolic functional group into a chitosan reactor.

[0016] Another objective of the present invention is to provide new uses for a highly soluble compound of a new structure prepared with the above-described structure.

[0017] The purpose of the present invention is not limited to the purposes stated above, and should be understood to include all purposes that can be inferred from the configuration of the invention described in the detailed description or claims of the present invention, or all purposes that can be achieved by the description or technical concept of the present invention.

[0018] To solve the problem of the present invention as described above, the present invention provides a novel highly soluble compound based on a short-chain fatty acid, comprising a highly soluble compound having the following chemical formula 1 as a repeating unit or a pharmaceutically acceptable salt thereof.

[0019] [Chemical Formula 1]

[0020]

[0021] In the above chemical formula 1,

[0022] R1, R2 is a hydrogen atom, a short-chain fatty acid group, or a phenolic functional group, respectively, and R1 and At least one of R2 is a short-chain fatty acid group or a phenolic functional group, and R1 and R2s may all be the same or different short-chain fatty acid groups or phenolic functional groups, and

[0023] n is an integer from 1 to 100,000.

[0024] According to a preferred embodiment of the present invention, the short-chain fatty acid group coupled to Formula 1 may be a substituent composed of a short-chain fatty acid selected from substituents composed of acetic acid, propionic acid, butyric acid, isobutyric acid, isovaleric acid, isocaproic acid, lactic acid, valeric acid, caprylic acid, 2-methylbutyric acid, 3-methylbutyric acid or aldehyde derivatives thereof, esters thereof, or acceptable salts thereof.

[0025] According to a preferred embodiment of the present invention, the phenolic functional group bound to Formula 1 may be a phenolic functional group derived from anthocyanin, gallic acid, hydroxybenzoic acids, caffeic acid, ferulic acid, protocatechuic acid, vanillic acid, syringic acid, pyrogallol, chlorogenic acid, or tannic acid.

[0026] According to a preferred embodiment of the present invention, a preferred example includes the case in which both a short-chain fatty acid group and a phenolic functional group are present in Formula 1.

[0027] In addition, the present invention comprises an anti-inflammatory composition containing, as an active ingredient, a highly soluble compound based on a short-chain fatty acid, which is composed of a highly soluble compound having the repeating unit of Formula 1 or a pharmaceutically acceptable salt thereof.

[0028] In addition, the present invention comprises a health food for improving inflammation comprising the above anti-inflammatory composition, and a pharmaceutical composition for preventing or treating inflammatory diseases comprising the above anti-inflammatory composition.

[0029] In addition, the present invention comprises a composition for improving, preventing, or treating neuroinflammatory or neurodegenerative diseases, comprising as an active ingredient a highly soluble compound based on a short-chain fatty acid, which is composed of a highly soluble compound having the repeating unit of Formula 1 or a pharmaceutically acceptable salt thereof.

[0030] In addition, the present invention includes a health food for improving mild cognitive impairment comprising the above composition.

[0031] In addition, the present invention comprises a pharmaceutical composition for improving, preventing, or treating neuroinflammatory or neurodegenerative diseases, comprising the above composition.

[0032] In addition, the present invention comprises a pharmaceutical composition for the prevention or treatment of Alzheimer's disease, Parkinson's disease, Lou Gehrig's disease (ALS), cognitive impairment, senile dementia, Lewy body dementia, frontotemporal dementia, Huntington's disease dementia, vascular dementia, myocardial infarction, arteriosclerosis, ischemia-reperfusion injury, type 2 diabetes, obesity, metabolic syndrome, rheumatoid arthritis, inflammatory bowel disease (IBD), chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, inhibition of photoaging, atopic dermatitis, anticancer, alleviation of anticancer side effects, anti-aging, macular degeneration (AMD), diabetic retinopathy, acute renal injury (AKI), and chronic kidney disease, which are associated with anti-inflammatory, oxidative stress, or neuroinflammation, and which contains as an active ingredient a highly soluble compound based on a short-chain fatty acid, comprising a highly soluble compound having the repeating unit of Formula 1 or a pharmaceutically acceptable salt thereof.

[0033] In addition, the present invention provides a method for preparing a highly soluble compound having the repeating unit of Formula 1, comprising the steps of: mixing a reaction product, which is a water-soluble substituent, with chitosan in a buffer, wherein the first reaction product is a short-chain fatty acid, or the second reaction product is one or more compounds selected from anthocyanin, gallic acid, hydroxybenzoic acids, caffeic acid, ferulic acid, protocatechuic acid, vanillic acid, syringic acid, pyrogallol, chlorogenic acid, or tannic acid, or mixing all of the first reaction product and the second reaction product; adding a crosslinking agent to the mixture and reacting; and centrifuging the reaction product.

[0034] According to a preferred embodiment of the present invention, the crosslinking agent may include 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide Hydrochloride.

[0035] The novel highly soluble compound according to the present invention is a novel highly soluble compound based on short-chain fatty acids, and in particular, by reacting the amine group of chitosan with the carboxyl group or phenolic functional group of the short-chain fatty acid to form a peptide bond, it can exhibit a new type of mechanism of action and physiological activity.

[0036] Therefore, the present invention exerts, for example, an anti-inflammatory effect, or more preferably, an anti-neuroinflammatory effect, thereby having excellent effects in improving inflammation and improving, preventing, or treating neuroinflammatory diseases.

[0037] Therefore, the novel highly soluble compounds of the present invention can be usefully utilized in related industries such as health foods, pharmaceuticals, and veterinary drugs.

[0038] The effects of the present invention are not limited to the effects described above, and should be understood to include all effects that can be inferred from the configuration of the invention described in the detailed description or claims of the present invention.

[0039] FIG. 1a is a diagram illustrating the state of a product prepared after the synthesis of a novel highly soluble compound having a short-chain fatty acid group in the repeating unit of Formula 1 in one embodiment of the present invention.

[0040] FIG. 1b is a diagram illustrating the state of a product prepared after the synthesis of a novel highly soluble compound containing a short-chain fatty acid group and a phenolic functional group having repeating units of Formula 1 in one embodiment of the present invention.

[0041] FIG. 2 is a photograph showing the state of a compound that appears during the purification process after the synthesis of a novel highly soluble compound having a repeating unit of Formula 1 in one embodiment of the present invention.

[0042] FIG. 3a is an NMR graph showing the results of the formation of a novel highly soluble compound (chitosan-propionic acid polysubstituted derivative) having repeating units of Formula 1, compared with the raw material before synthesis in Example 1 of the present invention and the novel highly soluble compound prepared after synthesis.

[0043] FIG. 3b is an NMR graph showing the results of the formation of a novel highly soluble compound containing a single-chain fatty acid group and a phenolic functional group having a repeating unit of Formula 1, compared with a raw material before synthesis and a novel compound prepared after synthesis in one embodiment of the present invention.

[0044] Figure 4a is FT-IR analysis data showing the results of the formation of a novel highly soluble compound (chitosan-propionic acid polysubstituted derivative) having repeating units of Formula 1, compared with the raw material before synthesis in Example 1 of the present invention.

[0045] Figure 4b is FT-IR analysis data showing the results of the production of a novel highly soluble compound (chitosan-propionate-gallic acid derivative) containing a single-chain fatty acid group and a phenolic functional group having a repeating unit of Formula 1, compared with the raw material before synthesis in one embodiment of the present invention.

[0046] Figure 5a is experimental data on the NO-inhibiting anti-inflammatory activity of a novel highly soluble compound having a repeating unit of Formula 1 synthesized in Example 1 of the present invention.

[0047] FIG. 5b is experimental data on the NO-inhibiting anti-inflammatory activity of a novel highly soluble compound containing a short-chain fatty acid group and a phenolic functional group having a repeating unit of Formula 1, synthesized in Example 8 of the present invention.

[0048] Figure 6a is experimental data on the anti-inflammatory activity of a novel highly soluble compound having a repeating unit of Formula 1 synthesized in Example 1 of the present invention, which inhibits inflammatory cytokine expression.

[0049] Figure 6b is experimental data on the anti-inflammatory activity of a novel highly soluble compound having a repeating unit of Formula 1 synthesized in Example 8 of the present invention, which inhibits inflammatory cytokine expression.

[0050] Figure 7a is experimental data on neuroprotective activity for a novel highly soluble compound having a repeating unit of Formula 1 synthesized in Example 1 of the present invention.

[0051] Figure 7b is experimental data on neuroprotective activity for a novel highly soluble compound containing a phenolic functional group having a repeating unit of Formula 1 synthesized in Example 8 of the present invention.

[0052] Figure 8 is experimental data related to neuroimmunomodulation through inhibition of reactive oxygen species for a novel highly soluble compound having a repeating unit of Formula 1 synthesized in one embodiment of the present invention.

[0053] Figure 9 is experimental data on the inhibition of phosphorylation of neuroimmunomodulation-related protein p38 by a novel highly soluble compound having a repeating unit of Formula 1 synthesized in one embodiment of the present invention.

[0054] Figure 10a is cytotoxicity test data for a novel highly soluble compound having a repeating unit of Formula 1 synthesized in Example 1 of the present invention.

[0055] FIG. 10b is cytotoxicity test data for a novel highly soluble compound having a repeating unit of Formula 1 synthesized in Example 8 of the present invention.

[0056] The present invention will be described in more detail below based on one embodiment.

[0057] The descriptions and configurations illustrated in the drawings in this specification are merely the most preferred embodiments of the invention and do not represent all technical aspects of the invention; therefore, it should be understood that various equivalents and modifications that may replace them may exist at the time of filing this application. The embodiments described in this invention should be understood as illustrative in all respects and not restrictive, and the scope of the invention is defined by the claims set forth below rather than by the detailed description, and all modifications or variations derived from the meaning and scope of the claims and equivalent concepts should be interpreted as being included within the scope of the invention.

[0058] Furthermore, the terminology used in this specification is used to appropriately describe preferred embodiments of the present invention, and may vary depending on the intent of the user or operator, or the conventions of the field to which the present invention belongs. Accordingly, the definitions of these terms should be based on the content throughout this specification. Throughout the specification, when a part is described as “comprising” or “containing” a certain component, unless specifically stated otherwise, this means that it may include additional components rather than excluding other components.

[0059] The present invention provides a novel highly soluble compound based on a short-chain fatty acid having the following chemical formula 1 as a repeating unit, or a pharmaceutically acceptable salt thereof.

[0060] [Chemical Formula 1]

[0061]

[0062] In the above formula,

[0063] R1, R2 is a hydrogen atom, a short-chain fatty acid group, or a phenolic functional group, respectively, and R1 and At least one of R2 is a short-chain fatty acid group or a phenolic functional group, and R1 and If all R2s are short-chain fatty acid groups or phenolic functional groups, they may be the same or different short-chain fatty acid groups or phenolic functional groups, and

[0064] n is an integer from 1 to 100,000.

[0065] According to a preferred embodiment of the present invention, more preferably, at least one of R1 and R2 in Formula 1 is a short-chain fatty acid group. Preferably, when n is 2 or more in the repeating unit, it may be even more preferable that R1 and R2 are the same substituent. However, when n is 2 or more, R1 and R2 may randomly include different short-chain fatty acid groups or phenolic functional groups in some parts, or a short-chain fatty acid group and a phenolic functional group, respectively.

[0066] According to a preferred embodiment of the present invention, a preferred case may be that at least one of R1 and R2 in Formula 1 is a phenolic functional group. In particular, a preferred example may be one in which both a short-chain fatty acid group and a phenolic functional group are substituents.

[0067] According to a preferred embodiment of the present invention, in the above chemical formula 1, n can be an integer from 1 to 1000, 1 to 100, or 1 to 10, and can be prepared as a substance with a relatively small molecular weight.

[0068] The novel highly soluble “compound” produced in the present invention is a pure chemical substance formed by the combination of atoms of two or more types of chemical elements, and can be separated into simple substances through a purification process or chemical reaction.

[0069] In this invention, the term “high-solubility” signifies excellent water solubility, specifically meaning that it dissolves well in various pharmaceutically acceptable solvents. Therefore, since it possesses properties that allow for easy utilization in formulations such as injections, liquids, and patches, it implies that it can be easily applied as pharmaceuticals or health foods.

[0070] According to a preferred embodiment of the present invention, as a preferred example, the short-chain fatty acid group in Formula 1 may be selected from substituents composed of short-chain fatty acids selected from acetic acid, propionic acid, butyric acid, isobutyric acid, 2-methylbutyric acid, and 3-methylbutyric acid. More preferably, R1 and At least one of R2 may be an acetic acid group, a propionic acid group, or a butyric acid group. In addition, as a preferred embodiment, the above R1 and R2 may be a short-chain fatty acid group selected from an acetic acid group, a propionic acid group, or a butyric acid group, all of which may be the same or different. The short-chain fatty acids (SCFAs) are major metabolic products of intestinal microorganisms and refer to fatty acids with six or fewer carbon atoms.

[0071] According to a preferred embodiment of the present invention, the phenolic functional group in Formula 1 may be selected from substituents consisting of anthocyanin, gallic acid, caffeic acid, or ferulic acid. More preferably, it may be a substituent consisting of anthocyanin or gallic acid.

[0072] According to a preferred embodiment of the present invention, the short-chain fatty acid may preferably be derived from natural products such as berries. In this case, for example, a berry extract obtained by fermenting it with a fermentation bacterium may be used.

[0073] According to a preferred embodiment of the present invention, a highly soluble compound having a repeating unit of Formula 1 according to the present invention may be prepared by a method comprising, for example, the step of mixing chitosan and a short-chain fatty acid in a buffer; the step of adding a crosslinking agent to the mixture and reacting it; and the step of centrifuging the reaction product. In this case, Formula 1 may have only a short-chain fatty acid as a substituent.

[0074] Another method may be prepared by a method comprising the steps of, for example, mixing chitosan in a buffer with a short-chain fatty acid as a first reactant and one or more compounds selected from the above-listed anthocyanin, gallic acid, caffeic acid, ferulic acid, hydroxybenzoic acids, protocatechuic acid, vanillic acid, syringic acid, pyrogallol, chlorogenic acid, or tannic acid as a second reactant in order to additionally introduce phenolic functional groups; adding a crosslinking agent to the mixture and reacting it; and centrifuging the reaction product.

[0075] The “buffer” or “buffer solution” of the present invention is generally a solution whose hydrogen ion concentration (pH) does not change significantly due to the common ion effect even when an acid or base is added.

[0076] The “chitosan” of the present invention is a linear polysaccharide composed of D-glucosamine and N-acetylglucosamine. According to a preferred embodiment of the present invention, naturally derived chitosan may be used.

[0077] According to a preferred embodiment of the present invention, the buffer may comprise MES (2-(N-molorial) ethanesulfonic acid), PBS (Phosphate-Buffered Saline), PIPES (Piperazine-N,N′-bis(2-ethanesulfonic acid)), MOPS (3-(N-morpholino)propanesulfonic acid) or HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), and preferably may be MES.

[0078] According to a preferred embodiment of the present invention, the crosslinking agent may include 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide Hydrochloride.

[0079] According to a preferred embodiment of the present invention, the reaction can be carried out by stirring for, for example, at 20 to 30°C, preferably at room temperature for 12 to 48 hours, more preferably for 20 to 30 hours.

[0080] According to a preferred embodiment of the present invention, the reaction may be carried out by adding a single short-chain fatty acid or a mixture of two or more different short-chain fatty acids as a reactant to chitosan in the above reaction, either simultaneously, or by adding one and reacting, and then reacting the other. Alternatively, the reaction may be carried out, for example, by adding two or more different phenolic functional groups as a reactant to chitosan in the above reaction, either simultaneously, or by adding one and reacting, and then reacting the other. Alternatively, the reaction may be carried out, for example, by adding a compound having a short-chain fatty acid group and a phenolic functional group as a reactant to chitosan in the above reaction, either simultaneously, or by adding one and reacting, and then reacting the other.

[0081] In the manufacturing method of the present invention, one or more short-chain fatty acids may be selected from those mentioned in the description of Chemical Formula 1 above.

[0082] According to the present invention, the novel highly soluble compound can be produced as a non-toxic substance when using naturally derived components. For example, chitosan, which is the core of the novel highly soluble compound of Formula 1, may be a natural product derived from crustaceans, etc. In addition, the compound introduced as a substituent in Formula 1, such as a short-chain fatty acid, anthocyanin, or gallic acid, which introduces a phenolic functional group, may preferably be derived from natural products such as berries.

[0083] According to a preferred embodiment of the present invention, the highly soluble compound of Formula 1 can be preferably used as a health food for improving inflammation or as a pharmaceutical composition for the prevention or treatment of inflammatory diseases.

[0084] In addition, the present invention can preferably be used as a composition for improving, preventing, or treating neuroinflammatory or neurodegenerative diseases, as the highly soluble compound of Formula 1 as described above is essentially effective for anti-inflammatory effects, particularly for neuroinflammation.

[0085] In particular, according to a preferred embodiment of the present invention, the composition of the present invention can exhibit excellent effects when used as a health food for improving mild cognitive impairment or as a pharmaceutical composition for the prevention or treatment of mild cognitive impairment.

[0086] According to the present invention, inflammatory diseases including inflammation, neuroinflammation, and neurodegeneration as a mechanism of action include autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, ankylosing spondylitis, and psoriatic arthritis; inflammatory bowel diseases (IBD) such as Crohn's disease and ulcerative colitis; skin inflammatory diseases such as psoriasis, eczema, and acne; respiratory inflammatory diseases such as asthma, chronic obstructive pulmonary disease (COPD), and allergic rhinitis; cardiovascular inflammatory diseases such as endocarditis, atherosclerosis, and vasculitis; multiple sclerosis; and Guillain-Barre syndrome. Examples include inflammatory diseases of the nervous system such as Alzheimer's disease, infectious inflammatory diseases such as sepsis, pneumonia, and hepatitis, inflammatory diseases of the musculoskeletal system such as synovitis, bursitis, and tendinitis, chronic inflammation-related metabolic syndrome, and obesity-related inflammation.

[0087] In addition, the highly soluble compound of Formula 1 according to the present invention is confirmed to have excellent neuronal corresponding activity and is effective against diseases related to oxidative stress, such as oxidative stress caused by ROS.

[0088] Therefore, the highly soluble compound of the present invention is effective in preventing or treating diseases related to inflammation, oxidative stress, or neuroinflammation, such as neurodegenerative diseases including Alzheimer's, Parkinson's, Lou Gehrig's disease (ALS), cognitive impairment, senile dementia, Lewy body dementia, frontotemporal dementia, Huntington's disease dementia, and vascular dementia; cardiovascular diseases including myocardial infarction, arteriosclerosis, and ischemia-reperfusion injury; metabolic diseases including type 2 diabetes, obesity, and metabolic syndrome; inflammatory diseases including rheumatoid arthritis and inflammatory bowel disease (IBD); lung diseases including chronic obstructive pulmonary disease (COPD) and pulmonary fibrosis; skin diseases including photoaging and atopic dermatitis; anticancer, alleviation of anticancer side effects, and anti-aging; and ophthalmic diseases including macular degeneration (AMD) and diabetic retinopathy, acute kidney injury (AKI), and chronic kidney disease.

[0089] According to a preferred embodiment of the present invention, the highly soluble compound of Formula 1 of the present invention is water-soluble, and its solubility in water may be, for example, 2.5 mg / mL or more.

[0090] A composition containing the novel highly soluble compound according to the present invention as an active ingredient can be used in formulations such as oral tablets, injections, and patches as a preparation that exhibits anti-inflammatory, particularly anti-neuroinflammatory, effects.

[0091] According to a preferred embodiment of the present invention, a composition comprising a highly soluble compound of Formula 1 can be manufactured into various formulations, such as health foods, pharmaceuticals, etc., and can also be manufactured into animal pharmaceuticals or feed additives as another example.

[0092] According to a preferred embodiment of the present invention, the highly soluble compound of the present invention can be formulated into a form including conventional excipients or additives, and can be manufactured into various oral preparations such as liquids, capsules, pills, granules, and extracts. In addition, since it has excellent water solubility or solubility in pharmaceutically acceptable solvents, it can be manufactured into injectables, liquids, etc. by conventional methods, and can also be applied as a patch, etc.

[0093] The formulation of the present invention can be manufactured into various forms of pharmaceuticals for the improvement, prevention, or treatment of inflammatory diseases or diseases caused by oxidative stress, such as health foods for improving inflammation, and can be formulated with a dosage of typically 10 to 600 mg. It can be taken 1 to 3 times a day, 1 to 2 tablets at a time, or as a liquid. In addition, patches other than oral or injectable formulations can also be administered at equivalent levels.

[0094]

[0095] Hereinafter, the present invention as described above will be explained in detail based on specific embodiments, but the present invention is not limited by the embodiments exemplified herein.

[0096]

[0097] Example 1: Preparation of a polysubstituted solubility compound of chitosan and propionic acid

[0098] The conjugate synthesis of chitosan and propionic acid was performed using raw materials. MES was used as the buffer and EDC as the crosslinking agent, and the reaction was carried out at room temperature.

[0099] Specifically, 40 mg of chitosan, 20 mg of propionate, and 80 mg of crosslinking agent (EDC) were added to 8 mL of 10 mM MES buffer, and the mixture was stirred at 600 rpm at room temperature for 24 hours. After confirming the completion of the reaction, the reaction mixture was precipitated in 25 mL of cold acetone and centrifuged at 2000 rpm for 4 minutes. After removing the supernatant, 10 mL of methanol and 10 mL of DMSO were added, sonicated for 3 minutes, and centrifuged; this centrifugation and purification process was repeated three times. The product was washed with 10 mL of methanol, and after removing the supernatant, concentrated under reduced pressure. Subsequently, 10 mL of distilled water was added, and the mixture was freeze-dried to obtain a chitosan-propionic acid-gallic acid bond-soluble compound as the final product. (Yield: 73%)

[0100] [Reaction Equation 1]

[0101]

[0102] The synthesis results of the product (HS) produced before, during, and after the reaction process and after the reaction was completed were obtained as the result shown in the imaged photograph of Fig. 1a.

[0103]

[0104] Example 2: Preparation of a Chitosan-Acetate Coupling Soluble Compound

[0105] The conjugate synthesis of chitosan and acetic acid was performed using raw materials. MES was used as the buffer and EDC as the crosslinking agent, and the reaction was carried out at room temperature.

[0106] Specifically, 40 mg of chitosan, 20 mg of acetate, and 80 mg of crosslinking agent (EDC) were added to 8 mL of 10 mM MES buffer, and the mixture was stirred at 600 rpm at room temperature for 24 hours. After confirming the completion of the reaction, the reaction mixture was precipitated in 25 mL of cold acetone and centrifuged at 2000 rpm for 4 minutes. After removing the supernatant, 10 mL of methanol and 10 mL of DMSO were added, sonicated for 3 minutes, and centrifuged; this centrifugation and purification process was repeated three times. Subsequently, the mixture was washed with 10 mL of methanol, the supernatant was removed, and the product was obtained by vacuum concentration. Finally, 10 mL of distilled water was added and freeze-dried to obtain a chitosan-acetate bonded polymer compound (a product in which both R1 and R2 in Chemical Formula 1 are polysubstituted with acetic acid groups). (Yield: 65%)

[0107]

[0108] Example 3: Preparation of a Chitosan-Butyric Acid Combined Soluble Compound

[0109] The conjugate synthesis of chitosan and n-butyric acid was performed using chitosan and n-butyric acid as raw materials. MES was used as the buffer and EDC as the crosslinking agent, and the reaction was carried out at room temperature.

[0110] Specifically, 40 mg of chitosan, 20 mg of butyrate, and 80 mg of a crosslinking agent (EDC) were added to 8 mL of 10 mM MES buffer, and the mixture was stirred at 600 rpm at room temperature for 24 hours. After confirming the completion of the reaction, the reaction mixture was precipitated in 25 mL of cold acetone and centrifuged at 2000 rpm for 4 minutes. After removing the supernatant, 10 mL of methanol and 10 mL of DMSO were added, sonicated for 3 minutes, and centrifuged; the above centrifugation and purification process was repeated 3 times. Subsequently, the mixture was washed with 10 mL of methanol, the supernatant was removed, and the product was obtained by vacuum concentration. Finally, 10 mL of distilled water was added and freeze-dried to obtain a chitosan-butyrate combined polymer compound (a product in which both R1 and R2 in Chemical Formula 1 are polysubstituted with butyrate groups). (The yield is 62%)

[0111]

[0112] Example 4: Preparation of a Chitosan-Propionic Acid Monosubstituted Soluble Compound

[0113] Conjugate monosubstituted derivatives of chitosan and propionic acid were synthesized using chitosan and propionic acid as raw materials. MES was used as the buffer and EDC as the crosslinking agent; however, to ensure monosubstituted selectivity, the reaction was carried out under conditions of low equivalent EDC, low temperature, short duration, and no additional single substances added.

[0114] Specifically, 40 mg of chitosan, 20 mg of propionate, and 20 mg of crosslinking agent (EDC) were added to 8 mL of 10 mM MES buffer, and the mixture was stirred at 600 rpm for 2 hours at 0–4 °C. After confirming the completion of the reaction, the reaction mixture was precipitated in 25 mL of cold acetone and centrifuged at 2000 rpm for 4 minutes. After removing the supernatant, 10 mL of methanol (DMSO omitted if necessary) was added, sonicated for 3 minutes, and centrifuged; this methanol washing / sonicating / centrifuging process was repeated a total of 3 times. After washing, the supernatant was removed, and the product was concentrated under reduced pressure. The product was then dissolved in 10 mL of distilled water and freeze-dried to obtain the chitosan-propionic acid monosubstituted polymer compound (HS-mono) as the final product. (Yield 61%)

[0115]

[0116] [Example 5] Preparation of Chitosan-Gallic Acid Combined Soluble Compound

[0117] As shown in Reaction Scheme 2 below, a chitosan-gallic acid conjugation compound was prepared using the EDC / NHS bioconjugation reaction mechanism with chitosan and gallic acid as starting materials.

[0118] Specifically, 40 mg of chitosan, 20 mg of gallic acid, and 80 mg of 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide Hydrochloride (EDC) were added to 8 mL of 10 mM MES buffer, and the mixture was stirred at 600 rpm at room temperature for 24 hours. After confirming the completion of the reaction, the reaction mixture was precipitated in 25 mL of cold acetone and centrifuged at 2000 rpm for 4 minutes. After removing the supernatant, 10 mL of methanol and 10 mL of DMSO were added, sonicated for 3 minutes, and centrifuged; this centrifugation and purification process was repeated three times. The product was washed with 10 mL of methanol, the supernatant was removed, and the solution was concentrated under reduced pressure. Subsequently, 10 mL of distilled water was added, and the mixture was freeze-dried to prepare the chitosan-gallic acid solid solution compound. (Yield: 71%)

[0119] [Reaction Equation 2]

[0120]

[0121]

[0122] [Example 6] Preparation of a Chitosan-Caffeic Acid Combined Soluble Compound

[0123] The conjugate synthesis of chitosan and caffeic acid was performed using raw materials. The reaction was carried out at room temperature using MES as the buffer and EDC / NHS as the crosslinking agent.

[0124] Specifically, 40 mg of chitosan, 20 mg of caffeic acid, 80 mg of the crosslinking agent EDC·HCl, and 40 mg of NHS were added to 8 mL of 10 mM MES buffer (pH 5.2), and the mixture was stirred at 600 rpm at room temperature (25 ℃) for 24 hours. After the reaction was complete, the reaction solution was precipitated in 25 mL of cold acetone and centrifuged at 2000 rpm for 4 minutes. After removing the supernatant, 10 mL of methanol and 10 mL of DMSO were added, sonicated for 3 minutes, and centrifuged; the same purification process was repeated three times. Subsequently, the product was washed with 10 mL of methanol and concentrated under reduced pressure to obtain the product, which was then dispersed in 10 mL of distilled water and freeze-dried to obtain a chitosan-caffeic acid combined polymer compound. (Yield 66%)

[0125]

[0126] [Example 7] Preparation of a Chitosan-Ferulic Acid Combined Soluble Compound

[0127] Conjugate synthesis of chitosan and ferulic acid was performed using raw materials. The reaction was carried out at room temperature using MES as the buffer and EDC / NHS as the crosslinking agent.

[0128] Specifically, 40 mg of chitosan, 20 mg of ferulic acid, 80 mg of EDC·HCl, and 40 mg of NHS were added to 8 mL of 10 mM MES (pH 5.2) and stirred at 600 rpm at room temperature for 24 hours. Subsequently, the purification process was repeated three times: precipitation in 25 mL of acetone → centrifugation at 2000 rpm for 4 minutes → ultrasonic treatment with 10 mL of methanol + 10 mL of DMSO (3 minutes) → centrifugation. The chitosan-ferulic acid conjugated polymer compound was obtained by washing with 10 mL of methanol → concentration under reduced pressure → redispersion in 10 mL of distilled water → freeze-drying. (Yield 64%)

[0129]

[0130] [Example 8] Preparation of a Chitosan-Propionic Acid-Gallic Acid Soluble Compound

[0131] As shown in Reaction Scheme 3 below, to confirm the regioselectivity of propionate and gallic acid toward chitosan, chitosan-gallic acid / propionate conjugation compounds were prepared using the EDC / NHS bioconjugation reaction mechanism. The above 'regioselectivity' refers to the position of propionate or gallic acid introduced into various free amine (-NH2) functional groups of the short-chain fatty acids of chitosan.

[0132] Specifically, 40 mg of chitosan, 20 mg of propionate, 20 mg of gallic acid, and 80 mg of 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide Hydrochloride (EDC) were added to 8 mL of 10 mM MES buffer, and the mixture was stirred at 600 rpm at room temperature for 24 hours. After confirming the end of the reaction, the reaction mixture was precipitated in 25 mL of cold acetone and centrifuged at 2000 rpm for 4 minutes. After removing the supernatant, 10 mL of methanol and 10 mL of DMSO were added, sonicated for 3 minutes, and then centrifuged; the above centrifugation and purification process was repeated 3 times. The product was washed with 10 mL of methanol, the supernatant was removed, and the solution was concentrated under reduced pressure. Then, 10 mL of distilled water was added and the mixture was freeze-dried to obtain a chitosan-propionic acid-gallic acid combined highly soluble compound (Yield: 67%).

[0133] [Reaction Equation 3]

[0134]

[0135] The synthesis results of the product (HSA) prepared before, during, and after the reaction process and after the reaction was completed were obtained as the result shown in the imaged photograph of Fig. 1b.

[0136]

[0137] [Example 9] Synthesis of Chitosan-Propionate-Caffeic Acid Combined Polymer Compound

[0138] To confirm the regioselectivity of propionate and caffeic acid toward chitosan, chitosan-caffeic acid / propionate conjugation compounds were prepared using the EDC / NHS bioconjugation reaction mechanism. The above 'regioselectivity' refers to the substitution positions of propionate or caffeic acid introduced into various free amine (-NH₂) functional groups of the short-chain fatty acids of chitosan.

[0139] Specifically, 40 mg of chitosan, 20 mg of propionate, 20 mg of caffeic acid, and 80 mg of 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide Hydrochloride (EDC) were added to 8 mL of 10 mM MES buffer, and stirred at 600 rpm at room temperature for 24 hours.

[0140] After the reaction was complete, the reaction solution was precipitated in 25 mL of cold acetone and centrifuged at 2000 rpm for 4 minutes. After removing the supernatant, 10 mL of methanol and 10 mL of DMSO were added, sonicated for 3 minutes, and centrifuged; this purification process was repeated three times. Subsequently, the solution was washed with 10 mL of methanol, the supernatant was removed, and the mixture was concentrated under reduced pressure. The product was dissolved in 10 mL of distilled water and freeze-dried to obtain a chitosan-propionic acid-caffeic acid conjugated polymer compound. (Yield: 65%)

[0141]

[0142] [Example 10] Synthesis of Chitosan-Propionate-Ferulic Acid Combined Polymer Compound

[0143] To confirm the regioselectivity of propionate and ferulic acid toward chitosan, chitosan-ferulic acid / propionate conjugation compounds were prepared using the EDC / NHS bioconjugation reaction mechanism. The above 'regioselectivity' refers to the substitution positions of propionate or ferulic acid introduced into various free amine (-NH₂) functional groups of the short-chain fatty acids of chitosan.

[0144] Specifically, 40 mg of chitosan, 20 mg of propionate, 20 mg of ferulic acid, and 80 mg of 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide Hydrochloride (EDC) were added to 8 mL of 10 mM MES buffer, and stirred at 600 rpm at room temperature for 24 hours.

[0145] After the reaction was complete, the reaction solution was precipitated in 25 mL of cold acetone and centrifuged at 2000 rpm for 4 minutes. After removing the supernatant, 10 mL of methanol and 10 mL of DMSO were added, sonicated for 3 minutes, and centrifuged; this purification process was repeated three times. Subsequently, the solution was washed with 10 mL of methanol, the supernatant was removed, and the mixture was concentrated under reduced pressure. The product was dissolved in 10 mL of distilled water and freeze-dried to obtain a chitosan-propionic acid-ferulic acid conjugated polymer compound. (Yield: 63%)

[0146]

[0147] [Experimental Example 1] Purification of Product and Identification of Synthetic Product (NMR)

[0148] (1) Example 1 Purification of product and identification of synthesized product (NMR)

[0149] Purification was performed on the polysubstituted solubility compound of propionic acid produced in Example 1 above. Purification was carried out in an electrolytic solvent of acetone and methanol in a 3:1 ratio.

[0150] The result is shown in the image of Fig. 2. Here, the purified state of the final conjugated synthetic highly soluble compound can be confirmed.

[0151] 1D-NMR was performed to identify novel compounds of the highly soluble synthetic material of Chemical Formula 1. The results are shown in Fig. 3a.

[0152] The samples used for 1D-NMR are data showing the synthesis results for various samples, including the raw materials used in the example, chitosan and propionate, and the sample (CHP) prepared in the example. The other samples being compared here are also chitosan-propion-gallic acid combined highly soluble compounds (CHPG) prepared by a method similar to that of the above example.

[0153] As shown in Fig. 3a, it was confirmed that the peak of propionic acid used as a raw material component appeared differently from that of the synthetic sample (CHP), confirming that it was manufactured as a novel polymer synthetic.

[0154] <Propionate의 메틸 신호(0.91.5 ppm), Chitosan의 특유한 스펙트럼 패턴을 기준으로 확인>

[0155] Spectral analysis of highly soluble compounds (HS) based on short-chain fatty acids: When Chitosan and Propionate are combined, an additional peak reflecting the new bond (specific chemical shift) should appear.

[0156] Upon confirming the changes in the peak positions and patterns of Chitosan and Propionate, it was found that both the Propionate signal (approx. 12.2 ppm) and the Chitosan signal (35 ppm) were maintained. It was confirmed that the chemical shift position or intensity of the existing signals changed slightly due to the binding of Chitosan and Propionate, with a signal associated with the new binding appearing at approximately 2–2.5 ppm. Therefore, it was confirmed that the synthesis of CHP was successfully achieved.

[0157] From the above NMR confirmation results, it was confirmed that a new highly soluble compound is prepared by reacting chitosan and propionate to form a peptide bond by reacting the amine group of chitosan with the carboxyl group of propionate and confirming that the propionate group is attached.

[0158]

[0159] (2) Example 8 Purification of product and identification of synthesized product (NMR)

[0160] Purification was performed on the highly soluble compounds produced in Examples 5 and 8 above. Purification was carried out in an electrolytic solvent of acetone and methanol in a 3:1 ratio.

[0161] The result is shown in the image of Fig. 2. Here, the purified state of the final conjugated synthetic highly soluble compound can be confirmed.

[0162] 1D-NMR was performed to confirm the novel synthesis of the polymer synthetic material of Chemical Formula 1. The results are shown in Fig. 3b.

[0163] The samples used for 1D-NMR are each raw material used and the chitosan-propionic acid combined solubility compound (CHP) of Example 1 and the chitosan-propion-gallic acid combined solubility compound (CHPG) of Example 3.

[0164] As shown in Fig. 3b, it is confirmed that the peak of gallic acid used as a raw material component does not appear in the composite (composite sample CHPG using gallic acid), and it is also confirmed that the peak of chitosan shows a significant change in each composite (CHP, CHPG), thus confirming that it was manufactured as a novel polymer composite.

[0165] <Gallic acid의 방향족 영역 (약 6.58 ppm)과 Propionate의 메틸 신호(0.91.5 ppm), Chitosan의 특유한 스펙트럼 패턴을 기준으로 확인>

[0166] The signal of CHP is preserved, and the signal of the aromatic region of gallic acid (around 6.5–8 ppm) was clearly observed in the HSA spectrum. Additionally, due to the introduction of gallic acid, a slight new signal appears between 6 and 8 ppm, which indicates that gallic acid was incorporated during the synthesis of HSA. This confirms that the basic structure was not damaged, as the signals of propionate (12.2 ppm) and chitosan (35 ppm) in HSA are also preserved.

[0167] From the above NMR confirmation results, it was confirmed that a new highly soluble compound is prepared by reacting chitosan with propionate and gallic acid to form peptide bonds through the reaction of the amine group of chitosan with the carboxyl group of propionate and gallic acid, and due to the effect of propionate and gallic acid being able to alternately bind.

[0168]

[0169] [Experimental Example 2] Purification of Product and Identification of Synthetic Product (IR)

[0170] (1) Example 1 Purification of product and identification of synthesized product (IR)

[0171] FT-IR analysis was performed to confirm whether the chitosan-propionic acid polysubstituted compound (HS) according to Example 1 above was synthesized. The results are as shown in Fig. 4a.

[0172] The samples were analyzed by classifying them into chitosan, sodium propionate, and synthesized chitosan-propionic acid substituent derivatives (HS).

[0173] The analysis was performed using the ATR (accessory) built into the JASCO FT / IR-4X instrument, and the measurement conditions were set to a range of 4000-650 cm⁻¹, a resolution of 4 cm⁻¹, and an average of 32 scans.

[0174] <Analysis of Experimental Results>

[0175] As shown in Fig. 4a, in the case of chitosan alone, the characteristics of OH / NH stretch at around 3300 cm⁻¹, Amide I (C=O stretch) at 1650 cm⁻¹, and Amide II (NH bend) at 1590 cm⁻¹ were confirmed.

[0176] In sodium propionate alone, distinct peaks of 1550 cm⁻¹ (ν_as COO) and 1400 cm⁻¹ (ν_s COO) were observed.

[0177] In the synthesized HS sample, the amide peak of chitosan and the COO peak of propionic acid were observed simultaneously, and in particular, the COO- asymmetric elongation band (1550 cm⁻¹) and symmetric elongation band (1400 cm⁻¹) overlapped with the chitosan backbone signal, confirming positional displacement and broadening phenomena.

[0178] This shows that a new chemical environment is formed by the combination of the amine group (-NH₂) or hydroxyl group (-OH) of chitosan and the carboxyl group (-COOH / COO-) of propionic acid.

[0179] Significance of Experimental Results

[0180] Through the above FT-IR analysis, it was confirmed that the binding between chitosan and propionic acid was successfully achieved and a chitosan-propionic acid derivative was synthesized.

[0181] From this result, it was confirmed that the chitosan derivative compound prepared in Example 1 above is not a simple physical mixture, but a novel compound synthesized based on chemical / non-covalent interactions.

[0182]

[0183] (2) Example 8 Purification of product and identification of synthesized product (IR)

[0184] FT-IR analysis was performed to confirm whether the chitosan-propionic acid combined compound prepared in Example 8 above was synthesized. The samples were classified into chitosan-propionic acid-gallic acid derivatives (HSA) synthesized from chitosan, sodium propionate, and gallic acid.

[0185] The analysis was performed using the ATR (accessory) built into the JASCO FT / IR-4X instrument, and the measurement conditions were set to a range of 4000-650 cm⁻¹, a resolution of 4 cm⁻¹, and an average of 32 scans.

[0186] <Analysis of Experimental Results>

[0187] As shown in Fig. 4b, in the propionate (SCFA) monotherapy group, the -COO asymmetric stretching vibration peak was identified at 1550 cm⁻¹ and the symmetric stretching vibration peak at 1400 cm⁻¹.

[0188] In the gallic acid-only group, the -OH broad band was observed at around 3300 cm⁻¹, the -C=O stretching peak was detected at 1700 cm⁻¹, and the aromatic C=C peak was detected at 1600 cm⁻¹.

[0189] In the chitosan-only group, -OH and -NH broad bands were observed at 3300 cm⁻¹, and a -COC peak was observed at 1070 cm⁻¹.

[0190] In the spectrum of the chitosan-propionic acid-gallic acid conjugate (HAS), compared to the SCFA alone group, the -COO (1550, 1400 cm⁻¹) peak shifted toward the lower wavenumber of about 7–12 cm⁻¹, and the peak intensity decreased.

[0191] The -OH stretching band (3300 cm⁻¹) of gallic acid was broadened and widened, which was confirmed by an increase in FWHM.

[0192] In addition, a new amide / ester-related band was observed around 1650-1630 cm⁻¹, suggesting the formation of amide bonds between the -NH₂ of chitosan and the -COOH of SCFA or gallic acid.

[0193] The above peak shift (Δν_as, Δν_s) and broadening phenomena reflect hydrogen bonding, non-covalent interactions, and the formation of amide bonds.

[0194] In particular, the shift of the -COO peak and the broadening of the -OH band indicate that binding and complex formation have occurred between the SCFA and polyphenol components.

[0195] Significance of Experimental Results

[0196] Through the above FT-IR analysis, it was confirmed that the binding between chitosan, propionic acid, and gallic acid was successfully formed, and a chitosan-propionic acid-gallic acid derivative was synthesized.

[0197] From these results, it was scientifically proven through FT-IR analysis that the chitosan derivative prepared in Example 8 was not a simple mixture, but rather a new chemical environment containing intermolecular hydrogen bonds and amide bonds was formed.

[0198]

[0199] [Experimental Example 3] Solubility Confirmation Experiment

[0200] (1) Confirmation of solubility of Example 1

[0201] The following experiment was performed to confirm the solubility of the highly soluble compound prepared in Example 1 above.

[0202] In this experiment, the solubility of the novel substance was evaluated at various concentrations (from 10 mg / mL to 1 mg / mL) to confirm its physical properties and analyze its potential for application. Each sample at each concentration was vigorously mixed for 1 minute and then left at room temperature (25°C) for 10 minutes. The degree of floating or sedimentation of the sample was measured by visually observing the sample and labeling complete dissolution as "completely dissolved (++)," partial dissolution (+), and insolubility (-) as cases where precipitation was clearly observed.

[0203] The results of the above experiment are as shown in the table below.

[0204] Concentration (mg / mL) Solubility State Airborne Emission Remarks 10 Insoluble (-) Partial precipitation observed 7 Partially soluble (+) Small amount of precipitation observed 5 Partially soluble (+) Small amount of precipitation observed 3 Completely soluble (++) No airborne emission Estimated at dissolution critical concentration 1 Completely soluble (++) No airborne emission

[0205] (2) Confirmation of solubility of Example 8

[0206] The following experiment was performed to confirm the solubility of the highly soluble compound prepared in Example 8 above.

[0207] The solubility of the novel substance was evaluated at various concentrations (from 10 mg / mL to 1 mg / mL) to confirm its physical properties and analyze its potential for application. Samples at each concentration were vigorously mixed for 1 minute and then left at room temperature (25°C) for 10 minutes. The degree of floating or sedimentation of the samples was measured by visually observing the sample and labeling complete dissolution as "Completely Dissolved (++)", partial dissolution (+) when some precipitation was observed, and insolubility (-) when precipitation was clearly observed.

[0208] The results of the above experiment are as shown in the table below.

[0209] Concentration (mg / mL) Solubility State Airborne Emission Remarks 10 Insoluble (-) Partial precipitation observed 7 Partially soluble (+) Small amount of precipitation observed 5 Partially soluble (+) Small amount of precipitation observed 2.5 Completely soluble (++) No airborne emission Estimated at dissolution critical concentration 1 Completely soluble (++) No airborne emission

[0210] [Experimental Example 4] Solubility Comparison Experiment

[0211] For the chitosan-propionate monosubstituted (HS-mono) prepared in Example 4 and the polysubstituted (HS-di) polymer compounds prepared in Example 1, the solubility was evaluated under the following conditions.

[0212] <Solvent / Temperature Conditions>

[0213] - Purified water (or 10 mM phosphate buffer solution pH 7.0), room temperature (25 ℃)

[0214] - The concentration ranges were set to 10, 7, 5, 3, and 1 mg / mL. Mixing: Samples of each concentration were vigorously mixed for 1 minute and then left at room temperature for 10 minutes.

[0215] Judgment Criteria

[0216] - Completely dissolved(++): Clear, no visible precipitate / suspended particles

[0217] - Partially Dissolved (+): Slight turbidity, small amount of precipitate or suspended matter observed

[0218] - Insoluble(-): Distinct precipitation or layer separation

[0219] - Observation Items: Dissolution status (++, +, -), presence or absence of floating or sedimentation

[0220] - Note: Under the same conditions, HS-mono (single substitution) is N-substitution centered (partial dissolution expected), while HS-di (multi-substitution) is N+O multi-substitution (high dissolution expected)

[0221] <Analysis of Experimental Results>

[0222] As a result of this experiment, the chitosan-propionate monosubstituted derivative (HS-mono) exhibited complete dissolution (++) at concentrations of 1 mg / mL or lower, but switched to partial dissolution (+) in the 3-10 mg / mL range, with a small amount of precipitation observed. This is interpreted as the dispersion stability being partially limited under high concentration conditions due to residual interchain hydrogen bonds and a lack of ionization sites in the monosubstituted structure.

[0223] In this experiment, the results of the evaluation of the solubility state according to the concentration of HS-mono (single substitution) according to Example 4 above are as shown in the following table.

[0224] Concentration (mg / mL) Solubility Aeration Remarks 10 Insoluble (-) Partial precipitation observed 7 Partially soluble (+) Small amount of precipitation observed 5 Partially soluble (+) Small amount of precipitation observed 3 Partially soluble (+) Fine precipitation observed 1 Completely soluble (++) No dispersion 0.5 Completely soluble (++) No dispersion / precipitation

[0225] In this experiment, as a solubility evaluation conducted for comparison with the above experimental results, the solubility of the HS-di (polysubstituted) substance of Example 1 was evaluated at different concentrations, and the results are shown in the table below.

[0226] Concentration (mg / mL) Solubility State Airborne Degree Remarks 10 Partially dissolved (+) Weak precipitation observed High solubility 7 Partially dissolved (+) Fine precipitation observed 5 Partially dissolved (+) Fine precipitation observed 3 Completely dissolved (++) No airborne / precipitation 1 Completely dissolved (++) No airborne / precipitation 0.5 Completely dissolved (++) No airborne / precipitation

[0227] Comparing the experimental results above, it was confirmed that the chitosan-propionate polysubstituted derivative (HS-di) maintained a state of complete dissolution (++) in the 0.5-3 mg / mL range, while only fine to weak precipitation was observed in the 5-10 mg / mL range. This is judged to be the result of the polysubstituted structure (Example 1) having relatively superior hydrophilicity compared to the monosubstituted structure (Example 4), and significantly improved dispersibility in aqueous solution due to the weakening of inter-chain bonds. Therefore, both chitosan derivatives having monochain fatty acid substituents in Examples 1 and 4 showed excellent water solubility. However, in the case of the polysubstituted derivative of Example 1, despite having the same SCFA-based structure, it significantly expanded the allowable dissolution concentration range under neutral conditions, thereby more clearly demonstrating its applicability to high-concentration liquid formulations.

[0228] Significance of Experimental Results

[0229] As shown in the experimental results, chitosan derivatives having short-chain fatty acid substituents are identified as highly soluble compounds.

[0230] However, derivatives with multiple single-chain fatty acid substituents extended the allowable solubility concentration range under neutral conditions compared to monosubstituted derivatives. This implies that dispersion stability in aqueous solution was significantly improved, attributed to increased hydrophilicity and weakened interchain bonding.

[0231] Therefore, the chitosan derivative having a short-chain fatty acid substituent of the present invention clearly supports the excellent applicability of high-concentration liquid formulations.

[0232]

[0233] [Experimental Example 5] Experiment on NO inhibition and anti-inflammatory effect

[0234] (1) NO inhibition of Example 1

[0235] The following experiment was performed to confirm the efficacy of the highly soluble compound prepared in Example 1 above.

[0236] To confirm the anti-inflammatory efficacy, the expression level of NO (nitric oxide) was measured using the Raw 264.7 cell line. NO is a key mediator of the inflammatory response, and excessive NO production is known to be a major cause of tissue damage and chronic inflammation. RAW 264.7 cells are a standard model for inflammatory response research, and the anti-inflammatory activity was verified by measuring the inhibitory effect of the novel substance on NO production in cells stimulated with LPS (lipopolysaccharide).

[0237] The experimental results for confirming such anti-inflammatory effects are shown in Fig. 5a. As a result of this experiment, all samples of the above examples at different concentrations showed anti-inflammatory effects, and in particular, NO production was inhibited to the highest level even at a concentration of 100 pg / ml. From this, it can be confirmed that it is effective in alleviating inflammatory responses even at low concentrations.

[0238]

[0239] (2) NO inhibition of Example 8

[0240] The following experiment was performed to confirm the efficacy of the highly soluble compound prepared in Example 8 above.

[0241] To confirm the anti-inflammatory efficacy, nitric oxide (NO) expression levels were measured using the Raw 264.7 cell line. NO is a key mediator of inflammatory responses, and excessive NO production is known to be a major cause of tissue damage and chronic inflammation. RAW 264.7 cells are a standard model for inflammatory response research, and the anti-inflammatory activity was verified by measuring the inhibitory effect of the novel substance on NO production in cells stimulated with lipopolysaccharide (LPS).

[0242] The experimental results obtained are as shown in Figure 5b. As a result of these experiments, NO production was inhibited even at a concentration of 100 pg / ml in all examples, confirming that it is effective in alleviating inflammatory responses even at low concentrations.

[0243]

[0244] [Experimental Example 6] Experiment on Anti-inflammatory Effect of Inhibiting Inflammatory Cytokine Expression

[0245] (1) Inhibition of inflammatory cytokine expression in Example 1

[0246] RAW 264.7 macrophages were used to confirm and compare the anti-inflammatory efficacy of the polymer compound (HS) prepared in Example 1 above.

[0247] Cells were cultured in DMEM medium containing 10% FBS and 1% penicillin / streptomycin and used in experiments at 70-80% confluent.

[0248] After pretreatment with HS at concentrations of 0.0001, 0.0005, and 0.0025 mg / mL for 24 hours, inflammation was induced by treatment with LPS (100 ng / ml) for 18 hours.

[0249] To confirm the anti-inflammatory effect of HS, the expression of iNOS, COX-2, IL-6, IL-1β, and TNF-α proteins was analyzed by Western blot. β-Actin was used as a control protein.

[0250] Statistical analysis was performed for each group using n=3 independent replicates, and p<0.05 was set as the statistical significance criterion.

[0251] The experimental results are shown in Fig. 6a.

[0252] <Analysis of Experimental Results>

[0253] Inhibition of iNOS and COX-2 expression

[0254] iNOS and COX-2 expression significantly increased upon LPS treatment, but decreased in a concentration-dependent manner in the HS treatment group. In particular, iNOS and COX-2 expression were significantly inhibited at 0.0025 mg / mL (p<0.05).

[0255] IL-1β and IL-6 inhibition

[0256] IL-1β and IL-6 expression significantly increased in the LPS-treated group, and expression levels decreased upon HS treatment. IL-1β was inhibited to a level close to the control group at high concentrations of HS (0.0025 mg / mL). A trend of attenuation was also observed for IL-6.

[0257] Regulation of TNF-α expression

[0258] A decreasing trend in TNF-α expression was observed in the HS treatment group, confirming that it suppressed the expression of inflammatory cytokines.

[0259] Significance of Experimental Results

[0260] The HS prepared in the above example inhibited iNOS and COX-2 expression in the LPS-induced inflammatory response of RAW 264.7 cells, exhibiting effects of inhibiting NO production and reducing inflammatory mediators.

[0261] In addition, it was confirmed that it contributes to the suppression of inflammatory signals and the maintenance of immune homeostasis by inhibiting the expression of inflammatory cytokines such as IL-1β, IL-6, and TNF-α.

[0262] These experimental results confirm that polymeric compounds of chitosan derivatives having short-chain fatty acid substituents are functional materials with anti-inflammatory efficacy, and demonstrate the potential for various industrial applications such as health functional foods and pharmaceutical compositions.

[0263]

[0264] (2) Inhibition of inflammatory cytokine expression in Example 8

[0265] RAW 264.7 mouse macrophages were used to confirm the anti-inflammatory efficacy of the chitosan-SCFA-phenolic acid conjugate (HSA) prepared in Example 8 above. The cells were cultured in DMEM medium containing 10% FBS and 1% penicillin / streptomycin and used in the experiment at 70-80% confluent.

[0266] After pretreatment with HSA at concentrations of 0.0001, 0.0005, and 0.0025 mg / mL for 24 hours, inflammation was induced by treatment with LPS (100 ng / ml) for 18 hours.

[0267] To confirm the anti-inflammatory effect of HSA, the expression of iNOS, COX-2, IL-1β, IL-6, and TNF-α was checked by Western blot, and β-Actin was used as a control protein.

[0268] Statistical analysis was performed for each group with independent replicates (n=3), and p<0.05 was set as the significance criterion.

[0269] The result is shown in Fig. 6b.

[0270] <Analysis of Experimental Results>

[0271] Inhibition of iNOS expression

[0272] iNOS expression was significantly increased in the LPS-treated group, but decreased in a concentration-dependent manner in the HSA-treated group. In particular, significant inhibition was observed compared to the LPS group at high concentrations (0.0025 mg / mL) (###, p<0.001).

[0273] COX-2 expression inhibition

[0274] COX-2 expression increased in the LPS-treated group, but significantly decreased in the high-concentration HSA group (0.0025 mg / mL) (###, p<0.001).

[0275] IL-1β expression

[0276] An increase was confirmed in the LPS-treated group, but no distinct decreasing trend was observed in the HSA-treated group. This suggests that the inhibitory effect on IL-1β is limited.

[0277] IL-6 expression inhibition

[0278] Expression increased significantly in the LPS-treated group, but a significant decrease was observed in the high-concentration HSA group (##, p<0.01).

[0279] TNF-α expression

[0280] Strong expression was observed in the LPS-treated group, but a tendency for the expression intensity to be attenuated was observed in the HSA-treated group.

[0281] Significance of Experimental Results

[0282] The HSA prepared in the above example significantly inhibited iNOS and COX-2 expression in an LPS-induced RAW 264.7 cell model, thereby effectively blocking the inflammatory mediating pathway.

[0283] In addition, inhibiting IL-6 expression alleviated inflammatory cytokine responses, and a decreasing trend in TNF-α was also confirmed.

[0284] These results confirm that the HSA of the present invention is a functional material with anti-inflammatory efficacy, and demonstrates the potential for various industrial applications such as health functional foods and pharmaceutical compositions.

[0285]

[0286] [Experimental Example 7] Experiment on the inhibitory effect of cell damage caused by oxidative stress

[0287] (1) Inhibition of cell damage caused by oxidative stress

[0288] To confirm other efficacy of the highly soluble compound prepared in Example 1 above, the following experiment was performed.

[0289] In this experiment, BV2 cell lines were used to evaluate cell damage caused by H₂O₂-induced oxidative stress and the protective effect against it using an MTT assay. H₂O₂ (hydrogen peroxide) is one of the reactive oxygen species (ROS) and is a common stimulus used in oxidative stress models; it depletes or disrupts the balance of intracellular antioxidant defense systems (e.g., SOD, glutathione), leading to cell damage and death. The cytoprotective effect was verified by confirming cell viability in the event of cell damage caused by H₂O₂.

[0290] The experimental results were obtained as shown in Figure 7a. As a result of these experiments, the expression of ROS significantly increased starting from a concentration of 1 ng / ml in all examples, confirming that it has the efficacy to inhibit cell damage and death caused by oxidative stress even at low concentrations in the ng / ml range.

[0291]

[0292] (2) Inhibition of cell damage caused by oxidative stress in Example 8

[0293] The following experiment was performed to confirm the efficacy of the highly soluble compound prepared in Example 8 above.

[0294] In this study, the cell damage of the examples caused by H₂O₂-induced oxidative stress and the protective effect were evaluated using the PC-12 cell line via the MTT assay.

[0295] H₂O₂ (hydrogen peroxide) is one of the reactive oxygen species (ROS) and is a stimulus group mainly used in oxidative stress models, which depletes or disrupts the balance of intracellular antioxidant defense systems (e.g., SOD, glutathione), leading to cell damage and death.

[0296] The cytoprotective effect was verified by confirming cell survival rates in cases of cell damage caused by H₂O₂.

[0297] The experimental results were obtained as shown in Figure 7b. As a result of these experiments, cell viability was significantly increased at a concentration of 5 ng / ml in all examples, confirming that it has the efficacy to inhibit cell damage and death caused by oxidative stress even at low concentrations in the ng / ml range.

[0298]

[0299] [Experimental Example 8] Experiment on the inhibitory effect of reactive oxygen species expression caused by oxidative stress

[0300] To confirm another efficacy of the highly soluble compound prepared in Example 1 above, the following experiment was performed.

[0301] In this experiment, the inhibition of reactive oxygen species expression levels in the example induced by H₂O₂-induced oxidative stress was evaluated using a BV2 cell line via an ROS assay. H₂O₂ (hydrogen peroxide) is one of the reactive oxygen species (ROS) and is a common stimulus used in oxidative stress models; it depletes or disrupts the balance of intracellular antioxidant defense systems (e.g., SOD, glutathione), leading to cell damage and death. The efficacy of inhibiting oxidative stress was confirmed by verifying the amount of ROS expression induced by H₂O₂.

[0302] The experimental results were obtained as shown in Figure 8. As a result of these experiments, cell viability significantly increased starting from a concentration of 1 ng / ml in all examples, and it was confirmed that it has the efficacy to significantly inhibit the expression of ROS even at low concentrations in the ng / ml range.

[0303]

[0304] [Experimental Example 9] Experiment on the inhibitory effect of neuroinflammation (protein phosphorylation)

[0305] To confirm another efficacy of the highly soluble compound prepared in the above example, the following experiment was performed.

[0306] In this experiment, BV2 cell lines were used to evaluate the regulation of the inflammatory response caused by cell damage in Example 1 due to H₂O₂-induced oxidative stress through western blot. H₂O₂ (hydrogen peroxide) is one of the reactive oxygen species (ROS) and is a stimulus commonly used in oxidative stress models; it depletes or disrupts the balance of intracellular antioxidant defense systems (e.g., SOD, glutathione), leading to cell damage and apoptosis. We investigated whether the phosphorylation of the p38 protein, the most representative protein in the MAPk pathway, caused by oxidative stress induced by H₂O₂ was inhibited.

[0307] The experimental results were obtained as shown in Figure 9. As a result of these experiments, cell viability was significantly increased starting from a concentration of 1 ng / ml in all examples, and it was confirmed that it has the efficacy to significantly inhibit the phosphorylation of p38 even at low concentrations in the ng / ml range.

[0308]

[0309] As a result of the above experiments, Chemical Formula 1, a highly soluble compound based on short-chain fatty acids represented by Chemical Formula 1, is a newly synthesized novel substance with excellent solubility, and it is confirmed to be effective against diseases related to inflammation, oxidative stress, and neuroinflammation, particularly because it has excellent antioxidant, anti-inflammatory, and neuroinflammation inhibitory effects and excellent neurocellular activity.

[0310]

[0311] [Experimental Example 10] Cytotoxicity Test MTT assay, RAW 264.7)

[0312] (1) Evaluation of cytotoxicity of Example 1

[0313] a) Experimental method

[0314] To evaluate the cytotoxicity of the compound (HS) prepared in Example 1 above, an MTT assay was performed using the mouse-derived macrophage cell line RAW 264.7. First, RAW 264.7 cells were cultured in DMEM medium (10% FBS, 1% penicillin-streptomycin) and maintained stably at 37°C under 5% CO₂ conditions. Subsequently, the cells were placed in a 96-well plate at a volume of 2.5 × 10⁶ 4 Cells were seeded at a density of cells / well and allowed to adhere for 24 hours to ensure sufficient proliferation and stabilization. To establish treatment groups, the compound HS of the present invention was added at concentrations of 0.0001, 0.0025, 0.005, 0.01, 0.025, 0.05, and 0.1 μg / mL, while cells treated only with the medium were designated as the control group. All samples were treated under identical conditions for 24 hours. After treatment, an MTT solution at a concentration of 0.2 mg / mL was added to each well and reacted for 3–4 hours to induce formazan production by intracellular mitochondrial enzymes. After the reaction was complete, the supernatant was removed, the generated formazan crystals were dissolved in DMSO, and the absorbance was measured at 570 nm. Statistical significance was tested using one-way ANOVA, and a p-value < 0.05 was considered statistically significant.

[0315] b) Experimental Results and Analysis

[0316] Cell viability was maintained in the range of approximately 95-103% at all test concentrations (0.0001-0.1 μg / mL), and no significant decrease was observed compared to the control group (see graph in Fig. 10a).

[0317] There were slight variations at some concentrations (e.g., 0.0025 μg / mL), but they were not statistically significant.

[0318] Even at a high concentration of 0.1 μg / mL, the survival rate was maintained at approximately 98-100%, confirming the absence of signs of acute cytotoxicity.

[0319] c) Significance based on results

[0320] The compound (HS) has a safety margin in which no cytotoxicity is detected up to 0.1 μg / mL in RAW 264.7 cells.

[0321] The concentration within this safety margin covers the range used in the previous anti-inflammatory efficacy test (LPS model), thus supporting the simultaneous satisfaction of efficacy and safety.

[0322]

[0323] (2) Evaluation of cytotoxicity of Example 8

[0324] ① Experimental Method

[0325] To evaluate the cytotoxicity of the compound (HSA) of Example 7 above, an MTT assay was performed using the mouse-derived macrophage cell line RAW 264.7. First, RAW 264.7 cells were cultured in DMEM medium (10% FBS, 1% penicillin-streptomycin) and maintained stably at 37°C under 5% CO₂ conditions. Subsequently, the cells were placed in a 96-well plate at a volume of 2.5 × 10⁶ 4Cells were seeded at a density of cells / well and allowed to adhere for 24 hours to ensure sufficient proliferation and stabilization. To establish treatment groups, the aforementioned synthetic compound HSA was added at concentrations of 0.0001, 0.0025, 0.005, 0.01, 0.025, 0.05, and 0.1 μg / mL, while cells treated only with the medium were designated as the control group. All samples were treated under identical conditions for 24 hours. After treatment, a 0.2 mg / mL MTT solution was added to each well and reacted for 3–4 hours to induce formazan production by intracellular mitochondrial enzymes. After the reaction was complete, the supernatant was removed, the generated formazan crystals were dissolved in DMSO, and the absorbance was measured at 570 nm. Statistical significance was tested using one-way ANOVA, and a p-value < 0.05 was considered statistically significant.

[0326] ② Experimental Results and Analysis

[0327] As shown in Figure 10b, cell viability was maintained at over 90% in all concentration ranges, and no significant cytotoxicity was observed even at high concentrations (0.1 μg / mL).

[0328] In particular, at low concentrations (0.0001–0.01 μg / mL), it showed a survival rate similar to the control group, and even at high concentrations (0.05–0.1 μg / mL), the cell survival rate was maintained at over 95%, confirming that there was almost no toxicity.

[0329] ③ Significance based on the result

[0330] HSA has a safety margin in which no cytotoxicity is detected up to 0.1 μg / mL in RAW 264.7 cells.

[0331] The concentration within this safety margin covers the range used in the previous anti-inflammatory efficacy test (LPS model), thus supporting the simultaneous satisfaction of efficacy and safety.

[0332]

[0333] As described above, specific embodiments of the present invention have been explained in detail but are not limited thereto. Those skilled in the art who understand the technical concept of the present invention will be able to easily propose other inventions that are similar or degenerate, or other embodiments included within the scope of the concept of the present invention, by adding, changing, or deleting other components within the same scope. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

[0334] The scope of the present invention is defined by the claims set forth below rather than by the description of the invention above, and all modifications or variations derived from the meaning and scope of the claims and equivalent concepts thereof should be interpreted as being included within the scope of the present invention.

Claims

1. A highly soluble compound comprising the following chemical formula 1 as a repeating unit, or a highly soluble compound based on a water-soluble substituent that is a pharmaceutically acceptable salt thereof: [Chemical Formula 1] In the above chemical formula 1, R1, R2 is a hydrogen atom, a short-chain fatty acid group, or a phenolic functional group, respectively, and R1 and At least one of R2 is a short-chain fatty acid group or a phenolic functional group, and R1 and R2s may all be the same or different short-chain fatty acid groups or phenolic functional groups, and n is an integer from 1 to 100,000.

2. A highly soluble compound based on a water-soluble substituent according to Claim 1, wherein the single-chain fatty acid group coupled to Formula 1 is a substituent composed of a single-chain fatty acid selected from substituents composed of acetic acid, propionic acid, butyric acid, isobutyric acid, lactic acid, valeric acid, caprylic acid, or aldehyde derivatives thereof, esters thereof, or acceptable salts thereof.

3. A highly soluble compound based on a water-soluble substituent according to Claim 1, wherein the phenolic functional group bonded to Formula 1 is a phenolic functional group derived from anthocyanin, gallic acid, hydroxybenzoic acids, caffeic acid, ferulic acid, protocatechuic acid, vanillic acid, syringic acid, pyrogallol, chlorogenic acid, or tannic acid.

4. A highly soluble compound based on a water-soluble substituent having both a short-chain fatty acid group and a phenolic functional group in Formula 1, according to Claim 1.

5. An anti-inflammatory composition containing the highly soluble compound based on a short-chain fatty acid of claim 1 as an active ingredient.

6. A health food for improving inflammation comprising an anti-inflammatory composition according to claim 4.

7. A composition for improving neuroinflammation or neurodegenerative diseases containing the highly soluble compound based on a short-chain fatty acid of claim 1 as an active ingredient.

8. A health food for improving mild cognitive impairment comprising the composition according to claim 7 as an active ingredient.

9. A pharmaceutical composition for the improvement, prevention, or treatment of neuroinflammation, neurodegenerative disease, or inflammatory disease comprising the composition according to claim 7 as an active ingredient.

10. A pharmaceutical composition for the prevention and treatment of Alzheimer's disease, Parkinson's disease, Lou Gehrig's disease (ALS), cognitive impairment, senile dementia, Lewy body dementia, frontotemporal dementia, Huntington's disease dementia, vascular dementia, myocardial infarction, arteriosclerosis, ischemia-reperfusion injury, type 2 diabetes mellitus, obesity, metabolic syndrome, rheumatoid arthritis, inflammatory bowel disease (IBD), chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, inhibition of photoaging, atopic dermatitis, anticancer, alleviation of anticancer side effects, anti-aging, macular degeneration (AMD), diabetic retinopathy, acute renal injury (AKI), and chronic renal disease associated with inflammation, oxidative stress, or neuroinflammation, comprising as an active ingredient a highly soluble compound containing the following chemical formula 1 as a repeating unit or a pharmaceutically acceptable salt thereof, wherein the composition is a highly soluble compound based on a short-chain fatty acid. [Chemical Formula 1] In the above chemical formula 1, R1, R2 is a hydrogen atom, a short-chain fatty acid group, or a phenolic functional group, respectively, and R1 and At least one of R2 is a short-chain fatty acid group or a phenolic functional group, and R1 and R2s may all be the same or different short-chain fatty acid groups or phenolic functional groups, and n is an integer from 1 to 100,000.

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