KR20090092179A - Composition containing bioavailable fucoidan for preventing or treating inflammatory diseases - Google Patents

Abstract

A composition for preventing and treating inflammatory diseases, which contains bioavailable low molecular fucoidan is provided to suppress the expression of cytokines such as TNF-alpha (tumor necrosis factor-alpha) and MMP-9 (matrix metalloproteinase-9). A composition for preventing and treating inflammatory diseases comprises a bioavailable low molecular fucoidan as an active ingredient. The bioavailable low molecular fucoidan has the sulfate content of 20-40%, average molecular weight of 500-5,000 Da, polydispersity index of 1-6, and viscosity of 0-200 centi poise. The component ratio of the bioavailable low molecular fucoidan is carbon:hydrogen:oxygen:nitrogen:sulfur = 20-30:3-7:45-60:0.1-1.0:5.7-20.

Description

Composition Containing Bioavailable Fucoidan for Preventing or Treating Inflammatory Diseases}

The present invention relates to a composition for the prevention and treatment of inflammatory diseases comprising bioavailable low molecular weight fucoidan as an active ingredient.

Inflammation is a very complex phenomenon between cells and inflammation-related components that occur in tissues in response to injury caused by trauma, infection, post-ischemic, toxic or autoimmune reactions (Nathan, C., Nature 420: 846-852 (2002). Usually this process leads to recovery and treatment after infection, but if these processes are not done properly, inflammation can cause persistent tissue damage by inflammatory cells or collagen.

Rheumatoid arthritis is a relatively common chronic inflammatory disease that can lead to joint destruction despite treatment with antirheumatic agents, immunosuppressants and corticosteroids (Cunnane, G., KM, Arch). Immunol Ther Exp 46: 1-7 (1998). Overcoming the limitations of existing therapies requires a more accurate understanding of the pathogenesis of rheumatoid arthritis and the development of new therapies. Recently, biological agents that block tumor necrosis factor-α (TNF-α) and innerleukin-1β (IL-1β), which have been shown to be important for the pathogenesis of rheumatoid arthritis, have been reported to be very effective as a result of treatment. (Cutolo , M., S., Ann Rheum Dis 65: 728-35 (2006). However, many patients who do not respond to these biological agents are still incomplete and require more effective or complementary drugs. Rheumatoid arthritis is an autoimmune disease, which is caused by the development of autoantibodies such as autoreactive T cells and rheumatoid factors by genetic and external factors, which induce inflammation in synovial tissue. Autoreactive T cells play a key role in inflammation of synovial tissue and are known to regulate inflammatory processes through interactions with macrophages, synovial cells, dendritic cells, and B cells in synovial tissue (Sweeney, SE, Int J Biochem Cell Biol 36: 372-8 (2004). Various cell surface molecules or secretory proteins are involved in the interaction between these cells, in particular the interactions of TRANCE / RANK, CD40 / CD40L, GITR / GITRL, and LTβ / LTβR belonging to the TNF ligand superfamily and the TNF receptor superfamily. Has been reported to play an important role (Cuzzocrea, S., E., Faseb J 19: 1253-65 (2005); Eissner, G., W., Cytokine). Growth Factor Rev 15: 353-66 (2004). Synovial cells and macrophages activated by T cells secrete a variety of inflammation-promoting cytokines and matrix metalloproteases (MMPs) that degrade the extracellular matrix, leading to synovitis and bone-cartilage destruction. Very important as a joint destruction mechanism (Yanni, G., A., Ann) Rheum Dis 53: 39-44 (1994).

Currently, the treatment of arthritis is focused on attenuating symptoms rather than radical treatment. Most of these treatments are nonspecific immune controls that do not distinguish between immune and disease-related immune phenomena and are likely to have side-effects of decreased immune function along with treatment of rheumatoid arthritis. It can be said to be large. Treatment methods for rheumatoid arthritis that are currently used or developed in Korea and abroad include 1) anti-inflammatory drugs using nonsteroidal anti-inflammatory drugs (NSAIDs) or steroids, and 2) antirheumatic drugs or disease modifying antirheumatic drugs: DMARD), 3) Biologic agents: antibodies or soluble receptors (ENBREL, etc.) that inhibit the function of TNF-α, one of cytokines, 4) Gene therapy: Antagonists of IL-1 and TNF-α ) Or gene therapy expressing anti-inflammatory cytokines such as IL-10, TGF-β1, and 5) oral type II collagen immunotherapy (Sweeney, SE, Int J Biochem). Cell Biol 36: 372-8 (2004), Sweeney, SE, Curr Opin Rheumatol 16: 231-7 (2004). However, these methods remain only to alleviate the symptoms of rheumatoid arthritis and are not a fundamental treatment, so further research is needed for the fundamental treatment of rheumatoid arthritis.

For thousands of years since ancient times, herbal medicines and plant extracts have been widely used in the medical field and have been recognized as a resource for health care in many Asian countries, and for the purpose of treating and preventing certain diseases from these herbs and plant extracts. Ingredient and drug efficacy studies have been actively conducted in recent years.

Fucoidan is a viscous polymer in the form of viscous polymer with a weight average molecular weight of 30,000-2,000,000 Daltons, and is mainly contained in mucus of mollusks such as abalone and squid, or brown algae such as seaweed and kelp, especially fucoidan extracted from seaweed spores. The weight average molecular weight is about 170,000 Daltons.

The function of fucoidan derived from brown algae has been studied in Japan since the 1990s, and has been shown to enhance immune and anti-tumor effects, inhibit blood sugar rise and inhibit blood coagulation, lower triglycerides and cholesterol, and promote antioxidant and gastric ulcer healing. Research has been conducted on the back. ( Scand . J. Urol . Nephrol . , 40 (1) (2006); Am . J. Hematol ., 78 (1) (2005); Journal of the Korean Chemical Society , 46 (2) (2002); Monthly food industry November issue (2005)

However, despite the continuous study of fucoidan and its processed materials more advantageous for bioavailability, there have been no reports on the effects of fucoidan on inflammatory diseases, especially arthritis.

Throughout this specification, many papers and patent documents are referenced and their citations are indicated. The disclosures of cited papers and patent documents are incorporated herein by reference in their entirety, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained.

The present inventors have tried to develop a composition for the prevention and treatment of inflammatory diseases, in particular arthritis, as a result of analyzing the progress of arthritis by feeding a high-molecular, medium-molecular and low-molecular fucoidan in arthritis model mice, whereas the polymer fucoidan promotes the development of arthritis. The bioavailable low molecular weight fucoidan has completed the present invention by confirming that it inhibits the progression of arthritis.

Accordingly, an object of the present invention is to provide a composition for the prevention and treatment of inflammatory diseases comprising bioavailable low molecular weight fucoidan as an active ingredient.

Other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings.

According to an aspect of the present invention, the present invention provides a composition comprising (a) 20-40% sulfuric acid group content, (b) a weight average molecular weight of 500-5,000 Da, (c) a polydispersity index of 1-6 and (d) 0-200 It provides a composition for the prevention and treatment of inflammatory diseases comprising a bioavailable low-molecular fucoidan having a viscosity of centi-poise as an active ingredient.

The present inventors have tried to develop a composition for the prevention and treatment of inflammatory diseases, in particular arthritis. As a result of analyzing the progress of arthritis by feeding a high-molecular, medium-molecular and low-molecular fucoidan to arthritis model mice, the polymer fucoidan promotes the development of arthritis. On the other hand, small molecule fucoidan was confirmed to inhibit the progression of arthritis.

The low molecular weight fucoidan used as an active ingredient in the composition for preventing and treating inflammatory diseases of the present invention has a very small molecular weight as compared with the naturally-derived high molecular weight fucoidan (typically 170,000 Da or more).

The composition of the present invention comprises bioavailable fucoidan excellent in physical properties and physiological activity as an active ingredient. As used herein, the term “bioavailability” as used in connection with fucoidan means a low molecular weight fucoidan having excellent absorption rate in water, high water solubility and higher physiological activity than natural fucoidan of natural (occurring).

As used herein, the term “low molecular weight fucoidan” or “low molecular weight fucoidan” means a sugar containing a sulfate group, preferably a fucoidan oligomer comprising a fucose, galactose, xylose and / or gulcuronic acid, including a sulfate group, Typically refers to fucoidan oligomers having a weight average molecular weight of 500-5,000 Da.

According to a preferred embodiment of the present invention, the bioavailable low molecular weight fucoidan used as the active ingredient has a weight average molecular weight of 500-2,000 Da, more preferably 500-1,800 Da weight average molecular weight, most preferably 700-1,400 Da It has an average molecular weight.

As used herein, the term “weight average molecular weight” refers to the weight average molecular weight of fucoidan molecules having various molecular weights that may be present in the fucoidan of the present invention. For example, when the fucoidan for preventing and treating inflammatory diseases of the present invention is obtained by treating an organic acid with fucoidan of high molecular weight, there may be fucoidan molecules having various molecular weights in the treatment product, which are present in the organic acid treatment product. The weight average molecular weight of the fucoidan molecules is the weight average molecular weight described herein. For example, when the weight average molecular weight of the fucoidan of the present invention is 1,500-3,000 Da, preferably the weight average molecular weight 3000-5000 Da: 1250-1500 Da: 600-750 Da: 140-210 Da = 30-60: It may be a mixture of fucoidan having 12-35: 5-20: 15-25.

Therefore, according to a preferred embodiment of the present invention, the bioavailable low molecular weight fucoidan of the present invention has a weight average molecular weight of 1,300-1,700 Da: 300-500 Da: 100-200 Da: 0-100 Da = 35-55: 10-30: 20-40: Fucoidan mixture having 1-10, more preferably 1,300-1,700 Da: 300-500 Da: 100-200 Da: 0-100 Da = 40-50: 15-25: 25-35: 3-8 Mixture.

The bioavailable low molecular weight fucoidan of the present invention can be prepared by hydrolyzing the fucoidan of the polymer and obtaining a product thereof. As used herein, the term “polymer fucoidan” is a polymeric fucoidan extracted from various marine organisms such as seaweeds such as seaweed, kelp, shellfish and rhubarb and abalone, squid and starfish. The low molecular fucoidan of the present invention can be prepared from the polymer fucoidan using various methods. For example, polymeric fucoidans can be prepared using acids, such as pyruvic acid, citric acid, acetic acid or mixtures thereof. Pyruvic acid, citric acid, acetic acid or mixtures thereof used as edible organic acids are very efficient for hydrolyzing fucoidan polysaccharides. Although pyruvic acid and citric acid are not a problem even when developing a hydrolyzate of fucoidan as a food, it acts on high molecular weight fucoidan and efficiently hydrolyzes it to provide a desired low molecular weight fucoidan, and can control the molecular weight of such fucoidan. I confirmed that it was a mountain. In addition, as acetic acid used for hydrolysis, the thing of the form of edible vinegar, such as vinegar, synthetic vinegar, spirit vinegar, and other vinegar, can be used. The vinegar is an edible vinegar, the main component of acetic acid, brewed vinegar includes citric acid, lactic acid and succinic acid in addition to acetic acid, the other vinegar is composed of acetic acid.

According to a preferred embodiment of the present invention, the sulfuric acid group content of the bioavailable low molecular weight fucoidan of the present invention is 20-35%, most preferably 20-30%. Sulfate is a very important part of fucoidan represents the physiological activity (Biochem Pharmacol , 65, 2003). Thus, high sulfate content improves the ability of the bioavailable low molecular weight fucoidan of the present invention to treat inflammatory diseases.

The bioavailable low molecular weight fucoidan of the present invention has a polydispersity of 1-6, preferably 1-5, more preferably 1-4, and most preferably 1-3. . This small polydispersity index shows that the size distribution of the low molecular fucoidan of the present invention is narrow. Accordingly, the low molecular fucoidan of the present invention is a fucoidan with uniform physical or physiological properties, and when it is commercialized, a low molecular fucoidan product of uniform quality can be obtained.

The bioavailable low molecular fucoidan of the present invention exhibits very low viscosity. Preferably, 0-100 centipoise, most preferably 0-50 centipoise. The low viscosity of low molecular fucoidan means that the low solubility of fucoidan in water is nearly 100.

According to a preferred embodiment of the present invention, the component ratio of the bioavailable low molecular fucoidan is carbon: hydrogen: oxygen: nitrogen: sulfur = 20-30: 3-7: 45-60: 0.1-1.0: 5.7-20, and more. Preferably the carbon: hydrogen: oxygen: nitrogen: sulfur is 23-25: 4.3-5.1: 52-58: 0.1-0.6: 6.0-10.

The bioavailable low molecular weight fucoidan of the present invention exhibits excellent efficacy in the prevention and treatment of inflammatory diseases as shown in the following examples.

In the present specification, the term “inflammatory disease” includes a general inflammatory disease, and according to a preferred embodiment of the present invention, the inflammatory disease is atherosclerotic coronary artery disease, arthritis, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease or glomerulonephritis disease More preferably arthritis.

According to a preferred embodiment of the present invention, the bioavailable low-molecular fucoidan of the present invention inhibits the expression of the proinflammatory cytokine, TNF-α (tumor necrosis factor-α) and matrix metal protease-9 (MMP-9), so that inflammation It shows the therapeutic efficacy of the disease.

The compositions of the present invention may be prepared from pharmaceutical compositions and food compositions.

When the composition of the present invention is prepared as a pharmaceutical composition, the composition of the present invention comprises (a) a therapeutically effective amount of low molecular fucoidan; And (b) a pharmaceutically acceptable carrier.

Bioavailable low molecular fucoidans can be prepared as pharmaceutically acceptable salts according to methods conventional in the art.

As the pharmaceutically acceptable salt, acid addition salts formed by free acid are useful. Acid addition salts are prepared by conventional methods, for example by dissolving a compound in an excess of aqueous acid solution and precipitating the salt using a water miscible organic solvent such as methanol, ethanol, acetone or acetonitrile. Equivalent molar amounts of the compound and acid or alcohol (eg, glycol monomethyl ether) in water can be heated and the mixture can then be evaporated to dryness or the precipitated salts can be suction filtered. In this case, organic acids and inorganic acids may be used as the free acid, hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid and tartaric acid may be used as the inorganic acid, and methanesulfonic acid, p -toluenesulfonic acid, acetic acid, trifluoroacetic acid, Citric acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, manderic acid, propionic acid, citric acid, lactic acid, glycolic acid, gluconic acid ( gluconic acid), galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanic acid, and hydroiodic acid.

Bases can also be used to make pharmaceutically acceptable metal salts. An alkali metal or alkaline earth metal salt is obtained by, for example, dissolving a compound in an excess alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and then evaporating and drying the filtrate. At this time, as the metal salt, it is particularly suitable to prepare sodium, potassium or calcium salt, and the corresponding silver salt is obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (for example, silver nitrate).

Pharmaceutically acceptable salts of small molecule fucoidans include salts of acidic or basic groups that may be present in fucoidan, unless otherwise indicated. For example, pharmaceutically acceptable salts include sodium, calcium and potassium salts of the hydroxy group, and other pharmaceutically acceptable salts of the amino group include hydrobromide, sulfate, hydrogen sulphate, phosphate, hydrogen phosphate, dihydrogen Phosphate, acetate, succinate, citrate, tartrate, lactate, mandelate, methanesulfonate (mesylate) and p -toluenesulfonate (tosylate) salts, and methods or processes for preparing salts known in the art It can be prepared through.

Pharmaceutical compositions comprising bioavailable low molecular fucoidans may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions, and such materials include lactose, dextrose, sucrose, sorbitol, mannitol Xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate , Talc, magnesium stearate and mineral oil, and the like, but are not limited thereto. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like.

The pharmaceutical composition comprising the bioavailable fucoidan according to the present invention may be prepared in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, oral formulations, external preparations, suppositories, and sterile injectable solutions, respectively, according to conventional methods. It can be formulated and used in the form. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, and capsules, and the like may include at least one excipient such as starch, calcium carbonate, sucrose, or the like in the extract or compound. It is prepared by mixing sucrose, lactose or gelatin. In addition to simple excipients, lubricants such as magnesium stearate or talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents, water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used. Suitable pharmaceutically acceptable carriers and formulations are Remington's Pharmaceutical Sciences (19th ed., 1995).

Preferred dosages of the compositions of the present invention vary depending on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art. However, for the desired effect, the pharmaceutical composition of the present invention is preferably administered at 0.0001 to 100 mg / kg, preferably at 0.001 to 10 mg / kg. Administration may be administered once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect.

The composition of the present invention can be administered orally or parenterally to mammals such as humans, and in the case of parenteral administration, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, topical administration, intraarticular injection, etc. Can be administered.

In the pharmaceutical composition of the present invention, the low molecular weight fucoidan is included at 0.1 to 50% by weight based on the total weight of the composition.

According to a preferred embodiment of the present invention, the composition of the present invention is a food composition.

That is, the present invention provides a health functional food comprising the low molecular fucoidan and food acceptable food additives exhibiting a therapeutic or prophylactic effect of a disease caused by inflammation. Examples of the food to which the bioavailable low molecular weight fucoidan of the present invention can be added include various foods, beverages, gums, teas, vitamin complexes, and health functional foods. At this time, the amount of the extract or compound in the food or beverage may be added at 0.01 to 15% by weight of the total food weight, the health beverage composition is 0.02 to 5 g, preferably 0.3 to 1 g based on 100 ml Can be added.

The health functional beverage composition of the present invention is not particularly limited to other ingredients except low molecular weight fucoidan as an essential ingredient in the indicated ratio, and may contain various flavors or natural carbohydrates, etc. as additional ingredients, as in general beverages. Examples of the natural carbohydrates described above include conventional saccharides such as monosaccharides (e.g. glucose or fructose, etc.), disaccharides (e.g. maltose or sucrose, etc.), and polysaccharides (e.g., dextrin or cyclodextrins, etc.). Party; And sugar alcohols such as xylitol, sorbitol and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract) and synthetic flavoring agents (e.g., saccharin or aspartame, etc.) can be advantageously used. The proportion of said natural carbohydrates is generally about 1-20 g, preferably about 5-12 g per 100 ml of the composition of the present invention.

In addition to the bioavailable low molecular weight fucoidan of the present invention, a variety of nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, such as flavoring agents, coloring and neutralizing agents (such as cheese, chocolate), pectic acid and salts thereof, Alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. In addition, the food composition of the present invention may contain fruit flesh for the production of natural fruit juice and fruit juice beverage and vegetable beverage.

The features and advantages of the present invention are summarized as follows:

(a) The bioavailable low molecular weight fucoidan of the present invention inhibits the expression of the proinflammatory cytokines TNF-α (tumor necrosis factor-α) and matrix metalprotease-9 (MMP-9) to indirectly block the induction of inflammatory responses. By doing so, there is a terrible effect on the prevention or treatment of inflammatory diseases.

(b) The composition containing the bioavailable low molecular weight fucoidan of the present invention as an active ingredient can be usefully used as a pharmaceutical and health functional food for the prevention and treatment of inflammatory diseases such as arteriosclerosis or arthritis.

1 is a graph comparing the incidence of arthritis when the high-molecular, low-molecular and low-molecular fucoidan was fed to arthritis model animals, and the low-molecular fucoidan reduced the incidence of arthritis while the high-molecular fucoidan promoted the development of arthritis. have.

2 is a graph comparing the expression level of anti-collagen antibody when macromolecule, medium molecule and low molecule fucoidan were fed to arthritis model animals. High molecular fucoidan increased antibody expression while low molecule fucoidan reduced antibody expression. can confirm.

Figures 3a and 3b shows that when the macromolecules, macromolecules, and low-molecular fucoidans are treated in macrophages, the macromolecules and the medium-molecule fucoidan promote the inflammatory response of macrophages, resulting in extracellular matrix degrading agents, MMP (matrix metalloproteinase) -9 and TNF-αa. While inducing expression, low molecular fucoidan did not induce an inflammatory response.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Example

Production Example  One: Fucoidan  Due to hydrolysis of organic acid Middle molecule  And Low molecular weight Fucoidan  Produce

1% (w / v) of polymer fucoidan having a weight average molecular weight of about 170,000 Da was added to pyruvic acid and acetic acid mixture (60/40, v / v) of edible organic acid, which was hydrolyzed at 100 ° C. for 4 hours. . The resulting mixture was concentrated under reduced pressure for 6 hours, filtered through a membrane filter having a molecular weight of 3,000 Da, followed by hot air drying to obtain a powder. This was used as a low molecular fucoidan in the experiment. Meanwhile, the hydrolyzate obtained by hydrolysis was filtered through a membrane filter having a molecular weight of 10,000 Da, and then filtered through a membrane filter of 3,000 Da, and the remaining supernatant was concentrated under reduced pressure for 6 hours, followed by hot air drying to obtain a powder. This was used as the middle molecule fucoidan in the experiment.

2N pyruvic acid + 2N acetic acid (60/40, v / v) Weight average molecular weight (Da) Polydispersity index Production amount (g) yield(%) Low Molecular Fucoidan 700-1,400 1.9 0.52 52 Medium molecule fucoidan 2,500-5,000 3.1 0.39 39

Production Example  2: produced by organic acid hydrolysis Low molecular weight Fucoidan  Weight average molecular weight

The molecular weight of the organic acid treated hydrolyzate obtained in Preparation Example 1 was determined using a column (PL aquagel, OH30 * 2, Waters Co. Ltd.), RI detector (waters 410 Differential Refractometer, Waters Co. Ltd.), and pump (waters 515 HPLC). pump, Waters Co. Ltd., and gel permeation chromatography (Waters Co. Ltd.) with an autosampler (waters 717 plus Autosampler, Waters Co. Ltd.), pH 7 buffer solution, at 35 ° C., per minute At a flow rate of ml, polyethylene glycol (PEG) was analyzed as a standard sample.

As a result of analysis, fucoidan has a molecular weight of about 170,000 Da, but when hydrolyzed using a mixture of pyruvic acid and acetic acid (60/40, v / v) at a concentration of 2 N, it is filtered through a molecular weight of 3,000 Da membrane filter. Weight average molecular weight averaged in the form of mixed molecular weight in the ratio of 1,300-1,700 Da: 300-500 Da: 100-200 Da: 0-100 Da = 35-55: 10-30: 20-40: 1-10 The molecular weight of 700-1,400 Da is shown. It is a bioavailable low molecular fucoidan used in this experiment.

On the other hand, the molecular weight fucoidan obtained in the preparation example is a mixed molecular weight of 2,500-4,000 Da: 800-1,500 Da: 550-750 Da: 100-200 Da = 50-70: 5-20: 1-10: 15-25 The weight average molecular weight averaged in the form showed a molecular weight of 2,500-5,000 Da.

Production Example  3: organic acid Hydrolyzed Fucoidan Sulfate  content

The sulfuric acid group contents of the bioavailable low molecular weight fucoidan and the medium molecular fucoidan prepared in Preparation Example 1 were measured.

Sulfuric acid content is 0.5-2 N hydrochloric acid solution and hydrolyzed the bioavailable fucoidan of the present invention at 110 ° C. for 4 hours, 0.2 ml of hydrolyzate was added to 3.8 ml of 4% TCA solution and BaCl ₂ -gelatin reagent (Difco Co., Ltd.). , USA) was added and allowed to stand at room temperature for 20 minutes and analyzed by measuring absorbance at 360 nm. K ₂ SO ₄ was used as the standard sample.

As a result of the experiment, the low molecular weight fucoidan and the high molecular weight fucoidan used in the present invention showed higher sulfate content, which plays an important role in physiological activity than high molecular weight fucoidan before hydrolysis. For example, when hydrolysis was performed using a high molecular weight fucoidan having 21% sulfuric acid group content, a low molecular weight fucoidan was obtained with a sulfuric acid group content of 27%. On the other hand, when hydrolyzed using a high molecular weight fucoidan having a 21% sulfuric acid group content, it was possible to obtain a medium molecule fucoidan with a sulfuric acid group content of 24%.

Production Example  4: organic acid Hydrolyzed Fucoidan  Major Elemental Analysis

Contents of the elements of carbon, hydrogen, oxygen, nitrogen and sulfur, which constitute the low molecular fucoidan and the medium molecule fucoidan prepared in Preparation Example 1, were analyzed using an elemental analyzer (EA1108, Fisons instrument, Italy). The results are summarized in Tables 2a and 2b.

Low molecular fucoidan elemental analysis division % Of element before hydrolysis  % Of element after hydrolysis  Carbon (C) 26.3 24.1  Hydrogen (H) 5.2 4.9 Oxygen (O) 48.3 54.9 Nitrogen (N) 0.8 0.3 Sulfur (S) 5.6 7.3 Ash 13.8 8.5 system 100 100

Medium molecule fucoidan elemental analysis division % Of element before hydrolysis  % Of element after hydrolysis  Carbon (C) 26.3 25.4  Hydrogen (H) 5.2 5.1 Oxygen (O) 48.3 52.1 Nitrogen (N) 0.8 0.4 Sulfur (S) 5.6 6.3 Ash 13.8 10.7 system 100 100

As shown in Table 2a, the low-molecular-weight fucoidan of the present invention showed a slightly different aspect of the main element content, and in particular, the content of oxygen and sulfur element was increased compared to before hydrolysis. This experimental result is consistent with the analysis result in which the content of sulfuric acid group of low molecular fucoidan is increased.

Production Example  5: organic acid Hydrolyzed Fucoidan  Water solubility and viscosity

The low molecular weight fucoidan prepared in Preparation Example 1 was each prepared at a concentration of 1-10% (w / v) using distilled water, and the viscosity according to the concentration was analyzed using a viscometer (Viscometer, LVT, Brookfield, USA). .

As a result of the experiment, the solution of the low molecular fucoidan used in the present invention can be seen that the water solubility is remarkably increased, the solution of the polymer fucoidan is very high suspension. In addition, the fucoidan used in the present invention showed a significantly lower viscosity compared to the polymer fucoidan, which shows that the low molecular weight fucoidan of the present invention is excellent in water solubility. For polymeric fucoidans, concentrations of 180, 300, 420, 510, and 580 centipoise at concentrations of 2 w / v%, 4 w / v%, 6 w / v%, 8 w / v% and 10 w / v%, respectively Viscosity was shown, and low molecular weight fucoidan was 20, 50, 60, 70 and 100 centimeters at concentrations of 2 w / v%, 4 w / v%, 6 w / v%, 8 w / v% and 10 w / v%, respectively. The viscosity of Poise is shown.

Experimental Example  One: Low molecular weight Fucoidan  Arthritis Treatment Effect and Macrophage Experiment

The low-molecular fucoidan obtained in Example 1 was immunized with collagen to mice that induced arthritis to measure the therapeutic effect of arthritis, and treated with RAW264.7 cells, which are mouse macrophage lines, to express MMP-9 and TNF-α. The effect on the experiment was as follows.

1-1. Sample Preparation

The low molecular weight fucoidan and the heavy molecule fucoidan obtained in Preparation Example 1 were resuspended in ethanol and used.

1-2. Arthritis model In mice Fucoidan  Treatment effect measurement

40 DBA mice (8-10 weeks old, Japan SLC, Inc, Japan) were divided into 5 groups (A, no arthritis-free group; B, arthritis-free group; C, arthritis) Induced, polymer fucoidan treatment group; D, induced arthritis and medium molecule fucoidan treatment group; E, induced arthritis and low molecule fucoidan treatment group). To induce arthritis, 100 μg (0.05M acetic acid, 50 μl) of type 2 collagen was immunized with 50 μl of adjuvant (Feund's complete adjuvant, Sigma., USA) in the tail of DBA mice and 50 μg in the left foot after 2 weeks. (0.05 M acetic acid, 25 μl) collagen was mixed with 25 μl adjuvant and subjected to secondary immunization. Fucoidan was orally administered 5 times / 1 week (300 mg / kg / time) zonde after the second vaccination. After the second vaccination, the incidence of arthritis was measured for 5 weeks at 5 days intervals by two people who did not know the classification of the experimental group. The measured values were obtained by observing the remaining three feet except the left foot after the second immunization, and the total score was 12 out of 10. The following criteria were used as a measure of the incidence of arthritis: 0 points, no edema or swelling; Mild edema and redness limited to one point, foot or ankle joint; 2 points, mild edema and redness across the ankle joint at the ankle joint; 3 points, moderate swelling and redness across the ankle joint at the ankle joint; 4 points, swelling and redness throughout the entire leg from the ankle, and joint stiffness.

1-3 Determination of Anti-Collagen Antibody Concentrations in Serum Using Enzyme Linked Immunosorbent Assay

Type 2 collagen used for immunization was dissolved in 0.05 M sodium carbonate coating buffer (pH9.6) at a concentration of 4 μg / μl and dispensed into 50-well plates (50 μl / well) and incubated at 4 ° C. for 16 hours. . Each well was washed three times with Tween 20, 0.05% dissolved in TBS, and then stored at room temperature for 30 minutes after the addition of 1% fetal bovine albumin, 5% sucrose and TBS mixture. After washing three times with the washing solution, the serum was diluted 50000 times or 100000 times in the wells were aliquoted and incubated for 1 hour at room temperature. Each well was washed three times with wash buffer solution and treated with 50 μl per well of anti-mouse IgG antibody with HRP and reacted in direct sunlight for 1 hour.

Each well was washed with a wash buffer solution and treated with a mixture of hydrogen peroxide and tetramethylbenzodine for 20 minutes under direct sunlight. 2 N sulfuric acid was added to each well to stop the reaction and absorbance (450 nm-540 nm) was measured.

1-4. Cell culture and treatment process

RAW 264.7, a mouse macrophage, was purchased from the American Type Cell Collection (American Type Culture Collection). RAW 264.7 cells were prepared in 10% fetal calf serum (FBS), 10 ml / L penicillin-streptomycin (10,000 units / ml penicillin; 10,000 mg / ml streptomycin, Sigma., USA) in liquid medium (Dulbecco's modified Eagle's medium (DMEM)). ), Medium supplemented with 1.5 g / L sodium bicarbonate, and 4.5 g / L glucose was supplied, 5% carbon dioxide, Culture was maintained in a humid state in a carbon dioxide cell incubator adjusted to 37 ℃.

In order to confirm the expression of MMP-9 and TNF-alpha by fucoidan from RAW 264.7 cell line, 5 × 10 ⁴ cells were dispensed per well of 96-plate. At this time, DMEM containing 0.1% fetal calf serum was used. The next day, polymer, medium and low molecular weight fucoidan were treated in cell culture medium at a concentration of 10-100 μg / ml, and the supernatants except cells were collected after 24 hours.

1-5. matrix Of Matrix Metalloprotease  Active measurement

Gel electrophoresis with gelatin was performed to measure the activity of matrix metalloproteinase-9 and -2 in cell culture supernatants. Gel was used sodium dodecyl sulfate-polyacrylamide gel containing 0.1% gelatin. Electrophoresis was performed on a 10% acrylamide gel by mixing the supernatant with a mixture of 4% SDS, 20% glycerol, 0.01% bromophenol and 0.125 M tris-chlorine.

After electrophoresis, the gel was washed with 2.5% Triton X-100 for 40 minutes to remove sodium dodecyl sulfate and 40 minutes with distilled water to remove Triton X-100. After washing, the gel was immersed in the reaction buffer solution (50 mM Tris-HCl, pH7.6, 0.15 M NaCl, 10 mM CaCl ₂ , 0.02% NaN ₃ ) at 37 ° C. for 14 hours to react the enzyme with the substrate. . The reaction gel was dyed with 0.1% Coomassie Brilliant Blue Egg 250 and decolorized with a mixture of 30% methanol, 10% acetic acid and 60% distilled water.

The decolorized gel was dried and the result was confirmed using a density meter (Image J software 1.37v, NIH, USA).

1-6. Enzyme-linked immunosorbent assay TNF -α measurement

To determine the concentration of TNF-α in cell culture supernatants, supernatants were dispensed into 96-well plates coated with anti-TNF-α antibodies (720 mg / ml in PBS, R & D systems) and 16 hours at 4 ° C. Incubated for 2 hours. Each well was washed three times with Tween 20, 0.05% dissolved in PBS, and then stored at room temperature for 1 hour after the addition of 1% fetal bovine albumin, 5% sucrose and PBS mixture. After washing three times with the washing solution, the supernatant to be measured was dispensed into the wells and incubated at room temperature for 2 hours.

Recombinant TNF-α (R & D systems) were set as baseline at a concentration of 10-125 pg / ml. Each well was washed three times with wash buffer solution and incubated for 2 hours at room temperature with biotin attached secondary antibody (R & D, USA) at a concentration of 20 ng / ml. Each well was washed three times with wash buffer solution and treated with streptavidin-HRP and allowed to react in direct sunlight for 20 minutes. Each well was washed with a wash buffer solution and treated with a mixture of hydrogen peroxide and tetramethylbenzodine for 20 minutes under direct sunlight. 2 N sulfuric acid was added to each well to stop the reaction, and the absorbance was measured at 450 nm.

Experiment result

The high-molecular, low-molecular and low-molecular fucoidan were fed to arthritis model mice and analyzed for the onset of arthritis, while the high-molecular fucoidan and low-molecular fucoidan reduced the incidence of arthritis. In particular, the low-molecular fucoidan was more effective in inhibiting the onset of disease, and compared with the B group (arthritis-induced arthritis, no treatment, positive control group) at 43 days after collagen immunization, the incidence was significantly decreased. (See Figure 1).

Collagen was immunized to induce arthritis, which is known to increase the concentration of anti-collagen antibodies in the serum of mice and is associated with an increase in the onset of these antibodies. Thus, 49 days after vaccination, the serum of each experimental group was obtained, diluted 50,000-fold or 100,000-fold, and the concentration of anti-collagen antibody was measured. As a result, it was confirmed that the serum concentration of anti-collagen antibody was significantly increased when the polymer fucoidan was treated and the concentration of the anti-collagen antibody was significantly decreased when the low molecular fucoidan was treated. No significant change was observed in the middle molecule fucoidan (see FIG. 2). These results are consistent with the results observed in the experiment of Figure 1 suggests that low-molecular fucoidan inhibits the immune response to collagen, thereby preventing the production of anti-collagen antibodies, thereby inhibiting the development of arthritis.

In the case of high molecular fucoidan, unlike the low molecular fucoidan, the incidence of arthritis was intensified. To determine the cause, macromolecules were treated with macromolecules, low molecular fucoidan, and cell responses were observed. As a result, it was found that the polymer fucoidan has an effect of causing an inflammatory response of macrophages to induce the expression of proinflammatory cytokines such as TNF-α and matrix degrading enzymes such as MMP-9 (see FIGS. 3A and 3B). These results suggest that the polymer fucoidan stimulates the body's immune system and consequently exacerbates the onset of arthritis. The low molecular fucoidan has no function of stimulating the immune system, while inhibiting the immune response for anti-collagen antibody production. It can be seen that this result was obtained.

It describes an example of the formulation of a pharmaceutical composition comprising the extract and the compounds of the present invention, the present invention is not intended to limit this but only to explain in detail.

Formulation example  One. Powder  Produce

Low Molecular Fucoidan 20 mg Lactose 100 mg Talc 10 mg

The above ingredients are mixed and filled in an airtight cloth to prepare a powder.

Formulation example  2. Preparation of Tablets

Low Molecular Fucoidan 10 mg Corn starch 100 mg Lactose 100 mg Magnesium Stearate 2 mg

After mixing the above components, tablets are prepared by tableting according to a conventional method for preparing tablets.

Formulation example  3. Encapsulated  Produce

Low Molecular Fucoidan 10 mg Crystalline cellulose 3 mg Lactose 14.8 mg Magnesium stearate 0.2 mg

According to a conventional capsule preparation method, the above ingredients are mixed and filled into gelatin capsules to prepare capsules.

Formulation example  4. Preparation of Injectables

Low Molecular Fucoidan 10 mg Mannitol 180 mg Sterile Distilled Water for Injection 2974 mg Na ₂ HPO ₄ 12H ₂ O 26 mg

According to the conventional method for preparing an injection, the amount of the above ingredient is prepared per ampoule (2 ml).

Formulation example  5. Liquid  Produce

Low Molecular Fucoidan 20 mg Iseong Hwadang 10 g Mannitol 5 g Purified water Quantity

According to the conventional method for preparing a liquid, each component is added and dissolved in purified water, lemon flavor is added, the above components are mixed, purified water is added, the whole is adjusted to 100 ml by adding purified water, and then filled in a brown bottle. The solution is prepared by sterilization.

Formulation example  6. Manufacture of health drinks

Low Molecular Fucoidan 100 mg Vitamin c 15 g Vitamin E (powder) 100 g Iron lactate 19.75 g Zinc oxide 3.5 g Nicotinic acid amide 3.5 g Vitamin A 0.2 g Vitamin B ₁ 0.25 g Vitamin B ₂ 0.3 g water dose

After mixing the above components according to the conventional healthy beverage manufacturing method, and stirred and heated at 85 ℃ for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 L container, sealed sterilization, and then refrigerated It is used to prepare a healthy beverage composition of the present invention.

Although the composition ratio is mixed with a component suitable for a favorite beverage in a preferred embodiment, the composition ratio may be arbitrarily modified according to regional and ethnic preferences such as demand hierarchy, demand country, and usage.

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Therefore, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Claims (13)

(a) 20-40% sulfate content, (b) weight average molecular weight of 500-5,000 Da, (c) polydispersity index of 1-6 and (d) viscosity of 0-200 centipoise A composition for the prevention and treatment of inflammatory diseases comprising a bioavailable low molecular weight fucoidan as an active ingredient. The composition of claim 1, wherein the bioavailable low molecular weight fucoidan has a weight average molecular weight of 500-2,000 Da. The composition of claim 2, wherein the bioavailable low molecular weight fucoidan has a weight average molecular weight of 700-1,400 Da. The fucoidan of claim 1, wherein the bioavailable low molecular weight fucoidan has a weight average molecular weight of 1,300-1,700 Da: 300-500 Da: 100-200 Da: 0-100 Da = 35-55: 10-30: 20-40: 1-10. A composition, characterized in that a mixture of. The composition of claim 1, wherein the sulfate content of the bioavailable low molecular weight fucoidan is 20-30%. The bioavailable composition according to claim 1, wherein the polydispersity index of the bioavailable low molecular fucoidan is 1-4. The composition of claim 1, wherein the bioavailable low molecular weight fucoidan has a viscosity of 0-50 centipoise. The method of claim 1, wherein the ratio of the components of the bioavailable low-molecular fucoidan is carbon: hydrogen: oxygen: nitrogen: sulfur = 20-30: 3-7: 45-60: 0.1-1.0: 5.7-20 Composition. 9. The composition of claim 8, wherein the ratio of the component of the bioavailable low-molecular fucoidan is carbon: hydrogen: oxygen: nitrogen: sulfur is 23-25: 4.3-5.1: 52-58: 0.1-0.6: 6.0-10. . The composition of claim 1, wherein the bioavailable small molecule fucoidan inhibits expression of the proinflammatory cytokine, TNF-α (tumor necrosis factor-α) and matrix metalprotease-9 (MMP-9). The composition of claim 1, wherein the inflammatory disease is selected from the group consisting of atherosclerotic coronary artery disease, arthritis, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease and glomerulonephritis disease. The composition of claim 11, wherein the inflammatory disease is arthritis. The composition of claim 1 wherein the composition is a food composition.