KR101565029B1 - Compositions for Prevention or Treating Autoimmune Thyroid Diseases - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating Autoimmune Thyroid Diseases comprising a federal ( nelumbo nucifera Gaerth) extract as an active ingredient, or a food composition for alleviating or improving autoimmune thyroid disease.
The composition of the present invention can effectively treat or ameliorate various pathological conditions caused by thyroid autoantibodies, such as thyroid ophthalmopathy, by significantly inhibiting the production of hyaluronan, differentiation of fats and inflammatory reaction have.

Description

TECHNICAL FIELD The present invention relates to a composition for preventing or treating autoimmune thyroid disease,

The present invention relates to a composition for the prevention or treatment of autoimmune thyroid disease, including thyroid ophthalmopathy comprising a federal ( nelumbo nucifera Gaerth) extract as an active ingredient.

Thyroid-related diseases include hyperthyroidism, a disease in which the thyroid gland functions excessively and thyroid hormone is excessively produced and the metabolism of the body is abnormally increased, and thyroid gland function, which is a disease in which the metabolism of the whole body is slowed by the deficiency of thyroid hormone There is hypopnea. In the case of hyperthyroidism, if left untreated for a long time, it may cause cardiac complications, resulting in arrhythmia or heart failure, and it causes aggravation of osteoporosis in postmenopausal women. Hypothyroidism is divided into primary hypothyroidism due to hypothyroidism and hypothyroid hypothyroidism due to pituitary hypothyroidism. In the case of hypothyroid hypothyroidism, genetic cause, thyroidectomy, radioiodine administration, chronic lymphocytic Thyroid tissue is destroyed by thyroid inflammation and secondary hypothyroidism occurs when hypothyroid function that stimulates secretion of thyroid hormone is lost. This decrease in thyroid function caused the metabolism to become too slow to cause constipation, weakness, swelling, fatigue, memory loss, excessive menstruation, muscle aches, poor resistance to the cold, poor sweating, decreased appetite, .

Recently, the number of patients with thyroid-related diseases has been steadily increasing. According to the National Health Insurance Corporation data, from 2002 to 2009, the number of hypothyroidism was 2.3 times from 128,000 to 289,000, and the number of hyperthyroidism was 173,000 To 233,000. The number of patients with autoimmune thyroid disease is expected to be much higher, even if there are no abnormalities.

Thyroid-associated ophthalmopathy is a chronic inflammatory disease of the extraocular muscle and connective tissue induced by autoimmune thyroid disease. About 90% of thyroid ophthalmopathy is found in patients with Graves' disease, also called Graves' ophthalmopathy (Graves' ophthalmopathy), and Hashimoto's thyroiditis also causes thyroid ophthalmopathy. Unlike hyperthyroidism, which removes thyroid gland or inhibits hormone production, thyroid gland is also treated with immunotherapy for medical treatment, but the effect is still at a level expected to alleviate symptoms.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have made extensive efforts to find effective natural therapeutic compositions for autoimmune thyroid disease. As a result, the extract of nelumbo nucifera Gaerth, an aquatic herbaceous plant belonging to the water lily family, significantly decreased the expression level of major marker / pathogenic agent for autoimmune thyroid disease, The present invention has been completed by discovering that the present invention is effective for prevention and treatment of autoimmune thyroid disease.

Accordingly, an object of the present invention is to provide a pharmaceutical composition for preventing or treating autoimmune thyroid diseases, or a food composition for alleviating or improving autoimmune thyroid disease.

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

According to one aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating autoimmune thyroid diseases, which comprises a federal ( nelumbo nucifera Gaerth) extract as an active ingredient.

The present inventors have made extensive efforts to find effective natural therapeutic compositions for autoimmune thyroid disease. As a result, the extract of nelumbo nucifera Gaerth, an aquatic herbaceous plant belonging to the water lily family, significantly decreased the expression level of major marker / pathogenic agent for autoimmune thyroid disease, And is effective in the prevention and treatment of autoimmune thyroid diseases.

The term " thyroid gland disorder " in the present invention means a disorder in which the secretion of thyroid hormone is excessive or deficient due to abnormal thyroid function. Accordingly, the thyroid disease treated with the composition of the present invention includes both hyperthyroidism and hypothyroidism.

Hyperthyroidism is a disorder in which the body's metabolism is abnormally increased due to abnormally activated thyroid function and excessive production of thyroid hormone. Hypothyroidism is caused by the deficiency of thyroid hormone, It is a disease that slows down the process.

As used herein, the term " Autoimmune Thyroid Diseases " refers to a disorder in which the thyroid hormone is excessively secreted or deficient by an immune response mediated by autoantibodies to the thyroid. When thyroid stimulating hormone receptor antibody (TRAb) is produced, the thyroid hormone is overproduced and progresses to hyperthyroidism. In contrast, thyroid stimulating blocking antibody (TSBAb), which destroys thyroid cells, When it is produced, it decreases hormone secretion and develops hypothyroidism.

The composition of the present invention can effectively treat or prevent autoimmune thyroid disease by controlling the autoimmune response by inhibiting or inactivating the production of autoantibodies.

As used herein, the term " treatment " includes (a) inhibiting the development of a disease, disorder or condition; (b) relief of the disease, disorder or condition; Or (c) eliminating the disease, disease or condition. The term " treatment " or " therapeutic agent " is used herein to refer to a therapeutic adjuvant or " therapeutic adjuvant " ≪ / RTI >

As used herein, the term " prophylactic " means inhibiting the development of a disease or disease in a subject who has never been diagnosed as having a disease or disease, but is likely to suffer from such disease or disease.

According to a specific embodiment of the present invention, the autoimmune thyroid disease to be treated or prevented with the composition of the present invention is thyroid-associated ophthalmopathy, Graves' disease, Hashimoto's thyroiditis, atrophic thyroiditis atrophic thyroiditis, painless thyroiditis, and postpartum thyroiditis.

According to a more specific embodiment of the present invention, the autoimmune thyroid disease treated or prevented with the composition of the present invention is thyroid-associated ophthalmopathy.

As used herein, the term " thyroid-associated ophthalmopathy " refers to a disorder in which the orbital orbital fibroblast cell is infiltrated by inflammatory reaction and lipolysis due to autoimmune thyroid disease. According to the present invention, the composition of the present invention inhibits the production of hyaluronan, which is one of the most important pathological mechanisms of thyroid ophthalmopathy, in a concentration-dependent manner, and significantly inhibits differentiation of fat and increases PPARγ , C / EBP? And C / EBP? As well as the phosphorylation of NF-κB p65, a marker of inflammatory response. By this multilateral experimental evidence, the present inventors have found that the composition of the present invention can be effectively used as an effective therapeutic agent for thyroid ophthalmopathy.

When the composition of the present invention is manufactured from a pharmaceutical composition, the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier. The pharmaceutically acceptable carriers to be contained in the pharmaceutical composition of the present invention are those conventionally used in the present invention and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, But are not limited to, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrups, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. It is not. The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington ' s Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention can be administered orally or parenterally, and in the case of parenteral administration, it can be administered by intravenous injection, subcutaneous injection, muscle injection, intraperitoneal injection, transdermal administration or the like.

The term " administering " or " administering " as used herein refers to the administration of an effective amount of a composition of the present invention directly to a subject so that the same amount is formed in the body of the subject.

As used herein, the term " subject " includes without limitation humans, mice, rats, guinea pigs, dogs, cats, horses, cows, pigs, monkeys, chimpanzees, baboons or rhesus monkeys. Preferably, the subject of the present invention is a human.

As used herein, the term " effective amount " means an amount of a composition that is effective enough to treat, or at least ameliorate, a condition in which a subject is expected to suffer or suffer from pain when administered to the subject.

The appropriate dosage of the pharmaceutical composition of the present invention may vary depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate, . The daily dosage of the pharmaceutical composition of the present invention is, for example, 0.001-100 mg / kg.

The pharmaceutical composition of the present invention may be formulated into a unit dose form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Or by intrusion into a multi-dose container. The formulations may be in the form of solutions, suspensions, syrups or emulsions in oils or aqueous media, or in the form of excipients, powders, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.

According to another aspect of the present invention, there is provided a food composition for alleviating or ameliorating an autoimmune thyroid disease, which comprises a federal ( nelumbo nucifera Gaerth) extract as an active ingredient.

Since the fed extract used in the present invention has already been described above, the description thereof is omitted in order to avoid excessive duplication.

When the composition of the present invention is prepared with a food composition, it includes not only the fed extract as an active ingredient but also a component that is ordinarily added at the time of food production, for example, a protein, a carbohydrate, a fat, a nutrient, . Examples of the above-mentioned carbohydrates are monosaccharides such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose, oligosaccharides and the like; And polysaccharides such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. Natural flavorings such as tau martin and stevia extract (e.g., rebaudioside A and glycyrrhizin) and synthetic flavorings (saccharine, aspartame, etc.) can be used as flavorings.

For example, when the food composition of the present invention is prepared as a drink, citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, juice, mulberry extract, jujube extract and licorice extract are added in addition to the active ingredient of the present invention .

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

(a) The present invention provides a pharmaceutical composition for preventing or treating autoimmune thyroid diseases comprising the nelumbo nucifera Gaerth extract as an active ingredient or a food composition for alleviating or improving autoimmune thyroid disease do.

(b) The composition of the present invention significantly inhibits the production of hyaluronan, the differentiation of fats and the inflammatory response, thereby effectively treating or preventing various pathological conditions caused by thyroid autoantibodies, such as thyroid ophthalmopathy Can be improved.

FIG. 1 is a graph showing the results of analysis of the efficacy of the fed extract of the present invention on the production of hyaluronan, which is one pathological mechanism of thyroid ophthalmopathy. Fibroblast cells were treated with 10 ng / ml of TNF-α for 24 h at concentrations of 10, 50 and 100 μg / ml, and the production of hyaluronic acid was analyzed by ELISA kit. Dependent inhibition of hyaluronic acid production.
FIG. 2 is a graph showing the effect of the fed extract of the present invention on lipid differentiation, which is one of the pathological mechanisms of thyroid ophthalmopathy, by oil-red o staining under the fed extract of each concentration (50, 100 μg / ml) .
FIG. 3 shows that the expression of PPARg, C / EBPa, and C / EBPb increased upon lipid differentiation under fed extracts at different concentrations (50 and 100 μg) and the decrease in NF-kB p65 phosphorylation was observed through Western blotting The results showed that the extract inhibited lipid differentiation and inflammatory response.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Experimental Method

Obtain an extract of Manganese mushroom

The federation used for the experiment was purchased directly from Muan, Jeollanam-do in August 2011, and the standard product (JKTM-2011-01) was stored in the sample room of the Jeollanam-do Oriental Industry Promotion Agency. The solution obtained by extracting 4.5 kg of shrimp with three times of MeOH for 3 hours was filtered, and the solvent was removed with a vacuum concentrator to obtain 765 g of an extract.

Orbital fibroblast differentiation from orbital adipose tissue of thyroid ophthalmopathy

Orbital decompression was performed in patients with thyroid ophthalmopathy and primary orbital fibroblasts were cultured from orbital fat tissue. For comparative experiments, normal orbital fibroblasts were obtained from the orbital fat tissue through upper eyelid surgery or orbital surgery in normal subjects without thyroid ophthalmopathy. The culture was performed by shaking the tissue with wescott scissors and then shaking collagenase II (3 mg / ml in HBSS, 1.5% BSA) for 1 hour at 37 ° C, The floating fat cells and fat globule were removed by centrifugation. The cells were washed twice with PBS and incubated in DMEM (Dulbecco's Modified Eagle's Medium) supplemented with 10% fetal bovine serum (FBS) and incubated in a CO ₂ incubator (5% CO ₂ , 37 ° C). The fused cells were detached with trypsin-EDTA solution and subcultured while the culture medium was changed at intervals of 2-3 days.

Adipogenesis

After orbital fibroblasts were divided into 6- well plates, when the cells reached confluence, lipid differentiation of the lipid precursor cells was induced. (10 mM, Cayman, Ann Arbor, MI, USA), 33 mM biotin, 17 mM pantothenic acid, 10 mg / ml transferrin and 10 mg / ml transferrin were added to the DMEM supplemented with 10% , 0.2 nM T3, 1 mM insulin (Boehringer-Mannheim, Mannheim, Germany) and 0.2 mM carbaprostaglandin (cPGI2; Calbiochem, La Jolla, CA, USA), 1 mM dexamethasone and 0.1 mM isobutyl methylxanthine. The lipid differentiation lasted for 10 days and the medium and drug were replaced every 2-3 days.

Hyaluronan ELISA

After orbital fibroblasts were divided into 12-well plates, the extracts were treated with 10-100 μg / ml of the extract for 24 hours, and stimulated with TNF-α. The cell culture medium was collected and the HA concentration was measured using a hyaluronan ELISA kit (Echelon, Salt Lake City, UT, USA). Absorbance was measured at 405 nm and the concentration was calculated.

Oil Red O staining of differentiated adipocytes

0.5% (w / v) of Oil Red O solution was prepared, diluted with sterilized distilled water at a ratio of 3: 2, and used as a staining reagent for adipocytes. After orbital fibroblasts were divided into 6-well plates, when the cells reached confluence, lipid differentiation of the lipid precursor cells was induced for 10 days. After the lipolysis was complete, the medium was removed, washed twice with PBS, and fixed with 3.7% (w / v) formalin for 1 h. After fixation, the Oil Red O working solution was treated for 1 h, washed twice with distilled water, and confirmed with a microscope (Olympus, Melville, NY, USA).

Western blotting

The aim of this study was to investigate the inhibition of lipid differentiation and inhibition of inflammation during induction of lipid differentiation in the orbital adipose tissue of thyroid ophthalmoplegia by detecting only the desired protein (antigen) in the protein mixture using an antibody reacting with the antigen epitope. The orbital adipose tissue cells were treated with extracts of soft bran extract (50, 100μg / ml) for 10 days. After obtaining cell lysate, proteolytic enzyme inhibitor and phosphatase inhibitor were added to the cell lysis buffer and then kept on ice for 20 minutes. After centrifugation at 12,000 rpm to obtain the supernatant, protein quantification was performed to make the same amount of protein in each experimental group. Proteins were separated using 10% SDS-PAGE, and the separated proteins were transferred to a PVDF membrane (Millipore, MA, USA). All non-specific binding sites in the membrane were blocked using 5% skim milk. After incubation for 24 h at 4 ° C, the secondary antibodies were incubated with chromogenic substrate for 1 h. The primary antibodies were incubated at 37 ° C for 1 h. The primary antibody, PPARγ, C / EBPα, C / EBPβ, p-P65, P65, HO- Respectively.

Experiment result

Federal inhibition of hyaluronan production

The efficacy of the federal hyaluronan for the production of hyaluronan, one of the pathological mechanisms of thyroid ophthalmopathy, was analyzed. After the fibroblasts were dispensed, the fed extract was treated with 10, 50 and 100 μg / ml for 24 hours, treated with 10 ng / ml TNF-α, and analyzed by ELISA kit for hyaluronic acid production in this cell culture medium , While the feds inhibited hyaluronic acid production in a concentration-dependent manner (Fig. 1).

Federal inhibition of adipogenesis and inflammation

The efficacy of the federal regimen for lipid differentiation and inflammatory response, one of the pathological mechanisms of thyroid ophthalmopathy, was analyzed. Fat-differentiation inhibition was confirmed by oil-red o staining at 50, 100 μg / ml of fed-batch extract. The expression of PPARg, C / EBPa and C / EBPb in lipid differentiation was decreased by the Western blotting, and the inflammatory response during lipid differentiation was suppressed by NF-kB p65 by phosphorylation inhibition observation (Figs. 2 and 3).

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (9)

A pharmaceutical composition for the prevention or treatment of thyroid-associated ophthalmopathy comprising the nelumbo nucifera Gaerth extract as an active ingredient.
delete delete 2. The composition of claim 1, wherein the composition inhibits hyaluronan production.
2. The method of claim 1, wherein the composition is selected from the group consisting of reducing the expression of PPARy (Peroxisome proliferator-activated receptor gamma), C / EBP alpha (CCAAT / enhancer-binding protein alpha) and C / EBP beta (CCAAT / enhancer- ≪ / RTI >
The composition of claim 1, wherein the composition inhibits phosphorylation of NF-κB (NF-kappa-B) p65.
A food composition for alleviating or ameliorating thyroid-associated ophthalmopathy comprising the nelumbo nucifera Gaerth extract as an active ingredient. delete delete

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