KR20190102909A - Composition for preventing, ameliorating or treating arthritis comprising low molecular weight peptide isolated from heat-treated snake venom as effective component - Google Patents

Abstract

The present invention relates to a composition for preventing, alleviating or treating arthritis containing a low molecular weight peptide isolated from heat-treated snake venom as an active component. The low molecular weight peptide (5kDa or less) isolated from heat-treated snake venom of the present invention is removed from an enzyme that causes allergy, shows no cytotoxicity, and has an excellent effect of treating rheumatoid arthritis or gouty arthritis. Therefore, the composition of the present invention containing the low molecular weight peptide isolated from heat-treated snake venom as the active component can be usefully used as an arthritis therapeutic agent which prevents allergy and side effects caused by snake venom and a material of health functional food for preventing or treating arthritis.

Description

Composition for preventing, ameliorating or treating arthritis comprising low molecular weight peptide isolated from heat-treated snake venom as effective component}

The present invention relates to a composition for the prevention, improvement or treatment of arthritis containing a low molecular peptide (5 kDa or less) isolated from heat treated snake venom as an active ingredient.

Joints are areas where bones meet. Joints are composed of cartilage, articular capsule, synovial membrane, ligaments, tendons, muscles, etc., so that the bones can move smoothly between bones and absorb shocks generated by movement. Arthritis is caused by inflammation of a joint due to various causes, a typical symptom caused by this is joint pain. However, just because of pain in the joints are not all arthritis, and swelling or heat accompanied by arthritis can be called. If you have arthritis, doctors can determine if it is acute or chronic, determine whether it is a problem with the joint itself or around the joint, whether it is a mechanical or inflammation problem, or whether it is a central or distal joint, to determine the cause of the arthritis Will be lowered.

Arthritis can be divided into various types depending on the cause and symptoms. Among them, rheumatoid arthritis is an unknown inflammatory disease characterized by multiple arthritis. Inflammation initially occurs in the synovial lining of the joint, but gradually the inflammation of the surrounding cartilage and bones causes joint destruction and deformation.

The exact cause of rheumatoid arthritis is not yet known, but it is known that the complex interaction between genetic and environmental factors plays a role. Among them, autoimmune phenomena are the main cause, and the living body itself recognizes itself as a nonmagnetic based on a recognition defect of self-nonmagnetic, and attacks its own tissue, causing abnormal immune response and causing inflammation in connective tissue. In other words, it is a refractory autoimmune disease that involves swelling, inflammation, stiffness, and pain in the joints and shows a symptom of multiple arthritis of the whole body.

In the drug therapy of chronic joint rheumatism, anti-inflammatory steroids, nonsteroidal anti-inflammatory agents, immunosuppressants, and disease-modifying antirheumatic agents are used. However, they only alleviate the symptoms, slow down the progress of the disease, and may cause side effects from long-term administration.

Gout, on the other hand, is a very common inflammatory arthritis in adult men. Gout is a disease in which the body's metabolism of purine (purine), which is ingested through food, increases the concentration of uric acid, a product of uric acid, which deposits in cartilage, tendons, and surrounding tissues of joints. to be.

Gout is known to be a clear and successfully treated disease, but it is often accompanied by other diseases such as hypertension and chronic renal failure. Patient effort is essential to improve prognosis in long-term care. Gout and hyperuricemia have clinical features such as hypertension, hyperlipidemia, hyperglycemia, abdominal obesity, and the criteria for metabolic syndrome, a complex complication that leads to an increased risk of adult diseases such as atherosclerotic heart disease and type 2 diabetes. However, metabolic syndrome is thought to be closely related. In Korea, 44% of gout patients reported metabolic syndrome. Gout usually occurs in the form of acute monoarthritis but may involve minor or rarely polyarthritis. Non-steroidal anti-inflammatory drugs (NSAIDs), which are used for the treatment of acute gout, are well known to inhibit inflammatory reactions. Colchicine has anti-inflammatory effects by inhibiting the activity and migration of white blood cells. Both steroids are effective drugs to treat gout attacks, and selective cyclooxygenase (COX-2) inhibitors are also known to have the same effect as conventional nonsteroidal anti-inflammatory drugs.

Snake venom, on the other hand, is a mixture consisting of a variety of components secreted from snake venom. Snake venom has been used to treat pain, inflammation and arthritis in India and China for thousands of years, and traditional Chinese medicine has shown that the poisonous toxic constituents of snake venom have a beneficial effect on the health of the body, cramps, and health. In the early 20th century, cobradog ( Naja naja atra vemon) was known to have drug-like analgesic activity.

In order to prepare snake venom as a safe therapeutic composition, it is necessary to remove allergens and inflammatory reactions of snake venom. Therefore, the inventors of the present invention have attempted to manufacture a snake venom for the treatment of arthritis from which allergens and inflammatory response inducing agents of low cost and high efficiency are removed.

Meanwhile, Korean Patent No. 1802514 discloses a composition for preventing or treating inflammatory diseases including snake venom, and Korean Patent No. 1647348 discloses gout prevention and treatment comprising SHP (Small Heterodimer Partner) as an active ingredient. Although a composition for the present disclosure is disclosed, there is no disclosure regarding the composition for the prevention, improvement or treatment of arthritis containing a low molecular peptide isolated from the heat treated snake venom of the present invention as an active ingredient.

The present invention is directed to the above-described demands, and provides a low molecular peptide of 5 kDa or less isolated and purified from a heat treated snake venom, wherein the low molecular peptide isolated and purified from the heat treated snake venom is arthritis, in particular rheumatoid The present invention has been completed by confirming that it is effective in treating arthritis or gouty arthritis.

In order to solve the above problems, the present invention provides a pharmaceutical composition for the prevention or treatment of arthritis containing a low molecular peptide isolated from the heat treated snake venom as an active ingredient.

In addition, the present invention provides a health functional food composition for the prevention or improvement of arthritis containing a low molecular peptide isolated from the heat treated snake venom as an active ingredient.

The present invention relates to a composition for the prevention, improvement or treatment of arthritis containing a low molecular peptide (5 kDa or less) isolated from the heat treated snake venom, the low molecular peptide isolated from the heat treated snake venom of the present invention The allergens causing allergy were eliminated, cytotoxicity was not shown, and the therapeutic effect of rheumatoid arthritis or gouty arthritis was excellent.

Figure 1 confirms the cytotoxicity of the low molecular peptides from the heat treated snake venom of the present invention. con is the negative control.

The present invention relates to a pharmaceutical composition for the prevention or treatment of arthritis containing a low molecular peptide isolated from the heat treated snake venom as an active ingredient.

In one embodiment of the present invention, the snake venom is taken from the salsa, but is not limited thereto.

The low molecular peptide refers to a peptide of 5 kDa or less, and only 5 kDa or less peptides are isolated and purified from a heat treated snake venom.

The arthritis is preferably gouty arthritis or rheumatoid arthritis, more preferably gouty arthritis, but is not limited thereto.

In one embodiment of the present invention, the small molecule peptide is

1) extracting the live snake venom from the snake and freeze-dried;

2) mixing the lyophilized snake venom of step 1) with a buffer solution and heat-treated with a hot bath at 80-120 ° C. for 3-8 minutes, followed by cooling;

3) filtering the cooled snake venom after the heat treatment of step 2) and centrifuging; And

4) removing the peptide or protein greater than 5kDa from the heat-treated snake venom centrifuged in step 3);

Preferably

 1) extracting the live snake venom from the snake and freeze-dried;

2) mixing the lyophilized snake venom of step 1) with a buffer solution, followed by heat treatment with a hot bath at 100 ° C. for 5 minutes, and then cooling 5 ° C. per minute to 20 ° C .;

3) filtering the snake venom cooled after the heat treatment of step 2) with a 0.045 μm filter, followed by a second filter with a 0.02 μm filter, followed by centrifugation; And

4) removing the peptide or protein in excess of 5kDa from the heat-treated snake venom centrifuged in step 3); but may be prepared, but is not limited thereto.

The composition of the present invention may further include a pharmaceutically acceptable carrier, excipient or diluent in addition to the active ingredient, and may be various oral or parenteral formulations. 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 capsules, powders, granules, tablets, pills, and the like, which may comprise at least one excipient such as starch, calcium carbonate, sucrose or lactose (at least one compound). lactose) and gelatin. In addition to simple excipients, lubricants such as magnesium stearate, talc and the like are also used. Liquid preparations for oral administration include suspensions, emulsions, syrups, aerosols, and the like, in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, preservatives, and the like. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and the suspension solvent, 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, glycero gelatin and the like can be used. For parenteral administration, it is desirable to select either external or intraperitoneal, rectal, intravenous, intramuscular, subcutaneous, intrauterine dura or cerebrovascular injections.

The pharmaceutical composition according to the invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, the effective amount of the level of the disease, the severity, the drug activity of the patient , Drug sensitivity, time of administration, route of administration and rate of release, duration of treatment, factors including concurrently used drugs, and other factors well known in the medical arts. The compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be single or multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect in a minimum amount without side effects, which can be easily determined by those skilled in the art.

The dosage of the composition of the present invention varies depending on the weight, age, sex, health condition, diet, time of administration, method of administration, rate of excretion and severity of the patient. The compositions of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy and biological response modifiers.

In addition, the present invention relates to a nutraceutical composition for the prevention or improvement of arthritis containing a low molecular peptide isolated from the heat treated snake venom as an active ingredient.

In one embodiment of the invention, the dietary supplement composition is preferably prepared in the form of powder, granules, pills, tablets, capsules, candy, syrup or beverage, but is not limited thereto.

When the health functional food composition of the present invention is used as a food additive, the composition may be added as it is or used with other food or food ingredients, and may be appropriately used according to a conventional method. The amount of the active ingredient to be mixed may be suitably determined according to the purpose of use (prevention, health or therapeutic treatment). Generally, in the preparation of food or beverages, the composition of the present invention is added in an amount of up to 15 parts by weight, preferably up to 10 parts by weight based on the raw materials. However, in the case of long-term intake for health and hygiene purposes or health control purposes, the amount may be below the above range, and the active ingredient may be used in an amount above the above range because there is no problem in terms of safety.

There is no particular limitation on the kind of food. Examples of foods to which the composition may be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, dairy products including gum, ice cream, various soups, beverages, teas, drinks, Alcoholic beverages and vitamin complexes, and includes all the health functional foods in the conventional sense.

When the composition of the present invention is used as a health beverage, various flavors, natural carbohydrates, and the like may be contained as additional components, as in general beverages. The above-mentioned natural carbohydrates are sugars such as monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as textine and cyclotenstrin, xylitol, sorbitol and erythritol. As the sweetening agent, natural sweetening agents such as tautin and stevia extract, synthetic sweetening agents such as saccharin and aspartame, and the like can be used. The ratio of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 g of the composition of the present invention. In addition to the above, the composition of the present invention includes various nutrients, vitamins, electrolytes, flavors, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols. And carbonation agents used in carbonated beverages. In addition, the composition of the present invention may contain a pulp for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components can be used independently or in combination. The proportion of such additives is not critical, but the composition of the present invention is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight.

Hereinafter, the present invention will be described in more detail with reference to Preparation Examples and Examples. These preparations and examples are only intended to explain the present invention more specifically, it is apparent to those skilled in the art that the scope of the present invention is not limited thereto.

Production Example  1. From heat treated snake venom Low molecular weight Peptide  Separation and Purification

(1) Snake Poisoning

After sealing the inlet of the sterilized glass beaker with a thin film and sterilizing it, the needle of the Korean salsa was used to penetrate the thin film on the upper part of the beer beaker and lightly pressed the poisonous part so that the venom solution flowed down the beer beaker along the glass wall. The collected snake venom was lyophilized at -65 ~ -75 ℃.

(2) heat treatment

The freeze-dried snake venom was dissolved in 0.5M PBS (pH 7.8) to prepare a salsa venom solution and sealed in a 500ml glass flask. The sealed flask was heat-treated by boiling water in a pot containing 100 ° C. boiling water for 5 minutes, and then put ice in the pot to decrease the temperature by 5 ° C. per minute, and finally cooled to 20 ° C. Heat treated snake venom increased the protein or peptide content with a molecular weight of 10 kDa or less. The snake venom cooled after the heat treatment was filtered with a 0.045 μm filter and then filtered with a 0.02 μm filter, and then centrifuged.

Production Example  2. Isolated and purified from heat treated snake venom Low molecular weight Peptide  Preparation of Pharmaceutical Composition

The low molecular weight peptide of Preparation Example 1 was diluted with NaCl solution, firstly filtered with 0.045㎛ filter, centrifuged, and then filtered secondly with 0.02㎛ filter to prepare a pharmaceutical composition. Removed.

Example  1. isolated from heat treated snake venom Low molecular weight Peptide  Cytotoxicity Check

The cytotoxicity of the low molecular peptides isolated from the heat treated snake venom of Preparation Example 1 was confirmed.

Toxicity-sensitive glial cells (BV2) were treated with low-molecular peptides isolated from the heat-treated snake venom of Preparation Example 1 diluted with sterile water and incubated for 24 hours to confirm cell viability. As a result, as shown in Figure 1, the low-molecular peptides isolated from the heat treated snake venom of Preparation Example 1 did not show cytotoxicity up to 50 µg / ml concentration.

Claims (6)

A pharmaceutical composition for preventing or treating arthritis, which contains a low molecular weight peptide isolated from a heat treated snake venom as an active ingredient. According to claim 1, wherein the snake venom is a pharmaceutical composition for the prevention or treatment of arthritis, characterized in that taken from the salsa. The pharmaceutical composition for preventing or treating arthritis of claim 1, wherein the low molecular peptide is 5 kDa or less. The pharmaceutical composition for preventing or treating arthritis according to claim 1, wherein the arthritis is gouty arthritis or rheumatoid arthritis. The method of claim 1, wherein the small molecule peptide
1) extracting the live snake venom from the snake and freeze-dried;
2) mixing the lyophilized snake venom of step 1) with a buffer solution and heat-treated with a hot bath at 80-120 ° C. for 3-8 minutes, followed by cooling;
3) filtering the cooled snake venom after the heat treatment of step 2) and centrifuging; And
4) removing the peptide or protein exceeding 5kDa from the centrifuged heat treated snake venom of step 3); pharmaceutical composition for preventing or treating arthritis, characterized in that it is prepared. Health functional food composition for the prevention or improvement of arthritis containing a low molecular peptide isolated from the heat treated snake venom as an active ingredient.

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