KR20180081008A - Anti-Inflammatory and Anti-Diabetic Composition Comprising Novel Compound, Oscarella stillans Extract Containing the Same, or a Fraction Thereof - Google Patents

Abstract

The present invention relates to a novel compound, to an extract of Oscarella stillans containing the same, or to an anti-inflammatory and anti-diabetic composition comprising a fraction thereof, wherein the composition exhibits modulatory activity of inflammatory mediator production and thus can be used for prevention and treatment of immunomodulation and inflammation-related diseases, and moreover, the composition can be used for prevention and treatment of diabetes as anti-diabetic activity is exhibited against type 1 and type 2 diabetes in a zebrafish model.

Description

An anti-inflammatory and anti-diabetic composition comprising a novel compound, an Oscarella stillans extract thereof, or a fraction thereof, a Fraction Thereof}

The present invention relates to anti-inflammatory and antidiabetic composition comprising a novel compound, Oscar Pasteurella styryl lance (Oscarella stillans) comprising the same extract, or fractions thereof.

Inflammation is an essential defensive response to deleterious stimuli, including pathogens, stimulants, and inflammatory cytokines that interact with other cells. Although the inflammatory process is a beneficial physiological response, excessive or persistent inflammation leads to severe diseases such as septic shock and autoimmune diseases such as rheumatoid arthritis. Macrophages in immune cells play an essential role in the regulation of host defense mechanisms. Lipopolysaccharide (LPS), an outer membrane component of Gram-positive bacteria, is known to induce macrophage activation. Upon exposure to LPS, macrophages are activated and release a variety of inflammatory mediators including TNF-α, IL-6, and nitric oxide (NO). Thus, inhibition of macrophage activation is considered a preferred strategy for the treatment of a variety of inflammatory diseases (Curr. Drug Targets Inflamm. Allergy 2005, 4, 281-286).

Diabetes is one of the most important geriatric diseases in the world. In recent years, with the rapid economic growth, the prevalence of diabetes in Korea has reached 10%, now more than 240 million people worldwide, and 308 million in 2025 And 60% of them will develop in Asia, according to the American Medical Association (JAMA) in 2009. In particular, the onset of diabetes mellitus has been pulled into the elderly, and it has become inevitable to progress to complications due to the extended life span. In general, almost 10 to 20 years after the onset of diabetes, almost all the organs in the body are damaged, causing diabetic retinopathy, diabetic cataract, diabetic nephropathy, diabetic neuropathy, Diabetic neuropathy, heart disease, cancer, and osteoporosis. Chronic diabetic nephropathy is the most important cause of hemodialysis treatment and end-stage renal failure, and diabetic cataract and retinopathy cause blindness and eventually death.

Currently, alloxan and streptozotocin have been used to induce diabetes in experimental animals during the study of insulin-dependent diabetes (type 1 diabetes) (Lee, JW, BI Seo, JH Park, SS Roh, YH Kim, and MR Kim Effect of Mantidis OOtheca and Mori fructus on treatment of osteoporosis in ovariectomized rats. Kor J Herbology. 2009. 24: 59-71). Alloxan selectively accumulates in the beta cells of the pancreas, inhibiting glucokinase in the beta cells and reducing insulin secretion (Lenzen, S .. Beta-cell apoptosis in type 2 diabetes: Quantitative and functional consequences. 34: 56-64, 10.1016 / S1262-3636 (08) 73396-2). Zebra fish ( Danio rerio ) is a small tropical freshwater fish from the Indian carp family. It has been known that the recovery of diabetes is confirmed by measuring the size of the pancreatic islets after administering the drug after inducing diabetes in zebrafish (Kim, HT, and CH Kim.) Zebrafish as a tool for functional genomics. Reprod, 2003 7: 69-80).

Marine organisms, on the other hand, are a valuable source of diverse bioactive compounds. The marine sponge of the Plakindae family includes seven genera Corticum, Oscarella, Placinolopha, Plakina, Plakinastrella, Plakortis and Pseudocorticium. Some secondary metabolites, including sterols, alkylpyrrole carboxaldehyde, sesterterpene glycosides and lysophospholipids, have been isolated in Oscarella sponges. The sponge collected in the Honda Bay of the Philippines was reported as Oscarella stillans , a new species of Plakinidae (Porifera, River: Demospongiae, neck: Homosclerophorida). Oscar Pasteurella styryl lance (Oscarella The bar does not study for chemical composition and biological activity of stillans).

Under this background, the present inventors have Oscar Pasteurella styryl lance (Oscarella The extract and fraction of the novel compounds, Oscar Pasteurella styryl lance (Oscarella stillans) comprising the same separate from stillans) and completed the present invention by finding that the efficacy in inflammatory diseases and diabetes.

To is an object of the present invention compound represented by the formula (I), a pharmaceutically acceptable salt thereof,, an inflammatory disease, including Oscar Pasteurella styryl lance (Oscarella stillans) containing the compound extract, or fractions thereof as an active ingredient Or a pharmaceutical composition for the prevention or treatment of diabetes.

[Chemical Formula 1]

Another object of the present invention are Pasteurella Oscar styryl lance (Oscarella to the compound, a pharmaceutically acceptable salt thereof, including the compound stillans) to provide an extract, or a fraction, an inflammatory disease or a food composition for preventing or improving diabetes comprising as an active ingredient thereof.

It is still another object of the present invention to provide a feed composition for preventing or ameliorating an inflammatory disease or diabetes, which comprises the above compound and a pharmaceutically acceptable salt thereof as an active ingredient.

It is still another object of the present invention to provide a method for the prophylaxis or treatment of inflammatory diseases or diabetes, comprising the step of administering the pharmaceutical composition to a subject.

Another object of the present invention is to provide a compound represented by the general formula (I) or a pharmaceutically acceptable salt thereof.

[Chemical Formula 1]

.

.

Another object of the present invention are Pasteurella Oscar styryl lance (Oscarella to the compound, a pharmaceutically acceptable salt thereof, including the compound stillans) to provide an extract, or an inflammatory disease or diabetes cosmetic composition for preventing or improving of the fraction containing thereof as an active ingredient.

Another object of the present invention are Pasteurella Oscar styryl lance (Oscarella to the compound, a pharmaceutically acceptable salt thereof, including the compound stillans) to provide an extract, or an inflammatory disease or a quasi-drug composition for preventing or improving diabetes comprising a fraction thereof as an active ingredient.

Hereinafter, the present invention will be described in more detail.

On the other hand, each description and embodiment disclosed herein can be applied to each other description and embodiment. That is, all combinations of the various elements disclosed herein are within the scope of the present invention. Further, the scope of the present invention can not be said to be limited by the following detailed description.

In addition, those of ordinary skill in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described in this application. Further, such equivalents are intended to be included in the present invention.

In some embodiments the present invention for solving the aforementioned problems is to Oscar Pasteurella styryl lance (Oscarella comprising a compound, pharmaceutically acceptable salt thereof, wherein the compound represented by general formula (1) stillans) provides an extract, or an inflammatory disease or a pharmaceutical composition for preventing or treating diabetes containing a fraction thereof as an active ingredient.

[Chemical Formula 1]

The term "compound represented by formula (1)" is referred to herein as compound 1, compound [1], and anthranilic acid derivative [1]. The compound can be isolated from or synthesized from sponge animal Oscarella stillans. If the compound is represented by the above formula (1), it can be used without being limited to the method of obtaining it. Such compounds may also include isomers which have an effect of preventing or treating inflammatory diseases or diabetes. Derivatives of the compound 1, for example, a derivative in which a COOH group is modified with an ester, or a substance in which a substituent is bonded to an NH ₂ group can be used as an equivalent range of the present invention for the prevention or treatment of an inflammatory disease or diabetes.

Compound 1 may be used for the prevention, treatment, or amelioration of an inflammatory disease or diabetes.

The compound can be used in the form of a pharmaceutically acceptable salt. The term " pharmaceutically acceptable salt " of the present invention means a concentration that is relatively non-toxic to a subject and has a harmless effective action, wherein the side effects caused by the salt are any all organic or inorganic salts of the compound that do not impair the beneficial efficacy of Compound An inorganic addition salt. In addition, the compound 1 may be used alone or in combination with another pharmaceutically active compound or in a set.

The pharmaceutically acceptable salts of the compound 1 of the present invention refer to salts prepared according to methods customary in the art, and such methods are known to those skilled in the art. In particular, the pharmaceutically acceptable salts include, but are not limited to, salts derived from inorganic and organic acids and bases which are pharmacologically or physiologically acceptable.

The acid addition salt is prepared by a conventional method, for example, by dissolving the compound in an excess amount of an acid aqueous solution, and precipitating the salt using a water-miscible organic solvent such as methanol, ethanol, acetone or acetonitrile. The same molar amount of the compound and the acid or alcohol (e.g., glycol monomethyl ether) in water may be heated and then the mixture may be evaporated to dryness, or the precipitated salt may be subjected to suction filtration. As the free acid, organic acids and inorganic acids can be used. As the inorganic acids, hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, tartaric acid and the like can be used. Examples of the organic acids include methanesulfonic acid, p- toluenesulfonic acid, acetic acid, trifluoroacetic acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, mandelic acid, propionic acid, citric acid, lactic acid, glycollic acid, gluconic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanillic acid, hydroiodic acid and the like can be used , But are not limited to these.

In addition, bases can be used to make pharmaceutically acceptable metal salts. The alkali metal salt or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess amount of an alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the non-soluble salt salt, and evaporating and drying the filtrate. At this time, it is pharmaceutically acceptable to produce sodium, potassium, or calcium salt, but not limited thereto. The corresponding silver salt can also be obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (e.g., silver nitrate).

The present invention includes both the compound 1 or a pharmaceutically acceptable salt thereof as well as a solvate or hydrate thereof exhibiting the same effect, which can be prepared therefrom, within the scope of the present invention.

As used herein, the term "Oscar Pasteurella styryl lance (Oscarella stillans)" is Plakinidae and a sponge (sponge) of the species (statement: Porifera, river:: Demospongiae, neck Homosclerophorida). It can be composed of associated thin tubes of 3 to 5 cm in length and about 2 mm in thickness, sometimes with short branches and having a very smooth and slippery surface. The color of living sponges can be dark yellow with yellowish green color in the water. These sponges can grow to a depth of 12m attached to protruding coral substrates on a vertical plane. In the present invention, the one collected in Honda Bay, Philippines is used, but the one collected or cultivated in another habitat may be used, or a commercially sold one may be used.

The term " extract " as used herein means an extract obtained by extracting Oscarella stillans . The Oscarella stillans extract is obtained by dissolving Oscarella stillans pulverized product in a volume of about 2 to 20 times, preferably about 3 to 5 times the dry weight of water, methanol, ethanol and butanol (C ₁ ) to 4 (C ₄ ), or a mixed solvent having a mixing ratio of about 1: 0.1 to 1:10 of them, is used as an elution solvent, and the extraction temperature is 20 to 100 The extraction can be carried out using extraction methods such as hot water extraction, cold extraction, reflux cooling extraction or ultrasonic extraction for about 1 hour to 4 days, Any method can be used without limitation as long as it is a method for extracting an active substance, specifically, a compound of formula (1).

More specifically, it may be the result of extracting twice with methanol at room temperature, and once with a mixture solvent of methanol and dichloromethane (1: 1) at room temperature. However, it may be a result of a prophylactic or therapeutic activity of an inflammatory disease or diabetes But are not limited to, extracts, diluted solutions or concentrates of the extracts, dried products obtained by drying the extracts, or these adjusted products or purified products.

The term " fraction " as used herein means a product obtained by a fractionation method for separating a specific component or a specific group from a mixture containing various constituents. Specifically, it may be water, a C ₁ to C ₄ alcohol, chloroform, ethyl acetate, hexane, butanol or a mixed solvent fraction thereof. The fraction of the present invention is not limited to the method as long as it is a method for obtaining a substance used for the prevention or treatment of an inflammatory disease or diabetes, particularly a fraction containing Compound 1. [ Specifically, the Oscarella stillans fraction of the present invention can be obtained by dissolving the above-mentioned Oscarella stillans extract in an aqueous solution of 1 (C ₁ ) to 4 (C ₄ ) carbon atoms such as water, methanol, ethanol and butanol And suspending the mixture in a polar solvent having a lower alcohol and fractionating the mixture with an organic solvent such as hexane or dichloromethane.

More specifically, the methanol extract of Oscarella stillans was suspended in 90% methanol and fractionated three times with hexane to obtain 4.01 g of hexane fraction. The remaining 90% methanol fraction was concentrated and then suspended in 70% methanol And repeatedly fractionated three times with a dichloromethane solvent to obtain 1.44 g of a dichloromethane fraction. The remaining 70% methanol fraction is concentrated, suspended in water, and repeatedly dispensed with butanol three times to obtain 1.39 g of a butanol fraction. The butanol fraction may be applied to a Sephadex LH-20 column and eluted with methanol to obtain a small fraction. Compound 1 can be isolated from these small fractions.

The term " inflammatory disease " as used herein means a disease caused by inflammation. The inflammatory disease is not particularly limited as long as the symptoms can be alleviated, alleviated, ameliorated or treated by the pharmaceutical composition of the present invention. Specific examples of the inflammatory disease include Crohn's disease, erythema, atopy, rheumatoid arthritis, Hashimoto's thyroiditis, Meniere's syndrome, Gillian-Barre syndrome, Meniere's syndrome, Gillian-Barre syndrome, Gillian-Barre syndrome, Gillian-Barrett's syndrome, But are not limited to, Guilian-Barre syndrome, Sjogren's syndrome, vitiligo, endometriosis, psoriasis, vitiligo, systemic scleroderma, or ulcerative colitis.

The present inventors have confirmed that Compound 1 of the present invention inhibits the inflammatory reaction upon inactivation of JNK, ERK, AP-1, and NF-kB and activation of ATF-3 (Example 3).

In the present invention, the term " diabetes " means a disease that occurs when insulin secretion is insufficient or insulin action and function are not sufficiently achieved. In case of this disease, excessive decomposition of glycogen, protein, Resulting in diabetes and ketoneuria, resulting in abnormal blood and electrolyte metabolism, and concomitant conditions such as circulatory disturbances and renal disorders, as well as blood concentration due to electrolyte loss. Insulin is secreted in the beta cells of the islets of the islets in the pancreas, secreted when the glucose level in the blood is increased, and secreted is suppressed when the blood glucose level is increased. The disease is divided into insulin - dependent diabetes mellitus (type I) and non - insulin dependent diabetes mellitus (type II). Diagnosis of diabetes is usually made by measuring blood glucose levels, which vary according to standards. In humans, diabetes is usually diagnosed when the blood glucose level is more than 200 mg / dl, and fasting is more than 140 mg / dl. Thus, the damaged pancreatic beta cells can be repaired or the pancreas can also be treated to increase or decrease the intracellular glucose uptake, thereby treating or preventing diabetes. The present inventors have found that when Compound 1 of the present invention is treated with zephyrate which has been induced by dihydrogenase-induced diabetes mellitus with alloxan or excessive insulin, the size of damaged pancreatic islets is increased. Therefore, Compound 1 or a pharmaceutically acceptable salt thereof It was confirmed that the pharmaceutical composition containing the compound can be effectively used for the prevention or treatment of diabetes (Example 4).

As used herein, the term " prevention " means any action that inhibits or delays the onset, spread and recurrence of a subject disease upon administration of the pharmaceutical composition, and " treatment " It means any act that improves or changes the symptoms.

The pharmaceutical composition according to the present application may contain, as an active ingredient, the compound represented by the formula (1) or a pharmaceutically acceptable salt thereof, and may further comprise a pharmaceutically acceptable carrier, diluent or excipient. Furthermore, it can be formulated into various forms such as powders, granules, tablets, capsules, oral formulations such as suspensions, emulsions, syrups and aerosols, injections of sterilized injection solutions and the like according to the respective application purposes .

The present invention as another aspect for solving the aforementioned problems is, containing the compound, a pharmaceutically acceptable salt thereof, Oscar Pasteurella styryl lance (Oscarella stillans) containing the compound extract, or fractions thereof as an active ingredient , A food composition for preventing or ameliorating an inflammatory disease or diabetes mellitus.

The terms used here are the same as those described above.

As used herein, the term " improvement " refers to any action that alleviates or alleviates the suspicion of the subject disease or symptoms of the subject by using a composition comprising Compound 1 as an active ingredient.

The food composition according to the present invention includes forms such as pills, powders, granules, infusions, tablets, capsules or liquid preparations. Foods to which the composition of the present invention can be added include, for example, , Beverages, gum, tea, vitamin complex, and health supplement foods.

There are no particular restrictions on other ingredients other than those containing Compound 1 as an essential ingredient that can be included in the food composition of the present invention, and may contain various herbal medicine extracts, food supplementary additives, natural carbohydrates, .

In addition, the food-aid additive includes food-aid additives customary in the art, for example, flavoring agents, flavoring agents, coloring agents, fillers, stabilizers and the like.

Examples of such natural carbohydrates include monosaccharides such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose and the like; And polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. In addition to the above, natural flavoring agents (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used as flavoring agents.

In addition to the above, the food composition of the present invention can be used as a food composition containing various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and fillers (cheese, chocolate etc.), pectic acid and its salts, Salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages and the like. In addition, it may contain natural fruit juice and pulp for the production of fruit juice drinks and vegetable drinks. These components may be used independently or in combination.

In the present invention, the health supplement food includes health functional food, health food and the like. The term "functional food" as used herein means the same term as "food for special health use" (FoSHU). In addition to nutrition, It means food. Here, the term "function (surname)" means that the structure and function of the human body have a beneficial effect for health use such as controlling nutrients or physiological action. The food of the present invention can be prepared by a method commonly used in the art and can be prepared by adding raw materials and ingredients which are conventionally added in the art. In addition, the formulations of the food can also be produced without restrictions as long as they are formulations recognized as food. The food composition of the present invention can be manufactured in various forms, and unlike general pharmaceuticals, it has advantages of being free from side effects that may occur when a food is used as a raw material for a long time, and is excellent in portability.

In another aspect of the present invention, there is provided a feed composition for preventing or ameliorating an inflammatory disease or diabetes comprising the compound or a pharmaceutically acceptable salt thereof as an active ingredient.

The terms used here are the same as those described above.

As used herein, the term " feed " means any natural or artificial diet, single meal, or the like ingredients described above for feeding, ingesting, digesting, or suitable for the animal. Specifically, the feed according to the present invention can be produced in various types of feeds known in the art, and preferably includes a concentrated feed, a feed and / or a special feed.

Concentrated feeds include seeds containing cereals such as wheat, oats, and corn, and by-products such as rice bran, wheat bran, barley, etc., Which is the by-product of the starch residue, which is the main component of the starch residue, which is the remaining product of starch minerals, sweet potatoes, potatoes, etc., which is a byproduct obtained by concentrating the fresh liquid product obtained from fish meal, fish meal, fish residue, The animal feed such as fish soluble, meat powder, blood meal, wheat meal, skim milk powder, cheese in milk, dried whey in the case of casein production in skim milk, dry yeast, yeast, Chlorella, algae, but not limited thereto.

Roughage includes root vegetables such as wild grasses, grasses and fodder, raw turnip for feed, turnip for feed, feed bit, turnip root, Silage, which is filled with lactic acid and fermented by lactic acid, hay, dried hay, herbage straw, straw of leguminous crops, leaves of leguminous plants, and the like. Special diets include mineral feeds such as fodder, rock salt, urea and its derivatives such as diuretic isobutane, nutrients such as diuretic isobutane, and nutrients that can be supplemented when only natural feed ingredients are mixed or supplemented with diets Feed additives, dietary supplements, which are substances added in small quantities, but are not limited thereto.

The composition for feed according to the present invention can be prepared by adding a compound represented by the formula (1) in an appropriate effective concentration range according to various feed production methods known in the art.

In another aspect of the present invention, the present invention provides a method of preventing or treating an inflammatory disease or diabetes, comprising the step of administering the pharmaceutical composition to a subject.

The terms used here are the same as those described above.

In the present invention, the term " individual " refers to all animals including humans who have developed or are capable of developing the target disease, and the individual can be effectively treated by administering the pharmaceutical composition of the present invention to a suspect individual.

The term " administering " as used herein means introducing the pharmaceutical composition of the present invention to a suspected individual of a target disease by any appropriate method, and the administration route may be administered through various routes of oral or parenteral administration, .

The pharmaceutical composition of the present invention can be administered in a pharmaceutically effective amount.

As used herein, the term " pharmaceutically effective amount " means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment and the effective dose level will vary depending on the species and severity, age, sex, , The activity of the drug, the sensitivity to the drug, the time of administration, the route of administration and rate of release, the duration of the treatment, factors including co-administered drugs, and other factors well known in the medical arts. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. And can be administered singly or multiply. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without adverse effect, and can be easily determined by those skilled in the art.

The pharmaceutical composition of the present invention is not particularly limited as long as it is an object for prevention or treatment of a target disease, and any of them can be applied. For example, non-human animals such as monkeys, dogs, cats, rabbits, guinea pigs, rats, mice, cattle, sheep, pigs and goats can be used, , Intraperitoneally, intrapulmonary, and intranasal, and may be administered by a suitable method, including localized administration, if necessary, for localized treatment. The preferred dosage of the pharmaceutical composition of the present invention varies depending on the condition and the weight of the individual, the degree of disease, the type of drug, the administration route and the period of time, but can be appropriately selected by those skilled in the art. For example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine dural or intracerebral injection.

Suitable total daily doses may be determined by the treatment within the scope of sound medical judgment and are generally in the range of 0.001 to 1000 mg / kg, preferably 0.05 to 200 mg / kg, more preferably 0.1 to 100 mg / kg can be administered once or several times a day.

In another aspect of the present invention, the present invention provides a compound represented by the formula (1) or a pharmaceutically acceptable salt thereof.

[Chemical Formula 1]

The terms used here are the same as those described above.

The present invention as still another aspect for solving the aforementioned problems is, containing the compound, a pharmaceutically acceptable salt thereof, Oscar Pasteurella styryl lance (Oscarella stillans) containing the compound extract, or fractions thereof as an active ingredient Which comprises administering to a mammal a therapeutically effective amount of a compound of the present invention.

The terms used here are the same as those described above.

The cosmetic composition according to the present invention can be used as a cosmetic composition in the form of a solution, an ointment for external use, a cream, a foam, a nutritional lotion, a softening water, a pack, a soft water, an emulsion, a makeup base, But are not limited to, emulsions, pastes, gels, lotions, powders, soaps, surfactant-containing cleansers, oils, powder foundations, emulsion foundations, wax foundations, patches and sprays.

In addition, the cosmetic composition of the present invention may further comprise at least one cosmetically acceptable carrier to be incorporated in a general skin cosmetic composition, and examples thereof include oil, water, a surfactant, a moisturizer, A thickening agent, a chelating agent, a coloring matter, an antiseptic, a perfume, and the like may be appropriately compounded, but the present invention is not limited thereto. The cosmetically acceptable carrier to be contained in the cosmetic composition of the present invention varies depending on the formulations.

When the formulation of the present invention is an ointment, a paste, a cream or a gel, the carrier component may be an animal oil, a vegetable oil, a wax, a paraffin, a starch, a tracer, a cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide Mixtures of these may be used.

When the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder or a mixture thereof may be used as the carrier component, Propellants such as fluorohydrocarbons, propane / butane or dimethyl ether.

When the formulation of the present invention is a solution or emulsion, a solvent, a dissolving agent or an emulsifying agent is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, 1,3-butyl glycol oil may be used, in particular fatty acid esters of cottonseed oil, peanut oil, corn oil, olive oil, castor oil and sesame oil, glycerol aliphatic esters, polyethylene glycols or sorbitan may be used have.

When the formulation of the present invention is a suspension, a carrier such as water, a liquid diluent such as ethanol or propylene glycol, a suspension such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, Crystalline cellulose, aluminum metahydroxide, bentonite, agar or tracant, etc. may be used.

When the formulation of the present invention is a soap, an alkali metal salt of a fatty acid, a fatty acid hemiester salt, a fatty acid protein hydrolizate, isethionate, a lanolin derivative, an aliphatic alcohol, a vegetable oil, glycerol, .

When the formulation of the present invention is an interface-active agent-containing cleansing, the carrier component is selected from the group consisting of aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters.

The present invention as still another aspect for solving the aforementioned problems is, containing the compound, a pharmaceutically acceptable salt thereof, Oscar Pasteurella styryl lance (Oscarella stillans) containing the compound extract, or fractions thereof as an active ingredient A composition for quasi-drugs for the prevention or amelioration of an inflammatory disease or diabetes.

The terms used here are the same as those described above.

The term " quasi-drug " in the present invention means a fiber, a rubber product or the like used for the purpose of treating, alleviating, treating or preventing a disease of a human or an animal, a weak action on the human body, Or products similar to those which are not machinery, preparations used for sterilization, insecticides and similar uses for the prevention of infections, for the purpose of diagnosis, treatment, alleviation, treatment or prevention of diseases of human beings or animals Machinery, or apparatus, and that is not an apparatus, machine, or apparatus of an article used for the purpose of giving pharmacological effects to the structure or function of a person or animal, It also includes supplies.

The novel compounds of the present invention, Oscar Pasteurella styryl lance (Oscarella stillans) extract, or an anti-inflammatory, including fractions thereof and an anti-diabetic composition immunomodulatory it exhibits a controlled activity of the inflammatory mediator production and inflammation-related diseases comprising the same And can also be used for prevention and treatment of diabetes because it exhibits antidiabetic activity against type 1 and type 2 diabetes in a zebrafish model.

Figure 1 shows the chemical structure of the novel anthranilic acid derivative [1].
Figure 2 shows selected HMBC correlations of the novel anthranilic acid derivatives [1].
3 is a view showing the synthesis route of the novel anthranilic acid derivative [1].
4 is a graph showing the cell proliferation rate of RAW 264.7 cells according to the concentration of compound [1] (0, 0.1, 1, 10, 50, 100 / / mL).
FIG. 5 is a graph showing the production of Nitrite and the expression of TNF-.alpha. And IL-6 when Compound [1] was treated at different concentrations in RAW 264.7 cells.
FIG. 6 shows the effect of compound [1] on NF-kB activation and IκB-α degradation in RAW 264.7 cells treated with LPS.
Figure 7 shows the effect of compound [1] on AP-1 activation and c-Jun and c-Fos expression control in RAW 264.7 cells treated with LPS.
Figure 8 shows the effect of compound [1] on ATF-3 activation in RAW 264.7 cells treated with LPS.
9 is a Western blot photograph showing the levels of p-ERK, ERK, p-p38, p38, p-JNK and JNK expression in RAW 264.7 cells according to the concentration of compound [1].
Fig. 10 shows the antidiabetic effect when compound [1] was administered in a short-term in a diabetic zebrafish model induced by alloxan.
Fig. 11 shows the antidiabetic effect of compound [1] on long-term administration in a alloxan-induced diabetic zebrafish model.
Figure 12 shows the antidiabetic effect of compound [1] in a diabetic zebrafish model induced by excessive insulin.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are intended to illustrate the present invention, but the scope of the present invention is not limited thereto.

Example  1: sponges Oscarella stillans from  new Anthranilic Acid  Derivatives [ 1]  Separation and structural confirmation

Sponges Oscarella stillans (Raw weight: 0.9 kg) was extracted with methanol twice at room temperature, and then extracted once with methanol and dichloromethane (1: 1) at room temperature. All the extracts were combined and concentrated to obtain an extract of the sea sponges.

The extract was suspended in 90% methanol and fractionated three times with hexane to obtain 4.01 g of hexane fraction. The remaining 90% methanol fraction was concentrated, then suspended in 70% methanol, repeatedly fractionated three times with dichloromethane solvent, 1.44 g. The remaining 70% methanol fraction was concentrated, suspended in water, and repeatedly dispensed with butanol three times to obtain 1.39 g of a butanol fraction.

The butanol fraction obtained above was applied to a Sephadex LH-20 column and eluted with methanol to obtain 8 small fractions. The third fraction was purified by reverse phase C-18 vacuum pressure column chromatography [25-30% MeOH (0.1% FTA) and reverse phase C-18 HPLC [(23% MeOH (0.1% FTA)] to give compound [1].

The structure of the compound [1] was identified by 2-amino-3- (3'-aminopropoxy) benzoic acid] Respectively. (Figs. 1 and 2)

Amino-3- (3'-aminopropoxy) benzoic acid]

Colorless amorphous solid; UV (MeOH) ? max (log ? ) 210 (4.55), 242 sh (3.83), 318 (3.55); IR ? _max 3500-2500 (br, NH and / or COOH), 1696.6 (C = O), 1195; ¹ H NMR (CD ₃ OD, 500 MHz) ? _{H 2.18 (2H, p, J} = 6.5 Hz, H-2 '), 3.19 (2H, t, J = 7.5 Hz, H-3'), 4.15 (2H, t, J = 6.0 Hz, H-1 ' ), 6.64 (1H, t, J = 8.0 Hz, H-5), 7.01 (1H, d, J = 8.0 Hz, H-4), 7.49 (1H, d, J = 8.0 Hz, H-6), _{(DMSO- d 6, 500 MHz)} ? _{H 2.05 (2H, p, J} = 6.3 Hz, H-2 '), 3.02 (2H, t, J = 7.3 Hz, H-3'), 4.05 (2H, t, J = 6.0 Hz, H-1 ' ), 6.47 (1H, t, J = 7.8 Hz, H-5), 6.92 (1H, d, J = 7.5 Hz, H-4), 7.32 (1H, d, J = 8.0 Hz, H-6), 8.07 (3H, br s, NH 3); ¹³ C NMR (CD ₃ OD, 125 MHz) ? _C -2 '), 38.9 (C-3'), 67.3 (C-1 '), 113.8 (C-1), 116.3 6), 141.5 (C-2), 148.6 (C-3), 171.9 (C-7); ESIMS m / z 211.1 [M + H] ⁺ , 233.1 [M + Na] ⁺ , 421.2 [2M + H] ⁺ , 443.2 [2M + Na] ⁺ ; FABMS m / z 211.1 [M + H] < ⁺ >; HREIMS m / z 210.1001 [M] +, (calcd for C 10 H 14 N 2 O 3, 210.1004).

Example  2: New Anthranilic Acid  Derivatives [ 1]  synthesis

3-Hydroxyanthranilic acid (0.47 g) was mixed with methanol (5.1 mL) and SOCl ₂ (0.11 mL) and reacted under reflux for 16 hours to obtain 3-hydroxyanthranilic acid methyl ester (3 (yield: 0.52 g), which was reacted with di-tert-butyl dicarbonate (0.89 mL) in 3N NaOH (15 mL) to give methyl 2- (tert- butoxycarbonylamino) (Tert-butoxycarbonylamino) -3-hydroxybenzoate, 0.58 g, compound 2 ] was obtained. An acetone anhydride solution (5.4 mL) of Compound 2 (0.29 g) was dissolved in K ₂ CO ₃ (Tert-butoxycarbonylamino) -3- [3 (3-bromopropyl) phthalimide, 0.5 g) and N- (3- bromopropyl) phthalimide - (1, 3-dioxoisoindolin-2-yl) propoxy] bozoate < / RTI > , 0.32 g, compound 3 } was obtained. Compound 3 was reacted with c-HCl (1.2 mL) at 100 for 48 hours with stirring, and then concentrated under reduced pressure.

The residue thus obtained was purified by column chromatography and recrystallization (acetone) to obtain the hydrochloride of the novel anthranilic acid derivative [1] (yield: 0.13 g). (Fig. 3)

Example  3: New Anthranilic Acid  Derivatives [ 1]  Evaluation of anti-inflammatory efficacy

(1) New Anthranilic Acid  Derivatives [ 1]  Toxicity assessment

RAW 264.7 cells were treated with compound [1] and MTT assay was performed to assess toxicity.

Rat macrophage RAW 264.7 cells were cultured in Dulbecco's modified Eagles medium (DMEM, Corning Life Sciences, USA) containing 10% heat inactivated FBS (ThermoFisher scientific, USA) and 5% antibiotics (ThermoFisher scientific, USA) And cultured in medium at 5% CO ₂ , 37. To measure cytotoxicity, RAW 264.7 cells were inoculated into 96-well culture plates at 2 x 10 ⁴ cells / mL and then treated with various concentrations (0, 0.1, 1, 10, 50, 100 / / [1] and then cultured for 24 hours.

Cell viability was confirmed by MTT [3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide] assay, which indicates the mitochondrial activity of living cells. MTT reduced by mitochondrial enzyme to formazan was measured by absorbance at 550 nm using a Molecular Device microplate reader.

All data were expressed as mean ± standard error, and statistical differences between the control and treatment groups were determined using Student's t-test. When the P value was less than 5%, it was judged statistically significant.

4 is a graph showing the cell proliferation rate of RAW 264.7 cells according to the concentration of compound [1] (0, 0.1, 1, 10, 50, 100 / / mL).

As shown in Fig. 4, cytotoxicity by compound [1] was not observed in RAW 264.7 cells. That is, it can be confirmed that the compound [1] does not show toxicity to cells even at a high concentration.

(2) New Anthranilic Acid  Derivatives [ 1]  Evaluation of anti-inflammatory effect

1) Evaluation of inhibitory effect of inflammatory mediators

RAW 264.7 cells were treated with compound [1] and then the degree of inflammation mediator Nitrite was measured using Griess reagent.

Raw mouse macrophage Raw 264.7 cells are cultured in 96 well plates. The culture conditions were 5% with Dulbecco's modified Eagles medium (DMEM, Corning Life Sciences, USA) containing 5% of inactivated FBS (ThermoFisher scientific, USA) and 5% CO ₂ , 37.

The cells are pretreated with compound [1] at various concentrations (0, 0.1, 1, and 10 μg / mL) for 2 hours, treated with 1 μg / mL of LPS, and cultured for 24 hours. The cultured medium was recovered and the degree of nitrite production was measured using a Griess reagent. All data were expressed as mean ± standard error, and statistical differences between the control and treatment groups were determined using Student's t-test. When the P value was less than 5%, it was determined to be statistically significant and marked with * on the graph.

FIG. 5A is a graph showing the production of Nitrite when RAW 264.7 cells were treated with Compound [1] at different concentrations.

As shown in Fig. 5A, it was confirmed that there was no influence on the production of Nitrite, an inflammatory mediator, in macrophage RAW 264.7 cells activated with LPS by the compound [1].

2) Evaluation of inhibitory effect on inflammatory cytokine expression

RAW 264.7 cells were treated with compound [1] and then subjected to ELISA analysis to measure the level of inflammatory cytokines TNF-? And IL-6.

Rat macrophage RAW 264.7 cells are cultured in 96-well plates. The culture conditions were 5% with Dulbecco's modified Eagles medium (DMEM, Corning Life Sciences, USA) containing 5% of inactivated FBS (ThermoFisher scientific, USA) and 5% CO ₂ , 37.

The cells were pretreated with compound [1] at various concentrations (0, 0.1, 1, and 10 μg / mL) for 2 hours, treated with 1 μg / mL of LPS and incubated for 24 hours. The production of TNF-? And IL-6 was measured using a mouse enzyme-linked immunosorbent assay (ELISA) kit (R & D Systems Inc., Minneapolis, MN, USA). All data were expressed as mean ± standard error, and statistical differences between the control and treatment groups were determined using Student's t-test. When the P value was less than 5%, it was determined to be statistically significant and marked with * on the graph.

FIG. 5B is a graph showing the expression of TNF-? And IL-6 when RAW 264.7 cells were treated with Compound [1] at different concentrations.

As shown in Fig. 5B, it was confirmed that TNF-? And IL-6 production, which are inflammatory cytokines, were inhibited in macrophage RAW 264.7 cells activated with LPS by the compound [1].

3) NF -? B pathway inhibition effect evaluation

Expression of inflammatory proteins is regulated through intracellular signaling mechanisms. In particular, inflammatory proteins induced by LPS are expressed through the NF-kB pathway. In order to evaluate the inhibitory effect of NF-? B pathway by the compound [1], confirmation of the expression of IkB, an upper signal protein of NF-? B, translocation into the nucleus of p65, a subunit of NF-kB, and p65 promoter activity Respectively. In addition, the effect of compound [1] on NF-kB activation was evaluated using a luciferase reporter assay.

Figure 6 shows the effect of NF-kB activation and I? B-? Of compound [1] on RAW 264.7 cells treated with LPS. And the effect on degradation.

As shown in FIG. 6, the effect of the compound [1] on NF-kB activation was almost unduly reduced by the treatment of the compound [1] with the luciferase activity almost 4-fold increased by LPS. Compound [1] inhibited p65 expression in a concentration dependent manner, decreased p65 nuclear migration, and inhibited LPS induced I? B degradation.

4) AP-1 activation and c- Jun  And c- Fos  Evaluation of Expression Modulating Effectiveness

A luciferase reporter assay and western blot analysis were performed to evaluate the ability of the compound [1] to regulate another transcription factor, AP-1 activation.

Compound [1] inhibited the activity of AP-1 induced by LPS in a concentration-dependent manner and lowered the nuclear migration of c-Jun and c-Fos by LPS treatment (Fig. 7)

5) ATF -3 Activation Control Effectiveness Evaluation

LPS induces ATF-3 activation in various cells including macrophages, and ATF-3 plays a crucial role as a negative regulator of LPS-induced inflammatory response. In order to confirm the effect of compound [1] on ATF-3 expression, various concentrations of compound [1] were applied, LPS was treated after 4 hours, and Western blot analysis was performed. In addition, the efficacy of ATF-3 short hairpin (sh) RNA was evaluated to confirm the relevance of ATF-3 in cells to which compound [1] was added.

As shown in FIG. 8, LPS significantly increased the expression of ATF-3 and Compound [1] additionally enhanced the expression of ATF-3. ATF-3 shRNAs are TNF-? And the inhibitory effect of the compound [1] on the production of IL-6. These results indicate that the inhibitory effect of compound [1] on pro-inflammatory cytokine production is due to the induction of ATF-3.

6) MAP Kinases  Evaluation of inhibition of phosphorylation

In order to evaluate the effect of inhibiting MAP Kinases phosphorylation by the compound [1], RAW 264.7 cells were pretreated with compound [1] (0, 0.1, 1, 10 ㎍ / Of LPS was not treated or treated and activated for 15 minutes. Then, the degree of phosphorylation of ERK, p38, and JNK in the macrophages was evaluated by Western blot analysis.

FIG. 9 is a Western blot photograph showing the levels of p-ERK, ERK, p-p38, p38, p-JNK and JNK expressed in RAW 264.7 cells according to the concentration of compound [1].

As shown in FIG. 9, it was confirmed that the compound [1] inhibited the phosphorylation of JNK and ERK1 / 2 by LPS treatment and did not affect the phosphorylation of p38.

In conclusion, the novel anthranilic acid derivatives [1] exhibit anti-inflammatory activity and immunoregulatory activity through the inhibition of TNF-? And IL-6 production in activated macrophages. This is due to JNK, ERK, AP -1, inactivation of NF-kB and activation of ATF-3.

Example  4: New Anthranilic Acid  Derivatives [ 1] Anti-diabetic  Efficacy evaluation

(One) Alloxan  Induced diabetes At the zebra fish  The compound [ 1]  Identification of antidiabetic effect by short-term administration

Five days post-fertilization zebrafish larvae were placed in 96 wells and stained with 25? M 2-NBDG for 12 hours. After exposure to 100 μM alloys for 15 minutes, the cells were replaced with 0.03% sea salt solution and exposed for up to 5 hours. Fluorescence microscopy (Olympus 1 × 70, Olympus, Japan) was used for direct pancreatic islet observation, and 25 μM 2-NBDG was re-stained for 1 h before fluorescence microscopy. Compound [1] was exposed to 0.5, 1, 5? M concentration for 1 hour after the first microscope photographing. Secondary microscopic photographs were taken to observe changes in the pancreatic islets caused by compound [ 1] , and 25? M 2-NBDG was re-stained for 1 h before fluorescence microscopy as in the first microscopic photograph. Images obtained through fluorescence microscopy were analyzed for the intensity of the pancreatic islets of the zebrafish and the intensity of the fluorescence intensity of the pancreatic islets using Focus Lite and Image J software.

As a result, the size of the pancreatic islets decreased with alooxa treatment, which was markedly increased by compound [1] and showed the strongest increase at 0.5 μM concentration. (Fig. 10)

As a result, the compound [1] In type 1 diabetes model It was confirmed that there was antidiabetic efficacy in the short-term administration.

(2) Alloxan  Induced diabetes At the zebra fish  The compound [ 1]  Identification of antidiabetic effect by long-term administration

Six days post-fertilization (5 day post fertilization) zebrafish larvae were placed in 96 wells and exposed to 100? M alloxic acid for 6 hours. Compound [1] was then exposed for 12 hours at 0.1, 1, and 10? M concentrations. Fluorescence microscopy (Olympus 1 × 70, Olympus, Japan) was used for direct pancreatic islet observation and 40 μM 2-NBDG was stained for 30 min before fluorescence microscopy and exposed to 0.03% saline solution for 20 min . Images obtained through fluorescence microscopy were analyzed for the intensity of the pancreatic islets of the zebrafish and the intensity of the fluorescence intensity of the pancreatic islets using Focus Lite and Image J software.

As a result, the size of pancreatic islets decreased by alooxan treatment was significantly increased at 0.1 μM concentration of compound [1], which showed better efficacy than glymefamide, a diabetic drug. Fluorescence intensity also showed the strongest increase at 0.1 μM concentration of compound [1]. (Fig. 11)

Therefore, it was confirmed that when compound [ 1 ] was administered to a type 1 diabetes model for a long time, excellent antidiabetic effect was obtained at a low concentration of 0.1? M.

(3) Diabetes caused by excessive insulin At the zebra fish  The compound [ 1] Anti-diabetic  Check the effect

Three days after fertilization (3 day post fertilization), zebrafish larvae were placed in 6 wells and exposed to 10 M insulin for 48 hours. Compound [1] was then exposed to a concentration of 0.5, 1, 5 g / mL for 48 hours. Fluorescence microscopy (Olympus 1 × 70, Olympus, Japan) was used for direct pancreatic islet observation and stained with 40 μM 2-NBDG for 30 min before fluorescence microscopy. Images obtained by fluorescence microscopy were analyzed for pancreatic islet size of zebrafish using Focus Lite software.

As shown in FIG. 12, the pancreatic islet size decreased by excessive insulin exposure was significantly increased at 1 and 5 g / mL of compound [1] compared to the untreated control, and fluorescence intensity was also increased to 5 M Of the total recovery.

Thus, compound [ 1 ] has excellent antidiabetic efficacy in a type 2 diabetes model induced by excessive insulin exposure.

From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are to be considered in all respects as illustrative and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the scope of the present invention as defined by the appended claims.

Claims (7)

To Oscar Pasteurella styryl lance (Oscarella comprising a compound, pharmaceutically acceptable salt thereof, wherein the compound represented by general formula (1) stillans) extract, or a fraction, an inflammatory disease or a pharmaceutical composition for preventing or treating diabetes containing as an active ingredient thereof:
[Chemical Formula 1]

. The pharmaceutical composition according to claim 1, wherein the extract is water, a C ₁ to C ₄ alcohol, dichloromethane, or a mixed solvent thereof. The pharmaceutical composition according to claim 1, wherein the fraction is water, a C ₁ to C ₄ alcohol, dichloromethane, chloroform, ethyl acetate, hexane, or a mixed solvent fraction thereof. To Oscar Pasteurella styryl lance (Oscarella comprising a compound, pharmaceutically acceptable salt thereof, wherein the compound represented by general formula (1) stillans) extract, or a fraction containing, as an active ingredient for food or inflammatory disease preventing or improving diabetes composition thereof:
[Chemical Formula 1]

. A feed composition for preventing or ameliorating an inflammatory disease or diabetes comprising a compound represented by the following formula (1) and a pharmaceutically acceptable salt thereof as an active ingredient:
[Chemical Formula 1]

. A method for the prophylaxis or treatment of an inflammatory disease or diabetes, comprising administering the pharmaceutical composition of claim 1 to a subject other than a human. 1. A compound represented by the following formula (1): < EMI ID =
[Chemical Formula 1]

.

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