KR101857350B1 - Novel Myricetin Derivative and Anti-inflammatory Composition Using the Same - Google Patents

Abstract

The present invention relates to a novel myristetine derivative isolated from Clausena xcavata extract and a novel myristate derivative thereof, based on the inhibitory activity of NO production in mouse macrophage cell line (RAW 264.7 cells) stimulated by LPS of the myrlicetin derivative As an active ingredient, an antiinflammatory composition.

Description

TECHNICAL FIELD The present invention relates to a novel myristatin derivative and an antiinflammatory composition using the same.

The present invention relates to a novel myristatin derivative and an anti-inflammatory composition using the same.

Due to recent developments in the economy, lifestyle diseases such as cancer, diabetes, hypertension, obesity, and vascular diseases are continuously increasing in the world due to changes in living environment and diet. In addition, due to various factors such as environmental changes due to rapid industrial development of modern society, and increased stress, inflammation induced by immunomodulation abnormality is persisted, and the incidence of related diseases is increasing.

The inflammatory response is a mechanism to repair and regenerate damaged tissue as a defense against external physical and chemical stimuli and various infectious agents including tissue damage (Zamora R et al., Mol. Med. 6: 347-373 (2000) ; Mariathasan S and Monack DM. Nat. Rev. Immunol. 7: 31-40 (2007); Lee HN et al., J. Food Sci. Technol. 43: 65-71 (2011)). During the inflammation reaction, plasma accumulates on the inflamed area, diluting the toxicities secreted by the bacteria, increasing the blood flow, and accompanied by symptoms such as erythema, pain, edema, and fever. In normal cases, the organism neutralizes or eliminates the onset factor through inflammatory reaction, regenerates the upper tissue and regenerates the normal structure and function, but chronic inflammatory reaction, which is continuously or excessively caused, causes tissue damage. This inflammatory response induces progression to stomach, colon, bladder, and prostate cancer, and causes various diseases such as rheumatoid arthritis and chronic infection (Hofseth LJ and Ying L. Biochim. Biophys. Acta 1765: 74-84 (2006 ; Yun HY et al., Rev. Neurobiol. 10: 291-316 (1996); Stuehr DJ et al., P. Natl. Acad Sci. USA 88: 7773-7777 (1991)).

(IL-1β), IL-6, IL-6, IL-6, IL-6, IL-6, IL-6, and IL-6 in the presence of inflammatory mediators such as macrophages and monocytes. 8, and IL-12 (Guha M and Mackman N. Cell Signal. 13: 85-94 (2001)). A typical example of an inflammatory response is activation of the toll like receptor (TLR) signaling pathway of macrophages by pathogen associated molecular patterns (PAMPs). Lipopolysaccharide (LPS), a type of endotoxin present in the outer membrane of gram-negative bacteria, belongs to the inflammatory mediator PAMPs present in external microorganisms. TLR4 recognizes LPS with the help of MD-2 or CD14 (Miyake K. Trends Microbiol. 12: 186-192 (2004)), and the lower signaling pathway (NF-κB), which is a transcription factor, which activates NF-κB (NF-κB). Food Sci. Technol. 41: 477-482 (2009)). NF-κB translocated to the nucleus was found to be an inflammatory cytokine (TNF-α, IL-1β, IL-6, IL-8 and IL-12), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 Nitric oxide (NO) induced by iNOS induces gene expression and exacerbates inflammation (Noh KH et al., J. Korean Soc. Food Sci. Nutr. 40: 625-634 (2011) ; Weisze et al., Biochem. J. 316: 209-215 (1996)). Nitric oxide, which is produced by iNOS in macrophages, reacts with superoxide to form peroxynitrite, which acts as a strong oxidizing agent and damages the cells, resulting in a variety of pathologies including inflammation and cancer (Gupta SC et al., Exp Biol Med., 236: 658-671, (2011); Riehemann et al., FEBS Lett., 442: 89-94 (1999)).

Although synthetic medicines such as ibuprofen, antihistamines, steroids, cortisone, immunosuppressants, and immunosuppressants are used, the therapeutic effect is temporary, there are many side effects such as simple symptom relief, hypersensitivity reaction and aggravation of the immune system, Treatment is difficult. Non-steroidal anti-inflammatory drugs (NSAIDS), which are now widely used as anti-inflammatory drugs, are known to cause serious side effects such as gastrointestinal disturbances, liver disorders, and renal insufficiency (Rainsford KD, Subcell biochem. , 42: 3-27, 2007, Guruprasad P. Aithal., Rheumatology., 7: 139-150, 2011, Praveen PN Rao et al., Pharmaceuticals., 3: 1530-1549, 2010).

Therefore, it is required to develop new drugs which have anti-inflammatory activity, have few side effects, and have a long-lasting effect. Techniques using natural substances with low toxicity are being developed. Korea Patent No. 101303198 discloses an anti-inflammatory effect of acorn extract. Korean Patent No. 100556524 discloses a camellia extract, Korean Patent No. 100536857 discloses a Daria extract, Korean Patent No. 101310980 discloses a Gugija extract, Although patent No. 101385768 discloses an antiinflammatory effect of Horseshoe chrysanthemum extract, studies using natural materials have been actively carried out, but development of commercially useful substances is not available.

The present invention discloses novel myrcetin derivatives having anti-inflammatory activity, isolated from Clausena excavata .

It is an object of the present invention to provide novel myrcetin derivatives.

It is another object of the present invention to provide an anti-inflammatory composition using the novel myristatin derivative.

Other and further objects of the present invention will be described below.

The present invention confirms that the 50% ethanol extract of Clausena xcavata and the new myrlicetin derivative, which is an active ingredient isolated from this 50% ethanol extract, have NO production inhibitory activity in LPS-stimulated mouse macrophage cell line (RAW 264.7 cells) . Here, Clausena X Cabata is a plant belonging to the family Rutaceae, which is native to Bangladesh, Bhutan, Cambodia, Vietnam and India.

In view of the above, the present invention in one aspect relates to a novel myristatin derivative, an isomer thereof, a prodrug thereof, a hydrate thereof, or a solvate thereof represented by the following Formula 1, and in another aspect the present invention relates to an anti-inflammatory composition comprising (i) Clausena xcavata extract or (ii) the novel myrcetine derivative, an isomer thereof, a prodrug thereof, a hydrate thereof, or a solvate thereof as an active ingredient, (I) an extract of Clausena xcavata or (ii) a disease caused by in vivo nitric oxide and its production including the above-mentioned novel myristate derivative, its isomer, its prodrug, its hydrate or its solvate as an active ingredient Or a pharmaceutically acceptable salt thereof.

[Chemical Formula 1]

As used herein, isomers include not only essentially pure diastereomers but also optical isomers, as well as conformation isomers (i.e. isomers differing only in the angle of one or more chemical bonds), position isomers, (Especially tautomers) or geometric isomers (e. G., Cis-trans isomers).

In the present specification, the term " prodrug "refers to a drug that chemically changes a drug to control its physical and chemical properties. Although the drug itself does not exhibit physiological activity, it does not exhibit chemical or enzymatic action Which means that the drug can be converted into the original drug and can be used to produce the drug.

In the present specification, the term "hydrate" means a compound having water bonded thereto, and it is meant to include an inclusion compound having no chemical bonding force between water and the compound.

"Solvate" as used herein means a compound formed between a molecule or ion of a solute and a molecule or ion of a solvent.

In the present specification, the term " Clausena xcavata extract "refers to an extract of Clausena xcavata stem, leaf, fruit, flower, root or the like to be extracted with water, a lower alcohol having 1 to 4 carbon atoms such as methanol, ethanol, (DMF), dimethylsulfoxide (DMSO), 1,3-butylene glycol, propylene glycol, or a mixture thereof, in the presence of an organic solvent, such as dichloromethane, chloroform, ethylene, acetone, hexane, ether, chloroform, ethyl acetate, Refers to an extract obtained by leaching using a solvent, an extract obtained by using a supercritical extraction solvent such as carbon dioxide, pentane, or a fraction obtained by fractionating the extract, and the extraction method may be performed by taking into account the polarity, extraction degree, Any method such as cold pressing, reflux, warming, ultrasonic irradiation, supercritical extraction and the like can be applied. In the case of the fractionated extract, the crude extract is suspended in a specific solvent, and the fractions obtained by mixing and leaving with a solvent having a different polarity are adsorbed on a column packed with silica gel or the like, and a hydrophobic solvent, a hydrophilic solvent or a mixture thereof Quot; means a fraction obtained by using a solvent as a mobile phase. Also, the meaning of the extract includes a concentrated liquid extract or a solid extract in which the extraction solvent is removed by a method such as freeze drying, vacuum drying, hot air drying, spray drying and the like. Preferably an extract obtained by using water, ethanol or a mixed solvent thereof as an extraction solvent, and more preferably an extract obtained by using a mixed solvent of water and ethanol as an extraction solvent.

In the present specification, the term " active ingredient "alone means an ingredient which exhibits the desired activity or which can exhibit activity together with a carrier which is not itself active.

As used herein, "anti-inflammatory" is meant to include improvement of an inflammatory disease (alleviation of symptoms), treatment, inhibition or delay of onset of such a disease as defined below.

In the present specification, the term "inflammatory disease" means an inflammatory reaction that is determined by an external physical or chemical stimulus or an infection of an external infectious source such as bacteria, fungi, viruses, various allergenic substances, or a local or systemic defense against autoimmunity Which may be defined as a pathological symptom. These inflammatory responses are caused by activation of various inflammatory mediators and enzymes associated with immune cells (e.g., iNOS, COX-2, etc.), secretion of inflammatory mediators (e.g., secretion of NO, TNF-a, IL-6, Cell migration, and tissue destruction, and manifest externally by symptoms such as erythema, pain, edema, fever, loss or loss of specific function of the body. The inflammatory disease may be acute, chronic, ulcerative, allergic or necrotic, so long as it is included in the definition of inflammatory diseases as above, it may be acute, chronic, ulcerative, allergic, Whether it is necrotic or not. Specifically, the inflammatory diseases include asthma, allergic and non-allergic rhinitis, chronic and acute rhinitis, chronic and acute gastritis or enteritis, ulcerative gastritis, acute and chronic nephritis, acute and chronic hepatitis, chronic obstructive pulmonary disease, Inflammatory bowel syndrome, inflammatory pain, migraine headache, headache, back pain, fibromyalgia, fascia disease, viral infection (e.g., C type infection), bacterial infection, fungal infection, burn, wound due to surgical or dental surgery, Rheumatoid arthritis, ankylosing spondylitis, Hodgkin's disease, pancreatitis, conjunctivitis, iritis, scleritis, uveitis, dermatitis (including atopic dermatitis), eczema, multiple sclerosis, etc. Will be included.

In the present specification, the term " a disease caused by in vivo nitric oxide and production "means a disease including inflammatory diseases listed above, asthma, Alzheimer's disease, Parkinson's disease, multiple sclerosis, sclerosis, to be. More specifically, reference can be made to the documents cited in Korean Patent Publication No. 1999-022174 and Korean Patent Publication No. 1999-022174, and Korean Patent Publication No. 1999-022174 and the documents cited therein Are considered part of this disclosure.

The anti-inflammatory composition or the like of the present invention may be contained in any amount (effective amount) as long as it can exhibit the activity of improving the inflammatory diseases intended to be treated according to the purpose of use, formulation, blending purpose, etc., An effective amount will be determined within the range of from 0.001% to 15% by weight based on the total weight of the composition. The term "effective amount" as used herein refers to an amount of an effective amount of a compound of the present invention to be administered to a mammal, preferably a human, to which the composition of the present invention is administered during a period of administration, / Delay, etc., of the active ingredient, which may be indicative of a medical or pharmacological effect. Such effective amounts can be determined experimentally within the ordinary skill of those skilled in the art.

The antiinflammatory composition of the present invention may further contain, in addition to the active ingredient, any compound or natural extract known to have safety and antiinflammatory activity for the purpose of raising or reinforcing antiinflammatory effects or the like. Specific examples of such compounds or extracts include MSM (dimethylsulfonylmethane), N-acetylglucosamine, glucosamine, and individual approval under the Health Functional Foods Act Complex extracts of CMO-containing complex extracts such as FAT (Fatty acid Complex) and Gassiogarifolia, complex extracts such as turmeric extract, chicken breast cartilage powder, rosehip powder, boswellia extract, beeswax alcohol, , Green lipped mussel extract oil, hop extract, and gold extract. Such compounds or natural extracts may be included in the anti-inflammatory compositions of the present invention in combination with one or more of their effectiveness.

The anti-inflammatory composition and the like of the present invention can be grasped as a food composition in a specific aspect.

The food composition of the present invention can be produced in any form and can be used in various forms such as beverages such as tea, juice, carbonated drink, ionic drink, processed oil such as milk and yogurt, gum, rice cake, Korean confectionery, A food, a health food, a food, a tablet, a capsule, a ring, a granule, a liquid, a powder, a slice, a paste, a syrup, a gel, a jelly and a bar. In addition, the food composition of the present invention may be classified into any product category as long as it meets the laws and regulations on the time of manufacture and distribution in the legal and functional category. For example, it may be a health functional food according to the Act on Health Functional Foods, or a confectionery, bean curd, fermented beverages, special-purpose foods, etc. according to each type of food in the food revolution (food standard, .

The food composition of the present invention may contain food additives in addition to the active ingredients thereof. Food additives are generally understood to be substances that are added to foods and mixed or infiltrated into food in the manufacture, processing or preservation of food, and their safety must be ensured since they are taken daily with food and for long periods. Food additives according to the Food Hygiene Act (food additives notification, food additive standards and standards) are limited by the classification of safe synthetic food additives as chemical synthetic products, natural additives and mixed preparations.

These functional food additives can be classified into sweeteners, flavors, preservatives, emulsifiers, acidifiers, and thickeners.

A sweetener is used to impart a sweet taste suitable for foods, and natural or synthetic sweeteners can be used. Preferably, natural sweeteners are used. Examples of natural sweeteners include sugar sweeteners such as corn syrup solids, honey, sucrose, fructose, lactose and maltose.

Flavors may be used to enhance taste or flavor, both natural and synthetic. Preferably, a natural one is used. When using natural ones, the purpose of nutritional fortification can be performed in addition to the flavor. Examples of natural flavoring agents include those obtained from apples, lemons, citrus fruits, grapes, strawberries, peaches, and the like, or those obtained from green tea leaves, Asiatica, Daegu, Cinnamon, Chrysanthemum leaves and Jasmine. Also, those obtained from ginseng (red ginseng), bamboo shoots, aloe vera, banks and the like can be used. The natural flavoring agent may be a liquid concentrate or a solid form of extract. Synthetic flavors may be used depending on the case, and synthetic flavors such as esters, alcohols, aldehydes, terpenes and the like may be used.

As the preservative, calcium sorbate, sodium sorbate, potassium sorbate, calcium benzoate, sodium benzoate, potassium benzoate and EDTA (ethylenediaminetetraacetic acid) can be used. As the emulsifier, acacia gum, carboxymethyl cellulose, Pectin and the like. As the acidulant, math, malic acid, fumaric acid, adipic acid, phosphoric acid, gluconic acid, tartaric acid, ascorbic acid, acetic acid, phosphoric acid and the like can be used. The acidulant may be added so that the food composition has a proper acidity for the purpose of inhibiting the growth of microorganisms other than the purpose of enhancing the taste.

Examples of the thickening agent include suspending agents, sedimentation agents, gel-forming agents, bulking agents and the like.

The food composition of the present invention may contain physiologically active substances or minerals which are known in the art and which are stable as a food additive in addition to the above-mentioned food additives in order to supplement and supplement functional and nutritional properties.

Examples of such physiologically active substances include catechins contained in green tea and the like, vitamins such as vitamin B1, vitamin C, vitamin E and vitamin B12, tocopherol, dibenzoyl thiamine, etc. Examples of minerals include calcium preparations such as calcium citrate, magnesium stearate , Iron preparations such as iron citrate, chromium chloride, potassium iodide, selenium, germanium, vanadium, zinc and the like.

The food composition of the present invention may contain an appropriate amount of the above-mentioned food additives according to the product type so as to achieve the purpose of addition thereof.

With regard to other food additives that can be included in the food composition of the present invention, reference may be made to the Food Code or the Food Additive Code.

The composition for anti-inflammation of the present invention and the like may be regarded as a pharmaceutical composition in another specific embodiment.

The pharmaceutical composition of the present invention may be prepared into oral formulations or parenteral formulations according to the route of administration by conventional methods known in the art, including pharmaceutically acceptable carriers in addition to the active ingredient. The term "pharmaceutically acceptable" as used herein means that the application (prescribing) subject does not have the above-mentioned toxicity that is adaptable without inhibiting the activity of the active ingredient.

When the pharmaceutical composition of the present invention is prepared into an oral formulation, it may be formulated into powder, granules, tablets, pills, sugar tablets, capsules, solutions, gels, syrups, suspensions, wafers And the like. Examples of suitable pharmaceutically acceptable carriers include sugars such as lactose, glucose, sucrose, dextrose, sorbitol, mannitol, xylitol, starch such as corn starch, potato starch and wheat starch, cellulose, methylcellulose, ethylcellulose, Cellulose derivatives such as sodium carboxymethyl cellulose and hydroxypropylmethyl cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate, mineral oil, malt, gelatin, talc, And the like. In case of formulation, a diluent such as a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant, and / or an excipient may be formulated according to need.

When the pharmaceutical composition of the present invention is prepared into a parenteral dosage form, it may be formulated in the form of an injection, transdermal drug delivery, nasal aspirate and suppository together with a suitable carrier according to methods known in the art. Examples of suitable carrier in the case of formulation with an injectable preparation include sterilized water, ethanol, polyol such as glycerol and propylene glycol, or a mixture thereof. Preferably, the carrier is selected from the group consisting of Ringer's solution, phosphate buffered saline containing triethanolamine, Water, or isotonic solution such as 5% dextrose may be used. When formulated with a transdermal drug, it can be formulated in the form of an ointment, a cream, a lotion, a gel, a solution for external use, a pasta, a liniment, or an air-roll. The nasal inhalant may be formulated in the form of an aerosol spray using a suitable propellant such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, etc. When formulated as a suppository, witepsol, tween 61, polyethylene glycols, cacao butter, laurin, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearates, and sorbitan fatty acid esters.

The formulation of pharmaceutical compositions is well known in the art and can be specifically described in Remington ' s Pharmaceutical Sciences (19th ed., 1995). This document is considered part of this specification.

The preferred dosage of the pharmaceutical composition of the present invention is 0.001 mg / kg to 10 g / kg per day, preferably 0.001 mg / kg to 1 g / day, depending on the patient's condition, body weight, sex, age, / kg < / RTI > The administration can be carried out once or several times a day. Such dosages should in no way be construed as limiting the scope of the invention.

In another specific embodiment, the composition for anti-inflammation of the present invention can be identified as a cosmetic composition. When the anti-inflammatory composition of the present invention is identified as a cosmetic composition, its use can be understood as a relief of inflammatory skin irritation.

The cosmetic composition of the present invention may contain, in addition to its active ingredient, conventional additives such as stabilizers, solubilizing agents, surfactants, vitamins, colorants and antioxidants, and carriers commonly used in cosmetic compositions.

The cosmetic composition of the present invention can be prepared into any of the formulations conventionally produced in the art and can be used in the form of solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, , Oil, powder foundation, emulsion foundation, wax foundation and spray, but is not limited thereto. More specifically, it can be manufactured in the form of a soft lotion, a nutritional lotion, a nutritional cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray or a powder.

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

When the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component. In the case of a spray, in particular, / Propane or dimethyl ether.

When the formulation of the present invention is a solution or an 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, , 3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan fatty acid esters.

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

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

The cosmetic composition of the present invention can be manufactured according to a method for producing a cosmetic composition which is conventionally performed in the art, except that it contains the active ingredient exhibiting anti-inflammatory activity and the like.

INDUSTRIAL APPLICABILITY As described above, according to the present invention, it is possible to provide an anti-inflammatory composition comprising a novel myristatin derivative and a novel myristatin derivative or the like as an active ingredient, and also to provide a novel myristatin derivative, It is possible to provide a composition for preventing or treating diseases caused by in vivo nitric oxide and production.

The composition for anti-inflammation of the present invention can be used as a food, a cosmetic, a medicine or the like for the purpose of improving an inflammatory disease or the like or for alleviating inflammatory skin irritation.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic illustration of a novel myristate derivative. FIG.
2 is ¹ H NMR data of a novel myristatin derivative.
3 is ¹³ C NMR data of a novel myristatin derivative.
4 is a COZY-NMR spectrum of a novel myristatin derivative.
5 is HMBC-NMR data of a novel myristatin derivative.
6 is HSQC-NMR data of a novel myristate derivative.
Figure 7 is ESI-MS data of the novel myristate derivative.
FIGS. 8 and 9 are results showing that the extract of Clausena xcavata and the new myrlicetin derivative have NO production inhibitory activity in LPS-stimulated mouse macrophage line (RAW 264.7 cells).
Figures 10 and 11 show the results of cytotoxicity of Clausena xcavata extract and the novel myristate derivative in LPS-stimulated mouse macrophage cell line (RAW 264.7 cells).

Hereinafter, the present invention will be described with reference to Examples and Experimental Examples. However, the scope of the present invention is not limited to these examples and experimental examples.

< Example > Clausena X Cabata  Preparation of extracts and isolation and identification of active compounds

< Example  1> Clausena X Cabata  Preparation of extract

The dried Clausena ( Clausena excavata ) 200g of leaf and stem crushed water was added with 1L of 50% ethanol and repeatedly soaked twice at room temperature for 24 hours and then filtered with a filter paper. The resulting 50% ethanol filtrate was concentrated under reduced pressure and then lyophilized to obtain a powdery extract.

< Example  2> Active compound separation

After the obtained extract was suspended in distilled water and then subjected to solvent fractionation with hexane, dichloromethane, ethyl acetate and butanol, the anti-inflammatory activity (NO production inhibitory activity in LPS-stimulated macrophages) of the extract and the fractions was measured 15% acetonitrile, 15 min; 15% acetonitrile, 35 min; 30% acetonitrile, 40 min.) Was added to the butanol fraction showing the highest activity as the mobile phase acetonitrile- (# 120-B-1 to # 120-B-4) were obtained by performing a reversed phase column (RP- _C18 ) preparative high performance liquid chromatography with 100% acetonitrile, (# 120-B-2-1 to # 120-B-2) were subjected to reverse phase column (RP- _C18 ) preparative high performance liquid chromatography by the gradient elution method using the same mobile phase, B-2-4) from the first fraction (# 120-B-2-1) To separate the water. Figure 1 shows the schematic diagram of the separation of active compounds.

< Example  3> Identification of active compounds

The separated active compounds were subjected to physicochemical and spectroscopic analyzes.

Yellow amorphous solid;

^{1 H-NMR (700 MHz,} methanol- d 4) δ 7.53 (2H, s, H-2 ', 6'), 6.38 (1H, d, J = 2.1 Hz, H-8), 6.24 (1H, d , J = 2.1 Hz, H- 6), 5.43 (1H, d, J = 1.4 Hz, H-1 "), 5.42 (2H, d, J = 1.4 Hz, H-1 ''',1"") , 4.21 (1H, dd, J = 3.5, 1.4 Hz, H-2 "), 4.20 (2H, dd, J = 3.5, 1.4 Hz, H- , J = 9.8, 3.5 Hz, H-3 ''', 3 ""), 3.91 (2H, dq, J = 9.8, 6.3 Hz, H-5''', 5 ""), 3.71 (1H, dd , J = 9.8, 3.5 Hz, H-3 "), 3.52 (2H, t, J = 9.8 Hz, H-4 ''',4""), 3.37 (1H, dq, J = 9.8, 6.3Hz, H-5 ''), 3.35 (1H, t, J = 9.8 Hz, H-4 ''), 1.35 (6H, d, J = 6.3 Hz, H- d, J = 6.3 Hz, H-6 ");

¹³ C-NMR (175 MHz, methanol- d ₄ )? 179.6 (C-4), 166.0 (C-7), 163.3 (C-5), 158.5 , 5 '), 143.1 (C-4'), 136.3 (C-3), 122.1 1), 102.4 (C-1 ''',1'''), 100.0 (C-6), 94.7 4 "), 72.1 (C-3 '', 3 '''), 72.2 (C-3'',5''), 71.9 , 71.1 (C-5 '', 5 '''), 18.2 (C-6'',6'''), 17.8 (C-6 '');

The active compound was found to be C ₃₃ H ₄₀ O ₂₀ from ESI-MS (757 [M + H] ⁺ ) and the ¹ H-NMR data and ¹³ C-NMR data and the COZY- From the NMR data, HMBC-NMR data and HSQC-NMR data, it was confirmed that the active compound had the structure of the above formula (1), and as a result of searching the existing literature, it was identified as a new compound not previously reported, ', 5'-tri-α-L-rhamnopyranside) was named as Myricetin 3,3'5'-tri-α-L-rhamnopyranside.

< Experimental Example > Anti-inflammatory activity experiment

In order to evaluate the intracellular anti-inflammatory activity of the Clausena xcavata extract of the above example and the above-identified active compound (# 120-B-2-1) isolated in mouse macrophage RAW264.7 cells, 5 × 10 ⁴ cells / well, and cultured at 37 ° C. and 5% CO ₂ for 24 hours. The samples were treated with 96-well plates for 1 hour, and treated with 1 μg / ml of LPS (Lipopolysacharide) for 24 hours. To evaluate the anti-inflammatory activity through the NO assay, 1% sulfanilamide and 0.1% N- (1-naphtyl) ethylenediamine dihydrochloride reagent were mixed in a ratio of 1: 1 using griess A and B reagents. After incubation at 37 ° C and 5% CO ₂ for 24 h, the supernatant of the culture medium was mixed with the griess (A + B) reagent at a ratio of 1: 1 and stored at room temperature for 10 min. And the inhibitory effect of NO production on the control group was measured by ELISA at 540 nm.

Cytotoxicity was measured by MTT assay. Specifically, the medium remaining in the 96-well plate was removed, and 100 μl of a 10% serum-free medium containing 5 mg / ml MTT solution was added to each well. The cells were incubated at 37 ° C in a 5% CO ₂ incubator for 2 hours. After removing the medium, DMSO was added to 100 μl / well and dissolved in a shaker for 15 minutes. The absorbance was measured at 540 nm using an ELISA reader to determine the cell viability and the toxicity of the sample was confirmed.

The results of the inhibition of NO production by the extract of Clausena xcavata and the active compound are shown in Figs. 8 and 9, and the cytotoxicity results thereof are shown in Fig. 10 and Fig.

The above-mentioned Clausena xcavata extract and the active compound inhibited NO production in a concentration-dependent manner. The IC _{50 values} were 63.74 / / ml and 281.24 각각, respectively, and did not show any cytotoxicity.

Claims (5)

A myristate derivative of the following formula 1, an isomer thereof, a hydrate thereof, or a solvate thereof.
[Chemical Formula 1]

An antiinflammatory composition comprising the myrcetin derivative of claim 1, an isomer thereof, a hydrate thereof, or a solvate thereof as an active ingredient.
3. The method of claim 2,
Wherein the composition is a food composition.
3. The method of claim 2,
Wherein the composition is a pharmaceutical composition.
3. The method of claim 2,
Wherein the composition is a cosmetic composition.

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