KR20230171532A - Guainolide-type sesquiterpen compounds isolated form Artemisiae Iwayomogii Herba and uses thereof - Google Patents

Abstract

The present invention relates to novel guaianolide-type sesquiterpenes compounds isolated from Han Injin and their uses, and includes iwayomogin A and iwayomogin B according to the present invention. B) has the effect of suppressing NO production and pro-inflammatory cytokine (TNF-α and IL-6) levels in microglial cells, so it can be useful for the prevention, improvement and treatment of inflammatory diseases, especially neuroinflammation. You can.

Description

Guainolide-type sesquiterpen compounds isolated form Artemisiae Iwayomogii Herba and uses thereof}

The present invention relates to a novel guaianolide-type sesquiterpenes compound isolated from Injin Han and a composition containing the same for preventing, improving, and treating inflammatory diseases.

Inflammation is a complex response to harmful pathogens expressed by immune cells. When immune cells are exposed to pathogens, including bacteria, viruses, or various toxic substances, activated immune cells secrete many factors that trigger an immune response. In the central nervous system (CNS), microglia, brain-resident immune cells, are known to play an important role in the pathological response of neuroinflammation leading to nerve damage. In a "resting" state, these macrophage-like cells monitor their surroundings. However, when microglia detect a threat, such as lipopolysaccharide (LPS), they become “activated” and induce an immune response. According to previous research, microglia stimulated and activated by endotoxin produce tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), and nitric oxide. , NO), excessive production and release of pro-inflammatory mediators such as pro-inflammatory cytokines and signaling molecules can have a negative effect, ultimately causing inflammation-mediated degenerative neurological diseases.

NO is synthesized by three types of nitric oxide synthase along with L-arginine and cofactors, and inducible NOS (iNOS) plays an important role in the NO production process. Since iNOS, one of the isozymes of NOS, is associated with various diseases, much research is being conducted targeting iNOS selective inhibitors to treat these diseases. Pro-inflammatory cytokines such as TNF-α and IL-6 are known to contribute to the progression and exacerbation of inflammation, and inhibition of the expression of TNF-α and IL-6 is associated with interaction with iNOS and subsequent regulation of NO production. It is believed to induce cessation of the overall inflammatory process, including the release of cytotoxic cytokines. Therefore, to date, natural products that can modulate pro-inflammatory mediators involved in the inflammatory pathway are considered powerful therapeutic candidates for neuroinflammation.

Meanwhile, Artemisia is one of the largest genera in the Compositae Family, containing about 500 species, and is commonly found in the Northern Hemisphere, especially Asia, North America, and Europe. In general, in oriental medicine, Artemisia species are used to treat malaria, hepatitis, and inflammatory diseases caused by bacterial, fungal, and viral infections. In particular, Artemisia iwayomogi Kitamura is a perennial herb with serrated yellow-green leaves and has been widely used as a medicinal plant in oriental medicine. It contains coumarin, phenolic compounds, fatty acids, ionones, monoterpenes, sesquiterpenes, and diterpenes. Various secondary metabolites, including , have been discovered and reported. However, no active compounds related to inflammation-mediated central nervous system disorders have yet been discovered in A. iwayomgi .

Accordingly, the present inventors isolated and identified a novel guaianolide-based sesquiterpine compound from the aerial parts of the heat killer, confirmed that the compound had an inhibitory effect on inflammation-causing mediators, and completed the present invention.

Republic of Korea Patent Publication No. 10-2021-0071678

The object of the present invention is to provide novel guaianolide sesquiterpenes compounds.

Additionally, an object of the present invention is to provide a pharmaceutical composition for preventing or treating inflammatory diseases.

Furthermore, an object of the present invention is to provide food and cosmetic compositions for preventing or improving inflammatory diseases.

Furthermore, an object of the present invention is to provide an anti-inflammatory composition.

In order to solve the above problems,

The present invention provides a guaianolide sesquiterpenes compound represented by the following formula (1), a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

[Formula 1]

.

In one embodiment, the compound may be iwayomogin A, which has a three-dimensional structure represented by the following formula (2):

[Formula 2]

.

In one embodiment, the compound may be iwayomogin B, which has a three-dimensional structure represented by the following formula (3):

[Formula 3]

.

In addition, the present invention provides a pharmaceutical composition for preventing or treating inflammatory diseases containing the guaianolide sesquiterpine compound represented by Formula 1 above as an active ingredient.

Additionally, the present invention provides a food composition for preventing or improving inflammatory diseases containing the guaianolide sesquiterpine compound represented by Formula 1 above as an active ingredient.

Additionally, the present invention provides a cosmetic composition for preventing or improving inflammatory diseases containing the guaianolide sesquiterpine compound represented by Formula 1 above as an active ingredient.

Additionally, the present invention provides an anti-inflammatory composition containing the guaianolide sesquiterpine compound represented by Formula 1 above as an active ingredient.

The present invention is a novel guaianolide-type sesquiterpenes compound from Injin Han, which separates iwayomogin A and iwayomogin B, which have stereoisomeric structures. and identified, the compound of the present invention not only inhibits NO production in microglial cells by inhibiting iNOS activity through direct binding to the iNOS enzyme, but also reduces the level of pro-inflammatory cytokines (TNF-α and IL-6). Since it has an inhibitory effect, it can be usefully used for the prevention, improvement, and treatment of inflammatory diseases, especially neuroinflammation.

Figure 1 is a diagram showing the chemical structures of Compound 1 (Iwayomogin A) and Compound 2 (Iwayomogin B) isolated from Haninjin of the present invention.
Figure 2 is a diagram showing the main correlations observed in the COZY (red line - indicated) and HMBC (arrow - indicated) NMR spectra of Compound 1 and Compound 2.
Figure 3 is a graph showing the main NOESY correlation and experimental and theoretical ECD spectra for compound 1 (A) and compound 2 (compound 2, B).
Figure 4 is a 3D and 2D diagram of molecular docking simulations obtained in the lowest energy conformation (RMSD <1.0) for the hydrogen bond interaction between compound 1 (A) and compound 2 (B) with iNOS (carbon: yellow/green; nitrogen: blue, oxygen: red, hydrogen bond interactions: indicated by pink dashes).
Figure 5 is a graph showing the inhibitory effect of compound 1 and compound 2 of the present invention on inflammatory cytokine release in BV-2 cells stimulated with LPS.

Hereinafter, the present invention will be described in detail.

In addition, the terminology used in this specification is a term used to appropriately express preferred embodiments of the present invention, and may vary depending on the intention of the user or operator or the customs of the field to which the present invention belongs. Therefore, definitions of these terms should be made based on the content throughout this specification. Throughout the specification, when a part is said to “include” a certain element, this means that it may further include other elements rather than excluding other elements, unless specifically stated to the contrary.

Throughout this specification, '%' used to indicate the concentration of a specific substance means (w/w) % for solid/solid, (w/v) % for solid/liquid, and Liquid/liquid is (v/v) %.

In one aspect, the present invention relates to a guaianolide sesquiterpenes compound represented by the following formula (1), a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

[Formula 1]

.

In one embodiment, the pharmaceutically acceptable salt is an acid addition salt formed by a pharmaceutically acceptable free acid. As used in the present invention, the term “pharmaceutically acceptable” means showing non-toxic properties to cells or humans exposed to the compound or its acid addition salt. The acid addition salts include inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid, aliphatic mono and dicarboxylates, phenyl-substituted alkanoates, hydroxy alkanoates and Obtained from non-toxic organic acids such as alkanedioates, aromatic acids, aliphatic and aromatic sulfonic acids. These pharmaceutically non-toxic salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide, and iodine. Ide, fluoride, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate , sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitro benzoate, hydroxybenzoate, methylbenzoate Toxybenzoate, phthalate, terephthalate, benzenesulfonate, toluenesulfonate, chlorobenzenesulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, hydroxybutyrate, glycolate, Includes malate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate or mandelate.

The acid addition salt according to the present invention can be prepared by a conventional method, for example, by dissolving the guaianolide sesquiterpine compound represented by Formula 1 in an excessive amount of aqueous acid solution, and dissolving this salt in a water-miscible organic solvent, such as It can be prepared by precipitation using methanol, ethanol, acetone, or acetonitrile. Additionally, this mixture can be prepared by evaporating the solvent or excess acid and drying it, or by suction-filtering the precipitated salt.

Additionally, a pharmaceutically acceptable metal salt can be prepared using a base. Alkali metal or alkaline earth metal salts are obtained, for example, by dissolving the compound in an excess of alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering off the undissolved compound salt, and evaporating and drying the filtrate. At this time, it is pharmaceutically appropriate to prepare sodium, calun, or calcium salts as metal salts. Additionally, the corresponding silver salt is obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (e.g., silver nitrate).

In one embodiment, the guaianolide sesquiterpine compound represented by Formula 1 of the present invention is not only a stereoisomer thereof or a pharmaceutically acceptable salt thereof, but also a possible solvate, hydrate, or hydrate that can be prepared therefrom. It may include both a semibody or a prodrug. In particular, the guaianolide sesquiterpine compound represented by Formula 1 may include isomers at all asymmetric carbons. In addition, when the compound of the present invention has an asymmetric carbon center in its substituent, it may exist as an R or S isomer, racemate, diastereo, isomer mixture, and individual diastereomers, and all of these isomers and mixtures thereof are present in the present invention. Included in the category of

In one embodiment, the compound represented by Formula 1 of the present invention may have a three-dimensional structure represented by Formula 2 or Formula 3 below, and the compound having a three-dimensional structure represented by Formula 2 below is Iwayomogin A ( iwayomogin A), a compound having a three-dimensional structure represented by the following formula (3) may be named iwayomogin B, respectively:

[Formula 2]

; and

[Formula 3]

.

In one embodiment of the present invention, the compounds represented by Formula 2 and Formula 3, respectively, were analyzed using an HR-DART mass spectrometer, and were confirmed to have the same molecular weight and molecular formula. In addition, through nuclear magnetic resonance (NMR) analysis, 1H NMR, 13C NMR, COZY ( ¹ H- ¹ H correlation spectroscopy), HSQC ( ¹ H- ¹³ C heteronuclear single quantum coherence), HMBC ( ¹ H- ¹³ C heteronuclear multiple As a result of obtaining bond correlation) and NOESY (nuclear overhauser enhancement spectroscopy) spectra and determining the molecular structure of each compound, it was found that guaianolide sesquiterpine has a structure in which 5-7-5 rings are sequentially connected (5/7/5 tricyclic system). They were identified as skeletal compounds. Finally, as a result of confirming the absolute arrangement through ECD spectrum and TDDFT method, the compound represented by Formula 2 (compound 1) is (3 R , 4 S , 5 S , 6 S , 7 R , 8 S ) -8 α -acetoxy- 3α ,4 α -dihydroxy-guaia-1,9,11(13)-triene-12,6 α -olide ((3 R ,4 S ,5 S ,6 S ,7 R ,8 S )-8 α -acetoxy-3 α ,4 α -dihydroxy-guaia-1,9,11(13)-trien-12,6 α -olide) and Iwayomogin It was named A (iwayomogin A), and the compound represented by Formula 3 (compound 2) is (3 R , 4 R , 5 S , 6 S , 7 R , 8 S )-8 α -acetoxy- 3 α ,4 ß -dihydroxy-guaia-1,9,11(13)-triene-12,6 α -olide ((3 R ,4 R ,5 S ,6 S ,7 R , 8 S )-8 α -acetoxy-3 α ,4 ß -dihydroxy-guaia-1,9,11(13)-trien-12,6 α -olide) and named as iwayomogin B. It was confirmed that the two compounds are enantiomers with different steric configurations at carbon 4.

In one embodiment, the compound of the present invention may be manufactured by synthesis, and preferably, may be obtained by extraction, separation and purification from Haninjin.

In the present invention, "haninjin (韓茵蔯)" is written as "Artemisiae Iwayomogii Herba" as a herbal medicine name, and refers to the aerial part of deowijigi ( Artemisia iwayomogi Kitamura).

In one embodiment, the method for isolating the compound of the present invention from Korean ginseng includes the steps of (a) preparing a Korean ginseng extract; (b) solvent fractionating the Korean Injin extract with ethyl acetate (EtOAc) and water to obtain an EtOAc fraction; and (c) purifying the compound represented by Formula 1 from the EtOAc fraction through column chromatography.

In step (a) above, “extract” refers to a preparation obtained by extracting Korean ginseng with an appropriate extraction solvent and concentrating the solvent by evaporating the solvent, but is not limited thereto, but is not limited to the extract obtained through the extraction process, or the diluted solution of the extract. Alternatively, it may be a dried product obtained by drying a concentrate or extract, or a crude product or purified product thereof. The Korean ginseng extract can be prepared using a general extraction method known in the art. The extraction method is not limited thereto, but any one of the methods such as hot water extraction, cold needle extraction, reflux cooling extraction, solvent extraction, steam distillation, ultrasonic extraction, dissolution, and compression can be selected and used, but the most Preferably, it may be a solvent extraction method. There is no limitation to the method for producing the extract of the present invention, and any known method can be used. For example, the extract included in the composition of the present invention can be prepared in powder form from the primary extract extracted by the solvent extraction method described above by additional processes such as reduced pressure distillation and freeze-drying or spray drying.

In step (a), any pharmaceutically acceptable organic solvent may be used as an appropriate solvent for extracting the extract from the Korean ginseng, and water or an organic solvent may be used, but is not limited thereto, e.g. For example, alcohols with 1 to 4 carbon atoms including purified water, methanol, ethanol, propanol, isopropanol, butanol, acetone, ether, Various solvents such as benzene, chloroform, ethyl acetate, methylene chloride, hexane, and cyclohexane can be used individually or in combination. Preferably, the extraction solvent used for the extraction may be one or more solvents selected from the group consisting of water and ethanol, more preferably 70 to 100% (v/v) ethanol aqueous solution, and even more preferably 70 to 100% (v/v) ethanol aqueous solution. Preferably, an 85 to 95% ethanol aqueous solution may be used.

In step (a), the step of preparing the Korean Jinjin extract may include mixing 8 to 15 times the volume of extraction solvent relative to the weight of the Korean Jinjin dried material and extracting it 1 to 3 times at 1 to 35 ° C. for 20 to 52 hours. .

In step (c), the column chromatography may mean open column chromatography, and one or more fillers selected from the group consisting of ion exchange resin, Sephadex, silica gel, and ODS are used. It may be performed, and preferably, ion exchange resin column chromatography, Sephadex column chromatography, and silica gel column chromatography may be performed sequentially.

In one embodiment according to the present invention, step (c), (c-1) 19 sequentially through chromatography using a column filled with ion exchange resin and an elution solvent mixed with acetone and water. Obtaining a fraction; (c-2) sequentially obtaining 13 fractions from the above fractions through chromatography using a column filled with Sephadex gel and an elution solvent containing a mixture of acetone and water; And (c-3) sequentially fractionating through chromatography using a column filled with silica gel and an elution solvent mixed with n-hexane, ethyl acetate, and methanol to obtain the compound represented by Formula 1. It may be.

In step (c-1), the ion exchange resin is a synthetic adsorption resin and may be an anion resin, for example, Diaion HP-20 resin, and the elution solvent is acetone and water at 30:70 (v/v). It may be eluted by a solvent concentration gradient that increases the mixing ratio of acetone, a non-polar solvent, from 100:0 (v/v).

In step (c-2), the Sephadex gel may be Sephadex LH-20 gel, and the elution solvent is a mixture of acetone and water at a ratio of 45:55 (v/v), which elutes without a solvent concentration gradient. It may be.

In step (c-3), the silica gel may have particles of 230-400 mesh, and the elution solvent is n-hexane, ethyl acetate, and methanol in a ratio of 60:35:5 (v/v). It may be eluted by a solvent concentration gradient from 0:95:5 (v/v).

In one embodiment, the compound of the present invention can inhibit nitric oxide (NO) production or secretion and can inhibit the expression, production or activity of iNOS protein or gene.

In one embodiment, the compound of the present invention may inhibit the expression, production or activity of the iNOS protein or gene by directly binding to the iNOS protein, thereby inhibiting NO production or secretion.

In one embodiment, the compound of the present invention can inhibit the expression or production of pro-inflammatory cytokines, and in particular, can inhibit the expression or production of proteins or genes of TNF-α and IL-6.

In one aspect, the present invention provides a guaianolide sesquiterpenes compound represented by the following formula (1), a stereoisomer thereof, or a pharmaceutically acceptable salt thereof for the prevention or treatment of inflammatory diseases comprising as an active ingredient: It relates to pharmaceutical compositions:

[Formula 1]

.

In one embodiment, the composition of the present invention can inhibit nitric oxide (NO) production or secretion and can inhibit the expression, production or activity of iNOS protein or gene.

In one embodiment, the composition of the present invention can inhibit the expression or production of pro-inflammatory cytokines, and in particular, can inhibit the expression or production of proteins or genes of TNF-α and IL-6.

In one embodiment, The inflammatory diseases include nerve inflammation, arthritis, rhinitis, hepatitis, keratitis, gastritis, enteritis, nephritis, bronchitis, pleurisy, peritonitis, spondylitis, pancreatitis, urethritis, cystitis, burn inflammation, dermatitis, allergy, atopy, periodontitis, gingivitis, otitis media, It may be any one or more selected from the group consisting of sore throat, arthritis, rheumatoid arthritis, tendinitis, tenosynovitis, and acute to chronic inflammatory diseases, and preferably may be neuroinflammation.

In one embodiment, The neuroinflammation may include inflammation-mediated degenerative neurological disease. The inflammation-mediated degenerative neurological disease may be included without limitation as long as it is a disease caused by inflammation of the nervous system, and may refer to a degenerative brain disease, such as dementia, Alzheimer's disease, and stroke. ), cerebral infarct, head trauma, cerebral arteriosclerosis, and Parkinson's disease, depression, and anxiety. It is not limited to this.

In one embodiment, the composition of the present invention may include the guaianolide sesquiterpine compound represented by Formula 1 at a concentration of 0.01 to 100 μM, preferably 0.05 to 50 μM, more preferably 0.1 μM. It can be included at a concentration of 20 μM to 20 μM, and it is more preferable to include it at a concentration of 5 to 15 μM.

In the present invention, the term "prevention" refers to any action that inhibits or delays the occurrence, spread, and recurrence of an inflammatory disease by administering the pharmaceutical composition according to the present invention, and "treatment" refers to any action that inhibits or delays the occurrence, spread, and recurrence of an inflammatory disease by administering the pharmaceutical composition according to the present invention. It refers to any action that improves or beneficially changes the symptoms of an inflammatory disease. Anyone with ordinary knowledge in the technical field to which the present invention pertains can refer to the data presented by the Korean Medical Association, etc. to know the exact criteria for diseases for which our composition is effective and to determine the degree of improvement, improvement, and treatment. will be.

In the present invention, the term "prevention" refers to any action that inhibits or delays the occurrence, spread, and recurrence of an inflammatory disease by administering the pharmaceutical composition according to the present invention, and "treatment" refers to any action that inhibits or delays the occurrence, spread, and recurrence of an inflammatory disease by administering the pharmaceutical composition according to the present invention. It refers to any action that improves or beneficially changes the symptoms of an inflammatory disease. Anyone with ordinary knowledge in the technical field to which the present invention pertains can refer to the data presented by the Korean Medical Association, etc. to know the exact criteria for diseases for which our composition is effective and to determine the degree of improvement, improvement, and treatment. will be.

The term "therapeutically effective amount" used in combination with an active ingredient in the present invention refers to an amount effective in preventing or treating inflammatory diseases, and the therapeutically effective amount of the composition of the present invention is determined by several factors, such as the method of administration. , may vary depending on the target area, patient condition, etc. Therefore, when used in the human body, the dosage must be determined as appropriate by considering both safety and efficiency. It is also possible to estimate the amount used in humans from the effective amount determined through animal testing. These considerations in determining an effective amount include, for example, Hardman and Limbird, eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed. (2001), Pergamon Press; and E.W. Martin ed., Remington's Pharmaceutical Sciences, 18th ed. (1990), Mack Publishing Co.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. As used in the present invention, the term "pharmaceutically effective amount" refers to an amount that is sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment and does not cause side effects, and the effective dose level is determined by the patient's Factors including health status, type of inflammatory disease, cause of inflammatory disease, severity, activity of drug, sensitivity to drug, method of administration, time of administration, route of administration and excretion rate, treatment period, combination or drugs used simultaneously, and It may be determined based on other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. Considering all of the above factors, it is important to administer an amount that can achieve maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art.

The pharmaceutical composition of the present invention may contain a carrier, diluent, excipient, or a combination of two or more commonly used in biological products. The carrier is not particularly limited as long as it is suitable for in vivo delivery of the composition, for example, Merck Index, 13th ed., Merck & Co. Inc. The compounds described in, saline solution, sterilized water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these ingredients can be mixed and used, and if necessary, other ingredients such as antioxidants, buffers, and bacteriostatic agents. Normal additives can be added. In addition, diluents, dispersants, surfactants, binders, and lubricants can be additionally added to formulate dosage forms such as aqueous solutions, suspensions, emulsions, etc., into pills, capsules, granules, or tablets. Furthermore, it can be preferably formulated according to each disease or ingredient using an appropriate method in the art or a method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990).

In one embodiment, the pharmaceutical composition may be one or more formulations selected from the group including oral formulations, topical formulations, suppositories, sterile injectable solutions, and sprays, with oral or injectable formulations being more preferable.

As used in the present invention, the term "administration" means providing a predetermined substance to an individual or patient by any appropriate method, and is administered parenterally (e.g., intravenously, subcutaneously, intraperitoneally) according to the desired method. Alternatively, it can be applied topically as an injection formulation) or orally administered, and the dosage range varies depending on the patient's weight, age, gender, health status, diet, administration time, administration method, excretion rate, and severity of the disease. Liquid preparations for oral administration of the composition of the present invention include suspensions, oral solutions, emulsions, syrups, etc., and in addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives are used. etc. may be included together. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, suppositories, etc. The pharmaceutical composition of the present invention may be administered by any device capable of transporting the active agent to target cells. Preferred administration methods and formulations include intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, and drip injection. Injections include aqueous solvents such as physiological saline solution and Ringer's solution, non-aqueous solvents such as vegetable oil, higher fatty acid esters (e.g., ethyl oleate, etc.), and alcohols (e.g., ethanol, benzyl alcohol, propylene glycol, glycerin, etc.). It can be manufactured using stabilizers to prevent deterioration (e.g., ascorbic acid, sodium bisulfite, sodium pyrosulphite, BHA, tocopherol, EDTA, etc.), emulsifiers, buffers for pH adjustment, and agents to prevent microbial growth. It may contain pharmaceutical carriers such as preservatives (e.g., phenylmercuric nitrate, thimerosal, benzalkonium chloride, phenol, cresol, benzyl alcohol, etc.).

As used in the present invention, the term "individual" refers to monkeys, cows, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, rats, rabbits, including humans who have or may develop the inflammatory disease. Or, it refers to all animals including guinea pigs, and the diseases can be effectively prevented or treated by administering the pharmaceutical composition of the present invention to the subject. The pharmaceutical composition of the present invention can be administered in combination with existing therapeutic agents.

The pharmaceutical composition of the present invention may further include pharmaceutically acceptable additives, wherein the pharmaceutically acceptable additives include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, and calcium hydrogen phosphate. , lactose, mannitol, taffy, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, Opadry, sodium starch glycolate, lead carnauba, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, Calcium stearate, white sugar, dextrose, sorbitol, and talc can be used. The pharmaceutically acceptable additive according to the present invention is preferably contained in an amount of 0.1 to 90 parts by weight based on the composition, but is not limited thereto.

The pharmaceutical composition of the present invention can also be provided in the form of an external preparation containing the guaianolide sesquiterpine compound represented by Formula 1 above as an active ingredient. When the pharmaceutical composition for preventing or treating inflammatory diseases of the present invention is used as an external skin agent, fatty substances, organic solvents, solubilizers, thickeners and gelling agents, softeners, antioxidants, suspending agents, stabilizers, and foaming agents are added. agent), fragrance, surfactant, water, ionic emulsifier, non-ionic emulsifier, filler, metal ion sequestrant, chelating agent, preservative, vitamin, blocking agent, wetting agent, essential oil, dye, pigment, hydrophilic activator, lipophilic It may contain adjuvants commonly used in the field of dermatology, such as active agents or lipid vesicles or any other ingredients commonly used in topical skin preparations. Additionally, the ingredients may be introduced in amounts commonly used in the field of dermatology.

When the pharmaceutical composition for preventing or treating inflammatory diseases of the present invention is provided as an external skin preparation, it may be in the form of an ointment, patch, gel, cream, or spray, but is not limited thereto.

In one aspect, the present invention provides a guaianolide sesquiterpenes compound represented by the following formula (1), a stereoisomer thereof, or a food-acceptable salt thereof as an active ingredient for preventing or improving inflammatory diseases. It relates to food compositions:

[Formula 1]

.

The food composition of the present invention, in addition to containing the guaianolide sesquiterpine compound represented by Chemical Formula 1 as an active ingredient, may contain various flavoring agents or natural carbohydrates as additional ingredients like a typical food composition. .

Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose, etc.; Disaccharides such as maltose, sucrose, etc.; and polysaccharides, such as common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. The above-described flavoring agents include natural flavoring agents (thaumatin), stevia extracts (e.g. rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.). The food composition of the present invention can be formulated in the same way as the pharmaceutical composition and used as a functional food or added to various foods. Foods to which the composition of the present invention can be added include, for example, beverages, meat, chocolate, foods, confectionery, pizza, ramen, other noodles, gum, candy, ice cream, alcoholic beverages, vitamin complexes, health supplements, etc. There is.

In addition, the food composition contains, in addition to the active ingredient compound, various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, colorants and thickening agents (cheese, chocolate, etc.), pectic acid, and salts thereof. , alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, the food composition of the present invention may contain pulp for the production of natural fruit juice, fruit juice beverages, and vegetable beverages.

The functional food composition of the present invention can be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. In the present invention, 'health functional food composition' refers to food manufactured and processed using raw materials or ingredients with functionality useful to the human body in accordance with Act No. 6727 on Health Functional Food, and refers to food that has It means taking it for the purpose of controlling nutrients or obtaining useful health effects such as physiological effects. The health functional food of the present invention may contain common food additives, and its suitability as a food additive is determined in accordance with the general provisions of the Food Additives Code and General Test Methods approved by the Food and Drug Administration, unless otherwise specified. Judgment is made according to specifications and standards. Items listed in the 'Food Additive Code' include, for example, chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; Natural additives such as dark pigment, licorice extract, crystalline cellulose, high-quality pigment, and guar gum; Examples include mixed preparations such as sodium L-glutamate preparations, noodle additive alkaline preparations, preservative preparations, and tar coloring preparations. For example, health functional foods in the form of tablets are prepared by granulating a mixture of the active ingredient of the present invention with excipients, binders, disintegrants, and other additives in a conventional manner, adding a lubricant, etc., and compression molding, or The mixture can be directly compression molded. Additionally, the health functional food in the form of tablets may contain flavoring agents, etc., if necessary. Among capsule-type health functional foods, hard capsules can be manufactured by filling a regular hard capsule with a mixture of the active ingredient of the present invention mixed with additives such as excipients, and soft capsules can be prepared by mixing the active ingredient of the present invention with additives such as excipients. It can be manufactured by filling the mixture with a capsule base such as gelatin. The soft capsule may contain plasticizers such as glycerin or sorbitol, colorants, preservatives, etc., if necessary. The health functional food in the form of a ring can be prepared by molding a mixture of the active ingredient of the present invention, excipients, binders, disintegrants, etc., using a known method. If necessary, it can be coated with white sugar or other coating agents. Alternatively, the surface can be coated with substances such as starch or talc. Health functional food in the form of granules can be manufactured into granules by mixing a mixture of excipients, binders, disintegrants, etc. of the active ingredients of the present invention by a known method, and may contain flavoring agents, flavoring agents, etc., if necessary. You can.

The health functional food composition according to the present invention can be manufactured in various forms according to conventional methods known in the art. General foods include, but are not limited to, beverages (including alcoholic beverages), fruits and their processed foods (e.g. canned fruit, bottled foods, jam, mamalade, etc.), fish, meat and their processed foods (e.g. ham, sausages, etc.) corned beef, etc.), bread and noodles (e.g. udon, buckwheat noodles, ramen, spagate, macaroni, etc.), fruit juice, various drinks, cookies, taffy, dairy products (e.g. butter, cheese, etc.), edible vegetable oil, margarine , can be produced by adding 5-hydroxymaltol of the present invention to vegetable proteins, retort foods, frozen foods, and various seasonings (e.g., soybean paste, soy sauce, sauce, etc.). In addition, nutritional supplements are not limited to this, but can be prepared by adding 5-hydroxymaltol of the present invention to capsules, tablets, pills, etc. In addition, the health functional food is not limited to this, but for example, the 5-hydroxymaltol itself of the present invention can be manufactured in the form of tea, juice, and drink and liquefied, granulated, encapsulated, and used for drinking (health beverage). It can be powdered and consumed. Additionally, in order to use 5-hydroxymaltol of the present invention in the form of a food additive, it can be prepared and used in the form of powder or concentrate. In addition, it can be prepared in the form of a composition by mixing the guaianolide sesquiterpine compound represented by Formula 1 of the present invention with a known active ingredient known to be effective in preventing or improving inflammatory diseases.

When the guaianolide sesquiterpine compound represented by Chemical Formula 1 of the present invention is used as a health drink, the health drink composition may contain various flavoring agents or natural carbohydrates as additional ingredients like ordinary drinks. . The above-mentioned natural carbohydrates include monosaccharides such as glucose and fructose; Disaccharides such as maltose and sucrose; polysaccharides such as dextrins and cyclodextrins; It may be a sugar alcohol such as xylitol, sorbitol, or erythritol. Sweeteners include natural sweeteners such as thaumatin and stevia extract; Synthetic sweeteners such as saccharin and aspartame can be used. The proportion of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g, per 100 mL of the composition of the present invention.

In addition, the guaianolide sesquiterpine compound represented by Formula 1 of the present invention may be contained as an active ingredient in a food composition for preventing or improving inflammatory diseases, and the amount is effective to achieve the effect of preventing or improving inflammatory diseases. The amount is not particularly limited, but is preferably 0.01 to 100% by weight based on the total weight of the entire composition. The health functional food composition of the present invention can be prepared by mixing the guaianolide sesquiterpine compound represented by Formula 1 above with other active ingredients known to be effective in inflammatory diseases.

In addition to the above, the health functional food of the present invention includes various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid, salts of pectic acid, alginic acid, salts of alginic acid, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, and preservatives. , may contain glycerin, alcohol, or carbonating agent. In addition, the health functional food of the present invention may contain pulp for the production of natural fruit juice, fruit juice beverage, or vegetable beverage. These ingredients can be used independently or in combination. The ratio of these additives is not very important, but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

In one aspect, the present invention provides a guaianolide sesquiterpenes compound represented by the following formula (1), a stereoisomer thereof, or a cosmetically acceptable salt thereof for the prevention or improvement of inflammatory diseases, comprising as an active ingredient: It relates to cosmetic compositions:

[Formula 1]

.

The “cosmetic composition” of the present invention can be prepared by comprising a cosmetically effective amount of the guaianolide sesquiterpine compound represented by Formula 1 of the present invention described above and a cosmetically acceptable carrier. .

In the present invention, the term “cosmetically effective amount” refers to an amount sufficient to achieve the inflammatory disease prevention or improvement effect of the composition of the present invention described above.

The appearance of the cosmetic composition contains a cosmetically or dermatologically acceptable medium or base. These are all formulations suitable for topical application, such as solutions, gels, solids, pasty anhydrous products, emulsions obtained by dispersing the oil phase in the water phase, suspensions, microemulsions, microcapsules, microgranules or ionic forms (liposomes) and It may be provided in the form of a non-ionic vesicular dispersion, or in the form of a cream, skin, lotion, powder, ointment, spray or concealer stick. These compositions can be prepared according to conventional methods in the art. The composition according to the invention can also be used in the form of a foam or in the form of an aerosol composition further containing a compressed propellant.

The cosmetic composition according to an embodiment of the present invention is not particularly limited in its formulation, and includes, for example, softening lotion, astringent lotion, nourishing lotion, nourishing cream, massage cream, essence, eye cream, eye essence, and cleansing lotion. It can be formulated into cosmetics such as cream, cleansing foam, cleansing water, pack, powder, body lotion, body cream, body oil, and body essence.

When the formulation of the cosmetic composition of the present invention is a paste, cream or gel, animal fiber, vegetable fiber, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide, etc. are used as carrier ingredients. This can be used.

When the formulation of the cosmetic composition of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder can be used as the carrier ingredient. In particular, when the cosmetic composition is a spray, chlorofluorohydride may be used additionally. May contain propellants such as carbon, propane/butane or dimethyl ether.

When the formulation of the cosmetic composition of the present invention is a solution or emulsion, a solvent, solvating agent or emulsifying agent is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene. These include fatty acid esters of glycol, 1,3-butylglycol oil, glycerol aliphatic esters, polyethylene glycol or sorbitan.

When the formulation of the cosmetic composition of the present invention is a suspension, the carrier component includes 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, Microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, or tracant may be used.

When the formulation of the cosmetic composition of the present invention is a surfactant-containing cleansing agent, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, and sarcosinate are used as carrier ingredients. , fatty acid amide ether sulfate, alkylamidobetaine, fatty alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, linoline derivative, or ethoxylated glycerol fatty acid ester can be used.

The cosmetic composition of the present invention includes skin, lotion, cream, essence, pack, foundation, color cosmetics, sunscreen, two-way cake, face powder, compact, makeup base, skin cover, eye shadow, lipstick, lip gloss, lip fix, and eyebrow pencil. , can be applied to cosmetics such as lotion and detergents such as shampoo and soap.

The cosmetic composition according to an embodiment of the present invention may further include functional additives and components included in general cosmetic compositions in addition to the guaianolide sesquiterpine compound represented by Formula 1 above. The functional additive may include ingredients selected from the group consisting of water-soluble vitamins, oil-soluble vitamins, polymer peptides, polymer polysaccharides, sphingolipids, and seaweed extract.

In addition to the above-mentioned functional additives, the cosmetic composition of the present invention may also contain components included in general cosmetic compositions, if necessary. Other ingredients included include oils and fats, moisturizers, emollients, surfactants, organic and inorganic pigments, organic powders, ultraviolet absorbers, preservatives, disinfectants, antioxidants, plant extracts, pH adjusters, alcohol, pigments, fragrances, and blood circulation agents. Examples include accelerators, cooling agents, limiting agents, and purified water.

In one aspect, the present invention relates to an anti-inflammatory composition comprising a guaianolide sesquiterpenes compound represented by the following formula (1), a stereoisomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient: :

[Formula 1]

.

In one embodiment, the anti-inflammatory composition may be one or more selected from the group consisting of pharmaceutical compositions, cosmetic compositions, and food compositions.

In the present invention, “anti-inflammatory” may be used interchangeably with “inhibiting or improving inflammation,” and may mean any action that alleviates the inflammatory response.

The present invention will be described in more detail through the following examples. However, the following examples are only for illustrating the content of the present invention and are not intended to limit the present invention.

Example 1. Separation and structural analysis of Korean Injin-derived compounds

1-1. Isolation of active compounds from Haninjin

Artemisiae Iwayomogii Herba, the above-ground part of Artemisia iwayomogi Kitamura (Asteraceae Compositae), was purchased from Gwangmyeongdang Pharmaceutical Co., Ltd. (Ulsan, Republic of Korea).

Dried Haninjin (3.0 kg) was extracted twice with 90% EtOH (ethanol aqueous solution, 30 L) at room temperature for 48 hours, and then the solvent was removed in vacuum at 45°C to obtain a 90% EtOH extract (300 g). The extract (300.0 g) was suspended in distilled water (0.6 L) and distributed three times with EtOAc (0.6 L). The EtOAc fraction (fraction dissolved in the EtOAc layer, 130.0 g) was loaded onto a column (11.3 × 55.6 cm) filled with Diaion HP-20 resin (Mitsubishi Chemical Industries, Ltd., Tokyo, JP), and acetone- as an elution solvent. Nineteen fractions (F1 to F19) were obtained through column chromatography (CC) using a water gradient system (acetone:H ₂ O=30:70 to 100:0, v/v). Among the 19 fractions, the F3 fraction was recrystallized in cold acetone, and then the supernatant of F3 (18.10 g) was loaded onto a column (5.5 × 61.0 cm) filled with Sephadex LH-20 gel (GE Healthecare, Sweden) and used as an elution solvent. It was eluted with acetone-water (acetone:H ₂ O=45:55, v/v) and 13 fractions (F3-1 to F3-13) were generated through CC. Among the 13 fractions, the F3-3 fraction was loaded on a column (5.3 × 34.0 cm) filled with silica gel (230-400 mesh, Merck, Germany), and n-hexane-ethyl acetate-methanol (n-hexane) was used as an elution solvent. Compound 1 (2.2 mg) and Compound 2 (5.6 mg) were purified by further fractionation through CC using :EtOAc:MeOH=60:35:5~0:95:5, v/v/v).

1-2. Data measurements for structural analysis of compounds 1 and 2

Data to analyze the structure and properties of Compound 1 and Compound 2 extracted, separated, and purified from Haninjin were measured. First, UV and FT-IR spectra were recorded using an OPTIZEN UV-Vis spectrophotometer and Agilent Cary 630 FT-IR (Agilent Technologies, CA, USA), respectively. Optical rotation was measured using a JASCO P-2000 polarimeter. NMR spectra were acquired by JEOL (JEOL, Tokyo, Japan) 500 MHz, and HR-DART-MS spectra were measured by a DART ion source (Ionsense, Tokyo, Japan) coupled to AccuTOF-TLC (JEOL). Circular dichroism spectra were measured by Chirascan-plus (Applied Photophysics Ltd., UK). The analysis results of Compound 1 and Compound 2 are summarized as follows, and the specific structural analysis process is shown in Examples 1-3 and 1-4 below.

Compound 1: Colorless solid; HR-DART-MS (positive mode) m/z = 338.1601 [M+NH ₄ ] ⁺ (calcd for C ₁₇ H ₂₄ NO ₆ , 338.1604) : +173°(c 0.01, MeOH); UV (MeOH) λ _max nm (log ε): 241 (3.85); IR (ATR) υ _max 3313, 2929, 1741, 1661 cm ^-1 ; ¹ H and ¹³ C NMR data, see Table 1.

Compound 2: Colorless solid; HR-DART-MS (positive mode) m/z = 338.1601 [M+NH ₄ ] ⁺ (calcd for C ₁₇ H ₂₄ NO ₆ , 338.1604) : +41°(c 0.01, MeOH); UV (MeOH) λ _max nm (log ε): 242 (3.86); IR (ATR) υ _max 3391, 2928, 1737 cm ^-1 ; ¹ H and ¹³ C NMR data, see Table 1.

Carbon number (Position) ^a Compound 1 ^c compound 2 ^c δ _H ^b δ _C δ _H ^b δ _C One - 143.4 - 143.5 2 6.21 t (2.5) 134.2 6.19 t (2.5) 134.9 3 3.95 d (3.0) 80.4 4.09 d (3.0) 83.4 4 - 80.2 - 82.2 5 3.20 d (11.5, 2.5) 56.3 3.05dd (11.0, 2.5) 59.6 6 4.35dd (11.5, 9.0) 78.7 4.41 (11.0, 9.5) 79.2 7 3.27 m 50.0 3.34m 49.5 8 5.50 d (11.5, 2.0) 74.0 5.47dd (11.0, 2.0) 74.0 9 5.52 br d 128.1 5.50 d (2.0) 129.8 10 - 132.5 - 132.3 11 - 138.6 - 138.6 12 - 171.6 - 171.7 13 5.76 d (3.0)
6.22 d (3.0) 123.5 5.78 d (3.0)
6.22 d (3.0) 123.6 14 2.00 s 25.4 2.03 s 26.0 15 1.32 s 21.8 1.54 s 23.1 COCH ₃ 2.17s 21.2 2.17s 21.2 OC=O - 172.0 - 172.0 ^a All assignments were based on ¹ H- ¹ H correlation spectroscopy (COSY), ¹ H- ¹³ C heteronuclear single quantum coherence spectroscopy (HSQC), ¹ H- ¹³ C heteronuclear multiple bond correlation (HMBC) results. ^b δ _H Multi ( J in Hz) ^c Measured in methanol- d ₄

1-3. Structural analysis of compound 1

Compound 1 was isolated as a colorless solid in HR-DART-MS (high-resolution direct analysis in real time mass spectroscopy, m/z 338.1601 [M+NH ₄ ] ⁺ ; calcd for C ₁₇ H ₂₄ O ₆ , 338.1604). The molecular formula was determined to be C ₁₇ H ₂₀ O ₆ based on the pseudomolecular ion peak. The infrared (IR) spectrum showed strong absorption bands at 1737 and 1660 cm ^-1 , confirming the presence of a γ-unsaturated lactone moiety.

^{The 1} H NMR spectrum of compound 1 is shown in Table 1, with δ _H 3.27 (1H, m), 4.35 (1H, dd, J = 11.5, 9.0 Hz), 5.76 (1H, d, J = 3.0 Hz) and 6.22. The presence of α-methylene-γ-lactone group was confirmed from the signal of (1H, d, J = 3.0Hz). Additionally, two olefin protons [ δH _5.52 (1H, br d), 6.21(1H, t, J = 2.5Hz)] and two oxymetine protons [ δH _3.95 (1H, d, J = 2.5Hz)] were observed in the ^1H NMR spectrum. 2.5 Hz), 5.50 (1H, dd, J = 11.5, 2.0 Hz)], 1 methine proton [ δ _H 3.20 (1H, dd, J = 11.5, 2.5 Hz)], 1 acetyl proton [ δ _H 2.17 ( 3H, s)], and two methyl proton [ δ _H 1.32(3H, s), 2.00(3H, s)] signals were identified.

As shown in Table 1, ^{the 13} C NMR spectrum contains a total of 17 skeletal carbon signals, including 15 skeletal carbon signals and two characteristic carbon signals of the acetyl group [δc 21.2 (COCH ₃ ), 172.0 (OC=O)]. It showed four carbon resonances, and through ¹ H ^-13 C HSQC (heteronuclear single quantum coherence) analysis, four sp2 quaternary carbons ( δ _c 132.5, 138.6, 143.4, 171.6) and two olefinic carbons ( δ _c 134.1). , methylene carbon ( δ _c 123.5), three oxymetine carbons ( δ _c 74.0, 78.7, 80.2), oxygenated quaternary carbon ( δ _c 80.4), two methine carbons ( δ _c 50.0, 59.3) and two methyl carbons ( δ _c 251). ^{The 1} H- ¹ H COSY (correlation spectroscopy) spectrum showed a correlation between H-2 and H-3 and a continuous correlation from H-5 to H-9 (Figure 2). The above results and ¹ H- ¹³ C HMBC (heteronuclear multiple bond correlation) signal (H-2/C-1; H-5/C-1, C-3, C-4; H-9/C-1, C-10; H-14/C-1, C-10, C-9; H-15/C-4, C-5), Compound 1 is a 5/7/5 tricyclic guaianolide. It was determined to be a sesquiterpene with a (guaianolide) skeleton (Figure 1). Meanwhile, a long-range correlation was observed between H-8 and the carbonyl carbon of the acetyl group, indicating that the O -acetyl group was located at C-8.

Nuclear overhauser enhancement spectroscopy (NOESY) spectrum analysis was performed to determine the three-dimensional structure of compound 1, and the results showed large coupling constants ( J _5,6 = 11.5 Hz, J _6,7 = 9.0 Hz, and J _{7, 8} = 11.5Hz), it was confirmed that H-5, H-6, H-7, and H-8 were trans -diaxial configured. Additionally, in the NOESY spectrum of compound 1, the correlations of H-6/H-8, H-6/H-15, and H-3/H-15 starting from β-oriented H-6 are H-3, It shows that H-6, H-8 and H-15 are connected to the β-side of the ring, and a cross-peak between H-5 and H-7 is observed, showing that H-5 and H-15 are connected to the β-side of the ring. It was shown that -7 is located on the α-side of the ring (Figure 3).

In order to determine the absolute configuration of Compound 1, the two enantiomeric models (3 R ,4 S ,5 S ,6 S ,7 R ,8) were obtained using the time-dependent density functional theory (TDDFT) method. S ) and (3 S , 4 R , 5 R , 6 R , 7 S , 8 R ) theoretical (calculated) ECD (experimental electronic circular dichroism) data and the experimental ECD spectrum of Compound 1 are compared. , the experimental ECD data of compound 1 were found to be consistent with the theoretical ECD data of the 3 R , 4 S , 5 S , 6 S , 7 R , 8 S- enantiomers (Figure 3).

Therefore, compound 1 is (3 R ,4 S ,5 S ,6 S ,7 R ,8 S )-8 α -acetoxy-3 α ,4 α -dihydroxy-guaia-1,9,11 (13)-triene-12,6 α -olide ((3 R ,4 S ,5 S ,6 S ,7 R ,8 S )-8 α- acetoxy-3 α ,4 α- dihydroxy-guaia- 1,9,11(13)-trien-12,6 α -olide) and was named iwayomogin A, and its structure is shown in Figure 1.

1-4. Structural analysis of compound 2

Compound 2 shows the same molecular ion peak ( m/z 338.1601 [M+NH ₄ ] ⁺ ; calcd for C ₁₇ H ₂₄ O ₆ , 338.1604) as Compound 1 in HR-DART-MS, and its molecular formula is Compound 1 and It was similarly determined to be C ₁₇ H ₂₀ O ₆ . The IR spectrum also showed strong absorption bands at 1741 and 1661 cm ^-1 , confirming the presence of a γ-unsaturated lactone group.

^{The 1} H and ¹³ C NMR spectra of compound 2 appeared almost similar to compound 1, but compared to the ¹ H NMR spectrum of compound 1, one methyl proton signal [ δH _1.54 (3H, s)] and two oxy proton signals were present. Generated methine signals [ δH _4.09 (1H, d, J = 3.0 Hz), and 4.41 (1H, d, J = 11.0, 9.0)] moved to the downfield region, and one methine signal [ δ _H 3.05 (1H, dd, J = 11.0, 2.5)] was shifted to the upfield area. As a result of comparing the ¹³ C NMR and HSQC data of Compound 2 with the data of Compound 1 (Table 1 and Figure 2), the C-5 methine signal ( δ _c 59.6) of Compound 2 was less deshielded than that of Compound 1. On the other hand, the H-5 methine signal was confirmed to be shielded. The carbon signals of C-3, C-4, C-6, and C-15 of compound 2 were shifted to the downfield region.

The major NOESY correlations of H-5/H-15, H-5/H-17, and H-6/H-8 indicated that compound 2 was a 4-epimer of compound 1 (Figure 3).

The absolute configuration of compound 2 is similar to the theoretical ECD spectra of (3 R ,4 R ,5 S ,6 S ,7 R ,8 S ) and (3 R ,4 S ,5 R ,6 R ,7 S ,8 R ) It was confirmed by comparison, and the experimental ECD data of compound 1 was found to be consistent with the theoretical ECD data of the 3 R , 4 R , 5 S , 6 S , 7 R , 8 S- enantiomer (Figure 3).

Therefore, compound 2 is (3 R , 4 R , 5 S , 6 S , 7 R , 8 S )-8 α -acetoxy-3 α ,4 ß -dihydroxy-guaia-1,9,11 (13)-triene-12,6 α -olide ((3 R ,4 R ,5 S ,6 S ,7 R ,8 S )-8 α -acetoxy-3 α ,4 ß -dihydroxy-guaia- It was determined to be 1,9,11(13)-trien-12,6 α -olide) and was named iwayomogin B, and its structure is shown in Figure 1.

Example 2. Inhibitory effect on NO production of compounds derived from Haninjin

NO is one of the key signaling molecules that mediate inflammatory processes. To confirm whether Iwayomogin A and Iwayomogin B (Compound 1 and Compound 2) of Example 1 exert regulatory activity on NO production, BV-2 microglial cells were treated with LPS and the treated NO concentration was measured in the supernatant of cells.

BV-2 microglia were maintained in Dulbecco's modified Eagle's medium (Hyclone Laboratories, Inc., Logan, UT, USA) supplemented with 10% fetal bovine serum (Hyclone Laboratories) and 1% penicillin-streptomycin (Hyclone Laboratories). , were cultured in a humidified atmosphere containing 5% CO ₂ at a temperature of 37°C. A 96-well plate was inoculated at a density of 3.0×10 ⁵ cells/mL and cultured for 24 hours. The next day, cells were pretreated with different concentrations of iwayomogin A and iwayomogin B, each from 0.1 to 20 μM, in serum-free medium for 1 h and then incubated with 100 ng/ml LPS (Sigma-Aldrich, St. Louis, MO, USA). The same volume of vehicle was treated in each of the control and LPS treatment groups. The supernatant of BV-2 cells seeded in 96-wells was collected and mixed with an equal volume of Griess reagent (1% sulfanilamide, 0.1% naphthylethylenediamine dihydrochloride, 2% phosphoric acid). After 10 minutes, the absorbance was measured at 540 nm using a microplate reader (Versamax™, Molecular Devices, LLC., San Jose, CA, USA). Sodium nitrite was used as a standard for calculating NO ^{2 -} concentration, and quercetin was used as a positive control. The IC ₅₀ value for inhibiting NO production in BV-2 cells was calculated from the absorbance measurement results and is shown in Table 2 below.

compound IC ₅₀ (μM) Iwayomogin A 10.59 Iwayomogin B 12.85 Quercetin 8.14

As a result, neither Iwayomogin A nor Iwayomogin B showed significant cytotoxicity, and showed excellent NO inhibitory activity at an IC ₅₀ value of 10.59 and 12.85 μM, respectively, which was almost similar to that of quercetin, a positive control. From these results, it was confirmed that Iwayomogin A and Iwayomogin B, which are guaianolide-based sesquiterpine compounds of the present invention, can be active compounds for anti-neuroinflammatory effects.

Example 3. Molecular docking analysis of compounds derived from Han In-jin

To further study the efficacy of the therapeutic agent that suppresses NO concentration by inhibiting the iNOS protein, an in silico molecular docking study was performed on Iwayomogin A and Iwayomogin B (Figure 4).

The crystal structure of iNOS was obtained from the RCSB PDB database (PDB code: 3E6T, resolution: 2.5 Å). Using Autodock 4.2 software (The Scripps Research Institute, La Jolla, CA, USA) and a grid box measuring 30 Å × 30 Å × 30 Å with 0.175 Å, proteins were prepared by removing all water molecules and adding polar hydrogen atoms. After confirming the absolute arrangement of Iwayomogin A and Iwayomogin B (compounds 1 and 2) through NOESY and ECD calculation data, the three-dimensional structure of the ligand was minimized using Chem3D Pro 14.0 software. After preparing the iNOS protein and the respective ligands of iwayomogin A and iwayomogin B, the hybrid Lamarckian Genetic Algorithm (LGA) was used to analyze the iNOS protein using AutoDock Tools 1.5.6. (The Scripps Research Institute, La Jolla, CA, USA) and AutoDock. Molecular docking calculations were performed via Vina software. 2D and 3D diagrams of protein-ligand complexes and 2D diagrams of protein-ligand interactions were generated using Maestro 12.9 software (Schrodinger, LLC, New York, NY, USA).

As a result, as shown in Figure 4, Iwayomogin A and Iwayomogin B have a strong binding affinity value (-8.0 kcal/mol) between iNOS proteins interacting with ALA-345 and HEM-901 involved in hydrogen bonding. ), and Iwayomogin A showed hydrogen interaction with GLN-257. These results suggest that Iwayomogin A and Iwayomogin B can reduce NO production in BV-2 microglial cells through inhibition of iNOS activity by direct binding to the iNOS enzyme.

Example 4. Inhibitory effect of Korean Injin-derived compounds on cytokines

To investigate the molecular mechanisms of iwayomogin A and iwayomogin B (compounds 1 and 2), which were identified as active candidate compounds against neuroinflammatory responses in Examples 2 and 3 above, BV-2 microglia stimulated with LPS The concentration of proinflammatory cytokines in the cell supernatant was measured depending on whether or not the compound was pretreated.

Specifically, a 12-well plate was inoculated at a density of 3.0×10 ⁵ cells/mL and then cultured for 24 hours. The next day, before LPS treatment, the cells were pretreated with Iwayomogin A or Iwayomogin B at concentrations of 0.1, 1, or 10 μM, respectively, for 1 hour, and then stimulated with LPS for 23 hours. After 23 hours of LPS stimulation, the supernatant of cells was collected and then assayed for TNF-α (BD Biosciences, Franklin Lakes) using enzyme-linked immunosorbent assay kits (ELISA kits) according to the manufacturer's protocol. , NJ, USA) and IL-6 (R&D systems, Minneapolis, MN, USA) were evaluated, and the results are shown in Figure 5. At this time, the data were analyzed by one-way ANOVA followed by Tukey's post hoc test, and compared to the control group, ###p<0.001; Compared to the LPS-only treatment group, significance was indicated as *p<0.05, **p<0.01, and ***p<0.001.

As a result, the levels of TNF-α and IL-6 were significantly increased in the group treated with LPS alone compared to the control group, but the levels of TNF-α and IL-6 were increased in a concentration-dependent manner by treatment with Iwayomogin A and Iwayomogin B. The level of was effectively reduced (Figure 5). These results mean that Iwayomogin A and Iwayomogin B of the present invention are effective in controlling neuroinflammation through simultaneous regulation of pro-inflammatory mediators.

So far, the present invention has been examined focusing on its preferred embodiments. A person skilled in the art to which the present invention pertains will understand that the present invention may be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative rather than a restrictive perspective. The scope of the present invention is set forth in particular in the claims rather than the foregoing description, and all differences within the equivalent scope should be construed as being included in the present invention.

Claims (15)

A guaianolide sesquiterpenes compound represented by the following formula (1), a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:
[Formula 1]
. According to paragraph 1,
The compound is a guaianolide sesquiterpine compound, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, wherein the compound is iwayomogin A having a three-dimensional structure represented by the following formula (2):
[Formula 2]
. According to paragraph 1,
The compound is a guaianolide sesquiterpine compound, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, wherein the compound is iwayomogin B having a three-dimensional structure represented by the following formula (3):
[Formula 3]
. According to paragraph 1,
The compound is a guaianolide sesquiterpine compound, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, isolated from Artemisiae Iwayomogii Herba. According to paragraph 1,
The guaianolide sesquiterpine compound, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, wherein the compound has an anti-inflammatory effect. According to paragraph 1,
The compound is a guaianolide sesquiterpine compound, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, which has the effect of inhibiting nitric oxide (NO) production or secretion and iNOS activity. According to paragraph 1,
The compound is a guaianolide sesquiterpine compound, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, which has the effect of inhibiting the expression or production of pro-inflammatory cytokines. A pharmaceutical composition for the prevention or treatment of inflammatory diseases comprising a guaianolide sesquiterpenes compound represented by the following formula (1), a stereoisomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient:
[Formula 1]
. According to clause 8,
The inflammatory diseases include nerve inflammation, arthritis, rhinitis, hepatitis, keratitis, gastritis, enteritis, nephritis, bronchitis, pleurisy, peritonitis, spondylitis, pancreatitis, urethritis, cystitis, burn inflammation, dermatitis, allergy, atopy, periodontitis, gingivitis, otitis media, A pharmaceutical composition that is at least one selected from the group consisting of sore throat, arthritis, rheumatoid arthritis, tendinitis, tenosynovitis, and acute to chronic inflammatory diseases. According to clause 8,
A pharmaceutical composition, wherein the inflammatory disease is neuroinflammation. According to clause 8,
A pharmaceutical composition that inhibits nitric oxide (NO) production or secretion and iNOS activity. According to clause 8,
A pharmaceutical composition that inhibits the expression or production of pro-inflammatory cytokines. A food composition for preventing or improving inflammatory diseases comprising a guaianolide sesquiterpenes compound represented by the following formula (1), a stereoisomer thereof, or a food-acceptable salt thereof as an active ingredient:
[Formula 1]
. A cosmetic composition for preventing or improving inflammatory diseases comprising a guaianolide sesquiterpenes compound represented by the following formula (1), a stereoisomer thereof, or a cosmetically acceptable salt thereof as an active ingredient:
[Formula 1]
. An anti-inflammatory composition comprising a guaianolide sesquiterpenes compound represented by the following formula (1), a stereoisomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient:
[Formula 1]
.