KR100991279B1 - A composition comprising the novel compound isolated from the extract of Parthenocissus tricuspidata for preventing and treating inflammatory disease - Google Patents

Abstract

 The present invention provides a novel compound 4,7,7'-trihydroxy-3,3 ', 4'-trimethoxylignan-9,9'-oxide (4,7,7'-) isolated from ivy extract. trihydroxy-3,3 ', 4'-trimethoxylignan-9,9'-olide (hereinafter referred to as' PT-MC-H') relates to a composition containing an active ingredient, in detail the PT-MC-H compound The composition of the present invention was found to significantly reduce the amount of LPS-induced NO production, p-IkBα protein expression, iNOS protein expression, and iNOS mRNA expression in RAW 264.7 cells, which are mouse macrophages. It can be usefully used as a pharmaceutical composition or health functional food.

4,7,7'-trihydroxy-3,3 ', 4'-trimethoxylignan-9,9'-olide, NO production, IkBα, p-IkBα, iNOS, inflammatory disease

Description

A composition comprising the novel compound isolated from the extract of Parthenocissus tricuspidata for preventing and treating inflammatory disease}

The present invention provides a composition for the prevention and treatment of inflammatory diseases containing a novel compound isolated from the ivy extract as an active ingredient.

1 Hawiger J., Innate Immunity and Inflammation: A Transcriptional Paradigm. Immunologic Research . 23 , pp. 99109, 2001

2 Gallo RL, Murakami M, Takaaki O, Zaiou M., Biology and clinical relevance of naturally occurring antimicrobial peptides. J. Allergy . Clin . Immunol . 110 , pp. 823-831. 2002

Graeme B. Ryan, MB, and Guido M., Acute Inflammation. American Journal of Pathology . 86 (1), pp. 185-274, 1977

Yamaki K, Thorlacius H, Xie X, Lindbom L, Hedqvist P, Raud J., Characteristics of histamine-induced leukocyte rolling in the undisturbed microcirculation of the rat mesentry. British J. Pharmacol . 123 , pp. 390-399, 1998

Document 5 Brocklehurst WE, Role of Kinins and Prostaglandins in Inflammation. Proc . Roy . Soc . Med. 64 , pp. 4-6, 1971

Esmon CT, The interactions between inflammation and coagulation. British Journal of Haematology. 131 , pp. 417430, 2005

7 Huang AL, Vita JA, Effects of Systemic Inflammation on Endothelium-Dependent Vasodilation. Trends, Cardiovasc . Med . 16 , pp. 1520, 2006

Renauld JC, New insights into the role of cytokines in asthma. J. Clin . Pathol . 54 , pp.577589, 2001

9 Blake GJ, Ridker PM, Tumor necrosis factor-α, inflammatory biomarkers, and atherogenesis. Eur . Heart J. 23 , pp.345347, 2002

[10] Moncada S, Palmer RM, Higgs EA, Nitric oxide: Physiology , pathophysiology , and pharmacology. Pharmacol . Rev. 43 , pp. 109-142, 1991

11 Mordan LJ, Burnett TS, Zang LK, Tom J, Cooney RV, Inhibitors of endogenous nitrogen oxide formation block the promotion of neoplastic transformation in C3H10T1 / 2 fibroblasts. Carcinogenesis . 14 , pp. 1555-1559, 1993

Lirk P, Hoffmann G, Rieder J, Inducible nitric oxide synthase. Time for reappraisal. Curr. Drug. Targets. Inflamm. Allergy. 1 , pp.89-108, 2002

[13] Hattori Y, Hattori S, Kasai K, Lipopolysaccharide activates Akt in vascular smooth muscle cells resulting in induction of inducible nitric oxide synthase through nuclear factor-kappa B activation. Eur . J. Pharmacol , 481 , pp. 153-158, 2003

[14] Kim SH, Johnson VJ, Shin TY, Sharma RP, Selenium attenuates lipopolysaccharide-induced oxidative stress responses through modulation of p38 MAPK and NF-κB signaling pathways. Exp . Biol . Medicine . pp.565-701, 2004

15 M. Robinson MJ, and Cobb MH, Mitogen-activated protein kinase pathways. Cur. Opi. Cell Biol. 9 , pp. 180-186, 1997

[16] Kyung Jin Lee, Chul Yung Choi, Young Chul Chung, Young Sup Kim, Si Yung Ryu, Seong Hwan Roh and Hye Gwang Jeong., Toxicology Letters , 147 , p271, 2004

17. Wang S et al., J. Ethnopharmacol . , 114 (3), pp 458-462, 2007

Heo et al., J. Immunol ., 179 (9), pp6305-6310, 2007

[19] Haiqi He, Kenneth J. Genovese, David J. Nisbet and Michael H. Kogut. Molecular Immunology 43, p783, 2006

Lee et al., J. Ethnopharmacol., 97, pp 561-566, 2005.

Inflammatory reactions are biological defenses caused by various factors, such as infection by tissues or damage to tissues, and perform an initial protective action to limit the damage to only infected or damaged areas. In most cases, this inflammatory response leads to the removal of pathogenic factors using intrinsic immune components and induction of specific adaptive immunity (Hawiger J., Innate Immunity and Inflammation: A Transcriptional Paradigm. Immunologic Research . 23 , pp. 99109, 2001). Rubs, tumors, heat, and pain, known as inflammation-associated features, are associated with the expansion of local blood flow due to the expansion of blood vessels by the action of inflammatory mediators and cytokines. Increased extravascular outflow of plasma components due to increased and decreased local blood flow rate, increased permeability of blood vessels, increased extravascular outflow of immune cells due to increased adhesion of vascular endothelial cells to circulating immune cells, and infection due to chemotaxis It is the result of successive immune responses such as increased migration to (Gallo RL, Murakami M, Takaaki O, Zaiou M., Biology and clinical relevance of naturally occurring antimicrobial peptides. J. Allergy. Clin. Immunol. 110 , pp . 823 -831 2002;.. Graeme B. Ryan , MB, and Guido M., Acute Inflammation American Journal of Pathology . 86 (1), pp. 185-274, 1977). Histamine produced in some cells as a response to tissue damage, and low molecular peptide kinin, which is inactivated in the blood and is activated by tissue damage, also contributes to inflammation-associated vasodilation and increased blood vessel permeability (Yamaki K, Thorlacius H, Xie X, Lindbom L, Hedqvist P, Raud J., Characteristics of histamine-induced leukocyte rolling in the undisturbed microcirculation of the rat mesentry.British J. Pharmacol . 123 , pp . 390-399, 1998; Brocklehurst WE, Role of Kinins and Prostaglandins in Inflammation. Proc. Roy. Soc. Med. 64, pp.4-6, 1971). Especially, bradykinin, a kind of kinin, works to stimulate the pain of the skin. On the other hand, dilation of blood vessels and increased permeability of blood vessels in damaged tissues also cause blood coagulation enzymes to leak into the tissues, followed by the chain reaction of these enzymes to produce insoluble fibrous strands, which is a major component of blood coagulation. The coagulated fiber strand act as deterrents to block the infection through the damaged tissue area that spread to other parts (Esmon CT, The interactions between inflammation and coagulation. British Journal of Haematology . 131 , pp. 417430, 2005). In general, acute inflammatory reactions progress rapidly and are maintained for a short period of time. Acute inflammation is accompanied by a systemic reaction called an acute phase reaction. On the other hand, chronic inflammation can be induced as a result of continuous immune activation associated with some diseases such as infections and autoimmune diseases, and the accumulation and activation of macrophages is evidence of chronic inflammatory response (Huang AL, Vita JA). , Effects of Systemic Inflammation on Endothelium- Dependent Vasodilation. Trends, Cardiovasc. Med. 16, pp.1520, 2006). However, persistent chronic inflammatory reactions can cause serious damage to host cells or tissues.

Inflammatory responses at the site of infection are initiated by the response of macrophages to pathogens. Reactive reactive oxygen species and reactive nitrogen species produced by macrophages activated by pathogens, inflammatory mediators such as prostaglandins, leukotrienes, and inflammatory mediators such as TNF-α, IL-6 and IL-8 (Renauld JC, New insights into the role of cytokines in asthma. J. Clin . Pathol . 54 , pp.577589, 2001; Blake GJ, Ridker PM, Tumour necrosis factor-α, inflammatory biomarkers, and atherogenesis. Eur. Heart J. 23, pp.345347, 2002). The activation of NF-κB, a transcription factor of genes involved in the production of these inflammatory mediators, is very important for the inflammation-related action of macrophages. Inflammatory genes such as inducible nitric oxide synthase (iNOS2), cyclooxygenase (COX-2), TNF-α, IL-6, and IL-8 are found in macrophages. It has been reported to be transcribed by κB.

Nitric oxide (NO) is a short-lasting free radical that is derived from L-arginine when the oxidation reaction of L-arginine occurs by catalysis of nitric oxide synthase (NOS). It is well known that nitric oxide is produced with L-citrulline. In vivo, nitric oxide exhibits various immune-related functions (Moncada S, Palmer RM, Higgs EA, Nitric oxide: Physiology, pathophysiology , and pharmacology . Pharmacol . Rev. 43 , pp.109-142, 1991), excessive nitric oxide production may be cytotoxic, leading to damage to host cells and tissues, as well as to various pathological phenomena such as atherosclerosis and carcinogenesis (Mordan LJ, Burnett TS, Zang LK, Tom J, Cooney RV, Inhibitors of endogenous nitrogen oxide formation block the promotion of neoplastic transformation in C3H10T1 / 2 fibroblasts Carcinogenesis 14, pp.1555-1559, 1993;.. Lirk P, Hoffmann G, Rieder J, Inducible nitric oxide synthase. Time for reappraisal. Curr. Drug. Targets. Inflamm. Allergy. 1, pp.89-108, 2002). Three types of nitric oxide synthase (NOS) are known to catalyze NO production, including neuronal nitric oxide synthase (neuronal NOS, nNOS, NOS1) and endothelial nitric oxide synthase (endothelial NOS, eNOS, NOS3). While constitutively expressed at all times, iNOS is inducible to be transcribed by the transcription factor NF-κB, which is activated when phagocytes are stimulated by the bacterial endotoxin lipopolysaccharide (LPS).

In macrophages, the transcription factor NF-κB is present in the cytoplasm in an inactive state by binding to its inhibitor, IκB, but is a signaling process induced when bacterial LPS binds to Toll-like receptors on the cell surface. NF-κB finally becomes activated as IκB is phosphorylated to dissociate from NF-κB / IκB and removed by proteasome digestion. NF-κB then migrates to the nucleus and induces transcription of various inflammation related genes. Activation of these NF-κB, the Akt signaling (Hattori Y, Hattori S, Kasai K, Lipopolysaccharide activates Akt in vascular smooth muscle cells resulting in induction of inducible nitric oxide synthase through nuclear factor-kappa B activation. Eur. J. Pharmacol, 481 , pp. 153-158, 2003) and ERK, c-jun- and p38-MAPK signaling pathways have been reported to be involved (Kim SH, Johnson VJ, Shin TY, Sharma RP, Selenium attenuates lipopolysaccharide-induced oxidative stress) .... responses through modulation of p38 MAPK and NF-κB signaling pathways Exp Biol Medicine pp.565-701, 2004;.... Robinson MJ, and Cobb MH, Mitogen-activated protein kinase pathways Cur Opi Cell Biol 9, pp. 180-186, 1997).

The expression regulation of iNOS in the transcriptional phase of the inflammatory response is of great importance in determining the duration and production of NO. Therefore, iNOS expression control mechanism and iNOS enzymatic activity have been used as a target of drug action in the study to isolate new anti-inflammatory drugs for the improvement of chronic inflammation-related diseases.

Therefore, the present inventors found a new drug that inhibits NO production that occurs when the mouse macrophage line RAW264.7 is treated with bacterial LPS, Parthenocissus Tricuspidata ) was used to investigate the inhibition of NO production in RAW264.7 cells. As a result, 4,7,7'-trihydroxy-3,3 ', 4'-trimethoxylignan-9,9'-oxide (hereinafter,' PT- ' It was first confirmed that the new compound identified as MC-H 'has a function of inhibiting the production of NO induced by LPS treatment in RAW264.7 cells and LPS- in RAW 264.7 cells, which are mouse macrophages. The present invention was completed by confirming that significantly reduced contents of p-IkBα protein expression, iNOS protein expression and iNOS mRNA expression as well as induced NO production.

In order to achieve the above object, 4,7,7'-trihydroxy-3,3 ', 4'-trimethoxylignan-9,9'-ol represented by the following structural formula (I) isolated from the ivy extract Provided is a lead (4,7,7'-trihydroxy-3,3 ', 4'-trimethoxylignan-9,9'-olide) compound or a pharmaceutically acceptable salt thereof.

(Ⅰ)

(Ⅰ)

The compounds of the present invention represented by the above formula (I) may be prepared with pharmaceutically acceptable salts and solvates according to conventional methods in the art.

As the pharmaceutically acceptable salt, acid addition salts formed by free acid are useful. Acid addition salts are prepared by conventional methods, for example by dissolving a compound in an excess of aqueous acid solution and precipitating the salt using a water miscible organic solvent such as methanol, ethanol, acetone or acetonitrile. Equal molar amounts of the compound and acid or alcohol (eg, glycol monomethylether) in water can be heated and the mixture can then be evaporated to dryness or the precipitated salts can be suction filtered.

In this case, organic acids and inorganic acids may be used as the free acid, hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, tartaric acid, etc. may be used as the inorganic acid, and methanesulfonic acid, p -toluenesulfonic acid, acetic acid, trifluoroacetic acid, Citric acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, manderic acid, propionic acid, citric acid, lactic acid, glycolic acid, gluconic acid ( gluconic acid), galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanic acid, and hydroiodic acid.

In addition, bases can be used to make pharmaceutically acceptable metal salts. An alkali metal or alkaline earth metal salt is obtained by, for example, dissolving a compound in an excess alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and then evaporating and drying the filtrate. At this time, as the metal salt, it is particularly suitable to prepare sodium, potassium or calcium salt, and the corresponding silver salt is obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (for example, silver nitrate).

Pharmaceutically acceptable salts of the compounds of the invention include salts of acidic or basic groups which may be present in the compounds of the invention, unless otherwise indicated. For example, pharmaceutically acceptable salts include sodium, calcium and potassium salts of the hydroxy group, and other pharmaceutically acceptable salts of the amino group include hydrobromide, sulfate, hydrogen sulphate, phosphate, hydrogen phosphate, dihydrogen Phosphate, acetate, succinate, citrate, tartrate, lactate, mandelate, methanesulfonate (mesylate) and p -toluenesulfonate (tosylate) salts. It can be prepared through.

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

The 'PT-MC-H' compound of the present invention can be obtained as follows.

Referring to the method for obtaining the ivy crude extract of the present invention, first, the ivy vines are collected, shaded, shredded and ground into a fine grinder, and then about 1 to 20 times the weight of the ivy sample, preferably about 1 to 5 times the volume of water and C1 to C4 lower alcohols such as methanol, ethanol, butanol and the like or a mixed solvent thereof, preferably methanol, more preferably 50 to 100% methanol, 10 to 100 Preferably, using an extraction method such as hot water extraction, cold needle extraction, reflux cooling extraction or ultrasonic extraction for 1 to 20 hours, preferably 5 to 15 hours at 50 ℃ to 90 ℃, preferably using a reflux extraction method, The supernatant was recovered by vacuum filtration, and then the above procedure was repeated 2 to 5 times, preferably 3 times, to collect the supernatant and concentrated under reduced pressure. All crude extracts can be obtained.

The methanol crude extract obtained above was mixed and fractionated with a mixed solvent of water and methylene chloride, and then concentrated under reduced pressure to obtain an ivy water soluble fraction or a methylene chloride soluble fraction of the present invention, and the methylene chloride soluble fraction was an eluting solvent. After performing column chromatography to separate into 36 fractions, the lower fraction can be purified by column chromatography in sequence to obtain the compound PT-MC-H of the present invention.

The present invention provides a pharmaceutical composition for the prevention and treatment of inflammatory diseases containing the novel compound 'PT-MC-H' or a pharmaceutically acceptable salt thereof isolated from the ivy extract obtained by the above method as an active ingredient. .

In addition, the ivy is a compound that has been separated from the ivy extract of the present invention as a medicinal herb that has been used for a long time or edible there is no problem such as toxicity and side effects.

The extract defined herein is characterized in that the crude extract of ivy, polar solvent soluble extract or non-polar solvent soluble extract.

The crude extract as defined herein is a solvent selected from water containing purified water, lower alcohols having 1 to 4 carbon atoms such as methanol, ethanol, butanol, or a mixed solvent thereof, preferably methanol, more preferably 50 to 100% methanol Contains extracts available for use.

The nonpolar solvent soluble extract as defined herein includes extracts soluble in methylene chloride, hexane, chloroform, dichloromethane or ethyl acetate, preferably methylene chloride.

Inflammatory diseases defined herein are inflammatory diseases include atopic dermatitis, arthritis, urethritis, cystitis, arteriosclerosis, allergic diseases, rhinitis, asthma, acute pain, chronic pain, periodontitis, gingivitis, inflammatory bowel disease, gout, myocardial infarction, congestion Sexual heart failure, hypertension, angina pectoris, gastric ulcer, cerebral infarction, Down syndrome, multiple sclerosis, obesity, diabetes, dementia, depression, schizophrenia, tuberculosis, sleep disorders, sepsis, burns or pancreatitis, preferably atopic dermatitis, arthritis, urethritis, cystitis or Periodontitis, more preferably periodontitis, urethritis or cystitis.

The composition for preventing and treating inflammatory diseases of the present invention comprises 0.1 to 50% by weight of the compound based on the total weight of the composition.

The pharmaceutical composition comprising the compound of the present invention may further include appropriate carriers, excipients and diluents commonly used in the preparation of pharmaceutical compositions.

Examples of carriers, excipients and diluents that can be included in the composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

Pharmaceutical dosage forms of the compounds of the present invention may be used in the form of their pharmaceutically acceptable salts, and may be used alone or in combination with other pharmaceutically active compounds as well as in a suitable collection.

Compositions comprising the compounds of the invention are each formulated in the form of oral dosage forms, external preparations, suppositories, and sterile injectable solutions, such as powders, granules, tablets, capsules, suspensions, emulsions, syrups and aerosols, according to conventional methods Can be used.

     More specifically, when formulating the composition, it can be prepared using a diluent or an excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant, and the like. Solid form preparations for oral administration include tablets, pills, powders, granules and capsules, and the like form at least one excipient such as starch, calcium carbonate, sucrose in the compound. ), Lactose, gelatin and the like can be mixed. In addition to the simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral use include suspensions, solvents, emulsions and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances and preservatives, in addition to the commonly used simple diluents, water and liquid paraffin. have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations and suppositories. As the non-aqueous solvent and suspending agent, vegetable oils such as propylene glycol, polyethylene glycol and olive oil, and injectable esters such as ethyl oleate may be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter and glycerogelatin can be used.

Preferred dosages of the compounds of the present invention depend on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, but may be appropriately selected by those skilled in the art. However, for the desired effect, the compound of the present invention may be administered in an amount of 0.0001 to 100 mg / kg, preferably in an amount of 0.001 to 100 mg / kg once to several times daily. The compound of the present invention in the composition may be formulated in an amount of 0.0001 to 50% by weight based on the total weight of the total composition.

The pharmaceutical composition of the present invention can be administered to mammals such as mice, mice, livestock, humans, etc. by various routes. All modes of administration can be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural and intracerebroventricular injections.

The present invention also provides a health functional food for preventing and improving inflammatory diseases containing the novel compound PT-MC-H or a pharmaceutically acceptable salt thereof as an active ingredient.

The compounds of the present invention can be used in various ways, such as drugs, food and beverages for the prevention and treatment of inflammatory diseases. Foods to which the compound of the present invention may be added include, for example, various foods, beverages, gums, teas, vitamin complexes and dietary supplements, and may be used in the form of powders, granules, tablets and capsules or beverages. have.

Since the compound of the present invention has little toxicity and side effects, it is a drug that can be used safely even for long-term administration for the purpose of prevention.

The compounds of the present invention may be added to food or beverages for the purpose of preventing and treating inflammatory diseases. At this time, the amount of the compound in the food or beverage is generally added to the health food composition of the present invention to 0.01 to 15% by weight of the total food weight, the health beverage composition is 0.02 to 30 g based on 100 ml, preferably Can be added in a ratio of 0.3 to 10 g.

The health beverage composition of the present invention, in addition to containing the compound as an essential ingredient in the indicated proportions, has no particular limitation on the liquid component and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. Examples of the above-mentioned natural carbohydrates include monosaccharides such as disaccharides such as glucose and fructose, for example polysaccharides such as maltose and sucrose, and conventional sugars such as dextrin and cyclodextrin. Sugar alcohols such as xylitol, sorbitol and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of said natural carbohydrates is generally about 1-20 g, preferably about 5-12 g per 100 ml of the composition of the present invention.

In addition to the above, the compounds of the present invention include various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese and chocolate), pectic acid and salts thereof, alginic acid and its Salts, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. In addition, the compounds of the present invention may contain flesh for the production of natural fruit juices and fruit juice beverages and vegetable beverages. These components can be used independently or in combination. The proportion of such additives is not so critical, but is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

As mentioned above, the novel compound PT-MC-H of the present invention is LPS-induced NO production, IkBα, p-IkBα, iNOS or COX-2 protein expression and iNOS mRNA expression in RAW 264.7 cells, which are mouse macrophages. By confirming that the content of expression is significantly reduced, the composition may be usefully used as a pharmaceutical composition or health functional food for the prevention and treatment of inflammatory diseases.

Hereinafter, the present invention will be described in detail with reference to the following examples and experimental examples.

However, the following Examples and Experimental Examples are only illustrative of the present invention, the contents of the present invention is not limited by the following Examples, Reference Examples and Experimental Examples.

Example  1. Ivy Crude extract  Produce

1-1. Ivy Crude extract  Produce

 In August 2002, ivy collected from pine trees in Daegu-si Palgongsan was collected, dried, and ground to a fine grinding machine. After pulverizing with fine powder, 40.5ℓ of 95% methanol was added to 10kg of fine powder sample and extracted at 80 ℃ for 10 hours, followed by vacuum filtration. To recover the supernatant. This process was repeated three times to collect the supernatant, and then concentrated under reduced pressure to obtain 1222.6 g of ivy methanol crude extract.

1-2. Preparation of Ivy Soluble Extracts

1222.6 g of the ivy methanol crude extract obtained in Example 1-1 was suspended in 2.1 L of water, followed by solvent fractionation with methylene chloride. First, 1 m of methylene chloride was added to the methanol soluble extract of ivy suspended in 2.1 L of water, and then dissolved, and only the components dissolved in the methylene chloride layer were separated and dried in vacuo. This process was repeated three times to give 127.6 g of methylene chloride soluble fraction.

Example  2. Isolation of New Compounds from Ivy Extracts

The methylene chloride soluble fraction obtained in Example 1-2 was subjected to flash column chromatography (YMC Gel ODS-A, 60 A, 220 mesh), that is, 230 to 240 in a column having a length of 35 cm and a diameter of 9 cm. Methylene chloride soluble fraction obtained in Example 1-2 was prepared by filling a mesh silica gel and eluting with 100% hexane to make the stationary phase uniform. g was adsorbed onto silica gel (70-230 mesh) and loaded onto the column. Then, hexane: ethyl acetate = 98: 2, 96: 5, 90: 10, 85: 15, 80: 20, 75: 25 in order to elute by elevating the polarity of the mobile phase (based on the TLC pattern) Fractions of PT-MC-1 to PT-MC-36 were fractionated.

2.0 g of PT-MC-20 fractions in the fractions were filled with RP C18 column chromatography, i.e. 57 cm in length and 3.5 cm in diameter, 22 cm with RP C18 (40-63 μM) and MeOH: H ₂ 0 = 2: 8 mixed solution was eluted ( After elution to make a uniform state, 2.0 g of the PT-MC-20 fraction was completely dissolved in 100% methanol and loaded. Subsequently, the ratio of MeOH was eluted, and the fractions were divided into PT-MC-20A and PT-MC-20R. Among them, PT-MC-20-B fractions were subjected to silicon open column chromatography; (SiO ₂ open colunm chromatography) .The column (ID 53cm X 2.3cm) was filled with silica gel (less than 70 mesh) about 15cm and 100% hexane. After elution to make the fixed phase uniform, 21.5 mg of PT-MC-20-B was adsorbed on a small amount of silica gel and loaded on a column to transfer a mixed solution of CH ₂ Cl ₂ : MeOH = 90: 1 to the mobile phase. Elution to compound 4,7,7'-trihydroxy-3,3 ', 4'-trimethoxylignan-9,9'-oxide (4,7,7'-trihydroxy- 11.3 mg of 3,3 ', 4'-trimethoxylignan-9,9'-olide) was obtained by separation and purification.

The structure of the compound was determined by nuclear magnetic resonance spectra and high resolution mass spectrometry data, and the assignment of 1H-NMR and 13C-NMR signals in the nuclear magnetic resonance spectra was COSY, TOCSY, etc. Was achieved through multidimensional nuclear magnetic resonance experiments.

Compound 1: 4,7,7'- Trihydroxy -3,3, ’4- Trimethoxylignan -9,9’- Old

Molecular Formula: C ₂₁ H ₂₄ O ₈

Molecular Weight: 404.15

Color: Light Yellow Amorphous Solid

Yield: 0.001%

R _f = 0.75 (Methylene chloride: methanol = 7: 1);

₁ H NMR (CDCl ₃ ): δ ppm 6.79 (s, 1H), 6.90-6.97 (m, 1H), 6.83-6.89 (m, 1H), 5.35 (d, J = 4.0Hz, 1H), 3.46 (dd , J = 8.5, 4.0 Hz, 1H), 6.90 (s, 1H), 6.90-6.97 (m, 1H), 6.75-6.80 (m, 1H), 5.33 (d, J = 4.0 Hz, 1H), 3.23- 3.26 (m, 1H), 4.04 (dd, J = 9.5, 4.5 Hz, 1H), 4.34 (dd, J = 9.5, 7.0 Hz, 1H), 3.91 (s, 9H), 5.61 (s, 1H), 5.68 (s, 1 H), 3.51 (s, 1 H);

₁₃ C NMR (CDCl ₃ ): δ ppm 132.2 (C-1), 118.3 (C-2), 146.0 (C-3), 145.2 (C-4), 114.7 (C-5), 108.0 (C-6 ), 84.6 (C-7), 53.3 (C-8), 177.0 (C-9), 131.0 (C-1 '), 117.9 (C-2'), 146.7 (C-3 '), 146.9 (C -4 '), 114.4 (C-5'), 107.7 (C-6 '), 83.3 (C-7'), 49.9 (C-8 '), 72.6 (C-9'), 56.0 (CO C H) ₃ );

FABMS ( m / z ): 372 (M-MeOH) ₊

Reference Example  1. Preparation of experimental materials

Reagents for cell culture, such as Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS), and streptomycin-penicillin, were purchased from Gibco BRL (Grand Island, USA), and SDS (Sodium Dodesyl Sulfate). , Acrylamide, Bis, Protein assay reagents were purchased from Bio-Rad (Hercules, USA), NP-40, CAPS, tween 20, protease inhibitors were purchased from Sigma (St. Louis, USA). The anti-IkBα, anti-p-Ikbα, or iNOS monoclonal antibody (mAb), the primary antibody used in the experiment, and the anti-rabbit or anti-mouse IgG horseradish peroxidase (HRP) -conjugated antibody, the secondary antibody, were used by SantaCruz Biotechnology. Santa Cruz, CA). Griess Reagent System was purchased from Promega (Madison, USA). All reagents used in the experiments were used above the analytical grade.

Reference Example  2. Cell Culture

Macrophage lines of mice, RAW 264.7 cells (KCLB), were treated with DMEM (Dulbecco's Modified Eagle Medium) medium containing 10% FBS and 1% penicillin-streptomycin (37%, 5% CO). Cultured under the conditions of ₂ was used in the following experiment.

Experimental Example  1. Cytotoxicity Measurement

In order to measure the cytotoxicity of the PT-MC-H compound obtained in Example 2, the following experiment was applied by applying the method described in the existing literature (Do Youn Jun, Seok Woo Rue, Kyu Hyun Han, Dennis Taub, Young Sup Lee, Young Seuk Bae and Young Ho Kim.Biochemical Pharmacology 66, p2291, 2003).

For cytotoxicity analysis, after dispensing Raw264.7 cells by adjusting the cell concentration to 1 × 10 ⁴ cells / well in a 96-well microtiter plate, the cells were cultured for 16 hours, and the PT of Example 2 was used. -MC-H compound was pretreated for 1 hour by concentration, treated with 0.1 μg / ml LPS, incubated for 12 hours, and then 50 μl of MTT [3- (4,5-dimethylthiazol-2-yl) -2,5- After diphenyl tetrazolium bromide] was added and reacted for 4 hours to form forzanane, 200 μl of the supernatant was removed, DMSO 150 μl / well was added to dissolve formazan and then ELISA reader (Variokan, Thermo Electron) was used. Measurement was made at a wavelength of 540 nm.

As a result, the cell survival rate for RAW264.7 cells was not significantly affected by the treatment of 6.25 or 12.5 μg / ml PT-MC-H compound as shown in FIG. 1, but the cell survival rate was 25 μg / ml. It was shown to be reduced by about 50% compared to this control, it was confirmed that there is a cytotoxic effect on macrophage RAW264.7 cells at 25 μg / ml concentration (see Figure 1). Therefore, the PT-MC-H compound was tested as follows at a concentration of 12.5 μg / ml or less showing no cytotoxicity to RAW264.7 cells.

Experimental Example  2. NO Measurement of production

In order to determine the amount of NO produced, experiments were carried out using the method described in Wang S et al., J. Ethnopharmacol . , 114 (3), pp458-462, 2007.

In order to measure NO production, the cells were diluted in a 96-well microtiter plate with a cell concentration of 2 × 10 ⁵ cells / well, and the raw 264.7 cells were dispensed and incubated for 16 hours, and the PT of Example 2 was used. -MC-H compound was pretreated for 1 hour by concentration, treated with 0.1 μg / ml LPS, incubated for 12 hours, 100 μl of the supernatant was collected and 100 μl of Greases Reagent was added and reacted at room temperature for 10 minutes. After absorbance was measured at 540 nm using an Automatic ELISA reader (precision microplate reader, Molecular Devices, BIO-TEX EL800uv-PC, USA). NO concentration in the culture was determined using a standard curve of concentration of sodium nitrite.

As a result, as shown in Figure 2 PT-MC-H compound showed an effect of inhibiting the NO production of the mouse macrophage RAW264.7 by almost 60% at 6.25 μg / ml concentration conditions (see Figure 2), RAW264 by LPS even when PT-MC-H compounds were added at a concentration of 2.5 μg / ml, 5 μg / ml or 10 μg / ml to identify the minimum concentration that could inhibit NO production in RAW24.7 cells. It was confirmed whether NO production of 7 cells was inhibited. As a result, as shown in FIG. 3A, NO production of RAW264.7 cells by LPS was inhibited in a concentration-dependent manner at 2.5 μg / ml, 5 μg / ml or 10 μg / ml concentration, resulting in 13%, 25% and 70% It was confirmed that each is inhibited (see Figure 3A), PT-MC-H compound confirmed by the MTT assay under the same concentration conditions was confirmed that no cytotoxicity as shown in Figure 3B (see Figure 3B).

Experimental Example  3. Weston Blot  Method ( Western blot analysis )

Western blot analysis was performed using Hu et al. (Heo et al., J. Immunol ., 179 (9), pp6305-6310, 2007) to determine IkBα, p-IkBα, or iNOS protein expression . Was performed.

4 × 10 ⁶ cells / well cells cultured by the method of Reference Example 2 were washed three times with TBS (ice-cold Tris buffered saline: 20 mM Tris-HCl, pH 8.0, 137 mM NaCl), followed by lysis buffer (lysis buffer: TBS, 1% NP-40, 1 mM sodium orthovanadata, 10 μg / ml aprotinin, 10 μg / ml leupeptin and 1 mM PMSF), crushed with an ultrasonic grinder, and reacted at 4 ° C. for 30 minutes and at 14000 rpm. The supernatant was collected by centrifugation for 20 minutes. The same amount of protein was separated by 7.5% SDS-PAG (sodium dodecyl sulfate-polyacrylamide gel electrophoresisE), and then the protein was transferred to an Immobilon-P membrane. The membrane was reacted for one hour in a blocking buffer (TBS solution containing 3% non-fat milk and 0.1% Tween 20) to block nonspecific binding of the antibody, followed by antibody to each protein (anti- iNOS and anti-COX2) were added and reacted for 1-2 hours. Subsequently, the cells were washed three times for 10 minutes with a TBST solution containing 0.1% Tween 20, and then reacted with a secondary antibody (anti-mouse or anti-rabbit IgG horseradish peroxidase-conjugated antibody) for 90 minutes to 2 hours. Subsequently, the protein was identified on the X-ray film (AGFA, Belgium) after the reaction with ECL system. Protein quantification of each sample was carried out by measuring the absorbance at 562 nm using a Bradford protein assay kit.

For reference, macrophages play an important role in the initiation and expansion of inflammatory responses. Macrophages that stimulate Toll-like receptors on the cell surface by bacterial LPS are expressed through intracellular signaling. It activates the transcription factor NF-κB, which in turn increases the expression of inducible nitric oxide synthase (iNOS) by the action of activated NF-κB. As a result, increased catabolism of iNOS enhances the production of NO, one of the inflammatory mediators from L-arginine (Haiqi He, Kenneth J. Genovese, David J. Nisbet and Michael H. Kogut). Molecular Immunology 43, p783, 2006).

As shown in FIG. 4, the phosphorylation of IκB protein associated with the activation of the transcription factor NF-κB was significantly increased by the treatment of LPS, but the concentration-dependent phosphorylation of IκB was observed in the presence of PT-MC-H compound. It showed a tendency to decrease significantly, and it was confirmed that the level of iNOS also decreased in a concentration dependent manner by PT-MC-H compound under the same conditions.

Experimental Example  4. Reverse Transcriptase Chain Reaction RT - PCR )

To determine whether the NO production inhibitory ability of the PT-MC-H compound is due to the inhibition of the expression of iNOS mRNA, the method described in the literature (Lee et al., J. Ethnopharmacol., 97, pp561-566, 2005) was used. RT-PCR was performed as follows.

Compound PT-MC-H (4,7,7-trihydroxy-3,3,4-trimethoxylignan-9,9-olide) of Example 2 in RAW264.7 cells at a concentration of 2.5-10 μg / ml for 1 hour After treatment and reaction, 0.1 μg / ml LPS was pretreated for 4 hours to induce NO production. After 4 hours of LPS treatment, cells were recovered by treatment with Trypsin-EDTA and total RNA was isolated using Trizole: Invitrogen, Carlsbad, CA, USA. The first strand cDNA was synthesized using MMLV-reverse transcriptase (Gibco BRL) from 2 μg of total RNA. The base sequence of the primer (Primer) is shown in Table 1 below.

GAPDH (349 bp) iNOS (449 bp) sense primer 5-CCACTGGCGTCTTCACCAC-3 5-ATGTCCGAAGCAAACATCAC-3 anti-
sense primer 5-CCTGCTTCACCACCTTTTG-3 ' 5-TAATGTCCAGGAAGTAGGTG-3

PCR was repeated 20 times in order of heating at 98 ° C. for 2 minutes and then reacting at 98 ° C. for 10 seconds, at 50 ° C. for 30 seconds, and at 72 ° C. for 1 minute, and then at 5 ° C. for 5 minutes. PCR products were identified by electrophoresis on 1.2% agarose gel and stained with ETBR (ethidium bromide) (0.5 μg / ml).

As shown in FIG. 5, iNOS mRNA expression level was significantly increased in RAW264.7 cells to which LPS was reacted without pretreatment of PT-MC-H compound, as compared to cells not treated with LPS, but PT-MC-H When the compound and LPS were treated together, it was confirmed that the iNOS mRNA level was reduced in a concentration-dependent manner of the PT-MC-H compound (see FIG. 5). Therefore, it was confirmed that NO production was inhibited because the gene expression of iNOS was inhibited by the PT-MC-H compound.

This result is shown by the novel compound 4,7,7'-trihydroxy-3,3 ', 4'-trimethoxylignan-9,9'-oxide (isolated from the ivy extract as shown in FIG. PT-MC-H) inhibits NF-κB activation, a transcription factor in RAW264.7 cells induced by LPS by reducing the phosphorylation of IκB, thereby inhibiting the transcription of iNOS induced by LPS. It was confirmed that the level could be lowered, and thus could have an effect of inhibiting NO production by LPS treatment.

Examples of the pharmaceutical compositions containing the compounds of the present invention will be described, but the present invention is not intended to be limited thereto, but is intended to be described in detail.

Formulation example  One. Powder  Produce

300 mg of the PT-MC-H compound of Example 2

Lactose 100 mg

Talc 10 mg

The above ingredients are mixed and filled in an airtight cloth to prepare a powder.

Formulation example  2. Preparation of Tablets

300 mg of the PT-MC-H compound of Example 2

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components, tablets are prepared by tableting according to a conventional method for preparing tablets.

Formulation example  3. Manufacture of capsule

300 mg of the PT-MC-H compound of Example 2

3 mg of crystalline cellulose

Lactose 14.8 mg

Magnesium Stearate 0.2 mg

According to a conventional capsule preparation method, the above ingredients are mixed and filled into gelatin capsules to prepare capsules.

Formulation example  4. Preparation of injections

300 mg of the PT-MC-H compound of Example 2

Mannitol 180 mg

Sterile distilled water for injection 2974 mg

Na ₂ HPO ₄ 12H ₂ O 26 mg

According to the conventional method for preparing an injection, the amount of the above ingredient is prepared per ampoule (2 ml).

Formulation example  5. Liquid  Produce

300 mg of the PT-MC-H compound of Example 2

10 g of isomerized sugar

5 g of mannitol

Purified water

According to the conventional method of preparing a liquid solution, each component is added to the purified water to dissolve, the lemon flavor is added appropriately, the above components are mixed, purified water is added, the whole is adjusted to 100 ml by the addition of purified water, and then filled into a brown bottle. The solution is prepared by sterilization.

Formulation example  6. Manufacture of health food

1000 mg of PT-MC-H compound of Example 2

Vitamin mixture proper amount

70 μg of Vitamin A Acetate

Vitamin E 1.0 mg

Vitamin B ₁ 0.13 mg

Vitamin B ₂ 0.15 mg

Vitamin B ₆ 0.5 mg

Vitamin B ₁₂ 0.2 μg

Vitamin C 10 mg

10 μg biotin

Nicotinic Acid 1.7 mg

50 μg folic acid

Calcium Pantothenate 0.5mg

Mineral mixture

Ferrous Sulfate 1.75 mg

Zinc Oxide 0.82 mg

Magnesium carbonate 25.3 mg

Potassium monophosphate 15 mg

Dibasic calcium phosphate 55 mg

Potassium Citrate 90 mg

Calcium Carbonate 100 mg

Magnesium chloride 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixture is comparatively mixed with a composition suitable for health food as a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional method for producing healthy foods , Granules can be prepared and used in the manufacture of health food compositions according to conventional methods.

Formulation example  7. Manufacture of health drinks

300 mg of the PT-MC-H compound of Example 2

15 g of vitamin C

100 g of vitamin E (powder)

Iron lactate 19.75 g

3.5 g of zinc oxide

Nicotinamide 3.5 g

0.2 g of vitamin A

Vitamin B ₁ 0.25 g

0.3 g of vitamin B ₂

Water quantification

After mixing the above components in accordance with the conventional healthy beverage manufacturing method, and stirred and heated at 85 ℃ for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 L container, sealed sterilization and refrigerated Used to prepare the healthy beverage composition of the invention.

Although the compositional ratio is relatively mixed with a component suitable for a favorite drink, it is also possible to arbitrarily modify the compounding ratio according to the regional or national preference such as the demand class, the demanding country, and the use purpose.

1 is a diagram showing the cytotoxicity of PT-MC-H compound,

(0, 6.25, 12.5, 25 ug / ml)

2 is a diagram showing a decrease in NO production of PT-MC-H compound,

(0, 2.5, 5, 10 ug / ml)

3 is a diagram showing the cytotoxicity of PT-MC-H compound,

(0, 2.5, 5, 10 ug / ml)

Figure 4 is a diagram showing the reduced production of inflammatory related proteins of PT-MC-H compound,

(IkBα, p-IkBα, iNOS)

5 is a diagram showing the inhibition of iNOS mRNA expression of PT-MC-H compound,

6 is a diagram schematically illustrating the inflammatory signaling of macrophage line Raw264.7.

<110> Kyungpook National University Industry-Academic Cooperation Foundation <120> A composition comprising the novel compound isolated from the          extract of Parthenocissus tricuspidata for preventing and          treating inflammatory disease <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> GAPDH-sense primer <400> 1 ccactggcgt cttcaccac 19 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> GAPDH-anti-sense primer <400> 2 cctgcttcac caccttttg 19 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> iNOS-sense primer <400> 3 atgtccgaag caaacatcac 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> iNOS-anti-sense-primer <400> 4 taatgtccag gaagtaggtg 20  

Claims (8)

Novel compound 4,7,7'-trihydroxy-3,3 ', 4'-trimethoxylignan-9,9'-ol, represented by the following structural formula (I), isolated from the extract of parthenocissus tricuspidata Lead (4,7,7'-trihydroxy-3,3 ', 4'-trimethoxylignan-9,9'-olide) or a pharmaceutically acceptable salt thereof:

(Ⅰ) Novel compound 4,7,7'-trihydroxy-3,3 ', 4'-trimethoxylignan-9,9'-oxide represented by Structural Formula (I) according to claim 1 isolated from the ivy extract For the prevention and treatment of periodontitis, urethritis or cystitis containing 4,7,7'-trihydroxy-3,3 ', 4'-trimethoxylignan-9,9'-olide) or a pharmaceutically acceptable salt thereof as an active ingredient Pharmaceutic composition. The pharmaceutical composition according to claim 2, wherein the extract is a crude extract of ivy, a polar solvent soluble extract or a nonpolar solvent soluble extract. delete The pharmaceutical composition according to claim 3, wherein the nonpolar solvent soluble extract comprises an extract soluble in methylene chloride, hexane, chloroform, dichloromethane or ethyl acetate. delete delete delete

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