KR102075482B1 - A composition for preventing or treating vasculitis comprising lignans from extract of Forsythia koreana flower - Google Patents

Abstract

The present invention relates to a composition for preventing or treating vascular inflammatory diseases containing Koreanaside A, and specifically, relates to: a pharmaceutical composition for preventing or treating vascular inflammatory diseases containing Koreanaside A; health functional food for preventing or alleviating vascular inflammatory diseases containing Koreanaside A; and a method for preventing or treating vascular inflammatory diseases, comprising a step of administering Koreanaside A to an individual other than a human. Koreanaside A, a lignan derived from a Forsythia koreana flower extract according to the present invention has excellent antioxidant activity and has an effect of inhibiting the expression of VCAM-1, thereby being able to be usefully used as a composition for preventing or treating vascular inflammatory diseases.

Description

A composition for preventing or treating vasculitis comprising lignans from extract of Forsythia koreana flower

The present invention relates to a composition for preventing or treating vascular inflammatory diseases, including lignan from forsythia flower extract, specifically, a pharmaceutical composition for preventing or treating vascular inflammatory diseases, including koreanaside A; The present invention relates to a method for preventing or treating vascular inflammatory disease, and the step of administering Coreanaside A to an individual except a human.

Early lesions of atherosclerosis, such as arteriosclerosis, are mainly caused by the inflammatory response of vascular endothelial cells (Ross R. Atherosclerosis--an inflammatory disease.N Engl J Med. 1999 Jan 14; 340 (2): 115-26 ). Normal vascular endothelial cells do not interact with the inflammatory cells and flow along the bloodstream, so there is little contact with the vascular wall. Inflammation of the vascular endothelial cells causes interaction with the inflammatory cells, causing the inflammatory cells to interact with the vascular wall. It will stick and cause an inflammatory reaction. During early arterial inflammation, endothelial cells are activated by various stimuli, including cytokines such as pathogens, oxidized lipids or tumor necrosis factor (TNF) -α, interleukin (IL) -1/3, and IL-6. Induced expression of Vascular Cell Adhesion Protein-1 (VCAM-1) protein, a specific adhesion receptor that then increases the successive circulating monocytes into plaque-forming foci. In inflamed vascular endothelial cells, rolling and adhered monocytes are metastasized continuously, resulting in diapedesis through the vascular wall. A substance that inhibits the expression of VCAM-1, which is known as a major mediator in this inflammatory response, has been used as a therapeutic agent for vascular diseases caused by inflammation (Preiss DJ, Sattar N. Vascular cell adhesion molecule-1: a viable therapeutic target for atherosclerosis; Int J Clin Pract. 2007 Apr; 61 (4): 697-701). Vascular inflammatory diseases caused by vasculitis include atherosclerosis, hypertension, angina pectoris, myocardial infarction, ischemic heart disease, heart failure, complications after cerebral angioplasty, cerebral infarction, cerebral hemorrhage and stroke.

On the other hand, Forsythia koreana is a deciduous shrub of the dicotyledonous genus Aureaceae , whose roots, stems, leaves, and fruits are used for medicinal purposes. In particular, forsythia fruit is called Yeon-kyo and is prescribed in cold, fever, purulent diseases, lymphadenitis, urinary tract, boil, nephritis, eczema, and is known to contain various medicinal ingredients. However, most of the research on pharmacological activity has been concentrated in the Fellowship, and active research on other parts of forsythia is necessary.

Against this background, the present inventors have studied to develop a composition for treating vascular inflammatory diseases. As a result, the lignans from forsythia flower extract, especially Koreanaside A, have an inhibitory effect on expression of VCAM-1, a factor involved in vascular inflammatory diseases. By confirming the superiority, the present invention was completed.

One object of the present invention is to provide a pharmaceutical composition for preventing or treating vascular inflammation comprising Coreanaside A.

It is another object of the present invention to provide a health functional food for preventing or improving vascular inflammation, including Koreanaside A.

Yet another object of the present invention is to provide a method for preventing or treating vascular inflammation, comprising administering Koreanaside A to a subject other than a human.

In order to achieve the above object, one embodiment of the present invention provides a pharmaceutical composition for preventing or treating vascular inflammation, including the corenaside A.

In the present invention, the term "Koreanaside A (koreanaside A)" may be represented by the following formula (1). Coreanaside A is a novel compound that has not been previously known, and its structure as well as what is present in forsythia flowers has also been first identified by the present inventors.

[Formula 1]

The Koreanaside A according to the present invention may be a lignan derived from forsythia flower extract.

In the present invention, the term "forsythia flower" means a flower of forsythia ( Forsythia koreana ). Although it is known that the extract of the forsythia flower has an antioxidant effect, it is not known about the vascular inflammation treatment effect, it was first identified by the present inventors.

The forsythia flowers may be purchased commercially, or may be used for harvested or grown in nature.

As used herein, the term "extract" refers to an extract obtained by extracting the forsythia flower, a diluent or concentrate of the extract, a dried product obtained by drying the extract, a crude or purified product of the extract, or a mixture thereof. Extracts of all formulations that can be formed using itself and extracts.

The method of extracting the forsythia flower is not particularly limited and may be extracted according to a method commonly used in the art. Non-limiting examples of the extraction method, hot water extraction method, ultrasonic extraction method, filtration method, reflux extraction method and the like, these may be performed alone or in combination of two or more methods.

In addition, the type of extraction solvent used to extract the forsythia flower is not particularly limited, and any solvent known in the art may be used. Non-limiting examples of the extraction solvent may include water, alcohols having 1 to 4 carbon atoms or mixed solvents thereof, and these may be used alone or in combination of one or more thereof. Specifically, methanol or ethanol aqueous solution may be used, the methanol or ethanol aqueous solution is 70 to 90% (v / v), more specifically 75 to 85% (v / v), most specifically 80% (v / v ) Methanol or ethanol, but is not limited thereto.

The lignan is forsythia flower extract water, a polar solvent such as alcohol having 1 to 4 carbon atoms; Nonpolar solvents such as hexane (Hexane) and ethyl acetate; Or a fraction obtained by fractionation with a mixed solvent thereof.

As used herein, the term "fraction" refers to the result obtained by performing the fractionation to separate a specific component or a specific component group from a mixture comprising various various components.

The fractionation method of obtaining the fractions is not particularly limited and may be performed according to a method commonly used in the art. Non-limiting examples of the fractionation method include a solvent fractionation method performed by treating various solvents, an ultrafiltration fractionation method performed through an ultrafiltration membrane having a constant molecular weight cut-off value, and various chromatography (size, charge, hydrophobicity). Or a chromatographic fractionation method for performing separation according to affinity), and combinations thereof.

In addition, as the type of fractionation solvent used to obtain the fraction, any solvent known in the art may be used. Non-limiting examples of the fractionation solvents include polar solvents such as water and alcohols having 1 to 4 carbon atoms; Nonpolar solvents such as hexane (Hexane) and ethyl acetate; Or these mixed solvents etc. are mentioned. These may be used alone or in combination of one or more, but are not limited thereto. Specifically, water, methanol, butanol, hexane, ethyl acetate or a mixed solvent thereof may be used.

Specifically, the fraction is the first fraction of the forsythia flower extract with a solvent containing butanol, the second fraction with a solvent containing water, and then the third fraction with a solvent containing methanol, the fourth fraction as a solvent containing acetone After fractionation may be carried out by a fifth fraction with a solvent comprising water. In addition, the first to fifth fractions may be repeated three to five times, specifically four times.

In this case, the first fraction may be a butanol fraction, the second fraction may be a water fraction obtained by fractionation with a solvent consisting of chloroform, methanol and water and then re-fractionation with a solvent consisting of ethyl acetate, butanol and water. The third fraction may be a methanol fraction obtained by fractionation with a solvent consisting of chloroform, methanol and water, and the fourth fraction may be an acetone fraction obtained by fractionation with a solvent consisting of acetone and water, and the fifth fraction is acetone, methanol And it may be a water fraction obtained by fractionation with a solvent consisting of water.

In an embodiment of the present invention, the forsythia flower extract was obtained using 80% methanol of dried forsythia flowers, and the extract was fractionated with butanol to obtain a first fraction, a butanol fraction, wherein the first fraction was chloroform and methanol. And fractionated with a solvent consisting of water and then repartitioned with a solvent consisting of ethyl acetate, butanol and water to obtain a second fraction. In addition, the second fraction was partitioned with a solvent consisting of chloroform, methanol, and water to obtain a third fraction, and the third fraction was fractionated with a solvent consisting of acetone and water to obtain a fourth fraction, and then, the fourth fraction was separated. Fractionation with a solvent consisting of acetone, methanol and water gave a fifth fraction.

As used herein, the term "lignan" is a part of polyphenols found in plants and is a steroid compound synthesized in cholesterol with a chemical structure similar to estrogen. It is found in over 55 plant groups and acts as a defense molecule against antibiotics, pathogens and bacteria. Lignan is a type of phytoestrogen, a phytoestrogen that plays a role similar to estrogen. Lignan is a single cell layer underneath the fibrous layer that is abundant in the skin of the fruit, tightly bound to fibrin. Epidemiological and physiological studies in the human body show that lignans can have a positive effect on the prevention of type II diabetes and cancer and reduce the incidence of estrogen-related cancers. It can also reduce or prevent osteoporosis in the human body. Types of lignans include enterlignan, enterodiol and enterolactone.

As used herein, the term "vascular inflammatory disease" means a disease characterized by inflammation of the blood vessel wall and thus tissue damage. Inflammation of the vessel wall causes ischemia in tissues that have been fed through the vessels, resulting in tissue damage. Since all types of blood vessels and blood vessels of all organs can be invaded, the symptoms and symptoms vary according to the location and characteristics of the involved vessels. It may occur primary without underlying disease, or secondary with rheumatic arthritis such as rheumatoid arthritis or systemic lupus erythematosus.

The vascular inflammatory disease is not limited thereto, but may be atherosclerosis, hypertension, angina pectoris, myocardial infarction, ischemic heart disease, heart failure, complications arising after angioplasty, angioplasty, cerebral infarction, cerebral hemorrhage and stroke.

The prevention or treatment of vascular inflammation may be achieved by inhibiting the expression of VCAM-1.

In one specific embodiment of the present invention, after treatment of forsythia flower extract-derived lignan to mouse vascular smooth muscle (MOVAS) cell line, the inhibitory effect on the expression of vascular cell adhesion molecule-1 (VCAM-1) induced by TNF-α As a result, it was confirmed that Koreanaside A effectively inhibited the expression of VCAM-1 (FIG. 3).

As used herein, the term "prevention" refers to any action that inhibits or delays the symptoms of vascular inflammation by administration of a composition comprising lignans derived from forsythia flower extract according to the present invention.

As used herein, the term "treatment" refers to any action in which a symptom of vascular inflammation is improved or beneficially changed by administration of a composition comprising forsythia flower extract-derived lignans.

As used herein, the term "pharmaceutical composition" means that is prepared for the purpose of preventing or treating a disease, and can be used by formulating in various forms according to each conventional method. For example, it may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, and the like, and may be used in the form of external preparations, suppositories, and sterile injectable solutions. In particular, it may be used in a form suitable for topical administration, for example, eye drops, creams, ointments, gels or lotions.

As used herein, the term “pharmaceutically acceptable salts” refers to salts that can be used pharmaceutically, even among salts in which cations and anions are bonded by electrostatic attraction. Salts, salts with inorganic acids, salts with organic acids, salts with basic or acidic amino acids, and the like. For example, the metal salt may be an alkali metal salt (sodium salt, potassium salt, etc.), alkaline earth metal salt (calcium salt, magnesium salt, barium salt, etc.), aluminum salt, or the like; Salts with organic bases include triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, N, N-dibenzylethylenediamine Salts and the like; Salts with inorganic acids may be salts with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, and the like; Salts with organic acids can be salts with formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid and the like; Salts with basic amino acids can be salts with arginine, lysine, ornithine and the like; Salts with acidic amino acids can be salts with aspartic acid, glutamic acid and the like.

The pharmaceutical composition of the present invention may include Koreannaside A or a pharmaceutically acceptable salt thereof in an amount of 0.01 to 80%, specifically 0.01 to 70%, and more specifically 0.01 to 60% by weight, based on the total weight of the composition. As long as it shows a prophylactic or therapeutic effect, it is not limited to this.

The composition of the present invention may be prepared in the form of a pharmaceutical composition for preventing or treating wounds further comprising a suitable carrier, excipient or diluent commonly used in the manufacture of the pharmaceutical composition, wherein the carrier is an unnatural carrier ( non-naturally occuring carriers).

In the present invention, carriers, excipients and diluents which may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, Calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and the solid preparations include at least one excipient such as starch, calcium carbonate, and the like in the above extracts and fractions thereof. , Sucrose or lactose, gelatin and the like are mixed and prepared. In addition to simple excipients, lubricants such as magnesium styrate and talc are also used. Liquid preparations for oral use may include various excipients such as wetting agents, sweeteners, fragrances, preservatives, etc., in addition to water and liquid paraffin, which are commonly used to include suspensions, solutions, emulsions, and syrups. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The pharmaceutical composition of the present invention may be administered orally, parenterally, rectally, topically, transdermally, intravenously, intramuscularly, intraperitoneally, subcutaneously, etc. according to the desired method, and the active ingredient of the pharmaceutical composition may be administered to the age of the subject to be administered. , Sex, weight, pathology and severity, route of administration or prescriber's judgment. Dosage determination based on these factors is within the level of one of skill in the art and its daily dosage may be, for example, from 0.1 mg / g / day to 100 mg / g / day, more specifically from 5 mg / g / day to 50 mg / day. g / day, and the frequency of administration of the composition of the present invention is not particularly limited, but may be administered once a day or several times in divided doses. The dosage does not limit the scope of the invention in any aspect.

Another aspect of the present invention provides a dietary supplement for preventing or improving vascular inflammation, including lignans from forsythia flower extract.

In the present invention, the terms "forsythia flower", "extract", "lignan", "vascular inflammation" are as described above.

Functional food is the same term as food for special health use (FOSHU), and refers to foods with high medical and medical effects that are processed to show efficient bioregulatory functions in addition to nutrition. In addition, the food may be prepared in various forms such as tablets, capsules, powders, granules, liquids, pills, etc. in order to obtain useful effects in inhibiting cancer metastasis.

The dietary supplement of the present invention may further comprise a food acceptable carrier.

There is no restriction | limiting in particular in the kind of food which can add the composition containing the lignan derived from forsythia flower extract of this invention, For example, there are various beverages, gum, tea, a vitamin complex, a health supplement, etc. The food composition may be added to other ingredients that do not interfere with the vascular inflammation treatment effect, the kind is not particularly limited. For example, various herbal extracts, food acceptable additives, natural carbohydrates, and the like may be contained as additional ingredients, such as conventional foods.

The food supplement additive is added to prepare the health functional food of each formulation can be used by those skilled in the art as appropriate. For example, various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic and natural flavors, colorants and fillers, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters , Stabilizers, preservatives, glycerin, alcohols, carbonation agents used in the carbonated beverage, and the like, but the kind is not limited by the above examples.

At this time, the content of the extract contained in the food is not particularly limited, but may be included in 0.01 to 100% by weight, more preferably 1 to 80% by weight relative to the total weight of the food composition.

When the food is a beverage, it may be included in a ratio of 1 to 30 g, preferably 3 to 20 g based on 100 ml. In addition, the composition may include additional ingredients that are commonly used in food compositions to improve the smell, taste, time and the like. For example, it may include vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, panthotenic acid, and the like. Also, minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn) and copper (Cu) may be included. It may also contain amino acids such as lysine, tryptophan, cysteine, valine and the like. Also, preservatives (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetic acid, etc.), fungicides (bleaching powder and highly bleaching powder, sodium hypochlorite, etc.), antioxidants (butylhydroxyanisol (BHA), butylhydroxytoluene ( BHT), etc.), colorants (such as tar pigment), colorants (such as sodium nitrite, sodium nitrite), bleach (sodium sulfite), seasonings (such as MSG glutamate), sweeteners (ducin, cyclate, saccharin, sodium, etc.) , Food additives such as flavorings (vanillin, lactones, etc.), swelling agents (alum, potassium D-tartrate, etc.), reinforcing agents, emulsifiers, thickeners (pigments), coatings, gum herbicides, foam inhibitors, solvents, improvers, etc. ) Can be added. The additive is selected according to the type of food and used in an appropriate amount.

The health functional food of the present invention can be prepared by a method commonly used in the art, and the preparation can be prepared by adding raw materials and ingredients commonly added in the art. In addition, unlike the general medicine has the advantage that there is no side effect that can occur when taking a long-term use of the drug as a raw material, and can be excellent in portability.

Yet another aspect of the present invention provides a method for preventing or treating vascular inflammation, comprising administering a forsythia flower extract-derived lignan to an individual except a human.

In the present invention, the terms "forsythia flower", "extract", "lignan", "vascular inflammation" are as described above.

As used herein, the term "individual" may mean any animal, including humans, which have or are likely to develop vascular inflammation. The animal may be a mammal such as, but not limited to, a human, a cow, a horse, a sheep, a pig, a goat, a camel, a antelope, a dog, a cat, and the like, which require treatment of similar symptoms.

The prophylactic or therapeutic method of the present invention may specifically include administering the composition to a subject having or at risk of developing vascular inflammation in a pharmaceutically effective amount.

As used herein, the term "administration" refers to introducing the pharmaceutical composition of the present invention to a patient in any suitable manner, wherein the route of administration of the composition of the present invention is oral or parenteral as long as the target tissue can be reached. Administration can be via a variety of routes.

Foranax A, a lignan from a forsythia flower extract, according to the present invention has excellent antioxidant activity and has an effect of inhibiting the expression of VCAM-1, and thus can be easily used as a composition for preventing or treating vascular inflammation.

1 is a diagram showing the molecular structure of six lignans derived from forsythia flower extract; 1, Koreanaside A; 2, Koreanaside B; 3, piligenin; 4, epipinoresinol; 5, pinoresinol; 6, Tynosposide A.
FIG. 2 is a graph showing ORAC values of six lignans derived from forsythia flower extracts in proportion to trolox, a radical scavenger; Trolox ORAC readings = 1.
3 is a graph showing the effect of forsythia flower extract derived lignans on vascular cell adhesion molecule-1 (VCAM-1) expression induced by TNF-α; (A) The expression level of VCAM-1 protein, β-actin protein expression level identified in MOVAS through Western blot analysis was used as a control; (B) VCAM-1 expression levels quantified by β-actin; 1, Koreanaside A; 2, Koreana B.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example 1. Isolation of Forsythia Flower Extract-Derived Compound

The dried forsythia flower (962 g) was extracted with 80% methanol (MeOH, 33 LХ2) for 24 hours at room temperature, which was filtered and concentrated. Thereafter, water (500 mL) was continuously poured into the obtained methanol extract, and extracted with n-butanol (n-BuOH, 450 mLH 2) and ethyl acetate (EtOAc, 500 mLH 2). The extracts were concentrated to give a residue of EtOAc fraction (FKE, 45g), n-BuOH fraction (FKB, 110g) and water fraction (FKW, 395g).

After obtaining of the fractions the compounds were separated using column chromatography.

Specifically, the EtOAc fraction was n -hexane-EtOAc (10: 1 → 2: 1 → 1: 1, 14 L each) using SiO ₂ cc (SiO ₂ Column chromatography, φ 12Х17 cm) → CHCl ₃ -MeOH Eluted with water (30: 3: 1 → 20: 3: 1 → 10: 3: 1 → 65: 35: 10, 15 L each) to obtain 12 fractions (FKE-1 to FKE-12).

Of the 12 fractions, FKE-10 fraction [1.9 g, eluent / total volume (Ve / Vt) 0.072-0.088] was converted to CHCl ₃ -EtOAc (10: 1 → 3) using SiO ₂ cc (φ 4.5Х15 cm). : 1, 5.6 L each), and 19 fractions (FKE-10-1 to FKE-10-19) were obtained.

Of the 19 fractions, FKE-10-12 fraction [78.1 mg, Ve / Vt 0.055-0.070] was eluted with acetone-water (1: 1, 480 mL) using ODS cc (Φ 2 × 5 cm). , Seven fractions (FKE-10-12-1 to FKE-10-12-7) were obtained. Then, phylligenin [23.2 mg, Ve / Vt 0.500-0.667, TLC (SiO ₂ ) 0.45, CHCl ₃ -EtOAc (6: 2), TLC in the FKE-10-12-4 fraction of the seven fractions (ODS) 0.51, acetone-water (2: 1-> 4: 2)].

In addition, among the 19 fractions, FKE-10-14 fraction [220.0 mg, Ve / Vt 0.736-0.780] was converted to acetone-water (2: 1 → 1: 1, respectively) using ODS cc (φ 2.5Х5 cm). 2.2 L), eleven fractions (FKE-10-14-1 to FKE-10-14-8) were obtained, and FKE-10-14-2 fraction [69.0 mg, Ve / Vt 0.736-0.780] was eluted with CHCl ₃ -EtOAc (5: 1, 500 mL) using an SiO ₂ (φ 2Х10 cm) column and three fractions (FKE-10-14-2-1 to FKE-10). -14-2-3). Thereafter, in the FKE-10-14-2-1 fraction of the three fractions, epipinoresinol [27.6 mg, Ve / Vt 0.000-0.275, TLC (SiO ₂ ) 0.50, CHCl ₃ -EtOAc (6: 2), TLC (ODS) 0.53, acetone-water (4: 2)].

In addition, among the 19 fractions, FKE-10-15 fraction [450.0 mg, Ve / Vt 0.780-0.860] was converted to acetone-water (3: 1 → 2: 1 → 1 :) using ODS cc (φ 3Х5 cm). 1, 350 mL each), and five fractions (FKE-10-15-1 to FKE-10-15-5) were obtained, and FKE-10-15-1 fraction [42.0 mg] out of the five fractions. , Ve / Vt 0.000-0.017] was eluted with n -hexane-EtOAc (1: 3, 550 mL) using SiO ₂ cc (φ 3Х30 cm), and 7 fractions (FKE-10-15-1-1) FKE-10-15-1-7) was obtained. Then, pinoresinol [FKE-10-15-1-4, 11.1 mg, Ve / Vt 0.855-0.909, TLC (SiO ₂ ) in the FKE-10-15-1-4 fraction of the seven fractions. 0.35, CHCl ₃ -EtOAc (6: 4), TLC (ODS) 0.42, acetone-water (2: 2)].

In addition, the n-BuOH fraction was purified using CH _{2 3} -MeOH-H ₂ O (30: 3: 1 → 20: 3: 1 → 10: 3: 1, 43 L each) using SiO ₂ cc (Φ 11Х15 cm). → eluted with EtOAc- n- BuOH-H ₂ O (4: 5: 1, 45 L), and 15 fractions (FKB-1 to FKB-15) were obtained, the FKB-4 fraction of which 15 fractions [ 11.5 g, Ve / Vt 0.154-0.363] was eluted with CHCl ₃ -MeOH-H ₂ O (25: 3: 1, 7.7 L) using SiO ₂ cc (φ 5.5Х15 cm) and 10 fractions (FKB) -4-1 to FKB-4-10).

FKB-4-6 fraction [4.9 g, Ve / Vt 0.316-0.411] of the 10 fractions was eluted with acetone-water (1: 2, 2 L) using ODS cc (Φ 5Х6 cm) and 4 Fractions (FKB-4-6-1 to FKB-4-6-4) were obtained, and FKB-4-6-1 fraction [2.2 g, Ve / Vt 0.000-0.571] of the four fractions was obtained by ODS cc ( Eluted with acetone-MeOH-water (1: 1: 4, 2.6 L) using Φ 4Х6 cm) and seven fractions (FKB-4-6-1-1 to FKB-4-6-1-7) Got. Then, Koreanaside A (koreanaside A) [219.8 mg, Ve / Vt 0.712-0.731, TLC (SiO ₂ ) 0.51, CHCl ₃ -MeOH-H ₂ in the FKB-4-6-1-4 fraction of the seven fractions. 0 (16: 6: 2), TLC (ODS) 0.20, acetone-MeOH-water (2: 2: 8)].

Also, FKB-4-7 fractions of the 10 fractions [1.2 g, Ve / Vt 0.413-0.531 ] a SiO ₂ cc using a _{(Φ 4Х15 cm) CHCl 3 -MeOH} -H 2 O (25: 3: 1 , 3.5 L), and 6 fractions (FKB-4-7-1 to FKB-4-7-6) were obtained. Thereafter, tinosposide A (FKB-4-7-6, 242.4 mg, Ve / Vt 1.000, TLC (SiO ₂ ) 0.45, CHCl ₃ in the FKB-4-7-6 fraction of the six fractions. -MeOH-H ₂ 0 (14: 6: 2), TLC (ODS) 0.55, acetone-water (4: 6)].

In addition, among the 12 fractions, FKB-8 fraction [2.9 g, Ve / Vt 0.594-0.693] was eluted with acetone-water (1: 1, 1.2 L) using ODS cc (Φ 6Х20 cm), 8 Fractions (FKB-8-1 to FKB-8-8) were obtained, and the FKB-8-1 fraction [2.0 g, Ve / Vt 0.000-0.203] of the eight fractions was converted into SiO ₂ cc (Φ 3.5Х17 cm). Elution with EtOAc- n- BuOH-H ₂ O (40: 3: 1 → 35: 3: 1 → 30: 3: 1 → 25: 3: 1 → 20: 3: 1, 1 L each) Twelve fractions (FKB-8-1-1 to FKB-8-1-12) were obtained.

FKB-8-1-11 fraction [762.5 mg, Ve / Vt 0.145-0.363] of the 12 fractions was purified using EtOAc ₂ cc (Φ 3.5Х16 cm) in EtOAc- n- BuOH-H ₂ O (11: 3). : 1, 3.2 L), eleven fractions (FKB-8-1-11-1 to FKB-8-1-11-10) were obtained, and FKB-8-1-11 of the ten fractions was obtained. Koreanaside B [FKB-8-1-11-5, 55.6 mg, Ve / Vt 0.380-0.473, TLC (SiO ₂ ) 0.45, EtOAc- n- BuOH-H ₂ O (16) : 6: 2), TLC (ODS) 0.82, acetone-MeOH (4: 2)].

Taken together, a total of six compounds were obtained from ethylacetate or butanol fractions of 80% methanol extract of forsythia flowers, including Coreanaside A, Coreanaside B, Piligenin, Epipinoresinol, Pinoresinol and Tynosposide A. . The structures of the six compounds are shown in FIG. 1.

In addition, as shown in Table 1, the analysis of ¹ H-NMR, ¹³ C-NMR, gHMBC, CD or HR-FAB-MS for Coreanaside A and Coreanaside B, the two compounds are We have confirmed that this is a new lignan that has not been reported previously.

One 2 δ _H ( J  in Hz) δ _C δ _H ( J  in Hz) δ _C One - 137.3 - 136.2 2 6.97 (d, 2.0) 111.5 6.99 (d, 1.6) 111.6 3 - 150.8 - 149.9 4 - 147.2 - 147.5 5 7.11 (d, 8.0) 117.8 7.12 (d, 7.6) 118.0 6 6.85 (dd, 8.0, 2.0) 119.7 6.92 (d, 7.6, 1.6) 120.4 7 4.64 (d, 4.0) 86.9 4.67 (d, 7.2) 84.9 8 3.00-3.08 (m) 55.2 2.48-2.58 (m) 53.9 9a 4.15 (dd, 8.0, 2.0) 72.6 4.23 (dd, 9.2, 4.4) 62.5 9b 3.79 (overlapped) - 3.93 (dd, 9.2, 7.2) - One' - 133.7 - 138.6 2' 6.75 (br. S) 120.0 6.88 (d, 1.6) 111.9 3 ' - 149.0 - 147.3 4' 6.75 (br. S) 116.1 6.72 (br. S) 116.1 5 ' - 147.3 - 149.1 6 ' 6.90 (br. S) 110.9 6.71 (br. D, 1.6) 120.9 7 ' 4.68 (d, 4.0) 87.3 4.45 (d, 8.4) 76.8 8' 3.00-3.08 (m) 55.4 1.85-1.91 (m) 49.9 9'a 4.15 (dd, 8.0, 2.0) 72.6 3.31 (overlapped) 71.8 9'b 3.79 (overlapped) - 3.21 (dd, 10.8, 6.0) - One'' 4.85 (d, 7.6) 102.7 4.88 (d, 7.6) 103.0 2'' 3.49 (overlapped) 74.8 3.48 (overlapped) 75.1 3 '' 3.75 (overlapped) 77.7 3.73 (overlapped) 78.0 4'' 3.14 (overlapped) 71.2 3.49 (overlapped) 71.5 5 '' 3.37 (overlapped) 78.0 3.39 (overlapped) 78.3 6''a 3.84 (overlapped) 62.4 3.86 (overlapped) 62.7 6 '' b 3.56 (overlapped) - 3.68 (d, 10.0, 6.8) - -OCH ₃ 3.81 (s) 56.7 3.86 (s) 56.9 -OCH ₃ 3.80 (s) 56.4 3.81 (s) 56.9

Example 2 Analysis of Antioxidant Activity of Novel Lignans Derived from Forsythia Flower Extract

Because reactive oxygen species (ROS) contribute to the inflammatory process and compounds with antioxidant activity have been reported to inhibit inflammatory factors such as iNOS, COX-2, p-p65 and VCAM-1 (Lee, Ah) Young, et al., 2016; Thi, Nhan Nguyen, et al., 2017), the antioxidant activity of the six lignans isolated through Example 1 was confirmed.

Antioxidant activity of the six compounds was carried out through oxygen radical absorbance capacity (ORAC) analysis, a very common and standardized chemical antioxidant test. ORAC confirmed the activity level compared to Trolox, a well known radical scavenger.

As a result, as shown in Figure 2, five compounds except the tynosposide A showed a high antioxidant effect, the ORAC of the five compounds was found to be 0.88 ± 0.13 to 1.02 ± 0.01. These results indicate that the tynosposide A does not have a hydroxyl group in the benzene ring, but five other compounds have an antioxidant effect by having at least one hydroxyl group in the benzene ring.

Example 3. Analysis of the inhibitory effect of VCAM-1 expression of forsythia flower extract-derived novel lignans

In order to confirm the effect of novel lignans on vascular inflammation, induced by TNF-α in mouse vascular smooth muscle (MOVAS) cell lines of novel lignans Koreanaside A and B of forsythia flower derived compounds obtained through Example 2 above The inhibitory effect on vascular cell adhesion molecule-1 (VCAM-1) expression was assessed.

Mouse vascular smooth muscle cells were pretreated with Coreanaside A or B (25 mg / mL) for 2 hours, and then stimulated with TNF-α (10ng / mL) for 24 hours and the expression of VCAM-1 was measured.

As a result, as shown in Fig. 3, Koreana B did not significantly inhibit the expression of VCAM-1 induced by TNF-α in MOVAS, while Koreana A effectively inhibited the expression of VCAM-1. .

Taken together, Koreanaside A, a novel lignan derived from forsythia flowers according to the present invention, has excellent antioxidant activity and inhibits the expression of VCAM-1 induced by TNF-α, and thus can be judged to have a therapeutic effect on vascular inflammation. .

In the present specification, those skilled in the art of the present disclosure can fully recognize and infer the details thereof, and the detailed descriptions thereof have been omitted, and the technical spirit or essential configuration of the present invention may be changed in addition to the specific examples described herein. More variations are possible within the scope of not doing so. Accordingly, the present invention may be practiced in a manner different from that specifically described and illustrated herein, which will be understood by those of ordinary skill in the art.

Claims (5)

Pharmaceutical composition for preventing or treating vascular inflammatory disease comprising Koreanaside A represented by Formula 1;

[Formula 1]

.

The method of claim 1,
The Koreanaside A is a forsythia flower extract water, a polar solvent such as alcohol having 1 to 4 carbon atoms; Nonpolar solvents such as hexane (Hexane) and ethyl acetate; Or obtained by fractionation with a mixed solvent thereof.
The method of claim 1,
The prevention or treatment of vascular inflammation is achieved by inhibiting the expression of VCAM-1, pharmaceutical composition.
Health functional foods for preventing or improving vascular inflammatory diseases, including Koreanaside A.
A method for preventing or treating vascular inflammatory disease comprising administering Koreanaside A to an individual except a human.

Images