KR20100038633A - Pharmaceutical compositions for prevention and treatment of influenza viral diseases containing sophora flavescens extracts, fractions, the isolated pterocarpan and flavonoid compounds therefrom, or the pharmaceutically acceptable salts as an active ingredient - Google Patents

Abstract

PURPOSE: A composition containing Sophora flavescens extract or compound isolated therefrom is provided to suppress neuraminidase and to prevent and treat influenza virus infection. CONSTITUTION: A pharmaceutical composition for preventing and treating influenza virus infectious diseases contains Sophora flavescens extract as an active ingredient. The Sophora flavescens extract is isolatd using water, alcohol of C1-C4, or mixture solvent thereof. The pharmaceutical composition also contains fraction from the Sophora flavescens extract as an active ingredient. The fraction is fractioned by hexane, chloroform, and ethyl acetate. The pharmaceutical composition contains pterocarpan compound of chemical formula 1 or its pharmaceutically acceptable salt as an active ingredient. The pterocarpan compound is isolated from Sophora flavescens extract or fraction.

Description

Pharmaceutical composition for the prevention and treatment of influenza virus infection disease containing as a active ingredient ginseng extract, ginseng fraction, terokapane and flavonoid compounds isolated therefrom or pharmaceutically acceptable salts thereof of influenza viral diseases containing Sophora flavescens extracts, fractions, the isolated pterocarpan and flavonoid compounds therefrom, or the 黄 acceptable salts as an active ingredient}

The present invention is a ginseng extract, a ginseng fraction, terokapane-based compounds and flavonoid-based compounds or pharmaceutically acceptable salts thereof isolated from the ginseng extract, pharmaceutical composition or influenza disinfection for the prevention and treatment of influenza virus infection disease It relates to a composition for.

Influenza virus is a highly contagious virus that causes acute respiratory disease and is one of the viruses that causes severe respiratory symptoms in children, the elderly, and patients with cardiopulmonary disease by causing a mass infection or epidemic in the world (Hien, TT et al. N) . Eng. J. Med., 350 , 1179, 2004). Influenza viruses belong to the orthomyxovirus taxonomically, and there are three types of A, B and C. In particular, the epidemic types are A and B. On the surface of these viruses, there are two types of surface antigens, the glycoproteins Hamagglutinin (HA) and Neuraminidase (NA), and there are eight segmented RNAs inside. Hemagglutinin is a trimer consisting of the head and the stem, the double head part of which is associated with most antigenic mutations, which binds to the terminal sialic acid residues on the surface of the host cell to attach the virus. In turn, the virus allows penetration into host cells (Chandrasekaran, A. et al. Nature biotechnology 26, 107, 2008). Neuraminidase is a mushroom-shaped tetramer with a head and stem shape, with active sites on the top surface of the head, and virus replicated and propagated in infected cells. Cleavage of alpha-ketosidic bonds linking neuraminic acid residues plays an important role in the release of viruses out of host cells and into respiratory mucosal cells (a. Mark, VI Nature review 6, 967, 2007. b. Huberman, K. et al. Virology 214 , 294, 1995).

The surface antigens of the virus cause mutations in the same subtype, with new antigenic variants appearing each year. In particular, avian influenza virus, which has recently been a problem among influenza viruses, infects various types of birds such as chickens, turkeys, ducks, and wild birds due to stool, and more than 80% of chickens die due to rapid spread. It is the virus disease that causes the biggest damage and threat to the poultry industry all over the world, and its ramifications are not limited to the poultry industry, and it has been reported to cause disease in humans due to infection with human body (Gubareva, LV et al. Lancet. 355 , 2000).

Therefore, the method of inhibiting the adsorption of influenza virus to epithelial cells, the method of inhibiting the invasion of cells, the method of inhibiting the transcription and replication of genes, the method of inhibiting protein synthesis, the release from cells for the purpose of treating viral diseases It is possible to think of a method for inhibiting these, each of which is a target of the antivirus.

Previously developed treatments for treating influenza viruses include four drugs: amatadine, rimantadine, zanamivir, and oseltamivir. It is approved by and has been used. However, M2 inhibitors Amantadine and Rimantadine, which have antiviral effects by blocking the uncoating of the virus by blocking the ion channel of M2 protein, a cell membrane protein essential for virus propagation, are influenza virus type A only. It has been reported to be effective and tolerate viruses for 40 years of use, with serious side effects in the nervous system and stomach (Bantia, S. et al. Antiviral Research 69 , 39, 2006). Since 1999, Zanamivir and Oseltamivir are inhibitors of neuraminidase, which play an important role in the propagation of viruses and have a low incidence of resistance and are reliably present in both influenza A and B influenza viruses. There are reports of treatment of viral infections with new drugs (Zhang, J. et al. Bioorg. Med. Chem. Lett. 16 , 3009, 2006).

However, zanamivir has high antiviral effects but has low bioavailability and rapid renal excretion (Ryan, DM et al. Antimicrob. Agents Chemother., 39, 2583, 1995). Seltamir has the side effect of severe nausea.

Antivirals developed to date have severe side effects and require much attention for their application. In addition, the development of a vaccine is less effective if the type of the virus and the virus of the vaccine does not match, there is an increasing need for the development of a new influenza virus agent excellent in the inhibitory effect and excellent stability.

On the other hand, the ginseng ( Sophora flavescens ) is a perennial herb that is distributed in Korea, Japan, China, and Siberia. In oriental medicine, dried roots are called red ginseng, which are used for flavoring, indigestion, neuralgia, hepatitis, jaundice, and hemorrhoids. In private, stems and leaves are used as antipyretic and analgesics. Ginseng has been mainly studied in Japan since 1957, and has been the main ingredient in alkaloids (Saito, K. et al., Planta Med ., 56: 487, 1990; Atta. R. et al., J. Nat. Prod ., 54: 929, 1991) and flavonoids (Iinuma, M. et al., Phytochemistry , 29, 2667, 1990; Shirataki, Y. et al., Chem. Pharm. Bull. , 38, 1712, 1990) It is known. Especially, in ginseng, the terokapane-based compound and flavonoid basic structure, which have been widely reported in legumes, are replaced with prenyl groups having 5 carbon isoprenyl and 10 carbon lavendulyl groups. It is well known to contain large amounts of flavonoids (Kim, SJ et al., Biol. Pharm. Bull., 26, 1348, 2003). However, activity studies on these have been shown to be cytotoxic (Kang, TH et al., J. Nat. Prod., 63, 680, 2000) and anti-inflammatory (Chi, YS et al., Biochem. Pharm., 62, 1185, 2001), but there is no known use of ginseng extracts to inhibit and prevent viral diseases, particularly influenza viruses.

Looking at the patent technology registered in relation to the conventional ginseng extract, ginseng extract (Korean patent registration No. 10-0380864) having an effect on the prevention and treatment of osteoporosis, a method of improving the shelf life of the bank using the ginseng extract (Korean Patent Registration No. 10 -0595055), cosmetic composition containing glyceryl caprylate and ginseng extract as preservatives (Korean Patent Registration No. 10-0722673), composition for improving sexual dysfunction, including the ginseng extract (Korean Patent Registration No. 10-0475647) ), Preylated pravonoid derivatives having anti-inflammatory action, and ginseng extract containing them (Korean Patent Registration No. 10-0384661). However, there is no known use of ginseng extract to suppress and prevent influenza virus.

Therefore, the present inventors while studying the ginseng extract, fractions thereof, or terokapane-based compounds and flavonoid-based compounds isolated therefrom, the ginseng extract, fractions or terokabane-based compounds and flavonoid-based compounds isolated therefrom are neura The present invention was completed by revealing the inhibitory activity against MiniDays and revealing their use for preventing and treating viral diseases or for disinfecting influenza.

An object of the present invention is a pharmaceutical composition for the prevention and treatment of influenza virus infection disease containing a ginseng extract, fractions thereof, terokapane-based compounds and flavonoid-based compounds or pharmaceutically acceptable salts thereof as an active ingredient It is to provide a composition for disinfecting influenza.

Another object of the present invention is to prevent and improve the health of influenza virus infection disease containing ginseng extract, fractions thereof, terokapane-based compounds and flavonoid-based compounds or pharmaceutically acceptable salts thereof as an active ingredient It is to provide a food composition.

In order to achieve the above object, the present invention is to prevent the influenza virus infection disease containing a ginseng extract, fractions thereof, terokane-based compounds and flavonoid-based compounds or pharmaceutically acceptable salts thereof as an active ingredient and Provided is a therapeutic pharmaceutical composition.

In another aspect, the present invention provides a composition for disinfecting influenza containing a ginseng extract, fractions thereof, terokapane-based compounds and flavonoid-based compounds or pharmaceutically acceptable salts thereof as an active ingredient.

In addition, the present invention provides a health food composition for the prevention or improvement of influenza virus infection diseases containing a ginseng extract, fractions thereof, terokapane-based compounds and flavonoid-based compounds or pharmaceutically acceptable salts thereof as an active ingredient. to provide.

The present invention relates to a pharmaceutical composition for the prevention and treatment of influenza virus infection disease containing as an active ingredient ginseng extract, fractions thereof, terokapane-based compounds and flavonoid-based compounds or pharmaceutically acceptable salts thereof. . Since the composition of the present invention exhibits an excellent inhibitory effect on neuraminidase, it may be usefully used for the prevention and treatment of influenza virus infection or for disinfection.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for the prevention and treatment of influenza virus infection disease containing a ginseng extract as an active ingredient.

The ginseng extract is preferably obtained by extracting from ginseng, the ginseng can be used without limitation, such as those grown or commercially available. The method for preparing the ginseng extract may use a conventional extraction method in the art, such as ultrasonic extraction, filtration and reflux extraction. Preferably, the dried ginseng extract may be an extract extracted with water, C ₁ to C ₃ alcohol or a mixed solvent thereof, more preferably, an extract extracted with C ₁ to C ₃ alcohol, and most preferably methanol Or an extract extracted with ethanol. For example, after cutting the dried dried ginseng roots into an extraction container, a lower alcohol of C ₁ to C ₃ or a mixed solvent thereof, preferably methanol or ethanol, and the mixture is left at room temperature for a certain period of time, followed by filtration to obtain an alcohol extract. have. At this time, the extraction is preferably left for 1 week at room temperature, after which it may be further subjected to methods such as concentration or lyophilization.

In addition, the ginseng extract may be an extract extracted with water, C ₁ ~ C ₄ alcohol or a mixed solvent thereof, more preferably methanol. At this time, the extraction solvent is preferably 2 to 20 times the dry weight of high ginseng. The extraction method can be any extraction method usually performed, and when using water, C ₁ ~ C ₄ alcohol or a mixed solvent thereof as the extraction solvent, it can be extracted by leaving for 2 to 20 days at room temperature. The extract obtained by the above method may be used as a extract of red ginseng through further steps such as filtration, purification and concentration using filter paper.

In another aspect, the present invention provides a pharmaceutical composition for the prevention and treatment of influenza virus infection diseases containing a high ginseng fraction as an active ingredient.

Preferably, the ginseng fraction is suspended by adding water to the ginseng extract, and may be obtained by sequentially fractionating using hexane, chloroform and ethyl acetate.

Furthermore, the present invention provides a pharmaceutical composition for the prevention and treatment of influenza virus infection disease containing a terokapane-based compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

이다.Wherein R ₁ is methoxy, hydroxy or

to be.

The terokapane-based compound of Formula 1 may be isolated from a ginseng extract or a ginseng fraction.

More specifically, the terokapane-based compound represented by Chemical Formula 1 may be a terokappine represented by the following Chemical Formula 3 (Pterocarpin), machiaine represented by the following Chemical Formula 4 (Maackiain), or tripolizine represented by the following Chemical Formula 5 (Trifolrhizin) )to be.

The present invention also provides a pharmaceutical composition for the prevention and treatment of influenza virus infection disease containing a flavonoid compound represented by the formula (2) or a pharmaceutically acceptable salt thereof as an active ingredient.

또는

이고; R₃ 및 R₄는 독립적으로 메톡시 또는 히드록시이고; R₅는 수소 또는 히드록시이다.Where R ₂ is

or

ego; R ₃ and R ₄ are independently methoxy or hydroxy; R ₅ is hydrogen or hydroxy.

Flavonoid-based compound of Formula 2 may be isolated from a ginseng extract or a ginseng fraction.

More specifically, the flavonoid compound represented by the formula (2) is Sophoraflavanone G (Sophoraflavanone G) represented by the formula (6), (-)-kuranonone ((-)-Kuraninone) represented by the formula (7), Lithuanone A represented by the following Chemical Formula 8, Kushenol A represented by the following Chemical Formula 9, Norkururarinol represented by the following Chemical Formula 10, and Kurarinol represented by the following Chemical Formula 11 Kurarinol) or Kushenol T represented by the following formula (12).

The method for separating the terokapane-based compound of Formula 1 and the flavonoid-based compound of Formula 2 according to the present invention from ginseng may be performed as follows.

The present invention,

Extracting ginseng with water, C ₁ -C ₄ alcohol or a mixed solvent thereof to obtain a ginseng extract (step 1);

Suspending by adding water to the ginseng extract obtained in the step 1, and sequentially fractionated using hexane, chloroform and ethyl acetate to obtain a ginseng fraction (step 2); And

Separation and purification of the ginseng fraction obtained in step 2 by silica gel chromatography to obtain a terokapane compound of Formula 1 and a flavonoid compound of Formula 2 (Step 3)

It provides a method for producing a compound represented by Formula 1 and Formula 2, including the separation.

A method for preparing a compound represented by Formula 1 and Formula 2 according to the present invention will be described below step by step.

Step 1 is a step of extracting the ginseng extract with water, C ₁ ~ C ₄ alcohol or a mixed solvent thereof. The ginseng can be used without limitation, such as cultivated or commercially available, it is used to clean and dry. The alcohol may be a lower alcohol such as methanol, ethanol, propanol, butanol, preferably methanol or ethanol, and most preferably methanol.

In step 1 according to the present invention, the step of obtaining the ginseng extract is dried in a dry ginseng and dried in an extraction container, and the appropriate amount of alcohol is added, it is left for 10 days at room temperature and then filtered with a filter paper Can be performed. This extraction process may be repeated several times, and then a method such as concentration or lyophilization may be additionally performed.

Step 2 according to the present invention is a step of obtaining the gosam extract by suspending by adding water to the extract of the ginseng obtained in step 1, sequentially using hexane, chloroform and ethyl acetate. At this time, a conventional fractional extraction method can be used, and preferably a fractionation funnel can be used. The ginseng fraction may be obtained as hexane ginseng fraction, chloroform ginseng fraction, ethyl acetate fraction.

Step 3 is a step of obtaining the terokapane compound of Formula 1 and the flavonoid compound of Formula 2 by separating and purifying the ginseng fraction obtained in step 2 by silica gel chromatography. More specifically, the ginseng fraction obtained in step 2 may be separated and purified using silica gel chromatography to obtain the terokapane compound of Formula 1 and the flavonoid compound of Formula 2. When performing silica gel chromatography for separating the compounds, it is preferable to use n -hexane, n-hexane ethyl acetate, a chloroform-acetone mixed solvent and methanol as the mobile phase, and n -hexane / acetone mixed in the additional chromatography. Solvents may be used. In this case, the volume ratio of the n -hexane / ethyl acetate mixed solvent used is preferably 50: 1 to 1: 5, and preferably 150: 1 to 1: 4 in the case of the chloroform and acetone mixed solvent. The chromatography may be performed once or several times until a single compound is purified, and may be concentrated and recrystallized as necessary.

As a result of measuring a Sophora inhibitory activity against nyura mini Days enzyme of the extract and fractions in accordance with the present invention, Sophora root hot-water extract and Sophora root ethanol extract were 500 μg / mL IC ₅₀ values, and 20.5 μg / mL of the IC ₅₀ of less than Value, and the red ginseng root ethyl acetate fraction showed an IC ₅₀ value of 15.2 μg / mL (see Table 1).

In addition, as a result of measuring the inhibitory activity against the neuraminidase enzyme of the compounds isolated from the ginseng extract or fractions, it showed a high inhibitory effect, confirming the excellent anti-influenza activity of the compounds of the present invention (see Table 2).

Therefore, the ginseng extract of the present invention, the ginseng fraction, the compound isolated therefrom exhibits inhibitory activity against the neuraminidase enzyme, so as to prevent or treat influenza virus-induced diseases caused by influenza virus or as a pharmaceutical composition for disinfection It can be usefully used as a composition for.

The present invention provides a terokapane compound represented by Formula 1 and a flavonoid compound represented by Formula 2, and pharmaceutically acceptable salts thereof, as well as possible solvates, hydrates, or racemates that can be prepared therefrom. It includes everything.

The terokapane-based compound represented by Chemical Formula 1 and the flavonoid-based compound represented by Chemical Formula 2 may be used in the form of a pharmaceutically acceptable salt, and may be used as a salt in a pharmaceutically acceptable free acid. Acid addition salts formed by these are useful. Acid addition salts include those derived from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid, and aliphatic mono- and dicarboxylates, phenyl-substituted alkanoates, hydroxyalkanoates, Dioleate, aromatic acid, aliphatic and aromatic sulfonic acids. Such pharmaceutically nontoxic salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide, and iodide. Id, fluoride, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suverate , Sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitro benzoate, hydroxybenzoate, meth Oxybenzoate, phthalate, terephthalate, benzenesulfonate, toluenesulfonate, chlorobenzenesul Nate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycolate, malate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1- Sulfonates, naphthalene-2-sulfonates or mandelate.

The acid addition salts according to the present invention can be dissolved in conventional methods, for example, by dissolving a derivative of the above formula (3) or (6) in an excess of an aqueous acid solution and dissolving the salt with a water miscible organic solvent such as methanol, ethanol, acetone or aceto. It can be prepared by precipitation using nitrile.

The same amount of terokabane compound represented by the formula (1) and flavonoid compound represented by the formula (2) and acid or alcohol in water, and then the mixture is evaporated to dryness or produced by suction filtration of the precipitated salt It may be.

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

The composition may be used as a pharmaceutical composition, and may be various oral or parenteral formulations. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid form preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, which form at least one excipient such as starch, calcium carbonate, sucrose or lactose (at least one compound). lactose) and gelatin. In addition to simple excipients, lubricants such as magnesium stearate, talc and the like are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups, and include various excipients such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. Can be. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester 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 composition of the present invention may be parenterally administered (for example, intravenously, subcutaneously, intraperitoneally or topically) or orally as desired, and 0.1 to 500 mg / kg body weight per day, preferably 1 It may be administered in portions of 1 to several times to be administered in an amount of ˜100 mg, most preferably 0.5-10 mg. The dosage for a particular patient may vary depending on the patient's weight, age, sex, health condition, diet, time of administration, method of administration, rate of excretion, severity of the disease, and the like.

As a result of toxicity experiments conducted by oral administration of the compound of the present invention, 50% lethal dose (LD ₅₀ ) by oral administration toxicity test is judged to be a safe substance of at least 1,000 mg / kg or more.

In addition, the present invention is a health food for the prevention and disinfection or improvement of influenza virus infection disease containing a ginseng extract, a ginseng fraction, terokapane-based compounds and flavonoid-based compounds or pharmaceutically acceptable salts thereof as an active ingredient To provide a composition.

The influenza virus infection disease may be a disease caused by influenza A, B or C virus. In addition, the disease caused by the influenza virus may be flu, cold, sore throat, bronchitis, or pneumonia, and particularly may be avian influenza which has a worldwide spreading effect.

The composition according to the present invention may be used for the purpose of preventing or improving influenza virus infectious diseases, extracts ginseng extract, ginseng fraction, compounds isolated therefrom, derivative compounds thereof or pharmaceutically acceptable salts thereof in food supplements such as food, beverages, etc. Can be added. In this case, when the ginseng extract, fractions, compounds isolated therefrom, derivative compounds thereof or pharmaceutically acceptable salts thereof as food additives, the amount of 0.01 to 20% by weight, preferably 0.1 to 5% by weight relative to the raw material Can be added. The blending amount of the active ingredient may be appropriately determined depending on the purpose of use (prevention, health or therapeutic treatment). However, in the case of long-term intake for health and hygiene purposes or health control purposes, the amount may be below the above range, and since there is no problem in terms of stability, the active ingredient may be used in an amount above the above range. The extracts, fractions, compounds isolated therefrom or derivatives thereof may be used with other foods or food ingredients and may be suitably used according to conventional methods.

There is no particular limitation on the kind of food. Examples of foods to which the extract, fraction, compound isolated therefrom, derivative compound thereof or pharmaceutically acceptable salt thereof may be added include meat, sausage, bread, chocolate, candy, snacks, confectionary, pizza, ramen, etc. There are dairy products including noodles, gums, ice creams, various soups, beverages, teas, drinks, alcoholic beverages and vitamin complexes, and include all healthy foods in the usual sense.

The food supplement additive of the present invention may use various flavors or natural carbohydrates. The natural carbohydrates are sugars such as glucose, monosaccharides such as fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, xylitol, sorbitol, and erythritol. As the sweetening agent, natural sweetening agents such as tautin and stevia extract, synthetic sweetening agents such as saccharin and aspartame, and the like can be used. The proportion of the natural carbohydrate is generally about 0.01 to 0.04 parts by weight, preferably 0.02 to 0.03 parts by weight, per 100 parts by weight of the composition according to the present invention.

In addition to the above, the composition according to the present invention includes various nutrients, vitamins, electrolytes, flavors, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, Alcohols, carbonating agents used in carbonated drinks, and the like. In addition, the composition according to the present invention may contain a pulp for the production of natural fruit juices and vegetable drinks. These components can be used independently or in combination, and the proportion of the additive is not critical, but is usually selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition according to the present invention.

Hereinafter, the present invention will be described in detail through Examples and Experimental Examples. However, the following examples are merely to illustrate the present invention, but the content of the present invention is not limited by the following examples.

Example 1: Preparation of Ginseng Extract

10 L of 100% ethanol (EtOH) was added to 1.0 kg of dried red ginseng root, which was left at room temperature for 1 week, filtered through a filter paper and concentrated to obtain an ethanol extract (380 g) of red ginseng root. In addition, 3 g of water was added to 200 g of red ginseng, and stirred at 70 ° C. for 6 hours to obtain a hot water extract (90 g).

Example 2: Preparation of Ginseng Fraction

The ethanol extract obtained in Example 1 was dissolved in 1000 mL of ethanol, fractionated with n -hexane, suspended in 1000 mL of water, fractionated with chloroform and ethyl acetate, and then n -hexane fraction ( n -hexane-soluble) 11 g, chloroform 14 g of a fraction (CHCl ₃ -soluble) and 67 g of an ethyl acetate fraction (EtOAc-soluble) were obtained, respectively.

Example 3: Isolation of Terokapane Compound and Flavonoid Compound from Ginseng

The ginseng used in this experiment was purchased 4 Kg of dry and fine products from Samo Pharm, a Chinese herbal medicine dealer near Jinju, Gyeongsangnam-do. The purchased ginseng was added to 10 l of methanol, and extracted repeatedly twice while standing at room temperature for 10 days. The extract was filtered through filter paper, and then concentrated under reduced pressure to obtain a brown oily methanol extract (450 g). The extract was suspended in 2.0 L of a mixed solution of water and methanol (9: 1 volume ratio), and then partitioned into n -hexane and chloroform in sequence, followed by 8 g of n -hexane gourd fraction, 150 g of chloroform ginseng fraction and 230 g. A methanol gourd fraction of was obtained.

Silica gel column chromatography (450 g of silica gel, 230 to 400 mesh) using 100% chloroform and a mixed solvent of chloroform and acetone (150: 1 to 1: 2 by volume) as a mobile phase with respect to 220 g of the chloroform gourd fraction obtained above. mesh)) to separate seven fractions (Fr. 1 to 7). The third fraction (Fr.3, 3 g) was eluted with increasing ratios of acetone in a mixed solvent of hexane and ethyl acetate (40: 1 to 1: 1 by volume) in order to elute the fraction under reduced pressure and concentrated. Mg of the compound of formula 6 was obtained.

In addition, silica gel column chromatography (230 g, 230 to 400 mesh) was carried out again using a mixed solvent of hexane and acetone (80: 1 to 2: 1 by volume) for the third fraction (Fr.3, 3.5 g) of the chloroform gourd fraction. ) To obtain 50 fractions (Fr. 3-1 ~ 50). 31 to 38 of these fractions (Fr.3-31 38) was concentrated under reduced pressure to obtain 250 mg of the compound of formula (9).

In addition, silica gel column chromatography (50 g, 230 to ˜3) was used for the third fraction (Fr. 3, 3.5 g) of the chloroform gourd fraction, using a mixed solvent of hexane and ethyl acetate (60: 1 to 1: 4 by volume) as a mobile phase. 400 mesh) to 35 fractions (Fr.3-1 ~ 35) was obtained. Double 11 to 16 fractions (Fr. 3-11 ~ 16) was concentrated under reduced pressure to separate 90 mg of the compound of formula (3).

In addition, silica gel column chromatography (50 g, 230 to ˜3) was used for the third fraction (Fr. 3, 3.5 g) of the chloroform gourd fraction, using a mixed solvent of hexane and ethyl acetate (60: 1 to 1: 4 by volume) as a mobile phase. 400 mesh) to 35 fractions (Fr.3-1 ~ 35) was obtained. Duplicate 23 to 26 fraction (Fr.3-23 ~ 26) under reduced pressure and concentrated to isolate 120 mg of the compound of formula (4).

In addition, a mixed solvent of hexane and ethyl acetate (60: 1 to 1: 1 by volume) was eluted with a sequential increase in the ethyl acetate ratio with respect to the fourth fraction (Fr.4, 1.8 g) of the chloroform gosam fraction 38 Fractions (Fr. 4-1 to 38) were obtained. 14-19 fractions (Fr.4-14-19) were concentrated under reduced pressure and 460 mg of the compound of formula 8 was obtained.

In addition, silica gel column chromatography (180 g, 230 to 400) was performed again with the mixed solvent of hexane and acetone (30: 1 to 1: 1 volume ratio) for the fifth fraction (Fr. 5, 5 g) of the chloroform gourd fraction. Mesh) to 40 fractions (Fr.5-1 ~ 40) was obtained. Of these 22 to 26 fractions (Fr.6-22 26) was concentrated under reduced pressure to obtain 530 mg of the compound of formula 7.

In addition, silica gel was added to the fifth fraction (Fr. 5, 5 g) of the chloroform Ginseng fraction with a solvent mixture of hexane and ethyl acetate (40: 1 to 1: 1 by volume) as the mobile phase, increasing the proportion of ethyl acetate sequentially. Column chromatography (50 g, 230-400 mesh) was carried out to 15 fractions (Fr.5-1 To 15). Double 7 to 10 fractions (Fr.5-7 10) was concentrated under reduced pressure to separate 120 mg of the compound of Formula 12.

In addition, silica gel column chromatography (80 g, 230 to 80 g) was used for the eighth fraction (Fr. 8, 1.6 g) of the methanol gourd fraction, using a mixed solvent of hexane and acetone (20: 1 to 1: 2 by volume) as a mobile phase. 30 meshes (Fr.8-1) To 30). Duplicate 21 to 23 fraction (Fr.8-21 23) was concentrated under reduced pressure to separate 340 mg of the compound of Formula 10.

In addition, silica gel column chromatography (150 g, 230-400) was carried out again with the mixed solvent of chloroform and acetone (80: 1 to 4: 1 by volume) for the ninth fraction (Fr. 9, 500 mg) of methanol gourd fraction. 20 fractions (Fr.9-1) To 20). Double 16-18 fraction (Fr.9-16 18) was concentrated under reduced pressure, and 70 mg of the compound of Formula 11 was isolated.

In addition, silica gel column chromatography (150 g, 230-400) was carried out again with the mixed solvent of chloroform and acetone (60: 1 to 1: 1 by volume) for the ninth fraction (Fr.9, 500 mg) of methanol gourd fraction. Mesh) to obtain 30 fractions (Fr. 9-1 to 30). Double 22-27 fractions (Fr.9-22 ~ 27) was concentrated under reduced pressure, and 170 mg of compound of Chemical Formula 5 was isolated.

Example 4 Structural Analysis of Flavonoid Compounds

The molecular weight and molecular formula of the compound obtained in Example 3 were determined using a VG high resolution GC / MS spectrometer, Election Ionization MS, Autospec-Ultima. In addition, ¹ H NMR, ¹³ C NMR, Homo-Cozy, HMQC (1H-Detected heteronuclear Multiple-Quantum Coherence), HMBC (Heteronuclear Multiple-) through nuclear magnetic resonance (NMR) analysis (Bruker AM 500) Bond Coherence) and DEPT (Distortionless Enhancement by Polarization) spectra were obtained to determine the molecular structure.

The measurement results are shown below.

As a result of comparing and analyzing the measurement results with those of the following publications, the compounds represented by the above Chemical Formulas 3 to 12 were 7-methoxy-4 ′, 5′-methylenedioxy-terokapan (7-Methoxy-4 ′, 5'-methylenedioxy-pterocarpan), 4 ', 5'-methylenedioxy-terokapan (4', 5'-Methylenedioxy-pterocarpan), macchiin-3- O- bD-glucopyranoside (Maackiain-3- O -bD-glucopyranoside), 5,7,2 ', 4'-tetrahydroxy-8-rapandulylflavanone (5,7,2', 4'-Tetrahydroxy-8-lavandulylflavanone), 5-methoxy- 7,2 ', 4'-trihydroxy-8-ravandulylflavanone (5-Methoxy-7,2', 4'-trihydroxy-8-lavandulylflavanone), 2'-methoxy-5,7,4´ 2'-Methoxy-5,7,4'-trihydroxy-8-lavandulylflavanone, 5,7,2'-trihydroxy-8-lavandulylflavanone (5'-Methoxy-5,7,4'-trihydroxy-8-lavandulylflavanone) , 7,2'-Trihydroxy-8-lavandulylflavanone), 5,7,2 ', 4'-trihydroxy-8- (5-hydroxy-2-isoprenyl-5-methyl-hexyl) -flavanone (5,7,2´, 4´-Trihydroxy-8- (5 -hydroxy-2-isoprenyl-5-methyl-hexyl) -flavanone), 5-methoxy-7,2 ', 4'-trihydroxy-8- (5-hydroxy-2-isoprenyl-5- Methyl-hexyl) -flavanone (5-Methoxy-7,2´, 4´-trihydroxy-8- (5-hydroxy-2-isoprenyl-5-methyl-hexyl) -flavanone), 5,7,2´- Trihydroxy-8- (5-hydroxy-2-isoprenyl-5-methyl-hexyl) -flavanone (5,7,2′-Trihydroxy-8- (5-hydroxy-2-isoprenyl-5- methyl-hexyl) -flavanone) (Park, KH et al., Biol. Pharm. Bull. 2006 ; Kim, YC et al., J. Nat. Prod. 2000 ; Hayashi, T. et al., J. Nat. Prod. 1999 ; Kawahara, H. et al., Bioorg. Med. Chem. Lett. 2007 ; Komiya, T. et al., Oncol. Rep. 2004 ).

Terokapane compound

Terokafine (7-methoxy-4 ', 5'-methylenedioxy-terokapan)

[Formula 3]

1) Physical property: Coloress needles (m.p. 112 ~ 115 ℃)

2) Molecular Weight: 289

3) Molecular formula: C ₁₇ H ₁₄ O ₅

4) ¹ H NMR (500 MHz, CDCl ₃ ) δ d 4.15 (1H, dd, J = 11.1, 5.0 Hz, H-2a), 3.58 (1H, dd, J = 11.1, 5.9 Hz, H-2b), 3.40 (1H, m, H-3), 5.40 (1H, d, J = 6.9 Hz, H-4), 7.31 (1H, d, J = 8.5 Hz, H-5), 6.55 (1H, dd, J = 8.5, 2.5 Hz, H-6), 6.40 (1H, s, H-8), 6.64 (1H, s, H-2 '), 6.39 (1H, d, J = 2.5 Hz, H-5') , 5.73 (2H, doublet of doublets, J = 13.8, 1.4 Hz, H-1 "), 3.71 (OCH ₃ ); ¹³ C NMR (125 MHz, CDCl ₃ ) δ 66.9 (C-2), 40.7 (C-3) , 78.9 (C-4), 132.1 (C-5), 110.0 (C-6), 157.0 (C-7), 102.1 (C-8), 112.8 (C-4a), 161.5 (C-8a), 118.4 (C-1 '), 105.1 (C-2'), 142.1 (C-3 '), 148.5 (C-4'), 94.2 (C-5 '), 154.7 (C-6'), 101.7 ( C-1 ″), 55.8 (OCH ₃ ).

Macchiin (4 ', 5'-methylenedioxy-terokapan)

[Formula 4]

1) Physical property: Orange needle type (m.p. 177 ℃)

2) Molecular Weight: 284

3) Molecular formula: C ₁₆ H ₁₂ O ₅

4) ¹ H NMR (500 MHz, CD ₃ OD) δ 4.18 (1H, dd, J = 10.9, 4.8 Hz, H-2a), 3.58 (1H, t (dd), J = 10.6 Hz, H-2b) , 3.44 (1H, m, H-3), 5.41 (1H, d, J = 7.0 Hz, H-4), 7.24 (1H, d, J = 8.4 Hz, H-5), 6.48 (1H, dd, J = 8.4, 2.4 Hz, H-6), 6.30 (1H, d, J = 2.4 Hz, H-8), 6.77 (1H, s, H-2 '), 6.36 (1H, s, H-5' ), 5.84 (2H, doublet of doublets, J = 13.0, 1.2 Hz, H-1 "); ¹³ C NMR (125 MHz, CD ₃ OD) δ 67.9 (C-2), 42.0 (C-3), 80.5 (C -4), 133.5 (C-5), 111.1 (C-6), 158.5 (C-7), 104.5 (C-8), 113.4 (C-4a), 160.5 (C-8a), 120.2 (C- 1 '), 106.3 (C-2'), 143.5 (C-3 '), 149.9 (C-4'), 94.6 (C-5 '), 156.0 (C-6'), 102.9 (C-1 " )

Tripolazine (Macchiine-3- O -β-D-glucopyranoside)

[Chemical Formula 5]

1) Physical property: white powder (m.p. 145 ~ 146 ℃)

2) Molecular Weight: 446

3) Molecular formula: C ₂₂ H ₂₂ O ₁₀

4) ¹ H NMR (500 MHz, DMSO- d ₆ ) δ 4.26 (1H, m, H-2a), 3.60 (1H, m, H-2b), 3.65 (1H, m, H-3), 5.56 ( 1H, d, J = 7.4 Hz, H-4), 7.36 (1H, s, H-5), 6.70 (1H, dd, J = 8.5, 2.4 Hz, H-6), 6.55 (1H, d, J = 2.4 Hz, H-8), 6.98 (1H, s, H-2 '), 6.52 (1H, s, H-5'), 5.92 (2H, d, J = 18.0 Hz, H-1 ") (1H, d, J = 7.5 Hz, G-1), 3.20 (1H, m, G-2), 3.25 (1H, m, G-3), 3.15 (1H, m, G-4), 3.29 ( 1H, m, G-5) , 3.48 (1H, m, G-6a), 3.69 (1H, m, G-6b) 13 C NMR (125 MHz, DMSO- d 6) δ 66.2 (C-2), 40.2 (C-3), 78.2 (C-4), 132.2 (C-5), 110.8 (C-6), 158.8 (C-7), 104.4 (C-8), 114.6 (C-4a), 156.6 (C-8a), 118.6 (C-1 '), 105.7 (C-2'), 141.5 (C-3 '), 147.9 (C-4'), 93.6 (C-5 '), 154.0 (C- 6 '), 101.4 (C-1 "), 100.7 (G-1), 73.6 (G-2), 76.9 (G-3), 70.1 (G-4), 77.4 (G-5), 61.0 (G -6).

Flavonoid Compounds

Sophoraflavanone G (5,7,2 ', 4'-tetrahydroxy-8-lavandulylflavanone)

[Formula 6]

1) Physical property: white powder (amorphous) (m.p. 178 ~ 180 ℃)

2) Molecular Weight: 424

3) Molecular formula: C ₂₅ H ₂₃ O ₆

4) ¹ H NMR (500 MHz, CD ₃ OD) δ 5.56 (1H, dd, J = 13.2, 2.7 Hz, H-2), 2.73 (1H, dd, J = 17.1, 2.8 Hz, H-3a), 2.97 (1H, dd, J = 17.1, 13.2 Hz, H-3b), 5.92 (1H, s, H-6), 6.37 (1H, d, J = 2.3 Hz, H-3 '), 6.35 (1H, m, H-5 '), 7.30 (1H, d, J = 8.2 Hz, H-6'), 2.85 (2H, m, H-1 "), 2.47 (1H, m, H-2"), 2.0 (2H, m, H-3 "), 4.90 (1H, m, H-4"), 1.48 (3H, s, H-6 "), 1.63 (3H, s, H-7"), 1.56 (3H , s, H-9 "), 4.55 (2H, br.s, H-10"); ¹³ C NMR (125 MHz, CD ₃ OD) δ 76.3 (d, C-2), 43.7 (t, C-3), 199.4 (s, C-4), 103.7 (s, C-4a), 163.6 ( s, C-5), 96.7 (d, C-6), 166.9 (s, C-7), 109.1 (s, C-8), 163.0 (s, C-8a), 118.8 (s, C-1 '), 157.1 (s, C-2'), 108.1 (d, C-3 '), 159.9 (s, C-4'), 103.8 (d, C-5 '), 129.1 (d, C-6 '), 28.4 (t, C-1 "), 48.7 (d, C-2"), 32.8 (t, C-3 "), 125.2 (d, C-4"), 132.4 (s, C-5 "), 18.3 (q, C-6"), 26.3 (q, C-7 "), 150.1 (s, C-8"), 19.6 (q, C-9 "), 111.6 (t, C-10 ").

(-)-Kuraninone (5-methoxy-7,2 ', 4'-trihydroxy-8-lavandulylflavanone)

[Formula 7]

1) Physical property: yellow powder (amorphous) (m.p. 117 ~ 121 ℃)

2) Molecular Weight: 438

3) Molecular formula: C ₂₆ H ₃₀ O ₆

4) ¹ H NMR (500 MHz, CD ₃ OD) δ 5.54 (1H, dd, J = 13.2, 2.8 Hz, H-2), 2.70 (1H, dd, J = 16.7, 2.8 Hz, H-3a), 2.87 (1H, dd, J = 16.7, 13.2 Hz, H-3b), 6.10 (1H, s, H-6), 6.36 (1H, d, J = 2.3 Hz, H-3 '), 6.34 (1H, m, H-5 '), 7.30 (1H, d, J = 8.2 Hz, H-6'), 2.62 (2H, m, H-1 "), 2.49 (1H, m, H-2"), 2.0 (2H, m, H-3 "), 4.96 (1H, m, H-4"), 1.45 (3H, s, H-6 "), 1.56 (3H, s, H-7"), 1.63 (3H , s, H-9 "), 4.57 (2H, s, H-10"), 3.80 (OCH ₃ ); ¹³ C NMR (125 MHz, CD ₃ OD) δ 75.9 (d, C-2), 46.0 (t, C-3), 194.3 (s, C-4), 106.2 (s, C-4a), 162.3 ( s, C-5), 93.7 (d, C-6), 165.3 (s, C-7), 110.0 (s, C-8), 165.1 (s, C-8a), 118.9 (s, C-1 '), 157.7 (s, C-2'), 108.1 (d, C-3 '), 159.9 (s, C-4'), 103.8 (d, C-5 '), 128.9 (d, C-6 '), 28.6 (t, C-1 "), 48.6 (d, C-2"), 32.8 (t, C-3 "), 125.2 (d, C-4"), 132.4 (s, C-5 "), 18.2 (q, C-6"), 26.3 (q, C-7 "), 150.2 (s, C-8"), 19.6 (q, C-9 "), 111.6 (t, C-10 "), 56.3 (OCH ₃ ).

Lich'anone A (2'-methoxy-5,7,4'-trihydroxy-8-lavandulylflavanone)

[Formula 8]

1) Physical property: Yellow powder (m.p. 155 ~ 156 ℃)

2) Molecular Weight: 438

3) Molecular formula: C ₂₆ H ₃₀ O ₆

4) ¹ H NMR (500 MHz, Acetone-d ₆ ) δ 5.51 (1H, dd, J = 13.2, 2.7 Hz, H-2), 2.93 (1H, dd, J = 13.3, 1.1 Hz, H-3a) , 2.90 (1H, dd, J = 13.3, 1.1 Hz, H-3b), 5.88 (1H, s, H-6), 6.42 (1H, d, J = 2.7 Hz, H-3 '), 6.39 (1H , m, H-5 '), 7.30 (1H, d, J = 8.4 Hz, H-6'), 2.58 (2H, m, H-1 "), 2.49 (1H, m, H-2"), 1.90 (2H, m, H-3 "), 4.85 (1H, m, H-4"), 1.35 (3H, s, H-6 "), 1.42 (3H, s, H-7"), 1.43 ( 3H, s, H-9 "), 4.45 (2H, s, H-10"), 3.69 (OCH ₃ ); ¹³ C NMR (125 MHz, Acetone-d ₆ ) δ 75.4 (d, C-2), 43.2 (t, C-3), 198.5 (s, C-4), 100.2 (s, C-4a), 163.4 (s, C-5), 93.7 (d, C-6), 165.6 (s, C-7), 103.6 (s, C-8), 165.6 (s, C-8a), 119.6 (s, C- 1 '), 159.0 (s, C-2'), 96.9 (d, C-3 '), 160.2 (s, C-4'), 103.8 (d, C-5 '), 129.1 (d, C- 6 '), 28.1 (t, C-1 "), 48.1 (d, C-2"), 32.3 (t, C-3 "), 124.8 (d, C-4"), 132.0 (s, C- 5 "), 18.2 (q, C-6"), 23.7 (q, C-7 "), 149.5 (s, C-8"), 19.5 (q, C-9 "), 111.5 (t, C- 10 "), 56.2 (OCH ₃ ).

Cushenol A (5,7,2′-trihydroxy-8-rabandulylflavanone)

[Formula 9]

1) Physical property: white powder (amorphous) (m.p. 175 ~ 178 ℃)

2) Molecular Weight: 408

3) Molecular formula: C ₂₅₆ H ₂₃ O ₅

4) ¹ H NMR (500 MHz, CD ₃ OD) δ 5.63 (1H, dd, J = 7.6, 1.4 Hz, H-2), 2.87 (2H, m, H-3), 5.93 (1H, s, H -6), 6.83 (1H, dd, J = 8.12, 0.9 Hz, H-3 '), 7.17 (1H, ddd, J = 15.0, 7.6, 0.9 Hz, H-4'), 6.90 (1H, ddd, J = 15.0, 7.6, 0.9 Hz, H-5 '), 7.54 (1H, dd, J = 7.6, 1.4 Hz, H-6'), 2.61 (2H, m, H-1 "), 2.48 (1H, m, H-2 "), 2.0 (2H, m, H-3"), 4.96 (1H, m, H-4 "), 1.46 (3H, s, H-6"), 1.55 (3H, s, H-7 ″), 1.64 (3H, s, H-9 ″), 4.59 (2H, m, H-10 ″); ¹³ C NMR (125 MHz, CD ₃ OD) δ 76.3 (d, C-2), 43.6 (t, C-3), 199.0 (s, C-4), 103.7 (s, C-4a), 163.7 ( s, C-5), 96.9 (d, C-6), 167.0 (s, C-7), 109.1 (s, C-8), 163.8 (s, C-8a), 127.6 (s, C-1 '), 155.6 (s, C-2'), 116.7 (d, 3 '), 130.6 (d, C-4'), 121.0 (d, C-5 '), 127.6 (d, C-6') , 28.4 (t, C-1 "), 48.7 (d, C-2"), 32.7 (t, C-3 "), 125.1 (d, C-4"), 132.5 (s, C-5 ") , 18.2 (q, C-6 "), 26.2 (q, C-7"), 150.1 (s, C-8 "), 19.7 (q, C-9"), 111.6 (t, C-10 ") .

Norcurinol (5,7,2 ', 4'-trihydroxy-8- (5-hydroxy-2-isoprenyl-5-methyl-hexyl) -flavanone)

[Formula 10]

1) Physical property: yellow powder (amorphous) (m.p. 102 ~ 104 ℃)

2) Molecular Weight: 442

3) Molecular formula: C ₂₅ H ₃₀ O ₇

4) ¹ H NMR (500 MHz, Acetone - d ₆ ) δ 5.68 (1H, dd, J = 13.2, 2.79 Hz, H-2), 3.13-3.08 (2H, m, H-3), 6.02 (1H, s, H-6), 6.49 (1H, m, H-3 '), 6.46 (1H, m, 3.7 Hz, H-5'), 7.39 (1H, d, J = 8.3 Hz, H-6 ') , 2.77 (2H, dd, J = 17.1, 2.88 Hz, H-1 "), 2.46 (1H, m, H-2"), 1.39 (2H, m, H-3 "), 1.26 (2H, m, H-4 "), 1.11 (3H, s, H-6"), 1.10 (3H, s, H-7 "), 1.66 (3H, s, H-9"), 4.61 (2H, m, H- 10 ") ¹³ C NMR (125 MHz, Acetone - d ₆ ) δ 75.8 (d, C-2), 43.0 (t, C-3), 198.6 (s, C-4), 118.3 (s, C-4a, ), 163.4 (s, C-5), 96.7 (d, C-6), 165.8 (s, C-7), 108.3 (s, C-8, s), 162.5 (s, C-8a), 108.4 (s, C-1 '), 156.7 (s, C-2'), 104.0 (d, C3 '), 159.8 (s, C-4'), 103.7 (d, C-5 '), 129.1 (d , C-6 '), 43.0 (t, C-1 "), 48.6 (d, C-2"), 28.6 (t, C-3 "), 28.1 (t, C-4"), 70.9 (s , C-5 "), 30.1 (q, C-6"), 29.9 (q, C-7 "), 149.6 (s, C-8"), 19.1 (q, C-9 "), 111.8 (t , C-10 ").

Curarinol (5-methoxy-7,2 ', 4'-trihydroxy-8- (5-hydroxy-2-isoprenyl-5-methyl-hexyl) -flavanone)

[Formula 11]

1) Physical property: Yellow powder (m.p. 166 ~ 169 ℃)

2) Molecular Weight: 456

3) Molecular formula: C ₂₆ H ₃₂ O ₇

4) ¹ H NMR (500 MHz, CD ₃ OD) δ 5.55 (1H, dd, J = 13.2, 2.7 Hz, H-2), 2.70-2.88 (2H, m, H-3), 6.13 (1H, s , H-6), 6.37 (1H, d, J = 2.3 Hz, H-3 '), 6.35 (1H, dd, J = 2.4, 3.7 Hz, H-5'), 7.32 (1H, d, J = 8.2 Hz, H-6 '), 2.66 (2H, dd, J = 2.0, 7.5 Hz, H-1 "), 2.42 (1H, m, H-2"), 1.26-1.40 (2H, m, H- 3 "), 1.25-1.40 (2H, m, H-4"), 1.02 (3H, s, H-6 "), 1.09 (3H, s, H-7"), 1.66 (3H, s, H- 9 "), 4.60 (2H, m, H-10"), 3.82 (OCH ₃ ) ¹³ C NMR (125 MHz, CD ₃ OD) δ 75.9 (d, C-2), 45.9 (t, C-3), 194.3 (s, C-4), 106.2 (s, C-4a,), 162.3 (s, C-5), 93.8 (d, C-6), 165.3 (s, C-7), 109.9 (s, C-8, s), 165.1 (s, C-8a), 118.8 (s, C-1 '), 157.1 (s, C-2'), 128.9.1 (d, C3 '), 159.9 (s, C-4 '), 103.8 (d, C-5'), 108.1 (d, C-6 '), 29.1 (t, C-1 "), 49.3 (d, C-2"), 28.5 (t, C-3 "), 43.2 (t, C-4"), 71.9 (s, C-5 "), 29.3 (q, C-6"), 29.6 (q, C-7 "), 150.0 (s, C-8 ″), 15.9 (q, C-9 ″), 112.0 (t, C-10 ″), 56.4 (OCH ₃ ).

Cushenol T (5,7,2′-trihydroxy-8- (5-hydroxy-2-isoprenyl-5-methyl-hexyl) -flavanone)

[Formula 12]

1) Physical property: yellow powder (amorphous)

2) Molecular Weight: 426

3) Molecular formula: C ₂₅ H ₃₀ O ₆

4) ¹ H NMR (500 MHz, CDCl ₃ ) δ 5.61 (1H, dd, J = 13.3, 2.7 Hz, H-2), 2.83 (1H, dd, J = 17.2, 2.8 Hz, H-3a), 2.97 (1H, dd, J = 17.2, 13.4 Hz, H-3b), 5.94 (1H, s, H-6), 6.80 (1H, d, J = 8.0 Hz, H-3 '), 7.18 (1H, m , H-4 '), 6.92 (1H, m, H-5'), 7.36 (1H, dd, J = 7.6, 1.3 Hz, H-6 '), 2.53 (2H, m, H-1 "), 2.15 (2H, m, H-2 "), 1.27 (2H, m, H-3"), 1.30 (2H, m, H-4 "), 1.03 (3H, s, H-6"), 1.09 ( 3H, s, H-7 "), 1.55 (3H, s, H-9"), 4.62 (2H, m, H-10 "); ¹³ C NMR (125 MHz, CDCl ₃ ) δ 76.1 (d, C-2), 42.1 (t, C-3), 197.6 (s, C-4), 103.3 (s, C-4a), 160.9 (s , C-5), 96.9 (d, C-6), 162.2 (s, C-7), 103.2 (s, C-8), 164.5 (s, C-8a), 125.6 (s, C-1 ' ), 153.6 (s, C-2 '), 117.1 (d, C-3'), 130.1 (d, C-4 '), 121.2 (d, C-5'), 127.2 (d, C-6 ' ), 27.9 (t, C-1 "), 47.7 (d, C-2"), 26.8 (t, C-2 "), 41.2 (t, C-3"), 72.8 (s, C-5 " ), 28.8 (q, C-6 "), 29.9 (q, C-7"), 148.8 (s, C-8 "), 111.5 (q, C-9"), 108.2 (t, C-10 " ).

Experimental Example 1 Determination of Neuraminidase Inhibitory Activity of Ginseng Extract and Fraction

In order to measure influenza inhibitory activity against the influenza virus neuraminidase of the ginseng extract of Example 1 and fractions thereof of the present invention, a method of Markus et al. ( Anal. Biochem. 250, 176, 1997) was used with some modification. As an enzyme source, neuraminidase (enzyme concentration, 0.1 ～ 0.11 U, from Clistridium Perfringgens, Sigma), and the substrate were synthesized using 2 ′-(4-trifluoromethyl) -α-D- N- acetyl-neuraminic acid.

The extract of Example 1 and its fractions were dissolved in methanol and diluted to 20 μg / mL. 10 μL of the diluted extract and fractions were added, and the substrate was placed in 10 μL of 2 ′-(4-trifluoromethyl) -α-DN-acetylneuraminic acid (final concentration, 0.2 mM) and 50 mM sodium acetate solution (pH = 5.0) was mixed with 70 μL, and 10 μL of the enzyme source neuraminidase (enzyme final concentration, 0.1˜0.11 U) was added and reacted at 37 ° C. for 15 minutes, followed by addition of 200 μL of 0.1 M glycine-sodium hydroxide solution. Stopped. The inhibitory activity of neuraminidase was measured by measuring absorption at 357 nm and emission at 499 nm with a fluorescence spectrometer.

The measurement results are shown in Table 1 below.

Inhibitory Activity of the Extracts and Fractions of the Invention on Neuraminidase Enzymes Extract, extract fraction Inhibitory activity ¹⁾ Ginseng Root Hydrothermal Extract > 500 μg / mL Ginseng Root Ethanol Extract 20.5 μg / mL Ginseng Root Ethyl Acetate Fraction 15.2 μg / mL NOTE 1 The results are shown as the average of two experiments.

As shown in Table 1, the ethanol extract and ethyl acetate fraction showed the best inhibitory effect.

Experimental Example 2 Determination of Neuraminidase Inhibitory Activity of Terokapane and Flavonoid Compounds Isolated from Ginseng Extract

Influenza inhibitory activity against neuraminidase was measured in the same manner as in Experimental Example 1 for 10 compounds isolated and identified from the extract of Ginseng of Example 1. The measurement results are shown in Table 2 below.

compound rescue IC ₅₀ One

1.4 ± 0.36 2

3.2 ± 1.1 3

237 ± 25.1 4

13.5 ± 1.08 5

15.1 ± 2.0 6

20.1 ± 0.8 7

14.8 ± 1.5 8

18.3 ± 1.2 9

17.0 ± 1.6 10

12.1 ± 2.4

As shown in Table 2, the ginseng extract of the present invention or terokapane-based compounds and flavonoid-based compounds isolated therefrom were found to have excellent inhibitory activity against neuraminidase enzyme.

Therefore, since the compounds of the present invention exhibit excellent inhibitory effects on neuraminidase, it is determined that the composition comprising the same may be useful for the prevention and treatment of influenza virus infection diseases or for influenza disinfection.

Experimental Example 3: Acute Toxicity Test of Compounds of the Invention

In order to determine the acute toxicity for the compounds of the present invention, the following experiment was performed.

SPF (specific pathogens free) 6-week-old C57BL / 6J mice were divided into four groups (three males and three males / experimental group) of 12 males and females, temperature 22 ± 3 ℃, humidity 55 ± 10%, illumination 12L / The animals were bred in 12D animal rooms. Mice were allowed to acclimate for about a week before being used in the experiment. Feed for experimental animals (for mice and rats, CheilJedang Co., Seoul, South Korea) and drinking water were sterilized and supplied freely.

The compounds of the present invention prepared in Examples 3 and 4 were prepared at a concentration of 50 mg / mL in 0.5% tween 80, and then 0.04 mL (100 mg / kg), 0.2 mL (500) per 20 g of mouse body weight. mg / kg) and 0.4 mL (1,000 mg / kg) orally. Samples were administered orally once and observed for side effects or lethality for 7 days after administration. That is, on the day of dosing, changes in general symptoms and the presence or absence of dead animals were observed at least once in the morning, at least once every afternoon from 1 hour, 4 hours, 8 hours, 12 hours, and the next day after administration.

As a result of the acute toxicity test as described above, all mice treated with the sample did not show any significant clinical symptoms, and there were no dead mice. Also, no change in toxicity was observed in weight change, blood test, blood biochemical test, autopsy findings, etc. Did.

Therefore, the compounds of the present invention did not show a change in toxicity in all mice up to 1,000 mg / kg, was determined to be a safe substance oral minimum dose (LD ₅₀ ) of 1,000 mg / kg or more.

Hereinafter, an example of a pharmaceutical preparation or health food containing a ginseng extract, a ginseng fraction, a terokane-based compound and a flavonoid compound or a pharmaceutically acceptable salt thereof separated therefrom will be described.

Formulation Example 1 Preparation of Pharmaceutical Formulation

1-1. Manufacture of powder

Red ginseng extract, fraction, terokapane compound and flavonoid compound or salt thereof 2 g

1 g lactose

After mixing the above components, the airtight cloth was filled to prepare a powder.

1-2. Manufacture of tablets

100 mg of red ginseng extract, fraction, terokapane compound and flavonoid compound or salt thereof

100 mg corn starch

Lactose 100 mg

2 mg magnesium stearate

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

1-3. Preparation of Capsules

100 mg of red ginseng extract, fraction, terokapane compound and flavonoid compound or salt thereof

100 mg corn starch

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components, the capsule was prepared by filling in gelatin capsules according to the conventional method for producing a capsule.

1-4. Preparation of Injection

Red ginseng extract, fraction, tercapaphan compound and flavonoid compound or salt thereof 10 ug / ml

Dilute hydrochloric acid BP until pH 3.5

Injectable sodium chloride BP up to 1 ml

The ginseng extract, fractions, terokapane-based compounds and flavonoid compounds or salts or salts thereof are dissolved in an appropriate volume of sodium chloride BP for injection, and the pH of the resulting solution is adjusted to pH 3.5 with dilute hydrochloric acid BP, for injection. The volume was adjusted with sodium chloride BP and mixed well. The solution was filled into a 5 ml Type I ampoule made of clear glass, encapsulated under an upper grid of air by dissolving the glass, and sterilized by autoclaving at 120 ° C. for at least 15 minutes to prepare an injection solution.

Preparation Example 2 Preparation of Health Food

2-1. Preparation of Cooking Seasonings

Red ginseng extract, fractions, terokapane-based compounds and flavonoid-based compounds or salts thereof was prepared in 10 to 10% by weight for health promotion cooking seasoning.

2-2. Preparation of Tomato Ketchup and Sauce

Health extract tomato ketchup or sauce was prepared by adding red ginseng extract, fraction, terokapane-based compound and flavonoid-based compound or 0.2 to 1.0 wt% of salt thereof to tomato ketchup or sauce.

2-3. Manufacture of Flour Food

0.1 to 5.0% by weight of red ginseng extract, fractions, terokapane compound and flavonoid compound or salts thereof are added to the flour, and bread, cake, cookies, crackers and noodles are prepared using the mixture to prepare foods for health promotion. It was.

2-4. Preparation of soups and gravy

Red ginseng extract, fractions, terokapane-based compound and flavonoid-based compounds or salts thereof were added to the soup and broth to prepare meat products for health promotion, soup and gravy of noodles.

2-5. Preparation of Ground Beef

Ginseng extract, fraction, terokapane-based compound and flavonoid-based compound or salt 10% by weight of the ground beef was added to prepare a ground beef for health promotion.

2-6. Manufacture of dairy products

0.1 to 1.0% by weight of red ginseng extract, fractions, terokapane-based compounds and flavonoid compounds or salts thereof were added to milk, and various dairy products such as butter and ice cream were prepared using the milk.

2-7. Manufacture of wire

Brown rice, barley, glutinous rice, and yulmu were alphad by a known method, and then dried and roasted to prepare a powder having a particle size of 60 mesh.

Black beans, black sesame seeds, and perilla were also steamed and dried by a known method, and then ground to a powder having a particle size of 60 mesh.

The red ginseng extract, fractions, terokapane and flavonoid compounds or salts thereof were concentrated under reduced pressure in a vacuum concentrator, dried by spraying and drying with a hot air dryer, and then pulverized with a particle size of 60 mesh to obtain a dry powder.

Cereals, seeds, and ginseng extracts, fractions, terokapane-based compounds and dried powders of the flavonoid-based compounds or salts thereof prepared above were prepared in the following ratio.

Cereals (30% by weight brown rice, 15% by weight radish, 20% by weight barley),

Seeds (7% by weight perilla, 8% by weight black beans, 7% by weight black sesame),

Dry powder of red ginseng extract, fraction, terokapane compound and flavonoid compound or salt thereof (1% by weight),

Ganoderma lucidum (0.5% by weight),

Foxglove (0.5 wt%)

2-8. Preparation of Carbonated Drinks

5-10% of sugar, 0.05-0.3% citric acid, 0.005-0.02% caramel, 0.1-1% of vitamin C are mixed, and 79-94% purified water is mixed to make syrup, and the syrup is 85-98 Sterilize for 20 ~ 180 seconds at ℃, mix with cooling water at a ratio of 1: 4, and inject 0.5 ~ 0.82% of carbon dioxide into carbonated beverages containing extracts of red ginseng extract, fractions, terocapan compounds and flavonoid compounds or salts thereof. Was prepared.

2-9. Manufacture of health drinks

Substances such as liquid fructose (0.5%), oligosaccharides (2%), sugars (2%), salts (0.5%), water (75%), and ginseng extracts, fractions, terokapane compounds and flavonoid compounds or salts thereof After homogeneous mixing and sterilization, and then packaged in a small packaging container such as glass bottles, plastic bottles to prepare a healthy drink.

2-10. Preparation of Vegetable Juice

Health extract vegetable juice was prepared by adding 0.5 g of red ginseng extract, fraction, terokapane compound and flavonoid compound or salt thereof to 1,000 ml of tomato or carrot juice.

2-11. Preparation of Fruit Juice

Health extract was prepared by adding 0.1 g of red ginseng extract, fraction, terokapane compound and flavonoid compound or salt thereof to 1,000 ml of apple or grape juice.

Claims (20)

Pharmaceutical composition for the prevention and treatment of influenza virus infection disease containing a ginseng extract as an active ingredient. The method of claim 1, wherein the ginseng extract is a pharmaceutical composition for the prevention and treatment of influenza virus infection disease, characterized in that the extract extracted with ginseng, water, C ₁ ~ C ₄ alcohol or a mixed solvent thereof. The pharmaceutical composition for preventing and treating influenza virus infection disease according to claim 2, wherein the alcohol is methanol or ethanol. According to claim 2, wherein the alcohol is a pharmaceutical composition for the prevention and treatment of influenza virus infection disease, characterized in that methanol. A pharmaceutical composition for the prevention and treatment of influenza virus infection diseases, comprising the extract of ginseng obtained from the extract of the extract of claim 2 as an active ingredient. 6. The pharmaceutical composition for preventing and treating influenza virus infectious diseases according to claim 5, wherein the fraction of ginseng is obtained by suspending the extract of ginseng in water and then fractionating it with hexane, chloroform and ethyl acetate. A pharmaceutical composition for preventing and treating influenza virus infection diseases containing a terokapane-based compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient: [Formula 1]

Wherein R ₁ is methoxy, hydroxy or

being. The method of claim 7, wherein the terokapane-based compound represented by the formula (1) is a terokappine (Pterocarpin) represented by the following formula (3), machiaine (Maackiain) represented by the following formula (4) and a tripolizine represented by the formula (5) Pharmaceutical composition for the prevention and treatment of influenza virus infection disease, characterized in that it is selected from the group consisting of (Trifolrhizin): [Formula 3]

[Formula 4]

[Chemical Formula 5]

. 8. The pharmaceutical composition for preventing and treating influenza virus infection disease according to claim 7, wherein the terokapane-based compound represented by Chemical Formula 1 is isolated from a ginseng extract or a ginseng fraction. A pharmaceutical composition for the prevention and treatment of influenza virus infection diseases containing a flavonoid compound represented by the following formula (2) or a pharmaceutically acceptable salt thereof as an active ingredient: [Formula 2]

Where R ₂ is

or

ego; R ₃ and R ₄ are independently methoxy or hydroxy; R ₅ is hydrogen or hydroxy. The method of claim 10, wherein the flavonoid compound represented by the formula (2) is Sophoraflavanone G represented by the formula (6), (-)-curanone ((-)-Kuraninone represented by the formula (7) ), Lithuanone A represented by the following Chemical Formula 8, Kushenol A represented by the following Chemical Formula 9, Norkururarinol represented by the following Chemical Formula 10, and Kura represented by the following Chemical Formula 11 Pharmaceutical composition for the prevention and treatment of influenza virus infection disease, characterized in that selected from the group consisting of Linole (Kurarinol) and Kusenol T represented by the following formula (12): [Formula 6]

[Formula 7]

[Formula 8]

[Formula 9]

[Formula 10]

[Formula 11]

[Formula 12]

. The pharmaceutical composition for preventing and treating influenza virus infection disease according to claim 10, wherein the flavonoid compound represented by Chemical Formula 2 is isolated from a ginseng extract or a ginseng fraction. The method of claim 7 or 10, wherein the compound of Formula 1 and Formula 2 Extracting ginseng with water, C ₁ -C ₄ alcohol or a mixed solvent thereof to obtain a ginseng extract (step 1); Suspending by adding water to the ginseng extract obtained in the step 1, and sequentially fractionated using hexane, chloroform and ethyl acetate to obtain a ginseng fraction (step 2); And The ginseng fraction obtained in step 2 is separated and purified by silica gel chromatography to obtain a terokapane compound of Formula 1 and a flavonoid compound of Formula 2 (Step 3), characterized in that the influenza is separated Pharmaceutical composition for the prevention and treatment of viral infection diseases. The pharmaceutical composition for preventing and treating influenza virus infection disease according to claim 13, wherein the alcohol of step 1 is methanol or ethanol. 14. The pharmaceutical composition for preventing and treating influenza virus infection disease according to claim 13, wherein the gourd fraction of step 2 is a hexane gourd fraction, a chloroform gourd fraction, or an ethyl acetate gourd fraction. 11. The method of claim 1, 5, 7, and 10, wherein the influenza virus infection disease is flu, cold, sore throat, bronchitis, pneumonia or avian influenza. Prophylactic and therapeutic pharmaceutical compositions. The pharmaceutical composition for preventing and treating influenza virus infection disease according to claim 16, wherein the pharmaceutical composition exhibits inhibitory activity against neuraminidase. Claim 1, 5, 7, and 10 of any one of the ginseng extract, ginseng fraction, terokapane-based compounds and flavonoid-based compounds or pharmaceutically acceptable salts thereof contained as an active ingredient Health food composition for the prevention or improvement of influenza virus infection disease. The health food composition of claim 18, wherein the influenza virus infection disease is flu, cold, sore throat, bronchitis, pneumonia, or bird flu. Claim 1, 5, 7, and 10 of the ginseng extract, the ginseng fraction, the terokapane-based compound and the flavonoid-based compound or pharmaceutically acceptable salts thereof separated therefrom as an active ingredient Influenza disinfection composition.