KR20050001899A - Compound showing anti-oxidant and anti-viral activity and extract of Chrysanthemum indicum comprising the same - Google Patents

Abstract

PURPOSE: Provided are a compound having an antioxidant or antivirus activity or its pharmaceutically acceptable salt, Chrysanthemum indicum extract containing the compound, a separation method of the compound from the extract, a pharmaceutical composition containing the compound for treating or preventing HIV or the disease due to the oxidation of cell components, an antioxidant food additive containing the compound and an antioxidant cosmetic containing the compound. CONSTITUTION: The compound is represented by the formula 1, wherein R1 and R2 are H or a caffeoyl group; and R3 is a specific caffeoyl group represented by the formula (a). The Chrysanthemum indicum extract is extracted by using water and an organic solvent. Preferably the organic solvent is at least one selected from the group consisting of an alcohol, ethyl acetate, dichloromethane and acetone. The compound of the formula 1 is separated from the extract by carrying out the column chromatography using a filler selected from the group consisting of silica gel, Sephadex, RP-18, polyamide, Toyopearl and XAD resin.

Description

Compound showing anti-oxidant and anti-viral activity and extract of Chrysanthemum indicum comprising the same}

The present invention is isolated from Chrysanthemum indicum and the compound having antioxidant and antiviral activity, containing the compound as an active ingredient and extracted with water or an organic solvent to separate the compound from the gamguk extract and gamguk extract It is about a method.

Oxygen, which was present in a stable state, was exposed to environmental and biochemical factors such as enzymes, reduction metabolism, chemicals, pollutants, and photochemical reactions, resulting in superoxide anion radicals (O _{2 '} ^- ) and hydroxyl radicals (hydroxyl radicals). Cellular components of the human body when converted to reactive oxygen species (ROS) such as radicals, OH _' ), hydrogen peroxide (H ₂ O ₂ ), singlet oxygen ( ¹ O ₂ ), etc. Phosphorus protein, lipids and DNA can be irreversibly destroyed. The action of free radicals is the antioxidant mechanisms such as superoxide dismutase (SOD), catalase, peroxidase, glutathione and vitamin C (vitamin C, ascorbic). acid), vitamin E (tocopherol) and the like can be minimized by the action of antioxidants. However, in the event of abnormal biological defenses or exposure to excessive free radicals, residual or excess free radicals cause lethal oxygen toxicity to the living body, and are non-selective and irreversible to cellular components such as lipids, proteins, sugars, and DNA. It is known to cause various diseases such as aging, cancer, stroke, Parkinson's disease, heart disease, ischemia, arteriosclerosis, skin disease, gastrointestinal disease, inflammation, rheumatism, autoimmune disease, and so on by inducing destruction. (Cross et al . , Ann. Intern. Med ., 1987, 107 , 526; Alderson et al . , Proc. Natl. Acad. Sci. USA , 1988, 85 , 2706).

As such, as oxidative stress is found to be an important cause of various diseases including aging, studies are being actively conducted to develop active oxygen scavenger which effectively removes free radicals in vivo. In particular, antioxidants such as superoxide dismutase, catalase, peroxidase, and glutathione that can regulate active oxygen species, and low molecular weight active oxygen scavengers derived from natural products such as vitimin C, vitamin E, and tocopherol Much research is being done on this. In addition, as the synthesized active oxygen scavenger, butylated hydroxy anosole (BHA), butylated hydroxy toluene (BHT) and nordihydroguaiaretic acid (NDGA) have been developed and used in medicine and food fields.

However, the synthesized reactive oxygen scavenger is toxic in vivo and has the disadvantage of causing allergy and tumors. Therefore, the development of natural reactive oxygen scavenger is more demanded, but the natural active oxygen scavenger known to date is safer than synthetic active oxygen scavenger, but in terms of its effect and production cost, it does not surpass that of synthetic active oxygen scavenger. It is true. Therefore, there is an urgent need for the development of a new natural active oxygen scavenger which is excellent in active oxygen scavenging activity and is safer and has low production cost.

Human immunodeficiency virus (hereinafter, abbreviated as "HIV") is a virus mainly attacking human lymphocytes and is surrounded by an envelope, and the host cell after reverse transcription of the RNA genome, a genetic material, into DNA. It is one of the retroviruses that has the property of inserting into the DNA. HIV has the most internal genetic information RNA and reverse transcriptase, the RNA and reverse transcriptase is surrounded by a shell protein, the outer membrane is a lipid membrane containing a glycoprotein called gp120 and gp41 Has a structure that forms. The gp120 protein is known to play a critical role in the virus' recognition and penetration of T-cells.

The pharmacological effects of anti-HIV drugs are mainly caused by inhibiting the life cycle of HIV, which is largely related to 1) inhibiting the action between gp120, the virus's glycoprotein, and CD4 receptors in host cells, or 2) converting RNA from the virus to proviral DNA. To inhibit the reverse transcriptase involved, 3) to inhibit the action of the viral regulatory protein (tat, rev), 4) to inhibit the degradation of the viral proprotein, or 5) to mature the glycoprotein of the virus. And inhibiting the action of glucose degrading enzyme (glucosidase) or mannose degrading enzyme (mannosidase), which are enzymes that degrade glucose or mannose of oligosaccharides.

As an HIV inhibitor developed to date, as a drug having a reverse transcriptase inhibitory activity such as 2) dideoxycytidine (DDC), dideoxyinosine (DDI), AZT (zidobudine, azidothymidine), dextran sulfate (dextran sulfate) ), Peptide T, ribavirin, castanospermine, GEQ 223 (GLQ 223), antisense oligonucleotides, protease inhibitors, etc. There is a situation. For example, AZT is somewhat effective in treating AIDS, but after 6 months of treatment, AZT-resistant HIV-1 strains appear and the drug does not appear anymore, and there are serious side effects such as granulocytopenia and anemia. In addition, headache, nausea, insomnia, etc. Central nervous system side effects. In addition, DDI, which is conditionally approved as a treatment for AIDS, may show serious side effects such as pancreatitis and peripheral neuropathy, and thus may be prescribed only to patients who do not show any effects on AZT or whose use of AZT has been discontinued due to toxicity.

Another therapeutic agent is a protease inhibitor of HIV. HIV forms precursor proteins such as gag protein and gag-pol protein with both envelope proteins and enzymes attached, and is then broken down by proteases in its own to produce envelope proteins, proteases, reverse transcriptases, and integrases. It was developed on the basis of the inhibition of the replication of the virus by inhibiting the protease involved in this process. As protease inhibitors, saquinavir, ritonavir, and indinavir have been developed and used as therapeutic agents for AIDS patients. However, it also has side effects such as diabetes, hemolysis, bleeding, hyperlipidemia, and fat metabolism disorder, and causes serious problems such as the appearance of resistant strains for the therapeutic agents due to the long-term use of the therapeutic agents.

The number of people with HIV and ADIS is increasing rapidly around the world, and for these treatments, cheap drugs that have fewer side effects than conventional drugs, are easy to take, and do not produce resistant strains against gene sites that are resistant to conventional drugs. Development is required. In order to solve the problems of adverse reactions and resistant strains of reverse transcriptase inhibitors or protease inhibitors developed as AIDS therapeutic agents, development of therapeutic agents by different mechanisms of action than conventional therapeutic agents is being actively conducted.

On the other hand, Chrysanthemum indicum is a perennial plant that is not only native to mountains but also cultivated in large quantities in Korea, and the herbaceous tree describes the liver as comfortable, clears the eyes, reduces heat and releases poison. have. In addition, various physiological effects such as antimicrobial, antiviral and anti-inflammatory effects have been reported (Hu et al., Journal of Natural Products , 1994, 57 , 42).

Accordingly, the present inventors have found that Ingra, an alcoholic extract of Gamguk, and dicapeoylquinic acid derivatives and flavonoid-based compounds isolated therefrom as one of the enzymes essential for HIV replication, have not yet been developed inhibitors that can inhibit the activity of enzymes. The present invention was completed by revealing the antioxidant activity as well as being able to be used as an inhibitor to HIV by showing an inhibitory activity against the integrase.

It is an object of the present invention is a compound having antioxidant and antiviral activity isolated from gakguk, the method containing the compound as an active ingredient and extracted with water or an organic solvent, characterized in that the method for separating the compound from the gakguk extract and gakguk extract To provide.

Another object of the present invention to provide a pharmaceutical composition, food additives or cosmetics for the antioxidant or antiviral containing the compound or a gamguk extract containing the same as an active ingredient.

In order to achieve the above object, the present invention provides a compound represented by the formula (1) and a pharmaceutically acceptable salt thereof.

In addition, the present invention provides a gamguk extract extracted with water or an organic solvent and having antioxidant and antiviral activity.

In addition, the present invention provides a method for separating the compound of Formula 1 to Formula 4 from the extract.

In another aspect, the present invention provides a pharmaceutical composition, food additives or cosmetics for antioxidant or anti-viral containing one or more compounds selected from the group consisting of the gamguk extract or the compound represented by Formula 1 to Formula 4 as an active ingredient.

Hereinafter, the present invention will be described in detail.

The present invention provides a compound represented by the following formula (1) and a pharmaceutically acceptable salt thereof.

In the above, R ₁ and R ₂ is hydrogen or a caffeoyl group, R ₃ is a caffeoyl group. Caffe oil group has the following structure.

The compound of formula 1 of the present invention is a 3,5- O- dicaffeoy- epi -quinic acid wherein R ₁ and R ₂ are hydrogen and caffeoyl groups, respectively. compound 1, referred to hereinafter abbreviated) or R ₁ and R ₂ are each hydrogen cafe five days group of 1,3-cafe oil-epi-quinolyl acid (1,3- O -dicaffeoy- epi -quinic acid) ( hereinafter " Abbreviated as compound 2 ') (see Table 1).

R ₁ R ₂ R ₃ Compound 1 H Cafe Oil Cafe Oil Compound 2 Cafe Oil H Cafe Oil

The compound of Compound 1 of the present invention is a white amorphous powder, which is initially yellow when sulfuric acid is developed on a TLC plate, and becomes red over time, and has been measured in methanol solution [α] ²¹ _D appears to be -166.0 ° and molecular weight is 516 in FABMS. Hydrogen and carbon NMR data of the compound 1 are as follows.

¹ H NMR (300 MHz, CD ₃ OD): δ 2.04 (m, 1H, H-6 _ax ), 2.11 (m, 2H, H-2), 2.28 (dd, 1H, J = 15.2, 2.7 Hz, H -6 _eq ), 3.91 (dd, 1H, J = 9.9, 3.4 Hz, H-4), 5.39 (m, 1H, H-5), 5.55 (m, 1H, H-3), 6.31, 6.43 (d , each 1H, J = 15.8 Hz, H-8 '), 6.78 (d, 2H, J = 8.2 Hz, H-5'), 6.96, 6.97 (dd, each 1H, J = 8.2, 2.0 Hz, H- 6 '), 7.06, 7.08 (d, each 1H, J = 2.0 Hz, H-2'), 7.59, 7.62 (d, each 1H, J = 15.8 Hz, H-7 ')

¹³ C NMR (75 MHz, CD ₃ OD): δ 36.6 (C-6), 39.7 (C-2), 71.5 (C-3), 72.1 (C-4), 73.5 (C-5), 75.4 ( C-1), 114.3 (C-2 '), 114.5, 115.0 (C-8'), 115.5 (C-5 '), 122.0 (C-6'), 126.9, 127.1 (C-1 '), 145.7 , 145.7 (C-7 '), 145.9, 146.0 (C-3'), 148.3, 148.5 (C-4 '), 168.1, 168.5 (C-9'), 180.4 (COOH)

In addition, the compound of Compound 2 of the present invention is a pale yellow amorphous powder, when the sulfuric acid in the TLC plate is initially yellow and then turns red over time, it can be seen that it is a dicapoyl derivative. The photoluminescence [α] ²¹ _D measured in methanol solution is found to be + 183.7 ° and the molecular weight is 516 in FABMS. Hydrogen and carbon NMR data of the compound 2 is as follows.

¹ H NMR (300 MHz, CD ₃ OD): δ 2.08 (dd, 1H, J = 13.6, 9.0 Hz, H-2 _ax ), 2.46 (m, 2H, H-6), 2.58 (dd, 1H, J = 13.7, 3.4 Hz, H-2 _eq ), 3.80 (dd, 1H, J = 8.0, 3.2 Hz, H-4), 4.31 (dd, 1H, J = 7.4, 3.8 Hz, H-5), 5.41 ( m, 1H, H-3), 6.28, 6.31 (d, each 1H, J = 15.9 Hz, H-8 '), 6.79 (d, 2H, J = 8.2 Hz, H-5'), 6.92 (dd, 2H, J = 8.2, 1.6 Hz, H-6 '), 7.07 (br s, 2H, H-2'), 7.58, 7.59 (d, each 1H, J = 15.9 Hz, H-7 ')

¹³ C NMR (75 MHz, CD ₃ OD): δ 34.7 (C-6), 35.9 (C-2), 68.5 (C-5), 70.6 (C-3), 71.9 (C-4), 80.0 ( C-1), 114.1 (C-2 '), 114.2 (C-8'), 115.5 (C-5 '), 122.1, 122.2 (C-6'), 126.8 (C-1 '), 145.8 (C -3 '), 146.6, 146.3 (C-7'), 148.6, 148.6 (C-4 '), 167.7, 167.1 (C-9'), 173.9 (COOH)

The compound represented by Formula 1 of the present invention may form a pharmaceutically acceptable salt, and such pharmaceutically acceptable salts include alkali metal hydroxides (eg sodium hydroxide, potassium hydroxide), alkali metal bicarbonates (eg bicarbonate) Inorganic bases such as sodium, potassium bicarbonate) and alkali metal carbonates (eg sodium carbonate, potassium carbonate, calcium carbonate) and organic bases such as primary and secondary tertiary amine amino acids. In addition, the compound represented by Formula 1 of the present invention may be in the form of solvates, especially hydrates, and hydration may occur during separation of the compound, or may occur over time due to the hygroscopicity of the compound.

In addition, the present invention provides a gamguk extract extracted with water or an organic solvent and having antioxidant and antiviral activity.

In the above, the organic solvent may be a solvent selected from the group consisting of alcohol, ethyl acetate, dichloromethane and acetone or a mixture thereof, preferably an alcohol. The alcohol can also be selected from the group consisting of methanol, ethanol, propanol and butanol, more preferably methanol or ethanol. In preparing the gamguk extract of the present invention, the gamguk may be used in its entirety and is preferably dried and dried in the shade.

The gamguk extract of the present invention is characterized in that it comprises one or more compounds selected from the group consisting of compounds represented by formulas (1) to (4), preferably compounds 1 to 11, as the active ingredient.

In the above, R ₁ is hydrogen or methyl group, R ₂ to R ₄ is hydrogen or caffeoyl group, R ₅ is caffeoyl group.

The compound of formula (2) is a 3,5 - dicafeoylquinic acid (3,5- O- dicaffeoylquinic acid) wherein R ₁ to R ₅ each have the functional groups described in Table 2, hereinafter abbreviated as 'compound 3', 1,5-cafe five days quinone acid (1,5-O -dicaffeoyquinic acid) (hereinafter abbreviated as "compound 4"), 4,5-cafe five days quinone acid (4,5-O -dicaffeoyquinic acid) ( hereinafter abbreviated as "compound 5"), methyl 3,5-cafe five days quinolyl carbonate (methyl 3,5-O -dicaffeoyquinate) (hereinafter abbreviated as "compound 6"), methyl 4,5-cafe five days quinolyl It is preferred to be methyl (methyl 4,5- O- dicaffeoyquinate) (hereinafter abbreviated as 'compound 7') or chlorogenic acid (hereinafter abbreviated as 'compound 8').

R ₁ R ₂ R ₃ R ₄ R ₅ Compound 3 H H Cafe Oil H Cafe Oil Compound 4 H Cafe Oil H H Cafe Oil Compound 5 H H H Cafe Oil Cafe Oil Compound 6 Me H Cafe Oil H Cafe Oil Compound 7 Me H H Cafe Oil Cafe Oil Compound 8 H H H H Cafe Oil

In the above, R ₁ is hydrogen or glucose.

In the compound of formula (3) of the present invention, luteolin (hereinafter abbreviated as 'Compound 9') in which R ₁ is hydrogen and luteolin 7- O- β-D-glucopyrano in which R ₁ is glucose It is preferred that it is the luteolin 7- O- β-D-glucopyranoside (hereinafter abbreviated as 'Compound 10') (see Table 3).

R ₁ Compound 9 H Compound 10

The compounds of the general formula (4) is Erie ohdik thiol 7- O -β-D- gluconic nose Llano side (eriodictyol 7- O -β-D- glucopyranoside) ( hereinafter abbreviated as "compound 11").

The gamgukchi extract of the present invention having antioxidant and antiviral activity may be prepared by extracting gamguk with an organic solvent such as alcohol, hydrous alcohol or ethyl acetate, for example, may be prepared by the following method. That is, after cutting or powdering the gamguk finely used or lyophilized, 60 to 100% lower alcohol aqueous solution such as methanol (MeOH), ethanol, ethylene glycol, propylene glycol, or glycerol per kg Or acetone is added in an amount of 0.1 to 5 L, preferably 0.5 to 1.0 L and left at room temperature for 4 to 5 days. The process may be repeated three or more times as necessary. The obtained extract is filtered and evaporated under reduced pressure to obtain an alcoholic extract of Persimmon. Ethyl acetate extract can be obtained by adding 1 to 5 L, preferably 1.5 to 2.0 L of water per kg of alcohol extract, and then sufficiently extracting with 0.1 to 5 L of ethyl acetate (EtOAc), preferably 1.0 to 1.5 L. have. The ethyl acetate extract may be prepared by omitting the alcohol extraction process and extracting the direct soup with ethyl acetate.

As a result of confirming the anti-oxidant and antiviral activity of the extract of persimmons obtained by the above method, the methanol extract of persimmons showed not only antioxidant activity (see Tables 5 and 6) but also anti-HIV activity (see Tables 7 and 8). In addition, the antioxidative and anti-HIV activities of the extracts of methanol extract of Gam-gukku (dichloromethane, ethyl acetate, butanol, water, etc.) were the most potent in the ethyl acetate and butanol extracts (see Table 4). Therefore, in order to use the gamguk extract of the present invention as an antioxidant and an anti-HIV agent, alcohol solvent extracts such as methanol, ethanol, and hydrous ethanol can be used, and when the ethyl acetate fraction and butanol fraction are used, very effective pharmacological action can be expected. .

The present invention also provides a method for separating the compound represented by the formula (1) to the formula (4) from the extract.

The compounds can be purified by performing column chromatography on ethyl acetate extract of Gam-guk using a filler selected from the group consisting of silica gel, Sephadex, RP-18, polyamide, Toyopearl and XAD resin. Column chromatography may be carried out several times by selecting an appropriate filler as necessary, and it is most preferable to perform appropriate combination of column chromatography using Sephadex, RP-18 and silica gel as fillers.

Through the column chromatography process, the dicafeoylquinic acid derivatives of the compounds 3 to 11 and the flavonoid-based compounds as well as the compounds 1 and 2, which are novel compounds, may be separated and purified from the ethyl acetate extract of Gamguk.

As described above, the extract of Gamguk according to the present invention and the compound of Compound 1 to Compound 11 isolated therefrom exhibit antioxidant and anti-HIV activity, so that the treatment or prevention of diseases caused by free radicals, maintenance of food and skin Not only is very useful in preventing the oxidative damage of the oxidized, but also useful for the treatment of diseases caused by HIV or for the relief of symptoms.

In another aspect, the present invention provides a pharmaceutical composition for the treatment of antioxidant or HIV containing one or more compounds selected from the group consisting of the gamguk extract or the compound represented by Formula 1 to Formula 4. as an active ingredient.

The pharmaceutical composition of the present invention contains a compound selected from the group consisting of Compound 1 to Compound 11 or an extract of Gam-guk as an active ingredient, and may contain conventional pharmacologically acceptable excipients. The pharmaceutical compositions of the present invention may comprise alone or one or more pharmaceutically acceptable carriers, which carriers are compatible with the active ingredients of the present invention and should not be harmful to the receptor. Pharmaceutical compositions of the invention can be determined by one of ordinary skill in the art based on common patient symptoms and disease severity. It may also be formulated in various forms, such as powders, tablets, capsules, solutions, injections, ointments, syrups, and the like, and may also be provided in unit-dose or multi-dose containers, such as sealed ampoules and bottles. .

The pharmaceutical composition of the present invention can be administered orally or parenterally. Routes of administration of the pharmaceutical compositions according to the invention are not limited to these, for example, oral, intravenous, intramuscular, intraarterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intestinal Sublingual, or topical administration is possible.

For such clinical administration, the pharmaceutical composition of the present invention can be formulated into a suitable formulation using known techniques. For example, during oral administration, it may be mixed with an inert diluent or an edible carrier, sealed in hard or soft gelatin capsules, or pressed into tablets. For oral administration, the active compound may be mixed with excipients and used in the form of intake tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers and the like. In addition, various formulations, such as for injection and parenteral administration, can be prepared according to techniques known in the art or commonly used techniques.

The pharmaceutical composition of the present invention can be usefully used for the treatment or prevention of diseases caused by oxidation of cellular components by free radicals. Such diseases include, but are not limited to, aging, cancer, multiple atherosclerosis, arthritis, Parkinson's disease, stroke, concussion, Alzheimer's disease, vascular disorders, Hyperlipidemia, myocardial infarction or cerebral infarction.

The pharmaceutical compositions of the present invention can be administered in various dosage forms, oral or parenteral, and can be used in the form of general pharmaceutical preparations. The pharmaceutical compositions of the present invention can be used to prepare pharmaceutical formulations according to conventional methods. In the preparation of the formulation, it is preferred that the active ingredient is mixed with the carrier, diluted with the carrier, or enclosed in a carrier in the form of a capsule, assay or other container. Thus, the formulation may be a tablet, pill, powder, sasay, elixir, suspension, emulsion, liquid, syrup, aerosol, soft or hard gelatin capsule, injectable solution or suspension, ointment, cream, gel or lotion, etc. It may be in the form of. Examples of suitable carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, Propylhydroxybenzoate, talc, magnesium stearate and mineral oil. The formulation may further comprise fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives and the like. Compositions of the invention can be formulated using methods well known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal.

An effective amount of the extract of Gamguk or the compound 1 to compound 11 separated therefrom included in the pharmaceutical composition of the present invention is 0.1 mg / kg to 500 mg / kg. Preferably a range of 500 to 5,000 mg is common, and some diseases may require higher daily dosages. In addition, the specific dose level for a particular patient may vary depending on the particular compound to be used, weight, age, sex, health condition, diet, time of administration, method of administration, rate of excretion, drug mixing and the severity of the disease.

The present invention also provides a food additive for antioxidant containing one or more compounds selected from the group consisting of the gamguk extract or the compound represented by the formula (1) to (4) as an active ingredient.

The persimmon extract according to the present invention and the active ingredient separated therefrom have excellent antioxidant activity and can effectively prevent the smell or flavor change of foods caused by oxidation, rancidity of fats and oils, and discoloration of foods. Therefore, by combining the extract and the compounds separated therefrom to a variety of conventional foods can be used to preserve these foods or to maintain the freshness and quality of foods for a long time.

In addition, the present invention provides cosmetics for antioxidants containing at least one compound selected from the group consisting of the gakguk extract or a compound represented by Formula 1 to Formula 4.

Cosmetics of the present invention can be used as it is, or as diluted as necessary or the compound separated from the persimmon soup. The persimmon extract and compounds isolated therefrom may be prepared in liquid or solid form using bases, adjuvants and additives commonly used in the cosmetic field. Cosmetics in liquid or solid form may include, but are not limited to, for example, cosmetics, creams, lotions, baths and the like. Bases, auxiliaries and additives commonly used in the cosmetic field are not particularly limited and may include, for example, water, alcohols, propylene glycol, stearic acid, glycerol, cetyl alcohol and liquid paraffin.

Cosmetics according to the present invention are very useful in preventing skin oxidative damage, for example, spots (brown spots), freckles, skin cracks, UV damage (sunburn), etc. It can be very useful to maintain the quality of cosmetics.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Preparation of Persimmon Extract

Gamguk, used as a herbal medicine in oriental medicine, was purchased from Andong chrysanthemum flower complex and used for experiment. 1.2 kg of dried persimmon soup was immersed in 18.4 L of methanol, cooled by immersion at room temperature for 4-5 days, and the solvent was dried under reduced pressure at 40 ° C. or lower to prepare methanol-X. The same procedure was repeated three times to extract a total methanol x 441.1 g. Methanol x 436 g was suspended in 2,500 ml of water, 2,000 ml of dichloromethane (CH ₂ Cl ₂ , 2,000 ml x 3), ethyl acetate (EtOAc, 2,000 ml x 3) and butanol (BuOH, 2,000 ml x 3) Each fraction was divided by repeating extraction three times in sequence according to the solvent polarity.

Example 2 Isolation of Antioxidant Active Substances from the Ethyl Acetate Fraction of Gamguk

Since the ethyl acetate fraction of Gam-guk was the strongest, 14.47 g of ethyl acetate fraction was subjected to column chromatography using Sephadex using methanol as a developing solvent. The obtained fractions were developed on a normal phase silica gel TLC (developing solvent: dichloromethane-methanol-water = 60: 20: 1), and the compounds having similar polarities were collected and divided into nine subfractions, respectively, named 940EA to 940EI. The small fraction 940EE (8.4 g) was divided into 11 small fractions (940EE1-940EE11) by column chromatography using ethanol as a developing solvent using Sephadex. Small fraction 940EE10 (600.7 mg) was subjected to column chromatography using reverse phase silica gel (LiChroprep RP-18). The solvent used was 50% methanol by gradually increasing the amount of methanol from 40% methanol, and was further divided into nine small fractions (940EE10a-940EE10i). Small fraction 940EE10c (422.2 mg) was subjected to column chromatography using Sephadex using ethanol as a developing solvent. After dividing into three small fractions, the second fraction was recrystallized in methanol to obtain Compound 4 (110.1 mg), and the filtrate was purified using preparative reverse phase silica gel (40% methanol) to give Compound 1 (162.8 mg) and Compound 2 ( 63.8 mg) was obtained purely. 3.92 g of subfraction 940EE7 were separated into eight subfractions (940EE7a-940EE7h) by column chromatography using ethanol as a developing solvent using Sephadex. The first fraction 940EE7a 36.5 mg was divided into preparative reverse phase TLC (45% methanol ) Was isolated 2.5 mg of compound 8. Small fraction 940EE7e 1642.4 mg was subjected to column chromatography using reverse phase silica gel. The solvent used was 70% methanol by gradually increasing the amount of methanol from 34% methanol, and 12 small fractions (940EE7e1-940EE7e12) were collected by integrating similar compounds in reverse phase TLC (developing solvent: methanol-water = 50: 50). Divided by. The first fraction, 940EE7e1, 26.3 mg, was isolated using preparative reverse phase TLC (40% methanol) to 10.8 mg of compound 11. Compound 3 (445 mg) was obtained by column chromatography (70% ethanol) using Sephadex, 968.5 mg of the third fraction, 940EE7e3.

Compound 7 (22.6 mg) was obtained by column chromatography using Sephadex with 28.1 mg of a small fraction of 940EE7e11 as the developing solvent of ethanol. In addition, compound 9 (2.5 mg) was obtained from the last fraction 940EE7e12 (7.1 mg). Small fraction 940EE7e10 143.3 mg was divided into four small fractions (940EE7e11a-940EE7e11d) using ethanol as a developing solvent and separated into four small fractions (940EE7e11a-940EE7e11d). Toyopearl HW-40 (Toyopearl HW-40) Column was performed. Methanol was used as the developing solvent, and pure compound 6 (126.2 mg) was obtained from the first eluted fraction of the four fractions.

Compound 10 (25.0 mg) formed by recrystallization of 156.8 mg of the subfractions 940EE7e7 and 940EE7e8 in methanol was subjected to a column (developing solvent: methanol) using Toyo Pearl to obtain pure Compound 5 (12.0 mg).

Example 3 Structure Determination of New Compound 1

For novel compound 1, the photoluminescence [α] ^D was measured in methanol solution, and NMR spectra of 300 MHz ( ¹ H) and 75 MHz ( ¹³ C) were measured, and the chemical shift of each peak was internal. It is shown as a relative value to the standard trimethylsilane.

Chemical properties of Compound 1 of the present invention are as follows.

White amorphous powder;

[α] ²¹ _D -166.0 ° ( c 0.27, MeOH);

Negative-ion mode (FABMS) m / z 515 [M−1] −;

¹ H NMR (300 MHz, CD ₃ OD): δ 2.04 (m, 1H, H-6 _ax ), 2.11 (m, 2H, H-2), 2.28 (dd, 1H, J = 15.2, 2.7 Hz, H -6 _eq ), 3.91 (dd, 1H, J = 9.9, 3.4 Hz, H-4), 5.39 (m, 1H, H-5), 5.55 (m, 1H, H-3), 6.31, 6.43 (d , each 1H, J = 15.8 Hz, H-8 '), 6.78 (d, 2H, J = 8.2 Hz, H-5'), 6.96, 6.97 (dd, each 1H, J = 8.2, 2.0 Hz, H- 6 '), 7.06, 7.08 (d, each 1H, J = 2.0 Hz, H-2'), 7.59, 7.62 (d, each 1H, J = 15.8 Hz, H-7 ')

¹³ C NMR (75 MHz, CD ₃ OD): δ 36.6 (C-6), 39.7 (C-2), 71.5 (C-3), 72.1 (C-4), 73.5 (C-5), 75.4 ( C-1), 114.3 (C-2 '), 114.5, 115.0 (C-8'), 115.5 (C-5 '), 122.0 (C-6'), 126.9, 127.1 (C-1 '), 145.7 , 145.7 (C-7 '), 145.9, 146.0 (C-3'), 148.3, 148.5 (C-4 '), 168.1, 168.5 (C-9'), 180.4 (COOH)

The compound is a white amorphous powder, which initially becomes yellow when sulfuric acid is developed on a TLC plate, and becomes red over time. The photoluminescence [α] ²¹ _D measured in methanol solution was found to be -166.0 °, and the molecular weight was found to be 516 in FABMS. ^{In 1} H NMR peaks due to double bonds are shown at δ 6.31 (d, J = 15.8 Hz) and 7.59 (d, J = 15.8 Hz), while peaks at other double bonds are at δ 6.43 (d, J = 15.8 Hz) and 7.62 (d, J = 15.8 Hz), indicating a double bond located in trans . The peaks at 6 'of the benzene ring appear at δ 6.96 and 6.97 ( J = 8.2, 2.0 Hz), respectively, and these peaks metastasize to peaks 2' at δ 7.06 and 7.08 ( J = 2.0 Hz). It was found that meta coupling was performed and ortho coupling was performed with the peak at δ 6.78 ( J = 8.2 Hz), which is the peak at 5 ′. This shows that the compound has two caffeoyl groups in which two benzene rings are substituted with the ABX system.

Meanwhile, multiple peaks at δ 5.55 and multiple peaks at δ 5.39 are peaks corresponding to one hydrogen, respectively, and their peak patterns are peaks 3 and 5 of quinic acid. Could know. Hydrogen number 3 of quinic acid has diaxial coupling with hydrogen number 4, and diaxial coupling and axial-equatorial coupling with hydrogen number 2 shows that the peak shape is broad multiple, unlike hydrogen number 5. Hydrogen 5 of quinic acid, unlike hydrogen 3, has axial-equatorial coupling with hydrogens 4 and 6, and appears as a relatively sharp multiple peak with six other hydrogens and equatorial-equatorial coupling. At δ 3.91, a peak corresponding to one hydrogen was represented as a doublet of doublet ( J = 9.86, 3.43 Hz), indicating that the hydrogen corresponds to the fourth peak of quinic acid. Thus, it was found that the compound was substituted with two caffeoyl groups 3 and 5. However, it was found that 3,5-dicafeoyl had a spectrum that was too well consistent with compound 3 when compared with the data published in the literature (Tolonen et al., Phytochemical Anal . 2002, 13 , 316). Comparing compound 1 to compound 3 (3,5-dicafeoyl), ¹ H-NMR showed that compound 1 showed peaks at 5 and 3 of the quinic acid divided at 5.39 and 5.55. On the other hand, compound 3 (3,5-dicafeoyl) was found to overlap the hydrogen at 3 and 5 at δ 5.42. In addition, it was found that the physical properties (solubility, color, luminous intensity, etc.) are different from 3,5-dicafeoil. Compound 1 is a white amorphous powder that dissolves in very small amounts in methanol, while 3,5-dicafeoyl (compound 3) is a pale yellow powder that dissolves very well in methanol. Beneficiation was also different. On the other hand, in a structure in which a quinone structure isomers of the optically active acid is kwinik acid epitaxial (epi) - compound by the present inventors in view of the separation rake acid only that document (such as Crose, J. Org Chem 1970, 35, 1904..) It was found that this epi. This once again proves that Compound 1 has a different optical activity than that of Compound 3 (-154.4 °), with the opticality measured at -166.0 ° in methanol.

^The peak by quinic acid at ¹³ C NMR is δ 36.6 (C-6), 39.7 (C-2), 71.5 (C-3), 72.1 (C-4), 73.5 (C-5), 75.4 (C- 1), 180.4 (COOH), it can be seen that there is a difference from the peak by 3,5-dicafeoylquinic acid. In HMQC, carbon 6 at δ 36.6 correlates with two hydrogens at δ 2.04 and 2.28, and the peak at δ 39.7 at carbon 2 correlates with hydrogen at δ 2.11. I could see that. Hydrogen 4 at δ 3.91 correlates with carbon at δ 72.1, and hydrogen 3 and 5 have a correlation with carbon at δ 71.5 and 73.5, respectively, to determine the exact location of quinic acid. From HMBC, hydrogens 3 and 5 at δ 5.55 and 5.39 correlate with carbonyl peaks of δ 168.1 and 168.5 attributable to the caffeoyl group. It could be confirmed. Therefore, in view of the above results, the compound was identified as 3,5- O- dicaffeoy epi- quinic acid, which is the first compound isolated in nature. (Carnat et al., Fitoterapia 2000, 71 , 587; Maruta et al. , J. Agric. Food. Chem 1995, 4 3, 2592; Scholz-Bottcher et al ., Liebigs Ann. Chem . 1991, 1029).

Example 4 Structure Determination of New Compound 2

In the same manner as in Example 3, the structure of New Compound 2 was analyzed.

Chemical properties of Compound 2 of the present invention are as follows.

Pale yellow amorphous powder;

[α] ²¹ _D + 183.7 ° ( c 1.94, MeOH);

Negative-ion mode (FABMS) m / z 515 [M−1] ⁻ ;

HRFABMS (negative-ion mode) m / z obsd. 515.1179 (calcd. For 515.1190, C ₂₅ H ₂₃ O ₁₂ );

¹ H NMR (300 MHz, CD ₃ OD): δ 2.08 (dd, 1H, J = 13.6, 9.0 Hz, H-2 _ax ), 2.46 (m, 2H, H-6), 2.58 (dd, 1H, J = 13.7, 3.4 Hz, H-2 _eq ), 3.80 (dd, 1H, J = 8.0, 3.2 Hz, H-4), 4.31 (dd, 1H, J = 7.4, 3.8 Hz, H-5), 5.41 ( m, 1H, H-3), 6.28, 6.31 (d, each 1H, J = 15.9 Hz, H-8 '), 6.79 (d, 2H, J = 8.2 Hz, H-5'), 6.92 (dd, 2H, J = 8.2, 1.6 Hz, H-6 '), 7.07 (br s, 2H, H-2'), 7.58, 7.59 (d, each 1H, J = 15.9 Hz, H-7 ')

¹³ C NMR (75 MHz, CD ₃ OD): δ 34.7 (C-6), 35.9 (C-2), 68.5 (C-5), 70.6 (C-3), 71.9 (C-4), 80.0 ( C-1), 114.1 (C-2 '), 114.2 (C-8'), 115.5 (C-5 '), 122.1,122.2 (C-6'), 126.8 (C-1 '), 145.8 (C -3 '), 146.6, 146.3 (C-7'), 148.6, 148.6 (C-4 '), 167.7, 167.1 (C-9'), 173.9 (COOH)

The compound was a pale yellow amorphous powder, which was initially yellow when colored sulfuric acid was developed on a TLC plate and became red as time passed. The photoluminescence [α] ²¹ _D measured in methanol solution was found to be + 183.7 ° and the molecular weight was 516 in FABMS. The compound has peaks at δ 6.31 (d, J = 15.9 Hz) and 7.59 (d, J = 15.8 Hz) at ¹ H NMR and peaks at other double bonds at δ 6.28 (d, J = 15.9 Hz) and 7.58 (d, J = 15.9 Hz), indicating that the double bond located in the trans ( trans ). The peaks at δ 6.92 (dd, 2H, J = 8.2, 1.6 Hz), 7.07 (br s, 2H), 6.79 (d, 2H, J = 8.2 Hz) indicate that the two benzene rings are substituted with the ABX system. Could know. This shows that the compound has two caffeoyl groups. The peak at quinic acid 4 is shown as doublet of doublet at δ 3.80, the peak at 3 is shown as multiplet at δ 5.41, and the peak at 5 is shown as multiplet at δ 4.31. Thus, it was found that this compound has a structure similar to 1,5-dicafeoylquinic acid (Compound 4). However, when compared to the literature, 1,5-dicafeoylquinic acid showed a spectrum consistent with Compound 4 in ¹ H-NMR (Tolonen et al., Phytochemical Anal . 2002, 13 , 316), and Compound 2 was 1, It was found that the epi structure was different from 5-dicafeoylquinic acid. Compound 2 has a chemical shit and peak shape similar to that of 1,5-dicafeoylquinic acid at 3, 5, and 4 of the quinic acid, but the CH ₂ peak at 6 of the quinic acid is δ. The peak at 2.46 is different from the 1,5-dicafeoylquinic acid, which appears as multiple peaks and separates two hydrogens. ^The peak attributable to quinic acid in ¹³ C NMR is δ 34.7 (C-6), 35.9 (C-2), 68.5 (C-5), 70.6 (C-3), 71.9 (C-4), 80.0 (C -1), 173.9 (COOH), which is found to move about 2-3 ppm higher than that of 1,5-dicafeoylquinic acid. In particular, 1 (Δ 2.8 ppm), 4 (Δ 1.8 ppm), 6 (Δ 3.0 ppm) and 7 (Δ 3.1 ppm) carbons of quinic acid are higher than those of 1,5-dicafeoylquinic acid. It can be seen from the magnetic field. Taken together, the compound was also identified as 1,3-dicafeoyl- epi -quinic acid (1,3- O- dicaffeoy- epi- quinic acid) because it also showed optical activity. (Carnat et al., Fitoterapia 2000, 71 , 587; Maruta et al. , J. Agric. Food. Chem 1995, 4 3, 2592; Scholz-Bottcher et al ., Liebigs Ann. Chem . 1991, 1029; Crose et al ., J. Org.Chem . 1970, 35 , 1904).

<Example 5> Free radical scavenging effect of the gamguk extract and isolated compounds

1) Experiment Method

The free radical scavenging action was fractionated in 190 μl of 100 μM 1,1-diphenyl-2-picryl hydrazyl (1,1-diphenyl-2-picryl hydrazyl; DPPH) ethanol solution. 10 μl of ethanol solution was added to each of the compounds extracted in Example 3 and the Example 3, reacted at 37 ° C. for 30 minutes, and the absorbance was measured at 515 nm to obtain a free radical scavenging effect as an IC ₅₀ value (Blois et al. , Nature , 1958, 181 , 1199). IC ₅₀ means the concentration (IC ₅₀ ) which shows a 50% inhibitory effect by calculating the free radical scavenging effect.

The effect of free radical scavenging of the extracts from Example 1 and each of the compounds isolated from Example 2 and the existing scavengers [ascorbic acid (vitamin C), α-tocopherol (vitamin E), resveratrol] In comparison, the results are shown in Table 4. Data presented are averages of three runs.

As a result, as shown in Table 4, the ethyl acetate fraction and the butanol fraction of Gam-guk showed high activity, and it was confirmed that most of the compounds separated from ethyl acetate showing high activity showed free radical scavenging activity. .

Test substance Free radical scavenging effect (IC ₅₀ ) Methanol extract > 50 Dichloromethane fraction > 50 Ethyl acetate fraction 9.89 ± 0.46 Butanol fraction 1.59 ± 0.66 Water fraction > 50 Compound 1 5.64 ± 0.11 Compound 2 5.76 ± 0.15 Compound 3 5.7 ± 0.06 Compound 4 5.88 ± 0.13 Compound 5 5.89 ± 0.21 Compound 6 5.53 ± 0.11 Compound 7 5.36 ± 0.13 Compound 8 12.81 ± 0.60 Compound 9 6.28 ± 0.31 Compound 10 6.19 ± 0.22 Compound 11 10.27 ± 0.55 Ascorbic acid (vitamin C) 5.53 ± 0.13 α-tocopherol (vitamin E) 9.42 ± 0.26 Resveratrol 17.05 ± 1.08

As a result of comparing the free radical scavenging effect of each of the compounds extracted in Example 1 and the compounds separated in Example 2 with conventional scavenging agents, the free radical scavenging effect of ethyl acetate extract of gamguk is known as an antioxidant. The free radical scavenging effect of vinic acid (vitamin C) and α-tocopherol (vitamin E) was similar, and the free radical scavenging effect of butanol fraction was similar to that of resveratrol. In addition, most of the compounds separated from the ethyl acetate fraction and ethyl acetate showed better or similar effects than ascorbic acid (vitamin C) and α-tocopherol (vitamin E), which are known as antioxidants, indicating that these compounds have antioxidant activities. Could.

Example 6 Xanthine / Xanthine Oxidase-Induced Superoxide Anion (O) _2' ^- ) Elimination effect

1) Experiment Method

To evaluate the efficacy of inhibiting the production of superoxide anions produced by the reaction of Xanthine and xanthine oxidase (XOD), Toda et al., Planta, Med. , 1991, 57 , 8) was carried out as follows. Specifically, 0.1 mM xanthine, 0.1 mM EDTA, 50 μg / ml bovine serum albumin (BSA), 25 mM nitroblue tetrazolium (NBT) and 40 mM Na ₂ CO ₃ solution, for each concentration. The diluted test compound solution and the solution containing 200 μl of the final volume containing 1.4 × 10 ⁻³ unit XOD were mixed and reacted at 25 ° C. for 20 minutes. After the reaction was stopped by adding 6.6 μl of 6 mM CuCl ₂ to the reaction solution, the absorbance of the resulting formazan at 560 nm was measured to compare the xanthine / xanthine oxidase-induced superoxide anion scavenging effect. Obtained _by IC ₅₀ value. IC ₅₀ means the concentration (IC ₅₀ ) which shows a 50% inhibitory effect by calculating the xanthine / xanthine oxidase induced superoxide radical (O _{2 ′} ⁻ ) scavenging effect.

The scavenging effect of xanthine / xanthine oxidase-induced superoxide radical (O _{2 ′} ^- ) scavenging effect of the gamguk extract fractionated in Example 1 and each of the compounds separated in Example 2 was used as an existing scavenger [vitamin C) , α-tocopherol (vitamin E), resveratrol] is shown in Table 5. Data presented are averages of three runs.

Test substance Xanthine / Xanthine oxidase-induced superoxide radical (O _{2 '} ^- ) scavenging effect (IC ₅₀ ) Methanol extract > 50 Dichloromethane fraction > 50 Ethyl acetate fraction 3.56 ± 0.38 Butanol fraction 9.12 ± 0.51 Water fraction > 50 Compound 1 2.90 ± 0.14 Compound 2 2.60 ± 0.45 Compound 3 3.82 ± 0.36 Compound 4 4.77 ± 0.32 Compound 5 3.29 ± 0.42 Compound 6 2.53 ± 0.44 Compound 7 3.04 ± 0.20 Compound 8 3.09 ± 0.61 Compound 9 4.14 ± 0.7 Compound 10 7.71 ± 0.73 Compound 11 3.89 ± 0.11 Ascorbic acid (vitamin C) > 50 α-tocopherol (vitamin E) > 50 Resveratrol > 50

The ethyl acetate fraction fractionated from the extract of Gam-gukki was compared with the existing scavengers by comparing the xanthine / xanthine oxidase-induced superoxide radical (O _{2 '} ^- ) scavenging effect of ethyl acetate fractions from the methanol extract of The butanol fraction showed very high activity, and the ethyl acetate fraction showed particularly high activity. In addition, since most of the compounds separated from ethyl acetate showed high antioxidant activity, it can be seen that they are the main component of the antioxidant effect. However, it has been found that most of the compounds previously known as antioxidants have no effect.

Example 7 Lipid Peroxide Inhibition Effects of Extracts of Persimmon Soup and Isolated Compounds

1) Experiment Method

Lipid peroxide is a substance produced by peroxidation of lipids through various oxidation reactions. Lipid peroxides are generated on cell membranes by oxidizing phospholipids of cell membranes containing a large amount of reactive fatty acids and free radicals. do. Accumulation of lipid peroxides on cell membranes results in local disorders in tissues, such as cell membrane fluidity and functionality, which impair cell function and alter cell structure. Therefore, the effects of inhibiting lipid peroxide production of the extracts of Gam-guk and the isolated compounds were measured as follows.

Inhibits the oxidation of linoleic acid by AAPH [2,2'-azobis (2-amidinopropane) dihydrochloride, 2,2'-azobis (2-amidinopropane) dihydrochloride] The ferric thiocyanateassay of Son et al. ( J. Agric. Food Chem. , 2002, 50, 468-472) was modified to evaluate the efficacy of the test. Specifically, a solution prepared by mixing 3 mg / ml linolenic acid, 15 mg / ml Tween-20, 0.2 M sodium phosphate buffer (pH 7.0), and 1.7 mM AAPH was mixed so that the final volume was 300 μl in a sample solution diluted in ethanol. The reaction was carried out at 37 ° C. for 150 minutes (final concentration of each sample in the reaction solution was 3.125 μg / ml). 0.1 ml of the reaction solution, 0.1 ml of 30% ammonium thiocyanate, and 0.1 ml of 2 × 10 ⁻² M ferric chloride (FeCl ₂ ) were mixed, and after 3 minutes, at 500 nm with a microplate reader. Absorbance was measured. Inhibition rate of lipid peroxide production (A,%) of the extracts and the isolated compounds was calculated by the following formula and the results are shown in Table 6, and the values presented are three times the average value.

A (%) = absorbance of the test sample / response of the control × 100

Test substance Inhibition rate of lipid peroxide formation (%) Methanol extract 10.9 Dichloromethane fraction 19.7 Ethyl acetate fraction 49.9 Butanol fraction 46.0 Water fraction 14.0 Compound 1 51.5 Compound 2 50.3 Compound 3 51.2 Compound 4 49.9 Compound 5 51.7 Compound 6 53.8 Compound 7 53.7 Compound 8 47.4 Compound 9 52.2 Compound 10 48.3 Compound 11 47.8 Ascorbic acid (vitamin C) 44.1 α-tocopherol (vitamin E) 61.2 Resveratrol 39.1 Trolox 61.6

Inhibition rate of lipid peroxide production of each of the extracts of the gamguk extract fractionated in Example 1 and each of the compounds isolated in Example 2 was determined by conventional scavengers (ascorbic acid (vitamin C), α-tocopherol (vitamin E), resveratrol), and troll. Trolox]. As can be seen from Table 6, it was found that the ethyl acetate fraction and the butanol fraction fractionated from the methanol extract of methanol and the methanol extract showed very high activity. In addition, most of the compounds isolated from ethyl acetate showed high activity, which was found to be superior to several compounds well known as antioxidants. Thus, the extracts and the isolated compounds showed lipid peroxidation inhibitory activity comparable to the control.

Example 8 Enzyme Activity Measurement and Activity Inhibitor Search

In order to measure the activity of integrase, it was determined by measuring the endonucleolytic cleavage activity of integrase using an oligonucleotide such as the end nucleotide sequence of viral DNA. The 3 'end of oligonucleotide A described in SEQ ID NO: 1 used for the measurement was labeled as follows. 5 μl (100 ng) of oligonucleotide A was added to 10 μl kinase buffer (0.5 M TrisHCl, pH 8.0, 0.1 M MgCl ₂ , 50 mM DTT, 1 mM EDTA) 4 μl, ³² P-ATP (5 mCi / ml) 30 1 μl and 1 μl of T4 DNA polynucleotide kinase (10 unit / μl) were added and reacted at 37 ° C. for 30 minutes. 1 μl of 0.5 M EDTA was added and heated at 85 ° C. for 15 minutes. 10 μl (200 ng) of oligonucleotide B described in SEQ ID NO: 2 complementary to oligonucleotide A was added thereto, soaked in boiling water, and cooled slowly overnight. Unreacted ³² P-ATP was separated and removed using a Sephadex G25 column. First discard the solution present in the prefilled column, wash the gel with 5 ml of TEN (10 mM TrisHCl, 1 mM EDTA, 100 mM NaCl), load the reaction (approximately 52 μl) with a micropipette on top and Two drops (approximately 100 μl) were collected in one fraction. After the reaction was completely absorbed, it was washed twice with 200 μl of TEN and 4 mL of TEN. 1 μl of each fraction was bound to whatman filter paper to measure the amount of radioactivity to identify the labeled oligonucleotide positions.

For cleavage activity, 3 pmol of labeled oligonucleotide substrate and 30 pmol of integrase were added to 10 μl of 10 mM TrisHCl, 1 mM MnCl ₂ and reacted at 34 ° C. for 90 minutes. The reaction was terminated by adding 4 μl of the reaction solution, and 2-4 μl of the reaction was loaded on a 20% polyacrylamide sequencing gel to determine that the substrate of 20 bases was converted to 18 bases. In order to confirm the optimum conditions of the reaction, the cleavage assay was repeatedly performed by varying the composition of the reaction buffer and the type and concentration of the divalent metal ion to establish the most suitable reaction conditions, and based on this, it could act as an inhibitor of the expression of enzyme activity. The reaction was carried out in the presence of compounds present to determine inhibitors by measuring the decrease in their activity.

Example 9 HIV Integrase Inhibitory Effects of Extracts from Persimmon and Extracts

The inhibitory effect of HIV integrase and the inhibitory activity against human immunodeficiency virus integrase of ethyl acetate extracts were investigated. The inhibitory activity of these compounds against human immunodeficiency virus integrase affects how the compound inhibits the degradation of oligonucleotide substrates containing radioisotopes in the compounds by immunodeficiency virus integrase, that is, reactions obtained after enzymatic reaction. It was determined by performing autoradiography on gels obtained by electrophoresis of by-products (Oh et al . , Mol. Cells , 1996, 6 , 96). Table 7 shows the results of inhibiting the HIV integrase inhibitory efficiency (%) of the extracts from the methanol extract and the methanol extract of Gam-guk and the inhibitory effects of the compounds isolated from ethyl acetate as IC ₅₀ values. IC ₅₀ means the concentration (IC ₅₀ ) which shows the inhibitory effect 50%.

Inhibitory Activity of Human Immunity Deficiency Virus Integrase on the Extracts of Ginseng and Extracts Fraction 100 μg / ml 25 μg / ml IC ₅₀ (μg / ml) Methanol extract 45-55% 30% less than > 100 Dichloromethane fraction 45-55% 30% less than > 100 Ethyl acetate fraction More than 70% 55-70% 14.1 ± 2.8 Butanol fraction 55-70% 45-55% 48.0 ± 13.3 Water fraction 30-45% - > 100 Compound 1 5.9 ± 2.1 Compound 2 10 ± 2.5 Compound 3 5.7 ± 3.6 Compound 4 6.3 ± 4.0 Compound 5 5.4 ± 2.6 Compound 6 9.1 ± 2.5 Compound 7 12.5 ± 3.1 Compound 8 41.7 ± 10.7 Compound 9 5.88 ± 1.2 Compound 10 16.9 ± 2.0 Compound 11 51.4 ± 8.4 L-chicoric acid ^a 7.4 ± 3.3 Curcumin 51.3 ± 3.5

In the above, ^a chicoric acid was prepared and used according to known literature (King et al., Journal of Medicinal Chemistry , 1999, 4 2 , 497).

In order to compare the integrase inhibitory effect of the extract of the present invention and the compounds isolated from the extract, chicory acid (L-chicoric acid) and curcumin, which are well known as antiviral substances, were used as control drugs. As shown in Table 7, it can be seen that the fraction of ethyl acetate or butanol of Gam-guk has an integrase inhibitory effect. In addition, the antiviral effect of Compound 1, a novel compound isolated from the ethyl acetate fraction of Gam-guk, showed significantly better integrase inhibitory activity than curcumin, and was slightly better than L-chicoric acid. Indicated. The other compounds also showed an excellent integrase inhibitory effect, so it was found that these compounds were the active ingredient in showing the inhibitory effect of integrase in Gamguk.

Example 10 Antiviral Activity and Cytotoxicity Test

In vitro HIV according to a known method (Tanaka et al ., J. Med. Chem ., 1991, 34 , 349) to investigate the antiviral activity of the methanol extract and the extract fractionated from methanol extract of the present invention -1 inhibitory effect test was performed. MT-4 cells were used as host cells to investigate the extent to which the compounds of the present invention inhibit the cytotoxicity of virus-infected MT-4 cells.

MT-4 cells were dispersed in the culture medium at a concentration of 1 × 10 ⁴ cells / ml, and then inoculated with HIV-1 to 500 TCID ₅₀ (the concentration at which 50% of the cells are infected) / well. Immediately after inoculation, 100 μl of cell dispersion was transferred to a flat microtiter plate containing a sample of the extract of the present invention and a compound isolated from the extract, and cultured at 37 ° C. for about 4 days to 5 days, and then antiviral activity using the MTT method. Was determined. At the same time, cytotoxicity was also determined by measuring the viability of mock-infected host cells by MTT method. Drug reference screening was performed using L-chicoric acid and curcumin as reference drugs. The test results are shown in Table 8.

Inhibitory Effects of Compounds Isolated from Persimmon Extracts on HIV Cell Lines compound EC _{50 (} μg / mL) ^a CC ₅₀ (μg / mL) ^b SI ^c Compound 1 76.9 119.7 1.6 Compound 3 64.32 135.10 2.10 Ethyl acetate fraction 18.3 32.3 1.8 Chicolic acid 54.33 > 150 > 2.76 Curcumin > 0.32 0.32 <1

In the above, ^a EC ₅₀ ; Concentrations that inhibit the proliferation of HIV-1 by 50%,

^b CC ₅₀ ; 50% cell damage concentration for MT-4 cells,

^c selectivity; Selectivity Index (CD ₅₀ / ED ₅₀ )

The antiviral effect of the compounds isolated from the ethyl acetate fraction of Gam-guk was tested using chicory acid and curcumin as control drugs. As a result, the new compound was superior or comparable to that of curcumin and chicory acid in HIV cell lines. These compounds showed that the ethyl acetate fraction of Gam-guk was an active ingredient in showing anti-HIV efficacy (Table 8).

Hereinafter, a specific disease to which the pharmaceutical composition comprising the sweetened extract of the present invention or the compound of Compounds 1 to 11 may be applied.

However, the following application examples are only for illustrating the present invention, and the content of the present invention is not limited to the following application examples.

<Application example 1> Cancer

Cancer is caused by a myriad of causes, but the most fundamental cause is the destruction of cells by free radicals and their inability to repair them, leading to cell dysfunction, which has been shown to metastasize to cancer (Ames, BN, Science , 1983, 221, 1256-1264). On the other hand, the effects of phenolic acid in fruits on liver cancer (Sun J et al ., J Agric Food Chem , 2002, 4; 50 (25), 7449-7454), and the effect of lycopene on tomatoes on breast cancer (Hadley CW et al ., Exp Biol Med , 2002, 227 (10), 869-80), effect of isoverbascoside on gastric cancer (Chen RC et al ., Acta Pharmacol Sin , 2002, 23 ( 11), 997-1001) and several known antioxidants are also effective against other cancers. Therefore, it can be seen that the antioxidant can be effectively used for the treatment and prevention of various types of cancer, the antioxidant pharmaceutical composition of the present invention excellent in antioxidant activity can be effectively used for the treatment and prevention of cancer.

Application Example 2 Aging

Free radicals, which are generated by the metabolic process in the normal course of metabolism, cause oxidative damage by non-selective and irreversible destruction of cell components such as lipids, proteins, sugars, or DNA. Harman, D, Free radical theory of aging , 1986, 3-49). In addition, it has been observed that lifespan is extended by reducing basal metabolic rate, ie oxygen consumption, such as by limiting diet or reducing exercise by varying experimental conditions (Medvedev, ZA, Biol Rev. , 1990, 65, 375-398; Loe , J. et al ., J. Biol. Chem ., 1971, 32, 103-121; Sohal, RS, Aging , 1982, 5, 21-24). In other words, the removal of free radicals is a way to delay aging and there are many antioxidants are currently developed to remove free radicals. Therefore, the antioxidant pharmaceutical composition of the present invention excellent in antioxidant activity may delay aging.

<Application Example 3> Coronary heart disease, hyperlipidemia and arteriosclerosis

Treatment with cholesterol biosynthetic enzyme inhibitors in patients with coronary heart disease and hypercholesterolemia reduces cholesterol content in low-density fats, but inhibits the biosynthesis of ubiquinone Q10 for low-density lipoproteins against peroxidation by free radicals. Because of its protective effect, it is not effective in the treatment of the disease, so the administration of the antioxidant cerivastatin or probucol to the patients rapidly reduced the content of low density lipoprotein (Lankin VZ et al . , Bull Exp Biol Med , 2002, 134 (1), 39-42). In addition, in clinical trials in which the antioxidant dihydropyridine calcium antagonist lacidipine was administered to atherosclerosis patients, blood pressure was lowered and cholesterol levels in the blood vessel wall were lowered, thereby reducing the size of atherosclerotic lesions (Haller H et. al ., Drugs RD , 2002, 3 (5), 311-23). That is, the antioxidant substance can be seen that it is an excellent substance in the treatment and prevention of vascular diseases related to cholesterol, such as coronary heart disease, hyperlipidemia and arteriosclerosis, the antioxidant pharmaceutical composition of the present invention excellent in antioxidant activity is coronary heart disease It can be used for the treatment or prevention of vascular diseases such as hyperlipidemia, arteriosclerosis.

Application Example 4 Multiple Sclerosis and Autoimmune Encephalomyelitis

In patients with multiple sclerosis and autoimmune encephalomyelitis disease models, a weakness was observed in clinical trials in which ALA (alpha lipoic acid), a type of antioxidant, was administered to mice. In other words, oxidative stress is a major cause of multiple sclerosis and autoimmune encephalomyelitis, and antioxidants can be used to treat such neurological disorders (Marracci GH et al ., J Neuroimmunol , 2002, 131 (1-2)). , 104-14). Therefore, the antioxidant pharmaceutical composition of the present invention having excellent antioxidant activity can be used for the treatment or prevention of neurological diseases such as multiple sclerosis and autoimmune encephalomyelitis.

Application Example 5 Stroke and Alzheimer's Disease

Oxidative stress, or free radicals, oxidizes the components of cells, leading to their dysfunction, and also impairs the function of nerve cells due to their dysfunction. This neuronal dysfunction causes stroke, shock, and the like. However, when these symptoms are not treated and given continuous oxidative stress, serious brain diseases such as stroke and Alzheimer's disease are caused. In addition, antioxidants that can remove free radicals can be used to treat brain diseases such as stroke and Alzheimer's disease (Perry G et al ., Comp Biochem Physiol C Toxicol Phaarmacol , 2002, 133 (4), 507-13; Cecchi C et al ., Free Radic Biol Med , 2002, 15:33 (10), 1372-9; Smith MA et al ., Free Radic Biol Med , 2002, 1:33 (9), 1194-9). Therefore, the antioxidant pharmaceutical composition of the present invention excellent in antioxidant activity can be used for the treatment or prevention of stroke and brain diseases such as Alzheimer's disease.

<Application Example 6> Enteritis

Excess peroxide by-products accumulate in leukocytes of enteritis patients, and cell damage by accumulated peroxide by-products is the cause of primary or secondary pathological mechanisms in the intestinal infection. That is, oxidative stress is a major cause of inflammatory enteritis by inducing intestinal infections (Kruidenier L et al ., Aliment Pharmacol Ther , 2002, 16 (12), 1997-2015). Therefore, the antioxidant pharmaceutical composition of the present invention excellent in antioxidant activity can be used for the treatment or prevention of diseases related to inflammation such as inflammatory enteritis.

As described above, the gamguk extract of the present invention and the compound isolated therefrom can be very useful for the treatment or prevention of diseases caused by free radicals, maintenance of food and prevention of damage caused by oxidation of the skin, It may also be usefully used for the treatment of diseases caused by HIV or for the relief of symptoms.

<110> Korea Institute of Science and Technology <120> Compound showing anti-oxidant and anti-viral activity and extract          of Chrysanthemum indicum configure the same <130> 3p-05-39B <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> Oligonucleotide A <400> 1 tgtggaaaat ctctagcagt 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide B which is complementary to oligonucleotide A <400> 2 actgctagag attttccaca 20

Claims (15)

A compound represented by the following formula (1) and a pharmaceutically acceptable salt thereof. <Formula 1> In the above, R ₁ and R ₂ is hydrogen or caffeyl group, R ₃ is a caffeoyl group having the following structure. The method of claim 1, wherein, R ₁ is hydrogen and R ₂ is a cafe five days caused 3,5-cafe oil-compound and characterized in that the quinone acid (3,5- O -dicaffeoy- epi -quinic acid) - epi Pharmaceutically acceptable salts thereof. A compound according to claim 1, wherein R ₁ is a caffeoyl group and R ₂ is 1,3-dicafeoyl- epi -quinic acid, wherein hydrogen is 1,3- O- dicaffeoy- epi- quinic acid. And pharmaceutically acceptable salts thereof. Extracted with water or organic solvents, it is an extract of gamguk with antioxidant and antiviral activity. The extract according to claim 4, wherein the organic solvent is a compound selected from the group consisting of alcohol, ethyl acetate, dichloromethane and acetone or a mixture thereof. 6. The extract of claim 5, wherein the alcohol is selected from the group consisting of methanol, ethanol, propanol, ethylene glycol, propylene glycol, glycerol and butanol. The extract according to claim 4, wherein the active ingredient comprises one or more compounds selected from the group consisting of compounds represented by Formulas 1 to 4. <Formula 1> In the above, R ₁ and R ₂ is hydrogen or a caffeoyl group, R ₃ is a caffeoyl group, <Formula 2> In the above, R ₁ is hydrogen or methyl group, R ₂ to R ₄ is hydrogen or caffeoyl group, R ₅ is caffeoyl group, <Formula 3> In the above, R ₁ is hydrogen or glucose <Formula 4> 8. The extract of claim 7, wherein the compound of formula 1 is 3,5-dicafeoyl-epi-quinic acid or 1,3-dicafeoyl-epi-quinic acid. 8. A compound according to claim 7, wherein the compound of formula 2 is 3,5-dicafeoylquinic acid, 1,5-dicafeoylquinic acid, 4,5-dicafeoylquinic acid, methyl 3,5-dicafeoylqui Extracts selected from the group consisting of nitrate, methyl 4,5-dicafeoylquinate and chlorogenic acid. 8. The extract of claim 7, wherein the compound of formula 3 is luteolin or luteolin 7- O- β-D-glucopyranoside. Compounds represented by Chemical Formulas 1 to 4 by performing column chromatography on ethyl acetate extract of Gam-guk using a filler selected from the group consisting of silica gel, Sephadex, RP-18, polyamide, Toyopearl and XAD resin How to separate it. A pharmaceutical composition for preventing or treating diseases or HIV caused by oxidation of a cellular component containing at least one compound selected from the group consisting of the gamguk extract of claim 4 or the compound represented by the formula (1) to (4) as an active ingredient. The pharmaceutical composition of claim 12, wherein the disease is selected from the group consisting of aging, cancer, complex arteriosclerosis, arthritis, Parkinson's disease, stroke, concussion, Alzheimer's disease, vascular disorders, hyperlipidemia, myocardial infarction and cerebral infarction. Composition. Antioxidant food additive containing at least one compound selected from the group consisting of the gamguk extract of claim 4 or the compound represented by Formula 1 to Formula 4. Antioxidant cosmetics for preventing the aging of the skin by oxidative damage containing at least one compound selected from the group consisting of the gamguk extract of claim 4 or the compound represented by Formula 1 to Formula 4.