KR100512096B1 - Compound showing anti-viral activity and extract of Chrysanthemum morifolium comprising the same - Google Patents

Abstract

The present invention relates to a compound having antiviral activity isolated from chrysanthemum, chrysanthemum extract and chrysanthemum extract, comprising the compound as an active ingredient and extracted with water or an organic solvent. Specifically, the compound represented by the formula (1) isolated from the chrysanthemum bud, the method of separating the compound from the chrysanthemum bud extract and chrysanthemum bud extract containing the compound and having antiviral activity and extracted with water or organic solvent It is about. Compounds and chrysanthemum bud extracts according to the invention can be usefully used for the treatment of diseases caused by HIV or for the relief of symptoms.

<Formula 1>

Description

Compound showing anti-viral activity and chrysanthemum extract comprising same {Compound showing anti-viral activity and extract of Chrysanthemum morifolium comprising the same}

The present invention the compound from chrysanthemum compound, containing such compounds as active ingredients and water, or chrysanthemums, characterized in that the extraction with an organic solvent bud extract and chrysanthemum flower bud extract is separated from the (Chrysanthemum morifolium) having antiviral activity It is about a method of separating.

Human immunodeficiency virus (hereinafter abbreviated as "HIV") is a virus mainly attacking human lymphocytes, surrounded by an envelope, and the DNA of the host cell after reverse transcription of the RNA genome, a genetic material, into DNA. It is one of the retroviruses that have the property of being inserted into. 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 the body 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.

Chrysanthemum morifolium , on the other hand, is a perennial plant that is imported from China and used in Chinese medicine. Herbal wood is described as relaxing the liver, clearing eyes, lowering heat and releasing poison. 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).

Therefore, the present inventors have found that the alcohol extract of the chrysanthemum bud and the compound isolated therefrom are inhibited by integrase, which has not been developed an inhibitor that can inhibit the activity of the enzyme as one of the enzymes essential for HIV replication. The present invention has been completed by revealing that it can be usefully used as an inhibitor against HIV.

An object of the present invention is a compound having an antiviral activity isolated from the chrysanthemum bud, the compound from the chrysanthemum bud extract and chrysanthemum bud extract containing the compound as an active ingredient and extracted with water or an organic solvent It is to provide a way to separate.

Another object of the present invention to provide an antiviral pharmaceutical composition containing the compound or chrysanthemum bud extract comprising 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.

The present invention also provides a chrysanthemum bud extract extracted with water or an organic solvent and having antioxidant and antiviral activity.

The present invention also provides a method of separating the compound of Formula 1 to Formula 4 from the chrysanthemum bud extract.

In another aspect, the present invention provides a pharmaceutical composition for anti-viral containing the chrysanthemum bud extract or at least one compound selected from the group consisting of compounds 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.

Compound represented by Formula 1 of the present invention (hereinafter, abbreviated as "Compound 1") can be seen that the color of the flavonoid series by the yellow color when the sulfuric acid in the TLC plate, [α] ²³ _D +30.8 ° ( c 0.25, 80% MeOH); UV (CH ₃ CN) λ _max (log ε) 270 (2.59), 325 (3.39); IR (KBr) ν _max 3408, 2929, 1604, 1500 cm ⁻¹ ; Negative-ion mode (FABMS) m / z 607 [M−1] ⁻ ; And HRFABMS (negative-ion mode) m / z obsd. 607.1078 (calcd. For 607.1088, C ₃₀ H ₂₃ O ₁₄ ); Has chemical properties. Hydrogen and carbon NMR data of the compound 1 is shown in Table 1 below.

¹ H NMR spectrum, ¹³ C NMR spectrum, and long-range ¹³ C- ¹ H COSY data of Compound 1 location ¹ H transition ^{a 13} C transition ^b C-H Correlation 2 6.54 (s) 167.3 3 104.5 C-2, 10, 1 ' 4 184.5 5 162.6 6 6.39 (br s) 101.8 C-5, 7, 8, 10 7 164.7 8 6.69 (br s) 96.8 C-6, 7, 9, 10 9 159.2 10 107.5 One' 123.4 2' 7.76 (d, 8.5) 130.2 C-2, 4 ', 6' 3 ' 6.85 (d, 8.5) 117.7 C-1 ', 4', 5 ' 4' 162.7 5 ' 6.85 (d, 8.5) 117.7 C-1 ', 3', 4 ' 6 ' 7.76 (d, 8.5) 130.2 C-2, 2 ', 4' One" 5.12 (d, 7.1) 101.3 C-7 2" 3.63 (t, 7.6) 74.8 C-1 ", 3" 3 " 3.75 (t, 9.2) 75.7 C-4 " 4" 5.08 (m) 73.9 C-3 ", 6", CO 5 " 4.10 (d, 9.2) 76.1 6 " 174.6 One"' 128.5 2"' 6.99 (d, 1.6) 116.1 C-3 "', 4"', 6 "', β 3 "' 146.8 4"' 149.6 5 "' 6.71 (d, 8.9) 117.3 C-1 "', 3"', 4 "' 6 "' 6.87 (br d, 8.9) 123.9 C-2 "', 4"', β α 6.26 (d, 15.8) 115.7 C-1 "', CO β 7.51 (d, 15.8) 148.1 C-2 "', 6"', α, CO CO 169.3

In the above, a; The numbers in parentheses represent the mating constants (in Hz, CD ₃ OD),

b; ¹³ C NMR data in DMSO- d ₆ .

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 chrysanthemum bud extract extracted with water or an organic solvent and having antiviral activity.

In the above, the organic solvent may be a compound selected from the group consisting of alcohol, ethyl acetate, dichloromethane and acetone or mixtures thereof, preferably alcohol. The alcohol may also be selected from the group consisting of methanol, ethanol, ethylene glycol, propylene glycol, glycerol, propanol and butanol, more preferably methanol or ethanol. In preparing the chrysanthemum bud extract of the present invention, the chrysanthemum bud may be used in its entirety, it is preferable to use dried in the shade.

The chrysanthemum bud 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 5, preferably Compounds 1 to 9, as active ingredients.

In the above, R ₁ is hydrogen, hydroxyl group, glucose or rhamnose, and R ₂ is hydrogen or hydroxyl group.

It is preferable that the compound of the formula (3) is a compound of compounds 3 to 7 in which R ₁ and R ₂ each have the functional groups shown in Table 2.

R ₁ R ₂ Compound 3 O -glucose H Compound 4 O -Rhamnos OH Compound 5 OH OH Compound 6 H H Compound 7 H OH

In the above, glucose and rhamnose have the following structure.

,

In the above, R is a caffe oil group, and the caffe oil group has a structure as follows (hereinafter, abbreviated as "Compound 8").

In the above, R is a caffeoyl group (hereinafter abbreviated as "Compound 9").

Chrysanthemum bud extract of the present invention having antiviral activity may be prepared by extracting the chrysanthemum bud with an organic solvent such as alcohol, hydrous alcohol or ethyl acetate, for example, may be prepared by the following method. That is, chrysanthemum buds are finely chopped, powdered, used as is or lyophilized, and then 0.1 to 60% aqueous solution of lower alcohol such as methanol (MeOH) or ethanol, or acetone, per kilogram of chrysanthemum buds is added thereto. 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 alcohol extract of chrysanthemum buds. 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 chrysanthemum buds directly with ethyl acetate.

As a result of confirming the antiviral activity of the chrysanthemum bud extract obtained by the above method, the methanol extract of the chrysanthemum bud showed anti-HIV activity (see Table 4). In addition, the inhibitory activity against HIV integrase of methanol extracts from chrysanthemum buds with dichloromethane, ethyl acetate, butanol, water, and the like was examined. 3). Therefore, in order to use the chrysanthemum bud extract of the present invention as an anti-HIV agent, alcohol solvent extracts such as methanol, ethanol, and hydrous ethanol can be used, and an effective pharmacological action can be expected by using an ethyl acetate fraction and a butanol fraction.

The present invention also provides a method for separating the compound represented by the formula (1) to (5) from the chrysanthemum bud extract.

The compounds can be purified by performing column chromatography on ethyl acetate extracts of chrysanthemum buds 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 compounds of the compounds 2 to 9 as well as the novel compound 1 may be separated and purified from the ethyl acetate extract of the chrysanthemum buds.

As described above, the extracts of the chrysanthemum buds according to the present invention and the compounds of the compounds 1 to 9 isolated therefrom exhibit anti-HIV activity, and thus may be useful for the treatment of diseases caused by HIV or for the relief of symptoms. Can be.

The present invention also provides a pharmaceutical composition for preventing or treating HIV, which contains one or more compounds selected from the group consisting of the chrysanthemum bud extract or the compound represented by Formula 1 to Formula 5 as an active ingredient.

The pharmaceutical composition of the present invention contains an extract of chrysanthemum buds or a compound selected from the group consisting of Compound 1 to Compound 9 purified therefrom 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 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 to mix the active ingredient with the carrier, to dilute it with the carrier, or to enclose it 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 chrysanthemum buds contained in the pharmaceutical composition of the present invention or the compounds 1 to 9 isolated therefrom 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.

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 Chrysanthemum Bud Extract

Chrysanthemum buds, which are used as herbal medicine in herbal medicine, were purchased in herbal medicine and used for experiments. 600 g of well-dried chrysanthemum buds were immersed in 3 L of methanol, filtered by cold 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 procedure was repeated three times to extract 90.87 g of total methanol. Methanol x 90.87 g is suspended in 1,000 ml of water, 500 ml of dichloromethane (CH ₂ Cl ₂ , 2,000 ml × 3), ethyl acetate (EtOAc, 2,000 ml × 3) and butanol (BuOH, 2,000 ml × 3) Each fraction was divided by repeating extraction three times in sequence according to the solvent polarity.

Example 2 Isolation of Anti-HIV Active Material from Ethyl Acetate Fraction of Chrysanthemum Buds

Since the ethyl acetate fraction of the chrysanthemum bud had the strongest HIV inhibitory activity, 5.37 g of the ethyl acetate fraction of the chrysanthemum bud 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 then the compounds having similar polarities were collected and divided into six subfractions, each named EA to EF. Compound 5 (107.8 mg) was obtained from small fraction EF. The third fraction EC (2,736.8 mg) was subjected to column chromatography using Sephadex. Ethanol was divided into six small fractions (ECa-ECf) using the developing solvent, and the final washing was sufficiently with methanol. Among them, the fourth fraction ECd (96.5 mg) was subjected to column chromatography using Sephadex and preparative reverse phase TLC using 60% methanol as a developing solvent to obtain pure Compound 1 (5.0 mg). .

Small fraction ECc (1,561.3 mg) was subjected to column chromatography using silica gel and divided into five fractions (ECc1-ECc5). The developing solvent was eluted by increasing the amount of methanol and water by increasing the polarity from a mixed solvent of dichloromethane-methanol-water (6: 2: 0.1), and finally washed with methanol sufficiently. Small fractions ECc2 was subjected to column chromatography using reverse phase silica gel (LiChroprep RP-18) using 50% methanol as a developing solvent, and then a dichloromethane-methanol (4: 1) mixed solvent was used as a developing solvent. Preparative column chromatography was performed on a TLC plate to obtain pure compound 3 (11.7 mg) and compound 4 (6.8 mg), respectively. Small fraction ECc3 was subjected to silica gel column chromatography with ethyl acetate-methanol-water = 4: 1: 0.2, and then column chromatography using reverse phase silica gel was performed using a mixed solvent of 30% methanol as a developing solvent. Pure compound 8 (29.8 mg) was obtained by carrying out. Small fraction ECc4 was subjected to column chromatography using reverse phase silica gel using 30% methanol as a developing solvent, to obtain pure compound 9 (11.8 mg). Column chromatography was performed on small fraction ECc5 using reverse phase silica gel in the order of 35%, 50% methanol, followed by column chromatography using silica gel under mixed solvent conditions of ethyl acetate-methanol-water (4: 1: 0.2). Photography was carried out to give pure compound 2 (34.4 mg). Small fraction ED (421.8 mg) was subjected to column chromatography using silica gel and divided into 6 fractions (EDa-EDf). The solvent used was eluted by increasing the amount of methanol by increasing the polarity from a mixed solvent of dichloromethane-methanol (20: 1). The solid formed from the small fraction EDb was washed with methanol sufficiently to obtain pure Compound 6 (27.5 mg). Pure fraction 7 (4.8 mg) was obtained by preparative reverse phase TLC using small fraction EDd as a developing solvent with 60% methanol.

Example 3 Structure Determination of New Compound 1

The structure of new compound 1 was determined. In the measurement of infrared (IR) spectrometer, the sample was mixed with KBr, and the optical intensity [α] ^D was measured in a MeOH solution, 23 ° C. NMR spectra of 500 MHz ( ¹ H) and 125 MHz ( ¹³ C) were measured and the chemical shift of each peak was expressed as a relative value to trimethylsilane, an internal standard. About 5 mg of compound 1 obtained was dissolved in 500 µl of methanol- d ₄ (99.5%) and the carbon nuclear magnetic resonance spectrum was measured to show the peak of characteristic compound (1). In addition, the mass spectrum (ESMS) of the compound (1) shows a peak of 607 ([M-1] ^- ).

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

pale yellow amorphous powder;

[α] ²³ _D + 30.8 ° ( c 0.25, 80% MeOH);

UV (CH ₃ CN) λ _max (log ε) 270 (2.59), 325 (3.39);

IR (KBr) ν _max 3408, 2929, 1604, 1500 cm ⁻¹ ;

Negative-ion mode (FABMS) m / z 607 [M−1] ⁻ ;

HRFABMS (negative-ion mode) m / z obsd. 607.1078 (calcd. For 607.1088, C ₃₀ H ₂₃ O ₁₄ );

¹ H NMR, ¹³ C NMR spectral data and long-range ¹³ C- ¹ H COSY results are shown in Table 1.

Compound 1 was found to be yellow in color when the sulfuric acid in the TLC plate was found to be a flavonoid-based compound, it can be seen that the peak shape in ¹ H NMR flavonoid derivatives. The peak at 6.85 ( J = 8.6 Hz) and the double peak at 7.76 ( J = 8.3 Hz) correspond to two hydrogen peaks, respectively, resulting in H-3 ', 5' and It was found that the peaks corresponded to H-2 'and 6'. The peak at δ 6.69 (br s, H-8) and the peak at δ 6.39 (br s, H-6) were attributed to the A ring of the flavonoids, and the single peak at δ 6.54 (H-3). It could be predicted that the basic skeleton of the flavonoids is apigenin. There is also an olefinic hydrogen with a transcoupling of the peak of δ 6.26 and a peak of 7.51 ( J = 15.8 Hz), δ 6.99 (d, J = 1.6Hz, H-2 ”'in the low-field region. ) And the peak at δ 6.87 (br d, J = 8.5 Hz, H-6 ″ ') are orthocoupled to each other, and the peak of δ 6.71 (d, J = 8.9 Hz, H-5 ″ ′). H-6 "'and meta coupling appeared, indicating that the caffeoyl group (caffeoyl group) is substituted. In the sugar equivalents, the peaks of δ 3.63 and 3.75 (t, 1H, H-2 ″, 3 ″), δ4, including an anmeric peak of δ 5.12 (d, 1H, J = 7.1 Hz). The peak shape of 10 (d, J = 9.2 Hz, H-5 ") and the peak shape of δ 5.08 (m, H-4") show that the peak resulting from H-4 "is the highest in the low field, It was expected that caffeoyl group was substituted.

^{In the 13} C NMR, the peaks of δ 73.9, 74.8, 75.7, 76.1, and 101.3 and carboxyl carbon at δ 172.9 showed that the sugar substituted with this compound was glucuronic acid. In the HMBC spectrum, anomeric hydrogen (H-1 ") has a peak of δ 163.3 (C-7), and a peak derived from H-4" has a peak of δ 169.3 (CO) and a cross peak. It was confirmed that the acid was substituted at position 7 of apigenin and the caffeoyl group at position 4 "of glucuronic acid.

As a result of comparing and analyzing the above results, Compound 1 was the first compound isolated in nature and used as apigenin 7- (4 "-caffeoyl) -β-D-glucuronide [apigenin 7- (4 "-caffeoyl) -β-D-glucuronide] (Huang et al. Phytochemistry 1999, 52 , 1701; Allais et al. Phytochemistry 1991, 30 , 3099; Ahmed et al. Phytochemistry 1989, 28 , 1751).

Example 4 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 immersed in 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 5 HIV Integrase Inhibitory Effects of Chrysanthemum Bud Extracts and Compounds Isolated from the Extracts

The inhibitory effect of HIV integrase and the extracts from methanol extract of chrysanthemum bud and extracts from methanol extract and inhibitory activity against human immunodeficiency virus integrase of the compounds isolated from ethyl acetate were observed. 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 3 shows the results of inhibiting the HIV integrase inhibition efficiency (%) of compounds extracted from methanol extracts and methanol extracts of chrysanthemum buds and the inhibitory effects of compounds separated from ethyl acetate in IC ₅₀ values. IC ₅₀ means the concentration (IC ₅₀ ) which shows the inhibitory effect 50%.

Inhibitory Activities of Extracts from Chrysanthemum Buds and Compounds Isolated from Extracts on Human Immunodeficiency Virus Integrase Fraction  100 μg / ml  25 μg / ml IC ₅₀ (μg / ml) Methanol extract 45-55% 30% less than Dichloromethane fraction 45-55% 30% less than Ethyl acetate fraction More than 70% 55-70% Butanol fraction 55-70% 45-55% Water fraction 30-45% - Compound 1 7.2 ± 3.4 Compound 2 > 100 Compound 3 > 100 Compound 4 15.1 ± 3.4 Compound 5 - Compound 6 > 100 Compound 7 5.88 ± 1.20 Compound 8 16.9 ± 2.0 Compound 9 5.4 ± 2.6 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, 42, 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 3, it was found that the ethyl acetate fraction and butanol fraction of the chrysanthemum buds had an excellent integrase inhibitory effect. In addition, the antiviral effect of Compound 1, a novel compound isolated from the ethyl acetate fraction of chrysanthemum buds, shows an integrase inhibitory effect that is significantly better than curcumin, and slightly superior to L-chicoric acid. The effect was shown. The other compounds also showed an excellent integrase inhibitory effect, so it was found that these compounds are active ingredients in indicating the inhibitory effect of integrase of chrysanthemum buds.

Example 6 Antiviral Activity and Cytotoxicity Test

In accordance with a known method (Tanaka et al ., J. Med. Chem ., 1991, 34 , 349) in order to determine the antiviral activity of the methanol extract of the chrysanthemum bud and extracts fractionated from the methanol extract of the present invention In vitro HIV-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 4.

Inhibitory Effects of Compounds from Chrysanthemum Bud Extracts on HIV Cell Lines compound EC _{50 (} μg / mL) ^a CC ₅₀ (μg / mL) ^b SI ^c Compound 1 42.1 > 150 > 3.56 Compound 8 64.32 135.10 2.10 Compound 9 100 123.79 1.20 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 on MT-4 cells,

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

The antiviral effect of the compounds isolated from the ethyl acetate fraction of the chrysanthemum bud was investigated using chicorylic acid and curcumin as a control drug. As a result, new compound 1 showed EC ₅₀ of 42.1 ㎍ / ml even in HIV cell line. It showed an excellent effect compared to curcumin and chicuric acid. In addition, Compound 8 and Compound 9 also showed significantly better anti-HIV efficacy than curcumin and similar efficacy to Chicolic acid (Table 4). Thus, it was confirmed that these compounds are effective ingredients for showing the anti-HIV efficacy of the ethyl acetate fraction of the chrysanthemum buds.

As described above, the chrysanthemum bud extract of the present invention and the compound isolated therefrom can 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-viral activity and extract of Chrysanthemum morifolium comprising the same <130> 3p-05-40B <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 (9)

A compound represented by the following formula (1) and a pharmaceutically acceptable salt thereof. <Formula 1> delete delete delete delete delete delete 1) obtaining ethyl acetate extract of chrysanthemum buds; 2) performing the column chromatography using a filler selected from the group consisting of silica gel, Sephadex, RP-18, polyamide, Toyopearl, and XAD resin; How to manufacture. HIV activity inhibitor containing the compound of claim 1 as an active ingredient.