KR20040067101A - Flavones, isolated from Artemisia absinthium and their anti-inflammatory constituents containing bioflavonoids - Google Patents

Abstract

PURPOSE: Flavones isolated from Artemisia absinthium and an anti-inflammatory composition containing the same compounds are provided, which compounds is useful for treatment of inflammatory disease such as rheumatic arthritis. CONSTITUTION: The flavones isolated from Artemisia absinthium, 5,6,3',5'-tetramethoxy 7,4'-hydroxy flavones represented by formula (1) are provided. The method for isolating and purifying the flavones isolated from Artemisia absinthium, 5,6,3',5'-tetramethoxy 7,4'-hydroxy flavones represented by formula (1) comprises the steps of: pulverizing Artemisia absinthium and inserting the pulverized Artemisia absinthium in alcohol; concentrating the extract of Artemisia absinthium under reduced pressure, and extracting the concentrate with chloroform and ethylacetate; sequentially subjecting the ethylacetate extract of Artemisia absinthium to silica gel chromatography, RP-18 resin chromatography, Sephadex LH-20 resin chromatography and HPLC.

Description

Compounds obtained from the leaves and anti-inflammatory agents containing them as active ingredients {Flavones, isolated from Artemisia absinthium and their anti-inflammatory constituents containing bioflavonoids}

The present invention relates to a compound obtained from the leaflets and an anti-inflammatory composition containing the same as an active ingredient. More specifically, the compounds 5, 6, 3 ', 5'-tetramethoxy 7, 4 isolated from the leaflets ( Artemisia absinthium ) Inflammatory diseases such as rheumatoid arthritis, utilizing the anti-inflammatory physiological activity of the '-hydroxy flavones (5, 6, 3', 5'- tetra methoxy 7, 4'- hydroxy flavone, p7F) and fractions containing them A therapeutic composition.

Inflammation is an immune response induced by damaged cells or tissues that interacts with local blood vessels and various immune cells in body fluids, causing enzyme activation, secretion of mediators, fluid infiltration, cell migration, and tissue destruction. It is a complex series of biological defense reactions [Arend, Semin Arthritis Rheum 30: 1-6, 2001]. Inflammatory responses are induced by bacterial and viral infections, local ischemia, immune responses by antigen-antibodies, and toxins or chemicals, and as a result, symptoms such as erythema, edema, fever, and pain appear. The inflammatory response is divided into three phases: a short-term acute phase with increased local vasoconstriction and capillary permeability, a delayed subacute phase with infiltration of leucocytes and phagocytes, and tissue degeneration and fibrosis. It is the proliferative chronic phase that occurs (Cuzzocrea, et al ., Pharmacol Rev 51: 135-159, 2001). The inflammatory response involves various immune cells, such as granulocytes (polymorphonuclear leucocytes), mast cells, macrophages and lymphocytes, and various mediators that release them play an important role in the regulation of the inflammatory response [Berenbaum et al ., Exp Cell Res 222: 379-384, 1996. Inflammatory cytokines secreted by macrophages and monocytes, mainly IL-1, TNF-α and IL-6, are known as important parameters in the early stages of inflammation, and phospholipase A ₂ in the next stage. Lipid mediators such as prostaglandin (PG), leucotriene, and many mediators such as reactive oxygen species (ROS) and NO (nitric oxide) produced through the pathway and cyclooxygenase (COX) pathway Are known as important action factors [Egon et al. , Bio Pharmacol 62: 795-801, 2001].

These inflammatory responses are life-dependent self-defense systems for external stimuli, varying depending on the stage of activity, growth and differentiation of immune cells. In this process, cytokines act as important factors in maintaining homeiostasis of immune function, and are involved in the interaction and regulation of immune cells during inflammation. In other words, it is involved in the growth and differentiation of various hematopoietic cells and lymphocytes, induces immune and inflammatory responses to invasion and tissue damage of various pathogens, and has a function of mediating the regulation of the overall immune response. [Dinarello, Chest 112: 321S- 329S, 1997]. Cytokines are divided into hematopoietins, interferon, TNF family, immunoglobulin super family, chemokine, etc., which are involved in hematopoiesis, depending on their function and structure, among which helpers ( helper) The cytokines produced by T cells are divided into Th1 and Th2 type cytokines according to their function. IL-1, IL-2, IL-6, interferon (IFN), and TNF (pro-inflammatory cytokines) belonging to Th1 type are involved in the immunity of macrophages, IL-4, IL-5, IL-10, IL-13 (anti-inflammatory cytokine), etc., are involved in the growth and differentiation of B cells into plasma cells that produce antibodies, or in inflammatory responses and autoimmune diseases caused by tissue damage. and Naoko, Trends in Cell Biology 10: 542-550, 2000]. Th1 and Th2 phenotypes are very important for maintaining the homeostasis of the overall immune system and are expressed by phenotypes such as antigen-presenting cells (APCs), surface proteins, or other types of cytokines. ) Can be changed (Orencole and Dinarello, Toxicology 158: 25-29, 1998). In general, the inflammatory response is characterized by the overexpression of pro-inflammatory cytokine, a Th1 cytokine, and due to the imbalance of inflammatory and anti-inflammatory cytokines It is known to cause forms of inflammatory diseases [Vane and Botting, Inflamm. res 47: S78-S87, 1998]. The secretion and production of inflammatory cytokines involved in the early inflammatory response is determined by programming gene expression in cells and stimulation from outside the cells during the growth and differentiation of immune cells. Pro-inflammatory cytokines activate transcription factors such as NF-κB and AP-1 to induce gene expression of proteins such as COX, NO, iNOS, and PG involved in the inflammatory response [Jue et al. , J Korean Med Sci 14: 231-238, 1999; Renard and Raes, Cell Biol Toxicol 15: 341-344, 1999].

The actions of cytokines, which act as important mediators of inflammation and immune responses, can be regulated in several ways, including how to inhibit the release of inflammatory cytokines by external stimuli and how they stimulate target cells. That is how it is controlled (Steven and Vera, Chest 117: 1162-1172, 2000). Inhibition of inflammatory cytokine production, such as IL-1 or TNF-α, can be induced by anti-inflammatory cytokine expression that down-regulates its expression by intracellular homeostasis or by cytokine inhibitors such as IL-1 receptor antagonists and soluble TNF-receptors. It is reported that the action of cytokines to stimulate the target cells may be regulated through the use of monoclonal antibodies, mutant cytokines, cytokine signaling regulators, etc. [Kox] , et al. , Intensive Care Med 26: S124-S128, 2000]. However, according to the ongoing research, various biological therapeutic agents due to the development of the above substances are still insufficient to expect many effects, and also the mechanisms of regulation of inflammatory and immune responses in which various active factors and cytokines work together The function and action of the regulatory factors that have been discovered so far is poorly understood. The abnormalities of these regulatory mechanisms cause various types of inflammatory diseases, which are necessary to understand the mechanisms of action of these cytokines and to regulate inflammation and immune function using their regulatory substances.

Therefore, the present inventors conducted a long study to develop a material showing a strong inflammatory inhibitory activity against inflammatory diseases such as rheumatoid arthritis and sepsis as a part of the development of a new type of anti-inflammatory agent, has isolated and screened the leaves from natural products From this extract of plants, a single substance that strongly inhibits the inflammatory response was isolated and purified, and the present invention was completed by knowing that the substance and the fraction containing the substance had anti-inflammatory effects.

Therefore, the present invention is anti-inflammatory effect of 5,6,3 ', 5'-tetramethoxy 7,4'-hydroxy flavone (p7F) and fractions containing this substance isolated from the leaves of natural plants The purpose is to develop a therapeutic for the treatment of inflammatory diseases such as rheumatoid arthritis and sepsis.

1 shows the HPLC spectra of compounds 5, 6, 3 ', 5'-tetramethoxy 7, 4'-hydroxy flavones (hereinafter referred to as p7F) according to the present invention.

2 shows the HMBC of p7F.

FIG. 3 compares the degree of cell proliferation that occurs after treatment with Con A, PMA, and PHA mitogens to D10S cells, which are IL-1 dependent mouse Th2 cell lines, and the degree of cell proliferation after IL-1β treatment. It is the result of analysis of IL-1 specificity.

4 is a result of measuring the degree of inhibition of proliferation seen in D10S cells treated with p7F in combination with IL-1β compared to the control group induced the proliferation of D10S cells by treatment with IL-1β.

FIG. 5 shows the gene expression level of pro-inflammatory cytokines such as IL-1β, TNF-α, and IL-6, which is treated with p7F in synovial cells of patients with rheumatoid arthritis having a clear inflammatory response. Is the result measured by the RT-PCR method.

FIG. 6 shows the gene expression level of anti-inflammatory cytokines such as IL-4 and IL-10, which is obtained by treating p7F in synovial cells of rheumatoid arthritis patients with inflammatory response. It is the result measured by the law.

FIG. 7 shows the gene expression level of inflammatory mediators such as ICAM-1, COX-2, and iNOS as treated by p7F in synovial cells of rheumatoid arthritis patients with a clear inflammatory response, measured by RT-PCR. The result is.

8 is a result of measuring the cytotoxicity resulting from treatment of p26F RAW 264.7 macrophages by MTS / PMS (microculture tetrazolium assay) method.

9 is a result of measuring the quantitative change of NO produced according to the concentration of p7F in RAW264.7 macrophages activated by LPS by concentration according to the Greases assay method.

FIG. 10 shows the removal of hydrogen peroxide into RAW 264.7 macrophages treated with different concentrations of p7F, followed by scavenging by the compound by measuring reactive green DCF fluorescence in living cells using a flow cytometer. It is the result which showed the degree of active oxygen becoming.

11 is a result of measuring the quantitative change of COX-2 (cyclooxygenase-2) protein produced according to concentration after p7F treatment in RAW 264.7 macrophages activated by LPS by concentration according to Western blotting method.

12 is a result of measuring the quantitative change of PGE2 produced according to the concentration after p7F treatment by RAW 264.7 macrophages activated by LPS by ELISA method.

The present invention is characterized by 5,6,3 ', 5'-tetramethoxy 7,4'-hydroxy flavone (p7F) separated and purified from Artemisia absinthium .

In addition, an anti-inflammatory composition containing the compound or the active fraction containing the same as an active ingredient is another feature.

Referring to the present invention in more detail as follows.

The present invention is compound 5,6,3 ', 5'-tetramethoxy 7,4'-hydroxy flavone (5,6,3', 5'-tetramethoxy 7,4'- isolated from Artemisia absinthium ) hydroxy flavone (p7F) and its fractions containing anti-inflammatory physiological activity, the present invention relates to a composition for the treatment of inflammatory diseases such as rheumatoid arthritis.

The process of separating and purifying the compound from the Artemisia absinthium according to the present invention is as follows.

1) pulverizing the leaflets and extracting the solvent aqueous solution at room temperature twice put 3 ~ 10 times the aqueous solution of alcohol compared to the leaf content;

2) concentrating the filtrate obtained in the extraction step under reduced pressure, suspending the concentrate (250 g) in distilled water, and then extracting and concentrating with an organic solvent such as chloroform and ethyl acetate;

3) developing the active fraction of ethyl acetate into a mixture of hexane, ethyl acetate, chloroform and methanol in chromatography using silica gel, concentrating the active fraction of chloroform: methanol (50; 1, V / V);

4) developing the active fraction with 60-100% MeOH in chromatography using RP-18 resin, concentrating the active fraction of 70% MeOH;

5) The active fraction was developed with a mixture of chloroform, hexane and methanol by chromatography using Sephadex LH-20 resin to obtain an active fraction from chloroform: hexane: methanol (2; 3; 1, V / V / V). Concentrating;

6) Isolate only the final active fraction using 75% methanol on preparative HPLC (J'sphere ODS-H80, 150 × 20 mm ID, S-4 μm, 80 μs, 254 nm, Simadzu LC-6AD). Concentrating by;

7) The final separation material was analyzed using 45% acetonitrile in analytical HPLC (J'sphere ODS-H80, 250 × 10 mm ID, S-4 μm, 80 μs, 254 nm, Simadzu LC-6AD). Purifying and powdering.

The powder was well soluble in methanol, ethanol, butanol, dimethyl sulfoxide (DMSO) but insoluble in water, hexane, chloroform and the like. As a result of ultraviolet spectrophotometric spectral analysis, the maximum absorption band was shown at 214 and 274 nm. Mass spectrometry (ESI-MS) showed a molecular weight of 374.32 and a molecular formula of C ₁₉ H ₁₈ O ₈ . These results were interpreted from the results of HMBC nuclear magnetic resonance spectrum analysis, and the structural formula is represented by the following Chemical Formula 1, 5,6,3 ', 5'-tetramethoxy 7,4'-hydroxy flavone (5,6,3', 5'-tetramethoxy 7,4'-hydroxy flavone, p7F).

The present invention relates to an anti-inflammatory composition containing the compound represented by the formula (1) or a fraction containing the compound is excellent in the treatment of inflammatory diseases such as rheumatoid arthritis and containing it as an active ingredient.

In addition to the active compositions according to the invention, pharmaceutically acceptable carriers or siblings may be used in the form of tablets, powders, granules, capsules, suspensions, emulsions or unitary or multiple dosage forms for parenteral administration. .

The effective dosage of the active ingredient represented by the active fraction may vary depending on the age, physical condition, weight, etc. of the patient, but is generally administered within the range of 1 to 100 mg / kg (weight) / day. In addition, administration is carried out once a day or divided several times a day within the effective dosage range per day.

Hereinafter, the present invention will be described in more detail based on the following examples, but the present invention is not limited thereto.

Example 1 Isolation and Purification of Compounds Showing Activity from the Leaves

After crushing 2 kg of Artemisia absinthium, the aqueous solution of alcohol was added 10 times compared to the content of the leaf of the leaf. The solvent extracted solution was concentrated under reduced pressure twice at room temperature, suspended in distilled water, and then extracted with ethyl acetate. The active fraction of ethyl acetate was developed by silica gel chromatography into a mixture of hexane, ethyl acetate, chloroform and methanol to obtain an active fraction of chloroform: methanol (50: 1, V / V) and concentrated. The active fractions were developed with 60-100% MeOH in RP-18 column chromatography to obtain active fractions in 70% MeOH. The active fractions were combined and concentrated, and the fractions obtained from chloroform: hexane: methanol (2: 3: 1; V / V / V) were separated by Sephadex LH-20 chromatography. Preparative HPLC (J'sphere ODS-H80, 150 × Concentrate only the final fractions with 75% methanol at 20 mm ID, S-4 μm, 80 μs, 254 nm, Simadzu LC-6AD), followed by analytical HPLC (J'sphere ODS-H80, 250 × 10 mm ID, S -4 μm, 80 μs, 254 nm, Simadzu LC-6AD, 45% acetonitrile, flow rate: 1.2 mL / min) to obtain the above pure compound at a residence time of 29 minutes and in the form of a pale yellow powder upon concentration under reduced pressure. Could get

Example 2 Structural Analysis of a Compound Showing Activity

Ultraviolet spectrophotometric analysis of the isolated compound showed the maximum absorption band at 214 and 274 nm, and mass spectrometry (ESI-MS, negative) showed a molecular weight of 373.32, resulting in a molecular weight of 374.32 and a molecular formula of C ₁₉ H ₁₈ O ₈ . Decided.

¹ H-NMR spectrum and ¹³ C-NMR spectrum of the isolated compound were measured. ^When measuring the ¹ H-NMR spectrum, four methoxy peaks were observed at 3.98 ppm, 3.94 ppm (2) and 3.78 ppm, and four aromatic hydrogens were observed at 7.41 ppm, 7.73 ppm, and 7.07 ppm. Three methoxy near 56.1 ppm and one methoxy peak at 59.1 ppm were observed in the ¹³ C-NMR spectrum. To verify the relationship between ¹ H and ¹³ C, heteronucelar multiple-bonding correlation (HMBC) was performed. 7.73 ppm of hydrogen was associated with 128.6 ppm and 131.6 ppm of carbon, and 7.41 ppm of hydrogen was associated with 167.8 ppm, 147 ppm, 140 ppm, 105.4 ppm, and 99.2 ppm.

Compounds isolated from these structural analysis results were 5,6,3 ', 5'-tetramethoxy 7,4'-hydroxy flavone (5,6,3', 5'-tetramethoxy 7,4'-hydroxy flavone, p7F).

Formula 1

Example 3: Determination of Inhibition of D10S Cell Proliferation by p7F

(Step 1) IL-1 Specificity-Analysis of IL-1 Specificity of D10S Cells, a Mouse Th2 Cell Line

IL-1 dependent D10S cells were used in the experiments to find substances from a variety of natural plants that exhibit anti-inflammatory effects by modulating the actions of mediators and cytokines of the inflammatory response. In order to determine whether the D10S cells used before the experiment specifically reacted only with IL-1 and not with other mitogens, mitogens of Con A, PMA and PHA and IL-1β were applied to D10S cells, respectively. The proliferative responses of the cells, which differed accordingly, were compared and analyzed. First, D10S cells were dispensed in a 96-well flat microtiter plate (Costar) to 2.5 × 10 ⁴ cells / well, then Con A 5 ng / ml, PMA 10 ng / ml, PHA 10 ng Cells were incubated for 48 hours in a 37%, 5% CO ₂ incubator treated with / ml of mitogen and IL-1β2 ng / ml. After incubation, tetrazolium complex MTS [3- (4,5-dimethylthiazol-2-l) -5- (3-carboxymeth-oxyphenyl) -2- (4-sulfophenyl

) 2H-tetrazolium] and 20 µl of a mixture of phenazine methosulfate (PMS), an electron binding reagent, are added to each well and incubated for another 4 hours, and the resulting color change of the medium is 490 nm using an ELISA reader. Measured at As a result, the IL-1β-treated D10S cells showed a high proliferative effect compared to the control group treated with RPMI 1640 added only 5% FBS. In contrast, no cell proliferation was achieved by mitogen treatment of Con A, PHA, and PMA [Fig. 3]. These results indicate that D10S cells respond specifically by IL-1.

(Step 2) Effect of p7F on proliferation of D10S cells

First, D10S cells were seeded in a 96-well flat microtatier plate (Costar) to 2.5 × 10 ⁴ cells / well, and then various concentrations of p7F 2.5, 5, 10, 15, 20, 25, 30 μg / ml Cells were incubated for 48 hours in a 37 ° C., 5% CO ₂ incubator with human IL-1β2 ng / ml. After incubation, 20 μl of tetrazolium complex MTS and an electron binding reagent PMS mixture were added to each well and incubated for another 4 hours, and the color change of the resulting medium was measured at 490 nm using an ELISA reader. The results are shown in FIG. This figure shows the inhibitory effect of IL-1 activity on the isolated and purified p7F. In order to observe the IL-1 specific response of D10S cells, only p7F was treated purely to show the degree of inhibition of cell proliferation. p7F and IL-1 were treated together, and the experiment was performed by dividing into two groups of the group showing the degree of inhibition of proliferation of IL-1-induced D10S cells by p7F. According to the proliferative capacity of D10S cells, p7F inhibited the proliferation of D10S cells induced by IL-1β in a concentration-dependent manner. These results confirm that p7F is a physiologically active substance that exhibits anti-inflammatory effect by inhibiting the activity of IL-1, one of pro-inflammatory cytokines.

Example 4: Analysis of anti-inflammatory efficacy on p7F

(Step 1) RNA Extraction from Synovial Cells in Patients with Rheumatoid Arthritis

Synovial cells treated with various concentrations of samples were collected by trypsin-EDTA and then easy-BLUE^TMTotal cellular RNA was extracted using the solution (Intron Biotech) and the extraction method was as follows. First, synovial cells (5 × 10) in patients with rheumatoid arthritis⁴After all the cells are collected, easy-BLUE to each cell^TM1 ml was added and mixed for about 30 seconds at room temperature. 200 µl of chloroform was added to the mixture, and the mixture was mixed well, followed by centrifugation for 10 minutes in a table top centrifuge (12,000 rpm) to separate 400 µl of the supernatant. An equal amount of isopropanol was added thereto and mixed for about 10 seconds, followed by centrifugation (12,000 rpm) for 10 minutes. RNA pellets were obtained and washed twice with ice-cold 70% ethanol. After removing ethanol, RNA was dissolved in distilled water treated with DEPC (diethylpyrocarbonate), and then the concentration and purity of the RNA were measured using a UV spectrophotometer. Distilled water and vitreous used to prevent RNase contamination were treated with DEPC or sterilized. Integrity of the extracted RNA was confirmed by 18S and 28S RNA bands by electrophoresis on a 1.2% denatured agarose gel.

(Step 2) Gene Expression Analysis of Inflammatory Cytokines by p7F

Treatment of synovial cells of rheumatoid arthritis patients with a pronounced inflammatory response to p7F (30 μg / ml) resulted in pro-inflammatory cytokines such as IL-1β, TNF-α and IL-6. Gene expression profile was measured by RT-PCR assay. First, cDNA was synthesized from RNA obtained from articular synovial cells, and PCR was performed using specific primers of Table 1 prepared for detecting mRNA of various inflammation-related cytokines and mediators.

Target Primer sequence Size (bp) IL-1 5'-primer 5'-CAG-CAG-TTG-GTC-ATC-TCT-TG-3 '(SEQ ID NO: 1) 3'-primer 5'-CCA-GCA-GGT-GAA-ACG-TCC-A-3 '(SEQ ID NO: 2) 802 TNF- 5'-primer 5'-GGA-AAG-GAC-ACC-ATG-AGC-AC-3 '(SEQ ID NO: 3) 3'-primer 5'-ATG-TTC-GTC-CTC-CTC-ACA-GG-3 '(SEQ ID NO: 4) 727 IL-6 5'-primer 5'-ATC-CTC-GAC-GGC-ATC-TCA-GCC-3 '(SEQ ID NO: 5) 3'-primer 5'-CTA-CAT-TTG-CCG-AAG-AGC-CCT-3 '(SEQ ID NO: 6) 462 GAPDH 5'-primer 5'-ACC-ACA-GTC-CAT-GCC-ATC-AC-3 '(SEQ ID NO: 7) 3'-primer 5'-TCC-ACC-ACC-CTG-TTG-CTG-TA-3 '(SEQ ID NO: 8) 500

M-MulV reverse transcriptase was used to synthesize cDNA from the extracted RNA. In order to separate Poly (A ⁺ ) mRNA from total RNA, 0.125 μM of oligo dT primer was used, and 5 μg of the obtained mRNA was MgCl ₂ 5 mM, 1 × RNA PCR buffer, dNTP mixture 1 mM, and 1 unit / μl of RNase inhibitor. Was added and reacted at 37 ° C. for 2 hours. Finally, cDNA was obtained by inactivating residual proteins by heating at 95 ° C. for 5 minutes. The synthesized cDNA was subjected to PCR amplification using the primers of Table 1, and the obtained PCR product was electrophoresed on 1.5% agarose gel and stained with EtBr (ethidium bromide) to confirm expression of a target gene.

In this experiment, after treatment with IL-1β (2 ng / ㎖) and TNF-α (10 ng / ㎖) and p7F (30 ㎍ / ㎖), inflammatory cytokines (TNF-α) appearing according to the above method , IL-1, IL-6) by comparing the expression levels of the genes were investigated the ability of the compound to regulate the inflammatory cytokine. As a result, inflammatory cytokines (TNF-α, IL-1β, IL-6) in cells treated with p7F only (group 2) compared to cells treated with DMEM medium added with 5% FBS (group 1) only Expression decreased to 0-20%. These inflammatory cytokine genes were markedly increased in comparison with the control group following stimulation of the inflammatory cytokines IL-1β and TNF-α (groups 3 and 5). In contrast, the expression of inflammatory cytokines was reduced in IL-1β and TNF-α treated cells (groups 4 and 6) after pretreatment of p7F compared to that in group 3 [FIG. 5]. In conclusion, expression of TNF-α, IL-1β, and IL-6 genes induced by IL-1β was inhibited by 11.4%, 21.9%, and 15.2%, respectively, following pretreatment of p7F. Gene expression was inhibited by 75.8%, 36% and 20%, respectively.

(Step 3) Gene expression analysis of anti-inflammatory cytokines by p7F

Gene expression patterns of anti-inflammatory cytokines, such as IL-4 and IL-10, were shown in RT-PCR when 30 μg / ml of p7F was treated in synovial cells of rheumatoid arthritis patients. Was measured. First, cDNA was synthesized from RNA obtained from articular synovial cells, and PCR was performed using specific primers of Table 2 prepared for detecting mRNA of anti-inflammatory cytokines. M-MulV reverse transcriptase was used to synthesize cDNA from the extracted RNA. First, 0.125 μM of an oligo dT primer was used to separate Poly (A ⁺ ) mRNA from total RNA. To 5 μg of the obtained mRNA, MgCl ₂ 5 mM, 1 × RNA PCR buffer, dNTP mixture 1 mM, and RNase inhibitor 1 unit / Μl was added and reacted at 37 ° C. for 2 hours. Finally, cDNA was obtained by inactivating residual proteins by heating at 95 ° C. for 5 minutes. The synthesized cDNA was subjected to PCR amplification reaction using the primers of Table 2, and the obtained PCR product was electrophoresed on 1.5% agarose gel and stained with EtBr to confirm expression of a target gene.

In this experiment, anti-inflammatory cytokine (IL-4, IL-10) genes were treated following treatment with IL-1β2 ng / ml, TNF-α10 ng / ml and p7F 30 ug / ml. The aim of this study was to determine the anti-inflammatory cytokine regulatory capacity of p7F by comparing their expression levels. As a result, in the cells treated with p7F only (Group 2), the expression of IL-4 and IL-10 was significantly increased by 100% or more, compared with cells treated with DMEM medium containing only 5% FBS (Group 1). It could be confirmed [Fig. 6]. In contrast, the expression of anti-inflammatory cytokines (IL-4, IL-10) genes in cells treated with the inflammatory cytokines IL-1β and TNF-α (group 3) resulted in inflammatory cytokines (TNF-). Unlike the α, IL-1β, IL-6) genes were confirmed that not increased.

Target Primer sequence Size (bp) IL-4 5'-primer 5'-ATG-GGT-CTC-ACC-TCC-CAA-CT-3 '(SEQ ID NO: 9) 3'-primer 5'-TTC-AGC-TCG-AAC-ACT-TTG-AA-3 '(SEQ ID NO: 10) 463 IL-10 5'-primer 5'-CTT-TCA-AAT-GAA-GGA-TCA-GC-3 '(SEQ ID NO: 11) 3'-primer 5'-AAA-CTC-ACT-CAT-GGC-TTT-GT-3 '(SEQ ID NO: 12) 322 GAPDH 5'-primer 5'-ACC-ACA-GTC-CAT-GCC-ATC-AC-3 '(SEQ ID NO: 7) 3'-primer 5'-TCC-ACC-ACC-CTG-TTG-CTG-TA-3 '(SEQ ID NO: 8) 500

(Step 4) Gene expression analysis of inflammatory mediators by p7F

RT-PCR measures the gene expression of inflammatory mediators such as ICAM-1, COX-2, and iNOS as treated with 30 μg / ml of p7F in synovial cells of patients with rheumatoid arthritis with a pronounced inflammatory response. It was. First, cDNA was synthesized from RNA obtained from joint synovial cells, and PCR was performed using specific primers of Table 3 prepared for detecting mRNA of various inflammation-related cytokines and mediators. In order to separate Poly (A ⁺ ) mRNA from total RNA, 0.125 μM of oligo dT primer was used, and 5 μg of the obtained mRNA was MgCl ₂ 5 mM, 1 × RNA PCR buffer, dNTP mixture 1 mM, and 1 unit / μl of RNase inhibitor. Was added and reacted at 37 ° C. for 2 hours. Finally, cDNA was obtained by inactivating residual proteins by heating at 95 ° C. for 5 minutes. The synthesized cDNA was subjected to PCR amplification using the primers of Table 3, and the obtained PCR product was electrophoresed on 1.5% agarose gel and stained with EtBr to confirm the expression of the target gene.

In this experiment, the gene expression levels of inflammatory mediators (ICAM-1, COX-2, iNOS) were compared after treatment with IL-1β2 ng / ml, TNF-α10 ng / ml and p4F 30 ug / ml. By doing so, we tried to find out the regulatory capacity of inflammatory mediators of p7F. As a result, the expression of inflammatory mediators (ICAM-1, COX-2, iNOS) was reduced by 0-10% in cells treated with only p7F (Group 2) compared with cells treated with only cells (Group 1). The genes of these inflammatory mediators significantly increased their expression following stimulation of the inflammatory cytokines IL-1β and TNF-α (groups 3 and 5). In contrast, the expression of inflammatory mediators was reduced in IL-1β and TNF-α-treated cells (groups 4 and 6) after p7F pretreatment [Fig. 7]. In summary, expression of ICAM-1, COX-2, and iNOS genes induced by IL-1β was inhibited by 21.5%, 46.4%, and 40.6%, respectively, following pretreatment of p7F, and the expression of these genes induced by TNF-α. Expression was inhibited 25.5%, 54.2% and 73% by p4F, respectively.

Target Primer sequence Size (bp) ICAM-1 5'-primer 5'-GGC-CTC-AGC-ACG-TAC-CTC-TA-3 '(SEQ ID NO: 13) 3'-primer 5'-TGC-TCC-TTC-CTC-TTG-GCT-TA-3 '(SEQ ID NO: 14) 350 COX-2 5'-primer 5'-AAA-AGA-ACG-TTC-GAC-TGA-ACT-G-3 '(SEQ ID NO: 15) 3'-primer 5'-AGT-ATT-CAA-ACA-TCT-TTA-CTT -TCG-TTC-3 '(SEQ ID NO: 16) 334 iNOS 5'-primer 5'-TTT-CCT-TAC-GAG-GCG-AAG-AA-3 '(SEQ ID NO: 17) 3'-primer 5'-TGA-AGT-GGT-GCA-CTC-AGC-A-3 '(SEQ ID NO: 18) 372 GAPDH 5'-primer 5'-ACC-ACA-GTC-CAT-GCC-ATC-AC-3 '(SEQ ID NO: 7) 3'-primer 5'-TCC-ACC-ACC-CTG-TTG-CTG-TA-3 '(SEQ ID NO: 8) 500

Example 5 Analysis of Antioxidant Activity of p7F

(Step 1) RAW 264.7 cytotoxicity assay of p7F

p7F was treated with RAW 264.7 macrophages and the resulting cytotoxicity was measured by MTS / PMS (microculture tetrazolium assay). First, RAW 264.7 cells were seeded in a 96-well flat microtiter plate (Costar) to 2.5 × 10 ⁴ cells / well, and then various concentrations of p4F 5, 10, 15, 20, 25, 50, 100 μg / The cells were treated and cultured for 48 hours at 37 ° C., 5% CO ₂ incubator. After incubation, 20 μl of a tetrazolium complex MTS and an electron binding reagent PMS mixture were added to each well for 4 hours, followed by incubation for 4 hours. The color change of the medium was 490 nm using an ELISA reader. Measured at Stimulation of RAW 264.7 cells at various concentrations (0-100 μg / ml) for 24 hours to determine the cytotoxicity of p7F showed no decrease in concentration-dependent cell viability [FIG. 8]. Based on these results, up to 100 μg / ml concentration of p7F was used for the experiment for 24 hours.

(Step 2) NO Production Inhibition Effect Analysis for p7F

After processing p7F by concentration in RAW 264.7 macrophages activated by LPS, the quantitative change of NO produced accordingly was measured according to the Greek assay. NO is a physiologically important secondary signaling agent secreted by many cells and regulates the activity of COX and synergistically acts on the inflammatory response [Evans & Stefanovic-Racic, 1996]. To examine the effect of p7F on NO production, one of the standard factors that can be judged as an indicator of the inflammatory response, 25, 50, 100 μg / ml and 100 ng / ml concentrations of LPS were applied to RAW 264.7 cells for 24 hours and After 48 hours of treatment, the culture of the cells was taken to determine the content of NO. As a result, no change in the NO content of the control group (treated with DMEM medium containing only 5% FBS) was observed, and it was confirmed that NO production was increased according to the LPS treatment. However, it was confirmed that NO production was inhibited in a concentration-dependent manner in cells pretreated with p7F at concentrations of 25, 50, and 100 μg / ml compared to the amount of NO produced by stimulation of LPS [FIG. 9].

(Step 3) Analysis of Reactive Oxygen Species (ROS) Efficacy of p7F

After adding hydrogen peroxide to RAW 264.7 macrophages treated with p7F by concentration, the active green cell was scavenged by p4F by measuring the reactive green DCF fluorescence in live cells using a flow cytometer. The degree of oxygen was measured. Reactive reactive oxygen, which is converted by energy metabolism, drug metabolism, stress, and chemicals, is known to cause various diseases and aging such as brain disease, heart disease and inflammation, rheumatoid arthritis, autoimmune disease, etc. [Cuzzocrea] et al ., 2001. In this experiment, DCFH-DA analysis (2 ', 7'-dichlorofluorescein diacetate assay) was performed to examine the scavenging effect of reactive oxygen species (ROS) according to p4F. Treatment of RAW 264.7 cells with 250 μM of H ₂ O ₂ for 10 minutes significantly increased fluorescence. On the other hand, in H ₂ O ₂ treated cells after 25,50 and 100 μg / ml pretreatment, the concentration-dependent decreases in the proportions of 61.2%, 89% and 94.5%, respectively, compared to the control group. [FIG. 10].

(Step 4) CO7-2 protein production inhibition effect analysis by p7F

COX-2 is an inflammatory mediator that selectively increases expression in cells activated by LPS and anti-inflammatory cytokines (IL-1 IL-2, TNF-α) and various inflammatory mediators, and the steroidal anti-inflammatory DEX (dexamethasone) selectively reduced (Han, 2001). Based on these findings, p7F was treated by concentration (25, 50, 100 μg / ml) in RAW 264.7 macrophages activated by LPS, and then the amount of COX-2 protein produced according to Western blot Measured. First, RAW 264.7 macrophages were stimulated with LPS (100 ng / ml), and then Western blot analysis was performed using an antibody that obtained the cytoplasm of cells treated with the compound and specifically reacted with COX-2 protein. As a result, expression of COX-2 protein was not found in the control group which did not process the sample, and expression of COX-2 protein was induced by treatment with LPS. However, in the cells treated with LPS and the compound at the same time, the expression of COX-2 protein was confirmed to decrease in a concentration-dependent manner compared to the LPS treatment group [Fig. 11].

Step 5) PGE by p7F ₂ Protein production inhibitory efficacy analysis

COX-2 is an inflammatory mediator, which selectively increases expression in cells activated by LPS and anti-inflammatory cytokines (IL-1 IL-2, TNF-α) and various inflammatory mediators and is a steroidal anti-inflammatory It is selectively reduced by DEX [Han, 2001]. Based on these findings, we treated p26F by concentration (25, 50, 100 μg / ml) in RAW 264.7 macrophages activated by LPS, and then measured the amount of COX-2 protein produced according to Western blotting. Measured accordingly. First, RAW 264.7 macrophages were stimulated with LPS (100 ng / ml), and then Western blots were performed using an antibody that obtained cytoplasm of cells treated with p7F and specifically reacted with COX-2 protein. As a result, expression of COX-2 protein was not found in the control group which did not process the sample, and expression of COX-2 protein was induced by treatment with LPS. However, in the cells treated with LPS and p7F at the same time, the expression of COX-2 protein was confirmed to be concentration-dependently reduced compared to the LPS treatment group [FIG. 12].

Example 6 Arthritis Therapeutic Effect

DBA / 1 mice were imported from Charles Liver, Japan, 5 weeks old, and adapted to the Yonsei experimental animal breeding room for 2 weeks, and then used for experiments from 7 weeks old (20 to 25 g).

In order to induce collagen induced arthritis (CIA), isolated chicken type II collagen [Sigma, St. Louis, MO, USA] was dissolved in 0.01 N acetic acid to a concentration of 2 mg / mL and stirred for 18 hours at 4 ℃ and then the same amount of Freund's supplement [Sigma, St. Louis, MO, USA] was mixed into an emulsion. 100 μl of this was intradermally injected near the base of the mouse tail and further injected in the same manner three weeks after the first injection.

The experimental animals were divided into three groups. Group 1 inoculated collagen with p7F (20 mg / kg) twice a week orally while inducing arthritis, and group 2 administered dexamethasone (1 mg / kg) twice a week intraperitoneally with collagen, 3 The group received no treatment with collagen, causing arthritis.

Arthritis incidence rate and arthritis index evaluation was performed as follows.

The incidence of arthritis at each time period was examined by visual observation of redness, swelling and malformation of the feet. In addition, the CIA score was determined by general visual inspection. In other words, when there is no evidence of arthritis such as redness or swelling, 0 points and 1 point cause redness with swelling on one or two toes, or minimal swelling, and 2 points have obvious redness or local upper extremity swelling. This was observed in several toes. Three points showed significant swelling and swelling up to the knee and the foot was not free to use. The scores of each foot were evaluated by four feet and summed up to a maximum of 12 points. In order to reduce the bias of the experimenter, scores were obtained by consensus in the presence of at least three members.

As a result, at the 5th week of the experiment, group 1 showed only one joint index on the forelimb and only two of them were normal [arthritis incidence 33%, mean arthritis index 0.33]. There was slight redness [one point for each joint index] and the other three were normal [arthritis incidence rate 40%, mean arthritis index 0.4]. 5, 4, 4, 4 points [arthritis incidence 100%, mean arthritis index 4.6].

As indicated above, p7F has been observed to have a clear therapeutic effect on arthritis induced in mice.

Example 7: Toxicity Test

Toxicity experiments were performed on the compounds of the present invention as follows.

p7F was dissolved in dimethylsulfoxide (DMSO), diluted with water, and then administered to the mice (10 mice per group) at 100 mg / kg each for 7 days. No rats died.

Example 8 Preparation of Tablets

10 g of active ingredients

70 g of lactose

15 g of crystalline cellulose

5 g of magnesium stearate

100 g in total

The ingredients were crushed finely and mixed to prepare tablets by the direct tableting method. The total amount of this tablet was 100 mg, of which 10 mg was an active ingredient.

Example 9 Preparation of Powder

10 g of active ingredients

50 g of corn starch

40 g of carboxy cellulose

100 g in total

The powder was prepared by pulverizing the above components and mixing. A capsule was prepared by putting 100 mg of powder into 5 hard capsules.

As described above, 5,6,3 ', 5'-tetramethoxy 7,4'-hydroxy flavone and a fraction containing the same, which are compounds separated and purified from Artemisia absinthium according to the present invention, inhibit inflammation. There is an action.

<110> KOREA RESEARCH INSTITUTE OF BIOSCIENCE AND BIOTECHNOLOGY <120> Flavones, isolated from Artemisia absinthium and their          anti-inflammatory constituents containing bioflavonoids <160> 18 <170> KopatentIn 1.71 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 5 'PRIMER <400> 1 cagcagttgg tcatctcttg 20 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> 3'-PRIMER <400> 2 ccagcaggtg aaacgtcca 19 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 5'-PRIMER <400> 3 ggaaaggaca ccatgagcac 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 3'-PRIMER <400> 4 atgttcgtcc tcctcacagg 20 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> 5'-PRIMER <400> 5 atcctcgacg gcatctcagc c 21 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> 3'-PRIMER <400> 6 ctacatttgc cgaagagccc t 21 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 5'-PRIMER <400> 7 accacagtcc atgccatcac 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 3'-PRIMER <400> 8 tccaccaccc tgttgctgta 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 5'-PRIMER <400> 9 atgggtctca cctcccaact 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 3'-PRIMER <400> 10 ttcagctcga acactttgaa 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 5'-PRIMER <400> 11 ctttcaaatg aaggatcagc 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 3'-PRIMER <400> 12 aaactcactc atggctttgt 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 5'-PRIMER <400> 13 ggcctcagca cgtacctcta 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 3'-PRIMER <400> 14 tgctccttcc tcttggctta 20 <210> 15 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> 5'-PRIMER <400> 15 aaaagaacgt tcgactgaac tg 22 <210> 16 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> 3'-PRIMER <400> 16 agtattcaaa catctttact ttcgttc 27 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 5'-PRIMER <400> 17 tttccttacg aggcgaagaa 20 <210> 18 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> 3'-PRIMER <400> 18 tgaagtggtg cactcagca 19

Claims (3)

5,6,3 ', 5'-tetramethoxy 7,4'-hydroxy flavone represented by the following formula (1). Formula 1 Next, an anti-inflammatory composition comprising 5,6,3 ', 5'-tetramethoxy 7,4'-hydroxy flavone represented by Formula 1 as an active ingredient. Formula 1 An anti-inflammatory composition comprising an active fraction obtained from the leaves of Artemisia absinthium as an active ingredient.