KR20080092263A - Cox-2 and inos proteins expression inhibitor - Google Patents

Abstract

A therapeutic composition comprising an extract of Tabebuia avellanedae is provided to inhibit prostaglandin E2 and NOx in macrophages from being generated by inhibiting expression of COX-2 and iNOS proteins in a transcription level, thereby showing anti-inflammatory and analgesic effects ultimately. An expression inhibitor of COX-2 and iNOS proteins comprises an extract of Tabebuia avellanedae, which is prepared by extracting the Tabebuia avellanedae with hot-water at a temperature of 90-95 deg.C for 2-3 hours. A composition for treating inflammatory diseases such as arthritis comprises the expression inhibitor of COX-2 and iNOS proteins as an effective ingredient.

Description

COX-2 and iNOS proteins expression inhibitor}

The present invention relates to inhibitors for the expression of COX-2 and iNOS proteins and compositions for the prevention and treatment of inflammatory diseases containing them.

In particular, the present invention relates to COX-2 protein expression inhibitors and iNOS protein expression inhibitors, which concentration-dependently reduce the production of prostaglandin E2 and nitric oxide, respectively, in macrophages. In addition, the present invention also includes a therapeutic composition for inflammatory diseases comprising such COX-2 and iNOS protein expression inhibitors.

Inflammation is a defense mechanism in vivo against external attacks caused by in vivo attack of external microorganisms or skin damage. However, chronic inflammation plays a central role in major inflammatory diseases such as bronchitis and arthritis. Often, in relation to inflammatory diseases, macrophage plays a central role in the inflammatory response, and macrophages initiate inflammatory reactions by producing arachidonic acid within the cell by external stimuli such as viruses and bacteria, It is known that -1 (cyclooxygenase-1) and COX-2 (cyclooxygenase-2) are expressed and are involved in the metabolism of arachidonic acid to secrete prostaglandin E ₂ (see FIG. 1).

Prostaglandins were first known as substances that help the uterine contractile function in the endometrium, but now they are attracting attention as inflammation-transmitting substances secreted by cell damage and increased pain during prostaglandin secretion. In addition, nitric oxide and TNF-α are known to be involved in the inflammatory response, and transcriptional level regulation and activation of inducible nitric oxide synthase (iNOS, hereinafter referred to as "iNOS") and phosphatase, etc. Regulation is known to be very important in relation to inflammatory responses.

In general, in the development of therapeutic and analgesic agents for inflammatory diseases, immune cells such as macrophages are stimulated with external stimulants such as lipopolysaccharide (LPS) to produce NO (nitric oxide), TNF-α, prostaglandin E2, etc. in the cells. After establishing a model to confirm whether or not the administration of an inflammatory disease treatment agent and / or analgesic candidate substance inhibits the production of NO (nitric oxide), TNF-α, prostaglandin E2, etc. in this model to anti-inflammatory A method of evaluating action and / or analgesic effects is adopted.

Recently, however, at the molecular level, target proteins involved in the production of NO (nitric oxide), TNF-α and prostaglandin E2 have been identified, and the expression levels of the target proteins are regulated or the activation levels of the expressed target proteins are regulated. There is an active research to find candidate anti-inflammatory and analgesic agents.

In other words, in the field of developing inflammatory diseases and / or analgesics such as arthritis, new drug candidates in LPS-stimulated macrophages inhibit the production of NO (nitric oxide), TNF-α and / or prostaglandin E2. In recent years, new drug research and development of molecular biological methods to find target proteins related to inflammatory diseases and to find new drug candidates that regulate the transcription and / or activation levels of these target proteins have been in the spotlight. I am getting it.

Meanwhile, as an anti-inflammatory agent, nonsteroidal anti-inflammatory drugs (NSAIDs) such as indomethacin and aspirin, which inhibit prostaglandin synthesis, have been mainly used, and steroid agents have been used as secondary prescriptions.

However, these drugs have a variety of side effects, particularly serious side effects of gastrointestinal disorders, liver and kidney dysfunction. For example, the use of nonsteroidal anti-inflammatory drugs inhibits the activity of COX enzyme, a target protein involved in the production of prostaglandin E2, which reduces the release of prostaglandins, thereby relieving inflammation and reducing pain, while non-steroidal anti-inflammatory drugs prevent gastric mucosa There is also a risk of producing gastric ulcers by reducing the production of secreted enzymes. Indomethacin, which is used for arthritis, is widely used as a patch rather than oral. As a result, these existing products have a problem that their use as a medicine is limited and may cause inconvenience in use.

Therefore, in the technical field to which the present invention belongs, it is possible to find a target protein associated with nitric oxide and / or prostaglandin production and to inhibit the expression of the target protein at the transcription level or to interact with the target protein. There is an urgent need to develop new drug candidates for the treatment of new anti-inflammatory and / or pain inhibitors.

Accordingly, the present inventors have been directed to inhibitors that inhibit the expression of COX-2 proteins known to be involved in the production of prostaglandin E2 and the expression of iNOS proteins known to be involved in the production of nitric oxide and / or activation inhibitors of these proteins. Explored.

As a result, hydrothermal extracts of Tabebuia avellanedae [Gapi-Taheebo; Trade name] inhibits COX-2 protein expression and iNOS protein expression at the transcription level. In addition, it was confirmed that the hot water extract of Tabebuia avellanedae inhibits the expression of COX-2 protein and iNOS protein, thereby inhibiting the production of prostaglandin E2 and nitric oxide.

In addition, the present inventors have confirmed that the hydrothermal extract of Tabebuia avellanedae has anti-inflammatory and analgesic effects in an animal model related to anti-inflammatory and analgesic agents, and thus the tahibo hydrothermal extract is used as an inhibitor of expression of COX-2 and iNOS proteins. The possibility of new inflammation-related metabolism as an indicator for the discovery of target proteins has also been identified.

Taheebo is a term used to refer to the natural tree or the tree itself, which is made from the inner bark of the Tabebuia avellanedae , a tree of Jacaranda, native to some parts of the Amazon. The history of Tahibo's use in folk medicine goes back 1500 years ago, and the ancient Inca empires are said to have drank Tahibo as tea for disease, wounds, and health.

Currently, the pharmaceutical industry and the medical community are actively conducting research to discover and extract herbal ingredients with new therapeutic effects from natural products.

In the current clinical trials, Tahibo is used in the treatment of cancer because it is effective in strengthening the immune system, increasing appetite, and strengthening blood. As a result of the analysis of Tahibo's ingredients in modern science, Tahibo was found to contain an anticancer substance called NFD of the kineon family. To date, NFD has been shown to prevent normal cells from becoming cancerous without harming normal cells in humans. In addition to lung cancer, Tahibo has been shown to inhibit the activity of 21 types of cancer cells, and research into the potential of anticancer drugs is being actively conducted. However, studies on the efficacy and physiological metabolism of Tahibo's NFD components were mainly related to the cancer development process and cancer treatment.

On the other hand, the pharmacological properties of the β-lapaconone of the quinone family obtained from the bark of Tahibo are known. Beta-rapacone inhibits the proliferation of cultured prostate cancer cells and apoptosis of cancer cells. It is known to cause). The study of the causes of metastasis and inhibition of prostate cancer cell proliferation and apoptosis by bepa-lapacon may provide important clues to the cause and treatment of cancer. Epidemiologic investigations have shown that the administration of NSAIDs reduces the risk of cancer. Cancer cells can be obtained by reducing the risk of cancer and the pharmacological mechanisms known for non-steroidal anti-inflammatory drugs. Research on the proliferation process and related metabolism have been actively conducted. As a result of these studies, it was found that overexpression of COX-2, which is involved in the pharmacological mechanisms of nonsteroidal anti-inflammatory drugs, causes tumor development in animal models and disruption of COX-2 metabolism reduces cancer development and progression. Based on these findings, a study was conducted on whether proliferation inhibitory and apoptosis effects of beta-rapacone-induced prostate cancer cells are related to the inhibition of COX-2 expression and activity. Inhibition of COX-2 expression and concomitant inhibition of production of prostaglandin E2 was confirmed to be consistent with the results that beta-rapacone interferes with cancer cell growth and induces apoptosis (Ref. Jae Hun Lee et al. ., Down-regulation of cyclooxygenase-2 and telomerase activity by β-lapachone in human prostate carcinoma cells, Pharmacological Research 51 (2005) 553-560).

However, studies on the efficacy and physiological metabolism of Tahibo's beta-rapacone components have been related to the role of beta-rapacone in the metabolism of cancer cells and to the inhibition and proliferation of cancer cell proliferation. The involvement of tahibo in metabolism and the role of tahibo in the inflammatory response of macrophages is unknown. In particular, the role of the Tahibo component in the expression of COX-2 and iNOS proteins involved in prostagladin E2 production and nitric oxide production in macrophages is unknown. However, it is known that Tahibo has been used for a long time as an analgesic and inflammatory therapy in Brazil as a folk remedy, and there are only reports that Tahibo appears to have an anti-inflammatory effect by binding to adenosine receptors in connection with adenosine metabolism.

In addition, a study on the efficacy of the Tabevia plant in relation to the role of the component of Tahibo in the inflammatory response has been conducted (36th Japanese Rheumatology Society, May 1992). There was no clarification or suggestion of the inflammatory response and its role in metabolism, only reports of weight gain and clinically effective rheumatism in rats treated with Tabevia plants.

In addition, US Patent Publication No. 6,274,176 (August 14., 2001) discloses an anti-inflammatory composition for relieving arthritis and gout, and the types and combinations of medicinal herbs constituting the mixed composition of at least seven edible herbs The pharmacological effect of is disclosed. However, the U.S. Patent Publication declaratively exemplifies in the summary and claims that Tabebuia impetiginosa , one of the Tabebuia plants, may constitute the seven edible herb mixture compositions, and the embodiment used as the specific mixture composition is not disclosed at all. There is no mention of the conduct of animal experiments and the identification of specific pharmacological effects. In particular, there is no disclosure or indication of the role of the Tahibo component in the expression of COX-2 and iNOS proteins involved in the production of prostagladin E2 and nitric oxide in macrophages.

In addition, there have been studies on the discovery of substances that exhibit anti-inflammatory effects by inhibiting COX-2 in macrophages and related mechanisms of action (final report of Chungbuk National University New Drug Development Project, NFD-37 that inhibits COX-2). Inflammation efficacy and mechanism of action), which is related to the anti-inflammatory efficacy and metabolism of furonaphthoquinone derived from alpha-viniferin extracted from native mirrors and its metabolism. It wasn't.

Thus, until now, it has been scientifically identified that the Tahibo component has analgesic and anti-inflammatory effects by inhibiting the expression of COX-2 protein and iNOS protein at the transcription level, thereby reducing prostaglandin E2 production and nitric oxide production in macrophages. One study was virtually absent.

The present invention has been made in response to the technical requirements as described above, as an inhibitor of the expression of COX-2 protein and iNOS protein involved in producing the inflammatory reactant prostaglandin E2 and nitric oxide, it provides an inhibitor consisting of Tahibo extract Its purpose is.

More specifically, an object of the present invention is to provide a composition for the prevention and treatment of inflammatory diseases, that is, analgesic composition and arthritis therapeutic composition containing a Tahibo extract as an active ingredient COX-2 protein and iNOS protein expression inhibitor.

It is also an object of the present invention to provide a health functional food effective for relieving pain and arthritis symptoms containing Tahibo extract as an active ingredient.

Furthermore, an object of the present invention is to provide an indicator for discovering a target protein of new inflammation-related metabolism as an inhibitor of expression of COX-2 and iNOS proteins consisting of tahibo extract.

COX-2 and iNOS protein expression inhibitors of the present invention consist of Tabebuia avellanedae extract. In addition, the composition for the prevention and treatment of inflammatory diseases of the present invention comprises a COX-2 and iNOS protein expression inhibitor consisting of the Tahibo extract as an active ingredient.

As described above, the composition for the prevention and treatment of inflammatory diseases of the present invention, that is, analgesic composition and arthritis therapeutic composition is inhibited expression of COX-2 and iNOS protein at the transcription level prostaglandin E2 and inflammatory reactants in macrophages and It inhibits the production of nitric oxide and ultimately exhibits anti-inflammatory or anti-inflammatory analgesic effects. In the following description, the tahibo extract is also referred to as Gapi-Taheebo.

As a result of careful consideration of the efficacy, anti-inflammatory effect, anti-inflammatory analgesic effect, and arthritis treatment effect of Tahibo as COX-2 and iNOS protein expression inhibitor, COX-2 and iNOS in an eluate diluted 1 to 10000 times of gapithabo The present invention was completed by observing the expression inhibitory effect at the transcription level of the protein. Therefore, the composition of the present invention preferably comprises 0.01% to 100% by weight of Tahibo extract based on the total weight of the composition.

On the other hand, in the therapeutic composition comprising the COX-2 and iNOS protein expression inhibitor of the present invention as an active ingredient, the Tahibo extract is cut or crushed to the appropriate size of the inner bark (bark) of Tahibo ( Tabebuia avellanedae ), heat The mixture is heated and heated at a temperature of about 90 ° C. to 95 ° C. for about 2 hours to 3 hours. In the present invention, such a hot water extract may be used as it is, or may be used as a purified extract after a conventional purification process including a filtration filter and chromatography. In addition, in the therapeutic composition of the present invention, the hot water extract and purified extract may be used as a powder by drying through a process such as distillation under reduced pressure, lyophilization, natural drying.

However, the extracts of the present invention are not limited to those obtained by the above method, but include those obtained by all known processing means used for preparing in an ingestible form. For example, of course, the ethanol extract extracted using ethanol is also included in the Tahibo extract.

In addition, Tabebuia avellanedae extract of the present invention that functions as an inhibitor of COX-2 and iNOS protein expression can be easily applied to food. The form of the food of the present invention is not particularly limited but may include drinks, beverages and the like. In addition, the tahibo extract of the present invention can be used in tea bags, capsules, powders, or tableting products, which are health functional foods.

The content of the tahibo extract in the food is not particularly limited, but in the case of general foods, it is preferable that the tahibo extract is contained in an amount of about 0.01 to 50% by weight relative to the final food weight, and in the case of the health functional food, the final food It is preferable that the tahibo extract is included from about 0.01 to 100% by weight based on the weight.

In addition, the therapeutic compositions of the Tahibo extract of the present invention may be formulated in a variety of oral dosage forms. Formulations for oral administration include, for example, tablets, pills, hard and soft capsules, solutions, suspensions, emulsifiers, syrups, granules, elixirs, and the like. Lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and / or glycine), glidants such as silica, talc, stearic acid and its magnesium or calcium salts and / or polyethylene glycols. . Tablets may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidine, optionally starch, agar, alginic acid or its Disintegrating or boiling mixtures such as sodium salts and / or absorbents, colorants, flavoring agents, and sweetening agents.

In addition, the therapeutic compositions of the Tahibo extract of the present invention may contain sterile and / or preservatives, stabilizers, emulsifiers or emulsifiers, auxiliaries such as salts and / or buffers for osmotic pressure control, and other therapeutically valuable substances. It may be formulated according to the conventional method of mixing, granulating or coating.

As described above, the Tahibo extract of the present invention inhibits COX-2 and iNOS protein expression at the transcription level.

In addition, the therapeutic composition containing the Tahibo extract of the present invention as an active ingredient inhibits the expression of COX-2 and iNOS proteins at the transcription level and ultimately inhibits the production of prostaglandin E2 and nitric oxide, which are inflammatory reactants in macrophages. Anti-inflammatory or anti-inflammatory analgesic efficacy.

In addition, the tahibo extract of the present invention may be used as an indicator for the discovery of target proteins of new inflammation-related metabolism as inhibitors of expression of COX-2 and iNOS proteins.

Hereinafter, the present invention will be described in detail according to embodiments which do not limit the present invention. The following examples of the present invention are not intended to limit or limit the scope of the present invention only to embody the present invention. Therefore, what can be easily inferred by the expert in the technical field to which this invention belongs from the detailed description and the Example of this invention is interpreted as belonging to the scope of the present invention.

Example 1.Preparation of Tahibo Extract

1 kg of inner bark (bark) (obtained from Nitive Health Food in Brazil) of Tahibo ( Tabebuia avellanedae ) is cut or ground to a suitable size and extracted by heating at 95 ° C. for 3 hours with a heated bath. The amount of the inner bark and the amount of water used in the hot water bath can be appropriately adjusted according to the desired concentration of the Tahibo extract. In addition, the extracted hot water extract is purified using a filtration filter. The purified extract was dried and concentrated under reduced pressure using a lyophilizer to obtain 35 g of an aqueous fraction of tahibo extract. The dried Tahibo extract was stored as a brown powder at room temperature.

Example 2. Cell Culture

In order to build an inflammatory response model as shown in Figure 1, a cell line RAW264.7 Macrophage (CRL-2278 ^TM ) provided by ATCC (Rockville, MD) was purchased from culture medium RPMI1640 (GIBCO) , Grand Island, NY) and culture dishes using 10% FBS (fetal bovine serum) (purchased from GIBCO, Grand Island, NY) and antibiotics (1/100 penicillinstreptomycin, Gibco-BRL cat 15140-122) (100 mm Dish, Nunc Cat 172958) and incubated at 37 ° C. in a 5% CO ₂ incubator.

Wash in serum-free RPMI medium (Rosewell Park Memorial Institute) in confluent state (cells sticking), then pipette without using Phosphate Buffered Saline (PBS) and PET After scraping off the bottom of the dish using the cells were collected and suspended, the cell count was measured with a hemocytometer and subcultured. However, there was a ± 10% error in viability measurement due to pipette damage in separating cells from the bottom of the dish. 10% DMSO was added as a freezing agent to the culture medium and stored at -196 ° C.

Example 3. Cell Viability Test

The tahibo extract obtained in Example 1 was prepared at each concentration (0, 50 µg / ml, 100 µg / ml, 200 µg / ml, 400 µg / ml), and then pre-cultured macrophages RAW264.7 (for 18 hours) 1 × 10 ⁶ cells / ml). Incubated for 24 hours after treatment. After incubation, 10 μl of MTT solution (10 mg / ml in phosphate buffered saline (PBS), pH 7.4) was added to the cultured cells, and after 3 hours, 15% SDS (in order to increase the solubility of formazan) Sodium dodecyl sulfate) was added to stop the reaction. Cytotoxicity studies of Tahibo extract were performed by conventional MTT assays (Lee, JY et al., 2007. Hydroquinone, a reactive metabolite of benzene, reduces macrophage-mediated immune responses.Mol Cells 23, 198-206), Optical density at 570 nm, ie absorbance, was measured using a Spectramax 250 microplate reader (FIG. 2). The data shown in FIG. 2 is an average value of the values measured through three repeated measurements.

As shown in FIG. 2, the tahibo extract did not show cytotoxicity even at a high concentration of 400 μg / ml.

Example 4-1. Inhibition of PEG2 Production

Cell suspensions with a cell concentration of 2 × 10 ⁶ cells / ml were inoculated in 2 ml / well units in a 24-well plate and incubated for 18 hours. The cell wash was then repeated twice with serum free medium (RPMI) to remove serum.

Lipopolysaccharide (LPS, sigma Cat # L4391) was diluted in RPMI medium to a concentration of 1 μg / ml, and then diluted LPS was administered to three wells in three wells. 500 μl of serum-free medium (RPMI) was added to the wells.Indometacin-administered group was diluted with 1M in RPMI1640 medium with Indometacin (Sigma cat. 065K1670). Used as a control. Tahibo was heated to 90 ℃ in a hot water bath for 2 hours to extract the aqueous solution to 1X, diluted 10 times, 100 times, 1000 times, 10000 times with RPMI 1640 medium and added 500μL to 24 wells.

After 6 hours after the addition of the samples having the components as shown in Figure 3 500μl of the culture was recovered. Cells and cultures were separated by centrifugation at a rate of 12000 RPM, and 400 μl supernatant was recovered from each group and used as a prostaglandin measurement sample.

The prostaglandin E2 EIA system kit (model name: Amersham RPN222) was purchased to confirm the presence of prostaglandin E2 in the supernatant recovered after LPS treatment of RAW264.7 macrophages.

First, to see non-specific binding (NSB), 100 μl of assay buffer is applied to the microplate, and 50 μl of assay buffer to the blank. Was administered.

The standard prostaglandin E2 group was sequentially diluted to 320 pg / ml, 160 pg / ml, 80 pg / ml, 40 pg / ml, 20 pg / ml, and 10 pg / m, and 50 µl was administered to each well. × 1) samples and samples diluted 100-fold, 1000-fold, 10000-fold were administered in 50 μl.

Next, 50 μl of the antibody was administered to the blank and standard prostaglandin E2 group and the measurement sample group, except for the nonspecific binding well, and then 50 μl of the conjugate was overall administered including the nonspecific binding well. .

Then, it was allowed to stand at room temperature for 1 hour, and then washed four times with a washing buffer, and 150 μl of TMB (substrate) was added to each well, followed by color development at room temperature for 30 minutes, followed by a stop solution (H ₂ SO). ₄ ) 100 μl was added to measure 450 nm absorbance to obtain a result as shown in FIG. 3.

According to the results of FIG. 3, the production of prostaglandin E2 decreases with increasing concentration of the composition comprising the tahibo extract of the present invention in macrophages stimulated to induce the secretion of prostaglandin E2 by LPS. It can be seen. Therefore, the Tahibo extract composition of the present invention inhibits the expression of COX-2 protein at the transcription level, inhibits the production of prostaglandin E2, an inflammatory reactant in macrophages, thereby inhibiting inflammatory reactions including arthritis and exhibiting analgesic effects. Confirmed.

Example 4-2. PEG2 production and COX-2 expression inhibition

In this example, a test similar to the test of Example 4-1 was performed to confirm that the tahibo extract of the present invention inhibited the production of prostaglandin E2 (PEG2) and simultaneously inhibited the expression of COX-2 protein at the transcription level.

First, the tahibo extract obtained in Example 1 was prepared for each concentration (0, 50 µg / ml, 100 µg / ml, 200 µg / ml, 400 µg / ml), and pre-cultured macrophages RAW264 for 18 hours. The Tahibo extract was treated at .7 (1 × 10 ⁶ cells / ml) at each concentration. And, 30 minutes after the tahibo extract treatment, the cells were treated with 2 μg / ml lipopolysaccharide (LPS). On the other hand, cells not treated with 2 μg / ml lipopolysaccharide (LPS) were used as negative controls. The cells were cultured for 24 hours after lipopolysaccharide treatment, and the supernatant was separated by centrifugation of the culture. In addition, the production amount of prostaglandin E2 for the obtained supernatant was measured using an ELISA kit (Amersham, Little Chalfont, Buckinghamshire, UK) according to a known method (Cho, JY et al., 2000. In vitro anti-inflammatory effects of cynaropicrin, a sesquiterpene lactone, from Saussurea lappa.Eur J Pharmacol 398, 399-407). The data shown in FIG. 4A is an average value of values measured through three repeated measurements.

As shown in Figure 4a, it was confirmed that the Tahibo extract of Example 1 inhibits the production of prostaglandin E2 by more than 90% at a concentration of 400 µg / ml. Therefore, it can be seen that the Tahibo extract has an effect of inhibiting the production and inflammatory response of prostaglandin E2 in a concentration-dependent manner.

Next, to evaluate cytokine mRNA expression level, TRIzol reagent (Gibco BRL) was prepared in macrophage RAW264.7 (1 × 10 ⁷ cells / ml) activated by lipopolysaccharide (LPS). Treatment according to company instructions yields total RNA. Total RNA was stored at -70 ° C. Then, the process of making a cDNA mixture using MuLV reverse transcriptase for the obtained mRNA was carried out as follows.

1 μg total RNA was incubated with oligo-dT15 at 70 ° C. for 5 min and mixed with 5 × 1 st-strand buffer. 10 mM dNTP and 0.1 M DTT were further treated at 37 ° C. for 5 minutes, followed by reaction for 60 minutes by the addition of MuLV reverse transcriptase (2 IU). The reactions were terminated at 70 ° C. for 10 minutes and total RNA was removed with RNase H. Then PCR incubation mixture [2 μl cDNA, 4 μM 5 ′ primer and 3 ′ primer, 10 × buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 0.1% Triton X-100), 250 μM dNTP, PCR was performed using 25 mM MgCl ₂ , 1 IU of Taq polymerase (Promega, USA) under the following conditions. On the other hand, the base sequences of the primers used in the PCR process (manufactured by Bione, Korea) are the same as SEQ ID NO: 3, 4, 5, 6:

Denaturation at 94 ° C. for 45 seconds;

Annealing at 55 ° C. to 60 ° C. for 45 seconds;

60 seconds extension at 72 ° C .; And

Last extension of 7 minutes at 72 ° C on the last of 30 cycles.

The PCR product was then electrophoresed on 1.5% agarose gel and the pattern analyzed (FIG. 4B).

As shown in Figure 4b, it can be seen that the Tahibo extract of Example 1 strongly inhibits the expression of COX-2 mRNA at a concentration of 400 µg / ml. Therefore, the Tahibo extract of Example 1 inhibits the expression of COX-2 protein at the transcription level, thereby inhibiting the production of prostaglandin E2, an inflammatory reactant in macrophages, and ultimately exhibits anti-inflammatory or anti-inflammatory analgesic efficacy. Can be.

Example 5 Nitric Oxide Production and iNOS Expression Inhibition

In this example, it was confirmed that the Tahibo extract of the present invention inhibits the production of nitric oxide (NO) in macrophages and inhibits the expression of iNOS (inducible nitric oxide synthase) protein at the transcription level. .

First, the tahibo extract obtained in Example 1 was prepared for each concentration (0, 50 µg / ml, 100 µg / ml, 200 µg / ml, 400 µg / ml), and pre-cultured macrophages RAW264 for 18 hours. The Tahibo extract was treated at .7 (1 × 10 ⁶ cells / ml) at each concentration. And, 30 minutes after the tahibo extract treatment, the cells were treated with 2 μg / ml lipopolysaccharide (LPS). On the other hand, cells not treated with 2 μg / ml lipopolysaccharide (LPS) were used as negative controls. Cells were incubated for 24 hours after lipopolysaccharide treatment, and the supernatant was separated by centrifugation of the culture. The production of nitrate ions (NO ₃ ⁻ ) as stable oxidation products in the obtained supernatant was measured. To this, 100 µl of the supernatant was added 100 µl of Griess reagent (0.1% N- (1-naphthyl) -ethylenediamine dihydrochloride dissolved in 1% sulfanylamide + 5% phosphoric acid). In addition, the amount of nitric oxide produced for the obtained supernatant was measured using an ELISA kit (Amersham, Little Chalfont, Buckinghamshire, UK) according to a known method (Cho, JY et al., 2000. In vitro anti-inflammatory effects of cynaropicrin, a sesquiterpene lactone, from Saussurea lappa.Eur J Pharmacol 398, 399-407). The data shown in FIG. 5A is an average value of values measured through three repeated measurements.

As shown in Figure 5a, it was confirmed that the Tahibo extract of Example 1 inhibits the generation of nitric oxide more than 70% at a concentration of 400㎍ / ml. Therefore, it can be seen that the Tahibo extract has an effect of inhibiting the production of nitric oxide and the inflammatory response in a concentration-dependent manner.

Next, macrophages RAW264.7 (1) activated by lipopolysaccharide (LPS) to evaluate mRNA expression levels of iNOS (inducible NO synthase) involved in the production of nitric oxide. TRIzol reagent (Gibco BRL) was treated according to the manufacturer's instructions in x10 ⁷ cells / ml to obtain total RNA. Total RNA was stored at -70 ° C. Then, the process of making a cDNA mixture using MuLV reverse transcriptase for the obtained mRNA was carried out as follows.

1 μg total RNA was incubated with oligo-dT15 at 70 ° C. for 5 min and mixed with 5 × 1 st-strand buffer. 10 mM dNTP and 0.1 M DTT were further treated at 37 ° C. for 5 minutes, followed by reaction for 60 minutes by the addition of MuLV reverse transcriptase (2 IU). The reactions were terminated at 70 ° C. for 10 minutes and total RNA was removed with RNase H. Then PCR incubation mixture [2 μl cDNA, 4 μM 5 ′ primer and 3 ′ primer, 10 × buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 0.1% Triton X-100), 250 μM dNTP, 25 mM MgCl ₂ , 1 IU Taq polymerase (Promega, USA)] using the PCR process was carried out under the following conditions. On the other hand, the base sequences of the primers used in the PCR (Bionia, Korea) are the same as SEQ ID NO: 1, 2, 5, 6:

Denaturation at 94 ° C. for 45 seconds;

Annealing at 55 ° C. to 60 ° C. for 45 seconds;

60 seconds extension at 72 ° C .; And

Last extension of 7 minutes at 72 ° C on the last of 30 cycles.

The PCR product was then electrophoresed on 1.5% agarose gel and the pattern analyzed (FIG. 5B).

As shown in Figure 5b, it can be seen that the Tahibo extract of Example 1 strongly inhibits the mRNA expression of iNOS at a concentration of 400 µg / ml. Thus, it was confirmed that the Tahibo extract of Example 1 inhibited the expression of iNOS protein at the transcription level, thereby inhibiting the production of nitric oxide, which is an inflammatory reactant in macrophages, and ultimately exhibiting anti-inflammatory or anti-inflammatory analgesic efficacy. .

Example 6. Cytotoxic Protective Effect

The tahibo extract obtained in Example 1 was prepared at each concentration (0, 50 µg / ml, 100 µg / ml, 200 µg / ml, 400 µg / ml), and then pre-cultured macrophages RAW264.7 (for 18 hours) 1 × 10 ⁶ cells / ml). After 30 minutes of the tahibo extract treatment, 2 μg / ml lipopolysaccharide (LPS) was treated and incubated for 24 hours. After incubation, 10 μl of MTT solution (10 mg / ml in phosphate buffered saline (PBS), pH 7.4) was added to the cultured cells, and after 3 hours, 15% SDS (in order to increase the solubility of formazan) Sodium dodecyl sulfate) was added to stop the reaction. Cytotoxic protective effect testing was performed by conventional MTT assays (Lee, JY et al., 2007. Hydroquinone, a reactive metabolite of benzene, reduces macrophage-mediated immune responses.Mol Cells 23, 198-206), 570nm Optical density at, i.e. absorbance, was measured using a Spectramax 250 microplate reader (FIG. 6). The data shown in FIG. 6 is an average value of values measured through three repeated measurements. As shown in Figure 6, the Tahibo extract of Example 1 inhibits the expression of iNOS protein at the transcription level, thereby inhibiting the production of nitric oxide, an inflammatory reactant in macrophages, and ultimately exhibiting anti-inflammatory or anti-inflammatory analgesic efficacy. In addition, it was confirmed to reduce the cytotoxicity caused by nitric oxide.

Example 7. Edema alleviation effect

The Tahibo extract composition of the present invention inhibits expression of COX-2 and iNOS proteins at the transcription level, thereby inhibiting the production of prostaglandin E2 and nitric oxide, which are inflammatory reactants in macrophages, thereby inhibiting edema in the ears of mice. The test was carried out as follows for efficacy.

1. Weight Change of ICR Mouse by Tahibo Extract

First, 6-week-old male ICR mice (B & K, Fremont, CA) were purchased to freely feed pelleted solid feed and water. The light was maintained at a 12 hour cycle and was bred according to the Guidelines for Breeding and Using Laboratory Animals (Revised 1985) published by the NIH.

The eight-week-old ICR mice were divided into four groups: the normal group (●), the vehicle administration group (▽), the indomethacin administration group (■), and the Tahibo extract administration group (◇) of Example 1, and the vehicle for 7 days. 5% CMC (carboxymethyl cellulose) was administered to the administration group, indomethacin was administered to the indomethacin administered group at a concentration of 1 mg / kg, and the tahibo extract of Example 1 was administered to the tahibo extract administration group at a concentration of 100 mg / kg. .

As a result, no specific body weight change of ICR mice was observed in the Tahibo extract administration group (FIG. 7A). Thus, the Tahibo extract of Example 1 was found to be suitable for animal model experiments.

2. Inhibitory Effect of Ear Edema on Mouse Ear by Tahibo Extract

The eight-week-old ICR mice were divided into four groups of normal group, vehicle group (control group), indomethacin group, and the Tahibo extract group of Example 1, twice a day for 7 days (10 am and 5 pm). 5% CMC (carboxymethyl cellulose) was administered to the vehicle-administered group, indomethacin was administered to the indomethacin-administered group at a concentration of 1 mg / kg, and tahibo of Example 1 at a concentration of 100 mg / kg to the tahibo extract-administered group. The extract was administered. Then, after the administration of each drug in the morning on the 7th day, 25 μl of 2% (w / v) arachidonic acid was applied to both surfaces of the left ear of the ICR mice of each group except the normal group. After the application of arachidonic acid, the thickness of the ears was measured using a dial thickness gauge at 1 hour and 4 hours, respectively.

In order to evaluate the anti-inflammatory efficacy of Tahibo extract, the effect of inhibiting ear edema by Tahibo extract was calculated by the following formula.

% Ratio to control = [(D _ET -N _ET ) / (C _ET -N _ET )] × 100

Meanwhile, in the above formula, D _ET is the ear thickness of each drug treatment group, N _ET is the ear thickness of the normal group, and C _ET is the ear thickness of the control group.

As a result of calculating the ear thickness measurement results of ICR mice in each group by the above formula, edema induced by arachidonic acid was suppressed by 80% at 1 hour after the tahibo extract of Example 1, 4 When time passed, it was confirmed that about 60% was suppressed (FIG. 7B). Thus, the tahibo extract composition of Example 1 inhibits the expression of COX-2 and iNOS proteins at the transcription level, thereby inhibiting the production of prostaglandin E2 and nitric oxide, which are inflammatory reactants in macrophages, resulting in edema in the ears of mice. It was found to have the effect of inhibiting.

Example 8 Animal Model Experiments Regarding Analgesic Efficacy

To specifically check whether the tahibo extract composition of the present invention exhibits anti-inflammatory analgesic efficacy by inhibiting the expression of COX-2 and iNOS proteins at the transcription level, inhibiting the production of prostaglandin E2 and nitric oxide, which are inflammatory reactants in macrophages. The following animal experiments were performed.

1. Breeding conditions of experimental animals

The test animals were supplied with ICR male mice (26 ± 3 g) and SD rats (150g ~ 180g) from Daehan Biolink Co., Ltd. to freely ingest solid food and water for experimental animals. The conditions for breeding were 21.6 ~ 24.9 ℃ and relative humidity was 42.8 ~ 52.4%. In addition, after only a week or so in the control room to adjust the contrast at a light intensity of 150 ~ 300 lux (Lux) 12 hours intervals to observe the visual symptoms were used only in the normal animals.

2. Preparation and Administration of Test Substance

The tahibo extract prepared in Example 1 was dissolved in distilled water at three doses of 2 mg / kg, 20 mg / kg and 200 mg / kg (based on the experimental animal body weight), respectively, and was forcibly administered to the experimental animal using an oral needle. (2 mg / kg / ml, 20 mg / kg / ml and 200 mg / kg / ml). Dosing volume was calculated according to the body weight of the experimental animals measured on the day of administration, the control group was administered only distilled water as a preparation solvent (10mg / kg / ml). Diclofenac (Diclofenac) was orally administered to the experimental animals suspended in distilled water as a preparation solvent at a dose of 25mg / kg.

3. Classification of experimental group and conditions for administration of test substance

The experimental animal group to which the test substance prepared above was administered was divided into a control group, Tahibo administration group, and diclofenac administration group, and the Tahibo administration group was further subdivided into three groups by dosage. Dosage and volume for each experimental group are shown in Table 1 below.

Table 1.

Military number Experimental group Dosage (mg / kg b. Wt.) Dosing volume (0.1 ml / 10 g b. Wt.) One Control 0 (Vehicle) 0.1 2 Tahibo 2 0.1 3 Tahibo 20 0.1 4 Tahibo 200 0.1 5 Diclofenac 25 0.1

4. Tahibo's Analgesic Effect Test

(1) Experiment on central nervous system analgesic effect

Thibo, an experimental substance, was orally administered to ICR male mice fasted 12 hours before the experiment. The pain response was carefully placed on a hot plate (Ugo Basile, Italy) maintained at 55 ° C and the time from the contact of the mouse on the hot plate to the licking or jumping of the hind paws (sec). It was measured and used as an indicator for central nervous system analgesic effect. Meanwhile, Diclofenac (25 mg / kg) was used as a control drug.

(2) Experiment on Peripheral Nerve Analgesic Effect

Thibo, an experimental substance, was orally administered to ICR male mice fasted 12 hours before the experiment. After 30 minutes of administration, 0.7% acetic acid-saline solution (0.1 mg / 10 g) was intraperitoneally injected into the mice, and 10 times after the injection, the number of writhing occurrences was measured. Meanwhile, Diclofenac (25 mg / kg) was used as a control drug.

5. Confirmation of the effect of Tahibo as an analgesic

(1) central nervous system analgesic effect

Thirty minutes after oral administration of the Tahibo extract, the hot plate pain response (to determine the central nervous system analgesic effect) was confirmed. The Tahibo 2mg / kg dose group showed similar results as the control group, but the Tahibo Extract 20mg / kg and The 200 mg / kg dose group showed excellent efficacy compared to the Diclofenac administration group, which is the analgesic control drug (see FIG. 8). In particular, the drug efficacy of Tahibo extract was shown to increase dose-dependently. In particular, the Tahibo extract dose of 200 mg / kg showed an analgesic effect almost equivalent to the control drug diclofenac (see Table 2 below). When orally administered 200 mg of Tahibo extract, the response time was 26.25 ± 1.83 seconds.

From these results, the Tahibo extract inhibits the expression of COX-2 and iNOS proteins at the transcription level, thereby inhibiting the production of prostaglandin E2 and nitric oxide, which are inflammatory reactants in macrophages, ultimately exhibiting central nervous system analgesic effects. do.

Table 2. Analgesic effects of Tahibo in hot plate test

(Validity level of significance: *: P <0.05; **: P <0.01)

(2) peripheral nerve analgesic effect

Thirty minutes after oral administration of the Tahibo extract, acetic acid was administered to the mice to confirm the peripheral nerve analgesic effect. The Tahibo extract 2mg / kg dose group showed similar results to the control group, but the Tahibo Extract 20mg / kg and 200mg In the / kg dose administration group, compared to the analgesic control drug Diclofenac (Diclofenac) and commercially available SK-Joins administration group showed excellent efficacy (see Fig. 9). In particular, the drug efficacy of Tahibo extract was shown to increase dose-dependently. In particular, the Tahibo dose of 200 mg / kg showed an analgesic effect almost equivalent to the reference SK-Join tablet (see Table 3 below). When oral administration of 200 mg tahibo extract was confirmed that the suppressive effect is significant to 37%.

From these results, the Tahibo extract inhibits the expression of COX-2 and iNOS proteins at the transcription level, thereby inhibiting the production of prostaglandin E2 and nitric oxide, which are inflammatory reactants in macrophages. do.

Table 3. Tahibo Effect on Abdominal Contractile Analgesia Induced by Acetic Acid

(Validity level of significance: *: P <0.05; **: P <0.01)

Example 9 Animal Model Experiments Regarding Efficacy of Arthritis Therapeutics

Whether the tahibo extract composition of the present invention inhibits the expression of COX-2 and iNOS proteins at the transcription level, thereby inhibiting the production of prostaglandin E2 and nitric oxide, which are inflammatory reactants in macrophages, and thus have anti-inflammatory (ie, arthritis therapeutic) efficacy. In order to confirm specifically, the following animal experiments were performed.

On the other hand, the breeding conditions of the experimental animals, the preparation and administration of the test substance, and the experimental group classification and test substance administration conditions were the same as in Example 8.

1. Experiments on Acute Foot Edema Induced by Carrageenan in Rats

Using 150-170g SD male rats (rats) fasted 12 hours before the experiment, 0.1% type IV lambda carrageenan-physiological saline solution (1% type IV lambda carrageenan-saline) as a inflammatory agent (inflammatory material) Aml was injected subcutaneously in the plantar central part of the right hind paw of the rat to cause acute foot edema. The edema increase rate was calculated by measuring the volume of the hind paw at 1 hour, 2 hours, 3 hours and 4 hours after the basement administration and comparing it with the volume of the foot before the basement administration.

2. Confirmation of Tahibo's Effect on Acute Foot Edema

After 60 minutes of oral administration of Tahibo extract to SD male rats, carrageenan, which is a inflammatory agent, was administered to the center of the plantar body, and the rate of inflammation increase until 1 hour, 2 hours, 3 hours, and 4 hours was calculated. The degree of inflammation inhibition was confirmed (see FIG. 10). The Tahibo extract 200 mg / kg dose group showed statistical significance compared to the control group SK-Join administration group (see Table 4 below). In particular, the drug efficacy of Tahibo extract was shown to increase dose-dependently. When oral administration of 200 mg tahibo extract significantly increased the edema growth rate (57.86 ± 2.43; 3 hours after the administration of the baseline drug), it was confirmed that the anti-edema effect was greatly exerted.

From these results, Tahibo extract inhibits the expression of COX-2 and iNOS proteins at the transcription level, thereby inhibiting the production of prostaglandin E2 and nitric oxide, which are inflammatory reactants in macrophages. It is believed to have a therapeutic effect on the species (acute arthritis).

Table 4. Tahibo Effects on Carrageenan-Induced Foot Edema

(Validity level of significance: *: P <0.05; **: P <0.01)

Although the present invention has been described in accordance with the above-described embodiments, the present invention is not limited to the disclosed embodiments. Those skilled in the art will appreciate that modifications can be made in accordance with the spirit of the invention, and such modifications also fall within the scope of the invention.

1 is a macrophage by the external stimulation to produce an arachidonic acid in the cell to start the inflammatory response, COX-1 (cyclooxygenase-1), COX-2 (cyclooxygenase-2) is expressed in metabolism of arachidonic acid It is a schematic diagram illustrating a model in which inflammation and arthritis are caused by the involvement of prostaglandin E ₂ as a metabolite of arachidonic acid by involvement.

Figure 2 is a graph showing the cell survival rate of macrophages at 24 hours after the tahibo extract of Example 1 was treated to macrophages.

3 is an experimental result graph showing that the production of prostaglandin E2 decreases in a concentration-dependent manner as the concentration of the composition containing the tahibo extract of the present invention is increased in macrophages stimulated to induce the secretion of prostaglandin E2 by LPS. to be.

4A is a graph showing experimental results showing that the tahibo extract of Example 1 reduced the production of prostaglandin E2 in a concentration-dependent manner.

Figure 4b is a photograph of the electrophoresis results showing that the Tahibo extract of Example 1 inhibits the expression of COX-2 protein involved in the production of prostaglandin E2 at the transcriptional level.

Figure 5a is a graph of the experimental results showing that the Tahibo extract of Example 1 reduces the production of nitric oxide in a concentration-dependent manner.

Figure 5b is a photograph of the electrophoresis results showing that the Tahibo extract of Example 1 inhibits the expression of iNOS protein involved in the production of nitric oxide at the transcriptional level.

6 is a graph showing the results of experiment showing that the tahibo extract of Example 1 has a cytotoxic effect of reducing the cytotoxicity by nitric oxide, that is, cytotoxicity.

Figure 7a is a graph of the experimental results showing that the Tahibo extract of Example 1 does not affect the weight change of the animal model ICR mouse.

Figure 7b is a graph of the experimental results showing that the tahibo extract of Example 1 inhibits the edema occurring in the ears of the animal model ICR mouse.

8 is a graph showing the results of experiments showing that the Tahibo extract of Example 1 suppresses central nerve pain.

9 is a graph showing the results of experiments showing that Tahibo extract of Example 1 suppresses peripheral nerve pain.

10 is a graph showing experimental results showing that the tahibo extract of Example 1 alleviates acute foot edema.

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Claims (17)

As an inhibitor of expression of COX-2 and iNOS proteins, An inhibitor of the expression of COX-2 and iNOS proteins, consisting of Tabebuia avellanedae extracts that inhibit the production of prostaglandin E2 and nitric oxide in macrophages by inhibiting expression of COX-2 and iNOS proteins at the transcription level. The method of claim 1, The tahibo extract is an inhibitor of the expression of COX-2 and iNOS proteins, characterized in that the hydrothermal extract extracted by heating the inner bark of Tahibo ( Taeboia avellanedae ) at a temperature of 90 ℃ to 95 ℃ for 2 hours to 3 hours. The method of claim 2, The hot water extract is an inhibitor of the expression of COX-2 and iNOS protein, characterized in that the dry powder is prepared through a drying process. Inflammatory disease therapeutic composition comprising an inhibitor of the expression of COX-2 and iNOS protein consisting of Tabebuia avellanedae extract. The method of claim 4, wherein The inflammatory disease is an inflammatory disease therapeutic composition, characterized in that arthritis. The method of claim 4, wherein Inflammatory disease therapeutic composition comprising 0.01 to 100% by weight of the Tahibo extract based on the total weight of the composition. The method of claim 4, wherein The tahibo extract is an inflammatory disease therapeutic composition, characterized in that the hot water extract extracted by heating the inner bark of Tahibo ( Taeboia avellanedae ) at a temperature of 90 ℃ to 95 ℃ for 2 hours to 3 hours. The method of claim 7, wherein The hot water extract is an inflammatory disease therapeutic composition, characterized in that it is manufactured in a dry powder form through a drying process. The method according to any one of claims 4 to 8, An inflammatory disease therapeutic composition, characterized in that it is formulated in a dosage form for oral administration such as tablets, pills, hard and soft capsules, solutions, suspensions, emulsifiers, syrups, granules, elixirs. An analgesic composition containing an inhibitor of the expression of COX-2 and iNOS protein consisting of Tabhibuia avellanedae extract as an active ingredient. The method of claim 10, Analgesic composition, characterized in that it comprises 0.01 to 100% by weight of the Tahibo extract relative to the total weight of the composition. The method of claim 10, The tahibo extract is an analgesic composition characterized in that the hot water extract extracted by heating the inner bark of Tahibo ( Tabebuia avellanedae ) in a hot water bath at a temperature of 90 ℃ to 95 ℃ for 2 hours to 3 hours. The method of claim 10, The hot water extract is an analgesic composition characterized in that it is manufactured in a dry powder form through a drying process. The method according to any one of claims 10 to 13, An analgesic composition characterized in that it is formulated in an oral dosage form such as tablets, pills, hard and soft capsules, liquids, suspensions, emulsifiers, syrups, granules, elixirs. Food for the improvement of inflammatory diseases comprising an inhibitor of the expression of COX-2 and iNOS protein consisting of Tabebuia avellanedae extract. The method of claim 15, The inflammatory disease is food, characterized in that arthritis. The method according to claim 15 or 16, The food is characterized in that the form of drinks, beverages, tea bags (tea bags), capsules, powders, or tablets.

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